AU2013202769A1 - Microorganisms and methods for the biosynthesis of aromatics, 2,4-pentadienoate and 1,3-butadiene - Google Patents

Abstract

The invention provides non-naturally occurring microbial organisms having a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-1ol or 1,3-butadiene pathway. The invention additionally provides methods of using such organisms to produce toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-1ol or 1,3-butadiene .

Description

AUSTRALIA Patents Act 1990 GENOMATICA, INC. COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Microorganisms and methods for the biosynthesis of aromatics, 2,4 pentadienoate and 1,3-butadiene The following statement is a full description of this invention including the best method of performing it known to us:- 2 MICROORGANISMS AND METHODS FOR THE BIOSYNTHESIS OF AROMATICS, 2,4-PENTADIENOATE AND 1,3-BUTADIENE BACKGROUND OF THE INVENTION The present invention relates generally to biosynthetic processes, and more specifically to organisms having toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic capability. Toluene is a common solvent that has replaced benzene due to benzene's greater carcinogenicity and is an industrial feedstock and used in the manufacture of TNT, polyurethane foam, benzaldehyde and benzoic acid. Toluene is a byproduct in the manufacture of gasoline and exists in small concentrations in crude oil. Benzene is often used as an intermediate to make other chemicals. Its most widely produced derivatives include styrene, which is used to make polymers and plastics, phenol for resins and adhesives, via cumene, and cyclohexane, which is used in the manufacture of Nylon. Benzene is also used to make some types of rubbers, lubricants, dyes, detergents, drugs, explosives, napalm and pesticides. Benzene production in the petroleum industry is made by various energy intensive processes including, catalytic reforming, toluene hydrodealkylation, toluene disproportionation, and steam cracking. Styrene is the precursor to polystyrene and numerous copolymers. Styrene based products include, acrylonitrile 1,3-butadiene styrene (ABS), styrene-1,3-butadiene (SBR) rubber, styrene-1,3-butadiene latex, SIS (styrene-isoprene-styrene), S-EB-S (styrene ethylene/butylene-styrene), styrene-divinylbenzene (S-DVB), and unsaturated polyesters. These materials are used in rubber, plastic, insulation, fiberglass, pipes, automobile and boat parts, food containers, and carpet backing. Styrene is most commonly produced by the catalytic dehydrogenation of ethylbenzene. Ethylbenzene is mixed in the gas phase with 10-15 times its volume in high temperature steam, and passed over a solid catalyst bed. Most ethylbenzene dehydrogenation catalysts are based on iron(III) oxide, promoted by several percent potassium oxide or potassium carbonate. Steam serves several roles in this reaction. It is the source of heat for powering the endothermic reaction, and it removes coke that tends to form on the iron oxide 3 catalyst through the water gas shift reaction. The potassium promoter enhances this decoking reaction. The steam also dilutes the reactant and products, shifting the position of chemical equilibrium towards products. A typical styrene plant consists of two or three reactors in series, which operate under vacuum to enhance the conversion and selectivity. Typical per pass conversions are ca. 65% for two reactors and 70-75% for three reactors. Over 25 billion pounds of 1,3-butadiene (or just butadiene or BD) are produced annually and is applied in the manufacture of polymers such as synthetic rubbers and ABS resins, and chemicals such as hexamethylenediamine and 1,4-butanediol. 1,3-butadiene is typically produced as a by-product of the steam cracking process for conversion of petroleum feedstocks such as naphtha, liquefied petroleum gas, ethane or natural gas to ethylene and other olefins. The ability to manufacture 1,3-butadiene from alternative and/or renewable feedstocks would represent a major advance in the quest for more sustainable chemical production processes One possible way to produce 1,3-butadiene renewably involves fermentation of sugars or other feedstocks to produce diols, such as 1,4-butanediol or 1,3-butanediol, which are separated, purified, and then dehydrated to 1,3-butadiene in a second step involving metal-based catalysis. Direct fermentative production of 1,3-butadiene from renewable feedstocks would obviate the need for dehydration steps and 1,3-butadiene gas (bp -4.4'C) would be continuously emitted from the fermenter and readily condensed and collected. Developing a fermentative production process would eliminate the need for fossil-based 1,3 butadiene and would allow substantial savings in cost, energy, and harmful waste and emissions relative to petrochemically-derived 1,3-butadiene. 2,4-Pentadienoate is a useful substituted butadiene derivative in its own right and a valuable intermediate en route to other substituted 1,3-butadiene derivatives, including, for example, 1-carbamoyl-1,3-butadienes which are accessible via Curtius rearrangement. The resultant N-protected-1,3-butadiene derivatives can be used in Diels alder reactions for the preparation of substituted anilines. 2,4-Pentadienoate can be used in the preparation of various polymers and co-polymers. Terephthalate (also known as terephthalic acid and PTA) is the immediate precursor of polyethylene terephthalate (PET), used to make clothing, resins, plastic bottles and even as a poultry feed additive. Nearly all PTA is produced from para-xylene by oxidation in air in a 4 process known as the Mid Century Process. This oxidation is conducted at high temperature in an acetic acid solvent with a catalyst composed of cobalt and/or manganese salts. Para xylene is derived from petrochemical sources and is formed by high severity catalytic reforming of naphtha. Xylene is also obtained from the pyrolysis gasoline stream in a naphtha steam cracker and by toluene disproportion. Cost-effective methods for generating renewable PTA have not yet been developed to date. PTA, toluene and other aromatic precursors are naturally degraded by some bacteria. However, these degradation pathways typically involve monooxygenases that operate irreversibly in the degradative direction. Hence, biosynthetic pathways for PTA are severely limited by the properties of known enzymes to date. A promising precursor for PTA is p-toluate, also known as p-methylbenzoate. P Toluate is an intermediate in some industrial processes for the oxidation ofp-xylene to PTA. It is also an intermediate for polymer stabilizers, pesticides, light sensitive compounds, animal feed supplements and other organic chemicals. Only slightly soluble in aqueous solution, p-toluate is a solid at physiological temperatures, with a melting point of 275 *C. Microbial catalysts for synthesizing this compound from sugar feedstocks have not been described to date. Thus, there exists a need for alternative methods for effectively producing commercial quantities of compounds such as styrene, 2,4-pentadienoate, 1,3-butadiene, p-toluate, terephthalate, benzene and toluene. The present invention satisfies this need and provides related advantages as well. SUMMARY OF THE INVENTION The invention provides non-naturally occurring microbial organisms having a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene- 1 ol or 1,3-butadiene pathway. The invention additionally provides methods of using such organisms to produce toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene .

5 The invention also provides non-naturally occurring microbial organisms having a (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M40P) pathway, a p- toluate pathway, a terephthalate pathway, a (2-hydroxy-4-oxobutoxy)phosphonate (2H40P) pathway, and/or a benzoate pathway. The invention additionally provides methods of using such organisms to produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, p- toluate, terephthalate, (2 hydroxy-4-oxobutoxy)phosphonate, or benzoate. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the conversion of phenylalanine to toluene via phenylacetate. Enzymes are A. phenylalanine aminotransferase and/or phenylalanine oxidoreductase (deaminating), B. phenylpyruvate decarboxylase, C. phenylacetaldehyde dehydrogenase and/or oxidase, D. phenylacetate decarboxylase, E. phenylacetaldehyde decarbonylase, and F. phenylpyruvate oxidase. Figure 2 shows the conversion of phenylalanine to benzene by phenylalanine benzene-lyase. Figure 3 shows pathways to styrene from benzoyl-CoA. Enzymes are: A. benzoyl CoA acetyltransferase, B. 3-oxo-3-phenylpropionyl-CoA synthetase, transferase and/or hydrolase, C. benzoyl-acetate decarboxylase, D. acetophenone reductase and E. 1 phenylethanol dehydratase, F. phosphotrans-3-oxo-3-phenylpropionylase, G. benzoyl-acetate kinase. Figure 4 shows the conversion of muconate stereoisomers to 1,3-butadiene. Enzymes are A. trans, trans-muconate decarboxylase, B. cis, trans-muconate cis-decarboxylase, C. cis, trans-muconate trans-decarboxylase, D. cis, cis-muconate decarboxylase, E. trans-2,4 pentadienoate decarboxylase, F. cis-2,4-pentadienoate decarboxylase. Figure 5 shows a schematic depiction of an exemplary pathway to (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate (2H3M40P) from glyceraldehyde-3-phosphate and pyruvate. G3P is glyceraldehyde-3-phosphate, DXP is 1-deoxy-D-xylulose-5-phosphate and 2ME4P is C-methyl-D-erythritol-4-phosphate. Enzymes are (A) DXP synthase; (B) DXP reductoisomerase; and (C) 2ME4P dehydratase.

6 Figure 6 shows a schematic depiction of an exemplary alternate shikimate pathway to p-toluate. Enzymes are: (A) 2-dehydro-3-deoxyphosphoheptonate synthase; (B) 3 dehydroquinate synthase; (C) 3-dehydroquinate dehydratase; (D) shikimate dehydrogenase; (E) Shikimate kinase; (F) 3-phosphoshikimate-2-carboxyvinyltransferase; (G) chorismate synthase; and (H) chorismate lyase. Compounds are: (1) (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate; (2) 2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2 carboxylate; (3) 1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate; (4) 5-hydroxy-4 methyl-3-oxocyclohex-1-ene-1-carboxylate; (5) 3,5-dihydroxy-4-methylcyclohex-1-ene-1 carboxylate; (6) 5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate; (7) 5 [(1 -carboxyeth- 1-en-i -yl)oxy] -4-methyl-3 -(phosphonooxy)cyclohex- 1 -ene- 1 -carboxylate; (8) 3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate; and (9)p toluate. Figure 7 shows an exemplary pathway for conversion ofp-toluate to terephthalic acid (PTA). Reactions A, B and C are catalyzed by p-toluate methyl-monooxygenase reductase, 4-carboxybenzyl alcohol dehydrogenase and 4-carboxybenzyl aldehyde dehydrogenase, respectively. The compounds shown are (1)p-toluic acid; (2) 4-carboxybenzyl alcohol; (3) 4-carboxybenzaldehyde and (4) terephthalic acid. Figure 8 shows an exemplary pathway to (2-hydroxy-4-oxobutoxy)phosphonate from erythrose-4-phosphate. Enzymes are: A. erythrose-4-phosphate dehydratase, B. (2,4 dioxobutoxy)phosphonate reductase. Compounds are: (1) erythrose-4-phosphate, (2) (2,4 dioxobutoxy)phosphonate and (3) (2-hydroxy-4-oxobutoxy)phosphonate. Figure 9 shows an alternate shikimate pathway from (2-hydroxy-4 oxobutoxy)phosphonate to benzoate. Enzymes are: A. 2-dehydro-3-deoxyphosphoheptonate synthase, B. 3-dehydroquinate synthase, C. 3-dehydroquinate dehydratase, D. shikimate dehydrogenase, E. shikimate kinase, F. 3-phosphoshikimate-2-carboxyvinyltransferase , G. chorismate synthase, H. chorismate lyase. Compounds are: 1. (2-hydroxy-4 oxobutoxy)phosphonate, 2. 2,4-dihydroxy-6-[(phosphonooxy)methyl]oxane-2 carboxylate, 3. 1,3-dihydroxy-5-oxocyclohexane-1-carboxylate, 4. 5-hydroxy-3 oxocyclohex-1-ene-1-carboxylate, 5. 3,5-dihydroxycyclohex-1-ene-1-carboxylate, 6. 5 hydroxy-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate, 7. 5-[(1-carboxyeth-1-en-1 yl)oxy]-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate, 8. 3-[(1-carboxyeth-1-en-1 yl)oxy]cyclohexa-1,5-diene-1-carboxylate, 9. benzoate.

7 Figure 10 shows pathways from benzoate and benzoyl-CoA to benzene. Enzymes are A. benzoyl-CoA synthetase, transferase and/or hydrolase, B. benzoate reductase, C. benzaldehyde decarbonylase, D. benzoyl-CoA reductase, E. benzoate decarboxylase, F. phosphotransbenzoylase, G. (benzoyloxy)phosphonate reductase (dephosphorylating), H. benzoate kinase. Figure 11 shows pathways from p-toluate (also called p-toluic acid) and p methylbenzoyl-CoA to toluene. Enzymes are A. p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase, B. p-toluate reductase, C. p-methylbenzaldehyde decarbonylase, D. p-methylbenzoyl-CoA reductase, E. p-toluate decarboxylase, F. phosphotrans-p methylbenzoylase, G. (p-methylbenzoyloxy)phosphonate reductase (dephosphorylating), H. p-toluate kinase. Figure 12 shows pathways to 2,4-pentadienoate from pyruvate. Enzymes are A. 4 hydroxy-2-oxovalerate aldolase, B. 4-hydroxy-2-oxovalerate dehydratase, C. 2 oxopentenoate reductase, D. 2-hydroxypentenoate dehydratase, E. 4-hydroxy-2-oxovalerate reductase, F. 2,4-dihydroxypentanoate 2-dehydratase, G. 4-hydroxypent-2-enoate dehydratase and H. 2,4-dihydroxypentanoate 4-dehydratase. Figure 13 shows pathways from alanine and omithine to 2,4-pentadienoate. Enzymes are A. AKP thiolase, B. AKP deaminase, C. acetylacrylate reductase , D. 4-hydroxypent-2 enoate dehydratase, E. AKP aminotransferase and/or dehydrogenase, F. 2-hydroxy-4 oxopentanoate dehydratase, G. 2,4-dihydroxypentanoate 2-dehydratase, H. 2,4 dioxopentanoate 2-reductase, I. 2-hydroxy-4-oxopentanoate reductase, J. AKP reductase, K. 2,4-dioxopentanoate 4-reductase, L. 2-amino-4-hydroxypentanoate aminotransferase and/or dehydrogenase, M. ornithine 4,5-aminomutase, N. 2,4-diaminopentanoate 4-aminotransferase and/or 4-dehydrogenase. AKP is 2-amino-4-oxopentanoate. Figure 14 shows additional pathways from omithine to 2,4-pentadienoate. Enzymes are A. ornithine 2,3-aminomutase, B. 3,5-diaminopentanoate deaminase, C. 5-aminopent-2 enoate deaminase, D. 3,5-diaminopentanoate aminotransferase and/or dehydrogenase, E. 3 amino-5-oxopentanoate deaminase, F. 5-oxopent-2-enoate reductase, G. 5-hydroxypent-2 enoate dehydratase, H. 5-aminopent-2-enoate aminotransferase and/or dehydrogenase, I. 3 amino-5-oxopentanoate reductase, J. 3-amino-5-hydroxypentanoate deaminase.

8 Figure 15 shows pathways from 3-hydroxypropanoyl-CoA and/or acrylyl-CoA to 2,4 pentadienoate. Enzymes are A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5 hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5 hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, H. 5 hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5 hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, K. 3 hydroxypropanoyl-CoA dehydratase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, 0. 3-oxopent-4-enoyl CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, Q. 5 hydroxypent-2-enoate dehydratase, R. 3-hydroxypent-4-enoyl-CoA dehydratase, S. 3 hydroxypent-4-enoate dehydratase. 3-HP-CoA is 3-hydroxypropanoyl-CoA. Figure 16 shows the formation of butadiene from 3-hydroxypent-4-enoate (3HP4) by 3-hydroxypent-4-enoate decarboxylase. Figure 17 shows the formation of butadiene from 3,5-dihydroxypentanoate by 3,5 dihydroxypentanoate decarboxylase and 3-butene-1-ol dehydratase. Dehydration of 3 butene-1-ol to butadiene can also occur via chemical catalysis. Figure 18 shows the formation of the 3-hydroxypent-4-enoate (3HP4) intermediate from 2,4-pentadienoate via 2,4-pentadienoate hydratase. Figure 19 shows pathways to butadiene, 3-hydroxypent-4-enoate (3HP4), 2,4 pentadienoate and 3-butene-1-ol from 3-HP-CoA and/or acrylyl-CoA. Enzymes are A. 3 hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase, F. 3-oxo-5 hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, G. 3,5-dihydroxypentanoyl CoA synthetase, transferase and/or hydrolase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5 dihydroxypentanoate dehydratase, K. 3-hydroxypropanoyl-CoA dehydratase, L. 3-oxo-5 hydroxypentanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4 enoyl-CoA reductase, 0. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P.

9 3-oxopent-4-enoate reductase, Q. 5-hydroxypent-2-enoate dehydratase, R. 3-hydroxypent-4 enoyl-CoA dehydratase, S. 3 -hydroxypent-4-enoate dehydratase, T. 3-hydroxypent-4-enoyl CoA transferase, synthetase or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase, V. 5 hydroxypent-2-enoate decarboxylase, W. 3-butene- 1 -ol dehydratase (or chemical conversion), X. 2,4-pentadiene decarboxylase, Y. 3-hydroxypent-4-enoate decarboxylase. 3 HP-CoA is 3-hydroxypropanoyl-CoA. Figure 20 shows pathways to 3-hydroxypent-4-enoate (3HP4), 2,4-pentadienoate and butadiene from succinyl-CoA. Enzymes are A. succinyl-CoA:acetyl-CoA acyltransferase, B. 3-oxoadipyl-CoA transferase, synthetase or hydrolase, C. 3-oxoadipate dehydrogenase, D. 2 fumarylacetate decarboxylase, E. 3-oxopent-4-enoate reductase, F. 3-hydroxypent-4-enoate dehydratase, G. 3-oxoadipyl-CoA reductase, H. 3-hydroxyadipyl-CoA transferase, synthetase or hydrolase, I. 3-hydroxyadipate dehydrogenase, J. 3-hydroxyhex-4-enedioate decarboxylase, K. 3-oxoadipate reductase, L. 2-fumarylacetate reductase., M. 3-hydroxypent 4-enoate decarboxylase, N. 2,4-pentadienoate decarboxylase. Figure 21 shows pathways to 3-butene-1-ol, butadiene and 2,4-pentadienoate from malonyl-CoA and acetyl-CoA. Enzymes for transformation of the identified substrates to products include: A. malonyl-CoA:acetyl-CoA acyltransferase, B. 3-oxoglutaryl-CoA reductase (ketone-reducing), C. 3-hydroxyglutaryl-CoA reductase (aldehyde forming), D. 3 hydroxy-5-oxopentanoate reductase, E. 3,5-dihydroxypentanoate dehydratase, F. 5 hydroxypent-2-enoate dehydratase, G. 3-hydroxyglutaryl-CoA reductase (alcohol forming), H. 3-oxoglutaryl-CoA reductase (aldehyde forming), I. 3,5-dioxopentanoate reductase (aldehyde reducing), J. 5-hydroxy-3-oxopentanoate reductase, K. 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming), L. 3,5-dioxopentanoate reductase (ketone reducing), M. 3,5-dihydroxypentanoate decarboxylase, N. 5-hydroxypent-2-enoate decarboxylase, 0. 3-butene-1-ol dehydratase (or chemical conversion), P. 2,4-pentadiene decarboxylase. Figure 22 shows the reverse TCA cycle for fixation of CO 2 on carbohydrates as substrates. The enzymatic transformations are carried out by the enzymes as shown. Figure 23 shows the pathway for the reverse TCA cycle coupled with carbon monoxide dehydrogenase and hydrogenase for the conversion of syngas to acetyl-CoA.

10 Figure 24 shows Western blots of 10 micrograms ACS90 (lane 1), ACS91 (lane2), Mta98/99 (lanes 3 and 4) cell extracts with size standards (lane 5) and controls of M. thermoacetica CODH (Moth_1202/1203) or Mtr (Moth 1197) proteins (50, 150, 250, 350, 450, 500, 750, 900, and 1000 ng). Figure 25 shows CO oxidation assay results. Cells (M. thermoacetica or E. coli with the CODH/ACS operon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extracts prepared. Assays were performed at 55 'C at various times on the day the extracts were prepared. Reduction of methylviologen was followed at 578 nm over a 120 sec time course. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed, in part, to the design and production of cells and organisms having biosynthetic production capabilities for toluene, benzene, styrene, 2,4 pentadienoate and 1,3-butadiene. The routes to toluene and benzene, Figures 1 and 2, begin with the naturally occurring amino acid phenylalanine and, thus, most organisms will be capable of serving as a host for the construction of a non-naturally occurring organism for the production of toluene and benzene. Strategies for enhancing phenylalanine production are known in the art (Yakandawala et al., App. Microbiol. Biotech. 78:283-291 (2008); Lee et al., US Patent 5,008,190). The route to styrene relies on an organism for generating benzoyl-CoA, as indicated in Figure 3. Benzoyl-CoA is a key metabolic intermediate of numerous biosynthetic and degradation pathways. Benzoyl-CoA is a key precursor of aromatic natural products such as antibiotics, aromas and defense signals. Biological pathways of benzoyl-CoA biosynthesis are known in the art (Boatright et al., Plant Physiol 135:1993-2011 (2004); Xiang et al., J Bacteriol. 185:399-404 (2003); Moore et al., J Nat.Prod 65:1956-1962 (2002)). Benzoyl CoA is also a common intermediate of anaerobic and aerobic aromatic compound degradation pathways (Gescher et al., J Bacteriol. 184:6301-6315 (2002); Philipp et al., FEMS Microbiol Lett. 212:139-143 (2002); Koch et al., Eur.J Biochem. 205:195-202 (1992)). This invention is also directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze 1,3-butadiene production, as shown in Figure 4. In some embodiments, pathways for the production of muconate are derived from central 11 metabolic precursors. Muconate is a common degradation product of diverse aromatic compounds in microbes. Several biocatalytic strategies for making cis,cis-muconate have been developed. Engineered E. coli strains producing muconate from glucose via shikimate pathway enzymes have been developed in the Frost lab (U.S. Patent 5,487,987 (1996); Niu et al., Biotechnol Prog., 18:201-211 (2002)). These strains are able to produce 36.8 g/L of cis,cis-muconate after 48 hours of culturing under fed-batch fermenter conditions (22% of the maximum theoretical yield from glucose). Muconate has also been produced biocatalytically from aromatic starting materials such as toluene, benzoic acid and catechol. Strains producing muconate from benzoate achieved titers of 13.5 g/L and productivity of 5.5 g/L/hr (Choi et al., J. Ferment. Bioeng. 84:70-76 (1997)). Muconate has also been generated from the effluents of a styrene monomer production plant (Wu et al., Enzyme and Microbiology Technology 35:598-604 (2004)). This invention is also directed, in part, to non-naturally occurring microorganisms that express genes encoding enzymes that catalyze 2,4-pentadienoate production, as shown in Figures 12-15. Any of these pathways can feed into a further 1,3-butadiene pathway by inclusion of the requisite 2,4-pentadienoate decarboxylase. Figure 12 shows the overall conversion of pyruvate to 2,4-pentadienoate by three pathways. Figure 13 shows the overall conversion of ornithine or alanine to 2,4-pentadienoate via common intermediate 2-amino-4 ketopentanoate (AKP). Figure 13 shows six routes to 2,4-pentadienoate from AKP, three of which intercept intermediates shown in Figure 12. Figure 14 shows four additional routes to 2,4-pentadienoate from ornithine. Figure 15 shows numerous routes to 2,4-pentadienoate from 3-hydroxypropanoyl-CoA (3-HP-CoA) and acryloyl-CoA. The invention is also directed, in part, to non-naturally occurring microbial organisms that express genes encoding enzymes that catalyze 1,3-butadiene production, as shown in Figures 16-17 and 19-21. Figure16 shows the decarboxylative dehydration of 3 hydroxypent-4-enoate (3HP4) to 1,3-butadiene, where 3HP4 is available via pathways shown in Figures 15 and 19. 3HP4, being important in its own right, is shown in Figure 18 via intermediate 2,4-pentadienoate via hydration, as well as the via the pathways of Figures 15, 19, and 20. Likewise, Figure 17 shows the tandem decarboxylative dehydration and elimination (further dehydration) of intermediate 3,5-dihydroxypentanoate, which is itself accessible through the pathways shown in Figure 15, 19, and 21.

12 Figure 19 shows pathways to 1,3-butadiene and 1,3-butadiene intermediates from 3 HP-CoA and acrylyl-CoA. Figure 20 shows pathways to 1,3-butadiene and 1,3-butadiene intermediates from succinyl-CoA. The requisite succinyl-CoA is a central metabolic intermediate, the yield of which can be enhanced via a reductive TCA cycle as described further herein. Finally, Figure 21 shows pathways to 1,3-butadiene and 1,3-butadiene intermediates from the condensation of malonyl-CoA and acetyl-CoA, the latter also benefitting from increased throughput via reductive TCA pathways described herein. The present invention is also directed to the design and production of cells and organisms having biosynthetic production capabilities for p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, toluene, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, or benzene. The results described herein indicate that metabolic pathways can be designed and recombinantly engineered to achieve the biosynthesis ofp-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, toluene, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, or benzene in Escherichia coli and other cells or organisms. Biosynthetic production ofp-toluate, terephthalate, (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate, toluene, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, or benzene can be confirmed by construction of strains having the designed metabolic genotype. These metabolically engineered cells or organisms also can be subjected to adaptive evolution to further augment p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, toluene, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, or benzene biosynthesis, including under conditions approaching theoretical maximum growth. The shikimate biosynthesis pathway in E. coli converts erythrose-4-phosphate to chorismate, an important intermediate that leads to the biosynthesis of many essential metabolites including 4-hydroxybenzoate. 4-Hydroxybenzoate is structurally similar to p toluate, an industrial precursor of terephthalic acid, and benzene. As disclosed herein, shikimate pathway enzymes are utilized to accept the alternate substrate, (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate (2H3M40P), and transform it to p-toluate or toluene, or the alternate substrate (2-hydroxy-4-oxobutoxy)phosphonate (2H40P) and transform it to benzoate or benzene. In addition, a pathway is used to synthesize the 2H3M40P or 2H40P precursor using enzymes from the non-mevalonate pathway for isoprenoid biosynthesis. Disclosed herein are strategies for engineering a microorganism to produce renewable p-toluate, terephthalate (PTA), toluene, benzoate, or benzene from carbohydrate feedstocks.

13 In the toluene series, glyceraldehyde-3-phosphate (G3P) and pyruvate are converted to 2 hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M40P) in three enzymatic steps (see Example III and Figure 5). The 2H3M40P intermediate is subsequently transformed to p toluate by enzymes in the shikimate pathway (see Example IV and Figure 6). p-Toluate can be further converted to PTA by a microorganism (see Example V and Figure 7). In the benzene series, 2H40P is prepared by dehydration and reduction of erythrose-4-phosphate (see Example VI and Figure 8). The 2H40P intermediate is subsequently transformed to benzoate by enzymes in the shikimate pathway (see Example VI and Figure 9). Benzoate and p-toluate are converted to benzene and toluene, respectively (see Example VII, and Figures 10 and 11). The conversion of G3P to p-toluate requires one ATP, two reducing equivalents (NAD(P)H), and two molecules of phosphoenolpyruvate, according to net reaction below. G3P + 2 PEP + ATP +2 NAD(P)H + 2 H+ -> p-Toluate + 4 Pi + ADP + 2 NAD(P) +

CO

2 + H 2 0 An additional ATP is required to synthesize G3P from glucose. The maximum theoretical p-toluate yield is 0.67 mol/mol (0.51 g/g) from glucose minus carbon required for energy. Under the assumption that 2 ATPs are consumed per p-toluate molecule synthesized, the predicted p-toluate yield from glucose is 0.62 mol/mol (0.46 g/g)p-toluate. Ifp-toluate is further converted to PTA by enzymes as described in Example III, the predicted PTA yield from glucose is 0.64 mol/mol (0.58 g/g). In this case, the oxidation ofp toluate to PTA generates an additional net reducing equivalent according to the net reaction: p-toluate + 02 + NAD+ - PTA + NADH + 2 H+ Enzyme candidates for catalyzing each step of the above pathways are described in the following sections. Successfully engineering pathways for the production of toluene, benzene, styrene, p-toluate, terephthalate, (2-hydroxy-3 -methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate or 1,3-butadiene entails identifying an appropriate set of enzymes with sufficient activity and specificity, cloning their corresponding genes into a production host, optimizing the expression of these genes in the 14 production host, optimizing fermentation conditions, and assaying for product formation following fermentation. As used herein, the term "non-naturally occurring" when used in reference to a microbial organism or microorganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate, toluene, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, or 1,3-butadiene biosynthetic pathway. A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring microorganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments thereof. Exemplary metabolic modifications are disclosed herein. As used herein, the term "isolated" when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial organism is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a microbial organism that is removed from some or all components as the microbial organism is found in non-naturally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments. Specific examples of isolated microbial organisms include partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.

15 As used herein, the terms "microbial," "microbial organism" or "microorganism" are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical. As used herein, the term "CoA" or "coenzyme A" is intended to mean an organic cofactor or prosthetic group (nonprotein portion of an enzyme) whose presence is required for the activity of many enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A functions in certain condensing enzymes, acts in acetyl or other acyl group transfer and in fatty acid synthesis and oxidation, pyruvate oxidation and in other acetylation. As used herein, the term "(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate," abbreviated herein as 2H3M40P, has the chemical formula as shown in Figure 5. Such a compound can also be described as 3-hydroxy-2-methyl butanal-4-phosphate. As used herein, the term "(2-hydroxy-4-oxobutoxy)phosphonate," abbreviated herein as 2H40P, has the chemical formula as shown in Figure 8 (compound 3). Such a compound can also be described as 3-hydroxybutanal-4-phosphate. As used herein, the term "p-toluate," having the molecular formula C 8

H

7 0 2 (see Figure 6, compound 9)(IUPAC name 4-methylbenzoate) is the ionized form ofp-toluic acid, and it is understood that p-toluate and p-toluic acid can be used interchangeably throughout to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled in the art that the specific form will depend on the pH. As used herein, the term "benzoate," having the molecular formula C 7

H

6 0 2 (see Figure 9, compound 9) is the ionized form of benzoic acid, and it is understood that benzoate and benzoic acid can be used interchangeably throughout to refer to the compound in any of it neutral or ionized forms, including any salt forms thereof. It is understood by those skilled in the art that the specific form will depend on the pH. As used herein, the term "terephthalate," having the molecular formula C 8

H

4 0 4 -2 (see Figure 7, compound 4)(IUPAC name terephthalate) is the ionized form of terephthalic acid, 16 also referred to as p-phthalic acid or PTA, and it is understood that terephthalate and terephthalic acid can be used interchangeably throughout to refer to the compound in any of its neutral or ionized forms, including any salt forms thereof. It is understood by those skilled understand that the specific form will depend on the pH. As used herein, the term "substantially anaerobic" when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen. "Exogenous" as it is used herein is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term "endogenous" refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species whereas "homologous" refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an encoding nucleic acid of the invention can utilize either or both a heterologous or homologous encoding nucleic acid. It is understood that when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced encoding nucleic acid or biosynthetic activity, as discussed above. It is further understood, as disclosed herein, that such more than one exogenous nucleic acids can be introduced into the host microbial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination thereof, and still be considered as more than one exogenous 17 nucleic acid. For example, as disclosed herein a microbial organism can be engineered to express two or more exogenous nucleic acids encoding a desired pathway enzyme or protein. In the case where two exogenous nucleic acids encoding a desired activity are introduced into a host microbial organism, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be introduced into a host organism in any desired combination, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism. The non-naturally occurring microbial organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. Generally, stable genetic alterations include modifications that persist greater than 10 generations, particularly stable modifications will persist more than about 25 generations, and more particularly, stable genetic modifications will be greater than 50 generations, including indefinitely. Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other organisms. For example, the E. coli metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.

18 An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of hydrolysis of epoxides. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor. Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for construction of the non-naturally occurring microorganism. An example of orthologs exhibiting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of elastase proteolysis and plasminogen proteolysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second example is the separation of mycoplasma 5'-3' exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa. In contrast, paralogs are homologs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. Paralogs 19 can originate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have distinct functions in the same species. Paralogs are proteins from the same species with significant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others. A nonorthologous gene displacement is a nonorthologous gene from one species that can substitute for a referenced gene function in a different species. Substitution includes, for example, being able to perform substantially the same or a similar function in the species of origin compared to the referenced function in the different species. Although generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced function, less structurally related but functionally similar genes and their corresponding gene products nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene. Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic capability, those skilled in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are present in the referenced microorganism that encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes. Orthologs, paralogs and nonorthologous gene displacements can be determined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal sequence identity and similarities 20 between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to indicate the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. Parameters for sufficient similarity to determine relatedness are computed based on well known methods for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or proteins can be expected to have a high similarity, for example, 25% to 100% sequence identity. Proteins that are unrelated can have an identity which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences. Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; xdropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept-16-1998) and the following parameters: Match: 1; mismatch: 2; gap open: 5; gap extension: 2; x dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences. In some embodiment, the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having a toluene pathway which includes at least one exogenous nucleic acid encoding a toluene pathway enzyme expressed in a sufficient amount to produce toluene. The toluene pathway is selected from (A) 1) one 21 or both of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, and 3) phenylacetaldehyde decarbonylase; (B) 1) one or more of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, 3) one or more of phenylacetaldehyde dehydrogenase and phenylacetaldehyde oxidase, and 4) phenylacetate decarboxylase; and (C) one or more of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate oxidase, and 3) phenylacetate decarboxylase, as shown in the alternate pathways in Figure 1. The non-naturally occurring microbial organism having the toluene pathway can include two exogenous nucleic acids each encoding a toluene pathway enzyme, three exogenous nucleic acids each encoding a toluene pathway enzyme, four exogenous nucleic acids each encoding a toluene pathway enzyme, or five exogenous nucleic acids each encoding a toluene pathway enzyme. An exemplary non-naturally occurring microbial organism having three exogenous nucleic acids can include an organism having genes encoding 1) phenylalanine aminotransferase and/or oxidoreductase (deaminating), 3) phenylpyruvate oxidase, and 5) phenylacetate decarboxylase. An exemplary non-naturally occurring organism having four exogenous nucleic acids can include an organism having exogenous genes encoding 1) phenylalanine aminotransferase, 2) phenylalanine oxidoreductase (deaminating), 3) phenylpyruvate decarboxylase, and 4) phenylacetaldehyde decarbonylase. An exemplary non-naturally occurring microbial organism having five exogenous nucleic acids can include an organism having genes encoding 1) phenylalanine aminotransferase, 2) phenylalanine oxidoreductase (deaminating), 3) phenylpyruvate decarboxylase, 4) phenylacetaldehyde dehydrogenase and/or oxidase, and 5) phenylacetate decarboxylase. Thus, in particular embodiments, a non-naturally occurring microbial organism can have a complete toluene pathway every gene encoding every enzyme in a complete toluene pathway. In some embodiments the non-naturally occurring microbial organism having a toluene pathway can include at least one exogenous nucleic acid that is a heterologous nucleic acid. Finally, the non-naturally occurring microbial organism having a toluene pathway can be in a substantially anaerobic culture medium. In some embodiments, the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having a benzene pathway which includes at least one exogenous nucleic acid encoding a benzene pathway enzyme expressed 22 in a sufficient amount to produce benzene. The benzene pathway can include a phenylalanine benzene-lyase as shown in Figure 2. The at least one exogenous nucleic acid can be phenylalanine benzene-lyase itself or a nucleic acid that affects the production of its precursor phenylalanine. In some embodiments the non-naturally occurring microbial organism having a benzene pathway has at least one exogenous nucleic acid that is a heterologous nucleic acid. In some embodiments, the non-naturally occurring microbial organism having a benzene pathway is in a substantially anaerobic culture medium. In some embodiments, the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having a styrene pathway which includes at least one exogenous nucleic acid encoding a styrene pathway enzyme expressed in a sufficient amount to produce styrene. The styrene pathway can be selected from (A) 1) benzoyl-CoA acetyltransferase, 2) one or more of 3-oxo-3-phenylpropionyl-CoA synthetase, transferase, and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetopheone reductase, and 5) 1-phenylethanol dehydratase; and (B) 1) benzoyl-CoA acetyltransferase, 2) phosphotrans 3-oxo-3-phenylpropionylase, 3) benzoyl-acetate kinase , 4) benzoyl-acetate decarboxylase, 5) acetopheone reductase, and 6) 1 -phenylethanol dehydratase, as indicated by the alternate pathways in Figure 3. In some embodiments, the non-naturally occurring microbial organism having a styrene pathway can include two exogenous nucleic acids each encoding a styrene pathway enzyme, three exogenous nucleic acids each encoding a styrene pathway enzyme, four exogenous nucleic acids each encoding a styrene pathway enzyme, five exogenous nucleic acids each encoding a styrene pathway enzyme, six exogenous nucleic acids each encoding a styrene pathway enzyme, and so on. An exemplary non-naturally occurring organism having five exogenous nucleic acids can include an organism having exogenous genes encoding 1) benzoyl-CoA acetyltransferase, 2) one of 3-oxo-3-phenylpropionyl-CoA synthetase, transferase, and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetophenone reductase, and 5) 1-phenylethanol dehydratase. An exemplary non-naturally occurring organism having six exogenous nucleic acids can include an organism having exogenous genes encoding 1) benzoyl-CoA acetyltransferase, 2) phosphotrans-3-oxo-3-phenylpropionylase, 3) benzoyl acetate kinase , 4) benzoyl-acetate decarboxylase, 5) acetopheone reductase, and 6) 1 phenylethanol dehydratase. In some embodiments the non-naturally occurring microbial organism having a styrene pathway has at least one exogenous nucleic acid that is a 23 heterologous nucleic acid. In some embodiments the non-naturally occurring microbial organism having a styrene pathway is in a substantially anaerobic culture medium. In some embodiments, the present invention provides a non-naturally occurring microbial organism that includes a microbial organism having a 1,3-butadiene pathway which includes at least one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene. The 1,3-butadiene pathway can be selected from (A) 1) trans, trans-muconate decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (B) 1) cis, trans-muconate cis-decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (C) 1) cis, trans-muconate trans-decarboxylase 2) cis-2,4-pentadienoate decarboxylase; and (D) 1) cis, cis-muconate decarboxylase and 2) cis-2,4-pentadienoate decarboxylase, as indicated in the alternate pathways in Figure 4. In some embodiments, the non-naturally occurring microbial organism having a 1,3 butadiene pathway can include two exogenous nucleic acids each encoding a 1,3-butadiene pathway enzyme. Thus, the two exogenous nucleic acids can encode a set selected from (A) 1) trans, trans-muconate decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (B) 1) cis, trans-muconate cis-decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (C) 1) cis, trans-muconate trans-decarboxylase 2) cis-2,4-pentadienoate decarboxylase; and (D) 1) cis, cis-muconate decarboxylase and 2) cis-2,4-pentadienoate decarboxylase, corresponding to the complete pathways shown in Figure 4. In some embodiments, the non-naturally occurring microbial organism having a 1,3-butadiene pathway has at least one exogenous nucleic acid that is a heterologous nucleic acid. In some embodiments, the non-naturally occurring microbial organism having a 1,3-butadiene pathway is in a substantially anaerobic culture medium. In some embodiments, the present invention provides a non-naturally occurring microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes capable of converting AKP to 2,4-pentadienoate selected from (A) 1) a 4-hydroxy-2-oxovalerate aldolase, 2) a 4-hydroxy-2-oxovalerate dehydratase, 3) a 2-oxopentenoate reductase, and 4) a 2-hydroxypentenoate dehydratase, as shown in steps A-D of Figure 12, (B) 1) an AKP deaminase, 2) an acetylacrylate reductase, and 3) a 4-hydroxypent-2-enoate dehydratase, as shown in steps B-D of Figure 13, (C) 1) an AKP aminotransferase and/or dehydrogenase, 2) a 24 2,4-dioxopentanoate-2-reductase, 3) a 2-hydroxy-4-oxopentanoate dehydratase, 4) an acetylacrylate reductase, and 5) a 4-hydroxypent-2-enoate dehydratase, as shown in steps E, H, F, C, and D of Figure 13, (D) 1) an AKP aminotransferase and/or dehydrogenase, 2) a 2,4 dioxopentanoate-4-reductase, 3) a 4-hydroxy-2-oxovalerate dehydratase, 4) a 2 oxopentenoate reductase, and 5) a 2-hydroxypentenoate dehydratase, as shown in steps E and K Figure 13, along with steps B-D of Figure 12, and (E) 1) an AKP reductase, 2) a 2-amino 4-hydroxypentanoate aminotransferase and/or dehydrogenase, 3) a 4-hydroxy-2-oxovalerate dehydratase, 4) a 2-oxopentenoate reductase, and 5) a 2-hydroxypentenoate dehydratase, also shown in steps J and L of Figure 13, along with steps B-D of Figure 12. In some embodiments, pathways of Figure 13 that include the intermediate 4-hydroxy-2-oxovalerate, shown in Figure 12, can also be directed through the 2,4-dihydroxypentanoate pathways shown in Figure 12 to provide 2,4-pentadienoate. For example, from 4-hydroxy-2 oxovalerate, pathways to 2,4-pentadienoate can include the enzymes in steps E, H, and D or steps E, F, and G, in Figure 12. In some embodiments, the present invention also provides a non-naturally occurring microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes selected from (A) 1) 4-hydroxy-2-oxovalerate aldolase, 2) 4-hydroxy-2-oxovalerate reductase, 3) 2,4 dihydroxypentanoate 2-dehydratase, and 4) 4-hydroxypent-2-enoate dehydratase, as shown in steps A, E, F, and G of Figure 12 and (B) 1) 4-hydroxy-2-oxovalerate aldolase, 2) 4-hydroxy 2-oxovalerate reductase, 3) 2,4-dihydroxypentanoate 4-dehydratase and 4) 2 hydroxypentenoate dehydratase, as shown in steps A, E, H, and D of Figure 12. In some embodiments, the present invention also provides a non-naturally occurring microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes selected from (A) 1) AKP aminotransferase and/or dehydrogenase, 2) 2,4-dioxopentanoate 2 reductase, 3) 2-hydroxy-4-oxopentanoate reductase, 4) 2,4-dihydroxypentanoate 2 dehydratase, and 5) 4-hydroxypent-2-enoate dehydratase, as shown in steps E, H, I, G, and D of Figure 13, and (B) 1) AKP aminotransferase and/or dehydrogenase, 2) 2,4 dioxopentanoate 2-reductase, 3) 2-hydroxy-4-oxopentanoate reductase, along with 4) 2,4- 25 dihydroxypetanoate-2-dehydratase and 5) 4-hydroxypent-2-enoate dehydratase or 4) 2,4 dihydroxypentanoate-4-dehydratase and 5) 2-hydroxypentenoate dehydratase, as shown in steps E, H, and I of Figure 13, along with steps F and G or H and D of Figure 12, respectively. That is to say, the double dehydration of 2,4-dihydroxypentanoate can be performed in any order. In some embodiments, the present invention also provides a non-naturally occurring microbial organism having an AKP pathway that includes at least one exogenous nucleic acid encoding an AKP pathway enzyme expressed in a sufficient amount to produce AKP. The AKP pathway includes an ornithine 4,5-aminomutase and a 2,4-diaminopentanoate 4 aminotransferase and/or 4-dehydrogenase, as shown in steps M and N of Figure 13. In some embodiments, the microbial organism having an AKP pathway includes two exogenous enzymes encoding an ornithine 4,5-aminomutase and a 2,4-diaminopentanoate 4 aminotransferase or 2,4-diaminopentanoate 4-dehydrogenase. In some embodiments, this AKP pathway can be added to any of the aforementioned 2,4-pentadienoate pathways and as indicated in Figure 13. Alternatively, AKP can be accessed from alanine by addition of an AKP thiolase, as shown in step A of Figure 13, and fed into the various 2,4-pentadienoate pathways described herein and shown in Figure 13, along with Figure 12. In some embodiments, the present invention also provides a non-naturally occurring microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes selected from (A) 1) ornithine 2,3-aminomutase, 2) 3,5-diaminopentanoate deaminase, and 3) 5 aminopent-2-enoate deaminase, as shown in steps A-C of Figure 14, (B) 1) ornithine 2,3 aminomutase, 2) 3,5-diaminopentanoate deaminase, 3) 5-aminopent-2-enoate aminotransferase and/or dehydrogenase, 4) 5-oxopent-2-enoate reductase, and 5) 5 hydroxypent-2-enoate dehydratase, as shown in steps A, B, H, F, and G of Figure 14, (C) 1) ornithine 2,3-aminomutase, 2) 3,5-diaminopentanoate aminotransferase and/or dehydrogenase, 3) 3-amino-5-oxopentanoate deaminase, 4) 5-oxopent-2-enoate reductase, and 5) 5-hydroxypent-2-enoate dehydratase as shown in steps A, D, E, F, and G of Figure 14, and (D) 1) ornithine 2,3-aminomutase, 2) 3,5-diaminopentanoate aminotransferase and/or dehydrogenase, 3) 3-amino-5-oxopentanoate reductase, and 4) 3-amino-5-hydroxypentanoate 26 deaminase, and 5) 5-hydroxypent-2-enoate dehydratase as shown in steps A, D, I, J, and G of Figure 14. In some embodiments, the present invention also provides a non-naturally occurring microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes selected from any of the numerous pathways shown in Figure 15 starting from 3-HP-CoA or acryloyl CoA. Exemplary pathways from 3-HP-CoA include the following enzyme sets (A) 1) 3 hydroxypropanoyl-CoA acetyltransferase, 2) 3-oxo-5-hydroxypentanoyl-CoA reductase, 3) 3,5-dihydroxypentanoyl-CoA dehydratase, 4) 5-hydroxypent-2-enoyl-CoA dehydratase, and 5) pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase, as shown in steps A-E of Figure 15, and (B) 1) 3-hydroxypropanoyl-CoA acetyltransferase, 2) 3-oxo-5 hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, 3) 3-oxo-5 hydroxypentanoate reductase, 4) 3,5-dihydroxypentanoate dehydratase, and 5) 5 hydroxypent-2-enoate dehydratase, as shown in steps A, F, I, J, and Q of Figure 15. One skilled in the art will recognize that enzyme sets defining pathways (A) and (B) from 3-HP CoA can be intermingled via reversible enzymes 3,5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, as shown by step G in Figure 15, and 5-hydroxypent-2-enoyl CoA synthetase, transferase and/or hydrolase, as shown by step H in Figure 15. Thus, a 3 HP-CoA to 2,4-pentadienoate pathway can include the enzymes in steps A, B, G, J, and Q, or steps A, B, C, H, and Q, or steps A, B, G, J, H, D, and E, or steps A, F, I, G, C, D, and E, or steps, A, F, I, G, C, H, and Q, or steps A, F, I, J, H, D, and E, each shown in Figure 15. Exemplary pathways from acryloyl-CoA include the following enzyme sets (C) 1) acryloyl-CoA acetyltransferase, 2) 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, 3) 3-oxopent-4-enoate reductase, 4) 3-hydroxypent-4-enoate dehydratase, as shown in steps M, 0, P, and S in Figure 15 and (D), 1) acryloyl-CoA acetyltransferase, 2) 3 oxopent-4-enoyl-CoA reductase, 3) 3-hydroxypent-4-enoyl-CoA dehydratase, and 4) pent 2,4-dienoyl-CoA synthetase, transferase and/or hydrolase, as shown in steps M, N, R, and E. One skilled in the art will recognize that enzyme sets defining pathways (A) and (B) from 3 HP-CoA and (C) and (D) from acryloyl-CoA can be intermingled via reversible enzymes 3 hydroxypropanoyl-CoA dehydratase, as shown in step K of Figure 15, and 3-oxo-5- 27 hydroxypentanoyl-CoA dehydratase, as shown in step L of Figure 15. Thus, step K can be added to any of the enumerated pathways from acryloyl-CoA to 2,4-pentadienoate providing 2,4-pentadienoate pathways such as steps K, M, N, R, and E or steps K, M, 0, P, and S. Step K can also be used a shuttle alternative to step A to provide 3-oxo-5-hydroxypentanoyl-CoA from 3-HP-CoA via steps K, M, and L. Thus, any of the aforementioned pathways utilizing the enzyme of step A can utilize the enzymes of steps K, M, and L, in its place. The same 3 oxo-5-hydroxypentanoyl-CoA intermediate can be accessed from acryloyl-CoA by pathways via the enzymes of steps K and A or M and L of Figure 15. Thus, acryloyl-CoA can be used to access all the enumerated pathways that would be accessible from 3-HP-CoA. Thus, for example, an acryloyl-CoA to 2,4-pentadienoate pathway can include enzymes from steps K, A, B, C, D, and E, or steps K, A, F, I, J and Q, or steps K, A, B, G, J, and Q, or steps K, A, B, G, J, H, D, and E, or steps K, A, B, C, H, and Q, or steps K, A, F, I, G, C, D, and E, or steps K, A, F, I, G, C, H, Q, or steps K, A, F, I, J, H, D and E, or steps M, L, B, C, D, and E, or steps M, L, F, I, J and Q, or steps M, L, B, G, J, and Q, or steps M, L, B, G, J, H, D, and E, or steps M, L, B, C, H, and Q, or steps M, L, F, I, G, C, D, and E, or steps M, L, F, I, G, C, H, Q, or steps M, L, F, I, J, H, D and E, all as shown in Figure 15. Similarly, 3-HP-CoA can feed into the enumerated acryloyl-CoA pathways via intermediate 3-oxopent-4-enoyl-CoA using the enzyme of step L. Thus, a 3-HP-CoA to 2,4-pentadienoate pathway can include enzymes from steps A, L, N, R, and E or steps A, L, 0, P, and S, each pathway being shown in Figure 15. In some embodiments, the present invention provides a non-naturally occurring microbial organism, that includes a microbial organism having a 2,4-pentadienoate pathway which includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate. The 2,4 pentadienoate pathway has a set of enzymes selected from: 1) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 2) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5 dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase 28 and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 3) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, G. 3,5 dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, C. 3,5 dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent 2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 4) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5 dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 5) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 6) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, H. 5 hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, D. 5-hydroxypent-2 enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 7) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5 hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 8) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, 29 H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, D. 5-hydroxypent 2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 9) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 10) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, H. 5 hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, D. 5-hydroxypent-2 enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 11) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, G. 3,5 dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, C. 3,5 dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent 2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 12) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5 dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 13) M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, R. 3 hydroxypent-4-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 14) M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3 hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, S. 3-hydroxypent-4-enoate dehydratase; and 30 15) M. acrylyl-CoA acetyltransferase, 0. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, S. 3-hydroxypent-4-enoate dehydratase; 16) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl CoA dehydratase, N. 3-oxopent-4-enoyl-CoA reductase, R. 3-hydroxypent-4-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 17) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl CoA dehydratase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, S. 3-hydroxypent-4-enoate dehydratase; and 18) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl CoA dehydratase, 0. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3 oxopent-4-enoate reductase, S. 3-hydroxypent-4-enoate dehydratase. In some embodiments, the non-naturally occurring microbial organism of the invention includes two, three, four, five, six, seven, or eight exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. In some embodiments, the non-naturally occurring microbial organism of the invention has at least one exogenous nucleic acid is a heterologous nucleic acid. In some embodiments, the non-naturally occurring microbial organism of the invention is in a substantially anaerobic culture medium. In some embodiments, the non-naturally occurring microbial organism of the invention further includes a 2,4-pentadiene decarboxylase to convert 2,4-pentadienoate to 1,3-butadiene. In some embodiments, a non-naturally occurring microbial organism includes a microbial organism having a 1,3-butadiene pathway which includes at least one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene. The 1,3-butadiene pathway has a set of enzymes selected from: 1) M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3 hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, Y. 3-hydroxypent-4-enoate decarboxylase; 31 2) M. acrylyl-CoA acetyltransferase, 0. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, Y. 3-hydroxypent-4-enoate decarboxylase; 3) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, N. 3 oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, Y. 3-hydroxypent-4-enoate decarboxylase; 4) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, 0. 3 oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, Y. 3-hydroxypent-4-enoate decarboxylase; 5) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, Y. 3-hydroxypent-4-enoate decarboxylase; 6) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, 0. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3 oxopent-4-enoate reductase, Y. 3-hydroxypent-4-enoate decarboxylase; In some embodiments, the non-naturally occurring microbial organism of the invention includes two, three, four, or five exogenous nucleic acids each encoding a 1,3 butadiene pathway enzyme. In some embodiments, the non-naturally occurring microbial organism of the invention includes at least one exogenous nucleic acid that is a heterologous nucleic acid. In some embodiments, the non-naturally occurring microbial organism of the invention is in a substantially anaerobic culture medium. In some embodiments, the present invention provides a non-naturally occurring microbial organism, that includes a microbial organism having a 1,3-butadiene pathway which includes at least one exogenous nucleic acid encoding a 3-butene-1-ol pathway enzyme expressed in a sufficient amount to produce 3-butene-1-ol. The 3-butene-1-ol pathway has a set of enzymes selected from: 1) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, U. 3,5 dihydroxypentanoate decarboxylase; 32 2) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5 dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 3) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, U. 3,5 dihydroxypentanoate decarboxylase; 4) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5 dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 5) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate decarboxylase; 6) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5 hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate decarboxylase; 7) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5 hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2 enoate decarboxylase; 8) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase; 9) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2 enoate decarboxylase; 10) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA 33 dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5 hydroxypent-2-enoate decarboxylase; 11) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate decarboxylase; 12) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 13) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl CoA synthetase, transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase; 14) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5 hydroxypent-2-enoate decarboxylase; 15) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl CoA dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate decarboxylase; 16) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3 oxo-5-hydroxypentanoyl-CoA dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, U. 3,5 dihydroxypentanoate decarboxylase; 17) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3 oxo-5-hydroxypentanoyl-CoA dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5 dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 34 18) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3 oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, U. 3,5 dihydroxypentanoate decarboxylase; 19) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3 oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5 dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 20) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3 oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate decarboxylase; In some embodiments, the non-naturally occurring microbial organism of the invention includes two, three, four, five, six, or seven, exogenous nucleic acids each encoding a 3-butene-1-ol pathway enzyme. In some embodiments, the non-naturally occurring microbial organism of the invention has at least one exogenous nucleic acid that is a heterologous nucleic acid. In some embodiments, the non-naturally occurring microbial organism of the invention is in a substantially anaerobic culture medium. In some embodiments, the non-naturally occurring microbial organism of the invention further includes a 3-butene-1-ol dehydratase to convert 3-butene-1-ol to 1,3-butadiene. In some embodiments, non-naturally occurring microbial organism of the invention can include two exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. In some embodiments, non-naturally occurring microbial organism of the invention can include three exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. For example, the non-naturally occurring microbial organism of the invention can include three exogenous nucleic acids encoding i) an AKP deaminase, ii) an acetylacrylate reductase, and iii) a 4-hydroxypent-2-enoate dehydratase, thus providing an alanine or omithine accessible pathway to 2,4-pentadienoate via AKP. One skilled in the art will recognize that this is merely exemplary and that three exogenous nucleic acids can be the basis of any 2,4-pentadienoate-producing non-naturally occurring organism in any of the enumerated pathways of Figure 12-15.

35 In some embodiments, the non-naturally occurring microbial organism of the invention microbial can include any four exogenous nucleic acids each encoding a 2,4 pentadienoate pathway enzyme. For example, a non-naturally occurring microbial organism can include four exogenous nucleic acids encoding i) a 4-hydroxy-2-oxovalerate aldolase, ii) a 4-hydroxy-2-oxovalerate dehydratase, iii) a 2-oxopentenoate reductase, and iv) a 2 hydroxypentenoate dehydratase, thus defining a complete pathway from pyruvate to 2,4 pentadienoate, as shown in Figure 12. One skilled in the art will recognize that this is merely exemplary and that four exogenous nucleic acids can be the basis of any 2,4-pentadienoate producing non-naturally occurring organism in any of the enumerated pathways of Figure 12 15. In still further embodiments, the non-naturally occurring microbial organism of the invention can include five exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. Exemplary non-naturally occurring microbial organism of the invention having five exogenous nucleic acids can include enzymes encoding (A) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2-reductase, iii) a 2 hydroxy-4-oxopentanoate dehydratase, iv) an acetylacrylate reductase, and v) a 4 hydroxypent-2-enoate dehydratase, as shown in steps E, H, F, C, and D in Figure 13, or (B) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-4-reductase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v) a 2 hydroxypentenoate dehydratase, as shown in steps E and K of Figure 13, along with steps B, C, and D of Figure 12, or i) an AKP reductase, ii) a 2-amino-4-hydroxypentanoate aminotransferase and/or dehydrogenase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2 oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase, as shown in steps J and L of Figure 13, along with steps B, C, and D of Figure 12. One skilled in the art will recognize that this is merely exemplary and that five exogenous nucleic acids can be the basis of any 2,4-pentadienoate-producing non-naturally occurring organism in any of the enumerated pathways of Figure 12-15. Thus, in some embodiments two, three, four, five, six, up to all of the enzymes in a 2,4-pentadienoate pathway can be provided insertion of exogenous nucleic acids. In some embodiments, the non-naturally occurring microbial organism of the invention has at least one exogenous nucleic acid is a heterologous nucleic acid. Moreover, in some embodiments, the non-naturally occurring microbial organism of the invention can be provided in a substantially anaerobic culture medium.

36 In some embodiments, the non-naturally occurring microbial organism of the invention can further include a 2,4-pentadienoate decarboxylase expressed in a sufficient amount to produce 1,3-butadiene by conversion of 2,4-pentadienoate to 1,3-butadiene. Thus, any 2,4-pentadienoate pathway of Figure 12 can form the basis of further production of 1,3 butadiene, as indicated by the conversion of cis or trans 2,4-pentadienoate to 1,3-butadiene in Figure 4. In some embodiments, the invention provides a non-naturally occurring microbial organism having a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product. For example, in a toluene pathway (Figure 1), such substrate to product is selected from the group consisting of phenylalanine to phenylpyruvate, phenylpyruvate to phenylacetaldehyde, phenylpyruvate to phenylacetate, phenylacetaldehyde to phenylacetate, phenylacetaldehyde to toluene, and phenylacetate to toluene. In a styrene pathway (Figure 3), such a substrate to product is selected from the group consisting of benzoyl-CoA to 3-oxo-3-phenylpropionyl-CoA, 3-oxo-3 phenylpropionyl-CoA to [(3-oxo-3-phenylpropionyl)oxy] phosphonate, [(3-oxo-3 phenylpropionyl)oxy] phosphonate to benzoyl-acetate, 3-oxo-3-phenylpropionyl-CoA to benzoyl-acetate, benzoyl-acetate to acetophenone, acetophenone to 1 -phenylethanol, and 1 phenylethanol to styrene. In a 1,3-butadiene pathway (Figure 4), such substrate to product is selected from trans, trans-muconate to trans-2,4-pentadienoate, cis, trans-muconate to trans 2,4-pentadienoate, cis,trans-muconate to cis-2,4-pentadienoate, cis,cis-muconate to cis-2,4 pentadienoate, trans-2,4-pentadienoate to 1,3-butadiene, and cis-2,4-pentadienoate to 1,3 butadiene. One skilled in the art will understand that these are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired product and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. In a 2,4-pentadienoate pathway, such a substrate to product is selected from pyruvate to 4-hydroxy-2-oxovalerate, 4-hydroxy-2-oxovalerate to 2-oxopentenoate, 2-oxopentenoate to 2-hydroxypentenoate, 2-hydroxypentenoate to 2,4-pentadienoate, AKP to acetylacrylate, acetylacrylate to 4-hydroxypent-2-enoate, 4-hydroxypent-2-enoate to 2,4-pentadienoate, AKP 37 to 2,4-dioxopentanoate, 2,4-dioxopentanoate to 4-hydroxy-2-oxovalerate, AKP to 2-amino-4 hydroxypentanoate, 2-amino-4-hydroxypentanoate to 4-hydroxy-2-oxovalerate, ornithine to 2,4-diaminopentanoate, 2,4-diaminopentanoate to AKP, alanine to AKP, and so on. The invention also provides a non-naturally occurring microbial organism, comprising a microbial organism having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising at least one exogenous nucleic acid encoding a (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate pathway enzyme expressed in a sufficient amount to produce (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, the (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate pathway comprising 2-C-methyl-D-erythritol-4-phosphate dehydratase (see Example III and Figure 5, step C). A non-naturally occurring microbial organism comprising a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway can further comprise 1 -deoxyxylulose-5 -phosphate synthase or 1 -deoxy-D-xylulose-5 -phosphate reductoisomerase (see Example III and Figure 5, steps A and B). Thus, a (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate can comprise 5 2-C-methyl-D-erythritol-4-phosphate dehydratase, 1 -deoxyxylulose-5 -phosphate synthase and 1 -deoxy-D-xylulose-5 -phosphate reductoisomerase. The invention also provides a non-naturally occurring microbial organism, comprising a microbial organism having a p-toluate pathway comprising at least one exogenous nucleic acid encoding a p-toluate pathway enzyme expressed in a sufficient amount to produce p toluate, the p-toluate pathway comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3 dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; or chorismate lyase (see Example IV and Figure 6, steps A-H). A non-naturally occurring microbial organism having ap-toluate pathway can further comprise a (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate pathway (Figure 5). A (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate pathway can comprise, for example, 2-C-methyl-D-erythritol-4 phosphate dehydratase, 1 -deoxyxylulose-5 -phosphate synthase or 1-deoxy-D-xylulose-5 phosphate reductoisomerase (Figure 5). The invention additionally provides a non-naturally occurring microbial organism, comprising a microbial organism having a terephthalate pathway comprising at least one exogenous nucleic acid encoding a terephthalate pathway enzyme expressed in a sufficient amount to produce terephthalate, the terephthalate pathway comprising p-toluate methyl- 38 monooxygenase reductase; 4-carboxybenzyl alcohol dehydrogenase; or 4-carboxybenzyl aldehyde dehydrogenase (see Example V and Figure 7). Such an organism containing a terephthalate pathway can additionally comprise a p-toluate pathway, wherein the p-toluate pathway comprises 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3 phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; or chorismate lyase (see Examples IV and V and Figures 6 and 7). Such a non-naturally occurring microbialorganism having a terephthalate pathway and a p-toluate pathway can further comprise a (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate pathway (see Example III and Figure 5). A (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate pathway can comprise, for example, 2-C-methyl-D erythritol-4-phosphate dehydratase, 1 -deoxyxylulose-5 -phosphate synthase or 1-deoxy-D xylulose-5 -phosphate reductoisomerase (see Example III and Figure 5). In some embodiments, the present invention provides a non-naturally occurring microbial organism having a toluene pathway comprising at least one exogenous nucleic acid encoding a toluene pathway enzyme expressed in a sufficient amount to produce toluene. The toluene pathway is selected from a set of pathway enzymes selected from: a) p-toluate decarboxylase; b) p-toluate reductase and p-methylbenzaldehyde decarbonylase; c) p-toluate kinase, (p-methylbenzoyloxy)phosphonate reductase, and p-methylbenzaldehyde decarbonylase; d) (p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), phosphotrans-p-methylbenzoylase, (p-methylbenzoyloxy)phosphonate reductase, and p methylbenzaldehyde decarbonylase; and e) (p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), p-methylbenzoyl-CoA reductase and p-methylbenzaldehyde decarbonylase. In some embodiments, the present invention provides a non-naturally occurring microbial organism having a (2-hydroxy-4-oxobutoxy)phosphonate pathway comprising at least one exogenous nucleic acid encoding a (2-hydroxy-4-oxobutoxy)phosphonate pathway enzyme expressed in a sufficient amount to produce (2-hydroxy-4-oxobutoxy)phosphonate. The (2-hydroxy-4-oxobutoxy)phosphonate pathway includes erythrose-4-phosphate dehydratase and (2,4-dioxobutoxy) phosphonate reductase. In some embodiments, the present invention provides a non-naturally occurring microbial organism having a benzoate pathway comprising at least one exogenous nucleic acid encoding a benzoate pathway enzyme expressed in a sufficient amount to produce 39 benzoate. The benzoate pathway includes 2-dehydro-3-deoxyphosphoheptonate synthase; 3 dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase. In some embodiments, the present invention provides a non-naturally occurring microbial organism having a benzene pathway comprising at least one exogenous nucleic acid encoding a benzene pathway enzyme expressed in a sufficient amount to produce benzene. The benzene pathway is selected from a set of pathway enzymes selected from: a) benzoate decarboxylase; b) benzoate reductase and benzaldehyde decarbonylase; c) benzoate kinase, (benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase; d) (benzoyl CoA synthetase, transferase and/or hydrolase), phosphotransbenzoylase, (benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase; and e) (benzoyl-CoA synthetase, transferase and/or hydrolase), benzoyl-CoA reductase and benzaldehyde decarbonylase. In an additional embodiment, the invention provides a non-naturally occurring microbial organism having a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, p- toluate, terephthalate, toluene, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, or benzene pathway, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding an enzyme or protein that converts a substrate to a product. For example, in a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, the substrates and products can be selected from the group consisting of glyceraldehyde-3-phosphate and pyruvate to 1 -deoxy-D-xylulose-5 -phosphate; 1 -deoxy-D-xylulose-5 -phosphate to C-methyl D-erythritol-4-phosphate; and C-methyl-D-erythritol-4-phosphate to (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate (see Example III and Figure 5). In another embodiment, a p-toluate pathway can comprise substrates and products selected from (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate to 2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2 carboxylate; 2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate to 1,3 dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate; 1,3-dihydroxy-4-methyl-5 oxocyclohexane-1-carboxylate to 5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylic acid; 5 -hydroxy-4-methyl-3 -oxocyclohex- 1 -ene- 1 -carboxylic acid to 3,5 -dihydroxy-4 methylcyclohex-1-ene-1-carboxylate; 3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate to 5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate; 5-hydroxy-4- 40 methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate to 5-[(1-carboxyeth-1-en-1-yl)oxy] 4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate; 5-[(1-carboxyeth-1-en-1-yl)oxy] 4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate to 3-[(1-carboxyeth-1-en-1 yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate; and 3-[(1-carboxyeth-1-en-1-yl)oxy]-4 methylcyclohexa-1,5-diene-1-carboxylate top-toluate (see Example IV and Figure 6). In still another embodiment, a terephthalate pathway can comprise substrates and products selected fromp-toluate to 4-carboxybenzyl alcohol; 4-carboxybenzyl alcohol to 4 carboxybenzaldehyde; and 4-carboxybenzaldehyde to and terephthalic acid (see Example V and Figure 7). In another embodiment, a toluene pathway can comprise substrates and products selected from p-toluate to toluene; p-toluate to p-methyl benzoyl-CoA; p-methyl benzoyl-CoA to p-methylbenzoyloxy phosphate or p-methylbenzaldehyde; p methylbenzoyloxy phosphonate to p-methylbenzaldehyde; and p-methylbenzaldehyde to toluene (see Example VII and Figure 11). In another embodiment, a 2H40P pathway can comprise substrates and products selected from erythrose-4-phosphate to (2,4 dioxobutoxy)phosphonate; and (2,4-dioxobutoxy)phosphonate to 2H40P (see Example VI and Figure 8). In another embodiment, a benzoate pathway can comprise substrates and products selected from (2-hydroxy-4-oxobutoxy)phosphonate to 2,4-dihydroxy-6 [(phosphonooxy)methyl]oxane-2-carboxylate; 2,4-dihydroxy-6 [(phosphonooxy)methyl]oxane-2-carboxylate to 1,3-dihydroxy-5-oxocyclohexane-1 carboxylate; 1,3-dihydroxy-5-oxocyclohexane-1-carboxylate to 5-hydroxy-3-oxocyclohex-1 ene-1-carboxylate; 5-hydroxy-3-oxocyclohex-1-ene-1-carboxylate to 3,5-dihydroxycyclohex 1-ene-1-carboxylate, 3,5-dihydroxycyclohex-1-ene-1-carboxylate to 5-hydroxy-3 (phosphonooxy)cyclohex-1-ene-1-carboxylate; 5-hydroxy-3-(phosphonooxy)cyclohex-1-ene 1-carboxylate to 5-[(1-carboxyeth-1-en-1-yl)oxy]-3-(phosphonooxy)cyclohex-1-ene-1 carboxylate; 5-[(1-carboxyeth-1-en-1-yl)oxy]-3-(phosphonooxy)cyclohex-1-ene-1 carboxylate to 3-[(1-carboxyeth-1-en-1-yl)oxy]cyclohexa-1,5-diene-1-carboxylate; and 3-[(1 carboxyeth-1-en-1-yl)oxy]cyclohexa-1,5-diene-1-carboxylate to benzoate (see Example VI and Figure 9). In another embodiment, a benzene pathway can comprise substrates and products selected from benzoate to benzene; benzoate to benzoyl-CoA, (benzoyloxy)phosphonate or benzaldehyde; benzoyl-CoA to (benzoyloxy)phosphonate or benzaldehyde; (benzoyloxy)phosphonate to benzaldehyde; and benzaldehyde to benzene. One skilled in the art will understand that these are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired product and for which 41 an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings herein. As disclosed herein, a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway is exemplified in Figure 5 (see Example III). Therefore, in addition to a microbial organism containing a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway that produces (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, the invention additionally provides a non naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme, where the microbial organism produces a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway intermediate, for example, 1 -deoxy-D-xylulose-5-phosphate or C-methyl-D-erythritol-4 phosphate. Similarly, the invention also provides a non-naturally occurring microbial organism containing a p-toluate pathway that produces p-toluate, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding a p toluate pathway enzyme, where the microbial organism produces a p-toluate pathway intermediate, for example, 2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2 carboxylate, 1,3-dihydroxy-4-methyl-5-oxocyclohexane-1-carboxylate, 5-hydroxy-4-methyl 3-oxocyclohex-1-ene-1-carboxylate, 3,5-dihydroxy-4-methylcyclohex-1-ene-1-carboxylate, 5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate, 5-[(1-carboxyeth-1-en 1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate, or 3-[(1-carboxyeth-1 en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1-carboxylate. Further, the invention additionally provides a non-naturally occurring microbial organism containing a terephthalate pathway enzyme, where the microbial organism produces a terephthalate pathway intermediate, for example, 4-carboxybenzyl alcohol or 4-carboxybenzaldehyde. Similarly, the invention also provides a non-naturally occurring microbial organism containing a toluene pathway that produces toluene, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding a toluene pathway enzyme, where the microbial organism produces a toluene pathway intermediate, for example, p-methylbenzoyl-CoA, (p-methylbenzoyloxy)phosphonate, or p methylbenzaldehyde. Similarly, the invention also provides a non-naturally occurring microbial organism containing a benzene pathway that produces benzene, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding a benzene 42 pathway enzyme, where the microbial organism produces a benzene pathway intermediate, for example, benzoyl-CoA, (benzoyloxy)phosphonate, and benzaldehyde (Figure 10). Similarly, the invention also provides a non-naturally occurring microbial organism containing a benzoate pathway that produces benzoate, wherein the non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding a benzoate pathway enzyme, where the microbial organism produces a benzoate pathway intermediate, for example, (2-hydroxy-4-oxobutoxy)phosphonate, 2,4-dihydroxy-6 [(phosphonooxy)methyl]oxane-2-carboxylate, 1,3-dihydroxy-5-oxocyclohexane-1 carboxylate, 5-hydroxy-3-oxocyclohex-1-ene-1-carboxylate, 3,5-dihydroxycyclohex-1-ene-1 carboxylate, 5-hydroxy-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate, 5-[(1-carboxyeth-1 en-1-yl)oxy]-3-(phosphonooxy)cyclohex-1-ene-1-carboxylate, and 3-[(1-carboxyeth-1-en-1 yl)oxy]cyclohexa-1,5-diene-1-carboxylate (Figure 9). Thus, the invention provides a non-naturally occurring microbial organism containing at least one exogenous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway, such as those shown in Figures 1-11. While generally described herein as a microbial organism that contains a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway, it is understood that the invention additionally provides a non naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce an intermediate of a toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway. For example, as disclosed herein, toluene, benzene, styrene, and 1,3-butadiene pathways are exemplified in Figures 1-23. Therefore, in addition to a microbial organism containing a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4- 43 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway that produces toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene , the invention additionally provides a non naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway enzyme, where the microbial organism produces a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway intermediate, for example, phenylpyruvate, phenylacetaldehyde, phenylacetate, 3-oxo-3-phenylpropionyl-CoA, [(3-oxo 3-phenylpropionyl)oxy] phosphonate, benzoyl-acetate, acetophenone, 1 -phenylethanol, DXP, 2ME4P, benzoyl-CoA, (benzoyloxy)phosphonate, benzaldehyde, trans-2,4-pentadienoate, and cis-2,4-pentadienoate, as well as any shikimate pathway intermediate shown in Figures 6 and 9. It is understood that any of the pathways disclosed herein, as described in the Examples and exemplified in the Figures, including the pathways of Figures 1-11, can be utilized to generate a non-naturally occurring microbial organism that produces any pathway intermediate or product, as desired. As disclosed herein, such a microbial organism that produces an intermediate can be used in combination with another microbial organism expressing downstream pathway enzymes to produce a desired product. However, it is understood that a non-naturally occurring microbial organism that produces a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene- 1 ol or 1,3-butadiene pathway intermediate can be utilized to produce the intermediate as a desired product. The invention is described herein with general reference to the metabolic reaction, reactant or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or catalyzing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to 44 the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, reference to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or proteins involved in the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding encoded enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the reactants and products of the reaction. The non-naturally occurring microbial organisms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes or proteins participating in one or more toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl 4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic pathway can be expressed. For example, if a chosen host is deficient in one or more enzymes or proteins for a desired biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous expression. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the deficient enzyme(s) or protein(s) to achieve toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthesis. Thus, a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired product such as toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene 45 Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fungus or any of a variety of other microorganisms applicable to fermentation processes. Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonasfluorescens, and Pseudomonas putida. Exemplary yeasts or fungi include species selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizobus oryzae, and the like. E. coli is a particularly useful host organisms since it is a well characterized microbial organism suitable for genetic engineering. Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to introduce metabolic and/or genetic modifications to produce a desired product. Depending on the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic pathway constituents of a selected host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one exogenously expressed toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic pathways. For example, toluene, benzene, p-toluate, terephthalate, (2-hydroxy 3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 46 1,3-butadiene pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a pathway can be expressed even if the host contains at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene can be included, such as phenylalanine aminotransferase and/or phenylalanine oxidoreductase (deaminating), phenylpyruvate decarboxylase, phenylacetaldehyde dehydrogenase and/or oxidase, phenylpyruvate oxidase, phenylacetate decarboxylase, phenylacetaldehyde decarbonylase, phenylalanine benzene-lyase, benzoyl-CoA acetyltransferase, 3-oxo-3-phenylpropionyl-CoA synthetase, transferase and/or hydrolase, benzoyl-acetate decarboxylase, acetophenone reductase, 1-phenylethanol dehydratase, phosphotrans-3-oxo-3-phenylpropionylase, benzoyl-acetate kinase, trans, trans-muconate decarboxylase, cis, trans-muconate cis-decarboxylase, cis, trans-muconate trans decarboxylase, cis, cis-muconate decarboxylase, trans-2,4-pentadienoate decarboxylase, and cis-2,4-pentadienoate decarboxylase. For example, all enzymes in a p-toluate pathway can be included, such as 2-dehydro 3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2 carboxyvinyltransferase; chorismate synthase; and chorismate lyase. In addition, all enzymes in a terephthalate pathway can be included, such as p-toluate methyl-monooxygenase reductase; 4-carboxybenzyl alcohol dehydrogenase; and 4-carboxybenzyl aldehyde dehydrogenase. Furthermore, all enzymes in a (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate pathway can be included, such as 2-C-methyl-D-erythritol-4 phosphate dehydratase, 1-deoxyxylulose-5 -phosphate synthase and 1-deoxy-D-xylulose-5 phosphate reductoisomerase.Likewise, all enzymes in a toluene pathway can be included, such as p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase, p-toluate reductase, p-methylbenzaldehyde decarbonylase, p-methylbenzoyl-CoA reductase, p-toluate decarboxylase, phosphotrans-p-methylbenzoylase, (p-methylbenzoyloxy)phosphonate reductase (dephosphorylating), and p-toluate kinase.

47 Likewise, all enzymes in a (2-hydroxy-4-oxobutoxy)phosphonate pathway can be included, such as erythrose-4-phosphate dehydratase and (2,4-dioxobutoxy)phosphonate reductase. Likewise, all enzymes in a benzozate pathway can be included, such as 2-dehydro-3 deoxyphosphoheptonate synthase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase, shikimate kinase, 3-phosphoshikimate-2-carboxyvinyltransferase, chorismate synthase, and chorismate lyase. Likewise, all enzymes in a benzene pathway can be included, such as benzoyl-CoA synthetase, transferase and/or hydrolase, benzoate reductase, benzaldehyde decarbonylase, benzoyl-CoA reductase, benzoate decarboxylase, phosphotransbenzoylase, (benzoyloxy)phosphonate reductase (dephosphorylating), and benzoate kinase. Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding nucleic acids to introduce in an expressible form will, at least, parallel the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway deficiencies of the selected host microbial organism. Therefore, a non-naturally occurring microbial organism of the invention can have one, two, three, four, five, six, seven, eight, that is, up to all nucleic acids encoding the enzymes or proteins constituting a toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize toluene, benzene, p toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthesis or that confer other useful functions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway precursors such as phenylalanine, phenylpyruvate, phenylacetaldehyde, phenylacetate, benzoyl-CoA, 3-oxo-3-phenylpropionyl CoA, [(3-oxo-3-phenylpropionyl)oxy]phosphonate, benzoyl acetate, acetophenone, 1- 48 phenylethanol, trans,trans-muconate, cis,trans-muconate, cis,cis-muconate, trans-2,4 pentadienoate, cis-2,4-pentadienoate, 4-hydroxy-2-oxovalerate, 2-oxopentenoate, 2 hydroxypentenoate, 2,4-dihydroxypentanoate, 4-hydroxypent-2-enoate, 2,4 diaminopentanoate, AKP, acetylacrylate, 2,4-dioxopentanoate, 2-hydroxy-4-oxo-pentanoate, 2-amino-4-hydroxypentanoate, 3,5-diaminopentanoate, 5 -aminopent-2-enoate, 3-amino-5 oxopentanoate, 5-oxopent-2-enoate, 5-hydroxypent-2-enoate, 3-amino-5-hydroxypentanoate, 3-HP-CoA, acryloyl-CoA, 3-oxo-5-hydroxypentanoyl-CoA, 3-oxo-5-hydroxypentanoate, 3,5 dihydroxypentanoate, 3,5-dihydroxypentanoyl-CoA, 5-hydroxypent-2-enoyl-CoA, 2,4 pentadienoyl-CoA, 3-oxopent-4-enoyl-CoA, 3-hydroxypent-4-enoyl-CoA, 3-oxopent-4 enoate, and 3-hydroxypent-4-enoate glyceraldehyde-3 -phosphate, pyruvate, (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate or p-toluate. Furthermore, as disclosed herein, multiple pathways can be included in a single organism such as the pathway to produce p-toluate (Figure 6), terephthalate (Figure 7) toluene (Figure 11) and (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate (Figure 5), as desired, or 2H40P (Figure 8), benzoate (Figure 9) and benzene (Figure 10). Generally, a host microbial organism is selected such that it produces the precursor of a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway, either as a naturally produced molecule or as an engineered product that either provides de novo production of a desired precursor or increased production of a precursor naturally produced by the host microbial organism. For example, cis,cis-muconate is produced naturally in a host organism such as E. coli. As a further example, glyceraldehyde-3-phosphate and phosphoenolpyruvate are produced naturally in a host organism such as E. coli. A host organism can be engineered to increase production of a precursor, as disclosed herein. In addition, a microbial organism that has been engineered to produce a desired precursor can be used as a host organism and further engineered to express enzymes or proteins of a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway. In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2- 49 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene- 1 ol or 1,3-butadiene . In this specific embodiment it can be useful to increase the synthesis or accumulation of a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway product to, for example, drive toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway reactions toward toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway enzymes or proteins. Over expression the enzyme or enzymes and/or protein or proteins of the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through exogenous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial organisms of the invention, for example, producing toluene, benzene, p toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene , through overexpression of one, two, three, four, five, six, seven, eight, that is, up to all nucleic acids encoding toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic pathway enzymes or proteins. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic pathway.

50 In particularly useful embodiments, exogenous expression of the encoding nucleic acids is employed. Exogenous expression confers the ability to custom tailor the expression and/or regulatory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by removing a negative regulatory effector or induction of the gene's promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring inducible promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism. It is understood that, in methods of the invention, any of the one or more exogenous nucleic acids can be introduced into a microbial organism to produce a non-naturally occurring microbial organism of the invention. The nucleic acids can be introduced so as to confer, for example, a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic pathway onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the biosynthetic capability to catalyze some of the required reactions to confer toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl 4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic capability. For example, a non naturally occurring microbial organism having a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, such as the combination of phenylalanine aminotransferase and/or phenylalanine oxidoreductase (deaminating) and phenylpyruvate decarboxylase, phenylalanine aminotransferase and/or phenylalanine oxidoreductase (deaminating) and phenylacetaldehyde dehydrogenase and/or oxidase, phenylalanine aminotransferase and/or phenylalanine oxidoreductase (deaminating) and phenylpyruvate oxidase, phenylpyruvate oxidase and phenylacetate decarboxylase, phenylalanine aminotransferase and/or phenylalanine 51 oxidoreductase (deaminating) and phenylacetate decarboxylase, phenylalanine aminotransferase and/or phenylalanine oxidoreductase (deaminating) and phenylacetaldehyde decarbonylase, phenylpyruvate decarboxylase and phenylacetaldehyde dehydrogenase, phenypyruvate decarboxylase and phenylacetate decarboxylase, phenylpyruvate decarboxylase and phenylacetaldehyde decarbonylase, and phenylacetaldehyde dehydrogenase and/or oxidase and phenylacetate decarboxylase, in a toluene pathway. Similarly, in a styrene pathway the combination of at least two exogenous nucleic acids can include benzoyl-CoA acetyltransferase and 3-oxo-3-phenylpropionyl-CoA synthetase, benzoyl-CoA acetyltransferase and benzoyl-acetate decarboxylase, benzoyl-CoA acetyltransferase and acetophenone reductase, benzoyl-CoA acetyltransferase and 1 phenylethanol dehydratase, benzoyl-CoA acetyltransferase and phosphotrans-3-oxo-3 phenylpropionylase, benzoyl-CoA acetyltransferase and benzoyl-acetate kinase, 3-oxo-3 phenylpropionyl-CoA synthetase and benzoyl-acetate decarboxylase, 3-oxo-3 phenylpropionyl-CoA synthetase and acetopheonone reductase, 3-oxo-3-phenylpropionyl CoA synthetase and 1 -phenylethanol dehydratase, and so on. Similarly, in a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway, a combination of the enzymes expressed can be a combination of 2-C-methyl-D-erythritol-4 phosphate dehydratase and 1-deoxyxylulose-5 -phosphate synthase, or 2-C-methyl-D erythritol-4-phosphate dehydratase and 1 -deoxy-D-xylulose-5 -phosphate reductoisomerase. In a p-toluate pathway, a combination of the enzymes expressed can be a combination of 2 dehydro-3-deoxyphosphoheptonate synthase and 3-dehydroquinate dehydratase; shikimate kinase and 3-phosphoshikimate-2-carboxyvinyltransferase; shikimate kinase and shikimate dehydrogenase and, and the like. Similarly, in a terephthalate pathway, a combination of the expressed enzymes can be p-toluate methyl-monooxygenase reductase and 4-carboxybenzyl alcohol dehydrogenase; or 4-carboxybenzyl alcohol dehydrogenase and 4-carboxybenzyl aldehyde dehydrogenase, and the like. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention. Similarly, it is understood that any combination of three or more enzymes or proteins of a biosynthetic pathway can be included in a non-naturally occurring microbial organism of the invention, and so forth, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired 52 product. Such combination of three enzymes can include, for example, 3-dehydroquinate synthase, shikimate dehydrogenase and shikimate kinase; shikimate kinase, chorismate synthase and chorismate lyase; 3-dehydroquinate dehydratase, chorismate synthase and chorismate lyase, and so forth, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. Similarly, any combination of four, five, six, or more enzymes or proteins of a biosynthetic pathway as disclosed herein can be included in a non-naturally occurring microbial organism of the invention, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in production of the corresponding desired product. In addition to the biosynthesis of toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene as described herein, the non-naturally occurring microbial organisms and methods of the invention also can be utilized in various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene other than use of the toluene, benzene, p toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene producers is through addition of another microbial organism capable of converting a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway intermediate to toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene . One such procedure includes, for example, the fermentation of a microbial organism that produces a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway intermediate. The toluene, benzene, p-toluate, terephthalate, (2- 53 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway intermediate can then be used as a substrate for a second microbial organism that converts the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway intermediate to toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene . The toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway intermediate can be added directly to another culture of the second organism or the original culture of the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway intermediate producers can be depleted of these microbial organisms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps. In other embodiments, the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for example, toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene. In these embodiments, biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the product of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene 54 also can be biosynthetically produced from microbial organisms through co-culture or co fermentation using two organisms in the same vessel, where the first microbial organism produces a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene intermediate and the second microbial organism converts the intermediate to toluene, benzene, p-toluate, terephthalate, (2-hydroxy 3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene . Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non-naturally occurring microbial organisms and methods of the invention together with other microbial organisms, with the co-culture of other non-naturally occurring microbial organisms having subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl 4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene . Sources of encoding nucleic acids for a toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway enzyme or protein can include, for example, any species where the encoded gene product is capable of catalyzing the referenced reaction. Such species include both prokaryotic and eukaryotic organisms including, but not limited to, bacteria, including archaea and eubacteria, and eukaryotes, including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, Mycobacterium tuberculosis, Agrobacterium tumefaciens, Bacillus subtilis, Synechocystis species, Arabidopsis thaliana, Zymomonas mobilis, Klebsiella oxytoca, Salmonella typhimurium, Salmonella typhi, Lactobacullus collinoides, Klebsiella pneumoniae, Clostridium pasteuranum, Citrobacter freundii, Clostridium butyricum, Roseburia inulinivorans, Sulfolobus solfataricus, Neurospora crassa, Sinorhizobium fredii, Helicobacter pylori, Pyrococcus furiosus, Haemophilus influenzae, Erwinia chrysanthemi, Staphylococcus aureus, Dunaliella salina, Streptococcus pneumoniae, Saccharomyces cerevisiae, Aspergillus nidulans, Pneumocystis carinii, Streptomyces coelicolor, species from the genera Burkholderia, Alcaligenes, 55 Pseudomonas, Shingomonas and Comamonas, for example, Comamonas testosteroni, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes. However, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite toluene, benzene, p toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic activity for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements of known genes, and the interchange of genetic alterations between organisms is routine and well known in the art. Accordingly, the metabolic alterations allowing biosynthesis of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those skilled in the art will know that a metabolic alteration exemplified in one organism can be applied equally to other organisms. In some instances, such as when an alternative toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic pathway exists in an unrelated species, toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthesis can be conferred onto the host species by, for example, exogenous expression of a paralog or paralogs from the unrelated species that catalyzes a similar, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among metabolic networks exist between different organisms, those skilled in the art will understand that the actual gene usage between different organisms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the teachings and methods of the invention can be applied to all microbial organisms using the cognate metabolic alterations to those exemplified herein to construct a microbial organism in a 56 species of interest that will synthesize toluene, benzene, p-toluate, terephthalate, (2-hydroxy 3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene . Methods for constructing and testing the expression levels of a non-naturally occurring toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene -producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). Exogenous nucleic acid sequences involved in a pathway for production of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, transfection, and ultrasound transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a mitochondrial leader sequence led to increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in yeast or other eukaryotic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins. An expression vector or vectors can be constructed to include one or more toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2- 57 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene- 1 ol or 1,3-butadiene biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein. In some embodiments, the present invention provides a method for producing toluene that includes culturing a non-naturally occurring microbial organism having a toluene pathway. The toluene pathway includes at least one exogenous nucleic acid encoding a toluene pathway enzyme expressed in a sufficient amount to produce toluene, under conditions and for a sufficient period of time to produce toluene. The toluene pathway can be selected from (A) 1) one or both of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, and 3) phenylacetaldehyde 58 decarbonylase; (B) 1) one or more of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, 3) one or more of phenylacetaldehyde dehydrogenase and phenylacetaldehyde oxidase, and 4) phenylacetate decarboxylase; and (C) 1) one or more of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate oxidase, and 3) phenylacetate decarboxylase, as indicated in the alternate pathways shown in Figure 1. In some embodiments, the method includes culturing the non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, methods of the invention for producing toluene include culturing a non-naturally microbial organism, that includes two exogenous nucleic acids each encoding a toluene pathway enzyme, three exogenous nucleic acids each encoding a toluene pathway enzyme, four exogenous nucleic acids each encoding a toluene pathway enzyme, five exogenous nucleic acids each encoding a toluene pathway, and so on. Exemplary organisms having four exogenous nucleic acids can encode 1) phenylalanine aminotransferase, 2) phenylalanine oxidoreductase (deaminating), 3) phenylpyruvate decarboxylase, and 4) phenylacetaldehyde decarbonylase. Exemplary organisms having five exogenous nucleic acids can encode 1) phenylalanine aminotransferase, 2) phenylalanine oxidoreductase (deaminating), 3) phenylpyruvate decarboxylase, 4) phenylacetaldehyde dehydrogenase and/or oxidase, and 5) phenylacetate decarboxylase. In some embodiments, methods of the invention for producing toluene can include culturing a non-naturally occurring microbial organism that has at least one exogenous nucleic acid that is a heterologous nucleic acid. In some embodiments, the present invention provides a method for producing benzene that includes culturing a non-naturally occurring microbial organism having a benzene pathway. The benzene pathway can include at least one exogenous nucleic acid encoding a benzene pathway enzyme expressed in a sufficient amount to produce benzene, under conditions and for a sufficient period of time to produce benzene. The benzene pathway can include a phenylalanine benzene-lyase, as shown in Figure 2. In some embodiments, the at least one exogenous nucleic acid is the phenylalanine benzene-lyase itself, while in alternate embodiments, the at least one exogenous nucleic acid can affect the production of the precursor metabolite phenylalanine. In some embodiments the at least one exogenous nucleic acid of the benzene pathway is a heterologous nucleic acid. In some embodiments, methods 59 of the invention for producing benzene can include culturing a non-naturally occurring microbial organism that is in a substantially anaerobic culture medium. In some embodiments, the present invention provides a method for producing styrene that includes culturing a non-naturally occurring microbial organism having a styrene pathway. The styrene pathway can include at least one exogenous nucleic acid encoding a styrene pathway enzyme expressed in a sufficient amount to produce styrene, under conditions and for a sufficient period of time to produce styrene. The styrene pathway can be selected from (A) 1) benzoyl-CoA acetyltransferase, 2) one or more of 3-oxo-3 phenylpropionyl-CoA synthetase, transferase, and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetopheone reductase, and 5) 1-phenylethanol dehydratase; and (B) 1) benzoyl-CoA acetyltransferase, 2) phosphotrans-3-oxo-3-phenylpropionylase, 3) benzoyl acetate kinase , 4) benzoyl-acetate decarboxylase, 5) acetopheone reductase, and 6) 1 phenylethanol dehydratase, as indicated in the alternate pathways in Figure 3. In some embodiments, methods of the invention for producing styrene can include culturing a non naturally occurring microbial organism that is in a substantially anaerobic culture medium. In some embodiments, methods of the invention for producing styrene include culturing a non-naturally microbial organism, that includes two exogenous nucleic acids each encoding a styrene pathway enzyme, three exogenous nucleic acids each encoding a styrene pathway enzyme, four exogenous nucleic acids each encoding a styrene pathway enzyme, five exogenous nucleic acids each encoding a styrene pathway enzyme, six exogenous nucleic acids each encoding a styrene pathway enzyme, and so on. An exemplary organism having five exogenous nucleic acids can encode 1) benzoyl-CoA acetyltransferase, 2) one of 3-oxo-3-phenylpropionyl-CoA synthetase, transferase, and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetopheone reductase, and 5) 1 -phenylethanol dehydratase. An exemplary organism having six six exogenous nucleic acids encode 1) benzoyl-CoA acetyltransferase, 2) phosphotrans-3-oxo-3-phenylpropionylase, 3) benzoyl-acetate kinase, 4) benzoyl-acetate decarboxylase, 5) acetopheone reductase, and 6) 1 -phenylethanol dehydratase. In some embodiments, methods of the present invention for producing styrene can include culturing a non-naturally occurring microbial organism in which at least one exogenous nucleic acid is a heterologous nucleic acid. In some embodiments, the present invention provides a method for producing 1,3 butadiene that includes culturing a non-naturally occurring microbial organism having a 1,3- 60 butadiene pathway. The 1,3-butadiene pathway includes at least one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3 butadiene, under conditions and for a sufficient period of time to produce 1,3-butadiene. The 1,3-butadiene pathway can be selected from (A) 1) trans, trans-muconate decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (B) 1) cis, trans-muconate cis-decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (C) 1) cis, trans-muconate trans-decarboxylase 2) cis-2,4-pentadienoate decarboxylase; and (D) 1) cis, cis-muconate decarboxylase and 2) cis 2,4-pentadienoate decarboxylase, as indicated by the alternate pathways in Figure 4. In some embodiments, the method of producing 1,3-butadiene can include culturing a non-naturally occurring microbial organism that is in a substantially anaerobic culture medium. In some embodiments, methods of the invention can include culturing a non-naturally occurring microbial organism that has two exogenous nucleic acids each encoding a 1,3 butadiene pathway enzyme. Exemplary organisms having two exogenous nucleic acids can include genes encoding a set of enzymes selected from (A) 1) trans, trans-muconate decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (B) 1) cis, trans-muconate cis decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (C) 1) cis, trans-muconate trans-decarboxylase 2) cis-2,4-pentadienoate decarboxylase; and (D) 1) cis, cis-muconate decarboxylase and 2) cis-2,4-pentadienoate decarboxylase. In some embodiments, methods of the invention can include culturing a non-naturally occurring microbial organism that has at least one exogenous nucleic acid that is a heterologous nucleic acid. The invention additionally provides a method for producing (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, comprising culturing the non-naturally occurring microbial organism containing a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway under conditions and for a sufficient period of time to produce (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate. Such a microbial organism can have a (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate pathway comprising at least one exogenous nucleic acid encoding a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway enzyme expressed in a sufficient amount to produce (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, the (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate pathway comprising 2-C-methyl-D-erythritol-4-phosphate dehydratase (see Example III and Figure 5, step C). A (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate pathway can optionally further comprise 1 -deoxyxylulose-5 - 61 phosphate synthase and/or 1-deoxy-D-xylulose-5-phosphate reductoisomerase (see Example III and Figure 5, steps A and B). In another embodiment, the invention provides a method for producing p-toluate, comprising culturing the non-naturally occurring microbial organism comprising ap-toluate pathway under conditions and for a sufficient period of time to produce p-toluate. Ap toluate pathway can comprise at least one exogenous nucleic acid encoding a p-toluate pathway enzyme expressed in a sufficient amount to produce p-toluate, the p-toluate pathway comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3 dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3 phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and/or chorismate lyase (see Example IV and Figure 6, steps A-H). In another embodiment, a method of the invention can utilize a non-naturally occurring microbial organism that further comprises a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway (see Example III and Figure 5). Such a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway can comprise 2-C-methyl D-erythritol-4-phosphate dehydratase, 1-deoxyxylulose-5 -phosphate synthase and/or 1 deoxy-D-xylulose-5-phosphate reductoisomerase (see Example III and Figure 5). The invention further provides a method for producing terephthalate, comprising culturing a non-naturally occurring microbial organism containing a terephthalate pathway under conditions and for a sufficient period of time to produce terephthalate. Such a terephthalate pathway can comprise at least one exogenous nucleic acid encoding a terephthalate pathway enzyme expressed in a sufficient amount to produce terephthalate, the terephthalate pathway comprising p-toluate methyl-monooxygenase reductase; 4 carboxybenzyl alcohol dehydrogenase; and/or 4-carboxybenzyl aldehyde dehydrogenase. Such a microbial organism can further comprise a p-toluate pathway, wherein the p-toluate pathway comprises 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3 phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and/or chorismate lyase (see Examples IV and V and Figures 6 and 7). In another embodiment, the non-naturally occurring microbial organism can further comprise a (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate pathway (see Example III and Figure 5). In some embodiments, the present invention provides a method for producing toluene, comprising culturing a non naturally occurring microbial organism having a toluene pathway comprising at least one 62 exogenous nucleic acid encoding a toluene pathway enzyme expressed in a sufficient amount to produce toluene. The toluene pathway can be selected from a set of pathway enzymes selected from: a) p-toluate decarboxylase; b)p-toluate reductase and p-methylbenzaldehyde decarbonylase; c) p-toluate kinase, (p-methylbenzoyloxy)phosphonate reductase, and p methylbenzaldehyde decarbonylase; d) (p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), phosphotrans-p-methylbenzoylase, (p-methylbenzoyloxy)phosphonate reductase, and p-methylbenzaldehyde decarbonylase; and e) (p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), p-methylbenzoyl-CoA reductase and p-methylbenzaldehyde decarbonylase. The non-naturally occurring microbial organism can be cultured under conditions and for a sufficient period of time to produce toluene. In a particular embodiment, the invention provides a non-naturally occurring microbial organism and methods of use, in which the microbial organism contains p-toluate, terephthalate or toluene, and (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate pathways. In some embodiments, the present invention provides a method for producing (2 hydroxy-4-oxobutoxy) phosphonate, comprising culturing a non-naturally occurring microbial organism having a (2-hydroxy-4-oxobutoxy) phosphonate pathway comprising at least one exogenous nucleic acid encoding a (2-hydroxy-4-oxobutoxy) phosphonate pathway enzyme expressed in a sufficient amount to produce (2-hydroxy-4-oxobutoxy) phosphonate. The (2-hydroxy-4-oxobutoxy) phosphonate pathway can include erythrose-4-phosphate dehydratase and (2,4-dioxobutoxy)phosphonate reductase. In some embodiments, the present invention provides a method for producing benzoate, comprising culturing a non-naturally occurring microbial organism having a benzoate pathway comprising at least one exogenous nucleic acid encoding a benzoate pathway enzyme expressed in a sufficient amount to produce benzoate. The benzoate pathway includes 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3 phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase. The method includes culturing the non-naturally occurring organism under conditions and for a sufficient period of time to produce benzoate. In some embodiments, the present invention provides a method for producing benzene, comprising culturing the non-naturally occurring microbial organism having a benzene pathway comprising at least one exogenous nucleic acid encoding a benzene 63 pathway enzyme expressed in a sufficient amount to produce benzene. The benzene pathway is selected from a set of pathway enzymes selected from: a) benzoate decarboxylase; b) benzoate reductase and benzaldehyde decarbonylase; c) benzoate kinase, (benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase; d) (benzoyl-CoA synthetase, transferase and/or hydrolase), phosphotransbenzoylase, (benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase; and e) (benzoyl-CoA synthetase, transferase and/or hydrolase), benzoyl-CoA reductase and benzaldehyde decarbonylase. The non naturally occurring microbial organism can be cultured under conditions and for a sufficient period of time to produce benzene. In a particular embodiment, the invention provides a non naturally occurring microbial organism and methods of use, in which the microbial organism contains (2-hydroxy-4-oxobutoxy) phosphonate, benzoate, and benzene pathways. In some embodiments, the present invention provides a method for producing 2,4 pentadienoate that includes culturing a non-naturally occurring microbial organism having a 2,4-pentadienoate pathway. The pathway includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate, under conditions and for a sufficient period of time to produce 2,4 pentadienoate. The 2,4-pentadienoate pathway selected from A) i) a 4-hydroxy-2 oxovalerate aldolase, ii) a 4-hydroxy-2-oxovalerate dehydratase, iii) a 2-oxopentenoate reductase, and iv) a 2-hydroxypentenoate dehydratase; B) i) an AKP deaminase, ii) an acetylacrylate reductase, and iii) a 4-hydroxypent-2-enoate dehydratase; C) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2-reductase, iii) a 2 hydroxy-4-oxopentanoate dehydratase, iv) an acetylacrylate reductase, and v) a 4 hydroxypent-2-enoate dehydratase; D) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-4-reductase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2 oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase; and E) i) an AKP reductase, ii) a 2-amino-4-hydroxypentanoate aminotransferase and/or dehydrogenase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v) a 2 hydroxypentenoate dehydratase. In some embodiments, the methods of the invention utilize a non-naturally occurring microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes selected 64 from (A) 1) 4-hydroxy-2-oxovalerate aldolase, 2) 4-hydroxy-2-oxovalerate reductase, 3) 2,4 dihydroxypentanoate 2-dehydratase, and 4) 4-hydroxypent-2-enoate dehydratase, as shown in steps A, E, F, and G of Figure 12 and (B) 1) 4-hydroxy-2-oxovalerate aldolase, 2) 4-hydroxy 2-oxovalerate reductase, 3) 2,4-dihydroxypentanoate 4-dehydratase and 4) 2 hydroxypentenoate dehydratase, as shown in steps A, E, H, and D of Figure 12. In some embodiments, the present methods of the invention utilize a non-naturally occurring microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes selected from (A) 1) AKP aminotransferase and/or dehydrogenase, 2) 2,4 dioxopentanoate 2-reductase, 3) 2-hydroxy-4-oxopentanoate reductase, 4) 2,4 dihydroxypentanoate 2-dehydratase, and 5) 4-hydroxypent-2-enoate dehydratase, as shown in steps E, H, I, G, and D of Figure 13, and (B) 1) AKP aminotransferase and/or dehydrogenase, 2) 2,4-dioxopentanoate 2-reductase, 3) 2-hydroxy-4-oxopentanoate reductase, along with 4) 2,4-dihydroxypetanoate-2-dehydratase and 5) 4-hydroxypent-2-enoate dehydratase or 4) 2,4 dihydroxypentanoate-4-dehydratase and 5) 2-hydroxypentenoate dehydratase, as shown in steps E, H, and I of Figure 13, along with steps F and G or H and D of Figure 12, respectively. That is to say, the double dehydration of 2,4-dihydroxypentanoate can be performed in any order. In some embodiments, the methods of the invention utilize a non-naturally occurring microbial organism having an AKP pathway that includes at least one exogenous nucleic acid encoding an AKP pathway enzyme expressed in a sufficient amount to produce AKP. The AKP pathway includes an ornithine 4,5-aminomutase and a 2,4-diaminopentanoate 4 aminotransferase and/or 4-dehydrogenase, as shown in steps M and N of Figure 13. In some embodiments, the microbial organism having an AKP pathway includes two exogenous enzymes encoding an ornithine 4,5-aminomutase and a 2,4-diaminopentanoate 4 aminotransferase or 2,4-diaminopentanoate 4-dehydrogenase. In some embodiments, this AKP pathway can be added to any of the aforementioned 2,4-pentadienoate pathways and as indicated in Figure 13. Alternatively, AKP can be accessed from alanine by addition of an AKP thiolase, as shown in step A of Figure 13, and fed into the various 2,4-pentadienoate pathways described herein and shown in Figure 13, along with Figure 12.

65 In some embodiments, the methods of the invention utilize a non-naturally occurring microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes selected from (A) 1) ornithine 2,3-aminomutase, 2) 3,5-diaminopentanoate deaminase, and 3) 5 aminopent-2-enoate deaminase, as shown in steps A-C of Figure 14, (B) 1) omithine 2,3 aminomutase, 2) 3,5-diaminopentanoate deaminase, 3) 5-aminopent-2-enoate aminotransferase and/or dehydrogenase, 4) 5-oxopent-2-enoate reductase, and 5) 5 hydroxypent-2-enoate dehydratase, as shown in steps A, B, H, F, and G of Figure 14, (C) 1) omithine 2,3-aminomutase, 2) 3,5-diaminopentanoate aminotransferase and/or dehydrogenase, 3) 3-amino-5-oxopentanoate deaminase, 4) 5-oxopent-2-enoate reductase, and 5) 5-hydroxypent-2-enoate dehydratase as shown in steps A, D, E, F, and G of Figure 14, and (D) 1) ornithine 2,3-aminomutase, 2) 3,5-diaminopentanoate aminotransferase and/or dehydrogenase, 3) 3-amino-5-oxopentanoate reductase, and 4) 3-amino-5-hydroxypentanoate deaminase, and 5) 5-hydroxypent-2-enoate dehydratase as shown in steps A, D, I, J, and G of Figure 14. In some embodiments, the methods of the invention utilize a non-naturally occurring microbial organism having a 2,4-pentadienoate pathway that includes at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate. The 2,4-pentadienoate pathway has a set of enzymes selected from any of the numerous pathways shown in Figure 15 starting from 3-HP-CoA or acryloyl CoA. Exemplary pathways from 3-HP include the following enzyme sets (A) 1) 3 hydroxypropanoyl-CoA acetyltransferase, 2) 3-oxo-5-hydroxypentanoyl-CoA reductase, 3) 3,5-dihydroxypentanoyl-CoA dehydratase, 4) 5-hydroxypent-2-enoyl-CoA dehydratase, and 5) pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase, as shown in steps A-E of Figure 15, and (B) 1) 3-hydroxypropanoyl-CoA acetyltransferase, 2) 3-oxo-5 hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, 3) 3-oxo-5 hydroxypentanoate reductase, 4) 3,5-dihydroxypentanoate dehydratase, and 5 5-hydroxypent 2-enoate dehydratase, as shown in steps A, F, I, J, and Q of Figure 15. One skilled in the art will recognize that enzyme sets defining pathways (A) and (B) from 3-HP-CoA can be intermingled via reversible enzymes 3,5-hydroxypentanoyl-CoA synthetase, transferase 66 and/or hydrolase, as shown by step G in Figure 15, and 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, as shown by step H in Figure 15. Thus, a 3-HP CoA to 2,4-pentadienoate pathway can include the enzymes in steps A, B, G, J, and Q, or steps A, B, C, H, and Q, or steps A, B, G, J, H, D, and E, or steps A, F, I, G, C, D, and E, or steps, A, F, I, G, C, H, and Q, or steps A, F, I, J, H, D, and E, each shown in Figure 15. Exemplary pathways from acryloyl-CoA include the following enzyme sets (C) 1) acryloyl-CoA acetyltransferase, 2) 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, 3) 3-oxopent-4-enoate reductase, 4) 3-hydroxypent-4-enoate dehydratase, as shown in steps M, 0, P, and S in Figure 15 and (D), 1) acryloyl-CoA acetyltransferase, 2) 3 oxopent-4-enoyl-CoA reductase, 3) 3-hydroxypent-4-enoyl-CoA dehydratase, and 4) pent 2,4-dienoyl-CoA synthetase, transferase and/or hydrolase, as shown in steps M, N, R, and E. One skilled in the art will recognize that enzyme sets defining pathways (A) and (B) from 3 HP-CoA and (C) and (D) from acryloyl-CoA can be intermingled via reversible enzymes 3 hydroxypropanoyl-CoA dehydratase, as shown in step K of Figure 15, and 3-oxo-5 hydroxypentanoyl-CoA dehydratase, as shown in step L of Figure 15. Thus, step K can be added to any of the enumerated pathways from acryloyl-CoA to 2,4-pentadienoate providing 2,4-pentadienoate pathways such as steps K, M, N, R, and E or steps K, M, 0, P, and S. Step K can also be used a shuttle alternative to step A to provide 3-oxo-5-hydroxypentanoyl-CoA from 3-HP-CoA via steps K, M, and L. Thus, any of the aforementioned pathways utilizing the enzyme of step A can utilize the enzymes of steps K, M, and L, in its place. The same 3 oxo-5-hydroxypentanoyl-CoA intermediate can be accessed from acryloyl-CoA by pathways via the enzymes of steps K and A or M and L of Figure 15. Thus, acryloyl-CoA can be used to access all the enumerated pathways that would be accessible from 3-HP-CoA. Thus, for example, an acryloyl-CoA to 2,4-pentadienoate pathway can include enzymes from steps K, A, B, C, D, and E, or steps K, A, F, I, J and Q, or steps K, A, B, G, J, and Q, or steps K, A, B, G, J, H, D, and E, or steps K, A, B, C, H, and Q, or steps K, A, F, I, G, C, D, and E, or steps K, A, F, I, G, C, H, Q, or steps K, A, F, I, J, H, D and E, or steps M, L, B, C, D, and E, or steps M, L, F, I, J and Q, or steps M, L, B, G, J, and Q, or steps M, L, B, G, J, H, D, and E, or steps M, L, B, C, H, and Q, or steps M, L, F, I, G, C, D, and E, or steps M, L, F, I, G, C, H, Q, or steps M, L, F, I, J, H, D and E, all as shown in Figure 15. Similarly, 3-HP-CoA can feed into the enumerated acryloyl-CoA pathways via intermediate 3-oxopent-4-enoyl-CoA using the enzyme of step L. Thus, a 3-HP-CoA to 2,4-pentadienoate pathway can include 67 enzymes from steps A, L, N, R, and E or steps A, L, 0, P, and S, each pathway being shown in Figure 15. In some embodiments, non-naturally occurring microbial organism used in methods of the invention can include two exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. In some embodiments, non-naturally occurring microbial organism of the invention can include three exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. For example, the non-naturally occurring microbial organism of the invention can include three exogenous nucleic acids encoding i) an AKP deaminase, ii) an acetylacrylate reductase, and iii) a 4-hydroxypent-2-enoate dehydratase, thus providing an alanine or ornithine accessible pathway to 2,4-pentadienoate via AKP. One skilled in the art will recognize that this is merely exemplary and that three exogenous nucleic acids can be the basis of any 2,4-pentadienoate-producing non-naturally occurring organism in any of the enumerated pathways of Figure 12-15. In some embodiments, the non-naturally occurring microbial organism used in methods of the invention microbial can include any four exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. For example, a non-naturally occurring microbial organism can include four exogenous nucleic acids encoding i) a 4-hydroxy-2 oxovalerate aldolase, ii) a 4-hydroxy-2-oxovalerate dehydratase, iii) a 2-oxopentenoate reductase, and iv) a 2-hydroxypentenoate dehydratase, thus defining a complete pathway from pyruvate to 2,4-pentadienoate, as shown in Figure 12. One skilled in the art will recognize that this is merely exemplary and that four exogenous nucleic acids can be the basis of any 2,4-pentadienoate-producing non-naturally occurring organism in any of the enumerated pathways of Figure 12-15. In still further embodiments, the non-naturally occurring microbial organism used in methods of the invention can include five exogenous nucleic acids each encoding a 2,4 pentadienoate pathway enzyme. Exemplary non-naturally occurring microbial organism of the invention having five exogenous nucleic acids can include enzymes encoding (A) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2-reductase, iii) a 2 hydroxy-4-oxopentanoate dehydratase, iv) an acetylacrylate reductase, and v) a 4 hydroxypent-2-enoate dehydratase, as shown in steps E, H, F, C, and D in Figure 13, or (B) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-4-reductase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v) a 2- 68 hydroxypentenoate dehydratase, as shown in steps E and K of Figure 13, along with steps B, C, and D of Figure 12, or i) an AKP reductase, ii) a 2-amino-4-hydroxypentanoate aminotransferase and/or dehydrogenase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2 oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase, as shown in steps J and L of Figure 13, along with steps B, C, and D of Figure 12. One skilled in the art will recognize that this is merely exemplary and that five exogenous nucleic acids can be the basis of any 2,4-pentadienoate-producing non-naturally occurring organism in any of the enumerated pathways of Figure 12-15. Thus, in some embodiments two, three, four, five, six, up to all of the enzymes in a 2,4-pentadienoate pathway can be provided insertion of exogenous nucleic acids. In some embodiments, the non-naturally occurring microbial organism of the invention has at least one exogenous nucleic acid is a heterologous nucleic acid. Moreover, in some embodiments, the methods employing non-naturally occurring microbial organism of the invention can utilize a substantially anaerobic culture medium. In some embodiments, the non-naturally occurring microbial organism used in methods of the invention can further include a 2,4-pentadienoate decarboxylase expressed in a sufficient amount to produce 1,3-butadiene by conversion of 2,4-pentadienoate to 1,3 butadiene. Thus, any 2,4-pentadienoate pathway of Figure 12 can form the basis of further production of 1,3 butadiene, as indicated by the conversion of cis or trans 2,4-pentadienoate to 1,3-butadiene in Figure 4. In some embodiments, the present invention provides a method for producing 2,4 pentadienoate, comprising culturing a non-naturally occurring microbial organism according to the aforementioned pathways described herein under conditions and for a sufficient period of time to produce 2,4-pentadienoate. In some such embodiments, the microbial organism includes two, three, four, five, six, seven, or eight exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. In some such embodiments, at least one exogenous nucleic acid is a heterologous nucleic acid. In some such embodiments thenon-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, the present invention provides a method for producing 1,3 butadiene that includes culturing a non-naturally occurring microbial organism according to the aforementioned pathways described herein, under conditions and for a sufficient period of time to produce 1,3-butadiene. In some such embodiments, the microbial organism includes two, three, four, five, six, seven, or eight exogenous nucleic acids each encoding a 1,3- 69 butadiene pathway enzyme. In some such embodiments, at least one exogenous nucleic acid is a heterologous nucleic acid. In some such embodiments, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, the present invention provides a method for producing 3 butene-1-ol which includes culturing a non-naturally occurring microbial organism according to the aforementioned pathway described herein, under conditions and for a sufficient period of time to produce 3-butene-1-ol. In some such embodiments, the microbial organism includes two, three, four, five, six, or seven exogenous nucleic acids each encoding a 3 butene-1-ol pathway enzyme. In some such embodiments, at least one exogenous nucleic acid is a heterologous nucleic acid. In some such embodiments, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some such embodiments, methods of the invention further include the chemical dehydration of 3-butene 1-ol to provide 1,3-butadiene. 3-butene-1-ol can be chemically dehydrated with formation of 1,3-butadiene, starting with pure 3-butene-1-ol isolated from the fermentation solution or starting with an aqueous or organic solutions of 3- butene-1-ol, isolated in work up of the fermentation solution. Such solutions of 3- butene-1-ol can also be concentrated before the dehydration step, for example by means of distillation, optionally in the presence of a suitable entrainer. The dehydration reaction can be carried out in liquid phase or in the gas phase. The dehydration reaction can be carried out in the presence of a catalyst, the nature of the catalyst employed depending on whether a gas-phase or a liquid-phase reaction is carried out. Suitable dehydration catalysts include both acidic catalysts and alkaline catalysts. Acidic catalysts, in particular can exhibit a decreased tendency to form oligomers. The dehydration catalyst can be employed as a homogeneous catalyst, a heterogeneous catalyst, or combinations thereof. Heterogeneous catalysts can be used in conjunction with a suitable support material. Such a support can itself be acidic or alkaline and provide the acidic or alkaline dehydration catalyst or a catalyst can be applied to an inert support. Suitable supports which serve as dehydration catalysts include natural or synthetic silicates such as mordenite, montmorillonite, acidic zeolites; supports which are coated with monobasic, dibasic or polybasic inorganic acids, such as phosphoric acid, or with acidic salts of inorganic acids, such as oxides or silicates, for example A1 2 0 3 , TiO 2 ; oxides and mixed 70 oxides such as y-A1 2 0 3 and ZnO-Al 2 0 3 mixed oxides of heteropolyacids. Alkaline substances which act both as dehydration catalyst and as a support a support material include alkali, alkaline earth, lanthanum, lanthoids or a combinations thereof as their oxides. A further class of materials that can effect dehydration are ion exchangers which can be used in either alkaline or acidic form. Suitable homogeneous dehydration catalysts include inorganic acids, such as phosphorus-containing acids such as phosphoric acid. Inorganic acids can be immobilized on the support material by immersion or impregnation. In some embodiments, dehydration reaction is carried out in the gas phase using conventional apparatuses known in the art, for example tubular reactors, shell-and-tube heat exchangers and reactors which comprise thermoplates as heat exchangers. In some embodiments, gas-phase dehydration can utilize isolated 3- butene-1-ol or solutions of butene-1-ol, the butene-1-ol being introduced into a reactor with fixed-bed catalysts. Thermal dehydration in the liquid phase can be carried out in a temperature range of between 200 'C and 350 'C, and in some embodiments between 250 and 3000 C. Suitable purification and/or assays to test for the production of toluene, benzene, p toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each engineered strain to be tested. For example, product and byproduct formation in the engineered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art. For example, the activity of phenylpyruvate decarboxylase can be measured using a coupled photometric assay with alcohol 71 dehydrogenase as an auxiliary enzyme as described by Weiss et al (Weiss et al., Biochem, 27:2197-2205 (1988). NADH- and NADPH-dependent enzymes such as acetophenone reductase can be followed spectrophotometrically at 340 nm as described by Schlieben et al (Schlieben et al, J. Mol. Biol., 349:801-813 (2005)). For typical hydrocarbon assay methods, see Manual on Hydrocarbon Analysis (ASTM Manula Series, A.W. Drews, ed., 6th edition, 1998, American Society for Testing and Materials, Baltimore, Maryland. P-toluate methyl monooxygenase activity can be assayed by incubating purified enzyme with NADH, FeSO 4 and the p-toluate substrate in a water bath, stopping the reaction by precipitation of the proteins, and analysis of the products in the supernatant by HPLC (Locher et al., J. Bacteriol. 173:3741-3748 (1991)). The toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene can be separated from other components in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art. Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention. For example, the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene producers can be cultured for the biosynthetic production of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene . For the production of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl 4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene , the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the 72 overall process. Such conditions can be obtained, for example, by first sparging the medium with nitrogen and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United States publication 2009/0047719, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or continuous manner, as disclosed herein. If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to maintain the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally occurring microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, mannose, fructose, sucrose and starch. Other sources of carbohydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such biomass feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, galactose, mannose, fructose and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene . In addition to renewable feedstocks such as those exemplified above, the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene microbial organisms of the invention also can be modified for growth on syngas as its source of carbon. In this specific embodiment, one or more proteins or enzymes 73 are expressed in the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene producing organisms to provide a metabolic pathway for utilization of syngas or other gaseous carbon source. Synthesis gas, also known as syngas or producer gas, is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues. Syngas is a mixture primarily of H 2 and CO and can be obtained from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high fuel to oxygen ratio. Although largely H 2 and CO, syngas can also include CO 2 and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, CO 2 . The Wood-Ljungdahl pathway catalyzes the conversion of CO and H 2 to acetyl-CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing CO 2 and C0 2

/H

2 mixtures through the same basic set of enzymes and transformations encompassed by the Wood-Ljungdahl pathway. H 2 dependent conversion of CO 2 to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of CO 2 and produce compounds such as acetate as long as hydrogen is present to supply the necessary reducing equivalents (see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation: 2 C0 2 + 4 H 2 + n ADP + n Pi -* CH 3 COOH + 2 H 2 0 + n ATP Hence, non-naturally occurring microorganisms possessing the Wood-Ljungdahl pathway can utilize CO 2 and H 2 mixtures as well for the production of acetyl-CoA and other desired products. The Wood-Ljungdahl pathway is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, formate 74 dehydrogenase, formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reactions in the carbonyl branch are catalyzed in order by the following enzymes or proteins: methyltetrahydrofolate:corrinoid protein methyltransferase (for example, AcsE), corrinoid iron-sulfur protein, nickel-protein assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the Wood-Ljungdahl enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete Wood-Ljungdahl pathway will confer syngas utilization ability. Additionally, the reductive (reverse) tricarboxylic acid cycle coupled with carbon monoxide dehydrogenase and/or hydrogenase activities can also be used for the conversion of CO, CO 2 and/or H 2 to acetyl-CoA and other products such as acetate. Organisms capable of fixing carbon via the reductive TCA pathway can utilize one or more of the following enzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase. Specifically, the reducing equivalents extracted from CO and/or H 2 by carbon monoxide dehydrogenase and hydrogenase are utilized to fix CO 2 via the reductive TCA cycle into acetyl-CoA or acetate. Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate kinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA can be converted to the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene precursors, glyceraldehyde-3 -phosphate, phosphoenolpyruvate, and pyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes of gluconeogenesis. Following the teachings and guidance provided herein for introducing a 75 sufficient number of encoding nucleic acids to generate a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the nucleic acids encoding the reductive TCA pathway enzymes or proteins absent in the host organism. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains the complete reductive TCA pathway will confer syngas utilization ability. In some embodiments, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a 1,3-butadiene pathway comprising at least one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene, said 1,3-butadiene pathway selected from: (A) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2 fumarylacetate decarboxylase; a 3-oxopent-4-enoate reductase; and a 3-hydroxypent-4-enoate decarboxylase; (B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate reductase; a 3 hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate decarboxylase; (C) a succinyl CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2 fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent 4-enoate decarboxylase; (D) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate decarboxylase; (E) a succinyl CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase; (F) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; a 76 3-hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate decarboxylase; (G) a succinyl CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA synthetase or a 3-hydroxyadipyl-CoA hydrolase; a 3 hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3 hydroxypent-4-enoate decarboxylase; and (H) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA synthetase or a 3-hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a 3 hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4 pentadienoate decarboxylase. In some aspects, the non-naturally occurring microbial organism comprises two, three, four, five, six or seven exogenous nucleic acids each encoding a 1,3-butadiene pathway enzyme. For example, the microbial organism can comprise exogenous nucleic acids encoding each of the enzymes selected from: (A) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase; a 3-oxopent-4 enoate reductase; and a 3-hydroxypent-4-enoate decarboxylase; (B) a succinyl-CoA:acetyl CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3 oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate reductase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4 pentadienoate decarboxylase; (C) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3 oxoadipate dehydrogenase; a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase; (D) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3 oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate reductase; a 3 hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4 pentadienoate decarboxylase; (E) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3 oxoadipate reductase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase; (F) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3 oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase; a 3 hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4- 77 pentadienoate decarboxylase; (G) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA synthetase or a 3-hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4 enedioate decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase; and (H) a succinyl CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA synthetase or a 3-hydroxyadipyl-CoA hydrolase; a 3 hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3 hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate decarboxylase. In some embodiments, the invention provides a non-naturally occurring microbial organism as described above, wherein said microbial organism further comprises: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof. In some aspects of the invention, the microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In some aspects of the invention, the microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In some embodiments, the invention provides a non-naturally occurring microbial orgnaism of as described above, wherein said microbial organism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial 78 organism comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase. In some aspects, the invention provides that the non-naturally occurring microbial organism as described herein, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. In another aspect, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, the invention provides a method for producing 1,3-butadiene, comprising culturing a non-naturally occurring microbial organism as described herein under conditions and for a sufficient period of time to produce 1,3-butadiene. In some embodiments, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a 2,4-pentadienoate pathway comprising at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate, said 2,4-pentadienoate pathway selected from: (A) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3 oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate reductase; and a 3-hydroxypent-4 enoate dehydratase; (B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate dehydratase; (C) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate dehydratase; and (D) a succinyl-CoA:acetyl CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3 hydroxyadipyl-CoA synthetase or a 3-hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate dehydratase. In some aspects, the microbial organism comprises two, three, four, five, or six exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. For example, 79 the microbial organism can comprise exogenous nucleic acids encoding each of the enzymes selected from: (A) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate reductase; and a 3 hydroxypent-4-enoate dehydratase; (B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3 oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate dehydratase; (C) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase ora 3 oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase; a 3 hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate dehydratase; and (D) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a 3 hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA synthetase or a 3-hydroxyadipyl CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate dehydratase. In some embodiments, the invention provides a non-naturally occurring microbial organism as described above, wherein said microbial organism further comprises: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof. In some aspects, the microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In some aspects, the microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA 80 synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In some aspects, the non-naturally occurring microbial orgnaism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase. In some aspects, the invention provides that the non-naturally occurring microbial organism as disclosed above, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. In some aspects, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, the invention provides a method for producing 2,4 pentadienoate, comprising culturing a non-naturally occurring microbial organism as described herein under conditions and for a sufficient period of time to produce 2,4 pentadienoate. In some embodiments, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a 1,3-butadiene pathway comprising at least one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene, said 1,3-butadiene pathway selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3 oxopentanoate reductase; a 3,5 -dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase; (B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene 1-ol dehydratase; (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5- 81 hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3 butene-1-ol dehydratase; (D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4 pentadiene decarboxylase; (E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3 butene-1-ol dehydratase; (F) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase; (G) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5 dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5 dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4 pentadiene decarboxylase; (H) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3 hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5 hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase; (I) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5 dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5 dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase; (J) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3 hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4 pentadiene decarboxylase; (K) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5 hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase; (L) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3 hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase; (M) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3 hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 -dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase; (N) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3- 82 hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 -dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase; and (0) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3 hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase. In some embodiments, the invention provides a non-naturally occurring microbial organism as described above, wherein said microbial organism comprises two, three, four, five, six or seven exogenous nucleic acids each encoding a 1,3-butadiene pathway enzyme. For example, in some aspects, the microbial organism comprises exogenous nucleic acids encoding each of the enzymes selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase; (B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3 oxopentanoate reductase; a 3,5 -dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase; (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase; (D) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase; (E) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase; (F) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase; (G) a malonyl-CoA:acetyl-CoA acyltransferase; a 3 oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase; (H) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5- 83 dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5 dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3-butene 1-ol dehydratase; (I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3 hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3 butene-1-ol dehydratase; (J) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5 hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase; (K) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3 hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3 butene-1-ol dehydratase; (L) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3 butene-1-ol dehydratase; (M) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4 pentadiene decarboxylase; (N) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3 butene-1-ol dehydratase; and (0) a malonyl-CoA:acetyl-CoA acyltransferase; a 3 oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase. In some embodiments, the invention provides a non-naturally occurring microbial organism as disclosed above, wherein said microbial organism further comprises: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 84 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof. In some aspects, the non naturally occurring microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In some aspects, the microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In some aspects, the microbial organism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase. In some aspects, the non-naturally occurring microbial organism as disclosed herein includes, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. In some aspects, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, the invention provides a method for producing 1,3-butadiene, comprising culturing a non-naturally occurring microbial organism as disclosed herein under conditions and for a sufficient period of time to produce 1,3-butadiene. In some embodiments, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a 2,4-pentadienoate pathway comprising at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate, said 2,4-pentadienoate pathway selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3 oxopentanoate reductase; a 3,5 -dihydroxypentanoate dehydratase; and a 5-hydroxypent-2- 85 enoate dehydratase; (B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase; (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase; (D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5 oxopentanoate reductase; a 3,5 -dihydroxypentanoate dehydratase; and a 5-hydroxypent-2 enoate dehydratase; and (E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase. In some aspects, the non-naturally occurring microbial organism as disclosed above comprises two, three, four, five or six exogenous nucleic acids each encoding a 2,4 pentadienoate pathway enzyme. For example, the microbial organism comprises exogenous nucleic acids encoding each of the enzymes selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase; (B) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase; (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase; (D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase; and (E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2 enoate dehydratase.

86 In some embodiments, the invention provides a non-naturally occurring microbial organism as disclosed above, wherein said microbial organism further comprises: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof. In some aspects, the microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In some aspects, the non-naturally occurring microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In some aspects, the microbial organism as disclose above comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase. In some aspects, the non-naturally occurring microbial organism as disclosed herein includes, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. In some aspects, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

87 In some embodiments, the invention provides a method for producing 2,4 pentadienoate, comprising culturing a non-naturally occurring microbial organism as disclosed above under conditions and for a sufficient period of time to produce 2,4 pentadienoate. In some embodiments, the invention provides a non-naturally occurring microbial organism, comprising a microbial organism having a 3-butene-1-ol pathway comprising at least one exogenous nucleic acid encoding a 3-butene-1-ol pathway enzyme expressed in a sufficient amount to produce 3-butene-1-ol, said 3-butene-1-ol pathway selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3 oxopentanoate reductase; a 3,5 -dihydroxypentanoate dehydratase; and a 5-hydroxypent-2 enoate decarboxylase; (B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5 hydroxy-3-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; (D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5 hydroxy-3-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; (F) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3 hydroxy-5-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (G) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; (H) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 hydroxy-5-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate 88 dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; and (J) a malonyl-CoA:acetyl CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl CoA reductase (alcohol forming); and a 3,5-dihydroxypentanoate decarboxylase. In some aspects, the non-naturally occurring microbial organism as dislosed above comprises two, three, four or five exogenous nucleic acids each encoding a 3-butene-1-ol pathway enzyme. For example, in some aspects, microbial organism comprises exogenous nucleic acids encoding each of the enzymes selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; (B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy-3 oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (C) a malonyl CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; (D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5 hydroxy-3-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; (F) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3 hydroxy-5-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (G) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3-hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; (H) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 hydroxy-5-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5-dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; and (J) a malonyl-CoA:acetyl- 89 CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl CoA reductase (alcohol forming); and a 3,5-dihydroxypentanoate decarboxylase. In some embodiments, the invention provides a non-naturally occurring microbial organism as disclosed above, wherein said microbial organism further comprises: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof. In some aspects, the non naturally occurring microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. In some aspects, the non-naturally occurring microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. In some aspects, the non-naturally occurring microbial comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase. In some aspects, the non-naturally occurring microbial organism as disclosed herein includes, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. In 90 some aspects, the non-naturally occurring microbial organism is in a substantially anaerobic culture medium. In some embodiments, the invention provides a method for producing 3-butene-1-ol, comprising culturing a non-naturally occurring microbial organism as disclosed abov e under conditions and for a sufficient period of time to produce 3-butene-1-ol. In some embodiments, the invention provides a method for producing 1,3-butadiene comprising, culturing a non-naturally occurring microbial organism that can produce 3 butene-1-ol as disclosed above under conditions and for a sufficient period of time to produce 3-butene-1-ol, and chemically converting said 3-butene-1-ol to 1,3-butadiene. It is understood that methods for chemically converting 3-butene-1-ol to 1,3-butadiene are well know in the art. This invention is also directed, in part to engineered biosynthetic pathways to improve carbon flux through a central metabolism intermediate en route to 2,4-pentadienoate, 3 butene-1-ol, or 1,3-butadiene. The present invention provides non-naturally occurring microbial organisms having one or more exogenous genes encoding enzymes that can catalyze various enzymatic transformations en route to 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene. In some embodiments, these enzymatic transformations are part of the reductive tricarboxylic acid (RTCA) cycle and are used to improve product yields, including but not limited to, from carbohydrate-based carbon feedstock. In numerous engineered pathways, realization of maximum product yields based on carbohydrate feedstock is hampered by insufficient reducing equivalents or by loss of reducing equivalents and/or carbon to byproducts. In accordance with some embodiments, the present invention increases the yields of 2,4-pentadienoate, 3-butene-1-ol, or 1,3 butadiene by (i) enhancing carbon fixation via the reductive TCA cycle, and/or (ii) accessing additional reducing equivalents from gaseous carbon sources and/or syngas components such as CO, C0 2 , and/or H 2 . In addition to syngas, other sources of such gases include, but are not listed to, the atmosphere, either as found in nature or generated. The C0 2 -fixing reductive tricarboxylic acid (RTCA) cycle is an endergenic anabolic pathway of CO 2 assimilation which uses reducing equivalents and ATP (Figure 22). One turn of the RTCA cycle assimilates two moles of CO 2 into one mole of acetyl-CoA, or four moles of CO 2 into one mole of oxaloacetate. This additional availability of acetyl-CoA 91 improves the maximum theoretical yield of product molecules derived from carbohydrate based carbon feedstock. Exemplary carbohydrates include but are not limited to glucose, sucrose, xylose, arabinose and glycerol. In some embodiments, the reductive TCA cycle, coupled with carbon monoxide dehydrogenase and/or hydrogenase enzymes, can be employed to allow syngas, C0 2 , CO, H 2 , and/or other gaseous carbon source utilization by microorganisms. Synthesis gas (syngas), in particular is a mixture of primarily H 2 and CO, sometimes including some amounts of C0 2 , that can be obtained via gasification of any organic feedstock, such as coal, coal oil, natural gas, biomass, or waste organic matter. Numerous gasification processes have been developed, and most designs are based on partial oxidation, where limiting oxygen avoids full combustion, of organic materials at high temperatures (500-1500'C) to provide syngas as a 0.5:1-3:1 H 2 /CO mixture. In addition to coal, biomass of many types has been used for syngas production and represents an inexpensive and flexible feedstock for the biological production of renewable chemicals and fuels. Carbon dioxide can be provided from the atmosphere or in condensed from, for example, from a tank cylinder, or via sublimation of solid CO 2 . Similarly, CO and hydrogen gas can be provided in reagent form and/or mixed in any desired ratio. Other gaseous carbon forms can include, for example, methanol or similar volatile organic solvents. The components of synthesis gas and/or other carbon sources can provide sufficient

CO

2 , reducing equivalents, and ATP for the reductive TCA cycle to operate. One turn of the RTCA cycle assimilates two moles of CO 2 into one mole of acetyl-CoA and requires 2 ATP and 4 reducing equivalents. CO and/or H 2 can provide reducing equivalents by means of carbon monoxide dehydrogenase and hydrogenase enzymes, respectively. Reducing equivalents can come in the form of NADH, NADPH, FADH, reduced quinones, reduced ferredoxins, reduced flavodoxins and thioredoxins. The reducing equivalents, particularly NADH, NADPH, and reduced ferredoxin, can serve as cofactors for the RTCA cycle enzymes, for example, malate dehydrogenase, fumarate reductase, alpha ketoglutarate:ferredoxin oxidoreductase (alternatively known as 2-oxoglutarate:ferredoxin oxidoreductase, alpha-ketoglutarate synthase, or 2-oxoglutarate synthase), pyruvate:ferredoxin oxidoreductase and isocitrate dehydrogenase. The electrons from these reducing equivalents can alternatively pass through an ion-gradient producing electron transport chain where they are passed to an acceptor such as oxygen, nitrate, oxidized metal 92 ions, protons, or an electrode. The ion-gradient can then be used for ATP generation via an ATP synthase or similar enzyme. The reductive TCA cycle was first reported in the green sulfur photosynthetic bacterium Chlorobium limicola (Evans et al., Proc. Natl. Acad. Sci. U.S.A. 55:928-934 (1966)). Similar pathways have been characterized in some prokaryotes (proteobacteria, green sulfur bacteria and thermophillic Knallgas bacteria) and sulfur-dependent archaea (Hugler et al., J Bacteriol. 187:3020-3027 (2005; Hugler et al., Environ. Microbiol. 9:81-92 (2007). In some cases, reductive and oxidative (Krebs) TCA cycles are present in the same organism (Hugler et al., supra (2007); Siebers et al., J Bacteriol. 186:2179-2194 (2004)). Some methanogens and obligate anaerobes possess incomplete oxidative or reductive TCA cycles that may function to synthesize biosynthetic intermediates (Ekiel et al., J Bacteriol. 162:905-908 (1985); Wood et al., FEMSMicrobiol. Rev. 28:335-352 (2004)). The key carbon-fixing enzymes of the reductive TCA cycle are alpha ketoglutarate:ferredoxin oxidoreductase, pyruvate:ferredoxin oxidoreductase and isocitrate dehydrogenase. Additional carbon may be fixed during the conversion of phosphoenolpyruvate to oxaloacetate by phosphoenolpyruvate carboxylase or phosphoenolpyruvate carboxykinase carboxykinase or by conversion of pyruvate to malate by malic enzyme. Many of the enzymes in the TCA cycle are reversible and can catalyze reactions in the reductive and oxidative directions. However, some TCA cycle reactions are irreversible in vivo and thus different enzymes are used to catalyze these reactions in the directions required for the reverse TCA cycle. These reactions are: (1) conversion of citrate to oxaloacetate and acetyl-CoA, (2) conversion of fumarate to succinate, and (3) conversion of succinyl-CoA to alpha-ketoglutarate. In the TCA cycle, citrate is formed from the condensation of oxaloacetate and acetyl-CoA. The reverse reaction, cleavage of citrate to oxaloacetate and acetyl-CoA, is ATP-dependent and catalyzed by ATP-citrate lyase, or citryl CoA synthetase and citryl-CoA lyase. Alternatively, citrate lyase can be coupled to acetyl CoA synthetase, an acetyl-CoA transferase, or phosphotransacetylase and acetate kinase to form acetyl-CoA and oxaloacetate from citrate. The conversion of succinate to fumarate is catalyzed by succinate dehydrogenase while the reverse reaction is catalyzed by fumarate reductase. In the TCA cycle succinyl-CoA is formed from the NAD(P) dependent 93 decarboxylation of alpha-ketoglutarate by the alpha-ketoglutarate dehydrogenase complex. The reverse reaction is catalyzed by alpha-ketoglutarate:ferredoxin oxidoreductase. An organism capable of utilizing the reverse tricarboxylic acid cycle to enable production of acetyl-CoA-derived products on 1) CO, 2) CO 2 and H 2 , 3) CO and CO 2 , 4) synthesis gas comprising CO and H 2 , and 5) synthesis gas or other gaseous carbon sources comprising CO, CO 2 , and H 2 can include any of the following enzyme activities: ATP-citrate lyase, citrate lyase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase, fumarase, malate dehydrogenase, acetate kinase, phosphotransacetylase, acetyl-CoA synthetase, acetyl-CoA transferase, pyruvate:ferredoxin oxidoreductase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, hydrogenase, and ferredoxin (see Figure 23). Enzymes and the corresponding genes required for these activities are described herein above. Carbon from syngas or other gaseous carbon sources can be fixed via the reverse TCA cycle and components thereof. Specifically, the combination of certain carbon gas-utilization pathway components with the pathways for formation of 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene from acetyl-CoA results in high yields of these products by providing an efficient mechanism for fixing the carbon present in carbon dioxide, fed exogenously or produced endogenously from CO, into acetyl-CoA. In some embodiments, a 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway in a non-naturally occurring microbial organism of the invention can utilize any combination of (1) CO, (2) CO 2 , (3) H 2 , or mixtures thereof to enhance the yields of biosynthetic steps involving reduction, including addition to driving the reductive TCA cycle. In some embodiments a non-naturally occurring microbial organism having an 2,4 pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway includes at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme. The at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a fumarate reductase, isocitrate dehydrogenase, aconitase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; and at least one exogenous enzyme selected from a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin, expressed in a sufficient amount to 94 allow the utilization of (1) CO, (2) C0 2 , (3) H 2 , (4) CO 2 and H 2 , (5) CO and CO 2 , (6) CO and

H

2 , or (7) CO, CO 2 , and H 2 . In some embodiments a method includes culturing a non-naturally occurring microbial organism having a 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway also comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme. The at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a fumarate reductase, isocitrate dehydrogenase, aconitase, and an alpha ketoglutarate:ferredoxin oxidoreductase. Additionally, such an organism can also include at least one exogenous enzyme selected from a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin, expressed in a sufficient amount to allow the utilization of (1) CO, (2) C0 2 , (3) H 2 , (4) CO 2 and H 2 , (5) CO and CO 2 , (6) CO and H 2 , or (7) CO, C0 2 , and H 2 to produce a product. In some embodiments a non-naturally occurring microbial organism having an 2,4 pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway further includes at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA. The at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a fumarate reductase, a pyruvate:ferredoxin oxidoreductase, isocitrate dehydrogenase, aconitase and an alpha ketoglutarate:ferredoxin oxidoreductase. In some embodiments a non-naturally occurring microbial organism having an 2,4 pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway includes at least one exogenous nucleic acid encoding an enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of carbon monoxide and/or hydrogen, thereby increasing the yield of redox-limited products via carbohydrate-based carbon feedstock. The at least one exogenous nucleic acid is selected from a carbon monoxide dehydrogenase, a hydrogenase, an NAD(P)H:ferredoxin oxidoreductase, and a ferredoxin. In some embodiments, the present invention provides a method for enhancing the availability of reducing equivalents in the presence of carbon monoxide or hydrogen thereby increasing the yield of redox-limited products via carbohydrate-based carbon feedstock, such as sugars or gaseous carbon sources, the method includes culturing this non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene.

95 In some embodiments, the non-naturally occurring microbial organism having an 2,4 pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway includes two exogenous nucleic acids, each encoding a reductive TCA pathway enzyme. In some embodiments, the non naturally occurring microbial organism having an 2,4-pentadienoate, 3-butene-1-ol, or 1,3 butadiene pathway includes three exogenous nucleic acids each encoding a reductive TCA pathway enzyme. In some embodiments, the non-naturally occurring microbial organism includes three exogenous nucleic acids encoding an ATP-citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase. In some embodiments, the non naturally occurring microbial organism includes three exogenous nucleic acids encoding a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase. In some embodiments, the non-naturally occurring microbial organisms having an 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway further include an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, and combinations thereof. In some embodiments, the non-naturally occurring microbial organism having an 2,4 pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway further includes an exogenous nucleic acid encoding an enzyme selected from carbon monoxide dehydrogenase, acetyl-CoA synthase, ferredoxin, NAD(P)H:ferredoxin oxidoreductase and combinations thereof. In some embodiments, the non-naturally occurring microbial organism having an 2,4 pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway utilizes a carbon feedstock selected from (1) CO, (2) CO 2 , (3) CO 2 and H 2 , (4) CO and H 2 , or (5) CO, CO 2 , and H 2 . In some embodiments, the non-naturally occurring microbial organism having an 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway utilizes hydrogen for reducing equivalents. In some embodiments, the non-naturally occurring microbial organism having an 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway utilizes CO for reducing equivalents. In some embodiments, the non-naturally occurring microbial organism having an 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway utilizes combinations of CO and hydrogen for reducing equivalents.

96 In some embodiments, the non-naturally occurring microbial organism having an 2,4 pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway further includes one or more nucleic acids encoding an enzyme selected from a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a pyruvate carboxylase, and a malic enzyme. In some embodiments, the non-naturally occurring microbial organism having an 2,4 pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway further includes one or more nucleic acids encoding an enzyme selected from a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA synthetase, and a succinyl-CoA transferase. In some embodiments, the non-naturally occurring microbial organism having an 2,4 pentadienoate, 3-butene-1-ol, or 1,3-butadiene pathway further includes at least one exogenous nucleic acid encoding a citrate lyase, an ATP-citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, and a ferredoxin. In some embodiments, the carbon feedstock and other cellular uptake sources such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in 1,3-butadiene or any 1,3-butadiene pathway intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as "uptake sources." Uptake sources can provide isotopic enrichment for any atom present in the product toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway or any intermediate en route thereto. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as "uptake sources." Uptake sources can provide isotopic enrichment for any atom present in the product toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene or toluene, benzene, p toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway intermediate including any toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene impurities generated in diverging away 97 from the pathway at any point. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, chloride or other halogens. In some embodiments, the uptake sources can be selected to alter the carbon-12, carbon-13, and carbon-14 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen-16, oxygen-17, and oxygen-18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and tritium ratios. In some embodiments, the uptake sources can selected to alter the nitrogen-14 and nitrogen-15 ratios. In some embodiments, the uptake sources can be selected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-3 1, phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be selected to alter the chlorine-35, chlorine-36, and chlorine-37 ratios. In some embodiments, a target isotopic ratio of an uptake source can be obtained via synthetic chemical enrichment of the uptake source. Such isotopically enriched uptake sources can be purchased commercially or prepared in the laboratory. In some embodiments, a target isotopic ratio of an uptake source can be obtained by choice of origin of the uptake source in nature. In some such embodiments, a source of carbon, for example, can be selected from a fossil fuel-derived carbon source, which can be relatively depleted of carbon 14, or an environmental carbon source, such as C0 2 , which can possess a larger amount of carbon-14 than its petroleum-derived counterpart. Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be integrated with separation techniques such as liquid chromatography (LC) and/or high performance liquid chromatography (HPLC). In some embodiments, the present invention provides toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene or a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4- 98 pentadienoate, 3-butene-lol or 1,3-butadiene intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that reflects an atmospheric carbon uptake source. In some such embodiments, the uptake source is CO 2 . In some embodiments, In some embodiments, the present invention provides toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene , intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that reflects petroleum-based carbon uptake source. In some embodiments, the present invention provides toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene or a toluene, benzene, p toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source. Such combination of uptake sources is one means by which the carbon-12, carbon-13, and carbon-14 ratio can be varied. Accordingly, given the teachings and guidance provided herein, those skilled in the art will understand that a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds of the invention when grown on a carbon source such as a carbohydrate. Such compounds include, for example, toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene and any of the intermediate metabolites in the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for example, inclusion of some or all of the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene biosynthetic pathways. Accordingly, the invention provides a non-naturally occurring microbial organism that produces and/or secretes toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene 99 when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway when grown on a carbohydrate or other carbon source. The toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene producing microbial organisms of the invention can initiate synthesis from an intermediate, for example, phenylalanine, phenylpyruvate, phenylacetaldehyde, phenylacetate, benzoyl-CoA, 3-oxo-3 phenylpropionyl-CoA, [(3-oxo-3-phenylpropionyl)oxy] phosphonate, benzoyl acetate, acetophenone, 1-phenylethanol, trans,trans-muconate, cis,trans-muconate, cis,cis-muconate, trans-2,4-pentadienoate, and cis-2,4-pentadienoate. As a further example, a (2-hydroxy-3 methyl-4-oxobutoxy)phosphonate pathway intermediate can be 1 -deoxy-D-xylulose-5 phosphate or C-methyl-D-erythritol-4-phosphate (see Example III and Figure 5). A p-toluate pathway intermediate can be, for example, 2,4-dihydroxy-5-methyl-6 [(phosphonooxy)methyl]oxane-2-carboxylate, 1,3-dihydroxy-4-methyl-5-oxocyclohexane-1 carboxylate, 5-hydroxy-4-methyl-3-oxocyclohex-1-ene-1-carboxylate, 3,5-dihydroxy-4 methylcyclohex-1-ene-1-carboxylate, 5-hydroxy-4-methyl-3-(phosphonooxy)cyclohex-1-ene 1-carboxylate, 5-[(1-carboxyeth-1-en-1-yl)oxy]-4-methyl-3-(phosphonooxy)cyclohex-1-ene 1-carboxylate, or 3-[(1-carboxyeth-1-en-1-yl)oxy]-4-methylcyclohexa-1,5-diene-1 carboxylate (see Example IV and Figure 6). A terephthalate intermediate can be, for example, 4-carboxybenzyl alcohol or 4-carboxybenzaldehyde (see Example V and Figure 7). As disclosed herein, p-toluate and benzoate are exemplary intermediates that can be the subject of a non-naturally occurring microbial organism. Such carboxylates can occur in ionized form or fully protonated form. Accordingly, the suffix "-ate," or the acid form, can be used interchangeably to describe both the free acid form as well as any deprotonated form, in particular since the ionized form is known to depend on the pH in which the compound is found. It is understood that propionate products accessible in accordance with the present invention include ester forms, such as 0-carboxylate and S-carboxylate esters. 0- and S carboxylates can include lower alkyl, that is C1 to C6, branched or straight chain carboxylates. Some such 0- or S-carboxylates include, without limitation, methyl, ethyl, n propyl, n-butyl, i-propyl, sec-butyl, and tert-butyl, pentyl, hexyl 0- or S-carboxylates, any of which can further possess an unsaturation, providing for example, propenyl, butenyl, pentyl, 100 and hexenyl 0- or S-carboxylates. O-carboxylates can be the product of a biosynthetic pathway. Exemplary 0-carboxylates accessed via biosynthetic pathways can include, without limitation, methyl propionate, ethyl propionate, and n-propyl propionate. Other biosynthetically accessible O-propionates can include medium to long chain groups, that is C7-C22, 0-propionate esters derived from fatty alcohols, such heptyl, octyl, nonyl, decyl, undecyl, lauryl, tridecyl, myristyl, pentadecyl, cetyl, palmitolyl, heptadecyl, stearyl, nonadecyl, arachidyl, heneicosyl, and behenyl alcohols, any one of which can be optionally branched and/or contain unsaturations. O-propionate esters can also be accessed via a biochemical or chemical process, such as esterification of a free carboxylic acid product or transesterification of an 0- or S-propionate. S-carboxylates are exemplified by CoA S-esters, cysteinyl S-esters, alkylthioesters, and various aryl and heteroaryl thioesters. The non-naturally occurring microbial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl 4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway enzyme or protein in sufficient amounts to produce toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene . It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce toluene, benzene, p toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene . Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene resulting in intracellular concentrations between about 0.1-200 mM or more. Generally, the intracellular concentration of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl 4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene is between about 3-150 mM, particularly between about 5-125 mM and more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular concentrations between and above 101 each of these exemplary ranges also can be achieved from the non-naturally occurring microbial organisms of the invention. In some embodiments, culture conditions include anaerobic or substantially anaerobic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in U.S. publication 2009/0047719, filed August 10, 2007. Any of these conditions can be employed with the non-naturally occurring microbial organisms as well as other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene producers can synthesize toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene at intracellular concentrations of 5-10 mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene producing microbial organisms can produce toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene intracellularly and/or secrete the product into the culture medium. In addition to the culturing and fermentation conditions disclosed herein, growth condition for achieving biosynthesis of toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene can include the addition of an osmoprotectant to the culturing conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant. Briefly, an osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. Osmoprotectants include, but are not limited to, betaines, amino acids, and the sugar trehalose. Non-limiting examples of such are glycine betaine, praline 102 betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio-2 methylproprionate, pipecolic acid, dimethylsulfonioacetate, choline, L-camitine and ectoine. In one aspect, the osmoprotectant is glycine betaine. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used. The amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about 10mM, no more than about 50mM, no more than about 100mM or no more than about 500mM. The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions. As described herein, one exemplary growth condition for achieving biosynthesis of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally occurring microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or substantially anaerobic conditions. Briefly, anaerobic conditions refers to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. Substantially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an N 2

/CO

2 mixture or other suitable non-oxygen gas or gases. The culture conditions described herein can be scaled up and grown continuously for manufacturing of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene . Exemplary growth procedures include, for 103 example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of toluene, benzene, p-toluate, terephthalate, (2 hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene . Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene will include culturing a non-naturally occurring toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene producing organism of the invention in sufficient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can be include, for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms of the invention can be cultured for hours, if suitable for a particular application. It is to be understood that the continuous and/or near continuous culture conditions also can include all time intervals in between these exemplary periods. It is further understood that the time of culturing the microbial organism of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose. Fermentation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene can be utilized in, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art. In addition to the above fermentation procedures using the toluene, benzene, p toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene 104 producers of the invention for continuous production of substantial quantities of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2 hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene , the toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene producers also can be, for example, simultaneously subjected to chemical synthesis procedures to convert the product to other compounds or the product can be separated from the fermentation culture and sequentially subjected to chemical conversion to convert the product to other compounds, if desired. To generate better producers, metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and U.S. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene . One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)). OptKnock is a metabolic modeling and simulation program that suggests gene deletion or disruption strategies that result in genetically stable microorganisms which overproduce the target product. Specifically, the framework examines the complete metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By coupling biochemical production with cell growth through strategically placed gene deletions or other functional gene disruption, the growth selection pressures imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains reverting to their wild-type states because the genes selected by OptKnock are to be completely removed from the genome. Therefore, this 105 computational methodology can be used to either identify alternative pathways that lead to biosynthesis of a desired product or used in connection with the non-naturally occurring microbial organisms for further optimization of biosynthesis of a desired product. Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock program relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) models. These constraints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA microarray experimental data. OptKnock also computes solutions to various metabolic problems by, for example, tightening the flux boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or deletions. OptKnock computational framework allows the construction of model formulations that allow an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, U.S. publication 2002/0168654, filed January 10, 2002, in International Patent No. PCT/US02/00660, filed January 10, 2002, and U.S. publication 2009/0047719, filed August 10, 2007. Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny@. This computational method and system is described in, for example, U.S. publication 2003/0233218, filed June 14, 2002, and in International Patent Application No. PCT/US03/18838, filed June 13, 2003. SimPheny@ is a computational system that can be used to produce a network model in silico and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby determining a range of allowed activities for the biological system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known stoichiometry of the included reactions as well as reaction thermodynamic and capacity constraints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the biological system or of its biochemical components.

106 These computational approaches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, thereby enhancing the precision with which physiological performance or phenotype can be predicted. Given the teachings and guidance provided herein, those skilled in the art will be able to apply various computational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired compound in host microbial organisms. Such metabolic modeling and simulation methods include, for example, the computational systems exemplified above as SimPheny@ and OptKnock. For illustration of the invention, some methods are described herein with reference to the OptKnock computation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation computational frameworks and methods well known in the art. The methods described above will provide one set of metabolic reactions to disrupt. Elimination of each reaction within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism. Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes encoding one or more enzymes that catalyze each reaction within the set of reactions. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction database having a relationship between enzymes and encoding genes. Once identified, the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set. One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction 107 by other genetic aberrations including, for example, mutation, deletion of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur. To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic modifications that can result in the biosynthesis, including growth-coupled biosynthesis of a desired product, an optimization method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration. Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous iteration that obligatorily couples product biosynthesis to growth. For example, if a previously identified growth-coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously considered in subsequent solutions. The integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in combination with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny@. The methods exemplified herein allow the construction of cells and organisms that biosynthetically produce a desired product, including the obligatory coupling of production of a target biochemical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the computational methods described herein allow the identification and implementation of metabolic modifications that are identified by an in silico method selected from OptKnock or SimPheny@. The set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.

108 As discussed above, the OptKnock methodology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum-growth phenotypes when subjected to long periods of growth selection. In other words, the approach leverages an organism's ability to self-optimize under selective pressures. The OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force a coupling between biochemical production and cell growth based on network stoichiometry. The identification of optimal gene/reaction knockouts requires the solution of a bilevel optimization problem that chooses the set of active reactions such that an optimal growth solution for the resulting network overproduces the biochemical of interest (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)). An in silico stoichiometric model of E. coli metabolism can be employed to identify essential genes for metabolic pathways as exemplified previously and described in, for example, U.S. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Patent No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint gene deletions leading to the growth-coupled production of a desired product. Further, the solution of the bilevel OptKnock problem provides only one set of deletions. To enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled production formation, an optimization technique, termed integer cuts, can be implemented. This entails iteratively solving the OptKnock problem with the incorporation of an additional constraint referred to as an integer cut at each iteration, as discussed above. As disclosed herein, a nucleic acid encoding a desired activity of a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate, (2-hydroxy-4 oxobutoxy)phosphonate, benzoate, styrene, 2,4-pentadienoate, 3-butene-lol or 1,3-butadiene pathway can be introduced into a host organism. In some cases, it can be desirable to modify an activity of a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway enzyme or protein to increase production of toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene . For example, known mutations that increase the activity of a protein or enzyme can be introduced into an encoding nucleic acid molecule.

109 Additionally, optimization methods can be applied to increase the activity of an enzyme or protein and/or decrease an inhibitory activity, for example, decrease the activity of a negative regulator. One such optimization method is directed evolution. Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high-throughput screening assays that allow the automated screening of many enzyme variants (for example, >104). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al., Biomol.Eng 22:11-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax. Biomol. Eng 22:1-9 (2005).; and Sen et al., Appl Biochem.Biotechnol 143:212-223 (2007)) to be effective at creating diverse variant libraries, and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes. Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example: selectivity/specificity, for conversion of non-natural substrates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH conditions; substrate or product tolerance, so that high product titers can be achieved; binding (Km), including broadening substrate binding to include non-natural substrates; inhibition (Ki), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increases enzymatic reaction rates to achieve desired flux; expression levels, to increase protein yields and overall pathway flux; oxygen stability, for operation of air sensitive enzymes under aerobic conditions; and anaerobic activity, for operation of an aerobic enzyme in the absence of oxygen. A number of exemplary methods have been developed for the mutagenesis and diversification of genes to target desired properties of specific enzymes. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of a toluene, benzene, p-toluate, terephthalate, (2-hydroxy-3-methyl-4- 110 oxobutoxy)phosphonate, (2-hydroxy-4-oxobutoxy)phosphonate, benzoate, styrene, 2,4 pentadienoate, 3-butene-lol or 1,3-butadiene pathway enzyme or protein. Such methods include, but are not limited to EpPCR, which introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions (Pritchard et al., J. Theor.Biol. 234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA), which is similar to epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats (Fujii et al., Nucleic Acids Res. 32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493 2497 (2006)); DNA or Family Shuffling, which typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes (Stemmer, Proc. Natl. Acad. Sci. U.S.A. 91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)); Staggered Extension (StEP), which entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec) (Zhao et al., Nat. Biotechnol. 16:258-261 (1998)); Random Priming Recombination (RPR), in which random sequence primers are used to generate many short DNA fragments complementary to different segments of the template (Shao et al., Nucleic Acids Res 26:681-683 (1998)). Additional methods include Heteroduplex Recombination, in which linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (Volkov et al, Nucleic Acids Res. 27:e18 (1999); and Volkov et al., Methods Enzymol. 328:456-463 (2000)); Random Chimeragenesis on Transient Templates (RACHITT), which employs Dnase I fragmentation and size fractionation of single stranded DNA (ssDNA) (Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined Extension on Truncated templates (RETT), which entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates (Lee et al., J. Molec. Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide Gene Shuffling (DOGS), in which degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods Mol. Biol. 352:191-204 (2007); Bergquist et al., Biomol. Eng. 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)); Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest (Ostermeier et al., Proc. Natl. Acad. Sci.

111 U.S.A. 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol. 17:1205-1209 (1999)); Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations (Lutz et al., Nucleic Acids Res. 29:E16 (2001)); SCRATCHY, which combines two methods for recombining genes, ITCHY and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. U.S.A. 98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which mutations made via epPCR are followed by screening/selection for those retaining usable activity (Bergquist et al., Biomol. Eng. 22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method that generates a pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage, which is used as a template to extend in the presence of "universal" bases such as inosine, and replication of an inosine containing complement gives random base incorporation and, consequently, mutagenesis (Wong et al., Biotechnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling, which uses overlapping oligonucleotides designed to encode "all genetic diversity in targets" and allows a very high diversity for the shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255 (2002)); Nucleotide Exchange and Excision Technology NexT, which exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation (Muller et al., Nucleic Acids Res. 33:e117 (2005)). Further methods include Sequence Homology-Independent Protein Recombination (SHIPREC), in which a linker is used to facilitate fusion between two distantly related or unrelated genes, and a range of chimeras is generated between the two genes, resulting in libraries of single-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460 (2001)); Gene Site Saturation MutagenesisTM (GSSMTM), in which the starting materials include a supercoiled double stranded DNA (dsDNA) plasmid containing an insert and two primers which are degenerate at the desired site of mutations (Kretz et al., Methods Enzymol. 388:3 11 (2004)); Combinatorial Cassette Mutagenesis (CCM), which involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acid sequence alterations (Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science 241:53-57 (1988)); Combinatorial Multiple Cassette Mutagenesis (CMCM), which is essentially similar to CCM and uses epPCR at high mutation rate to identify hot spots and hot regions and then extension by CMCM to cover a defined 112 region of protein sequence space (Reetz et al., Angew. Chem. Int. Ed Engl. 40:3589-3591 (2001)); the Mutator Strains technique, in which conditional ts mutator plasmids, utilizing the mutD5 gene, which encodes a mutant subunit of DNA polymerase III, to allow increases of 20 to 4000-X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required (Selifonova et al., Appl. Environ. Microbiol. 67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260:359-3680 (1996)). Additional exemplary methods include Look-Through Mutagenesis (LTM), which is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of selected amino acids (Rajpal et al., Proc. Natl. Acad. Sci. U.S.A. 102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene (Tunable GeneReassembly T M

(TGR

T M ) Technology supplied by Verenium Corporation), in Silico Protein Design Automation (PDA), which is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics, and generally works most effectively on proteins with known three-dimensional structures (Hayes et al., Proc. Natl. Acad. Sci. U.S.A. 99:15926-15931 (2002)); and Iterative Saturation Mutagenesis (ISM), which involves using knowledge of structure/function to choose a likely site for enzyme improvement, performing saturation mutagenesis at chosen site using a mutagenesis method such as Stratagene QuikChange (Stratagene; San Diego CA), screening/selecting for desired properties, and, using improved clone(s), starting over at another site and continue repeating until a desired activity is achieved (Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)). Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with adaptive evolution techniques, as described herein. EXAMPLE I Pathways to Benzene and Toluene This example shows pathways from phenylalanine to toluene, phenylalanine to benzene and benzoyl-CoA to styrene.

113 Pathways for enzymatic conversion of phenylalanine are shown in Figure 1. The first step entails conversion of phenylalanine to phenylpyruvate, a transformation that can be accomplished by an aminotransferase or a deaminating oxidoreductase. Phenylpyruvate is then decarboxylated to phenylacetaldehyde in Step B of Figure 1. Toluene may be produced directly from phenylacetaldehyde by decarbonylation (Figure 1, Step E), or indirectly via a phenylacetate intermediate (Figure 1, Steps C and D). Phenylacetate can be oxidized to phenylacetate (Figure 1, Step C) by either a phenylacetaldehyde dehydrogenase or a phenylacetaldehyde oxidase. An alternate pathway is the direct oxidative decarboxylation of phenylpyruvate to phenylacetate by phenylpyruvate oxidase (Figure 1, StepF). A one-step pathway for enzymatic conversion of phenylalanine to benzene is shown in Figure 2. The conversion of phenylalanine and water to benzene, pyruvate and ammonia is catalyzed by an enzyme with phenylalanine benzene-lyase activity. Enzymatic pathways to styrene from benzoyl-CoA are shown in Figure 3. Benzoyl CoA is a common metabolic intermediate of numerous biosynthetic and degradation pathways. Pathways involving the biosynthesis of benzoyl-CoA, and also the generation of benzoyl-CoA as a degradation product, are known in the art. In the proposed styrene pathways, benzoyl-CoA and acetyl-CoA are first converted to 3-oxo-3-phenylpropionyl-CoA by a beta-ketothiolase (Figure 3, Step A). The CoA moiety of 3-oxo-3-phenylpropionyl-CoA is then released by a CoA hydrolase, transferase or synthase (Figure 3, Step B). Alternately, 3-oxo-3-phenylpropionyl-CoA is converted to benzoyl-acetate in two enzymatic steps by a phosphotrans-3-oxo-3-phenylpropionylase and benzoyl-acetate kinase (Figure 3, Steps F and G). Once formed, benzoyl-acetate is decarboxylated, reduced and dehydrated to form styrene (Figure 3, Steps C, D and E). Enzymes for catalyzing the transformations shown in Figures 1-3 are categorized by EC number (Table 1) and described further below. Label Function Step 1.1.1.a Oxidoreductase (oxo to alcohol) 3D 1.2.1.a Oxidoreductase (aldehyde to acid) IC 1.2.3.a Aldehyde oxidase 1 C/F 1.4.1.a Oxidoreductase (aminating/deaminating) 1A 2.3.1.a Acyltransferase (transferring phosphate group to CoA) 3F 2.3.1 .b Beta-ketothiolase 3A 2.6.1.a Aminotransferase 1A 114 2.7.2.a Phosphotransferase, carboxyl group acceptor (kinase) 3G 2.8.3.a Coenzyme-A transferase 3B 3.1.2.a Thiolester hydrolase (CoA specific) 3B 4.1.1.a Carboxy-lyase 1B/D, 3C 4.1.99.a Decarbonylase 1E 4.1.99.b Lyase 2 4.2.1.a Hydro-lyase 3E 6.2.1.a Acid-thiol ligase 3B 1.1.1 .a Oxidoreductase (oxo to alcohol): The reduction of acetophenone to 1 phenylethanol (Step D of Figure 3) is catalyzed by a ketone reductase with acetophenone reductase activity. Enzymes with this activity have been characterized in numerous organisms, and product formation is generally stereoselective. An exemplary enzyme with this activity is the R-specific short-chain dehydrogenase/reductase of Lactobacillus brevis, which has been extensively studied, structurally characterized and re-engineered to prefer NADH to NADPH as a cosubstrate (Schlieben et al., J. Mol.Biol. 349:801-813 (2005)). Additional enzymes with acetophenone reductase activity are encoded by adhF1 of Pseudomonasfluorescens (Hildebrandt et al., Appl. Microbiol Biotechnol. 59:483-487 (2002)), adh of Thermus thermophilus (Pennacchio et al., Appl. Environ. Microbiol 74:3949 3958 (2008)) and LSADH of Leifsonia sp. S749 (Inoue et al., Biosci. Biotechnol. Biochem. 70:418-426 (2006)). An S-specific enzyme was characterized in the ethylbenzene degradation pathway of the denitrifying bacterium Aromatoleum aromaticum EbN1 (Kniemeyer et al., Arch. Microbiol. 176:129-135 (2001)). This enzyme, encoded by ped, favors the reductive direction at low pH (4), while the oxidative direction is favored at neutral pH. Gene GenBank Accession No. GI No. Organism LVIS_0347 YP_794544.1 116333017 Lactobacillus brevis adhF1 AAL79772.1 18860822 Pseudomonasfluorescens adhTt YP_003977.1 46198310 Thermus thermophilus LSADH BAD99642.1 67625613 Leifsonia sp. S749 ped YP_158329.1 56476740 Aromatoleum aromaticum EbN1 A variety of alcohol dehydrogenase enzymes catalyze the reduction of a ketone to an alcohol functional group. These enzymes are also suitable for catalyzing the reduction of acetophenone. Two such enzymes in E. coli are encoded by malate dehydrogenase (mdh) and 115 lactate dehydrogenase (ldhA). The lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high activities on 2-ketoacids of various chain lengths including lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (Steinbuchel et al., Eur. J. Biochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun. 77:586-591 (1977)). An additional oxidoreductase is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the human heart which has been cloned and characterized (Marks et al., J. Biol. Chem. 267:15459-15463 (1992)). Alcohol dehydrogenase enzymes of C. beijerinckii (Ismaiel et al., J.Bacteriol. 175:5097-5105 (1993)) and T. brockii (Lamed et al., Biochem. J. 195:183-190 (1981); Peretz et al., Biochemistry 28:6549-6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase, or alternatively, 2-butanol dehydrogenase, catalyzes the reduction of MEK to form 2-butanol. Exemplary MEK reductase enzymes can be found in Rhodococcus ruber (Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and Pyrococcusfuriosus (van der et al., Eur. J. Biochem. 268:3062-3068 (2001)). Gene GenBank Accession No. GI No. Organism Mdh AAC76268.1 1789632 Escherichia coli IdhA NP_415898.1 16129341 Escherichia coli Ldh YP_725182.1 113866693 Ralstonia eutropha bdh AAA58352.1 177198 Homo sapiens adh AAA23199.2 60592974 Clostridium beijerinckii NRRL B593 adh P14941.1 113443 Thermoanaerobacter brockii HTD4 adhA AAC25556 3288810 Pyrococcusfuriosus sadh CAD36475 21615553 Rhodococcus ruber 1.2.1 .a Oxidoreductase (aldehyde to acid): Oxidation of phenylacetaldehyde to phenylacetate is catalyzed by phenylacetaldehyde dehydrogenase (Step C of Figure 1), an enzyme in the EC class 1.2.1. NAD+-dependent phenylacetaldehyde dehydrogenase enzymes (EC 1.2.1.39) have been characterized in E. coli, Pseudomonas putida and Antirrhinum majus 116 (Long et al., Plant J59:256-265 (2009); Arias et al., Environ.Microbiol 10:413-432 (2008); Ferrandez et al., FEBS Lett. 406:23-27 (1997)). NAD+-dependent aldehyde dehydrogenase enzymes with high activity on phenylacetaldehyde have also been characterized in mammals such as Bos taurus, Rattus norvegicus and Homo sapiens (Klyosov, Biochemistry 35:4457 4467 (1996a); Lindahl et al., JBiol.Chem. 259:11991-11996 (1984)). Two aldehyde dehydrogenases found in human liver, ALDH-1 and ALDH-2, have broad substrate ranges for a variety of aliphatic, aromatic and polycyclic aldehydes with demonstrated activity on phenylacetaldehyde (Klyosov, Biochemistry 35:4457-4467 (1996b)). The NADP-dependent benzaldehyde dehydrogenase of Pseudomonas putida encoded by badh also demonstrated activity on phenylacetaldehyde (Yeung et al., Biochim.Biophys.Acta 1784:1248-1255 (2008)). NAD+-dependent aldehyde dehydrogenase enzymes with high activity on phenylacetaldehyde have also been characterized in mammals such as Bos taurus, Rattus norvegicus and Homo sapiens (Klyosov, Biochemistry 35:4457-4467 (1996a); Lindahl et al., JBiol.Chem. 259:11991-11996 (1984)). Two aldehyde dehydrogenases found in human liver, ALDH- 1 and ALDH-2, have broad substrate ranges for a variety of aliphatic, aromatic and polycyclic aldehydes with demonstrated activity on phenylacetaldehyde (Klyosov, Biochemistry 35:4457-4467 (1996b)). Gene GenBank Accession No. GI No. Organism feaB AAC74467.2 87081896 Escherichia coli peaE ABR57205.1 150014683 Pseudomonas putida BALDH ACM89738.1 223452696 Antirrhinum majus ALDH-2 P05091.2 118504 Homo sapiens badh P39849.1 731175 Pseudomonas putida 1.2.3.a Aldehyde oxidase: An 0 2 -dependent aldehyde oxidase enzyme can be employed to convert phenylacetaldehyde or phenylpyruvate to phenylacetate (Steps C and F of Figure 1). Phenylacetaldehyde oxidase enzymes convert phenylacetaldehyde, water and 02 to phenylacetate and hydrogen peroxide. Exemplary phenylacetaldehyde oxidase enzymes are found in Methylobacillus sp., Pseudomonas sp., Streptomyces moderatus, Cavia porcellus and Zea mays (Koshiba et al., Plant Physiol 110:781-789 (1996)). The two flavin- and molybdenum- containing aldehyde oxidases of Zea mays are encoded by zmAO-1 and zmAO 2 (Sekimoto et al., JBiol.Chem. 272:15280-15285 (1997)). Phenylacetaldehyde oxidase 117 activity has also been demonstrated in the indole-3-acetaldehyde oxidase (EC 1.2.3.7) of Avena sativa, although the genes associated with this activity have not been identified to date and bear no significant homology to the aldehyde oxidase genes of Zea mays (Rajagopal, Physiol.Plant 24:272-281 (1971)). Additional phenylacetaldehyde oxidases can be inferred by sequence homology to the Z. mays genes and are shown below. Gene GenBank Accession No. GI No. Organism zmAO-1 NP_001105308.1 162458742 Zea mays zmAO-2 BAA23227.1 2589164 Zea mays Aox1 054754.2 20978408 Mus musculus ALDOl_ORYSJ Q7XH05.1 75296231 Oryza sativa AAO3 BAA82672.1 5672672 Arabidopsis thaliana XDH DAA24801.1 296482686 Bos taurus Phenylpyruvate oxidase enzymes convert phenylpyruvate and 02 to phenylacetate, C02 and water (Step F of Figure 1). The 4-hydroxyphenylpyruvate oxidase (EC 1.2.3.13) from Arthrobacter globiformis was shown catalyze the 02-dependent oxidation of phenylpyruvate to phenylacetate during tyrosine catabolism (Blakley, Can.J Microbiol 23:1128-1139 (1977)). This enzymatic activity was demonstrated in cell extracts; however, the gene encoding this enzyme has not been identified to date. 1.4.1 .a Oxidoreductase (deaminating): The NAD(P)+-dependent oxidation of phenylalanine to phenylpyruvate (Step A of Figure 1) is catalyzed by phenylalanine oxidoreductase (deaminating), also called phenylalanine dehydrogenase. NAD+-dependent phenylalanine dehydrogenase enzymes encoded by pdh genes have been characterized in Bacillus badius, Lysinibacillus sphaericus and Thermoactinomyces intermedius (Yamada et al., Biosci.Biotechnol.Biochem. 59:1994-1995 (1995); Takada et al., JBiochem. 109:371-376 (1991); Okazaki et al., Gene 63:337-341 (1988)). Gene GenBank Accession No. GI No. Organism pdh BAA08816.1 1228936 Bacillus badius pdh AAA22646.1 529017 Lysinibacillus sphaericus pdh P22823.1 118598 Thermoactinomyces intermedius 118 2.3.1 .a Acyltransferase (phosphotransacylase): An enzyme with phosphotransbenzoylase activity is required to phosphorylate 3-oxo-3-phenylpropionyl-CoA to [(3-oxo-3-phenylpropionyl)oxy]phosphoate (Step F of Figure 3). An enzyme with this activity has not been characterized to date. Exemplary phosphate-transferring acyltransferases include phosphotransacetylase (EC 2.3.1.8) and phosphotransbutyrylase (EC 2.3.1.19). The pta gene from E. coli encodes a phosphotransacetylase that reversibly converts acetyl-CoA into acetyl-phosphate (Suzuki, Biochim.Biophys.Acta 191:559-569 (1969)). Phosphotransacetylase enzymes in several organisms also catalyze the conversion of propionyl-CoA to propionylphosphate. Such enzymes include the pta gene products of E. coli (Hesslinger et al., Mol.Microbiol 27:477-492 (1998)), Bacillus subtilis (Rado et al., Biochim.Biophys.Acta 321:114-125 (1973)), Clostridium kluyveri (Stadtman, 1:596-599 (1955)), and Thermotoga maritima (Bock et al., JBacteriol. 181:1861-1867 (1999)). Theptb gene from C. acetobutylicum encodes phosphotransbutyrylase, an enzyme that reversibly converts butyryl-CoA into butyryl-phosphate (Wiesenborn et al., Appl Environ.Microbiol 55:317-322 (1989); Walter et al., Gene 134:107-111 (1993)). Additionalptb genes are found in butyrate-producing bacterium L2-50 (Louis et al., JBacteriol. 186:2099-2106 (2004)) and Bacillus megaterium (Vazquez et al., Curr.Microbiol 42:345-349 (2001)). Gene GenBank Accession No. GI No. Organism pta NP_416800.1 71152910 Escherichia coli pta P39646 730415 Bacillus subtilis pta A5N801 146346896 Clostridium kluyveri pta Q9X0L4 6685776 Thermotoga maritime ptb NP_349676 34540484 Clostridium acetobutylicum ptb AAR19757.1 38425288 butyrate-producing bacterium L2 50 ptb CAC07932.1 10046659 Bacillus megaterium 2.3.1 .b Beta-ketothiolase. A beta-ketothiolase enzyme is required to convert benzoyl CoA and acetyl-CoA to 3-oxo-3-phenylpropionyl-CoA (Step A of Figure 3). This transformation is not known to occur naturally. Suitable beta-ketothiolase enzymes include 3 oxoadipyl-CoA thiolase (EC 2.3.1.174), 3-oxopimeloyl-CoA:glutaryl-CoA acyltransferase (EC 2.3.1.16), 3-oxovaleryl-CoA thiolase and acetoacetyl-CoA thiolase (EC 2.1.3.9).

119 3-Oxoadipyl-CoA thiolase (EC 2.3.1.174) converts beta-ketoadipyl-CoA to succinyl CoA and acetyl-CoA, and is a key enzyme of the beta-ketoadipate pathway for aromatic compound degradation. The enzyme is widespread in soil bacteria and fungi including Pseudomonas putida (Harwood et al., J Bacteriol. 176:6479-6488 (1994)) and Acinetobacter calcoaceticus (Doten et al., J Bacteriol. 169:3168-3174 (1987)). The gene products encoded by pcaF in Pseudomonas strain B13 (Kaschabek et al., J Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera et al., Proc.Natl.Acad.Sci U.S.A 95:6419-6424 (1998)), paaE in Pseudomonas fluorescens ST (Di et al., Arch.Microbiol 188:117-125 (2007)), and paaJ from E. coli (Nogales et al., Microbiology 153:357-365 (2007)) also catalyze this transformation. Several beta-ketothiolases exhibit significant and selective activities in the oxoadipyl-CoA forming direction including bkt from Pseudomonas putida, pcaF and bkt from Pseudomonas aeruginosa PA01, bkt from Burkholderia ambifaria AMMD, paaJ from E. coli, and phaD from P. putida. Gene name GI# GenBank Accession # Organism paaJ 16129358 NP_415915.1 Escherichia coli pcaF 17736947 AAL02407 Pseudomonas knackmussii (B13) phaD 3253200 AAC24332.1 Pseudomonas putida pcaF 506695 AAA85138.1 Pseudomonas putida pcaF 141777 AAC37148.1 Acinetobacter calcoaceticus paaE 106636097 ABF82237.1 Pseudomonasfluorescens bkt 115360515 YP_777652.1 Burkholderia ambifaria AMMD bkt 9949744 AAG06977.1 Pseudomonas aeruginosa PA01 pcaF 9946065 AAG03617.1 Pseudomonas aeruginosa PA01 3-Oxopimeloyl-CoA thiolase catalyzes the condensation of glutaryl-CoA and acetyl CoA into 3-oxopimeloyl-CoA (EC 2.3.1.16). An enzyme catalyzing this transformation is encoded by genes bktB and bktC in Ralstonia eutropha (formerly known as Alcaligenes eutrophus) (Slater et al., JBacteriol. 180:1979-1987 (1998); Haywood et al., FEMS Microbiology Letters 52:91-96 (1988)). The sequence of the BktB protein is known but the sequence of the BktC protein has not been reported to date. The pim operon of Rhodopseudomonas palustris also encodes a beta-ketothiolase, encoded by pimB, predicted to catalyze this transformation in the degradative direction during benzoyl-CoA degradation 120 (Harrison et al., Microbiology 151:727-736 (2005)). A beta-ketothiolase enzyme in S. aciditrophicus was identified by sequence homology to bktB (430% identity, evalue = 1 e-93). Gene name GI# GenBank Accession # Organism bktB 11386745 YP_725948 Ralstonia eutropha pimB 39650633 CAE29156 Rhodopseudomonas palustris syn_02642 85860483 YP_462685.1 Syntrophus aciditrophicus Beta-ketothiolase enzymes catalyzing the formation of 3-oxovalerate from acetyl CoA and propionyl-CoA may also be able to catalyze the formation of 3-oxo-3 phenylpropionyl-CoA. Zoogloea ramigera possesses two ketothiolases that can form p ketovaleryl-CoA from propionyl-CoA and acetyl-CoA and R. eutropha has a P -oxidation ketothiolase that is also capable of catalyzing this transformation (Gruys et al., (1999)). The sequences of these genes or their translated proteins have not been reported, but several genes in R. eutropha, Z. ramigera, or other organisms can be identified based on sequence homology to bktB from R. eutropha and are listed below. Gene name GI# GenBank Accession # Organism phaA 113867452 YP_725941.1 Ralstonia eutropha h16_A1713 113867716 YP_726205.1 Ralstonia eutropha pcaF 116694155 YP_728366.1 Ralstonia eutropha h16_B1369 116695312 YP_840888.1 Ralstonia eutropha h16_A0I70 113866201 YP_724690.1 Ralstonia eutropha h16_A0462 113866491 YP_724980.1 Ralstonia eutropha h16_AI528 113867539 YP_726028.1 Ralstonia eutropha h16_B0381 116694334 YP_728545.1 Ralstonia eutropha h16_B0662 116694613 YP_728824.1 Ralstonia eutropha h16_B0759 116694710 YP_728921.1 Ralstonia eutropha h16_B0668 116694619 YP_728830.1 Ralstonia eutropha h16_A1720 113867723 YP_726212.1 Ralstonia eutropha h16_A1887 113867867 YP_726356.1 Ralstonia eutropha phbA 135759 P07097.4 Zoogloea ramigera bktB 194289475 YP_002005382.1 Cupriavidus taiwanensis 121 Rmet_1362 94310304 YP_583514.1 Ralstonia metallidurans Bphy_0975 186475740 YP_001857210.1 Burkholderia phymatum Additional enzymes include beta-ketothiolases that are known to convert two molecules of acetyl-CoA into acetoacetyl-CoA (EC 2.1.3.9). Exemplary acetoacetyl-CoA thiolase enzymes include the gene products of atoB from E. coli (Martin et al., Nat.Biotechnol 21:796-802 (2003)), thIA and thiB from C. acetobutylicum (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007); Winzer et al., JMol.Microbiol Biotechnol 2:531 541 (2000)), and ERG1O from S. cerevisiae (Hiser et al., JBiol. Chem. 269:31383-31389 (1994)). Gene name GI# GenBank Accession # Organism atoB 16130161 NP_416728 Escherichia coli thIA 15896127 NP_349476.1 Clostridium acetobutylicum thiB 15004782 NP_149242.1 Clostridium acetobutylicum ERG1O 6325229 NP_015297 Saccharomyces cerevisiae 2.6.1 .a Aminotransferase: A phenylalanine aminotransferase or transaminase in the EC class 2.6.1 is required to convert phenylalanine to phenylpyruvate (Step A of Figure 1). A variety of enzymes catalyze this transformation, including phenylacetate aminotransferase, aromatic amino acid aminotransferase, tryptophan aminotransferase, aspartate aminotransferase, branched chain amino acid aminotransferase and others. Enzymes with phenylalanine aminotransferase activity in E. coli are encoded by tyrB, ilvE and aspC (Lee Peng et al., JBacteriol. 139:339-345 (1979); Powell et al., Eur.JBiochem. 87:391-400 (1978)). Exemplary enzymes with phenylalanine aminotransferase activity in other organisms include the aromatic amino acid aminotransferase of S. cerevisiae encoded by ARO9 (Iraqui et al., Mol.Gen.Genet. 257:238-248 (1998)) and the tryptophan aminotransferase of Arabidopsis thaliana, encoded by TAA1 (Tao et al., Cell 133:164-176 (2008)).

122 Gene GenBank Accession No. GI No. Organism tyrB AAC77024.1 1790488 Escherichia coli ilvE AAT48207.1 48994963 Escherichia coli aspC NP_415448.1 16128895 Escherichia coli ARO9 NP_012005.1 6321929 Saccharomyces cerevisiae TAA1 NP_177213.1 15223183 Arabidopsis thaliana 2.7.2.a Phosphotransferase: Phosphotransferase enzymes in the EC class 2.7.2 convert carboxylic acids to phosphonic acids with concurrent hydrolysis of one ATP. In Step G of Figure 3, a phosphotransferase is required to convert [(3-oxo-3 phenylpropionyl)oxy]phosphonate and ADP to benzoyl-acetate and ATP. An enzyme with this exact activity has not been characterized to date. Exemplary enzymes include butyrate kinase (EC 2.7.2.7), isobutyrate kinase (EC 2.7.2.14), aspartokinase (EC 2.7.2.4), acetate kinase (EC 2.7.2.1) and gamma-glutamyl kinase (EC 2.7.2.11). Aspartokinase catalyzes the ATP-dependent phosphorylation of aspartate and participates in the synthesis of several amino acids. The aspartokinase III enzyme in E. coli, encoded by lysC, has a broad substrate range that includes the aromatic compound aspartic acid 1 -benzyl ester, and the catalytic residues involved in substrate specificity have been elucidated (Keng et al., Arch.Biochem.Biophys. 335:73-81 (1996)). Two additional kinases in E. coli include acetate kinase and gamma-glutamyl kinase. The E. coli acetate kinase, encoded by ackA (Skarstedt et al., JBiol.Chem. 251:6775-6783 (1976)), phosphorylates propionate in addition to acetate (Hesslinger et al., Mol.Microbiol 27:477-492 (1998)). The E. coli gamma-glutamyl kinase, encoded by proB (Smith et al., JBacteriol. 157:545-551 (1984)), phosphorylates the gamma carbonic acid group of glutamate. Butyrate kinase carries out the reversible conversion of butyryl-phosphate to butyrate during acidogenesis in C. acetobutylicum (Cary et al., Appl.Environ.Microbiol 56:1576-1583 (1990)). This enzyme is encoded by either of the two buk gene products (Huang et al., JMol.Microbiol Biotechnol 2:33-38 (2000)). Other butyrate kinase enzymes are found in C. butyricum and C. tetanomorphum (TWAROG et al., J Bacteriol. 86:112-117 (1963)). A related enzyme, isobutyrate kinase from Thermotoga maritime, was expressed in E. coli and crystallized (Diao et al., JBacteriol. 191:2521-2529 (2009); Diao et al., Acta Crystallogr.D.Biol.Crystallogr. 59:1100-1102 (2003)).

123 Gene name GI# GenBank Accession # Organism lysC 16131850 NP_418448.1 Escherichia coli ackA 16130231 NP_416799.1 Escherichia coli proB 16128228 NP_414777.1 Escherichia coli buki 15896326 NP_349675 Clostridium acetobutylicum buk2 20137415 Q97111 Clostridium acetobutylicum buk2 6685256 Q9X278.1 Thermotoga maritima 2.8.3.a CoA transferase: CoA transferases catalyze the reversible transfer of a CoA moiety from one molecule to another. Step B of Figure 3 is catalyzed by an enzyme with 3 oxo-3-phenylpropionyl-CoA transferase activity. In this transformation, benzoyl-acetate is formed from 3-oxo-3-phenylpropionyl-CoA by the transfer of the CoA to a CoA acceptor such as acetate, succinate or others. Exemplary CoA transferase enzymes that react with similar substrates include cinnamoyl-CoA transferase (EC 2.8.3.17) and benzylsuccinyl-CoA transferase. Cinnamoyl-CoA transferase, encoded byfldA in Clostridium sporogenes, transfers a CoA moiety from cinnamoyl-CoA to a variety of aromatic acid substrates including phenylacetate, 3-phenylpropionate and 4-phenylbutyrate (Dickert et al., Eur.J Biochem. 267:3874-3884 (2000)). Benzylsuccinyl-CoA transferase utilizes succinate or maleate as the CoA acceptor, forming benzylsuccinate from benzylsuccinyl-CoA. This enzyme was characterized in the reverse direction in denitrifying bacteria Thauera aromatica, where it is encoded by bbsEF (Leutwein et al., JBacteriol. 183:4288-4295 (2001)). Gene GenBank Accession No. GI No. Organism fldA AAL18808.1 16417587 Clostridium sporogenes bbsE AAF89840.1 9622535 Thauera aromatica bbsF AAF89841.1 9622536 Thauera aromatica Additional CoA transferase enzymes with diverse substrate ranges include succinyl CoA transferase, 4-hydroxybutyryl-CoA transferase, butyryl-CoA transferase, glutaconyl CoA transferase and acetoacetyl-CoA transferase. The gene products of cat], cat2, and cat3 of Clostridium kluyveri have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively (Seedorf et al., Proc.Natl.Acad.Sci U.S.A 105:2128-2133 (2008); Sohling et al., JBacteriol. 178:871-880 (1996)). Similar CoA 124 transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., JBiol.Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., JBiol.Chem. 279:45337-45346 (2004)). The glutaconyl-CoA-transferase (EC 2.8.3.12) from the anaerobic bacterium Acidaminococcusfermentans reacts with glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., Eur.JBiochem. 226:41-51 (1994)). The genes encoding this enzyme are gctA and gctB. This enzyme exhibits reduced but detectable activity with several alternate substrates including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA, crotonyl-CoA and acrylyl-CoA (Buckel et al., Eur.JBiochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mack et al., Eur.JBiochem. 226:41-51 (1994)). Glutaconate CoA-transferase activity has also been detected in Clostridium sporosphaeroides and Clostridium symbiosum. Acetoacetyl-CoA transferase utilizes acetyl-CoA as the CoA donor. This enzyme is encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Korolev et al., Acta Crystallogr.D.Biol.Crystallogr. 58:2116-2121 (2002); Vanderwinkel et al., Biochem.Biophys.Res.Commun. 33:902-908 (1968)). This enzyme has a broad substrate range (Sramek et al., Arch.Biochem.Biophys. 171:14-26 (1975)) and has been shown to transfer the CoA moiety from acetyl-CoA to a variety of substrates, including isobutyrate (Matthies et al., App/ Environ.Microbiol 58:1435-1439 (1992)), valerate and butanoate (Vanderwinkel et al., Biochem.Biophys.Res.Commun. 33:902-908 (1968)). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl.Environ.Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl.Environ.Microbiol 56:1576-1583 (1990); Wiesenborn et al., Appl.Environ.Microbiol 55:323-329 (1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71:58 68 (2007)).

125 Gene GenBank Accession No. GI No. Organism cat] P38946.1 729048 Clostridium kluyveri cat2 P38942.2 172046066 Clostridium kluyveri cat3 EDK35586.1 146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034 Trichomonas vaginalis G3 Tbl1.02.0290 XP_828352 71754875 Trypanosoma brucei gctA CAA57199.1 559392 Acidaminococcusfermentans gctB CAA57200.1 559393 Acidaminococcusfermentans gctA ACJ24333.1 212292816 Clostridium symbiosum gctB ACJ24326.1 212292808 Clostridium symbiosum atoA P76459.1 2492994 Escherichia coli K12 atoD P76458.1 2492990 Escherichia coli K12 actA YP_226809.1 62391407 Corynebacterium glutamicum cg0592 YP_224801.1 62389399 Corynebacterium glutamicum ctfA NP_149326.1 15004866 Clostridium acetobutylicum ctfB NP_149327.1 15004867 Clostridium acetobutylicum Clostridium ctfA AAP42564.1 31075384 saccharoperbutylacetonicum Clostridium ctfB AAP42565.1 31075385 saccharoperbutylacetonicum 3.1.2.a CoA hydrolase: 3-Oxo-3-phenylpropionyl-CoA can be hydrolyzed to its corresponding acid by a CoA hydrolase or thioesterase in the EC class 3.1.2 (Step B of Figure 3). Exemplary CoA thioesters that hydrolyze aromatic substrates include benzoyl-CoA hydrolase, 4-hydroxybenzoyl-CoA hydrolase (EC 3.1.2.23) and phenylacetyl-CoA hydrolase (EC 3.1.2.25). The Azoarcus evansii gene orf1 encodes an enzyme with benzoyl-CoA hydrolase activity that participates in benzoate metabolism (Ismail, Arch.Microbiol 190:451 460 (2008)). This enzyme, when heterologously expressed in E. coli, demonstrated activity on a number of alternate substrates. Additional benzoyl-CoA hydrolase enzymes were identified in benzonate degradation gene clusters of Magnetospirillum magnetotacticum, Jannaschia sp. CCS 1 and Sagittula stellata E-37 by sequence similarity (Ismail, Arch.Microbiol 190:451-460 (2008)). The 4-hydroxybenzoyl-CoA hydrolase of 126 Pseudomonas sp. CBS3 exhibits activity on the alternate substrates benzoyl-CoA and p methylbenzoyl-CoA, and has been heterologously expressed and characterized in E. coli (Song et al., Bioorg.Chem. 35:1-10 (2007)). Additional enzymes with aryl-CoA hydrolase activity include the palmitoyl-CoA hydrolase of Mycobacterium tuberculosis (Wang et al., Chem.Biol. 14:543-551 (2007)) and the acyl-CoA hydrolase of E. coli encoded by entH (Guo et al., Biochemistry 48:1712-1722 (2009)). GenBank Accession Gene GI No. Organism No. orf1 AAN39365.1 23664428 Azoarcus evansii MagnO3011230 ZP_00207794 46200680 Magnetospirillum magnetotacticum Jann_0674 YP_508616 89053165 Jannaschia sp. CCS1 SSE37_24444 ZP_01745221 126729407 Sagittula stellata EF569604.1:4745..5170 ABQ44580.1 146761194 Pseudomonas sp. CBS3 Rv0098 NP_214612.1 15607240 Mycobacterium tuberculosis entH AAC73698.1 1786813 Escherichia coli Several additional CoA hydrolases with broad substrate ranges are suitable for hydrolyzing benzoyl-CoA and/or p-methylbenzoyl-CoA. For example, the enzyme encoded by acotl2 from Rattus norvegicus brain (Robinson et al., Biochem.Biophys.Res.Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. The human dicarboxylic acid thioesterase, encoded by acot8, exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., JBiol.Chem. 280:38125-38132 (2005)). The closest E. coli homolog to this enzyme, tesB, can also hydrolyze a range of CoA thiolesters (Naggert et al., JBiol Chem 266:11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana R., Biochem Int 26:767-773 (1992)). Additional enzymes with hydrolase activity in E. coli include ybgC, paaI, and ybdB (Kuznetsova, et al., FEMS Microbiol Rev, 2005, 29(2):263-279; Song et al., J Biol Chem, 2006, 281(16):11028-38).

127 Gene name GenBank Accession # GI# Organism acot12 NP_570103.1 18543355 Rattus norvegicus tesB NP_414986 16128437 Escherichia coli acot8 CAA15502 3191970 Homo sapiens acot8 NP_570112 51036669 Rattus norvegicus tesA NP_415027 16128478 Escherichia coli ybgC NP_415264 16128711 Escherichia coli paaI NP_415914 16129357 Escherichia coli ybdB NP_415129 16128580 Escherichia coli 4.1.la. Carboxy-lyase: Decarboxylase enzymes in the EC class 4.1.1 are required to catalyze several transformations in Figures 1-3. Conversion of phenylpyruvate to phenylacetaldehyde (Step B of Figure 1) is catalyzed by a keto-acid decarboxylase. Decarboxylation of phenylacetate to toluene (Step D of Figure 1) is catalyzed by an enzyme with phenylacetate decarboxylase activity. A 3-oxoacid decarboxylase is required to decarboxylate benzoyl-acetate to acetophenone (Step C of Figure 7). Decarboxylases (also known as carboxy lyases) catalyze the loss of carbon dioxide from an organic compound or a cellular metabolite possessing a carboxylic acid function. Decarboxylases are prevalent in nature and can require either pyridoxal phosphate or pyruvate as a co-factor, although many require no bound co-factors. Over 50 decarboxylase enzymes have been reported and characterized by biochemical and/or analytical methods. A phenylacetate decarboxylase is required to catalyze the decarboxylation of phenylacetate to toluene (Step D of Figure 1). Such an activity has not been detected in characterized decarboxylase enzymes to date. An enzyme catalyzing a similar reaction is 4 hydroxyphenylacetate decarboxylase (EC 4.1.1.83), which naturally decarboxylates 4 hydroxyphenylacetate to p-cresol. Characterized 4-hydroxyphenylacetate decarboxylase enzymes from Clostridium difficile and Clostridium scatologenes are composed of two subunits and require activation by a specific activating enzyme (Yu et al., Biochemistry 45:9584-9592 (2006); Andrei et al., Eur.JBiochem. 271:2225-2230 (2004)). These enzymes, encoded by csdABC in C. scatologenes and hpdABC in C. difficile, have been heterologously expressed in E. coli. Another suitable enzyme is the arylmalonate decarboxylase (EC 128 4.1.1.76) of Enterobacter cloacae, which has activity on the structurally related substrate, 2 phenylpropionate (Yatake et al., Appl.Microbiol Biotechnol. 78:793-799 (2008)). The sequence of this enzyme is available in the publication by Yatake et al; however this enzyme has not been assigned a GenBank Accession number to date. A related enzyme from Bordetella bronchiseptica (98% amino acid identity) encoded by AMDABORBR was recently crystallized (Kuettner et al., JMol.Biol. 377:386-394 (2008)). Gene name GenBank Accession # GI# Organism csdA ABB05048.1 77863917 Clostridium scatologenes csdB ABB05046.1 77863915 Clostridium scatologenes csdC ABB05047.1 77863916 Clostridium scatologenes hpdA CAD65891.1 28300943 Clostridium difficile hpdB CAD65889.1 28300939 Clostridium difficile hpdC CAD65890.1 28300941 Clostridium difficile AMDABORBR Q051 15.1 728844 Bordetella bronchiseptica The conversion of phenylpyruvate to phenylacetaldehyde (Figure 1, Step B) is catalyzed by an enzyme with phenylpyruvate decarboxylase activity. Several keto-acid decarboxylase enzymes have demonstrated activity on phenylpyruvate including phenylpyruvate decarboxylase (EC 4.1.1.43), pyruvate decarboxylase (EC 4.1.1.1), branched chain alpha-ketoacid decarboxylase (EC 4.1.1.72) and benzylformate decarboxylase (EC 4.1.1.7). An exemplary phenylpyruvate decarboxylase is encoded by the ipdC gene of Azospirillum brasilense (Spaepen et al., JBacteriol. 189:7626-7633 (2007)). Phenylpyruvate is the favored substrate of this enzyme. The Saccharomyces cerevisiae enzymes encoded by the genes PDCl, PDC5, PDC6 and AR010 also exhibit phenylpyruvate decarboxylase activity (Dickinson et al., JBiol.Chem. 278:8028-8034 (2003)). Other enzymes with phenylpyruvate decarboxylase activity include the pyruvate dehydrogenase enzyme Zygosaccharomyces bisporus (Neuser et al., Biol.Chem. 381:349-353 (2000)), the branched chain 2-oxoacid decarboxylase enzymes of Mycobacterium tuberculosis H37Rv and Lactococcus lactis (Gocke et al., Adv.Synth.Catal. 349:1425-1435 (2007); Werther et al., J Biol. Chem. 283:5344-5354 (2008)), and the benzylformate decarboxylase enzyme of Pseudomonas putida (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). Another suitable enzyme is the pyruvate decarboxylase from Zymomonas mobilus, as this enzyme has 129 a broad substrate range and has been a subject of directed engineering studies to alter the substrate specificity (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). Protein GenBank ID GI Number Organism ipdC P51852.1 1706320 Azospirillum brasilense PDC1 P06169.7 30923172 Saccharomyces cerevisiae PDC5 P16467.4 1352225 Saccharomyces cerevisiae PDC6 P26263.3 118389 Saccharomyces cerevisiae AROJ 0 Q06408.1 50400299 Saccharomyces cerevisiae pdcl CAB65554.1 6689662 Zygosaccharomyces bisporus RvO853c NP_215368.1 15607993 Mycobacterium tuberculosis H37Rv kdcA AAS49166.1 44921617 Lactococcus lactis mdlC P20906.2 3915757 Pseudomonas putida pdc P06672.1 118391 Zymomonas mobilis The decarboxylation of benzoyl-acetate to acetophenone (Step C of Figure 3) is catalyzed by an enzyme with benzoyl-acetate decarboxylase activity. An ATP-dependent enzyme with this activity, acetophenone carboxylase, operates in the reverse (carboxylation) direction during ethylbenzene degradation in Aromatoleum aromaticum EbN1 (Rabus et al., Arch.Microbiol 178:506-516 (2002)). This enzyme is composed of five subunits encoded by apcl-5 and is ATP-dependent (AMP forming) in the direction of carboxylation. Similar enzymes are found by sequence homology in Azotobacter vinelandii and Rubrobacter xylanophilus. Gene name GenBank Accession # GI# Organism apcl YP_158351.1 56476762 Aromatoleum aromaticum EbNJ apc2 YP_158350.1 56476761 Aromatoleum aromaticum EbNJ apc3 YP_158349.1 56476760 Aromatoleum aromaticum EbNJ apc4 YP_158348.1 56476759 Aromatoleum aromaticum EbNJ apc5 YP_158347.1 56476758 Aromatoleum aromaticum EbNJ apcl YP_002798345.1 226943272 Azotobacter vinelandii apc2 AC077369.1 226718198 Azotobacter vinelandii apc3 YP_002798343.1 226943270 Azotobacter vinelandii 130 apc4 YP_002798342.1 226943269 Azotobacter vinelandii apcl YP_644617.1 108804680 Rubrobacter xylanophilus apc2 YP_644616.1 108804679 Rubrobacter xylanophilus apc3 YP_644615.1 108804678 Rubrobacter xylanophilus apc4 YP_644614.1 108804677 Rubrobacter xylanophilus apc5 YP_644613.1 108804676 Rubrobacter xylanophilus Alternatively, a 3-oxoacid decarboxylase can convert benzoyl-acetate to acetophenone. An exemplary 3-oxoacid decarboxylase is acetoacetate decarboxylase (EC 4.1.1.4), which naturally converts acetoacetate into acetone and CO 2 . The enzyme from Clostridium acetobutylicum, encoded by adc, has a broad substrate range and has been shown to catalyze the desired decarboxylation of benzoyl-acetate to acetophenone (Rozzel et al., JAm.Chem.Soc. 106:4937-4941 (1984); Benner et al., JAm.Chem.Soc. 103:993-994 (1981); Autor et al., JBiol.Chem. 245:5214-5222 (1970)). A related acetoacetate decarboxylase has been characterized in Clostridium beijerinckii (Ravagnani et al., Mol.Microbiol 37:1172-1185 (2000)). The acetoacetate decarboxylase from Bacillus polymyxa, characterized in cell-free extracts, also has a broad substrate specificity for 3-keto acids and can decarboxylate 3 oxopentanoate (Matiasek et al., Curr.Microbiol 42:276-281 (2001)). The gene encoding this enzyme has not been identified to date and the genome sequence of B. polymyxa is not yet available. Another adc is found in Clostridium saccharoperbutylacetonicum (Kosaka, et al., Biosci.Biotechnol Biochem. 71:58-68 (2007)). Additional genes in other organisms, including Clostridium botulinum and Bacillus amyloliquefaciens FZB42, can be inferred by sequence homology. Protein GenBank ID GI Number Organism adc NP_149328.1 15004868 Clostridium acetobutylicum adc AAP42566.1 31075386 Clostridium saccharoperbutylacetonicum adc YP_001310906.1 150018652 Clostridium bejerinckii CLLA2135 YP_001886324.1 187933144 Clostridium botulinum RBAM_030030 YP_001422565.1 154687404 Bacillus amyloliquefaciens 131 4.1.99.a Decarbonylase: A decarbonylase enzyme is required to convert phenylacetaldehyde to toluene (Step E of Figure 1). Decarbonylase enzymes catalyze the final step of alkane biosynthesis in plants, mammals, insects and bacteria (Dennis et al., Arch.Biochem.Biophys. 287:268-275 (1991)). Non-oxidative decarbonylases transfom aldehydes into alkanes with the concurrent release of CO, whereas oxidative decarboxylases are cytochrome P450 enzymes that utilize NADPH and 02 as cofactors and release C0 2 , water and NADP. Exemplary decarbonylase enzymes include octadecanal decarbonylase (EC 4.1.99.5), sterol desaturase and fatty aldehyde decarbonylase. A cobalt-porphyrin containing decarbonylase was purified and characterized in the algae Botryococcus braunii; however, no gene is associated with this activity to date (Dennis et al., Proc.Natl.Acad.Sci. U.S.A 89:5306-5310 (1992)). A copper-containing decarbonylase from Pisum sativum was also purified and characterized (Schneider-Belhaddad et al., Arch.Biochem.Biophys. 377:341-349 (2000)). The CER1 gene of Arabidopsis thaliana encodes a fatty acid decarbonylase involved in epicuticular wax formation (US 6,437,218). Additional fatty acid decarbonylases are found in Medicago truncatula, Vitis vinfera and Oryza sativa (US Patent Application 2009/0061493). Protein GenBank ID GI Number Organism CER] NP_850932 145361948 Arabidopsis thaliana MtrDRAFT AC153128g2v2 ABN07985 124359969 Medicago truncatula VITISV_029045 CAN60676 147781102 Vitis vinfera OSJNBa0004N05.14 CAE03390.2 38345317 Oryza sativa Oxidative decarbonylase enzymes are encoded by the CYP4G2v1 and CYP4Glgene products of Musca domestica and Drosophila melanogaster (US Patent Application 2010/0136595). Additional enzymes with oxidative decarbonylase activity can be identified in other organisms, for example Mamestra brassicae, Helicoverpa zea and Acyrthosiphon pisum, by sequence homology.

132 Protein GenBank ID GI Number Organism CYP4G2v1 ABV48808.1 157382740 Musca domestica CYP4G1 NP_525031.1 17933498 Drosophila melanogaster CYP4G25 BAD81026.1 56710314 Antheraea yamamai CYP4M6 AAM54722.1 21552585 Helicoverpa zea LOC100164072 XP_001944205.1 193650239 Acyrthosiphon pisum 4.1.99.b Lyase: The conversion of phenylalanine to benzene in Figure 2 is catalyzed by an enzyme with phenylalanine benzene-lyase activity. The required novel activity is similar to that of tyrosine phenol-lyase (EC 4.1.99.2), which reversibly interconverts tyrosine and phenol, with concomitant release of pyruvate and ammonia. The enzyme from Pantoea agglomerans (formerly Erwinia herbicola), encoded by tutA, reacts with a range of substituted derivatives including dihydroxyphenylalanine (Foor et al., Appl.Environ.Microbiol 59:3070-3075 (1993)). This enzyme was heterologously expressed in E. coli and utilized to synthesize dihydroxyphenylalanine from catechol, pyruvate and ammonia. Additional tyrosine phenol-lyase enzymes include are found in Citrobacter intermedius (Nagasawa et al., Eur.JBiochem. 117:33-40 (1981)), Citrobacterfreundii (Phillips et al., Biochim.Biophys.Acta 1647:167-172 (2003)) and Symbiobacterium thermophilum (Hirahara et al., Appl.Microbiol Biotechnol. 39:341-346 (1993)). The Citrobacterfreundii enzyme has been structurally characterized and residues involved in substrate binding were identified (Milic et al., JBiol.Chem. 283:29206-29214 (2008)). Phenylalanine is not a natural substrate of any characterized tyrosine phenol-lyase enzyme; rather it has been shown to act as inhibitor of some enzymes. Directed evolution or other protein engineering approaches well known in the art will likely be required to gain this activity and improve performance. Protein GenBank ID GI Number Organism tutA AAA66390.1 806897 Pantoea agglomerans tpl AB175028.1 114451977 Citrobacterfreundii tpl BAA00763.1 216675 Citrobacter intermedius tpl YP_076671.1 51893980 Symbiobacterium thermophilum 133 An enzyme catalyzing a similar reaction is tryptophan indole-lyase (EC 4.1.99.1), also called tryptophanase, which converts tryptophan and water to indole and pyruvate. Exemplary tryptophan indole-lyase enzymes include the gene products of tnaA of E. coli, tnal of Symbiobacterium thermophilum and tnaA of Enterobacter aerogenes (Kogan et al., Acta Crystallogr.D.Biol.Crystallogr. 60:2073-2075 (2004); Kawasaki et al., Biosci.Biotechnol.Biochem. 59:1938-1943 (1995); Suzuki et al., Agric.Biol.Chem. 55:3059 3066 (1991)). Protein GenBank ID GI Number Organism tnaA NP_418164.4 90111643 Escherichia coli tnal BAA24688.1 2842554 Symbiobacterium thermophilum tnaA Q59342.1 2501228 Enterobacter aerogenes 4.2.1.a Dehydratase: Step E of Figure 3 employs a dehydratase (EC 4.1.2.-) to convert 1 -phenylethanol to styrene. Exemplary enzymes for catalyzing this reaction include fumarase (EC 4.2.1.2), citramalate hydratase (EC 4.2.1.34) and dimethylmaleate hydratase (EC 4.2.1.85). Fumarase enzymes naturally catalyze the reversible dehydration of malate to fumarate. Although the suitability of 1 -phenylethanol as a substrate for fumarase enzymes has not been described in the literature to date, a wealth of structural information is available for this enzyme and researchers have successfully engineered the enzyme to alter activity, inhibition and localization (Weaver, 61:1395-1401 (2005)). E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions. FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is the only enzyme active during aerobic growth (Tseng et al., 183:461-467 (2001); Woods et al., 954:14-26 (1988); Guest et al., J Gen Microbiol 131:2971-2984 (1985)). Additional enzymes are found in Campylobacterjejuni (Smith et al., Int.J Biochem.Cell Biol 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch.Biochem.Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al., 89:1923 1931 (1981)). Similar enzymes with high sequence homology includefum1 from Arabidopsis thaliana and fumC from Corynebacterium glutamicum. The mmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., 270:207-213 (2007)). Citramalate hydrolyase naturally dehydrates 2 methylmalate to mesaconate. This enzyme has been studied in Methanocaldococcus 134 jannaschii in the context of the pyruvate pathway to 2-oxobutanoate, where it has been shown to have a broad substrate specificity (Drevland et al., JBacteriol. 189:4391-4400 (2007)). This enzyme activity was also detected in Clostridium tetanomorphum, Morganella morganii, Citrobacter amalonaticus where it is thought to participate in glutamate degradation (Kato and Asano, Arch.Microbiol 168:457-463 (1997)). The M. jannaschii protein sequence does not bear significant homology to genes in these organisms. Dimethylmaleate hydratase is a reversible Fe 2+-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB in Eubacterium barkeri (Alhapel et al., supra; Kollmann-Koch et al., Hoppe Seylers.Z.Physiol Chem. 365:847-857 (1984)). Protein GenBank ID GI Number Organism fumA NP_416129.1 16129570 Escherichia coli fumB NP_418546.1 16131948 Escherichia coli fumC NP_416128.1 16129569 Escherichia coli fumC 069294 9789756 Campylobacterjejuni fumC P84127 75427690 Thermus thermophilus fumH P14408 120605 Rattus norvegicus fum1 P93033 39931311 Arabidopsis thaliana fumC Q8NRN8 39931596 Corynebacterium glutamicum mmcB YP_001211906 147677691 Pelotomaculum thermopropionicum mmcC YP_001211907 147677692 Pelotomaculum thermopropionicum leuD Q58673.1 3122345 Methanocaldococcus jannaschii dmdA ABC88408 86278276 Eubacterium barkeri dmdB ABC88409.1 86278277 Eubacterium barkeri 6.2.1 .a Acid-thiol ligase: The conversion of 3-oxo-3-phenylpropionyl-CoA to benzoyl-acetate (Step B of Figure 3) can be catalyzed by a CoA synthetase or acid-thiol ligase in the EC class 6.2.1. ATP-forming CoA synthetases catalyzing this exact transformation have not been characterized to date; however, several enzymes with broad substrate specificities have been described in the literature. The ADP-forming acetyl-CoA 135 synthetase (ACD, EC 6.2.1.13) from Archaeoglobusfulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including isobutyrate, isopentanoate, and fumarate (Musfeldt et al., JBacteriol. 184:636-644 (2002)). A second reversible ACD in Archaeoglobusfulgidus, encoded by AF1983, was also shown to have a broad substrate range with high activity on aromatic compounds phenylacetate and indoleacetate (Musfeldt et al., supra). The enzyme from Haloarcula marismortui, annotated as a succinyl-CoA synthetase, accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch.Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl CoA (preferred substrate) and phenylacetyl-CoA (Brasen and Schonheit, Arch.Microbiol 182:277-287 (2004)). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A.fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Brasen and Schonheit, Arch.Microbiol 182:277-287 (2004); Musfeldt and Schonheit, JBacteriol. 184:636-644 (2002)). An additional enzyme is encoded by sucCD in E. coli, which naturally catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). The acyl CoA ligase from Pseudomonas putida has been demonstrated to work on several aliphatic substrates including acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds such as phenylacetic and phenoxyacetic acids (Fernandez-Valverde et al., Appl.Environ.Microbiol. 59:1149-1154 (1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) from Rhizobium leguminosarum could convert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-, cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into their corresponding monothioesters (Pohl et al., JAm.Chem.Soc. 123:5822-5823 (2001)). GenBank Accession Gene GI No. Organism No. AF128 NP_070039.1 11498810 Archaeoglobusfulgidus AF1983 NP_070807.1 11499565 Archaeoglobusfulgidus scs YP_135572.1 55377722 Haloarcula marismortui 136 GenBank Accession Gene GI No. Organism No. PAE3250 NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2 sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia coli paaF AAC24333.2 22711873 Pseudomonas putida matB AAC83455.1 3982573 Rhizobium leguminosarum EXAMPLE II Pathways to 1,3-Butadiene from Muconate Isomers This example shows pathways from muconate isomers to 1,3-butadiene. Figure 4 shows the conversion of muconate isomers to 1,3-butadiene by decarboxylase enzymes. Cis,cis-muconate, cis,trans-muconate or trans,trans-muconate is first decarboxylated to either cis-2,4-pentadienoate or trans-2,4-pentadienoate (Steps A, B, C and D of Figure 4). 2,4-Pentadienoate is subsequently decarboxylated to form 1,3-butadiene (Steps E, F of Figure 4). It is understood that the decarboxylation of any muconate isomer can serve as part of a pathway to 1,3-butadiene. The biological production of cis,cis-muconate is well-known in the art (Draths and Frost. J Am Chem Soc. 116:399-400 (1994); Niu et al. Biotechnol Prog. 18:201-211 (2002); Bang and Choi. J Ferm Bioeng. 79(4):381-383 (1995)). Isomers of muconate can be interconverted by muconate isomerase enzymes in the EC class 5.2.1. It is further understood that decarboxylation of either isomer of 2,4-pentadienoate will form 1,3-butadiene. Isomers of 2,4-pentadienoate can alternatively be formed from starting materials other than muconate (e.g., introduction of second double bond via dehydrogenation of pent-2-enoate or pent-4-enoate; removal of CoA from 2,4-pentadienoyl-CoA via a hydrolase, synthetase, or transferase, dehydrogenation of 2,4-pentadienal or 2,4-pentadienol via an aldehyde or aldehyde/alcohol dehydrogenase, respectively). Isomers of 2,4 pentadienoate can be interconverted by isomerase enzymes in the EC class: 5.2.1.

137 Numerous decarboxylase enzymes have been characterized and shown to decarboxylate structurally similar substrates to muconate or 2,4-pentadienoate isomers. Exemplary enzymes include sorbic acid decarboxylase, aconitate decarboxylase (EC 4.1.1.16), 4-oxalocrotonate decarboxylase (EC 4.1.1.77), cinnamate decarboxylase and ferulic acid decarboxylase. These enzymes are applicable for use in the present invention to decarboxylate muconate and/or 2,4-pentadienoate as shown in Figure 4. One decarboxylase enzyme with closely related function is sorbic acid decarboxylase which converts sorbic acid to 1,3-pentadiene. Sorbic acid decarboxylation by Aspergillus niger requires three genes: padAl, ohbAl, and sdrA (Plumridge et al. Fung. Genet. Bio, 47:683-692 (2010). PadAl is annotated as a phenylacrylic acid decarboxylase, ohbAl is a putative 4-hydroxybenzoic acid decarboxylase, and sdrA is a sorbic acid decarboxylase regulator. Additional species have also been shown to decarboxylate sorbic acid including several fungal and yeast species (Kinderlerler and Hatton, Food Addit Contam., 7(5):657-69 (1990); Casas et al., Int J Food Micro., 94(1):93-96 (2004); Pinches and Apps, Int. J. Food Microbiol. 116: 182-185 (2007)). For example, Aspergillus oryzae and Neosartorya fischeri have been shown to decarboxylate sorbic acid and have close homologs to padA1, ohbA1, and sdrA. Gene name GenBanklD GI Number Organism padAl XP_001390532.1 145235767 Aspergillus niger ohbA1 XP_001390534.1 145235771 Aspergillus niger sdrA XP_001390533.1 145235769 Aspergillus niger padAl XP_001818651.1 169768362 Aspergillus oryzae ohbA1 XP_001818650.1 169768360 Aspergillus oryzae sdrA XP_001818649.1 169768358 Aspergillus oryzae padAl XP_001261423.1 119482790 Neosartoryafischeri ohbA1 XP_001261424.1 119482792 Neosartoryafischeri sdrA XP_001261422.1 119482788 Neosartoryafischeri Aconitate decarboxylase is another useful enzyme for this invention. This enzyme catalyzes the final step in itaconate biosynthesis in a strain of Candida and also in the filamentous fungus Aspergillus terreus. (Bonnarme et al. JBacteriol. 177:3573-3578 (1995); Willke and Vorlop, AppI Microbiol. Biotechnol 56:289-295 (2001)) Aconitate decarboxylase 138 has been purified and characterized from Aspergillus terreus. (Dwiarti et al., J Biosci. Bioeng. 94(1): 29-33 (2002)) The gene and protein sequence for the cis-aconitic acid decarboxylase (CAD) enzyme were reported previously (EP 2017344 Al; WO 2009/014437 Al), along with several close homologs listed in the table below. Gene name GenBanklD GI Number Organism CAD XP_001209273 115385453 Aspergillus terreus XP_001217495 115402837 Aspergillus terreus XP_001209946 115386810 Aspergillus terreus BAE66063 83775944 Aspergillus oryzae XP_001393934 145242722 Aspergillus niger XP_391316 46139251 Gibberella zeae XP_001389415 145230213 Aspergillus niger XP_001383451 126133853 Pichia stipitis YP_891060 118473159 Mycobacterium smegmatis NP_961187 41408351 Mycobacterium avium subsp. pratuberculosis YP_880968 118466464 Mycobacterium avium ZP_01648681 119882410 Salinispora arenicola 4-Oxalocronate decarboxylase catalyzes the decarboxylation of 4-oxalocrotonate to 2 oxopentanoate. This enzyme has been isolated from numerous organisms and characterized. Genes encoding this enzyme include dmpH and dmpE in Pseudomonas sp. (strain 600) (Shingler et al., 174:711-724 (1992)), xylII and xylIII from Pseudomonas putida (Kato et al., Arch.Microbiol 168:457-463 (1997); Stanley et al., Biochemistry 39:3514 (2000); Lian et al., JAm.Chem.Soc. 116:10403-10411 (1994)) and ReutB5691 and ReutB5692 from Ralstonia eutropha JMP134 (Hughes et al., 158:79-83 (1984)). The genes encoding the enzyme from Pseudomonas sp. (strain 600) have been cloned and expressed in E. coli (Shingler et al., 174:711-724 (1992)).

139 Gene name GenBanklD GI Number Organism dmpH CAA43228.1 45685 Pseudomonas sp. CF600 dmpE CAA43225.1 45682 Pseudomonas sp. CF600 xylII YP_709328.1 111116444 Pseudomonas putida xylIII YP_709353.1 111116469 Pseudomonas putida ReutB5691 YP_299880.1 73539513 Ralstonia eutropha JMP134 ReutB5692 YP_299881.1 73539514 Ralstonia eutropha JMP134 Finally, a class of decarboxylases has been characterized that catalyze the conversion of cinnamate (phenylacrylate) and substituted cinnamate derivatives to their corresponding styrene derivatives. These enzymes are common in a variety of organisms and specific genes encoding these enzymes that have been cloned and expressed in E. coli include: pad 1 from Saccharomyces cerevisae (Clausen et al., Gene 142:107-112 (1994)), pdc from Lactobacillus plantarum (Barthelmebs et al., 67:1063-1069 (2001); Qi et al., Metab Eng 9:268-276 (2007); Rodriguez et al., JAgric.Food Chem. 56:3068-3072 (2008)), pofK (pad) from Klebsiella oxytoca (Uchiyama et al., Biosci.Biotechnol.Biochem. 72:116-123 (2008); Hashidoko et al., Biosci.Biotech.Biochem. 58:217-218 (1994)), Pedicoccus pentosaceus (Barthelmebs et al., 67:1063-1069 (2001)), and padC from Bacillus subtilis and Bacillus pumilus (Shingler et al., 174:711-724 (1992)). A ferulic acid decarboxylase from Pseudomonasfluorescens also has been purified and characterized (Huang et al., JBacteriol. 176:5912-5918 (1994)). Enzymes in this class are stable and do not require either exogenous or internally bound co-factors, thus making these enzymes ideally suitable for biotransformations (Sariaslani, Annu.Rev.Microbiol. 61:51-69 (2007)). Gene name GenBanklD GI Number Organism pad] BAG32372.1 188496949 Saccharomyces cerevisae pdc AAC45282.1 1762616 Lactobacillus plantarum pofK (pad) BAF65031.1 149941607 Klebsiella oxytoca padC AAC46254.1 2394282 Bacillus subtilis pad CAC16793.1 11322457 Pedicoccus pentosaceus pad CAC18719.1 11691810 Bacillus pumilus 140 Each of the decarboxylases listed above represents a possible suitable enzyme for the desired transformations shown in Figure 4. If the desired activity or productivity of the enzyme is not observed in the desired conversions (e.g., muconate to 2,4-pentadienoate, 2,4 pentadienoate to butadiene), the decarboxylase enzymes can be evolved using known protein engineering methods to achieve the required performance. Importantly, it was shown through the use of chimeric enzymes that the C-terminal region of decarboxylases appears to be responsible for substrate specificity (Barthelmebs, L.; Divies, C.; Cavin, J.-F. 2001. Expression in Escherichia coli of Native and Chimeric Phenolic Acid Decarboxylases with Modified Enzymatic Activities and Method for Screening Recombinant E. coli Strains Expressing These Enzymes, Appl. Environ. Microbiol. 67, 1063-1069.). Accordingly, directed evolution experiments to broaden the specificity of decarboxylases in order to gain activity with muconate or 2,4-pentadienoate can be focused on the C-terminal region of these enzymes. Some of the decarboxylases required to catalyze the transformations in Figure 4 may exhibit higher activity on specific isomers of muconate or 2,4-pentadienoate. Isomerase enzymes can be applied to convert less desirable isomers of muconate and 2,4-pentadienoate into more desirable isomers for decarboxylation. Exemplary isomerases that catalyze similar transformations and thus represent suitable enzymes for this invention include maleate cis trans isomerase (EC 5.2.1.1), maleylacetone cis-trans isomerase (EC 5.2.1.2) and fatty acid cis-trans isomerase. Maleate cis-trans isomerase converts fumarate to maleate. This enzyme is encoded by the maiA gene from Alcaligenesfaecalis (Hatakeyama, et al., 1997, Gene Cloning and Characterization of Maleate cis-trans Isomerase from Alcaligenes faecalis, Biochem. Biophys. Research Comm. 239, 74-79) or Serratia marcescens (Hatakeyama et al., Biosci. Biotechnol. Biochem. 64:1477-1485 (2000)). Similar genes that can be identified by sequence homology include those from Geobacillus stearothermophilus, Ralstonia pickettii 12D, and Ralstonia eutropha H16. Additional maleate cis-trans isomerase enzymes are encoded by the enzymes whose amino acid sequences are provided as sequence ID's 1 through 4 in ref (Mukouyama et al., US Patent 6,133,014). Maleylacetone cis,trans-isomerase catalyzes the conversion of 4-maleyl-acetoacetate to 4-fumaryl-acetyacetate, a cis to trans conversion. This enzyme is encoded by maiA in Pseudomonas aeruginosa (Fernandez Canon et al., JBiol.Chem. 273:329-337 (1998))) and Vibrio cholera (Seltzer, JBiol.Chem. 248:215-222 (1973)). A similar enzyme was identified by sequence homology in E. coli 0157. The cti gene product catalyzes the conversion of cis- unsaturated fatty acids (UFA) to 141 trans- UFA. The enzyme has been characterized in P. putida (Junker et al., JBacteriol. 181:5693-5700 (1999)). Similar enzymes are found in Shewanella sp. MR-4 and Vibrio cholerae. Gene name GenBanklD GI Number Organism maiA BAA23002.1 2575787 Alcaligenesfaecalis maiA BAA96747.1 8570038 Serratia marcescens maiA BAA77296 4760466 Geobacillus stearothermophilus RpicJ2DDRAFT_0600 ZP_02009633 153888491 Ralstonia pickettii 12D maiA YP_725437 113866948 Ralstonia eutropha H16 maiA NP_250697 15597203 Pseudomonas aeruginosa maiA NP_230991 15641359 Vibrio cholerae maiA EDU73766 189355347 Escherichia coli 0157 cti AAD41252 5257178 Pseudomonas putida cti YP_732637 113968844 Shewanella sp. MR-4 cti ZP_04395785 229506276 Vibrio cholerae EXAMPLE III Exemplary Pathway for Producing (2-Hydroxy-3-methyl-4-oxobutoxy)phosphonate This example describes an exemplary pathway for producing the terephthalic acid (PTA) precursor (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate (2H3M40P). The precursor to the p-toluate and PTA pathways is 2H3M40P. This chemical can be derived from central metabolites glyceraldehyde-3-phosphate (G3P) and pyruvate in three enzymatic steps as shown in Figure 5. The first two steps are native to E. coli and other organisms that utilize the methylerythritol phosphate (non-mevalonate) pathway for isoprenoid biosynthesis. Pyruvate and G3P are first condensed to form 1-deoxy-D-xylulose 5-phosphate (DXP) by DXP synthase. Subsequent reduction and rearrangement of the carbon backbone is catalyzed by DXP reductoisomerase. Finally, a novel diol dehydratase transforms 2-C-methyl-D-erythritol-4-phosphate to the p-toluate precursor 2H3M40P. A. 1- Deoxyxylulose-5-phosphate (DXP) synthase. Pyruvate and G3P are condensed to form DXP by DXP synthase (EC 2.2.1.7). This enzyme catalyzes the first step in the non- 142 mevalonate pathway of isoprenoid biosynthesis. The enzyme requires thiamine diphosphate as a cofactor, and also requires reduced FAD, although there is no net redox change. A crystal structure of the E. coli enzyme is available (Xiang et al., J. Biol. Chem. 282:2676 2682 (2007)). Other enzymes have been cloned and characterized in M. tuberculosis (Bailey et al., Glycobiology 12:813-820 (2002) and Agrobacterium tumefaciens (Lee et al., J. Biotechnol. 128:555-566 (2007)). DXP synthase enzymes from B. subtilis and Synechocystis sp. PCC 6803 were cloned into E. coli (Harker and Bramley, FEBS Lett. 448:115-119 (1999)). Gene GenBank Accession No. GI No. Organism dxs AAC73523.1 1786622 Escherichia coli dxs P0A554.1 61222979 M. tuberculosis dxs11 AAP56243.1 37903541 Agrobacterium tumefaciens dxs P54523.1 1731052 Bacillus subtilis sll1945 BAA17089.1 1652165 Synechocystis sp. PCC 6803 B. 1 -Deoxy-D-xylulose-5-phosphate reductoisomerase (EC 1.1.1.267). The NAD(P)H-dependent reduction and rearrangement of 1-deoxy-D-xylulose-5-phosphate (DXP) to 2-C-methyl-D-erythritol-4-phosphate is catalyzed by DXP reductoisomerase (DXR, EC 1.1.1.267) in the second step of the non-mevalonate pathway for isoprenoid biosynthesis. The NADPH-dependent E. coli enzyme is encoded by dxr (Takahashi et al., Proc. Natl. Acad. Sci. USA 95:9879-9884 (1998)). A recombinant enzyme from Arabidopsis thaliana was functionally expressed in E. coli (Carretero-Paulet et al., Plant Physiol. 129:1581-1591 (2002)). DXR enzymes from Zymomonas mobilis and Mycobacterium tuberculosis have been characterized and crystal structures are available (Grolle et al., FEMS Microbiol. Lett. 191:131-137 (2000)); Henriksson et al., Acta Crystallogr. D. Biol. Crystallogr. 62:807-813 (2006)). Most characterized DXR enzymes are strictly NADPH dependent, but the enzymes from A. thaliana and M. tuberculosis react with NADH at a reduced rate (Argyrou and Blanchard, Biochemistry 43:4375-4384 (2004)); Rohdich et al., FEBSJ. 273:4446-4458 (2006)).

143 Gene GenBank Accession No. GI No. Organism dxr AAC73284.1 1786369 Escherichia coli dxr AAF73140.1 8131928 Arabisopsis thaliana dxr CAB60758.1 6434139 Zymomonas mobilis dxr NP_217386.2 57117032 Mycobacterium tuberculosis C. 2-C-Methyl-D-erythritol-4-phosphate dehydratase. A diol dehydratase is required to convert 2-C-methyl-D-erythritol-4-phosphate into the p-toluate precursor (Altmiller and Wagner, Arch. Biochem. Biophys. 138:160-170 (1970)). Although this transformation has not been demonstrated experimentally, several enzymes catalyze similar transformations including dihydroxy-acid dehydratase (EC 4.2.1.9), propanediol dehydratase (EC 4.2.1.28), glycerol dehydratase (EC 4.2.1.30) and myo-inositose dehydratase (EC 4.2.1.44). Diol dehydratase or propanediol dehydratase enzymes (EC 4.2.1.28) capable of converting the secondary diol 2,3-butanediol to 2-butanone are excellent candidates for this transformation. Adenosylcobalamin-dependent diol dehydratases contain alpha, beta and gamma subunits, which are all required for enzyme function. Exemplary gene candidates are found in Klebsiella pneumoniae (Tobimatsu et al., Biosci. Biotechnol. Biochem. 62:1774 1777 (1998); Toraya et al.,. Biochem. Biophys. Res. Commun. 69:475-480 (1976)), Salmonella typhimurium (Bobik et al., J Bacteriol. 179:6633-6639 (1997)), Klebsiella oxytoca (Tobimatsu et al., J Biol. Chem. 270:7142-7148 (1995)) and Lactobacillus collinoides (Sauvageot et al., FEMS Microbiol. Lett. 209:69-74 (2002)). Methods for isolating diol dehydratase gene candidates in other organisms are well known in the art (see, for example, U.S. Patent No. 5,686,276). Gene GenBank Accession No. GI No. Organism pddA BAA08099.1 868006 Klebsiella oxytoca pddB BAA08100.1 868007 Klebsiella oxytoca pddC BAA08101.1 868008 Klebsiella oxytoca pduC AAB84102.1 2587029 Salmonella typhimurium pduD AAB84103.1 2587030 Salmonella typhimurium 144 pduE AAB84104.1 2587031 Salmonella typhimurium pduC CAC82541.1 18857678 Lactobacullus collinoides pduD CAC82542.1 18857679 Lactobacullus collinoides pduE CAD01091.1 18857680 Lactobacullus collinoides pddA AAC98384.1 4063702 Klebsiella pneumoniae pddB AAC98385.1 4063703 Klebsiella pneumoniae pddC AAC98386.1 4063704 Klebsiella pneumoniae Enzymes in the glycerol dehydratase family (EC 4.2.1.30) can also be used to dehydrate 2-C-methyl-D-erythritol-4-phosphate. Exemplary gene candidates encoded by gldABC and dhaBl23 in Klebsiellapneumoniae (WO 2008/137403) and (Toraya et al., Biochem. Biophys. Res. Commun. 69:475-480 (1976)), dhaBCE in Clostridiumpasteuranum (Macis et al., FEMS Microbiol Lett. 164:21-28 (1998)) and dhaBCE in Citrobacterfreundii (Seyfried et al., J Bacteriol. 178:5793-5796 (1996)). Variants of the B12-dependent diol dehydratase from K. pneumoniae with 80- to 336-fold enhanced activity were recently engineered by introducing mutations in two residues of the beta subunit (Qi et al., J Biotechnol. 144:43-50 (2009)). Diol dehydratase enzymes with reduced inactivation kinetics were developed by DuPont using error-prone PCR (WO 2004/056963). Gene GenBank Accession No. GI No. Organism gidA AAB96343.1 1778022 Klebsiella pneumoniae gldB AAB96344.1 1778023 Klebsiella pneumoniae gldC AAB96345.1 1778024 Klebsiella pneumoniae dhaBl ABR78884.1 150956854 Klebsiella pneumoniae dhaB2 ABR78883.1 150956853 Klebsiella pneumoniae dhaB3 ABR78882.1 150956852 Klebsiella pneumoniae dhaB AAC27922.1 3360389 Clostridium pasteuranum dhaC AAC27923.1 3360390 Clostridium pasteuranum 145 dhaE AAC27924.1 3360391 Clostridium pasteuranum dhaB P45514.1 1169287 Citrobacterfreundii dhaC AAB48851.1 1229154 Citrobacterfreundii dhaE AAB48852.1 1229155 Citrobacterfreundii If a B 12-dependent diol dehydratase is utilized, heterologous expression of the corresponding reactivating factor is recommended. B12-dependent diol dehydratases are subject to mechanism-based suicide activation by substrates and some downstream products. Inactivation, caused by a tight association with inactive cobalamin, can be partially overcome by diol dehydratase reactivating factors in an ATP-dependent process. Regeneration of the B12 cofactor requires an additional ATP. Diol dehydratase regenerating factors are two subunit proteins. Exemplary candidates are found in Klebsiella oxytoca (Mori et al., J Biol. Chem. 272:32034-32041 (1997)), Salmonella typhimurium (Bobik et al., J Bacteriol. 179:6633-6639 (1997); Chen et al., J Bacteriol. 176:5474-5482 (1994)), Lactobacillus collinoides (Sauvageot et al., FEMS Microbiol. Lett. 209:69-74 (2002)), and Klebsiella pneumonia (WO 2008/137403). Gene GenBank Accession No. GI No. Organism ddrA AAC15871.1 3115376 Klebsiella oxytoca ddrB AAC15872.1 3115377 Klebsiella oxytoca pduG AAL20947.1 16420573 Salmonella typhimurium pduH AAL20948.1 16420574 Salmonella typhimurium pduG YP_002236779 206579698 Klebsiella pneumonia pduH YP_002236778 206579863 Klebsiella pneumonia pduG CAD01092 29335724 Lactobacillus collinoides pduH CAD01093 29335725 Lactobacillus collinoides B 12-independent diol dehydratase enzymes utilize S-adenosylmethionine (SAM) as a cofactor, function under strictly anaerobic conditions, and require activation by a specific activating enzyme (Frey et al., Chem. Rev. 103:2129-2148 (2003)). The glycerol 146 dehydrogenase and corresponding activating factor of Clostridium butyricum, encoded by dhaBl and dhaB2, have been well-characterized (O'Brien et al., Biochemistry 43:4635-4645 (2004); Raynaud et al., Proc. Natl. A cad. Sci USA 100:5010-5015 (2003)). This enzyme was recently employed in a 1,3-propanediol overproducing strain of E. coli and was able to achieve very high titers of product (Tang et al., Appl. Environ. Microbiol. 75:1628-1634 (2009)). An additional B12-independent diol dehydratase enzyme and activating factor from Roseburia inulinivorans was shown to catalyze the conversion of 2,3-butanediol to 2 butanone (US publication 2009/09155870). Gene GenBank Accession No. GI No. Organism dhaBl AAM54728.1 27461255 Clostridium butyricum dhaB2 AAM54729.1 27461256 Clostridium butyricum rdhtA ABC25539.1 83596382 Roseburia inulinivorans rdhtB ABC25540.1 83596383 Roseburia inulinivorans Dihydroxy-acid dehydratase (DHAD, EC 4.2.1.9) is a B12-independent enzyme participating in branched-chain amino acid biosynthesis. In its native role, it converts 2,3 dihydroxy-3-methylvalerate to 2-keto-3-methyl-valerate, a precursor of isoleucine. In valine biosynthesis, the enzyme catalyzes the dehydration of 2,3-dihydroxy-isovalerate to 2 oxoisovalerate. The DHAD from Sulfolobus solfataricus has a broad substrate range, and activity of a recombinant enzyme expressed in E. coli was demonstrated on a variety of aldonic acids (Kim and Lee, J Biochem. 139:591-596 (2006)). The S. solfataricus enzyme is tolerant of oxygen unlike many diol dehydratase enzymes. The E. coli enzyme, encoded by ilvD, is sensitive to oxygen, which inactivates its iron-sulfur cluster (Flint et al., J Biol. Chem. 268:14732-14742 (1993)). Similar enzymes have been characterized in Neurospora crassa (Altmiller and Wagner, Arch. Biochem. Biophys. 138:160-170 (1970)) and Salmonella typhimurium (Armstrong et al., Biochim. Biophys. Acta 498:282-293 (1977)). Gene GenBank Accession No. GI No. Organism ilvD NP_344419.1 15899814 Sulfolobus solfataricus ilvD AAT48208.1 48994964 Escherichia coli ilvD NP_462795.1 16767180 Salmonella typhimurium ilvD XP_958280.1 85090149 Neurospora crassa 147 The diol dehydratase myo-inosose-2-dehydratase (EC 4.2.1.44) is another exemplary candidate. Myo-inosose is a six-membered ring containing adjacent alcohol groups. A purified enzyme encoding myo-inosose-2-dehydratase functionality has been studied in Klebsiella aerogenes in the context of myo-inositol degradation (Berman and Magasanik, J Biol. Chem. 241:800-806 (1966)), but has not been associated with a gene to date. The myo inosose-2-dehydratase of Sinorhizobiumfredii was cloned and functionally expressed in E. coli (Yoshida et al., Biosci .Biotechnol. Biochem. 70:2957-2964 (2006)). A similar enzyme from B. subtilis, encoded by iolE, has also been studied (Yoshida et al., Microbiology 150:571-580 (2004)). Gene GenBank Accession No. GI No. Organism io/E P42416.1 1176989 Bacillus subtilis io/E AAX24114.1 60549621 Sinorhizobiumfredii EXAMPLE IV Exemplary Pathway for Synthesis of p-Toluate from (2-Hydroxy-3-methyl-4 oxobutoxy)phosphonate by Shikimate Pathway Enzymes This example describes exemplary pathways for synthesis ofp-toluate using shikimate pathway enzymes. The chemical structure ofp-toluate closely resembles p-hydroxybenzoate, a precursor of the electron carrier ubiquinone. 4-Hydroxybenzoate is synthesized from central metabolic precursors by enzymes in the shikimate pathway, found in bacteria, plants and fungi. The shikimate pathway is comprised of seven enzymatic steps that transform D-erythrose-4 phosphate (E4P) and phosphoenolpyruvate (PEP) to chorismate. Pathway enzymes include 2-dehydro-3-deoxyphosphoheptonate (DAHP) synthase, dehydroquinate (DHQ) synthase, DHQ dehydratase, shikimate dehydrogenase, shikimate kinase, 5-enolpyruvylshikimate-3 phosphate (EPSP) synthase and chorismate synthase. In the first step of the pathway, erythrose-4-phosphate and phosphoenolpyruvate are joined by DAHP synthase to form 3 deoxy-D-arabino-heptulosonate-7-phosphate. This compound is then dephosphorylated, dehydrated and reduced to form shikimate. Shikimate is converted to chorismate by the 148 actions of three enzymes: shikimate kinase, 3-phosphoshikimate-2-carboxyvinyltransferase and chorismate synthase. Subsequent conversion of chorismate to 4-hydroxybenzoate is catalyzed by chorismate lyase. The synthesis of p-toluate proceeds in an analogous manner as shown in Figure 6. The pathway originates with PEP and 2H3M40P, a compound analogous to E4P with a methyl group in place of the 3-hydroxyl group of E4P. The hydroxyl group of E4P does not directly participate in the chemistry of the shikimate pathway reactions, so the methyl substituted 2H3M40P precursor is expected to react as an alternate substrate. Directed or adaptive evolution can be used to improve preference for 2H3M40P and downstream derivatives as substrates. Such methods are well-known in the art. Strain engineering strategies for improving the efficiency of flux through shikimate pathway enzymes are also applicable here. The availability of the pathway precursor PEP can be increased by altering glucose transport systems (Yi et al., Biotechnol. Prog. 19:1450 1459 (2003)). 4-Hydroxybenzoate-overproducing strains were engineered to improve flux through the shikimate pathway by means of overexpression of a feedback-insensitive isozyme of 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase (Barker and Frost, Biotechnol. Bioeng. 76:376-390 (2001)). Additionally, expression levels of shikimate pathway enzymes and chorismate lyase were enhanced. Similar strategies can be employed in a strain for overproducing p-toluate. A. 2-Dehydro-3-deoxyphosphoheptonate synthase (EC 2.5.1.54). The condensation of erythrose-4-phosphate and phosphoenolpyruvate is catalyzed by 2-dehydro-3 deoxyphosphoheptonate (DAHP) synthase ( EC 2.5.1.54). Three isozymes of this enzyme are encoded in the E. coli genome by aroG, aroF and aroH and are subject to feedback inhibition by phenylalanine, tyrosine and tryptophan, respectively. In wild-type cells grown on minimal medium, the aroG, aroF and aroH gene products contributed 80%, 20% and 1% of DAHP synthase activity, respectively (Hudson and Davidson, J. Mol. Biol. 180:1023-1051 (1984)). Two residues of AroG were found to relieve inhibition by phenylalanine (Kikuchi et al., Appl. Environ. Microbiol. 63:761-762 (1997)). The feedback inhibition of AroF by tyrosine was removed by a single base-pair change (Weaver and Herrmann, J. Bacteriol. 172:6581-6584 (1990)). The tyrosine-insensitive DAHP synthase was overexpressed in a 4 hydroxybenzoate-overproducing strain of E. coli (Barker and Frost, Biotechnol. Bioeng. 76:376-390 (2001)). The aroG gene product was shown to accept a variety of alternate 4- 149 and 5-carbon length substrates (Sheflyan et al., J. Am. Chem. Soc. 120(43):11027-11032 (1998); Williamson et al., Bioorg. Med. Chem. Lett. 15:2339-2342 (2005)). The enzyme reacts efficiently with (3S)-2-deoxyerythrose-4-phosphate, a substrate analogous to erythrose-4-phosphate but lacking the alcohol at the 2-position ( Williamson et al., supra 2005). Enzymes from Helicobacter pylori and Pyrococcus furiosus also accept this alternate substrate (Schofield et al., Biochemistry 44:11950-11962 (2005)); Webby et al., Biochem. J. 390:223-230 2005)) and have been expressed in E. coli. An evolved variant of DAHP synthase, differing from the wild type E. coli AroG enzyme by 7 amino acids, was shown to exhibit a 60-fold improvement in Kcat/KM (Ran and Frost, J Am. Chem. Soc. 129:6130 6139 (2007)). Gene GenBank Accession No. GI No. Organism aroG AAC73841.1 1786969 Escherichia coli aroF AAC75650.1 1788953 Escherichia coli aroH AAC74774.1 1787996 Escherichia coli aroF Q9ZMU5 81555637 Helicobacter pylori PF1690 NP_579419.1 18978062 Pyrococcusfuriosus B. 3-Dehydroquinate synthase (EC 4.2.3.4). The dephosphorylation of substrate (2)(2,4-dihydroxy-5-methyl-6-[(phosphonooxy)methyl]oxane-2-carboxylate) to substrate (3)(1,3-dihydroxy-4-methylcylohex-1-ene-1-carboxylate) as shown in Figure 2 is analogous to the dephosphorylation of 3-deoxy-arabino-heptulonate-7-phosphate by 3-dehydroquinate synthase. The enzyme has been characterized in E. coli (Mehdi et al., Methods Enzymol. 142:306-314 (1987), B. subtilis (Hasan and Nester, J Biol. Chem. 253:4999-5004 (1978)) and Mycobacterium tuberculosis H37Rv (de Mendonca et al., J Bacteriol. 189:6246-6252 (2007)). The E. coli enzyme is subject to inhibition by L-tyrosine (Barker and Frost, Biotechnol. Bioeng. 76:376-390 2001)). Gene GenBank Accession No. GI No. Organism aroB AAC76414.1 1789791 Escherichia coli aroB NP_390151.1 16079327 Bacillus subtilis aroB CAB06200.1 1781064 Mycobacterium tuberculosis 150 C. 3-Dehydroquinate dehydratase (EC 4.2.1.10). 3-Dehydroquinate dehydratase, also termed 3-dehydroquinase (DHQase), naturally catalyzes the dehydration of 3-dehydroquinate to 3-dehydroshikimate, analogous to step C in the p-toluate pathway of Figure 2. DHQase enzymes can be divided into two classes based on mechanism, stereochemistry and sequence homology (Gourley et al., Nat. Struct. Biol. 6:521-525. (1999)). Generally the type 1 enzymes are involved in biosynthesis, while the type 2 enzymes operate in the reverse (degradative) direction. Type 1 enzymes from E. coli (Kinghom et al., Gene 14:73-80. 1981)), Salmonella typhi (Kinghom et al., supra 1981; Servos et al., J Gen. Microbiol. 137:147-152 (1991)) and B. subtilis (Warburg et al., Gene 32:57-66 1984)) have been cloned and characterized. Exemplary type II 3-dehydroquinate dehydratase enzymes are found in Mycobacterium tuberculosis, Streptomyces coelicolor (Evans et al., FEBS Lett. 530:24-30 (2002)) and Helicobacterpylori (Lee et al., Proteins 51:616-7 (2003)). Gene GenBank Accession No. GI No. Organism aroD AAC74763.1 1787984 Escherichia coli aroD P24670.2 17433709 Salmonella typhi aroC NP_390189.1 16079365 Bacillus subtilis aroD POA4Z6.2 61219243 Mycobacterium tuberculosis aroQ P15474.3 8039781 Streptomyces coelicolor aroQ Q48255.2 2492957 Helicobacterpylori D. Shikimate dehydrogenase (EC 1.1.1.25). Shikimate dehydrogenase catalyzes the NAD(P)H dependent reduction of 3-dehydroshikimate to shikimate, analogous to Step D of Figure 2. The E. coli genome encodes two shikimate dehydrogenase paralogs with different cofactor specificities. The enzyme encoded by aroE is NADPH specific, whereas the ydiB gene product is a quinate/shikimate dehydrogenase which can utilize NADH (preferred) or NADPH as a cofactor (Michel et al., J Biol. Chem. 278:19463-19472 (2003)). NADPH dependent enzymes from Mycobacterium tuberculosis (Zhang et al., J Biochem. Mol. Biol. 38:624-631 (2005)), Haemophilus influenzae (Ye et al., J Bacteriol. 185:4144-4151 (2003)) and Helicobacter pylori (Han et al., FEBS J 273:4682-4692 (2006)) have been functionally expressed in E. coli.

151 Gene GenBank Accession No. GI No. Organism aroE AAC76306.1 1789675 Escherichia coli ydiB AAC74762.1 1787983 Escherichia coli aroE NP_217068.1 15609689 Mycobacterium tuberculosis aroE P43876.1 1168510 Haemophilus influenzae aroE AAW22052.1 56684731 Helicobacterpylori E. Shikimate kinase (EC 2.7.1.71). Shikimate kinase catalyzes the ATP-dependent phosphorylation of the 3-hydroxyl group of shikimate analogous to Step E of Figure 2. Two shikimate kinase enzymes are encoded by aroK (SKI) and aroL (SK2) in E. coli (DeFeyter and Pittard, J. Bacteriol. 165:331-333 (1986); Lobner-Olesen and Marinus, J Bacteriol. 174:525-529 (1992)). The Km of SK2, encoded by aroL, is 100-fold lower than that of SKI, indicating that this enzyme is responsible for aromatic biosynthesis (DeFeyter et al., supra 1986). Additional shikimate kinase enzymes from Mycobacterium tuberculosis (Gu et al., J Mol. Biol. 319:779-789 (2002)); Oliveira et al., Protein Expr. Purif 22:430-435 (2001)(doi: 10.1006/prep.2001.1457, doi;S 1046-5928(01)91457-3, pii), Helicobacter pylori (Cheng et al., J Bacteriol. 187:8156-8163 (2005)) and Erwinia chrysanthemi (Krell et al., Protein Sci. 10:1137-1149 (2001)) have been cloned in E. coli. Gene GenBank Accession No. GI No. Organism aroK YP_026215.2 90111581 Escherichia coli aroL NP_414922.1 16128373 Escherichia coli aroK CAB06199.1 1781063 Mycobacterium tuberculosis aroK NP_206956.1 15644786 Helicobacterpylori SK CAA32883.1 42966 Erwinia chrysanthemi F. 3-Phosphoshikimate-2-carboxyvinyltransferase (EC 2.5.1.19). 3 Phosphoshikimate-2-carboxyvinyltransferase, also known as 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS), catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate to the 5-hydroxyl of shikimate-3-phosphate. The enzyme is encoded 152 by aroA in E. coli (Anderson et al., Biochemistry 27:1604-1610 (1988)). EPSPS enzymes from Mycobacterium tuberculosis (Oliveira et al., Protein Expr. Purif 22:430-435 (2001)), Dunaliella salina (Yi et al., J Microbiol. 45:153-157 (2007)) and Staphylococcus aureus (Priestman et al., FEBS Lett. 579:728-732 (2005)) have been cloned and functionally expressed in E. coli. Gene GenBank Accession No. GI No. Organism aroA AAC73994.1 1787137 Escherichia coli aroA AAA25356.1 149928 Mycobacterium tuberculosis aroA AAA71897.1 152956 Staphylococcus aureus aroA ABM68632.1 122937807 Dunaliella salina G. Chorismate synthase (EC 4.2.3.5). Chorismate synthase is the seventh enzyme in the shikimate pathway, catalyzing the transformation of 5 -enolpyruvylshikimate-3 -phosphate to chorismate. The enzyme requires reduced flavin mononucleotide (FMN) as a cofactor, although the net reaction of the enzyme does not involve a redox change. In contrast to the enzyme found in plants and bacteria, the chorismate synthase in fungi is also able to reduce FMN at the expense of NADPH (Macheroux et al., Planta 207:325-334 (1999)). Representative monofunctional enzymes are encoded by aroC of E. coli (White et al., Biochem. J 251:313-322 (1988)) and Streptococcus pneumoniae (Maclean and Ali, Structure 11:1499-1511 (2003)(doi:SO969212603002648, pii). Bifunctional fungal enzymes are found in Neurospora crassa (Kitzing et al., J Biol. Chem. 276:42658-42666 (2001)) and Saccharomyces cerevisiae (Jones et al., Mol. Microbiol. 5:2143-2152 (1991)). GenBank Accession Gene No. GI No. Organism aroC NP_416832.1 16130264 Escherichia coli aroC ACH47980.1 197205483 Streptococcus pneumoniae U25818.1:19..1317 AAC49056.1 976375 Neurospora crassa ARO2 CAA42745.1 3387 Saccharomyces cerevisiae 153 H. Chorismate lyase (EC 4.1.3.40). Chorismate lyase catalyzes the first committed step in ubiquinone biosynthesis: the removal of pyruvate from chorismate to form 4 hydroxybenzoate. The enzymatic reaction is rate-limited by the slow release of the 4 hydroxybenzoate product (Gallagher et al., Proteins 44:304-311 (2001)), which is thought to play a role in delivery of 4-hydroxybenzoate to downstream membrane-bound enzymes. The chorismate lyase of E. coli was cloned and characterized and the enzyme has been crystallized (Gallagher et al., supra 2001; Siebert et al., FEBSLett. 307:347-350 (1992)). Structural studies implicate the G90 residue as contributing to product inhibition (Smith et al., Arch. Biochem. Biophys. 445:72-80 (2006)). Modification of two surface-active cysteine residues reduced protein aggregation (Holden et al., Biochim. Biophys. Acta 1594:160-167 (2002)). A recombinant form of the Mycobacterium tuberculosis chorismate lyase was cloned and characterized in E. coli (Stadthagen et al., J Biol. Chem. 280:40699-40706 2005)). Gene GenBank Accession No. GI No. Organism ubiC AAC77009.2 87082361 Escherichia coli Rv2949c NP_217465.1 15610086 Mycobacterium tuberculosis B-F. Multifunctional AROM protein. In most bacteria, the enzymes of the shikimate pathway are encoded by separate polypeptides. In microbial eukaryotes, five enzymatic functions are catalyzed by a polyfunctional protein encoded by a pentafunctional supergene (Campbell et al., Int. J Parasitol. 34:5-13 (2004)). The multifunctional AROM protein complex catalyzes reactions analogous to reactions B-F of Figure 2. The AROM protein complex has been characterized in fungi including Aspergillus nidulans, Neurospora crassa, Saccharomyces cerevisiae and Pneumocystis carinii (Banerji et al., J Gen. Microbiol. 139:2901-2914 (1993); Charles et al., Nucleic Acids Res. 14:2201-2213 (1986); Coggins et al., Methods Enzymol. 142:325-341 (1987); Duncan, K., Biochem. J 246:375-386 (1987)). Several components of AROM have been shown to function independently as individual polypeptides. For example, dehydroquinate synthase (DHQS) forms the amino-terminal domain of AROM, and can function independently when cloned into E. coli (Moore et al., Biochem. J. 301 ( Pt 1):297-304 (1994)). Several crystal structures of AROM components from Aspergillus nidulans provide insight into the catalytic mechanism (Carpenter et al., Nature 394:299-302 (1998)).

154 Gene GenBank Accession No. GI No. Organism AROM P07547.3 238054389 Aspergillus nidulans AROM P08566.1 114166 Saccharomyces cerevisiae AROM P07547.3 238054389 Aspergillus nidulans AROM Q12659.1 2492977 Pneumocystis carinii EXAMPLE V Exemplary Pathway for Enzymatic Transformation of p-Toluate to Terephthalic Acid This example describes exemplary pathways for conversion ofp-toluate to terephthalic acid (PTA). P-toluate can be further transformed to PTA by oxidation of the methyl group to an acid in three enzymatic steps as shown in Figure 3. The pathway is comprised of a p-toluate methyl-monooxygenase reductase, a 4-carboxybenzyl alcohol dehydrogenase and a 4 carboxybenzyl aldehyde dehydrogenase. In the first step, p-toluate methyl-monooxyngenase oxidizes p-toluate to 4-carboxybenzyl alcohol in the presence of 02. The Comamonas testosteroni enzyme (tsaBM), which also reacts with 4-toluene sulfonate as a substrate, has been purified and characterized (Locher et al., J Bacteriol. 173:3741-3748 (1991)). 4 Carboxybenzyl alcohol is subsequently converted to an aldehyde by 4-carboxybenzyl alcohol dehydrogenase (tsaC). The aldehyde to acid transformation is catalyzed by 4 carboxybenzaldehyde dehydrogenase (tsaD). Enzymes catalyzing these reactions are found in Comamonas testosteroni T-2, an organism capable of utilizing p-toluate as the sole source of carbon and energy (Junker et al., J Bacteriol. 179:919-927 (1997)). Additional genes to transform p-toluate to PTA can be found by sequence homology, in particular to proteobacteria in the genera Burkholderia, Alcaligenes, Pseudomonas, Shingomonas and Comamonas (US Patent No. 6,187,569 and US publication 2003/0170836). Genbank identifiers associated with the Comamonas testosteroni enzymes are listed below.

155 Gene GenBank Accession No. GI No. Organism tsaB AAC44805.1 1790868 Comamonas testosteroni tsaM AAC44804.1 1790867 Comamonas testosteroni tsaC AAC44807.1 1790870 Comamonas testosteroni tsaD AAC44808.1 1790871 Comamonas testosteroni EXAMPLE VI Synthesis of benzoate from 2H40P This example shows the synthesis of benzoate from 2H40P by shikimate pathway enzymes (Figure 9) and an exemplary pathway for producing the benzoate pathway precursor 2H40P (Figure 8). Like p-toluate, the chemical structure of benzoate resembles p-hydroxybenzoate, a product of the shikimate pathway described above in Example IV. In this Example shikimate pathway enzymes are utilized to synthesize benzoate from the pathway precursor (2-hydroxy 4-oxobutoxy)phosphonate (2H40P) by the pathway shown in Figure 9. The reactivity of shikimate pathway enzymes on 2H40P and the alternate substrates used in benzoate formation are optionally optimized by directed or adaptive evolution to improve preference for 2H40P and downstream derivatives as substrates. Such methods are well-known in the art and described herein. An exemplary and novel pathway for synthesizing the benzoate pathway precursor (2 hydroxy-4-oxobutoxy)phosphonate (2H40P) is shown in Figure 8. In this pathway, 2H40P is derived from the central metabolite erythrose-4-phosphate in two enzymatic steps. In the first step, a diol dehydratase transforms erythrose-4-phosphate to (2,4 dioxobutoxy)phosphonate (24DBP). The 2-keto group of 24DBP is subsequently reduced to the alcohol of 2H40P by an oxidoreductase with 24DBP reductase activity. Exemplary enzymes for Steps A and B of Figure 8 are presented below. A. Erythrose-4-phosphate dehydratase: Diol dehydratase enzymes in the EC class 4.2.1 are used to convert erythrose-4-phosphate into 24DBP (Figure 4, Step A). Although 156 enzymes catalyzing this transformation have not been indicated, several enzymes catalyze similar transformations including dihydroxy-acid dehydratase (EC 4.2.1.9), propanediol dehydratase (EC 4.2.1.28), glycerol dehydratase (EC 4.2.1.30) and myo-inositose dehydratase (EC 4.2.1.44). Exemplary diol dehydratase enzymes are described above in Example III. Diol dehydratase or propanediol dehydratase enzymes (EC 4.2.1.28) capable of converting the secondary diol 2,3-butanediol to 2-butanone can be used for this transformation. Adenosylcobalamin- or B12-dependent diol dehydratases contain alpha, beta and gamma subunits, all of which are used for enzyme function. Exemplary genes are found in Klebsiella pneumoniae (Tobimatsu et al., Biosci. Biotechnol. Biochem. 62:1774-1777 (1998); Toraya et al.,. Biochem. Biophys. Res. Commun. 69:475-480 (1976)), Salmonella typhimurium (Bobik et al., J Bacteriol. 179:6633-6639 (1997)), Klebsiella oxytoca (Tobimatsu et al., J Biol. Chem. 270:7142-7148 (1995)) and Lactobacillus collinoides (Sauvageot et al., FEMS Microbiol. Lett. 209:69-74 (2002)). Methods for isolating diol dehydratase genes in other organisms are well known in the art as exemplified in U.S. Patent No. 5,686,276, which is incorporated herein by reference in its entirety. Gene GenBank Accession No. GI No. Organism pddA BAA08099.1 868006 Klebsiella oxytoca pddB BAA08100.1 868007 Klebsiella oxytoca pddC BAA08101.1 868008 Klebsiella oxytoca pduC AAB84102.1 2587029 Salmonella typhimurium pduD AAB84103.1 2587030 Salmonella typhimurium pduE AAB84104.1 2587031 Salmonella typhimurium pduC CAC82541.1 18857678 Lactobacullus collinoides pduD CAC82542.1 18857679 Lactobacullus collinoides pduE CAD01091.1 18857680 Lactobacullus collinoides pddA AAC98384.1 4063702 Klebsiella pneumoniae pddB AAC983 85.1 4063703 Klebsiella pneumoniae pddC AAC98386.1 4063704 Klebsiella pneumoniae Enzymes in the glycerol dehydratase family (EC 4.2.1.30) can also be used to dehydrate erythrose-4-phosphate. Exemplary genes include gldABC and dhaB123 in Klebsiella pneumoniae (WO 2008/137403) and (Toraya et al., Biochem. Biophys. Res.

157 Commun. 69:475-480 (1976)), dhaBCE in Clostridiumpasteuranum (Macis et al., FEMS Microbiol Lett. 164:21-28 (1998)) and dhaBCE in Citrobacterfreundii (Seyfried et al., J Bacteriol. 178:5793-5796 (1996)). Variants of the B12-dependent diol dehydratase from K. pneumoniae with 80- to 336-fold enhanced activity were recently engineered by introducing mutations in two residues of the beta subunit (Qi et al., J Biotechnol. 144:43-50 (2009)). Diol dehydratase enzymes with reduced inactivation kinetics were developed by DuPont using error-prone PCR (WO 2004/056963). Gene GenBank Accession No. GI No. Organism gldA AAB96343.1 1778022 Klebsiella pneumoniae gldB AAB96344.1 1778023 Klebsiella pneumoniae gldC AAB96345.1 1778024 Klebsiella pneumoniae dhaBl ABR78884.1 150956854 Klebsiella pneumoniae dhaB2 ABR78883.1 150956853 Klebsiella pneumoniae dhaB3 ABR78882.1 150956852 Klebsiella pneumoniae dhaB AAC27922.1 3360389 Clostridium pasteuranum dhaC AAC27923.1 3360390 Clostridium pasteuranum dhaE AAC27924.1 3360391 Clostridium pasteuranum dhaB P45514.1 1169287 Citrobacterfreundii dhaC AAB48851.1 1229154 Citrobacterfreundii dhaE AAB48852.1 1229155 Citrobacterfreundii If a B 12-dependent diol dehydratase is utilized, heterologous expression of the corresponding reactivating factor is useful. B 12-dependent diol dehydratases are subject to mechanism-based suicide activation by substrates and some downstream products. Inactivation, caused by a tight association with inactive cobalamin, can be partially overcome by diol dehydratase reactivating factors in an ATP-dependent process. Regeneration of the B12 cofactor is ATP-dependent. Diol dehydratase regenerating factors are two-subunit proteins. Exemplary genes are found in Klebsiella oxytoca (Mori et al., J Biol. Chem. 272:32034-32041 (1997)), Salmonella typhimurium (Bobik et al., J Bacteriol. 179:6633 6639 (1997); Chen et al., J Bacteriol. 176:5474-5482 (1994)), Lactobacillus collinoides (Sauvageot et al., FEMS Microbiol. Lett. 209:69-74 (2002)), and Klebsiella pneumonia (WO 2008/137403).

158 Gene GenBank Accession No. GI No. Organism ddrA AAC15871.1 3115376 Klebsiella oxytoca ddrB AAC15872.1 3115377 Klebsiella oxytoca pduG AAL20947.1 16420573 Salmonella typhimurium pduH AAL20948.1 16420574 Salmonella typhimurium pduG YP_002236779 206579698 Klebsiella pneumonia pduH YP_002236778 206579863 Klebsiella pneumonia pduG CAD01092 29335724 Lactobacillus collinoides pduH CAD01093 29335725 Lactobacillus collinoides B12-independent diol dehydratase enzymes utilize S-adenosylmethionine (SAM) as a cofactor, function under strictly anaerobic conditions, and require activation by a specific activating enzyme (Frey et al., Chem. Rev. 103:2129-2148 (2003)). The glycerol dehydrogenase and corresponding activating factor of Clostridium butyricum, encoded by dhaBl and dhaB2, have been well-characterized (O'Brien et al., Biochemistry 43:4635-4645 (2004); Raynaud et al., Proc. Natl. A cad. Sci USA 100:5010-5015 (2003)). This enzyme was recently employed in a 1,3-propanediol overproducing strain of E. coli and was able to achieve very high titers of product (Tang et al., Appl. Environ. Microbiol. 75:1628-1634 (2009)). An additional B12-independent diol dehydratase enzyme and activating factor from Roseburia inulinivorans was shown to catalyze the conversion of 2,3-butanediol to 2 butanone (US publication 2009/09155870). Gene GenBank Accession No. GI No. Organism dhaBl AAM54728.1 27461255 Clostridium butyricum dhaB2 AAM54729.1 27461256 Clostridium butyricum rdhtA ABC25539.1 83596382 Roseburia inulinivorans rdhtB ABC25540.1 83596383 Roseburia inulinivorans Dihydroxy-acid dehydratase (DHAD, EC 4.2.1.9) is a B12-independent enzyme participating in branched-chain amino acid biosynthesis. In its native role, it converts 2,3 dihydroxy-3-methylvalerate to 2-keto-3-methyl-valerate, a precursor of isoleucine. In valine biosynthesis, the enzyme catalyzes the dehydration of 2,3-dihydroxy-isovalerate to 2 oxoisovalerate. The DHAD from Sulfolobus solfataricus has a broad substrate range, and activity of a recombinant enzyme expressed in E. coli was demonstrated on a variety of 159 aldonic acids (Kim and Lee, J Biochem. 139:591-596 (2006)). The S. solfataricus enzyme is tolerant of oxygen unlike many diol dehydratase enzymes. The E. coli enzyme, encoded by ilvD, is sensitive to oxygen, which inactivates its iron-sulfur cluster (Flint et al., J Biol. Chem. 268:14732-14742 (1993)). Similar enzymes have been characterized in Neurospora crassa (Altmiller and Wagner, Arch. Biochem. Biophys. 138:160-170 (1970)) and Salmonella typhimurium (Armstrong et al., Biochim. Biophys. Acta 498:282-293 (1977)). Gene GenBank Accession No. GI No. Organism ilvD NP_344419.1 15899814 Sulfolobus solfataricus ilvD AAT48208.1 48994964 Escherichia coli ilvD NP_462795.1 16767180 Salmonella typhimurium ilvD XP_958280.1 85090149 Neurospora crassa The diol dehydratase myo-inosose-2-dehydratase (EC 4.2.1.44) is another exemplary enzyme. Myo-inosose is a six-membered ring containing adjacent alcohol groups. A purified enzyme encoding myo-inosose-2-dehydratase functionality has been studied in Klebsiella aerogenes in the context of myo-inositol degradation (Berman and Magasanik, J Biol. Chem. 241:800-806 (1966)), but has not been associated with a gene to date. The myo inosose-2-dehydratase of Sinorhizobiumfredii was cloned and functionally expressed in E. coli (Yoshida et al., Biosci .Biotechnol. Biochem. 70:2957-2964 (2006)). A similar enzyme from B. subtilis, encoded by iolE, has also been studied (Yoshida et al., Microbiology 150:571-580 (2004)). Gene GenBank Accession No. GI No. Organism iolE P42416.1 1176989 Bacillus subtilis iolE AAX24114.1 60549621 Sinorhizobiumfredii B. (2,4-Dioxobutoxy)phosphonate reductase: An enzyme with (2,4 dioxobutoxy)phosphonate reductase activity is used to convert 24DBP to 2H40P. Although this compound is not a known substrate of alcohol dehydrogenase enzymes, several enzymes in the EC class 1.1.1 catalyze similar reactions. Exemplary enzymes that reduce ketones of phosphorylated substrates include glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), 3 phosphoglycerate dehydrogenase (EC 1.1.1.95) and erythronate-4-phosphate dehydrogenase (EC 1.1.1.290). Glycerol-3-phosphate dehydrogenase catalyzes the NAD(P)H-dependent 160 reduction of dihydroxyacetone-phosphate to glycerol-3-phosphate. This enzyme is encoded by gpsA of E. coli (Kito et al., JBiol.Chem. 244:3316-3323 (1969)). 3-Phosphoglycerate dehydrogenase catalyzes the first step of serine biosynthesis in several organisms including E. coli, where it is encoded by the gene serA (Tobey et al., JBiol.Chem. 261:12179-12183 (1986)). Erythronate-4-phosphate dehydrogenase (EC 1.1.1.290) catalyzes the reversible reduction of 2-oxo-3-hydroxy-4-phosphobutanoate to erythronate-4-phosphate. This enzyme, encoded by pdxB of E. coli, normally operates in the reverse direction in the context of pyridoxal 5'-phosphate biosynthesis (Schoenlein et al., JBacteriol. 171:6084-6092 (1989)). A similar enzyme encoded by pdxB in Pseudomonas aeruginosa has been characterized and heterologously expressed in E. coli (Ha et al., Acta Crystallogr.Sect.F.Struct.Biol.Cryst.Commun. 62:139-141 (2006)). Gene GenBank Accession No. GI No. Organism gpsA AAC76632.1 1790037 Escherichia coli serA AAC75950.1 1789279 Escherichia coli pdxB AAC75380.1 1788660 Escherichia coli pdxB AAG04764.1 9947319 Pseudomonas aeruginosa A wide variety of alcohol dehydrogenase enzymes catalyze the reduction of a ketone to an alcohol functional group. Two such enzymes from E. coli are encoded by malate dehydrogenase (mdh) and lactate dehydrogenase (/dhA). The lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high activities on 2-ketoacids of various chain lengths including lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (Steinbuchel et al., Eur.JBiochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta (Suda et al., Arch.Biochem.Biophys. 176:610-620 (1976); Suda et al., Biochem.Biophys.Res.Commun. 77:586-591 (1977)). An additional oxidoreductase is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the human heart which has been cloned and characterized (Marks et al., JBiol.Chem. 267:15459-15463 (1992)). Alcohol dehydrogenase enzymes of C. beijerinckii (Ismaiel et al., JBacteriol. 175:5097-5105 (1993)) and T. brockii (Lamed et al., Biochem.J 195:183-190 (1981); Peretz et al., Biochemistry. 28:6549-6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase, or alternatively, 2-butanol dehydrogenase, catalyzes the reduction of MEK to form 2-butanol. Exemplary enzymes can be found in Rhodococcus ruber (Kosjek et al., 161 Biotechnol Bioeng. 86:55-62 (2004)) and Pyrococcusfuriosus (van der et al., Eur.JBiochem. 268:3062-3068 (2001)). Gene GenBank Accession No. GI No. Organism mdh AAC76268.1 1789632 Escherichia coli ldhA NP_415898.1 16129341 Escherichia coli ldh YP_725182.1 113866693 Ralstonia eutropha bdh AAA58352.1 177198 Homo sapiens adh AAA23199.2 60592974 Clostridium beijerinckii NRRL B593 adh P14941.1 113443 Thermoanaerobacter brockii HTD4 adhA AAC25556 3288810 Pyrococcusfuriosus sadh CAD36475 21615553 Rhodococcus ruber A number of organisms can catalyze the reduction of 4-hydroxy-2-butanone to 1,3 butanediol, including those belonging to the genus Bacillus, Brevibacterium, Candida, and Klebsiella among others, as described by Matsuyama et al. ( (1995)). A mutated Rhodococcus phenylacetaldehyde reductase (Sar268) and a Leifonia alcohol dehydrogenase have also been shown to catalyze this transformation at high yields (Itoh et al., Appl.Microbiol Biotechnol. 75:1249-1256 (2007)). Homoserine dehydrogenase (EC 1.1.1.13) catalyzes the NAD(P)H-dependent reduction of aspartate semialdehyde to homoserine. In many organisms, including E. coli, homoserine dehydrogenase is a bifunctional enzyme that also catalyzes the ATP-dependent conversion of aspartate to aspartyl-4-phosphate (Stames et al., Biochemistry 11:677-687 (1972)). The functional domains are catalytically independent and connected by a linker region (Sibilli et al., JBiol.Chem. 256:10228-10230 (1981)) and both domains are subject to allosteric inhibition by threonine. The homoserine dehydrogenase domain of the E. coli enzyme, encoded by thrA, was separated from the aspartate kinase domain, characterized, and found to exhibit high catalytic activity and reduced inhibition by threonine (James et al., Biochemistry 41:3720-3725 (2002)). This can be applied to other bifunctional threonine kinases including, for example, hom of Lactobacillus plantarum (Cahyanto et al., Microbiology 152:105-112 (2006)) and Arabidopsis thaliana. The monofunctional 162 homoserine dehydrogenases encoded by hom6 in S. cerevisiae (Jacques et al., Biochim.Biophys.Acta 1544:28-41 (2001)) and hom2 in Lactobacillus plantarum (Cahyanto et al., Microbiology 152:105-112 (2006)) have been functionally expressed and characterized in E. coli. Gene GenBank Accession No. GI No. Organism thrA AAC73113.1 1786183 Escherichia coli K12 akthr2 081852 75100442 Arabidopsis thaliana hom6 CAA89671 1015880 Saccharomyces cerevisiae hom] CAD64819 28271914 Lactobacillus plantarum hom2 CAD63186 28270285 Lactobacillus plantarum EXAMPLE VII Pathways to Benzene and Toluene This Example shows pathways from benzoate and benzoyl-CoA to benzene, and p toluate and p-methylbenzoyl-CoA to toluene. Pathways for enzymatic production of benzene from benzoate or benzoyl-CoA are shoen in Figure 10. Benzoate and benzoyl-CoA are naturally occurring metabolic intermediates common to diverse aromatic degradation pathways. Benzoate can also be produced by the alternate shikimate pathway route described herein. Several routes from benzoate and/or benzoyl-CoA to benzene are shown in Figure 10. First, benzene can be produced directly from benzoate via decarboxylation (Figure 10, path E). Alternately, the acid moiety can be reduced to an aldehyde either directly by a benzoic acid reductase (Figure 10, path B) or indirectly via benzoyl-CoA and/or (benzoyloxy)phosphonate intermediates (Figure 10, paths A and D, path B, path H and G, or path A, F, and G). Benzaldehyde can be subsequently decarbonylated to form benzene (Figure 10, path C). Similar pathways for enzymatic production of toluene from p-toluate and/or p methylbenzoyl-CoA are shown in Figure 11. p-Toluate can be produced by the alternate shikimate pathway as described herein. Routes for converting p-toluate (also known as p methylbenzoate) and/or p-methylbenzoyl-CoA to toluene are analogous to the transformation of benzoate or benzoyl-CoA to benzene, described above. Enzymes for catalyzing the 163 transformations shown in Figures 10 and 11 are categorized by EC number and described further below. Label Function Fig-Path 1.2.1 .b Oxidoreductase (acyl-CoA to aldehyde) 6/7-D 1.2.1.d Oxidoreductase (phosphorylating/dephosphorylating) 6/7-G 1.2.1.e Oxidoreductase (acid to aldehyde) 6/7-B Acyltransferase (transferring phosphate group to CoA; 6/7-F 2.3.1.a phosphotransacylase) 2.7.2.a Phosphotransferase, carboxyl group acceptor (kinase) 6/7-H 2.8.3.a Coenzyme-A transferase 6/7-A 3.1.2.a Thiolester hydrolase (CoA specific) 6/7-A 4.1.1.a Carboxy-lyase 6/7-E 4.1.99.a Decarbonylase 6/7-C 6.2.1.a Acid-thiol ligase 6/7-A 1.2.1 .b Oxidoreductase (acyl-CoA to aldehyde): An enzyme with benzoyl-CoA reductase activity is used to convert benzoyl-CoA into benzaldehyde (Path D of Figure 10). Similarly, the reduction ofp-methylbenzoyl-CoA to p-methylbenzaldehyde (also called p tolualdehyde) is catalyzed by an enzyme with p-methylbenzoyl-CoA reductase activity (Path D of Figure 11). Although enzymes with these activities have not been characterized, an enzyme catalyzing a similar reaction is cinnamoyl-CoA reductase (EC 1.2.1.44). This enzyme catalyzes the NAD(P)H-dependent reduction of cinnamoyl-CoA and substituted aromatic derivatives such as coumaroyl-CoA and feruloyl-CoA. The enzyme has been characterized in organisms including Arabidopsis thaliana (Lauvergeat et al., Phytochemistry 57:1187-1195 (2001)), Triticum aestivum (Ma, JExp.Bot. 58:2011-2021 (2007)) and Panicum virgatum (Escamilla-Trevino et al., New Phytol. 185:143-155 (2010)). The enzymes from A. thaliana and P. virgatum were characterized and heterologously expressed in E. coli.

164 GenBank Accession Gene GI No. Organism No. TACCRI ABE01883.1 90902167 Triticum aestivum AtCCR1 AAU45042.1 52355804 Arabidopsis thaliana AtCCR2 AAG53687.1 12407990 Arabidopsis thaliana PvCCRJ ACZ74580.1 270315096 Panicum virgatum PvCCR2 ACZ74585.1 270315106 Panicum virgatum Several other well-characterized acyl-CoA reductases reduce an acyl-CoA to its corresponding aldehyde. Exemplary enzymes include fatty acyl-CoA reductase (EC 1.2.1.50), succinyl-CoA reductase (EC 1.2.1.76), acetyl-CoA reductase (EC 1.2.1.10) and butyryl-CoA reductase. Exemplary fatty acyl-CoA reductases enzymes are encoded by acr1 of Acinetobacter calcoaceticus (Reiser et al., 179:2969-2975 (1997)) and Acinetobacter sp. M-1 (Ishige et al., Appl.Environ.Microbiol. 68:1192-1195 (2002)). Enzymes with succinyl CoA reductase activity are encoded by sucD of Clostridium kluyveri (Sohling et al., J Bacteriol. 178:871-880 (1996a); Sohling et al., 178:871-80 (1996)) and sucD of P. gingivalis (Takahashi et al., JBacteriol. 182:4704-4710 (2000)). Additional succinyl-CoA reductase enzymes participate in the 3-hydroxypropionate/4-hydroxybutyrate cycle of thermophilic archaea such as Metallosphaera sedula (Berg et al., Science. 318:1782-1786 (2007)) and Thermoproteus neutrophilus (Ramos-Vera et al., JBacteriol. 191:4286-4297 (2009)). The M. sedula CoA reductase, encoded by Msed 0709, is NADPH-dependent and also has malonyl-CoA reductase activity. The T. neutrophilus enzyme is active with both NADPH and NADH. The acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde to their corresponding CoA-esters (Powlowski et al., 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Koo et al., Biotechnol Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA to butyraldehyde, in solventogenic organisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71:5 8-68 (2007)). The acyl-CoA reductase encoded by ald in Clostridium beijerinckii has been indicated to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes (Toth et al., Appl 165 Environ.Microbiol 65:4973-4980 (1999)). This enzyme exhibits high sequence homology to the CoA-dependent acetaldehyde dehydrogenase enzymes of Salmonella typhimurium and E. coli encoded by eutE (Toth et al., Appl Environ.Microbiol 65:4973-4980 (1999)). GenBank Accession Gene GI No. Organism No. acri YP_047869.1 50086359 Acinetobacter calcoaceticus acri AAC45217 1684886 Acinetobacter baylyi acri BAB85476.1 18857901 Acinetobacter sp. Strain M-1 MSED_0709 YP_001190808.1 146303492 Metallosphaera sedula Tneu_0421 ACB39369.1 170934108 Thermoproteus neutrophilus sucD P38947.1 172046062 Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonas gingivalis bphG BAA03892.1 425213 Pseudomonas sp adhE AAV66076.1 55818563 Leuconostoc mesenteroides bld AAP42563.1 31075383 Clostridium saccharoperbutylacetonicum ald AAT66436 9473535 Clostridium beijerinckii eutE AAA80209 687645 Salmonella typhimurium eutE P77445 2498347 Escherichia coli An additional CoA reductase enzyme is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archael bacteria (Berg et al., 318:1782-1786 (2007); Thauer, 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus spp (Alber et al., 188:8551-8559 (2006); Hugler et al., 184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula (Alber et al., 188:8551-8559 (2006); Berg et al., 318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., 188:8551-8559 (2006)). This enzyme has also been indicated to catalyze the conversion of 166 methylmalonyl-CoA to its corresponding aldehyde (WO/2007/141208). Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional genes can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius. Gene GenBank Accession No. GI No. Organism MSED_0709 YP_001190808.1 146303492 Metallosphaera sedula mcr NP_378167.1 15922498 Sulfolobus tokodaii asd-2 NP_343563.1 15898958 Sulfolobus solfataricus Saci_2370 YP_256941.1 70608071 Sulfolobus acidocaldarius 1.2.1 .d Oxidoreductase (phosphorylating/dephosphorylating) (10/11 G): The reductions of (benzoyloxy)phosphonate to benzaldehyde (Path G of Figure 10) and (p methylbenzoyloxy) phosphonate to p-methylbenzaldehyde (Path G of Figure 11) are catalyzed by enzymes with phosphonate reductase activities. Although enzymes catalyzing these conversions have not been identified to date, similar transformations catalyzed by glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), aspartate-semialdehyde dehydrogenase (EC 1.2.1.11) acetylglutamylphosphate reductase (EC 1.2.1.38) and glutamate-5-semialdehyde dehydrogenase (EC 1.2.1.) are well documented. Aspartate semialdehyde dehydrogenase (ASD, EC 1.2.1.11) catalyzes the NADPH-dependent reduction of 4-aspartyl phosphate to aspartate-4-semialdehyde. ASD participates in amino acid biosynthesis and recently has been studied as an antimicrobial target (Hadfield et al., Biochemistry 40:14475-14483 (2001)). The E. coli ASD structure has been solved (Hadfield et al., JMol.Biol. 289:991-1002 (1999)) and the enzyme has been shown to accept the alternate substrate beta-3-methylaspartyl phosphate (Shames et al., JBiol.Chem. 259:1533 1 15339 (1984)). The Haemophilus influenzae enzyme has been the subject of enzyme engineering studies to alter substrate binding affinities at the active site (Blanco et al., Acta Crystallogr.D.Biol.Crystallogr. 60:1388-1395 (2004)). Other ASD genes/enzymes are found in Mycobacterium tuberculosis (Shafiani et al., JAppl Microbiol 98:832-83 8 (2005)), Methanococcusjannaschii (Faehnle et al., JMol.Biol. 353:1055-1068 (2005)), and the infectious microorganisms Vibrio cholera and Helicobacterpylori (Moore et al., Protein Expr.Purif 25:189-194 (2002)). A related enzyme is acetylglutamylphosphate reductase (EC 167 1.2.1.38), an enzyme that naturally reduces acetylglutamylphosphate to acetylglutamate-5 semialdehyde, found in S. cerevisiae (Pauwels et al., Eur.JBiochem. 270:1014-1024 (2003)), B. subtilis (O'Reilly et al., Microbiology 140 ( Pt 5):1023-1025 (1994)), E. coli (Parsot et al., Gene. 68:275-283 (1988)), and other organisms. Additional phosphate reductase enzymes of E. coli include glyceraldehyde 3-phosphate dehydrogenase encoded by gapA (Branlant et al., Eur.JBiochem. 150:61-66 (1985)) and glutamate-5-semialdehyde dehydrogenase encoded by proA (Smith et al., JBacteriol. 157:545-551 (1984b)). Genes encoding glutamate-5 semialdehyde dehydrogenase enzymes from Salmonella typhimurium (Mahan et al., J Bacteriol. 156:1249-1262 (1983)) and Campylobacterjejuni (Louie et al., Mol. Gen. Genet. 240:29-35 (1993)) were cloned and expressed in E. coli. Protein GenBank ID GI Number Organism asd NP_417891.1 16131307 Escherichia coli asd YP_248335.1 68249223 Haemophilus influenzae asd AAB49996 1899206 Mycobacterium tuberculosis VC2036 NP_231670 15642038 Vibrio cholera asd YP_002301787.1 210135348 Helicobacterpylori ARG5,6 NP_010992.1 6320913 Saccharomyces cerevisiae argC NP_389001.1 16078184 Bacillus subtilis argC NP_418393.1 16131796 Escherichia coli gapA POA9B2.2 71159358 Escherichia coli proA NP_414778.1 16128229 Escherichia coli proA NP_459319.1 16763704 Salmonella typhimurium proA P53000.2 9087222 Campylobacterjejuni 1.2.1 .e Oxidoreductase (acid to aldehyde): Direct conversion of benzoate to benzaldehyde or p-toluate to p-methylbenzaldehyde (Path B of Figures 10 and 11) is catalyzed by a carboxylic acid reductase. Exemplary enzymes include carboxylic acid reductase, alpha-aminoadipate reductase and retinoic acid reductase. Carboxylic acid reductase (CAR) catalyzes the magnesium, ATP and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes (Venkitasubramanian et al., JBiol.Chem. 282:478-485 (2007)). The natural substrate of this enzyme is benzoate and the enzyme 168 exhibits broad acceptance of aromatic substrates including p-toluate (Venkitasubramanian et al., Biocatalysis in Pharmaceutical and Biotechnology Industries. CRC press (2006)). The enzyme from Nocardia iowensis, encoded by car, was cloned and functionally expressed in E. coli (Venkitasubramanian et al., JBiol.Chem. 282:478-485 (2007)). CAR requires post translational activation by a phosphopantetheine transferase (PPTase) that converts the inactive apo-enzyme to the active holo-enzyme (Hansen et al., Appl.Environ.Microbiol 75:2765-2774 (2009)). Expression of the npt gene, encoding a specific PPTase, improved activity of the enzyme. A similar enzyme found in Streptomyces griseus is encoded by the griC and griD genes. This enzyme has been indicated to convert 3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzaldehyde, as deletion of either griC or griD led to accumulation of extracellular 3-acetylamino-4-hydroxybenzoic acid, a shunt product of 3 amino-4-hydroxybenzoic acid metabolism (Suzuki, et al., J Antibiot. 60(6):380-387 (2007)). The S. griseus PPTase is likely encoded by SGR_665, as predicted by sequence homology to the Nocardia iowensis npt gene. Gene GenBank Accession No. GI No. Organism car AAR91681.1 40796035 Nocardia iowensis npt AB183656.1 114848891 Nocardia iowensis griC YP_001825755.1 182438036 Streptomyces griseus griD YP_001825756.1 182438037 Streptomyces griseus SGR_665 YP_001822177.1 182434458 Streptomyces griseus An enzyme with similar characteristics, alpha-aminoadipate reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis pathways in some fungal species. This enzyme naturally reduces alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group is first activated through the ATP-dependent formation of an adenylate that is then reduced by NAD(P)H to yield the aldehyde and AMP. Like CAR, this enzyme utilizes magnesium and is activated by a PPTase. Enzymes for AAR and its corresponding PPTase are found in Saccharomyces cerevisiae (Morris et al., Gene 98:141-145 (1991)), Candida albicans (Guo et al., Mol.Genet.Genomics 269:271-279 (2003)), and Schizosaccharomyces pombe (Ford et al., Curr.Genet. 28:131-137 (1995)). The AAR from S. pombe exhibited significant activity when expressed in E. coli (Guo et al., Yeast 21:1279-1288 (2004)). The AAR from Penicillium chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate substrate, but did not react with adipate, L-glutamate or diaminopimelate (Hijarrubia et al., J 169 Biol.Chem. 278:8250-8256 (2003)). The gene encoding the P. chrysogenum PPTase has not been identified to date and no high-confidence hits were identified by sequence comparison homology searching. Gene GenBank Accession No. GI No. Organism LYS2 AAA34747.1 171867 Saccharomyces cerevisiae L YS5 P50113.1 1708896 Saccharomyces cerevisiae LYS2 AAC02241.1 2853226 Candida albicans LYS5 AA026020.1 28136195 Candida albicans Lysip P40976.3 13124791 Schizosaccharomyces pombe Lys7p Q10474.1 1723561 Schizosaccharomyces pombe Lys2 CAA74300.1 3282044 Penicillium chrysogenum 2.3.1 .a Acyltransferase (phosphotransacylase): An enzyme with phosphotransbenzoylase activity is used to interconvert benzoyl-CoA and (benzoyloxy)phosphonate (Path F of Figure 10). A similar enzyme with phosphotrans-p methylbenzoylase activity interconverts p-methylbenzoyl-CoA and (p-methylbenzoyloxy) phosphonate (Path F of Figure 11). Exemplary phosphate-transferring acyltransferases include phosphotransacetylase (EC 2.3.1.8) and phosphotransbutyrylase (EC 2.3.1.19). The pta gene from E. coli encodes a phosphotransacetylase that reversibly converts acetyl-CoA into acetyl-phosphate (Suzuki, Biochim.Biophys.Acta 191:559-569 (1969)). This enzyme can also convert propionyl-CoA propionylphosphate (Hesslinger et al., Mol.Microbiol 27:477 492 (1998)). Other phosphate acetyltransferases that exhibit activity on propionyl-CoA are found in Bacillus subtilis (Rado et al., Biochim.Biophys.Acta 321:114-125 (1973)), Clostridium kluyveri (Stadtman, 1:596-599 (1955)), and Thermotoga maritima (Bock et al., J Bacteriol. 181:1861-1867 (1999)). Similarly, the ptb gene from C. acetobutylicum encodes phosphotransbutyrylase, an enzyme that reversibly converts butyryl-CoA into butyryl phosphate (Wiesenborn et al., Appl Environ.Microbiol 55:317-322 (1989); Walter et al., Gene 134:107-111 (1993)). Additional ptb genes are found in butyrate-producing bacterium L2-50 (Louis et al., JBacteriol. 186:2099-2106 (2004)) and Bacillus megaterium (Vazquez et al., Curr.Microbiol 42:345-349 (2001)).

170 Gene GenBank Accession No. GI No. Organism pta NP_416800.1 71152910 Escherichia coli pta P39646 730415 Bacillus subtilis pta A5N801 146346896 Clostridium kluyveri pta Q9XOL4 6685776 Thermotoga maritima ptb NP_349676 34540484 Clostridium acetobutylicum ptb AAR19757.1 38425288 butyrate-producing bacterium L2 50 ptb CAC07932.1 10046659 Bacillus megaterium 2.7.2.a Phosphotransferase, carboxyl group acceptor (kinase): Kinase or phosphotransferase enzymes transform carboxylic acids to phosphonic acids with concurrent hydrolysis of one ATP. Such an enzyme is used to convert benzoate to (benzoyloxy)phosphonate (Figure 10, Path H) and p-toluate to (p methylbenzoyloxy)phosphonate (Figure 11, Path H). These exact transformations have not been demonstrated to date. Exemplary enzymes include butyrate kinase (EC 2.7.2.7), isobutyrate kinase (EC 2.7.2.14), aspartokinase (EC 2.7.2.4), acetate kinase (EC 2.7.2.1) and gamma-glutamyl kinase (EC 2.7.2.11). Butyrate kinase catalyzes the reversible conversion of butyryl-phosphate to butyrate during acidogenesis in Clostridial species (Cary et al., Appl.Environ.Microbiol 56:1576-1583 (1990)). The Clostridium acetobutylicum enzyme is encoded by either of the two buk gene products (Huang et al., JMol.Microbiol Biotechnol 2:33-38 (2000)). Other butyrate kinase enzymes are found in C. butyricum and C. tetanomorphum (TWAROG et al., JBacteriol. 86:112-117 (1963)). A related enzyme, isobutyrate kinase from Thermotoga maritima, was expressed in E. coli and crystallized (Diao et al., JBacteriol. 191:2521-2529 (2009); Diao et al., Acta Crystallogr.D.Biol.Crystallogr. 59:1100-1102 (2003)). Aspartokinase catalyzes the ATP dependent phosphorylation of aspartate and participates in the synthesis of several amino acids. The aspartokinase III enzyme in E. coli, encoded by lysC, has a broad substrate range and the catalytic residues involved in substrate specificity have been elucidated (Keng et al., Arch.Biochem.Biophys. 335:73-81 (1996)). Two additional kinases in E. coli are acetate kinase and gamma-glutamyl kinase. The E. coli acetate kinase, encoded by ackA (Skarstedt et al., JBiol.Chem. 251:6775-6783 (1976)), phosphorylates propionate in addition to acetate (Hesslinger et al., Mol.Microbiol 27:477-492 (1998)). The E. coli gamma-glutamyl kinase, 171 encoded by proB (Smith et al., JBacteriol. 157:545-551 (1984a)), phosphorylates the gamma carbonic acid group of glutamate. Gene GenBank ID GI Number Organism buki NP_349675 15896326 Clostridium acetobutylicum buk2 Q97111 20137415 Clostridium acetobutylicum buk2 Q9X278.1 6685256 Thermotoga maritima lysC NP_418448.1 16131850 Escherichia coli ackA NP_416799.1 16130231 Escherichia coli proB NP_414777.1 16128228 Escherichia coli 2.8.3.a CoA transferase (10/11 A): CoA transferases catalyze the reversible transfer of a CoA moiety from one molecule to another. Path A of Figure 10 is catalyzed by an enzyme with benzoyl-CoA transferase activity. In this transformation, benzoyl-CoA is formed from benzoate by the transfer of the CoA group from a CoA donor such as acetyl-CoA, succinyl CoA or others. p-Methylbenzoyl-CoA transferase catalyzes a similar reaction from p-toluate in Path A of Figure 11. Exemplary CoA transferase enzymes that react with similar substrates include cinnamoyl-CoA transferase (EC 2.8.3.17) and benzylsuccinyl-CoA transferase. Cinnamoyl-CoA transferase, encoded byfldA in Clostridium sporogenes, transfers a CoA moiety from cinnamoyl-CoA to a variety of aromatic acid substrates including phenylacetate, 3-phenylpropionate and 4-phenylbutyrate (Dickert et al., Eur.J Biochem. 267:3874-3884 (2000)). Benzylsuccinyl-CoA transferase utilizes succinyl-CoA or maleyl-CoA as the CoA donor, forming benzylsuccinyl-CoA from benzylsuccinate. This enzyme was characterized in the denitrifying bacteria Thauera aromatica, where it is encoded by bbsEF (Leutwein et al., JBacteriol. 183:4288-4295 (2001)). Gene GenBank Accession No. GI No. Organism fldA AAL18808.1 16417587 Clostridium sporogenes bbsE AAF89840.1 9622535 Thauera aromatica bbsF AAF89841.1 9622536 Thauera aromatica Additional CoA transferase enzymes with diverse substrate ranges include succinyl CoA transferase, 4-hydroxybutyryl-CoA transferase, butyryl-CoA transferase, glutaconyl CoA transferase and acetoacetyl-CoA transferase. The gene products of cat], cat2, and cat3 172 of Clostridium k/uyveri have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively (Seedorf et al., Proc.Nat.Acad.Sci U.S.A 105:2128-2133 (2008); Sohling et al., JBacteriol. 178:871-880 (1996b)). Similar CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., JBiol.Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., JBiol.Chem. 279:45337-45346 (2004)). The glutaconyl-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium Acidaminococcusfermentans reacts with glutaconyl-CoA and 3 butenoyl-CoA (Mack et al., Eur.JBiochem. 226:41-51 (1994)). The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA, crotonyl-CoA and acrylyl-CoA (Buckel et al., Eur.JBiochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mack et al., Eur.JBiochem. 226:41-51 (1994)). Glutaconate CoA-transferase activity has also been detected in Clostridium sporosphaeroides and Clostridium symbiosum. Acetoacetyl-CoA transferase utilizes acetyl-CoA as the CoA donor. This enzyme is encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Korolev et al., Acta Crystallogr.D.Biol.Crystallogr. 58:2116-2121 (2002); Vanderwinkel et al., Biochem.Biophys.Res.Commun. 33:902-908 (1968)). This enzyme has a broad substrate range (Sramek et al., Arch.Biochem.Biophys. 171:14-26 (1975)) and has been shown to transfer the CoA moiety from acetyl-CoA to a variety of substrates, including isobutyrate (Matthies et al., App/ Environ.Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al., Biochem.Biophys.Res.Commun. 33:902-908 (1968)) and butanoate (Vanderwinkel et al., Biochem.Biophys.Res.Commun. 33:902-908 (1968)). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., Appl.Environ.Microbiol 68:5186 5190 (2002)), Clostridium acetobutylicum (Cary et al., Appl.Environ.Microbiol 56:1576-1583 (1990); Wiesenborn et al., Appl.Environ.Microbiol 55:323-329 (1989)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71:5 8-68 (2007)). GenBank Accession Gene GI No. Organism No. cat] P38946.1 729048 Clostridium kluyveri cat2 P38942.2 172046066 Clostridium kluyveri cat3 EDK35586.1 146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034 Trichomonas vaginalis G3 Tbl].02.0290 XP_828352 71754875 Trypanosoma brucei 173 gctA CAA57199.1 559392 Acidaminococcusfermentans gctB CAA57200.1 559393 Acidaminococcusfermentans gctA ACJ24333.1 212292816 Clostridium symbiosum gctB ACJ24326.1 212292808 Clostridium symbiosum atoA P76459.1 2492994 Escherichia coli K12 atoD P76458.1 2492990 Escherichia coli K12 actA YP_226809.1 62391407 Corynebacterium glutamicum cg0592 YP_224801.1 62389399 Corynebacterium glutamicum ctfA NP_149326.1 15004866 Clostridium acetobutylicum ctfB NP_149327.1 15004867 Clostridium acetobutylicum ctfA AAP42564.1 31075384 Clostridium saccharoperbutylacetonicum ctfB AAP42565.1 31075385 Clostridium saccharoperbutylacetonicum 3.1.2.a CoA hydrolase (10/11 A): Benzoyl-CoA and p-methylbenzoyl-CoA can be hydrolyzed to their corresponding acids by CoA hydrolases or thioesterases in the EC class 3.1.2 (Path A of Figures 10 and 11). Exemplary CoA thioesters that hydrolyze benzoyl-CoA and/or similar substrates include 4-hydroxybenzoyl-CoA hydrolase (EC 3.1.2.23) and phenylglyoxal-CoA hydrolase (EC 3.1.2.25). The Azoarcus evansii gene orf1 encodes an enzyme with benzoyl-CoA hydrolase activity that participates in benzoate metabolism (Ismail, Arch.Microbiol 190:451-460 (2008)). This enzyme, when heterologously expressed in E. coli, demonstrated activity on a number of alternate substrates. Additional benzoyl CoA hydrolase enzymes were identified in benzonate degradation gene clusters of Magnetospirillum magnetotacticum, Jannaschia sp. CC S 1 and Sagittula stellata E-37 by sequence similarity (Ismail, Arch.Microbiol 190:451-460 (2008)). The 4-hydroxybenzoyl CoA hydrolase of Pseudomonas sp. CBS3 accepts benzoyl-CoA and p-methylbenzoyl-CoA as substrates and has been heterologously expressed and characterized in E. coli (Song et al., Bioorg.Chem. 35:1-10 (2007)). Additional enzymes with demonstrated benzoyl-CoA hydrolase activity include the palmitoyl-CoA hydrolase of Mycobacterium tuberculosis (Wang et al., Chem.Biol. 14:543-551 (2007)) and the acyl-CoA hydrolase of E. coli encoded by entH (Guo et al., Biochemistry 48:1712-1722 (2009)).

174 GenBank Accession Gene GI No. Organism No. orf1 AAN39365.1 23664428 Azoarcus evansii MagnO3011230 ZP_00207794 46200680 Magnetospirillum magnetotacticum Jann_0674 YP_508616 89053165 Jannaschia sp. CCS1 SSE37_24444 ZP_01745221 126729407 Sagittula stellata EF569604.1:4745..5170 ABQ44580.1 146761194 Pseudomonas sp. CBS3 Rv0098 NP_214612.1 15607240 Mycobacterium tuberculosis entH AAC73698.1 1786813 Escherichia coli Several CoA hydrolases with broad substrate ranges are suitable enzymes for hydrolyzing benzoyl-CoA and/or p-methylbenzoyl-CoA. For example, the enzyme encoded by acotl2 from Rattus norvegicus brain (Robinson et al., Biochem.Biophys.Res.Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. The human dicarboxylic acid thioesterase, encoded by acot8, exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., JBiol.Chem. 280:38125-38132 (2005)). The closest E. coli homolog to this enzyme, tesB, can also hydrolyze a range of CoA thiolesters (Naggert et al., JBiol Chem 266:11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana R., Biochem Int 26:767-773 (1992)). Gene name GI# GenBank Accession # Organism acot12 18543355 NP_570103.1 Rattus norvegicus tesB 16128437 NP_414986 Escherichia coli acot8 3191970 CAA15502 Homo sapiens acot8 51036669 NP_570112 Rattus norvegicus tesA 16128478 NP_415027 Escherichia coli ybgC 16128711 NP_415264 Escherichia coli paaI 16129357 NP_415914 Escherichia coli ybdB 16128580 NP_415129 Escherichia coli 4.1.la. Carboxy-lyase (6/7 E): Decarboxylase enzymes in the EC class 4.1.1 are used to convert benzoate to benzene (Path E of Figure 10) and p-toluate to toluene (Path E of 175 Figure 11). Exemplary enzymes that react with these or similar substrates include benzene carboxylase, vanillate decarboxylase, cinnamate decarboxylase, aminobenzoate decarboxylase and a variety of hydroxybenzoate decarboxylases. Decarboxylase enzymes can be oxidative or nonoxidative, depending on the cofactors utilized (Lupa et al., Genomics 86:342-351 (2005a)). An enzyme predicted to have benzoate carboxylase activity was identified in a Clostridia bacterium enrichment culture clone BF (Abu et al., Environ.Microbiol (2010)). GenBank Accession Gene No. GI No. Organism Clostridia bacterium abcA ADJ94002.1 300245889 enrichment culture clone BF Clostridia bacterium abcD ADJ94001.1 300245887 enrichment culture clone BF A number of characterized decarboxylases with demonstrated activity on hydroxylated aromatics such as 4-hydroxybenzoate, 2,3-dihydroxybenzoate, 3,4 dihydroxybenzoate, 2,6-dihydroxybenzoate and 4,5-dihydroxyphthalate can also exhibit activity on alternate substrates such as p-toluate or benzoate. Exemplary hydroxybenzoate decarboxylase enzymes include the 4,5-dihydroxyphthalate decarboxylase of Comamonas testosteroni (Nakazawa et al., Appl.Environ.Microbiol 36:264-269 (1978)), the 2,3 dihydroxybenzoate decarboxylase of Aspergillus niger (Kamath et al., Biochem.Biophys.Res.Commun. 145:586-595 (1987)) and the 3-octaprenyl-4 hydroxybenzoate decarboxylase of E. coli (Zhang et al., JBacteriol. 182:6243-6246 (2000)). Exemplary 4-hydroxybenzoate decarboxylases are encoded by shdBD and ubiD of Sedimentibacter hydroxybenzoicus (formerly Clostridium hydroxybenzoicum) and ubiD of Enterobacter cloacae P240 (Matsui et al., Arch.Microbiol 186:21-29 (2006a); He et al., Eur.J Biochem. 229:77-82 (1995)). The 4-hydroxybenzoate decarboxylase from the facultative anaerobe, Enterobacter cloacae, encoded by ubiD, has been tested for activity on multiple substrates and was shown to be induced by both 4-hydroxybenzoic acid and 4-aminobenzoic acid (Matsui et al., Arch.Microbiol 186:21-29 (2006b)). The bsdBCD genes of Bacillus subtilis encode a reversible non-oxidative 4-hydroxybenzoate/vanillate decarboxylase (Lupa et al., Can.JMicrobiol 54:75-81 (2008)). This enzyme was heterologously expressed in E.

176 coli. Similar decarboxylases have been indicated in several other organisms (Lupa et al., Genomics 86:342-351 (2005b)) and genes for some of these are listed below. GenBank Accession Gene GI No. Organism No. phtD Q59727.1 3914354 Comamonas testosteroni dhbD CAK48106.1 134075758 Aspergillus niger ubiD NP_418285.1 16131689 Escherichia coli Sedimentibacter shdD AAY6785 1.1 67462198 hydroxybenzoicus Sedimentibacter shdB AAY67850.1 67462197 hydroxybenzoicus Sedimentibacter ubiD AAD50377.1 5739200 hydroxybenzoicus ubiD BAE97712.1 110331749 Enterobacter cloacae P240 bsdB CAB12157.1 2632649 Bacillus subtilis bsdC CAB12158.1 2632650 Bacillus subtilis bsdD CAB12159.1 2632651 Bacillus subtilis STM292 NP_461842.1 16766227 Salmonella typhimurium LT2 STM2922 NP_461843.1 16766228 Salmonella typhimurium LT2 STM2923 NP_461844.1 16766229 Salmonella typhimurium LT2 kpdB YP_002236894.1 206580833 Klebsiella pneumoniae 342 kpdC YP_002236895.1 206576360 Klebsiella pneumoniae 342 kpdD YP_002236896.1 206579343 Klebsiella pneumoniae 342 pad] NP_311620.1 15832847 Escherichia coli 0157 yclC NP_311619.1 15832846 Escherichia coli 0157 yclD NP_311618.1 15832845 Escherichia coli 0157 An additional class of decarboxylases has been characterized that catalyze the decarboxylation of cinnamate (phenylacrylate) and substituted cinnamate derivatives. These enzymes are common in a variety of organisms and specific genes encoding these enzymes 177 that have been cloned and expressed in E. coli include pad 1 from Saccharomyces cerevisae (Clausen et al., Gene 142:107-112 (1994)), pdc from Lactobacillus plantarum (Barthelmebs et al., 67:1063-1069 (2001); Qi et al., Metab Eng 9:268-276 (2007); Rodriguez et al., JAgric.Food Chem. 56:3068-3072 (2008)), pofK (pad) from Klebsiella oxytoca (Uchiyama et al., Biosci.Biotechnol.Biochem. 72:116-123 (2008); Hashidoko et al., Biosci.Biotech.Biochem. 58:217-218 (1994)), Pedicoccus pentosaceus (Barthelmebs et al., 67:1063-1069 (2001)), and padC from Bacillus subtilis and Bacillus pumilus (Shingler et al., 174:711-724 (1992)). A ferulic acid decarboxylase from Pseudomonasfluorescens also has been purified and characterized (Huang et al., JBacteriol. 176:5912-5918 (1994)). Enzymes in this class have been shown to be stable and do not require either exogenous or internally bound co-factors, thus making these enzymes suitable for biotransformations (Sariaslani, Annu.Rev.Microbiol. 61:51-69 (2007)). Protein GenBank ID GI Number Organism pad] AAB64980.1 1165293 Saccharomyces cerevisae pdc AAC45282.1 1762616 Lactobacillus plantarum pad BAF65031.1 149941608 Klebsiella oxytoca padC NP_391320.1 16080493 Bacillus subtilis pad YP_804027.1 116492292 Pedicoccus pentosaceus pad CAC18719.1 11691810 Bacillus pumilus 4.1.99.a Decarbonylase: A decarbonylase enzyme is used to convert benzaldehyde to benzene (Path C of Figure 10) and p-methylbenzaldehyde to toluene (Path C of Figure 11). Decarbonylase enzymes catalyze the final step of alkane biosynthesis in plants, mammals, insects and bacteria (Dennis et al., Arch.Biochem.Biophys. 287:268-275 (1991)). Non oxidative decarbonylases transform aldehydes into alkanes with the concurrent release of CO. Exemplary decarbonylase enzymes include octadecanal decarbonylase (EC 4.1.99.5), sterol desaturase and fatty aldehyde decarbonylase. The CERI gene of Arabidopsis thaliana encodes a fatty acid decarbonylase involved in epicuticular wax formation (US 6,437,218). Additional fatty acid decarbonylases are found in Medicago truncatula, Vitis vinfera and Oryza sativa (US Patent Application 2009/0061493). A cobalt-porphyrin containing decarbonylase was purified and characterized in the algae Botryococcus braunii; however, no gene has been associated with this activity to date (Dennis et al., Proc.Natl.Acad.Sci. U.S.A 178 89:5306-5310 (1992)). A copper-containing decarbonylase from Pisum sativum was also purified and characterized, but is not yet associated with a gene (Schneider-Belhaddad et al., Arch.Biochem.Biophys. 377:341-349 (2000)). Gene GenBank Accession No. GI No. Organism CER] NP_850932 145361948 Arabidopsis thaliana MtrDRAFT AC53128g2v2 ABN07985 124359969 Medicago truncatula VITISV_029045 CAN60676 147781102 Vitis vinfera OSJNBa0004N05.14 CAE03390.2 38345317 Oryza sativa Alternately, an oxidative decarbonylase can convert an aldehyde into an alkane. Oxidative decarbonylases are cytochrome P450 enzymes that utilize NADPH and 02 as cofactors and release C0 2 , water and NADP. This activity was demonstrated in the CYP4G2vJ and CYP4Glgene products of Musca domestica and Drosophila melanogaster (US Patent Application 2010/0136595). Additional enzymes with oxidative decarbonylase activity can be identified by sequence homology in other organisms such as Mamestra brassicae, Helicoverpa zea and Acyrthosiphon pisum. Protein GenBank ID GI Number Organism CYP4G2v1 ABV48808.1 157382740 Musca domestica CYP4G1 NP_525031.1 17933498 Drosophila melanogaster CYP4G25 BAD81026.1 56710314 Antheraea yamamai CYP4M6 AAM54722.1 21552585 Helicoverpa zea LOC100164072 XP_001944205.1 193650239 Acyrthosiphon pisum 6.2.1 .a Acid-thiol ligase (8A, 1 IB): The ATP-dependent activation of benzoate to benzoyl-CoA or p-toluate to p-methylbenzoyl-CoA (Path A of Figures 10 and 11) is catalyzed by a CoA synthetase or acid-thiol ligase. AMP-forming CoA ligases activate the aromatic acids to their corresponding CoA derivatives, whereas ADP-forming CoA ligases are generally reversible. Exemplary AMP-forming benzoyl-CoA ligases from Thauera aromatica and Azoarcus sp. strain CIB have been characterized (Lopez Barragan et al., J Bacteriol. 186:5762-5774 (2004); Schuhle et al., JBacteriol. 185:4920-4929 (2003)). Alternately, AMP-forming CoA ligases that react with structurally similar substrates can have activity on benzoate orp-toluate. The AMP-forming cyclohexanecarboxylate CoA- 179 ligase from Rhodopseudomonas palustris, encoded by aliA, is well-characterized, and alteration of the active site has been shown to impact the substrate specificity of the enzyme (Samanta et al., Mol.Microbiol 55:1151-1159 (2005)). This enzyme also functions as a cyclohex-1-ene-1-carboxylate CoA-ligase during anaerobic benzene ring degradation (Egland et al., Proc.Natl.Acad.Sci U.S.A 94:6484-6489 (1997)). Additional exemplary CoA ligases include two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al., Biochem.J395:147-155 (2006); Wang et al., Biochem.Biophys.Res.Commun. 360:453 458 (2007)); Wang et al., Biochem. Biophys. Res. Commun. 360:453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco et al., JBiol.Chem. 265:7084-7090 (1990)), and the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower et al., JBacteriol. 178:4122-4130 (1996)). GenBank Accession Gene No. GI No. Organism bc/A Q8GQN9.1 75526585 Thauera aromatica bzdA AAQ08820.1 45649073 Azoarcus sp. strain CIB aliA AAC23919 2190573 Rhodopseudomonas palustris phl CAJ15517.1 77019264 Penicillium chrysogenum ph/B ABS 19624.1 152002983 Penicillium chrysogenum paaF AAC24333.2 22711873 Pseudomonas putida bioW NP_390902.2 50812281 Bacillus subtilis ADP-forming CoA ligases catalyzing these exact transformations have not been characterized to date; however, several enzymes with broad substrate specificities have been described in the literature. The ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) from Archaeoglobusfulgidus, encoded by AF 1211, was shown to operate on a variety of linear and branched-chain substrates including isobutyrate, isopentanoate, and fumarate (Musfeldt et al., JBacteriol. 184:636-644 (2002)). A second reversible ACD in Archaeoglobusfulgidus, encoded by AF1983, was also indicated to have a broad substrate range with high activity on aromatic compounds phenylacetate and indoleacetate (Musfeldt et al., supra). The enzyme from Haloarcula marismortui, annotated as a succinyl-CoA synthetase, accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch.Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from 180 hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen and Schonheit, Arch.Microbiol 182:277-287 (2004)). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A. fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Brasen and Schonheit, Arch.Microbiol 182:277-287 (2004); Musfeldt and Schonheit, JBacteriol. 184:636-644 (2002)). An additional enzyme is encoded by sucCD in E. coli, which naturally catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). The acyl CoA ligase from Pseudomonasputida has been indicated to work on several aliphatic substrates including acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds such as phenylacetic and phenoxyacetic acids (Fernandez-Valverde et al., Appl.Environ.Microbiol. 59:1149-1154 (1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) from Rhizobium leguminosarum could convert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-, cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into their corresponding monothioesters (Pohl et al., JAm.Chem.Soc. 123:5822-5823 (2001)). GenBank Accession Gene GI No. Organism No. AF128 NP_070039.1 11498810 Archaeoglobusfulgidus AF1983 NP_070807.1 11499565 Archaeoglobusfulgidus scs YP_135572.1 55377722 Haloarcula marismortui PAE3250 NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2 sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia coli paaF AAC24333.2 22711873 Pseudomonas putida matB AAC83455.1 3982573 Rhizobium leguminosarum 181 EXAMPLE VIII Pathways to 2,4-pentadienoate from pyruvate, ornithine and alanine This example shows pathways from pyruvate, ornithine and alanine to 2,4 pentadienoate. Figure 12 shows the conversion of pyruvate to 2,4-pentadienoate. This conversion can be accomplished in four enzymatic steps. Pyruvate and acetaldehyde are first condensed to 4-hydroxy-2-oxovalerate by 4-hydroxy-2-ketovalerate aldolase (Step A of Figure 12). The 4 hydroxy-2-oxovalerate product is next dehydrated to 2-oxopentenoate (Step B of Figure 12). Subsequent reduction and dehydration of 2-oxopentenoate yields 2,4-pentadienoate (Steps C/D of Figure 12). Figure 13 shows pathways from alanine or ornithine to 2,4-pentadienoate. In Step A of Figure 13, alanine and acetyl-CoA are joined by AKP thiolase to form AKP. In one pathway, AKP is deaminated to acetylacrlate (Step B). The 4-oxo group of acetylacrylate is then reduced and dehydrated to 2,4-pentadienoate (Steps C/D). In an alternative pathway, AKP is converted to 2,4-dioxopentanoate by an aminotransferase or dehydrogenase (Step E). Reduction of the 2- or 4-oxo group of 2,4-dioxopentanoate yields 2-hydroxy-4-oxopentanoate (Step H) or 4-hydroxy-2-oxovalerate (Step K), respectively. 4-Hydroxy-2-oxovalerate can alternately be formed by the reduction of AKP to 2-amino-4-hydroxypentanoate (Step J) followed by transamination or oxidative deamination (Step L). Once formed, 4-hydroxy-2 oxovalerate can be converted to 2,4-pentadienoate in three enzymatic steps as shown in Figure 12 (Steps B/C/D of Figure 12). The 2-hydroxy-4-oxopentanoate intermediate can undergo dehydration to acetylacrylate (Step F) followed by reduction and dehydration (Steps C/D). An alternate entry point into the pathways from AKP shown in Figure 13 is ornithine. An ornithine aminomutase is first required to convert ornithine to 2,4-diaminopentanoate (Step M). The 2,4-diaminopentanoate intermediate is then converted to AKP by transamination or oxidative deamination (Step N). It is understood that either the D- or L- stereoisomer of alanine or ornithine can serve serve as the precursor or intermediate to a 2,4-pentadienoate pathway shown in Figure 13.

182 The D- and L- stereoisomers of alanine or omithine are readily interconverted by alanine racemase or ornithine racemase enzymes. Enzymes for catalyzing the transformations shown in Figures 5 and 6 are categorized by EC number (Table 2) and described further below. Label Function Step 1C, 1.1.1.a Oxidoreductase (oxo to alcohol) 2C/H/K/J 1.4.1.a Oxidoreductase (aminating/deaminating) 2E/L/N 2.6.1.a Aminotransferase 2E/L/N 4.1.3.a Lyase 1A 4.2.1.a Hydro-lyase 1B/D, 2D/F 4.3.1.a Ammonia-lyase 2B 5.1.1.a D/L Racemase 2A/M 5.4.3.a Aminomutase 2M Other AKP thioloase 2A 1.1.1 .a Oxidoreductase (oxo to alcohol): A number of transformations in Figures 5 and 6 involve the reduction of a ketone to an alcohol. In Step C of Figure 12, 2 oxopentenoate is reduced to 2-hyroxypentenoate. A similar transformation is the reduction of the keto-acid 2,4-dioxopentanoate to its corresponding hydroxy-acid, 2-hydroxy-4 oxopentanoate (Step H of Figure 13). Steps C, J and K of Figure 13 entail reduction of the 4 oxo groups of AKP, 2,4-dioxopentanoate and acetylacrylate to their corresponding alcohols. These transformations are catalyzed by oxidoreductase enzymes in the EC class 1.1.1. Several exemplary alcohol dehydrogenases convert a ketone to an alcohol functional group. Two such enzymes from E. coli are encoded by malate dehydrogenase (mdh) and lactate dehydrogenase (ldhA). In addition, lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high activities on 2-ketoacids of various chain lengths includings lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (Steinbuchel et al., Eur.JBiochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha hydroxyadipate is catalyzed by 2-ketoadipate reductase, an enzyme found in rat and in human placenta (Suda et al., Arch.Biochem.Biophys. 176:610-620 (1976); Suda et al., 183 Biochem.Biophys.Res.Commun. 77:586-591 (1977)). An additional candidate oxidoreductase is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the human heart which has been cloned and characterized (Marks et al., JBiol.Chem. 267:15459-15463 (1992)). Alcohol dehydrogenase enzymes of C. beijerinckii (Ismaiel et al., JBacteriol. 175:5097-5105 (1993)) and T. brockii (Lamed et al., Biochem.J 195:183-190 (1981); Peretz et al., Biochemistry. 28:6549-6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase catalyzes the reduction of MEK to 2-butanol. Exemplary MEK reductase enzymes can be found in Rhodococcus ruber (Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and Pyrococcusfuriosus (van der et al., Eur.JBiochem. 268:3062-3068 (2001)). Gene GenBank ID GI Number Organism mdh AAC76268.1 1789632 Escherichia coli ldhA NP_415898.1 16129341 Escherichia coli ldh YP_725182.1 113866693 Ralstonia eutropha bdh AAA58352.1 177198 Homo sapiens adh AAA23199.2 60592974 Clostridium beijerinckii NRRL B593 adh P14941.1 113443 Thermoanaerobacter brockii HTD4 sadh CAD36475 21615553 Rhodococcus ruber adhA AAC25556 3288810 Pyrococcusfuriosus 1.4.1 .a Oxidoreductase (deaminating): Enzymes in the EC class 1.4.1 catalyze the oxidative deamination of amino groups with NAD+, NADP+ or FAD as acceptor. Such an enzyme is required to catalyze the oxidative deamination of AKP to 2,4-dioxopentanoate (Figure 13, Step E), 2-amino-4-hydroxypentanoate to 4-hydroxy-2-oxovalerate (Figure 13, Step L) and 2,4-diaminopentanoate to AKP (Figure 13, Step N). The conversion of 2,4 diaminopentanoate to AKP (Step N of Figure 13) is catalyzed by 2,4-diaminopentanoate dehydrogenase (EC 1.4.1.12). 2,4-Diaminopentanoate dehydrogenase enzymes have been characterized in organisms that undergo anaerobic fermentation of ornithine, such as the ord gene product of Clostridium sticklandii (Fonknechten, JBacteriol. In Press: (2009)). Additional 2,4-diaminopentanoate dehydrogenase gene candidates can be inferred in other organisms by sequence similarity to the ord gene product. A related enzyme, 3,5- 184 diaminohexanoate dehydrogenase (EC 1.4.1.11), catalyzes the oxidative deamination of 3,5 diaminohexanoate to 5-amino-3-oxohexanoate. The gene encoding this enzyme, kdd, was recently identified in Fusobacterium nucleatum (Kreimeyer et al., JBiol.Chem. 282:7191 7197 (2007)). The enzyme has been purified and characterized in other organisms that ferment lysine but the genes associated with these enzymes have not been identified to date (Baker et al., JBiol.Chem. 247:7724-7734 (1972); Baker et al., Biochemistry 13:292-299 (1974)). Candidates in Myxococcus xanthus, Porphyromonas gingivalis W83 and other sequenced organisms can be inferred by sequence homology. Gene GenBank ID GI Number Organism ord CAQ42978.1 226885213 Clostridium sticklandii Hore_21120 YP_002509852.1 220932944 Halothermothrix orenii CD0442 YP_001086913.1 126698016 Clostridium difficile kdd AAL93966.1 19713113 Fusobacterium nucleatum mxan_4391 ABF87267.1 108462082 Myxococcus xanthus pg_1069 AAQ66183.1 34397119 Porphyromonas gingivalis The substrates AKP and 2-amino-4-hydroxypentanoate (Steps E and L of Figure 13), are similar to alpha-amino acids and may serve as alternate substrates for amino acid dehydrogenase enzymes such as glutamate dehydrogenase (EC 1.4.1.2), leucine dehydrogenase (EC 1.4.1.9), and aspartate dehydrogenase (EC 1.4.1.21). Glutamate dehydrogenase catalyzes the reversible NAD(P)+ dependent conversion of glutamate to 2 oxoglutarate. Exemplary enzymes are encoded by gdhA in Escherichia coli (McPherson et al., Nucleic.Acids Res. 11:5257-5266 (1983); Korber et al., JMol.Biol. 234:1270-1273 (1993)), gdh in Thermotoga maritima (Kort et al., Extremophiles 1:52-60 (1997); Lebbink et al., JMol.Biol. 280:287-296 (1998); Lebbink et al., JMol.Biol. 289:357-369 (1999)), and gdhA1 in Halobacterium salinarum (Ingoldsby et al., Gene. 349:237-244 (2005)). Additional glutamate dehydrogenase enzymes have been characterized in Bacillus subtilis (Khan et al., Biosci.Biotechnol Biochem. 69:1861-1870 (2005)), Nicotiana tabacum (Purnell et al., Planta 185 222:167-180 (2005)), Oryza sativa (Abiko et al., Plant Cell Physiol 46:1724-1734 (2005)), Haloferax mediterranei (Diaz et al., Extremophiles. 10:105-115 (2006)) and Halobactreium salinarum (Hayden et al., FEMS Microbiol Lett. 211:37-41 (2002)). The Nicotiana tabacum enzyme is composed of alpha and beta subunits encoded by gdh1 and gdh2 (Purnell et al., Planta 222:167-180 (2005)). An exemplary leucine dehydrogenase is encoded by ldh of Bacillus cereus. This enzyme reacts with a range of substrates including leucine, isoleucine, valine, and 2-aminobutanoate (Stoyan et al., JBiotechnol 54:77-80 (1997); Ansorge et al., Biotechnol Bioeng. 68:557-562 (2000)). The aspartate dehydrogenase from Thermotoga maritime, encoded by nadX, is involved in the biosynthesis of NAD (Yang et al., JBiol.Chem. 278:8804-8808 (2003)). GGene GenBank ID GI Number Organism gdhA P00370 118547 Escherichia coli gdh P96110.4 6226595 Thermotoga maritima gdhA1 NP_279651.1 15789827 Halobacterium salinarum rocG NP_391659.1 16080831 Bacillus subtilis gdhl AAR11534.1 38146335 Nicotiana tabacum gdh2 AAR11535.1 38146337 Nicotiana tabacum GDH Q852M0 75243660 Oryza sativa GDH Q977U6 74499858 Haloferax mediterranei GDH P29051 118549 Halobactreium salinarum GDH2 NP_010066.1 6319986 Saccharomyces cerevisiae ldh P0A393 61222614 Bacillus cereus nadX NP_229443.1 15644391 Thermotoga maritima 2.6.1 .a Aminotransferase: Several transformations in Figure 13 are catalyzed by aminotransferase or transaminase enzymes, including the conversion of AKP to 2,4 dioxopentanoate (Step E), 2-amino-4-hydroxypentanoate to 4-hydroxy-2-oxovalerate (Step L) and 2,4-diaminopentanoate to AKP (Step N). Several aminotransferases convert amino acids and derivatives to their corresponding 2-oxoacids. Such enzymes are particularly well suited to catalyze the transformations depicted in Steps E and L of Figure 13 (i.e. AKP 186 aminotransferase and 2-amino-4-hydroxypentanoate aminotransferase). Selection of an appropriate amino acid aminotransferase for these transformations may depend on the stereochemistry of the substrate. When the substrate is in the D-configuration, a D-amino acid aminotransferase (EC 2.6.1.21) can be utilized, whereas the L-stereoisomer is the preferred substrate of an L-amino acid aminotransferase such as aspartate aminotransferase (EC 2.6.1.1). Aspartate aminotransferase naturally transfers an oxo group from oxaloacetate to glutamate, forming alpha-ketoglutarate and aspartate. Aspartate aminotransferase activity is catalyzed by, for example, the gene products of aspC from Escherichia coli (Yagi et al., 100:81-84 (1979); Yagi et al., 113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi et al., 92:35-43 (1982)) and ASP5 from Arabidopsis thaliana (Kwok et al., 55:595 604 (2004); de la et al., 46:414-425 (2006); Wilkie et al., Protein Expr.Purif 12:381-389 (1998)). The enzyme from Rattus norvegicus has been shown to transaminate alternate substrates such as 2-aminohexanedioic acid and 2,4-diaminobutyric acid (Recasens et al., Biochemistry 19:4583-4589 (1980)). Aminotransferases that work on other L-amino-acid substrates may also be able to catalyze these transformation. Valine aminotransferase catalyzes the conversion of valine and pyruvate to 2-ketoisovalerate and alanine. The E. coli gene, avtA, encodes a similar enzyme (Whalen et al., JBacteriol. 150:739-746 (1982)), which also catalyzes the transamination of a-ketobutyrate to generate a-aminobutyrate, although the amino donor in this reaction has not been identified (Whalen et al., JBacteriol. 158:571-574 (1984)). Another enzyme candidate is alpha-aminoadipate aminotransferase (EC 2.6.1.39), an enzyme that participates in lysine biosynthesis and degradation in some organisms. This enzyme interconverts 2-aminoadipate and 2-oxoadipate, using alpha ketoglutarate as the amino acceptor. Gene candidates are found in Homo sapiens (Okuno et al., Enzyme Protein 47:136-148 (1993)) and Thermus thermophilus (Miyazaki et al., Microbiology 150:2327-2334 (2004)). The Thermus thermophilus enzyme, encoded by lysN, is active with several alternate substrates including oxaloacetate, 2-oxoisocaproate, 2 oxoisovalerate, and 2-oxo-3-methylvalerate. Gene GenBank ID GI Number Organism aspC NP_415448.1 16128895 Escherichia coli AA T2 P23542.3 1703040 Saccharomyces cerevisiae ASP5 P46248.2 20532373 Arabidopsis thaliana got2 P00507 112987 Rattus norvegicus 187 avtA YP_026231.1 49176374 Escherichia coli lysN BAC76939.1 31096548 Thermus thermophilus AadAT-II Q8N5Z0.2 46395904 Homo sapiens If the substrate is present in the D-stereoisomer, transamination can be catalyzed by D-aminotransferase (EC 2.6.1.21), also known as D-amino acid aminotransferase and D alanine aminotransferase (DAAT). This class of enzyme is noted for its broad substrate specificity, which is species-specific. The D-aminotransferase from Bacillus species YM-1, encoded by dat, has been cloned, sequenced (Tanizawa et al., JBiol.Chem. 264:2450-2454 (1989)) and the crystal structure has been solved (Peisach et al., Biochemistry 37:4958-4967 (1998)). This enzyme has also been engineered to alter the substrate specificity (Gutierrez et al., Eur.JBiochem. 267:7218-7223 (2000); Gutierrez et al., Protein Eng 11:53-58 (1998)). Additional gene candidates are found in Bacillus lichenformis ATCC 10716 (Taylor et al., Biochim.Biophys.Acta 1350:38-40 (1997)), Staphylococcus haemolyticus (Pucci et al., J Bacteriol. 177:336-342 (1995)) and Bacillus subtilis (Martinez-Carrion et al., JBiol.Chem. 240:3538-3546 (1965)). Gene GenBank ID GI Number Organism dat P19938 118222 Bacillus sp. YM-1 dat P54692 1706292 Bacillus lichenformis ATCC 10716 dat P54694 1706294 Staphylococcus haemolyticus dat 007597.1 3121979 Bacillus subtilis The conversion of 2,4-diaminopentanoate to AKP (Step N of Figure 13) is catalyzed by an enzyme with 2,4-diaminopentanoate aminotransferase activity. Although this activity is has not been characterized in enzymes to date, several enzymes catalyze a similar transformation, the conversion of 2,4-diaminobutanoate to aspartate-4-semialdehyde. Exemplary enzyme candidates include beta-alanine aminotransferase (EC 2.6.1.18), diaminobutyrate aminotransferase (EC 2.6.1.46 and EC 2.6.1.76) and gamma-aminobutyrate (GABA) aminotransferase (EC 2.6.1.19). An exemplary diaminobutyrate aminotransferase enzyme is encoded by the dat gene products in Acinetobacter baumanii and Haemophilus influenza (Ikai et al., JBacteriol. 179:5118-5125 (1997); Ikai et al., Biol Pharm.Bull. 21:170- 188 173 (1998)). In addition to its natural substrate, 2,4-diaminobutyrate, the A. baumanii DAT transaminates the terminal amines of lysine, 4-aminobutyrate and omithine. Additional diaminobutyrate aminotransferase gene candidates include the ectB gene products of Marinococcus halophilus and Halobacillus dabanensis (Zhao et al., Curr Microbiol 53:183 188 (2006); Louis et al., Microbiology 143 (Pt 4):1141-1149 (1997)) and the pvdH gene product of Pseudomonas aeruginosa (Vandenende et al., JBacteriol. 186:5596-5602 (2004)). Diaminobutyrate aminotransferase enzymes that utilize alpha-ketoglutarate as an amino acceptor are included in the EC class 2.6.1.76. Such enzymes are found in Acinetobacter baumanii, The beta-alanine aminotransferase of Pseudomonasfluorescens also accepts 2,4 diaminobutyrate as a substrate (Hayaishi et al., JBiol Chem 236:781-790 (1961)); however, this activity has not been associated with a gene to date. Gamma-aminobutyrate aminotransferase naturally interconverts succinic semialdehyde and glutamate to 4 aminobutyrate and alpha-ketoglutarate. Generally, GABA aminotransferases react with a broad range of alternate substrates (Schulz et al., 56:1-6 (1990); Liu et al., 43:10896-10905 (2004)). The two GABA transaminases in E. coli are encoded by gabT (Bartsch et al., J Bacteriol. 172:7035-7042 (1990)) and puuE (Kurihara et al., JBiol.Chem. 280:4602-4608 (2005)). The gabT gene product has been shown to have broad substrate specificity (Schulz et al., 56:1-6 (1990); Liu et al., 43:10896-10905 (2004)). GABA aminotransferases in Mus musculus and Sus scrofa have been shown to react with a range of alternate substrates (Cooper, Methods Enzymol. 113:80-82 (1985)). Gene GenBank ID GI Number Organism dat P56744.1 6685373 Acinetobacter baumanii dat P44951.1 1175339 Haemophilus influenzae ectB AAB57634.1 2098609 Marinococcus halophilus ectB AAZ57191.1 71979940 Halobacillus dabanensis pvdH AAG05801.1 9948457 Pseudomonas aeruginosa gabT NP_417148.1 16130576 Escherichia coli puuE NP_415818.1 16129263 Escherichia coli Abat NP_766549.2 37202121 Mus musculus gabT YP_257332.1 70733692 Pseudomonasfluorescens Abat NP_999428.1 47523600 Sus scrofa 189 4.1.3.a Lyase: The condensation of pyruvate and acetaldehyde to 4-hydroxy-2 oxovalerate (Step A of Figure 12) is catalyzed by 4-hydroxy-2-oxovalerate aldolase (EC 4.1.3.39). This enzyme participates in pathways for the degradation of phenols, cresols and catechols. The E. coli enzyme, encoded by mhpE, is highly specific for acetaldehyde as an acceptor but accepts the alternate substrates 2-ketobutyrate or phenylpyruvate as donors (Pollard et al., Appl Environ Microbiol 64:4093-4094 (1998)). Similar enzymes are encoded by the cmtG and todH genes of Pseudomonas putida (Lau et al., Gene 146:7-13 (1994); Eaton, JBacteriol. 178:1351-1362 (1996)). In Pseudomonas CF600, this enzyme is part of a bifunctional aldolase-dehydrogenase heterodimer encoded by dmpFG (Manjasetty et al., Acta Crystallogr.D.Biol Crystallogr. 57:582-585 (2001)). The dehydrogenase functionality interconverts acetaldehyde and acetyl-CoA, providing the advantage of reduced cellular concentrations of acetaldehyde, toxic to some cells. Gene GenBank ID GI Number Organism mhpE AAC73455.1 1786548 Escherichia coli cmtG AAB62295.1 1263190 Pseudomonasputida todH AAA61944.1 485740 Pseudomonas putida dmpG CAA43227.1 45684 Pseudomonas sp. CF600 dmpF CAA43226.1 45683 Pseudomonas sp. CF600 4.2.1 .a Dehydratase: Dehydration of 4-hydroxy-2-oxovalerate to 2-oxopentenoate (Step B of Figure 12) is catalyzed by 4-hydroxy-2-oxovalerate hydratase (EC 4.2.1.80). A similar enzyme is required to catalyze the dehydration of 4-hydroxypent-2-enoate to 2,4 pentadienoate (Step D of Figure 13). 4-Hydroxy-2-oxovalerate hydratase participates in aromatic degradation pathways and is typically co-transcribed with a gene encoding an enzyme with 4-hydroxy-2-oxovalerate aldolase activity. Exemplary gene products are encoded by mhpD of E. coli (Ferrandez et al., JBacteriol. 179:2573-2581 (1997); Pollard et al., Eur JBiochem. 251:98-106 (1998)), todG and cmtF of Pseudomonas putida (Lau et al., Gene 146:7-13 (1994); Eaton, JBacteriol. 178:1351-1362 (1996)), cnbE of Comamonas sp. CNB-1 (Ma et al., Appl Environ Microbiol 73:4477-4483 (2007)) and mhpD of Burkholderia xenovorans (Wang et al., FEBS J272:966-974 (2005)). A closely related enzyme, 2 oxohepta-4-ene-1,7-dioate hydratase, participates in 4-hydroxyphenylacetic acid degradation, 190 where it converts 2-oxo-hept-4-ene-1,7-dioate (OHED) to 2-oxo-4-hydroxy-hepta-1,7-dioate using magnesium as a cofactor (Burks et al., JAm.Chem.Soc. 120: (1998)). OHED hydratase enzyme candidates have been identified and characterized in E. coli C (Roper et al., Gene 156:47-51 (1995); Izumi et al., JMol.Biol. 370:899-911 (2007)) and E. coli W (Prieto et al., J Bacteriol. 178:111-120 (1996)). Sequence comparison reveals homologs in a wide range of bacteria, plants and animals. Enzymes with highly similar sequences are contained in Klebsiella pneumonia (910% identity, eval = 2e- 138) and Salmonella enterica (910% identity, eval = 4e-138), among others. Gene GenBank ID GI Number Organism mhpD AAC73453.2 87081722 Escherichia coli cmtF AAB62293.1 1263188 Pseudomonas putida todG AAA61942.1 485738 Pseudomonas putida cnbE YP_001967714.1 190572008 Comamonas sp. CNB-1 mhpD Q13VUO 123358582 Burkholderia xenovorans hpcG CAA57202.1 556840 Escherichia coli C hpaH CAA86044.1 757830 Escherichia coli W hpaH ABR80130.1 150958100 Klebsiella pneumoniae Sari_01896 ABX21779.1 160865156 Salmonella enterica Enzyme candidates for catalyzing the dehydration of 2-hydroxypentenoate (Figure 12, Step D) or 2-hydroxy-4-oxopentanoate (Figure 13, Step F) include fumarase (EC 4.2.1.2), citramalate hydratase (EC 4.2.1.34) and dimethylmaleate hydratase (EC 4.2.1.85). Fumarase enzymes naturally catalyze the reversible dehydration of malate to fumarate. Although the ability of fumarase to react with 2-hydroxypentenoate or 2-hydroxy-4-oxopentanoate as substrates has not been described in the literature, a wealth of structural information is available for this enzyme and other researchers have successfully engineered the enzyme to alter activity, inhibition and localization (Weaver, 61:1395-1401 (2005)). E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions. FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is the only active enzyme in aerobic growth (Tseng et al., 183:461-467 (2001); Woods et al., 954:14-26 (1988); Guest et al., J Gen Microbiol 131:2971-2984 (1985)). Additional enzyme candidates are found in Campylobacterjejuni 191 (Smith et al., Int.JBiochem.Cell Biol 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch.Biochem.Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al., 89:1923-1931 (1981)). Similar enzymes with high sequence homology includefum] from Arabidopsis thaliana and fumC from Corynebacterium glutamicum. The mmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., 270:207-213 (2007)). Citramalate hydrolyase naturally dehydrates 2 methylmalate to mesaconate. This enzyme has been studied in Methanocaldococcus jannaschii in the context of the pyruvate pathway to 2-oxobutanoate, where it has been shown to have a broad substrate specificity (Drevland et al., JBacteriol. 189:4391-4400 (2007)). This enzyme activity was also detected in Clostridium tetanomorphum, Morganella morganii, Citrobacter amalonaticus where it is thought to participate in glutamate degradation (Kato et al., Arch.Microbiol 168:457-463 (1997)). The M. jannaschii protein sequence does not bear significant homology to genes in these organisms. Dimethylmaleate hydratase is a reversible Fe2+-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB in Eubacterium barkeri (Alhapel et al., supra;Kollmann-Koch et al., Hoppe Seylers.Z.Physiol Chem. 365:847-857 (1984)). Gene GenBank ID GI Number Organism fumA NP_416129.1 16129570 Escherichia coli fumB NP_418546.1 16131948 Escherichia coli fumC NP_416128.1 16129569 Escherichia coli fumC 069294 9789756 Campylobacterjejuni fumC P84127 75427690 Thermus thermophilus fumH P14408 120605 Rattus norvegicus fum1 P93033 39931311 Arabidopsis thaliana fumC Q8NRN8 39931596 Corynebacterium glutamicum mmcB YP_001211906 147677691 Pelotomaculum thermopropionicum mmcC YP_001211907 147677692 Pelotomaculum thermopropionicum leuD Q58673.1 3122345 Methanocaldococcus jannaschii dmdA ABC88408 86278276 Eubacterium barkeri dmdB ABC88409.1 86278277 Eubacterium barkeri 4.3.1 .a Ammonia-lyase: An ammonia lyase enzyme is required to catalyze the deamination of 2-amino-4-oxopentanoate (AKP) to acetylacrylate in Step B of Figure 13. An enzyme catalyzing this exact transformation has not been identified. However the AKP is 192 structurally similar to aspartate, the native substrate of aspartase (EC 4.3.1.1.). Aspartase is a widespread enzyme in microorganisms, and has been characterized extensively (Viola, 74:295-341 (2000)). The E. coli enzyme has been shown to react with a variety of alternate substrates including aspartatephenylmethylester, asparagine, benzyl-aspartate and malate (Ma et al., 672:60-65 (1992)). In addition, directed evolution was been employed on this enzyme to alter substrate specificity (Asano et al., 22:95-101 (2005)). The crystal structure of the E. coli aspartase, encoded by aspA, has been solved (Shi et al., 36:9136-9144 (1997)). Enzymes with aspartase functionality have also been characterized in Haemophilus influenzae (Sjostrom et al., Biochim.Biophys.Acta 1324:182-190 (1997)), Pseudomonasfluorescens (Takagi et al., JBiochem. 96:545-552 (1984)), Bacillus subtilis (Sjostrom et al., 1324:182 190 (1997)) and Serratia marcescens (Takagi et al., 161:1-6 (1985)). Gene GenBank ID GI Number Organism aspA NP_418562 90111690 Escherichia coli K12 subsp. MG1655 aspA P44324.1 1168534 Haemophilus influenzae aspA P07346.1 114273 Pseudomonasfluorescens ansB P26899.1 251757243 Bacillus subtilis aspA P33109.1 416661 Serratia marcescens Another enzyme candidate for catalyzing the deamination of AKP is 3 methylaspartase (EC 4.3.1.2). This enzyme, also known as beta-methylaspartase and 3 methylaspartate ammonia-lyase, naturally catalyzes the deamination of threo-3 methylasparatate to mesaconate. The 3-methylaspartase from Clostridium tetanomorphum has been cloned, functionally expressed in E. coli, and crystallized (Asuncion et al., 57:731 733 (2001); Asuncion et al., JBiol Chem. 277:8306-8311 (2002); Botting et al., 27:2953 2955 (1988); Goda et al., 31:10747-10756 (1992)). In Citrobacter amalonaticus, this enzyme is encoded by BAA28709 (Kato and Asano, Arch.Microbiol 168:457-463 (1997)). 3 Methylaspartase has also been crystallized from E. coli YG1002 (Asano et al., FEMS MicrobiolLett. 118:255-258 (1994)) although the protein sequence is not listed in public databases such as GenBank. Sequence homology can be used to identify additional candidate genes, including CTC_02563 in C. tetani and ECs0 761 in Escherichia coli 015 7:H7.

193 Gene GenBank ID GI Number Organism mal A AB24070.1 259429 Clostridium tetanomorphum BAA28709 BAA28709.1 3184397 Citrobacter amalonaticus CTC_02563 NP_783085.1 28212141 Clostridium tetani ECs0761 BAB34184.1 13360220 Escherichia coli 0157:H7 5.1.1.a Racemase: Racemase enzymes in the EC class 5.1.1 isomerize D- and L amino acids. Such an enzyme may be required to increase the bioavailability of D-alanine and/or D-ornithine and thus enhance the conversion of alanine to AKP (Step A of Figure 13) or ornithine to 2,4-diaminopentanoate (Step M of Figure 13). Enzymes with alanine racemase (EC 5.1.1.1) and ornithine racemase (EC 5.1.1.12) activity have been characterized. Alanine racemase interconverts the L and D stereoisomers of alanine. Escherichia coli has two alanine aminomutase enzymes, encoded alr and dadX(Lilley et al., Gene 129:9-16 (1993); Wild et al., Mol Gen Genet. 198:315-322 (1985)). The vanT gene of Enterococcus gallinarum also exhibited alanine racemase activity when expressed in E. coli (Arias et al., Microbiology 146 ( Pt 7):1727-1734 (2000)). Additional alanine racemase enzyme candidates have been characterized in Bacillus subtilis and Mycobacterium tuberculosis (Pierce et al., FEMS Microbiol Lett. 283:69-74 (2008); Strych et al., FEMSMicrobiol Lett. 196:93-98 (2001)). Interconversion of D-omithine and L-omithine is catalyzed by ornithine racemase. The enzyme encoded by the orr gene product of C. sticklandii was purified and characterized (Fonknechten, JBacteriol. In Press: (2009)). Additional ornithine racemase gene candidates can be identified by sequence similarity in organisms such as Clostridium difficile and Fusobacterium periodonticum. Gene GenBank ID GI Number Organism alr NP_418477.1 16131879 Escherichia coli dadX AAC74274.1 1787439 Escherichia coli van T Q9X3P3.1 20140922 Enterococcus gallinarum yncD NP_389646.1 16078827 Bacillus subtilis alr NP_338056.1 15843019 Mycobacterium tuberculosis CDC1551 alr NP_217940.1 15610559 Mycobacterium tuberculosis 194 Gene GenBank ID GI Number Organism H37Rv orr CAQ4298 1.1 226885219 Clostridium sticklandii CdifA_020200002638 ZP_05349631.1 255305459 Clostridium difficile FUSPEROL_00295 ZP_06025693.1 262066081 Fusobacterium periodonticum 5.4.3.a Aminomutase: Omithine aminomutase (EC 5.4.3.5) catalyzes the conversion of ornithine to 2,4-diaminopentanoate (Step M of Figure 13). A B12-dependent enzyme with this activity, encoded by oraSE of Clostridium sticklandii, has been cloned, sequenced and expressed in E. coli (Chen et al., JBiol.Chem. 276:44744-44750 (2001)). This enzyme preferentially reacts with the D-stereoisomer of ornithine (Fonknechten, JBacteriol. In Press: (2009)). Ornithine aminomutase enzymes have not been characterized in other organisms to date. Similar enzymes in organisms such as Alkaliphilus oremlandii and Clostridium difficile can be identified by sequence similarity. Lysine aminomutase catalyzes two similar transformations: the interconversin of lysine with 2,5-diaminohexanoate (EC 5.4.3.4), and 3,6-diaminohexanoate with 3,5-diaminohexanoate (EC 5.4.3.3). This enzyme participates in the fermentation of lysine to acetate and butyrate and has been characterized in Clostridium sticklandii (Berkovitch et al., Proc.Nat.Acad.Sci. U.S.A 101:15 870-15 875 (2004)) and Porphyromonas gingivalis (Tang et al., Biochemistry 41:8767-8776 (2002)). Gene GenBank ID GI Number Organism oraE AAK72502.1 17223685 Clostridium sticklandii oraS AAK72501.1 17223684 Clostridium sticklandii oraE (Clos_1695) YP_001513231.1 158320724 Alkaliphilus oremlandii oraS (Clos 1696) YP_001513232.1 158320725 Alkaliphilus oremlandii oraE ZP_05349629.1 255305457 Clostridium difficile oraS ZP_05349628.1 255305456 Clostridium difficile kamD AAC79717.1 3928904 Clostridium sticklandii kamE AAC79718.1 3928905 Clostridium sticklandii kamD NP_905288.1 34540809 Porphyromonas gingivalis W83 kamE NP_905289.1 34540810 Porphyromonas gingivalis W83 195 Other: 2-Amino-4-oxopentanoate (AKP) is formed from alanine and acetyl-CoA by AKP thioloase (Step A in Figure 13). AKP thiolase (AKPT, no EC number) is a pyridoxal phosphate-dependent enzyme that participates in ornithine degradation in Clostridium sticklandii (Jeng et al., Biochemistry 13:2898-2903 (1974); Kenklies et al., Microbiology 145 ( Pt 4):819-826 (1999)). A gene cluster encoding the alpha and beta subunits of AKPT (or-2 (ortA) and or-3 (ortB)) was recently described and the biochemical properties of the enzyme were characterized (Fonknechten, JBacteriol. In Press: (2009)). The enzyme is capable of operating in both directions and reacts with the D- isomer of alanine. Enzyme engineering or directed evolution can enable the enzyme to function with L-alanine as a substrate providing additional pathway versatility with regards to the primary substrate. Alternately, co expression of an alanine racemase enzyme may enhance substrate availability. Enzymes with high sequence homology are found in Clostridium difficile, Alkaliphilus metalliredigenes QYF, Thermoanaerobacter sp. X514, and Thermoanaerobacter tengcongensis MB4 (Fonknechten, JBacteriol. In Press: (2009)). Gene GenBank ID GI Number Organism ortA CAQ42979.1 226885215 Clostridium sticklandii ortB CAQ42980.1 GI: 226885217 Clostridium sticklandii ortA YP_001086914.1 126698017 Clostridium difficile 630 ortB YP_001086915.1 126698018 Clostridium difficile 630 Amet_2368 YP_001320181.1 150390132 Alkaliphilus metalliredigenes QYF Amet_2369 YP_001320182.1 150390133 Alkaliphilus metalliredigenes QYF Teth514_1478 YP_001663101.1 167040116 Thermoanaerobacter sp. X514 Teth514_1479) YP_001663102.1 167040117 Thermoanaerobacter sp. X514 TTE1235 NP_622858.1 20807687 Thermoanaerobacter tengcongensis MB4 thrC NP_622859.1 20807688 Thermoanaerobacter tengcongensis MB4 196 EXAMPLE IX Exemplary Hydrogenase and CO Dehydrogenase Enzymes for Extracting Reducing Equivalents from Syngas and Exemplary Reductive TCA Cycle Enzymes Enzymes of the reductive TCA cycle useful in the non-naturally occurring microbial organisms of the present invention include one or more of ATP-citrate lyase and three C0 2 fixing enzymes: isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase, pyruvate:ferredoxin oxidoreductase. The presence of ATP-citrate lyase or citrate lyase and alpha-ketoglutarate:ferredoxin oxidoreductase indicates the presence of an active reductive TCA cycle in an organism. Enzymes for each step of the reductive TCA cycle are shown below. ATP-citrate lyase (ACL, EC 2.3.3.8), also called ATP citrate synthase, catalyzes the ATP-dependent cleavage of citrate to oxaloacetate and acetyl-CoA. ACL is an enzyme of the RTCA cycle that has been studied in green sulfur bacteria Chlorobium limicola and Chlorobium tepidum. The alpha(4)beta(4) heteromeric enzyme from Chlorobium limicola was cloned and characterized in E. coli (Kanao et al., Eur. J Biochem. 269:3409-3416 (2002). The C. limicola enzyme, encoded by aclAB, is irreversible and activity of the enzyme is regulated by the ratio of ADP/ATP. A recombinant ACL from Chlorobium tepidum was also expressed in E. coli and the holoenzyme was reconstituted in vitro, in a study elucidating the role of the alpha and beta subunits in the catalytic mechanism (Kim and Tabita, J Bacteriol. 188:6544-6552 (2006). ACL enzymes have also been identified in Balnearium lithotrophicum, Sulfurihydrogenibium subterraneum and other members of the bacterial phylum Aquificae (Hugler et al., Environ. Microbiol. 9:81-92 (2007)). This acitivy has been reported in some fungi as well. Exemplary organisms include Sordaria macrospora (Nowrousian et al., Curr. Genet. 37:189-93 (2000), Aspergillus nidulans , Yarrowia lipolytica (Hynes and Murray, Eukaryotic Cell, July: 1039-1048, (2010) and Aspergillus niger (Meijer et al. J Ind. Microbiol. Biotechnol. 36:1275-1280 (2009). Other candidates can be found based on sequence homology. Information related to these enzymes is tabulated below: 197 Protein GenBank ID GI Number Organism ac/A BAB21376.1 12407237 Chlorobium limicola ac/B BAB21375.1 12407235 Chlorobium limicola ac/A AAM72321.1 21647054 Chlorobium tepidum ac/B AAM72322.1 21647055 Chlorobium tepidum aclA AB150076.1 114054981 Balnearium lithotrophicum aciB AB150075.1 114054980 Balnearium lithotrophicum aclA ABI50085.1 114055040 Sulfurihydrogenibium subterraneum aciB ABI50084.1 114055039 Sulfurihydrogenibium subterraneum aclA AAX76834.1 62199504 Sulfurimonas denitrificans aciB AAX76835.1 62199506 Sulfurimonas denitrificans ad] XP_504787.1 50554757 Yarrowia lipolytica acl2 XP_503231.1 50551515 Yarrowia lipolytica SPBC1 703.07 NP_596202.1 19112994 Schizosaccharomyces pombe SPAC22A12.16 NP_593246.1 19114158 Schizosaccharomyces pombe ad] CAB76165.1 7160185 Sordaria macrospora acl2 CAB76164.1 7160184 Sordaria macrospora acA CBF86850.1 259487849 Aspergillus nidulans aciB CBF86848 259487848 Aspergillus nidulans In some organisms the conversion of citrate to oxaloacetate and acetyl-CoA proceeds through a citryl-CoA intermediate and is catalyzed by two separate enzymes, citryl-CoA synthetase (EC 6.2.1.18) and citryl-CoA lyase (EC 4.1.3.34) (Aoshima, M., Appl. Microbiol. Biotechnol. 75:249-255 (2007). Citryl-CoA synthetase catalyzes the activation of citrate to citryl-CoA. The Hydrogenobacter thermophilus enzyme is composed of large and small subunits encoded by ccsA and ccsB, respectively (Aoshima et al., Mol. Micrbiol. 52:751-761 198 (2004)). The citryl-CoA synthetase of Aquifex aeolicus is composed of alpha and beta subunits encoded by sucC1 and sucD1 (Hugler et al., Environ. Microbiol. 9:81-92 (2007)). Citryl-CoA lyase splits citryl-CoA into oxaloacetate and acetyl-CoA. This enzyme is a homotrimer encoded by ccl in Hydrogenobacter thermophilus (Aoshima et al., Mol. Microbiol. 52:763-770 (2004)) and aq_150 in Aquifex aeolicus (Hugler et al., supra (2007)). The genes for this mechanism of converting citrate to oxaloacetate and citryl-CoA have also been reported recently in Chlorobium tepidum (Eisen et al., PNAS 99(14): 9509-14 (2002). Protein GenBank ID GI Number Organism ccsA BAD17844.1 46849514 Hydrogenobacter thermophilus ccsB BAD17846.1 46849517 Hydrogenobacter thermophilus sucCi AAC07285 2983723 Aquifex aeolicus sucD1 AAC07686 2984152 Aquifex aeolicus ccl BAD17841.1 46849510 Hydrogenobacter thermophilus aq_150 AAC06486 2982866 Aquifex aeolicus CT0380 NP_661284 21673219 Chlorobium tepidum CT0269 NP_661173.1 21673108 Chlorobium tepidum CT1834 AAM73055.1 21647851 Chlorobium tepidum Oxaloacetate is converted into malate by malate dehydrogenase (EC 1.1.1.37), an enzyme which functions in both the forward and reverse direction. S. cerevisiae possesses three copies of malate dehydrogenase, MDH1 (McAlister-Henn and Thompson, J Bacteriol. 169:5157-5166 (1987), MDH2 (Minard and McAlister-Henn, Mol. Cell. Biol. 11:370-380 (1991); Gibson and McAlister-Henn, J Biol. Chem. 278:25628-25636 (2003)), and MDH3 (Steffan and McAlister-Henn, J Biol. Chem. 267:24708-24715 (1992)), which localize to the mitochondrion, cytosol, and peroxisome, respectively. E. coli is known to have an active malate dehydrogenase encoded by mdh.

199 Protein GenBank ID GI Number Organism MDH1 NP_012838 6322765 Saccharomyces cerevisiae MDH2 NP_014515 116006499 Saccharomyces cerevisiae MDH3 NP_010205 6320125 Saccharomyces cerevisiae Mdh NP_417703.1 16131126 Escherichia coli Fumarate hydratase (EC 4.2.1.2) catalyzes the reversible hydration of fumarate to malate. The three fumarases of E. coli, encoded byfumA,fumB andfumC, are regulated under different conditions of oxygen availability. FumB is oxygen sensitive and is active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is active under aerobic growth conditions (Tseng et al., J Bacteriol. 183:461-467 (2001);Woods et al., Biochim. Biophys. Acta 954:14-26 (1988); Guest et al., J Gen. Microbiol. 131:2971-2984 (1985)). S. cerevisiae contains one copy of a fumarase-encoding gene, FUM1, whose product localizes to both the cytosol and mitochondrion (Sass et al., J Biol. Chem. 278:45109-45116 (2003)). Additional fumarase enzymes are found in Campylobacterjejuni (Smith et al., Int. J Biochem. Cell. Biol. 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch. Biochem. Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al., J Biochem. 89:1923-1931 (1981)). Similar enzymes with high sequence homology includefum1 from Arabidopsis thaliana and fumC from Corynebacterium glutamicum. The MmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol. Lett. 270:207-213 (2007)). Protein GenBank ID GI Number Organism fumA NP_416129.1 16129570 Escherichia coli fumB NP_418546.1 16131948 Escherichia coli fumC NP_416128.1 16129569 Escherichia coli FUM1 NP_015061 6324993 Saccharomyces cerevisiae fumC Q8NRN8.1 39931596 Corynebacterium glutamicum 200 Protein GenBank ID GI Number Organism fumC 069294.1 9789756 Campylobacterjejuni fumC P84127 75427690 Thermus thermophilus fumH P14408.1 120605 Rattus norvegicus MmcB YP_001211906 147677691 Pelotomaculum thermopropionicum MmcC YP_001211907 147677692 Pelotomaculum thermopropionicum Fumarate reductase catalyzes the reduction of fumarate to succinate. The fumarate reductase of E. coli, composed of four subunits encoded byfrdABCD, is membrane-bound and active under anaerobic conditions. The electron donor for this reaction is menaquinone and the two protons produced in this reaction do not contribute to the proton gradient (Iverson et al., Science 284:1961-1966 (1999)). The yeast genome encodes two soluble fumarate reductase isozymes encoded by FRDS1 (Enomoto et al., DNA Res. 3:263-267 (1996)) and FRDS2 (Muratsubaki et al., Arch. Biochem. Biophys. 352:175-181 (1998)), which localize to the cytosol and promitochondrion, respectively, and are used during anaerobic growth on glucose (Arikawa et al., FEMS Microbiol. Lett. 165:111-116 (1998)). Protein GenBank ID GI Number Organism FRDS1 P32614 418423 Saccharomyces cerevisiae FRDS2 NP_012585 6322511 Saccharomyces cerevisiae frdA NP_418578.1 16131979 Escherichia coli frdB NP_418577.1 16131978 Escherichia coli frdC NP_418576.1 16131977 Escherichia coli frdD NP_418475.1 16131877 Escherichia coli The ATP-dependent acylation of succinate to succinyl-CoA is catalyzed by succinyl CoA synthetase (EC 6.2.1.5). The product of the LSC1 and LSC2 genes of S. cerevisiae and 201 the sucC and sucD genes of E. coli naturally form a succinyl-CoA synthetase complex that catalyzes the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP, a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). These proteins are identified below: Protein GenBank ID GI Number Organism LSCJ NP_014785 6324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683 Saccharomyces cerevisiae sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia coli Alpha-ketoglutarate:ferredoxin oxidoreductase (EC 1.2.7.3), also known as 2 oxoglutarate synthase or 2-oxoglutarate:ferredoxin oxidoreductase (OFOR), forms alpha ketoglutarate from C02 and succinyl-CoA with concurrent consumption of two reduced ferredoxin equivalents. OFOR and pyruvate:ferredoxin oxidoreductase (PFOR) are members of a diverse family of 2-oxoacid:ferredoxin (flavodoxin) oxidoreductases which utilize thiamine pyrophosphate, CoA and iron-sulfur clusters as cofactors and ferredoxin, flavodoxin and FAD as electron carriers (Adams et al., Archaea. Adv. Protein Chem. 48:101-180 (1996)). Enzymes in this class are reversible and function in the carboxylation direction in organisms that fix carbon by the RTCA cycle such as Hydrogenobacter thermophilus, Desu/fobacter hydrogenophilus and Chlorobium species (Shiba et al. 1985; Evans et al., Proc. Natl. Acad. ScI. U.S.A. 55:92934 (1966); Buchanan, 1971). The two-subunit enzyme from H. thermophilus, encoded by korAB, has been cloned and expressed in E. coli (Yun et al., Biochem. Biophys. Res. Commun. 282:589-594 (2001)). A five subunit OFOR from the same organism with strict substrate specificity for succinyl-CoA, encoded byforDABGE, was recently identified and expressed in E. coli (Yun et al. 2002). The kinetics of C02 fixation of both H. thermophilus OFOR enzymes have been characterized (Yamamoto et al., Extremophiles 14:79-85 (2010)). A C02-fixing OFOR from Chlorobium thiosu/fatophilum has been purified and characterized but the genes encoding this enzyme have not been identified to date. Enzyme candidates in Chlorobium species can be inferred by sequence similarity to the H. thermophilus genes. For example, the Chlorobium limicola genome encodes two similar proteins. Acetogenic bacteria such as Moorella thermoacetica are 202 predicted to encode two OFOR enzymes. The enzyme encoded by Moth_0034 is predicted to function in the C02-assimilating direction. The genes associated with this enzyme, Moth_0034 have not been experimentally validated to date but can be inferred by sequence similarity to known OFOR enzymes. OFOR enzymes that function in the decarboxylation direction under physiological conditions can also catalyze the reverse reaction. The OFOR from the thermoacidophilic archaeon Sulfolobus sp. strain 7, encoded by ST2300, has been extensively studied (Zhang et al. 1996. A plasmid-based expression system has been developed for efficiently expressing this protein in E. coli (Fukuda et al., Eur. J. Biochem. 268:5639-5646 (2001)) and residues involved in substrate specificity were determined (Fukuda and Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002)). The OFOR encoded by Ape1472/Ape1473 from Aeropyrum pernix str. KI was recently cloned into E. coli, characterized, and found to react with 2-oxoglutarate and a broad range of 2-oxoacids (Nishizawa et al., FEBS Lett. 579:2319-2322 (2005)). Another exemplary OFOR is encoded by oorDABC in Helicobacterpylori (Hughes et al. 1998). An enzyme specific to alpha-ketoglutarate has been reported in Thauera aromatica (Dorner and Boll, J, Bacteriol. 184 (14), 3975-83 (2002). A similar enzyme can be found in Rhodospirillum rubrum by sequence homology. A two subunit enzyme has also been identified in Chlorobium tepidum (Eisen et al., PNAS 99(14): 9509-14 (2002)). Protein GenBank ID GI Number Organism korA BAB21494 12583691 Hydrogenobacter thermophilus korB BAB21495 12583692 Hydrogenobacter thermophilus forD BAB62132.1 14970994 Hydrogenobacter thermophilus forA BAB62133.1 14970995 Hydrogenobacter thermophilus forB BAB62134.1 14970996 Hydrogenobacter thermophilus forG BAB62135.1 14970997 Hydrogenobacter thermophilus forE BAB62136.1 14970998 Hydrogenobacter thermophilus Clim_0204 ACD89303.1 189339900 Chlorobium limicola Clim_0205 ACD89302.1 189339899 Chlorobium limicola 203 Clim_1123 ACD90192.1 189340789 Chlorobium limicola Clim_1124 ACD90193.1 189340790 Chlorobium limicola Moth_1984 YP_430825.1 83590816 Moorella thermoacetica Moth_1985 YP_430826.1 83590817 Moorella thermoacetica Moth_0034 YP_428917.1 83588908 Moorella thermoacetica ST2300 NP_378302.1 15922633 Sulfolobus sp. strain 7 Ape1472 BAA80470.1 5105156 Aeropyrum pernix Ape] 473 BAA8047 1.2 116062794 Aeropyrum pernix oorD NP_207383.1 15645213 Helicobacterpylori oorA NP_207384.1 15645214 Helicobacterpylori oorB NP_207385.1 15645215 Helicobacterpylori oorC NP_207386.1 15645216 Helicobacterpylori CT0163 NP_661069.1 21673004 Chlorobium tepidum CT0162 NP_661068.1 21673003 Chlorobium tepidum korA CAA12243.2 19571179 Thauera aromatica korB CAD27440.1 19571178 Thauera aromatica RruA2721 YP_427805.1 83594053 Rhodospirillum rubrum RruA2722 YP_427806.1 83594054 Rhodospirillum rubrum Isocitrate dehydrogenase catalyzes the reversible decarboxylation of isocitrate to 2 oxoglutarate coupled to the reduction of NAD(P). IDH enzymes in Saccharomyces cerevisiae and Escherichia coli are encoded by IDP1 and icd, respectively (Haselbeck and McAlister-Henn, J Biol. Chem. 266:2339-2345 (1991); Nimmo, H.G., Biochem. J. 234:317 2332 (1986)). The reverse reaction in the reductive TCA cycle, the reductive carboxylation 204 of 2-oxoglutarate to isocitrate, is favored by the NADPH-dependent C0 2 -fixing IDH from Chlorobium limicola and was functionally expressed in E. coli (Kanao et al., Eur. J Biochem. 269:1926-1931 (2002)). A similar enzyme with 95% sequence identity is found in the C. tepidum genome in addititon to some other candidates listed below. Protein GenBank ID GI Number Organism Icd AC184720.1 209772816 Escherichia coli IDP1 AAA34703.1 171749 Saccharomyces cerevisiae Idh BAC00856.1 21396513 Chlorobium limicola Icd AAM71597.1 21646271 Chlorobium tepidum icd NP_952516.1 39996565 Geobacter sulfurreducens icd YP_393560. 78777245 Sulfurimonas denitrificans In H. thermophilus the reductive carboxylation of 2-oxoglutarate to isocitrate is catalyzed by two enzymes: 2-oxoglutarate carboxylase and oxalosuccinate reductase. 2 Oxoglutarate carboxylase (EC 6.4.1.7) catalyzes the ATP-dependent carboxylation of alpha ketoglutarate to oxalosuccinate (Aoshima and Igarashi, Mol. Microbiol. 62:748-759 (2006)). This enzyme is a large complex composed of two subunits. Biotinylation of the large (A) subunit is required for enzyme function (Aoshima et al., Mol. Microbiol. 51:791-798 (2004)). Oxalosuccinate reductase (EC 1.1.1.-) catalyzes the NAD-dependent conversion of oxalosuccinate to D-threo-isocitrate. The enzyme is a homodimer encoded by icd in H. thermophilus. The kinetic parameters of this enzyme indicate that the enzyme only operates in the reductive carboxylation direction in vivo, in contrast to isocitrate dehydrogenase enzymes in other organisms (Aoshima and Igarashi, J Bacteriol. 190:2050-2055 (2008)). Based on sequence homology, gene candidates have also been found in Thiobacillus denitrificans and Thermocrinis albus. Protein GenBank ID GI Number Organism cfiA BAF34932.1 116234991 Hydrogenobacter thermophilus cifB BAF3493 1.1 116234990 Hydrogenobacter thermophilus 205 Protein GenBank ID GI Number Organism Icd BAD02487.1 38602676 Hydrogenobacter thermophilus Tbd_1556 YP_315314 74317574 Thiobacillus denitrificans Tbd_1555 YP_315313 74317573 Thiobacillus denitrificans Tbd_0854 YP_314612 74316872 Thiobacillus denitrificans Thal_0268 YP_003473030 289548042 Thermocrinis albus Thal_0267 YP_003473029 289548041 Thermocrinis albus Thal_0646 YP_003473406 289548418 Thermocrinis albus Aconitase (EC 4.2.1.3) is an iron-sulfur-containing protein catalyzing the reversible isomerization of citrate and iso-citrate via the intermediate cis-aconitate. Two aconitase enzymes are encoded in the E. coli genome by acnA and acnB. AcnB is the main catabolic enzyme, while AcnA is more stable and appears to be active under conditions of oxidative or acid stress (Cunningham et al., Microbiology 143 (Pt 12):3795-3805 (1997)). Two isozymes of aconitase in Salmonella typhimurium are encoded by acnA and acnB (Horswill and Escalante-Semerena, Biochemistry 40:4703-4713 (2001)). The S. cerevisiae aconitase, encoded by ACO, is localized to the mitochondria where it participates in the TCA cycle (Gangloff et al., Mol. Cell. Biol. 10:3551-3561 (1990)) and the cytosol where it participates in the glyoxylate shunt (Regev-Rudzki et al., Mol. Biol. Cell. 16:4163-4171 (2005)). Protein GenBank ID GI Number Organism acnA AAC7438.1 1787531 Escherichia coli acnB AAC73229.1 2367097 Escherichia coli acnA NP_460671.1 16765056 Salmonella typhimurium HP0779 NP_207572.1 15645398 Helicobacter pylori 26695 H16_B0568 CAJ95365.1 113529018 Ralstonia eutropha DesfrDRAFT_3783 ZP_07335307.1 303249064 Desulfovibriofructosovorans JJ Suden_1040 ABB44318.1 78497778 Sulfurimonas denitrificans 206 Protein GenBank ID GI Number Organism (acnB) Hydth_0755 ADO45152.1 308751669 Hydrogenobacter thermophilus CT0543 (acn) AAM71785.1 21646475 Chlorobium tepidum Clim_2436 YP_001944436.1 189347907 Chlorobium limicola Clim_0515 ACD89607.1 189340204 Chlorobium limicola acnB NP_459163.1 16763548 Salmonella typhimurium ACO1 AAA34389.1 170982 Saccharomyces cerevisiae Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the reversible oxidation of pyruvate to form acetyl-CoA. The PFOR from Desulfovibrio africanus has been cloned and expressed in E. coli resulting in an active recombinant enzyme that was stable for several days in the presence of oxygen (Pieulle et al., J Bacteriol. 179:5684-5692 (1997)). Oxygen stability is relatively uncommon in PFORs and is believed to be conferred by a 60 residue extension in the polypeptide chain of the D. africanus enzyme. Two cysteine residues in this enzyme form a disulfide bond that prtotects it against inactivation in the form of oxygen. This disulfide bond and the stability in the presence of oxygen has been found in other Desulfovibrio species also (Vita et al., Biochemistry, 47: 957-64 (2008)). The M. thermoacetica PFOR is also well characterized (Menon and Ragsdale, Biochemistry 36:8484 8494 (1997)) and was shown to have high activity in the direction of pyruvate synthesis during autotrophic growth (Furdui and Ragsdale, J Biol. Chem. 275:28494-28499 (2000)). Further, E. coli possesses an uncharacterized open reading frame, ydbK, encoding a protein that is 510% identical to the M. thermoacetica PFOR. Evidence for pyruvate oxidoreductase activity in E. coli has been described (Blaschkowski et al., Eur. J Biochem. 123:563-569 (1982)). PFORs have also been described in other organisms, including Rhodobacter capsulatas (Yakunin and Hallenbeck, Biochimica et Biophysica Acta 1409 (1998) 39-49 (1998)) and Choloboum tepidum (Eisen et al., PNAS 99(14): 9509-14 (2002)). The five subunit PFOR from H. thermophilus, encoded by porEDABG, was cloned into E. coli and shown to function in both the decarboxylating and C0 2 -assimilating directions (Ikeda et al. 2006; Yamamoto et al., Extremophiles 14:79-85 (2010)). Homologs also exist in C.

207 carboxidivorans P7. Several additional PFOR enzymes are described in the following review (Ragsdale, S.W., Chem. Rev. 103:2333-2346 (2003)). Finally, flavodoxin reductases (e.g., fqrB from Helicobacterpylori or Campylobacterjejuni) (St Maurice et al., J Bacteriol. 189:4764-4773 (2007)) or Rnf-type proteins (Seedorf et al., Proc. NatI. Acad. Sci. U.S.A. 105:2128-2133 (2008); and Herrmann, J Bacteriol 190:784-791 (2008)) provide a means to generate NADH or NADPH from the reduced ferredoxin generated by PFOR. These proteins are identified below. Protein GenBank ID GI Number Organism DesfrDRAFT_0121 ZP_07331646.1 303245362 Desulfovibriofructosovorans JJ Por CAA70873.1 1770208 Desulfovibrio africanus por YP_012236.1 46581428 Desulfovibrio vulgaris str. Hildenborough Dde_3237 ABB4003 1.1 78220682 DesulfoVibrio desulfuricans G20 Ddes_0298 YP_002478891.1 220903579 Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774 Por YP_428946.1 83588937 Moorella thermoacetica YdbK NP_415896.1 16129339 Escherichia coli nifJ (CT1628) NP_662511.1 21674446 Chlorobium tepidum CJE1649 YP_179630.1 57238499 Campylobacterjejuni nifJ ADE85473.1 294476085 Rhodobacter capsulatus porE BAA95603.1 7768912 Hydrogenobacter thermophilus porD BAA95604.1 7768913 Hydrogenobacter thermophilus porA BAA95605.1 7768914 Hydrogenobacter thermophilus porB BAA95606.1 776891 Hydrogenobacter thermophilus porG BAA95607.1 7768916 Hydrogenobacter thermophilus FqrB YP_001482096.1 157414840 Campylobacterjejuni HP1164 NP_207955.1 15645778 Helicobacterpylori RnfC EDK33306.1 146346770 Clostridium kluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri RnfG EDK33308.1 146346772 Clostridium kluyveri RnfE EDK33309.1 146346773 Clostridium kluyveri 208 RnfA EDK33310.1 146346774 Clostridium kluyveri RnfB EDK333 11.1 146346775 Clostridium kluyveri The conversion of pyruvate into acetyl-CoA can be catalyzed by several other enzymes or their combinations thereof. For example, pyruvate dehydrogenase can transform pyruvate into acetyl-CoA with the concomitant reduction of a molecule of NAD into NADH. It is a multi-enzyme complex that catalyzes a series of partial reactions which results in acylating oxidative decarboxylation of pyruvate. The enzyme comprises of three subunits: the pyruvate decarboxylase (El), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). This enzyme is naturally present in several organisms, including E. coli and S. cerevisiae. In the E. coli enzyme, specific residues in the E l component are responsible for substrate specificity (Bisswanger, H., J. Biol. Chem. 256:815-82 (1981); Bremer, J., Eur. J. Biochem. 8:535-540 (1969); Gong et al., J. Biol. Chem. 275:13645-13653 (2000)). Enzyme engineering efforts have improved the E. coli PDH enzyme activity under anaerobic conditions (Kim et al., J. Bacteriol. 190:3851-3858 (2008); Kim et al., Appl. Environ. Microbiol. 73:1766-1771 (2007); Zhou et al., Biotechnol. Lett. 30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtilis complex is active and required for growth under anaerobic conditions (Nakano et al., J. Bacteriol. 179:6749-6755 (1997)). The Klebsiella pneumoniae PDH, characterized during growth on glycerol, is also active under anaerobic conditions (5). Crystal structures of the enzyme complex from bovine kidney (18) and the E2 catalytic domain from Azotobacter vinelandii are available (4). Yet another enzyme that can catalyze this conversion is pyruvate formate lyase. This enzyme catalyzes the conversion of pyruvate and CoA into acetyl-CoA and formate. Pyruvate formate lyase is a common enzyme in prokaryotic organisms that is used to help modulate anaerobic redox balance. Exemplary enzymes can be found in Escherichia coli encoded by pflB (Knappe and Sawers, FEMS.Microbiol Rev. 6:383-398 (1990)), Lactococcus lactis (Melchiorsen et al., Appl Microbiol Biotechnol 58:338-344 (2002)), and Streptococcus mutans (Takahashi-Abbe et al., Oral.Microbiol Immunol. 18:293-297 (2003)). E. coli possesses an additional pyruvate formate lyase, encoded by tdcE, that catalyzes the conversion of pyruvate or 2-oxobutanoate to acetyl-CoA or propionyl-CoA, respectively (Hesslinger et al., Mol. Microbiol 27:477-492 (1998)). Both pflB and tdcE from E. coli require the presence of pyruvate formate lyase activating enzyme, encoded by pflA. Further, a short protein encoded by yfiD in E. coli can associate with and restore activity to oxygen-cleaved pyruvate formate lyase (Vey et al., 209 Proc.Natl. Acad. Sci. U.S.A. 105:16137-16141 (2008). Note thatpflA andpflB from E. coli were expressed in S. cerevisiae as a means to increase cytosolic acetyl-CoA for butanol production as described in WO/2008/080124]. Additional pyruvate formate lyase and activating enzyme candidates, encoded by pfl and act, respectively, are found in Clostridium pasteurianum (Weidner et al., J Bacteriol. 178:2440-2444 (1996)). Further, different enzymes can be used in combination to convert pyruvate into acetyl-CoA. For example, in S. cerevisiae, acetyl-CoA is obtained in the cytosol by first decarboxylating pyruvate to form acetaldehyde; the latter is oxidized to acetate by acetaldehyde dehydrogenase and subsequently activated to form acetyl-CoA by acetyl-CoA synthetase. Acetyl-CoA synthetase is a native enzyme in several other organisms including E. coli (Kumari et al., J. Bacteriol. 177:2878-2886 (1995)), Salmonella enterica (Starai et al., Microbiology 151:3793-3801 (2005); Starai et al., J. Biol. Chem. 280:26200-26205 (2005)), and Moorella thermoacetica (described already). Alternatively, acetate can be activated to form acetyl-CoA by acetate kinase and phosphotransacetylase. Acetate kinase first converts acetate into acetyl-phosphate with the accompanying use of an ATP molecule. Acetyl phosphate and CoA are next converted into acetyl-CoA with the release of one phosphate by phosphotransacetylase. Both acetate kinase and phosphotransacetlyase are well-studied enzymes in several Clostridia and Methanosarcina thermophila. Yet another way of converting pyruvate to acetyl-CoA is via pyruvate oxidase. Pyruvate oxidase converts pyruvate into acetate, using ubiquione as the electron acceptor. In E. coli, this activity is encoded by poxB. PoxB has similarity to pyruvate decarboxylase of S. cerevisiae and Zymomonas mobilis. The enzyme has a thiamin pyrophosphate cofactor (Koland and Gennis, Biochemistry 21:4438-4442 (1982)); O'Brien et al., Biochemistry 16:3105-3109 (1977); O'Brien and Gennis, J. Biol. Chem. 255:3302-3307 (1980)) and a flavin adenine dinucleotide (FAD) cofactor. Acetate can then be converted into acetyl-CoA by either acetyl-CoA synthetase or by acetate kinase and phosphotransacetylase, as described earlier. Some of these enzymes can also catalyze the reverse reaction from acetyl-CoA to pyruvate. For enzymes that use reducing equivalents in the form of NADH or NADPH, these reduced carriers can be generated by transferring electrons from reduced ferredoxin. Two enzymes catalyze the reversible transfer of electrons from reduced ferredoxins to NAD(P)f, ferredoxin:NAD+ oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP oxidoreductase (FNR, 210 EC 1.18.1.2). Ferredoxin:NADP oxidoreductase (FNR, EC 1.18.1.2) has a noncovalently bound FAD cofactor that facilitates the reversible transfer of electrons from NADPH to low potential acceptors such as ferredoxins or flavodoxins (Blaschkowski et al., Eur. J Biochem. 123:563-569 (1982); Fujii et al., 1977). The Helicobacterpylori FNR, encoded by HP1164 (fqrB), is coupled to the activity of pyruvate:ferredoxin oxidoreductase (PFOR) resulting in the pyruvate-dependent production of NADPH (St et al. 2007). An analogous enzyme is found in Campylobacterjejuni (St et al. 2007). A ferredoxin:NADP oxidoreductase enzyme is encoded in the E. coli genome byfpr (Bianchi et al. 1993). Ferredoxin:NAD+ oxidoreductase utilizes reduced ferredoxin to generate NADH from NAD+. In several organisms, including E. coli, this enzyme is a component of multifunctional dioxygenase enzyme complexes. The ferredoxin:NAD+ oxidoreductase of E. coli, encoded by hcaD, is a component of the 3-phenylproppionate dioxygenase system involved in involved in aromatic acid utilization (Diaz et al. 1998). NADH:ferredoxin reductase activity was detected in cell extracts of Hydrogenobacter thermophilus strain TK-6, although a gene with this activity has not yet been indicated (Yoon et al. 2006). Finally, the energy-conserving membrane associated Rnf-type proteins (Seedorf et al., Proc. Natl. Acad. Sci. U.S.A. 105:2128-2133 (2008); Herrmann et al., J Bacteriol. 190:784-791 (2008)) provide a means to generate NADH or NADPH from reduced ferredoxin. Additional ferredoxin:NAD(P)+ oxidoreductases have been annotated in Clostridium carboxydivorans P7. Protein GenBank ID GI Number Organism HP1164 NP_207955.1 15645778 Helicobacterpylori RPA3 954 CAE29395.1 39650872 Rhodopseudomonas palustris fpr BAH29712.1 225320633 Hydrogenobacter thermophilus yumC NP_391091.2 255767736 Bacillus subtilis CJE0663 AAW35824.1 57167045 Campylobacterjejuni fpr P28861.4 399486 Escherichia coli hcaD AAC75595.1 1788892 Escherichia coli LOC100282643 NP_001149023.1 226497434 Zea mays RnfC EDK33306.1 146346770 Clostridium kluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri RnfG EDK33308.1 146346772 Clostridium kluyveri 211 RnfE EDK33309.1 146346773 Clostridium kluyveri RnfA EDK33310.1 146346774 Clostridium kluyveri RnfB EDK333 11.1 146346775 Clostridium kluyveri CcarbDRAFT_2639 ZP_05392639.1 255525707 Clostridium carboxidivorans P7 CcarbDRAFT_2638 ZP_05392638.1 255525706 Clostridium carboxidivorans P7 CcarbDRAFT_2636 ZP_05392636.1 255525704 Clostridium carboxidivorans P7 CcarbDRAFT 5060 ZP_05395060.1 255528241 Clostridium carboxidivorans P7 CcarbDRAFT_2450 ZP_05392450.1 255525514 Clostridium carboxidivorans P7 CcarbDRAFT 1084 ZP_05391084.1 255524124 Clostridium carboxidivorans P7 Ferredoxins are small acidic proteins containing one or more iron-sulfur clusters that function as intracellular electron carriers with a low reduction potential. Reduced ferredoxins donate electrons to Fe-dependent enzymes such as ferredoxin-NADP oxidoreductase, pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OFOR). The H. thermophilus genefdx1 encodes a [4Fe-4S]-type ferredoxin that is required for the reversible carboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR, respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). The ferredoxin associated with the Sulfolobus so/fataricus 2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S] type ferredoxin (Park et al. 2006). While the gene associated with this protein has not been fully sequenced, the N-terminal domain shares 93% homology with the zfx ferredoxin from S. acidocaldarius. The E. coli genome encodes a soluble ferredoxin of unknown physiological function,fdx. Some evidence indicates that this protein can function in iron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additional ferredoxin proteins have been characterized in Helicobacter pylori (Mukhopadhyay et al. 2003) and Campylobacterjejuni (van Vliet et al. 2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been cloned and expressed in E. coli (Fujinaga and Meyer, Biochemical and Biophysical Research Communications, 192(3): (1993)). Acetogenic bacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7 and Rhodospirillum rubrum are predicted to encode several ferredoxins, listed in the table below.

212 Protein GenBank ID GI Number Organism fdxl BAE02673.1 68163284 Hydrogenobacter thermophilus M 11214.1 AAA83524.1 144806 Clostridium pasteurianum Zfx AAY79867.1 68566938 Sulfolobus acidocalarius Fdx AAC75578.1 1788874 Escherichia coli hp_0277 AAD07340.1 2313367 Helicobacterpylori fdxA CAL34484.1 112359698 Campylobacterjejuni Moth_0061 ABC18400.1 83571848 Moorella thermoacetica Moth_1200 ABC19514.1 83572962 Moorella thermoacetica Moth_1888 ABC20188.1 83573636 Moorella thermoacetica Moth_2112 ABC20404.1 83573852 Moorella thermoacetica Moth_1037 ABC19351.1 83572799 Moorella thermoacetica CcarbDRAFT_4383 ZP_05394383.1 255527515 Clostridium carboxidivorans P7 CcarbDRAFT_2958 ZP_05392958.1 255526034 Clostridium carboxidivorans P7 CcarbDRAFT_2281 ZP_05392281.1 255525342 Clostridium carboxidivorans P7 CcarbDRAFT_5296 ZP_05395295.1 255528511 Clostridium carboxidivorans P7 CcarbDRAFT_1615 ZP_05391615.1 255524662 Clostridium carboxidivorans P7 CcarbDRAFT_1304 ZP_05391304.1 255524347 Clostridium carboxidivorans P7 cooF AAG29808.1 11095245 Carboxydothermus hydrogenoformans fdxN CAA35699.1 46143 Rhodobacter capsulatus RruA2264 ABC23064.1 83576513 Rhodospirillum rubrum RruA1916 ABC22716.1 83576165 Rhodospirillum rubrum RruA2026 ABC22826.1 83576275 Rhodospirillum rubrum cooF AAC45122.1 1498747 Rhodospirillum rubrum fdxN AAA26460.1 152605 Rhodospirillum rubrum Alvin_2884 ADC63789.1 288897953 Allochromatium vinosum DSM 180 fdx YP_002801146.1 226946073 Azotobacter vinelandii DJ CKL_3790 YP_001397146.1 153956381 Clostridium kluyveri DSM 555 ferI NP_949965.1 39937689 Rhodopseudomonas palustris CGA009 fdx CAA12251.1 3724172 Thauera aromatica 213 CHY_2405 YP_361202.1 78044690 Carboxydothermus hydrogenoformans fer YP_359966.1 78045103 Carboxydothermus hydrogenoformans fer AAC83945.1 1146198 Bacillus subtilis fdx1 NP_249053.1 15595559 Pseudomonas aeruginosa PA01 yfhL AP_003148.1 89109368 Escherichia coli K-12 Succinyl-CoA transferase catalyzes the conversion of succinyl-CoA to succinate while transferring the CoA moiety to a CoA acceptor molecule. Many transferases have broad specificity and can utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3-ketopentanoate, valerate, crotonate, 3 mercaptopropionate, propionate, vinylacetate, and butyrate, among others. The conversion of succinate to succinyl-CoA can be carried by a transferase which does not require the direct consumption of an ATP or GTP. This type of reaction is common in a number of organisms. The conversion of succinate to succinyl-CoA can also be catalyzed by succinyl-CoA:Acetyl-CoA transferase. The gene product of cat] of Clostridium kluyveri has been shown to exhibit succinyl-CoA: acetyl-CoA transferase activity (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996)). In addition, the activity is present in Trichomonas vaginalis (van Grinsven et al. 2008) and Trypanosoma brucei (Riviere et al. 2004). The succinyl-CoA:acetate CoA-transferase from Acetobacter aceti, encoded by aarC, replaces succinyl-CoA synthetase in a variant TCA cycle (Mullins et al. 2008). Similar succinyl-CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al. 2008), Trypanosoma brucei (Riviere et al. 2004) and Clostridium kluyveri (Sohling and Gottschalk, 1996c). The beta-ketoadipate:succinyl-CoA transferase encoded by pcaI and pcaJ in Pseudomonas putida is yet another candidate (Kaschabek et al. 2002). The aforementioned proteins are identified below. Protein GenBank ID GI Number Organism cat] P38946.1 729048 Clostridium kluyveri TVAG_395550 XP_001330176 123975034 Trichomonas vaginalis G3 Tbl].02.0290 XP_828352 71754875 Trypanosoma brucei 214 pcaI AAN69545.1 24985644 Pseudomonas putida pcaJ NP_746082.1 26990657 Pseudomonas putida aarC ACD85596.1 189233555 Acetobacter aceti An additional exemplary transferase that converts succinate to succinyl-CoA while converting a 3-ketoacyl-CoA to a 3-ketoacid is succinyl-CoA:3:ketoacid-CoA transferase (EC 2.8.3.5). Exemplary succinyl-CoA:3:ketoacid-CoA transferases are present in Helicobacterpylori (Corthesy-Theulaz et al. 1997), Bacillus subtilis, and Homo sapiens (Fukao et al. 2000; Tanaka et al. 2002). The aforementioned proteins are identified below. Protein GenBank ID GI Number Organism HPAGJ_0676 YP_627417 108563101 Helicobacterpylori HPAGJ_0677 YP_627418 108563102 Helicobacterpylori ScoA NP_391778 16080950 Bacillus subtilis ScoB NP_391777 16080949 Bacillus subtilis OXCT1 NP_000427 4557817 Homo sapiens OXCT2 NP_071403 11545841 Homo sapiens Converting succinate to succinyl-CoA by succinyl-CoA:3:ketoacid-CoA transferase requires the simultaneous conversion of a 3-ketoacyl-CoA such as acetoacetyl-CoA to a 3 ketoacid such as acetoacetate. Conversion of a 3-ketoacid back to a 3-ketoacyl-CoA can be catalyzed by an acetoacetyl-CoA: acetate: CoA transferase. Acetoacetyl-CoA: acetate: CoA transferase converts acetoacetyl-CoA and acetate to acetoacetate and acetyl-CoA, or vice versa. Exemplary enzymes include the gene products of atoAD from E. coli (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007), ctfAB from C. acetobutylicum (Jojima et al., Appl Microbiol Biotechnol 77:1219-1224 (2008), and ctfAB from Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71:5 8-68 (2007)) are shown below.

215 Protein GenBank ID GI Number Organism AtoA NP_416726.1 2492994 Escherichia coli AtoD NP_416725.1 2492990 Escherichia coli CtfA NP_149326.1 15004866 Clostridium acetobutylicum CtfB NP_149327.1 15004867 Clostridium acetobutylicum CtfA AAP42564.1 31075384 Clostridium saccharoperbutylacetonicum CtfB AAP42565.1 31075385 Clostridium saccharoperbutylacetonicum Yet another possible CoA acceptor is benzylsuccinate. Succinyl-CoA:(R) Benzylsuccinate CoA-Transferase functions as part of an anaerobic degradation pathway for toluene in organisms such as Thauera aromatica (Leutwein and Heider, J Bact. 183(14) 4288-4295 (2001)). Homologs can be found in Azoarcus sp. T, Aromatoleum aromaticum EbN1, and Geobacter metallireducens GS-15. The aforementioned proteins are identified below. Protein GenBank ID GI Number Organism bbsE AAF89840 9622535 Thauera aromatic Bbsf AAF89841 9622536 Thauera aromatic bbsE AAU45405.1 52421824 Azoarcus sp. T bbsF AAU45406.1 52421825 Azoarcus sp. T bbsE YP_158075.1 56476486 Aromatoleum aromaticum EbNJ bbsF YP_158074.1 56476485 Aromatoleum aromaticum EbNJ Gmet_1521 YP_384480.1 78222733 Geobacter metallireducens GS-15 Gmet_1522 YP_384481.1 78222734 Geobacter metallireducens GS-15 Additionally, ygfH encodes a propionyl CoA:succinate CoA transferase in E. coli (Haller et al., Biochemistry, 39(16) 4622-4629). Close homologs can be found in, for 216 example, Citrobacteryoungae ATCC 29220, Salmonella enterica subsp. arizonae serovar, and Yersinia intermedia ATCC 29909. The aforementioned proteins are identified below. Protein GenBank ID GI Number Organism ygfH NP_417395.1 16130821 Escherichia coli str. K-12 substr. MG1655 CIT292_04485 ZP_03838384.1 227334728 Citrobacter youngae A TCC 29220 SARI_04582 YP_001573497.1 161506385 Salmonella enterica subsp. arizonae serovar yinte000l_14430 ZP_04635364.1 238791727 Yersinia intermedia A TCC 29909 Citrate lyase (EC 4.1.3.6) catalyzes a series of reactions resulting in the cleavage of citrate to acetate and oxaloacetate. The enzyme is active under anaerobic conditions and is composed of three subunits: an acyl-carrier protein (ACP, gamma), an ACP transferase (alpha), and a acyl lyase (beta). Enzyme activation uses covalent binding and acetylation of an unusual prosthetic group, 2'-(5"-phosphoribosyl)-3-'-dephospho-CoA, which is similar in structure to acetyl-CoA. Acylation is catalyzed by CitC, a citrate lyase synthetase. Two additional proteins, CitG and CitX, are used to convert the apo enzyme into the active holo enzyme (Schneider et al., Biochemistry 39:9438-9450 (2000)). Wild type E. coli does not have citrate lyase activity; however, mutants deficient in molybdenum cofactor synthesis have an active citrate lyase (Clark, FEMS Microbiol. Lett. 55:245-249 (1990)). The E. coli enzyme is encoded by citEFD and the citrate lyase synthetase is encoded by citC (Nilekani and SivaRaman, Biochemistry 22:4657-4663 (1983)). The Leuconostoc mesenteroides citrate lyase has been cloned, characterized and expressed in E. coli (Bekal et al., J Bacteriol. 180:647-654 (1998)). Citrate lyase enzymes have also been identified in enterobacteria that utilize citrate as a carbon and energy source, including Salmonella typhimurium and Klebsiella pneumoniae (Bott, Arch. Microbiol. 167: 78-88 (1997); Bott and Dimroth, Mol. Microbiol. 14:347-356 (1994)). The aforementioned proteins are tabulated below.

217 Protein GenBank ID GI Number Organism citF AAC73716.1 1786832 Escherichia coli Cite AAC73717.2 87081764 Escherichia coli citD AAC73718.1 1786834 Escherichia coli citC AAC73719.2 87081765 Escherichia coli citG AAC73714.1 1786830 Escherichia coli citX AAC73715.1 1786831 Escherichia coli citF CAA71633.1 2842397 Leuconostoc mesenteroides Cite CAA71632.1 2842396 Leuconostoc mesenteroides citD CAA71635.1 2842395 Leuconostoc mesenteroides citC CAA71636.1 3413797 Leuconostoc mesenteroides citG CAA71634.1 2842398 Leuconostoc mesenteroides citX CAA71634.1 2842398 Leuconostoc mesenteroides citF NP_459613.1 16763998 Salmonella typhimurium cite AAL19573.1 16419133 Salmonella typhimurium citD NP_459064.1 16763449 Salmonella typhimurium citC NP_459616.1 16764001 Salmonella typhimurium citG NP_459611.1 16763996 Salmonella typhimurium citX NP_459612.1 16763997 Salmonella typhimurium citF CAA56217.1 565619 Klebsiella pneumoniae cite CAA56216.1 565618 Klebsiella pneumoniae citD CAA56215.1 565617 Klebsiella pneumoniae citC BAH66541.1 238774045 Klebsiella pneumoniae citG CAA56218.1 565620 Klebsiella pneumoniae citX AAL60463.1 18140907 Klebsiella pneumoniae 218 Acetate kinase (EC 2.7.2.1) catalyzes the reversible ATP-dependent phosphorylation of acetate to acetylphosphate. Exemplary acetate kinase enzymes have been characterized in many organisms including E. coli, Clostridium acetobutylicum and Methanosarcina thermophila (Ingram-Smith et al., J Bacteriol. 187:2386-2394 (2005); Fox and Roseman, J Biol. Chem. 261:13487-13497 (1986); Winzer et al., Microbioloy 143 (Pt 10):3279-3286 (1997)). Acetate kinase activity has also been demonstrated in the gene product of E. coli purT (Marolewski et al., Biochemistry 33:2531-2537 (1994). Some butyrate kinase enzymes (EC 2.7.2.7), for example buk1 and buk2 from Clostridium acetobutylicum, also accept acetate as a substrate (Hartmanis, M.G., J Biol. Chem. 262:617-621 (1987)). Protein GenBank ID GI Number Organism ackA NP_416799.1 16130231 Escherichia coli Ack AAB18301.1 1491790 Clostridium acetobutylicum Ack AAA72042.1 349834 Methanosarcina thermophila purT AAC74919.1 1788155 Escherichia coli buki NP_349675 15896326 Clostridium acetobutylicum buk2 Q97111 20137415 Clostridium acetobutylicum The formation of acetyl-CoA from acetylphosphate is catalyzed by phosphotransacetylase (EC 2.3.1.8). The pta gene from E. coli encodes an enzyme that reversibly converts acetyl-CoA into acetyl-phosphate (Suzuki, T., Biochim. Biophys. Acta 191:559-569 (969)). Additional acetyltransferase enzymes have been characterized in Bacillus subtilis (Rado and Hoch, Biochim. Biophys. Acta 321:114-125 (1973), Clostridium kluyveri (Stadtman, E., Methods Enzymol. 1:5896-599 (1955), and Thermotoga maritima (Bock et al., J Bacteriol. 181:1861-1867 (1999)). This reaction is also catalyzed by some phosphotranbutyrylase enzymes (EC 2.3.1.19) including the ptb gene products from Clostridium acetobutylicum (Wiesenbom et al., App. Environ. Microbiol. 55:317-322 (1989); Walter et al., Gene 134:107-111 (1993)). Additionalptb genes are found in butyrate producing bacterium L2-50 (Louis et al., J Bacteriol. 186:2099-2106 (2004) and Bacillus megaterium (Vazquez et al., Curr. Microbiol. 42:345-349 (2001).

219 Protein GenBank ID GI Number Organism Pta NP_416800.1 71152910 Escherichia coli Pta P39646 730415 Bacillus subtilis Pta A5N801 146346896 Clostridium kluyveri Pta Q9XOL4 6685776 Thermotoga maritima Ptb NP_349676 34540484 Clostridium acetobutylicum Ptb AAR19757.1 butyrate-producing bacterium 38425288 L2-50 Ptb CAC07932.1 10046659 Bacillus megaterium The acylation of acetate to acetyl-CoA is catalyzed by enzymes with acetyl-CoA synthetase activity. Two enzymes that catalyze this reaction are AMP-forming acetyl-CoA synthetase (EC 6.2.1.1) and ADP-forming acetyl-CoA synthetase (EC 6.2.1.13). AMP forming acetyl-CoA synthetase (ACS) is the predominant enzyme for activation of acetate to acetyl-CoA. Exemplary ACS enzymes are found in E. coli (Brown et al., J. Gen. Microbiol. 102:327-336 (1977)), Ralstonia eutropha (Priefert and Steinbuchel, J Bacteriol. 174:6590 6599 (1992)), Methanothermobacter thermautotrophicus (Ingram-Smith and Smith, Archaea 2:95-107 (2007)), Salmonella enterica (Gulick et al., Biochemistry 42:2866-2873 (2003)) and Saccharomyces cerevisiae (Jogl and Tong, Biochemistry 43:1425-1431 (2004)). ADP forming acetyl-CoA synthetases are reversible enzymes with a generally broad substrate range (Musfeldt and Schonheit, J Bacteriol. 184:636-644 (2002)). Two isozymes of ADP forming acetyl-CoA synthetases are encoded in the Archaeoglobusfulgidus genome by are encoded by AF1211 and AF1983 (Musfeldt and Schonheit, supra (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) also accepts acetate as a substrate and reversibility of the enzyme was demonstrated (Brasen and Schonheit, Arch. Microbiol. 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetate, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen and Schonheit, supra (2004)). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A. fulgidus, H. marismortui and P. aerophilum have all been 220 cloned, functionally expressed, and characterized in E. coli (Brasen and Schonheit, supra (2004); Musfeldt and Schonheit, supra (2002)). Additional candidates include the succinyl CoA synthetase encoded by sucCD in E. coli (Buck et al., Biochemistry 24:6245-6252 (1985)) and the acyl-CoA ligase from Pseudomonas putida (Fernandez-Valverde et al., AppI. Environ. Microbiol. 59:1149-1154 (1993)). The aforementioned proteins are tabulated below. Protein GenBank ID GI Number Organism acs AAC77039.1 1790505 Escherichia coli acoE AAA21945.1 141890 Ralstonia eutropha acs] ABC87079.1 86169671 Methanothermobacter thermautotrophicus acs] AAL23099.1 16422835 Salmonella enterica ACS] Q01574.2 257050994 Saccharomyces cerevisiae AF1211 NP_070039.1 11498810 Archaeoglobusfulgidus AF1983 NP_070807.1 11499565 Archaeoglobusfulgidus scs YP_135572.1 55377722 Haloarcula marismortui PAE3250 NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2 sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia coli paaF AAC24333.2 22711873 Pseudomonas putida Conversion of acetyl-CoA to malonyl-CoA can be carried out by an acetyl-CoA carboxylase enzyme. These enzymes contain multiple subunits. Three of such enzymes are provided below.

221 Accession Gene Organism number GI Number accA Escherichia coli K-12 AAC73296.1 1786382 accB Escherichia coli K-12 AAC76287.1 1789653 accC Escherichia coli K-12 AAC76288.1 1789654 accD Escherichia coli K-12 AAC75376.1 1788655 accA Salmonella enterica CAD08690.1 16501513 accB Salmonella enterica CAD07894.1 16504441 accC Salmonella enterica CAD07895.1 16504442 accD Salmonella enterica CAD07598.1 16503590 YMR207C Saccharomyces cerevisiae NP_013934.1 6323863 YNR016C Saccharomyces cerevisiae NP_014413.1 6324343 YGR037C Saccharomyces cerevisiae NP_011551.1 6321474 YKL182W Saccharomyces cerevisiae NP_012739.1 6322666 YPL231W Saccharomyces cerevisiae NP_015093.1 6325025 The product yields per C-mol of substrate of microbial cells synthesizing reduced fermentation products such as 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene, are limited by insufficient reducing equivalents in the carbohydrate feedstock. Reducing equivalents, or electrons, can be extracted from synthesis gas components such as CO and H 2 using carbon monoxide dehydrogenase (CODH) and hydrogenase enzymes, respectively. The reducing equivalents are then passed to acceptors such as oxidized ferredoxins, oxidized quinones, oxidized cytochromes, NAD(P)+, water, or hydrogen peroxide to form reduced ferredoxin, reduced quinones, reduced cytochromes, NAD(P)H, H 2 , or water, respectively. Reduced ferredoxin and NAD(P)H are particularly useful as they can serve as redox carriers for various Wood-Ljungdahl pathway and reductive TCA cycle enzymes. Herein below the enzymes and the corresponding genes used for extracting redox from synags components are described. CODH is a reversible enzyme that interconverts CO and CO 2 at the expense or gain of electrons. The natural physiological role of the CODH in 222 ACS/CODH complexes is to convert CO 2 to CO for incorporation into acetyl-CoA by acetyl CoA synthase. Nevertheless, such CODH enzymes are suitable for the extraction of reducing equivalents from CO due to the reversible nature of such enzymes. Expressing such CODH enzymes in the absence of ACS allows them to operate in the direction opposite to their natural physiological role (i.e., CO oxidation). In M. thermoacetica, C. hydrogenoformans, C. carboxidivorans P7, and several other organisms, additional CODH encoding genes are located outside of the ACS/CODH operons. These enzymes provide a means for extracting electrons (or reducing equivalents) from the conversion of carbon monoxide to carbon dioxide. The M. thermoacetica gene (Genbank Accession Number: YP_430813) is expressed by itself in an operon and is believed to transfer electrons from CO to an external mediator like ferredoxin in a "Ping-pong" reaction. The reduced mediator then couples to other reduced nicolinamide adenine dinucleotide phosphate (NAD(P)H) carriers or ferredoxin-dependent cellular processes (Ragsdale, Annals of the New York Academy of Sciences 1125: 129-136 (2008)). The genes encoding the C. hydrogenoformans CODH-II and CooF, a neighboring protein, were cloned and sequenced (Gonzalez and Robb, FEMS Microbiol Lett. 191:243-247 (2000)). The resulting complex was membrane-bound, although cytoplasmic fractions of CODH-II were shown to catalyze the formation of NADPH suggesting an anabolic role (Svetlitchnyi et al., JBacteriol. 183:5134-5144 (2001)). The crystal structure of the CODH-II is also available (Dobbek et al., Science 293:1281-1285 (2001)). Similar ACS-free CODH enzymes can be found in a diverse array of organisms including Geobacter metallireducens GS-15, Chlorobium phaeobacteroides DSM 266, Clostridium cellulolyticum H 10, Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774, Pelobacter carbinolicus DSM 2380, and Campylobacter curvus 525.92. Protein GenBank ID GI Number Organism CODH (putative) YP_430813 83590804 Moorella thermoacetica CODH-II (CooS-II) YP_358957 78044574 Carboxydothermus hydrogenoformans CooF YP_358958 78045112 Carboxydothermus hydrogenoformans CODH (putative) ZP_05390164.1 255523193 Clostridium carboxidivorans P7 223 CcarbDRAFT_0341 ZP_05390341.1 255523371 Clostridium carboxidivorans P7 CcarbDRAFT_1756 ZP_05391756.1 255524806 Clostridium carboxidivorans P7 CcarbDRAFT_2944 ZP_05392944.1 255526020 Clostridium carboxidivorans P7 CODH YP_384856.1 78223109 Geobacter metallireducens GS-15 Cpha266_0148 YP_910642.1 119355998 Chlorobium (cytochrome c) phaeobacteroides DSM 266 Cpha266_0149 YP_910643.1 119355999 Chlorobium (CODH) phaeobacteroides DSM 266 Ccel_0438 YP_002504800.1 220927891 Clostridium cellulolyticum Hi0 Ddes_0382 YP_002478973.1 220903661 Desulfovibrio desulfuricans (CODH) subsp. desulfuricans str. ATCC 27774 Ddes_0381 (CooC) YP_002478972.1 220903660 Desulfovibrio desulfuricans subsp. desulfuricans str. ATCC 27774 Pcar_0057 YP_355490.1 7791767 Pelobacter carbinolicus DSM (CODH) 2380 Pcar_0058 YP_355491.1 7791766 Pelobacter carbinolicus DSM (CooC) 2380 Pcar_0058 YP_355492.1 7791765 Pelobacter carbinolicus DSM (HypA) 2380 CooS (CODH) YP_001407343.1 154175407 Campylobacter curvus 525.92 In some cases, hydrogenase encoding genes are located adjacent to a CODH. In Rhodospirillum rubrum, the encoded CODH/hydrogenase proteins form a membrane-bound enzyme complex that has been indicated to be a site where energy, in the form of a proton gradient, is generated from the conversion of CO and H 2 0 to CO 2 and H 2 (Fox et al., J Bacteriol. 178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and its adjacent genes have been proposed to catalyze a similar functional role based on their similarity to the R. rubrum CODH/hydrogenase gene cluster (Wu et al., PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-I was also shown to exhibit intense CO oxidation and CO 2 reduction activities when linked to an electrode (Parkin et al., JAm.Chem.Soc. 129:10328- 224 10329 (2007)). The protein sequences of exemplary CODH and hydrogenase genes can be identified by the following GenBank accession numbers. Protein GenBank ID GI Number Organism CODH-I YP_360644 78043418 Carboxydothermus (CooS-I) hydrogenoformans CooF YP_360645 78044791 Carboxydothermus hydrogenoformans HypA YP_360646 78044340 Carboxydothermus hydrogenoformans CooH YP_360647 78043871 Carboxydothermus hydrogenoformans CooU YP_360648 78044023 Carboxydothermus hydrogenoformans CooX YP_360649 78043124 Carboxydothermus hydrogenoformans CooL YP_360650 78043938 Carboxydothermus hydrogenoformans CooK YP_360651 78044700 Carboxydothermus hydrogenoformans CooM YP_360652 78043942 Carboxydothermus hydrogenoformans CooC YP_360654.1 78043296 Carboxydothermus hydrogenoformans CooA-1 YP_360655.1 78044021 Carboxydothermus hydrogenoformans CooL AAC45118 1515468 Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum rubrum CooU AAC45120 1515470 Rhodospirillum rubrum CooH AAC45121 1498746 Rhodospirillum rubrum CooF AAC45122 1498747 Rhodospirillum rubrum CODH (CooS) AAC45123 1498748 Rhodospirillum rubrum 225 CooC AAC45124 1498749 Rhodospirillum rubrum CooT AAC45125 1498750 Rhodospirillum rubrum CooJ AAC45126 1498751 Rhodospirillum rubrum Native to E. coli and other enteric bacteria are multiple genes encoding up to four hydrogenases (Sawers, G., Antonie Van Leeuwenhoek 66:57-88 (1994); Sawers et al., J Bacteriol. 164:1324-1331 (1985); Sawers and Boxer, Eur.JBiochem. 156:265-275 (1986); Sawers et al., JBacteriol. 168:398-404 (1986)). Given the multiplicity of enzyme activities, E. coli or another host organism can provide sufficient hydrogenase activity to split incoming molecular hydrogen and reduce the corresponding acceptor. E. coli possesses two uptake hydrogenases, Hyd-1 and Hyd-2, encoded by the hyaABCDEF and hybOABCDEFG gene clusters, respectively (Lukey et al., How E. coli is equipped to oxidize hydrogen under different redox conditions, JBiol Chem published online Nov 16, 2009). Hyd-1 is oxygen tolerant, irreversible, and is coupled to quinone reduction via the hyaC cytochrome. Hyd-2 is sensitive to 02, reversible, and transfers electrons to the periplasmic ferredoxin hybA which, in turn, reduces a quinone via the hybB integral membrane protein. Reduced quinones can serve as the source of electrons for fumarate reductase in the reductive branch of the TCA cycle. Reduced ferredoxins can be used by enzymes such as NAD(P)H:ferredoxin oxidoreductases to generate NADPH or NADH. They can alternatively be used as the electron donor for reactions such as pyruvate ferredoxin oxidoreductase, AKG ferredoxin oxidoreductase, and 5,10-methylene-H4folate reductase. Protein GenBank ID GI Number Organism HyaA AAC74057.1 1787206 Escherichia coli HyaB AAC74058.1 1787207 Escherichia coli HyaC AAC74059.1 1787208 Escherichia coli HyaD AAC74060.1 1787209 Escherichia coli HyaE AAC74061.1 1787210 Escherichia coli HyaF AAC74062.1 1787211 Escherichia coli 226 Protein GenBank ID GI Number Organism HybO AAC76033.1 1789371 Escherichia coli HybA AAC76032.1 1789370 Escherichia coli HybB AAC76031.1 2367183 Escherichia coli HybC AAC76030.1 1789368 Escherichia coli HybD AAC76029.1 1789367 Escherichia coli HybE AAC76028.1 1789366 Escherichia coli HybF AAC76027.1 1789365 Escherichia coli HybG AAC76026.1 1789364 Escherichia coli The hydrogen-lyase systems of E. coli include hydrogenase 3, a membrane-bound enzyme complex using ferredoxin as an acceptor, and hydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4 are encoded by the hyc and hyf gene clusters, respectively. Hydrogenase 3 has been shown to be a reversible enzyme (Maeda et al., Appl Microbiol Biotechnol 76(5):1035-42 (2007)). Hydrogenase activity in E. coli is also dependent upon the expression of the hyp genes whose corresponding proteins are involved in the assembly of the hydrogenase complexes (Jacobi et al., Arch.Microbiol 158:444-451 (1992); Rangarajan et al., J Bacteriol. 190:1447-1458 (2008)). Protein GenBank ID GI Number Organism HycA NP_417205 16130632 Escherichia coli HycB NP_417204 16130631 Escherichia coli HycC NP_417203 16130630 Escherichia coli HycD NP_417202 16130629 Escherichia coli HycE NP_417201 16130628 Escherichia coli HycF NP_417200 16130627 Escherichia coli 227 HycG NP_417199 16130626 Escherichia coli HycH NP_417198 16130625 Escherichia coli HycI NP_417197 16130624 Escherichia coli Protein GenBank ID GI Number Organism HyfA NP_416976 90111444 Escherichia coli HyfB NP_416977 16130407 Escherichia coli HyfC NP_416978 90111445 Escherichia coli HyfD NP_416979 16130409 Escherichia coli HyfE NP_416980 16130410 Escherichia coli HyfF NP_416981 16130411 Escherichia coli HyfG NP_416982 16130412 Escherichia coli HyfH NP_416983 16130413 Escherichia coli Hyfi NP_416984 16130414 Escherichia coli HyfJ NP_416985 90111446 Escherichia coli HyfR NP_416986 90111447 Escherichia coli Protein GenBank ID GI Number Organism HypA NP_417206 16130633 Escherichia coli HypB NP_417207 16130634 Escherichia coli HypC NP_417208 16130635 Escherichia coli HypD NP_417209 16130636 Escherichia coli HypE NP_417210 226524740 Escherichia coli HypF NP_417192 16130619 Escherichia coli 228 The M. thermoacetica hydrogenases are suitable for a host that lacks sufficient endogenous hydrogenase activity. M. thermoacetica can grow with CO 2 as the exclusive carbon source indicating that reducing equivalents are extracted from H 2 to enable acetyl CoA synthesis via the Wood-Ljungdahl pathway (Drake, H. L., J Bacteriol. 150:702-709 (1982); Drake and Daniel, Res. Microbiol. 155:869-883 (2004); Kellum and Drake, J Bacteriol. 160:466-469 (1984)) (see Figure 22). M. thermoacetica has homologs to several hyp, hyc, and hyf genes from E. coli. The protein sequences encoded for by these genes are identified by the following GenBank accession numbers. Proteins in M. thermoacetica whose genes are homologous to the E. coli hyp genes are shown below. Protein GenBank ID GI Number Organism Moth_2175 YP_431007 83590998 Moorella thermoacetica Moth_2176 YP_431008 83590999 Moorella thermoacetica Moth_2177 YP_431009 83591000 Moorella thermoacetica Moth_2178 YP_431010 83591001 Moorella thermoacetica Moth_2179 YP_431011 83591002 Moorella thermoacetica Moth_2180 YP_431012 83591003 Moorella thermoacetica Moth_2181 YP_431013 83591004 Moorella thermoacetica Proteins in M. thermoacetica that are homologous to the E. coli Hydrogenase 3 and/or 4 proteins are listed in the following table. Protein GenBank ID GI Number Organism Moth_2182 YP_431014 83591005 Moorella thermoacetica Moth_2183 YP_431015 83591006 Moorella thermoacetica Moth_2184 YP_431016 83591007 Moorella thermoacetica Moth_2185 YP_431017 83591008 Moorella thermoacetica 229 Moth_2186 YP_431018 83591009 Moorella thermoacetica Moth_2187 YP_431019 83591010 Moorella thermoacetica Moth_2188 YP_431020 83591011 Moorella thermoacetica Moth_2189 YP_431021 83591012 Moorella thermoacetica Moth_2190 YP_431022 83591013 Moorella thermoacetica Moth_2191 YP_431023 83591014 Moorella thermoacetica Moth_2192 YP_431024 83591015 Moorella thermoacetica In addition, several gene clusters encoding hydrogenase functionality are present in M. thermoacetica and their corresponding protein sequences are provided below. Protein GenBank ID GI Number Organism Moth_0439 YP_429313 83589304 Moorella thermoacetica Moth_0440 YP_429314 83589305 Moorella thermoacetica Moth_0441 YP_429315 83589306 Moorella thermoacetica Moth_0442 YP_429316 83589307 Moorella thermoacetica Moth_0809 YP_429670 83589661 Moorella thermoacetica Moth_0810 YP_429671 83589662 Moorella thermoacetica Moth_0811 YP_429672 83589663 Moorella thermoacetica Moth_0812 YP_429673 83589664 Moorella thermoacetica Moth_0814 YP_429674 83589665 Moorella thermoacetica Moth_0815 YP_429675 83589666 Moorella thermoacetica Moth_0816 YP_429676 83589667 Moorella thermoacetica Moth_1193 YP_430050 83590041 Moorella thermoacetica Moth_1194 YP_430051 83590042 Moorella thermoacetica 230 Moth_1195 YP_430052 83590043 Moorella thermoacetica Moth_1196 YP_430053 83590044 Moorella thermoacetica Moth_1717 YP_430562 83590553 Moorella thermoacetica Moth_1718 YP_430563 83590554 Moorella thermoacetica Moth_1719 YP_430564 83590555 Moorella thermoacetica Moth_1883 YP_430726 83590717 Moorella thermoacetica Moth_1884 YP_430727 83590718 Moorella thermoacetica Moth_1885 YP_430728 83590719 Moorella thermoacetica Moth_1886 YP_430729 83590720 Moorella thermoacetica Moth_1887 YP_430730 83590721 Moorella thermoacetica Moth_1888 YP_430731 83590722 Moorella thermoacetica Moth_1452 YP_430305 83590296 Moorella thermoacetica Moth_1453 YP_430306 83590297 Moorella thermoacetica Moth_1454 YP_430307 83590298 Moorella thermoacetica Ralstonia eutropha H16 uses hydrogen as an energy source with oxygen as a terminal electron acceptor. Its membrane-bound uptake [NiFe]-hydrogenase is an "02-tolerant" hydrogenase (Cracknell, et al. Proc Nat Acad Sci, 106(49) 20681-20686 (2009)) that is periplasmically-oriented and connected to the respiratory chain via a b-type cytochrome (Schink and Schlegel, Biochim. Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. J Biochem. 248, 179-186 (1997)). R. eutropha also contains an 0 2 -tolerant soluble hydrogenase encoded by the Hox operon which is cytoplasmic and directly reduces NAD+ at the expense of hydrogen (Schneider and Schlegel, Biochim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J Bact. 187(9) 3122-3132(2005)). Soluble hydrogenase enzymes are additionally present in several other organisms including Geobacter sulfurreducens (Coppi, Microbiology 151, 1239-1254 (2005)), Synechocystis str. PCC 6803 (Germer, J Biol. Chem., 284(52), 36462-36472 (2009)), and Thiocapsa roseopersicina (Rakhely, Appl. Environ. Microbiol. 70(2) 722-728 (2004)). The Synechocystis enzyme is capable of generating NADPH from hydrogen. Overexpression of both the Hox operon from Synechocystis str. PCC 6803 and the 231 accessory genes encoded by the Hyp operon from Nostoc sp. PCC 7120 led to increased hydrogenase activity compared to expression of the Hox genes alone (Germer, J Biol. Chem. 284(52), 36462-36472 (2009)). Protein GenBank ID GI Number Organism HoxF NP_942727.1 38637753 Ralstonia eutropha H16 HoxU NP_942728.1 38637754 Ralstonia eutropha H16 HoxY NP_942729.1 38637755 Ralstonia eutropha H16 HoxH NP_942730.1 38637756 Ralstonia eutropha H16 HoxW NP_942731.1 38637757 Ralstonia eutropha H16 HoxI NP_942732.1 38637758 Ralstonia eutropha H16 HoxE NP_953767.1 39997816 Geobacter sulfurreducens HoxF NP_953766.1 39997815 Geobacter sulfurreducens HoxU NP_953765.1 39997814 Geobacter sulfurreducens HoxY NP_953764.1 39997813 Geobacter sulfurreducens HoxH NP_953763.1 39997812 Geobacter sulfurreducens GSU2717 NP_953762.1 39997811 Geobacter sulfurreducens HoxE NP_441418.1 16330690 Synechocystis str. PCC 6803 HoxF NP_441417.1 16330689 Synechocystis str. PCC 6803 Unknown NP_441416.1 16330688 Synechocystis str. PCC function 6803 HoxU NP_441415.1 16330687 Synechocystis str. PCC 6803 HoxY NP_441414.1 16330686 Synechocystis str. PCC 6803 232 Unknown NP_441413.1 16330685 Synechocystis str. PCC function 6803 Unknown NP_441412.1 16330684 Synechocystis str. PCC function 6803 HoxH NP_441411.1 16330683 Synechocystis str. PCC 6803 HypF NP_484737.1 17228189 Nostoc sp. PCC 7120 HypC NP_484738.1 17228190 Nostoc sp. PCC 7120 HypD NP_484739.1 17228191 Nostoc sp. PCC 7120 Unknown NP_484740.1 17228192 Nostoc sp. PCC 7120 function HypE NP_484741.1 17228193 Nostoc sp. PCC 7120 HypA NP_484742.1 17228194 Nostoc sp. PCC 7120 HypB NP_484743.1 17228195 Nostoc sp. PCC 7120 Hox1E AAP50519.1 37787351 Thiocapsa roseopersicina HoxIF AAP50520.1 37787352 Thiocapsa roseopersicina HoxlU AAP50521.1 37787353 Thiocapsa roseopersicina HoxlY AAP50522.1 37787354 Thiocapsa roseopersicina HoxlH AAP50523.1 37787355 Thiocapsa roseopersicina Several enzymes and the corresponding genes used for fixing carbon dioxide to either pyruvate or phosphoenolpyruvate to form the TCA cycle intermediates, oxaloacetate or malate are described below. Carboxylation of phosphoenolpyruvate to oxaloacetate is catalyzed by phosphoenolpyruvate carboxylase. Exemplary PEP carboxylase enzymes are encoded by ppc in E. coli (Kai et al., Arch. Biochem. Biophys. 414:170-179 (2003), ppcA in Methylobacterium extorquens AM] (Arps et al., J Bacteriol. 175:3776-3783 (1993), and ppc in Corynebacterium glutamicum (Eikmanns et al., Mol. Gen. Genet. 218:330-339 (1989).

233 Protein GenBank ID GI Number Organism Ppc NP_418391 16131794 Escherichia coli ppcA AAB58883 28572162 Methylobacterium extorquens Ppc ABB53270 80973080 Corynebacterium glutamicum An alternative enzyme for converting phosphoenolpyruvate to oxaloacetate is PEP carboxykinase, which simultaneously forms an ATP while carboxylating PEP. In most organisms PEP carboxykinase serves a gluconeogenic function and converts oxaloacetate to PEP at the expense of one ATP. S. cerevisiae is one such organism whose native PEP carboxykinase, PCK1, serves a gluconeogenic role (Valdes-Hevia et al., FEBS Lett. 258:313 316 (1989). E. coli is another such organism, as the role of PEP carboxykinase in producing oxaloacetate is believed to be minor when compared to PEP carboxylase, which does not form ATP, possibly due to the higher Km for bicarbonate of PEP carboxykinase (Kim et al., Apple. Environ. Microbiol. 70:1238-1241 (2004)). Nevertheless, activity of the native E. coli PEP carboxykinase from PEP towards oxaloacetate has been recently demonstrated in ppc mutants of E. coli K-12 (Kwon et al., J Microbiol. Biotechnol. 16:1448-1452 (2006)). These strains exhibited no growth defects and had increased succinate production at high NaHCO 3 concentrations. Mutant strains of E. coli can adopt Pck as the dominant C02-fixing enzyme following adaptive evolution (Zhang et al. 2009). In some organisms, particularly rumen bacteria, PEP carboxykinase is quite efficient in producing oxaloacetate from PEP and generating ATP. Examples of PEP carboxykinase genes that have been cloned into E. coli include those from Mannheimia succiniciproducens (Lee et al., Biotechnol. Bioprocess Eng. 7:95-99 (2002)), Anaerobiospirillum succiniciproducens (Laivenieks et al., Appl. Environ. Microbiol. 63:2273-2280 (1997), and Actinobacillus succinogenes (Kim et al. supra). The PEP carboxykinase enzyme encoded by Haemophilus influenza is effective at forming oxaloacetate from PEP. Protein GenBank ID GI Number Organism PCKJ NP_013023 6322950 Saccharomyces cerevisiae pck NP_417862.1 16131280 Escherichia coli pckA YP_089485.1 52426348 Mannheimia succiniciproducens 234 Protein GenBank ID GI Number Organism pckA 009460.1 3122621 Anaerobiospirillum succiniciproducens pckA Q6W6X5 75440571 Actinobacillus succinogenes pckA P43923.1 1172573 Haemophilus influenza Pyruvate carboxylase (EC 6.4.1.1) directly converts pyruvate to oxaloacetate at the cost of one ATP. Pyruvate carboxylase enzymes are encoded by PYC1 (Walker et al., Biochem. Biophys. Res. Commun. 176:1210-1217 (1991) and PYC2 (Walker et al., supra) in Saccharomyces cerevisiae, and pyc in Mycobacterium smegmatis (Mukhopadhyay and Purwantini, Biochim. Biophys. Acta 1475:191-206 (2000)). Protein GenBank ID GI Number Organism PYC1 NP_011453 6321376 Saccharomyces cerevisiae PYC2 NP_009777 6319695 Saccharomyces cerevisiae Pyc YP_890857.1 118470447 Mycobacterium smegmatis Malic enzyme can be applied to convert CO 2 and pyruvate to malate at the expense of one reducing equivalent. Malic enzymes for this purpose can include, without limitation, malic enzyme (NAD-dependent) and malic enzyme (NADP-dependent). For example, one of the E. coli malic enzymes (Takeo, J Biochem. 66:379-387 (1969)) or a similar enzyme with higher activity can be expressed to enable the conversion of pyruvate and CO 2 to malate. By fixing carbon to pyruvate as opposed to PEP, malic enzyme allows the high-energy phosphate bond from PEP to be conserved by pyruvate kinase whereby ATP is generated in the formation of pyruvate or by the phosphotransferase system for glucose transport. Although malic enzyme is typically assumed to operate in the direction of pyruvate formation from malate, overexpression of the NAD-dependent enzyme, encoded by maeA, has been demonstrated to increase succinate production in E. coli while restoring the lethal Apfl-AldhA phenotype under anaerobic conditions by operating in the carbon-fixing direction (Stols and Donnelly, Appl. Environ. Microbiol. 63(7) 2695-2701 (1997)). A similar observation was 235 made upon overexpressing the malic enzyme from Ascaris suum in E. coli (Stols et al., Appl. Biochem. Biotechnol. 63-65(1), 153-158 (1997)). The second E. coli malic enzyme, encoded by maeB, is NADP-dependent and also decarboxylates oxaloacetate and other alpha-keto acids (Iwakura et al., J. Biochem. 85(5):1355-65 (1979)). Protein GenBank ID GI Number Organism maeA NP_415996 90111281 Escherichia coli maeB NP_416958 16130388 Escherichia coli NAD-ME P27443 126732 Ascaris suum The enzymes used for converting oxaloacetate (formed from, for example, PEP carboxylase, PEP carboxykinase, or pyruvate carboxylase) or malate (formed from, for example, malic enzyme or malate dehydrogenase) to succinyl-CoA via the reductive branch of the TCA cycle are malate dehydrogenase, fumarate dehydratase (fumarase), fumarate reductase, and succinyl-CoA transferase. The genes for each of the enzymes are described herein above. Enzymes, genes and methods for engineering pathways from succinyl-CoA to various products into a microorganism are now known in the art. The additional reducing equivalents obtained from CO and/or H 2 , as disclosed herein, improve the yields of 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene when utilizing carbohydrate-based feedstock. For example, 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene can be produced from succinyl-CoA via pathways exemplified in Figure 20. Exemplary enzymes for the conversion succinyl-CoA to 2,4-pentadienoate, 3-butene-1-ol, or 1,3-butadiene include succinyl-CoA:acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, synthetase or hydrolase, 3-oxoadipate dehydrogenase, 2-fumarylacetate decarboxylase, 3-oxopent-4-enoate reductase, 3 hydroxypent-4-enoate dehydratase, 3-oxoadipyl-CoA reductase, 3-hydroxyadipyl-CoA transferase, synthetase or hydrolase, 3-hydroxyadipate dehydrogenase, 3-hydroxyhex-4 enedioate decarboxylase, 3-oxoadipate reductase, 2-fumarylacetate reductase, 3-hydroxypent 4-enoate decarboxylase, 2,4-pentadienoate decarboxylase.

236 Enzymes, genes and methods for engineering pathways from glycolysis intermediates to various products into a microorganism are known in the art. The additional reducing equivalents obtained from CO and H 2 , as described herein, improve the yields of all these products on carbohydrates. EXAMPLE X Methods for Handling CO and Anaerobic Cultures This example describes methods used in handling CO and anaerobic cultures. A. Handling of CO in small quantities for assays and small cultures. CO is an odorless, colorless and tasteless gas that is a poison. Therefore, cultures and assays that utilized CO required special handling. Several assays, including CO oxidation, acetyl-CoA synthesis, CO concentration using myoglobin, and CO tolerance/utilization in small batch cultures, called for small quantities of the CO gas that were dispensed and handled within a fume hood. Biochemical assays called for saturating very small quantities (<2 mL) of the biochemical assay medium or buffer with CO and then performing the assay. All of the CO handling steps were performed in a fume hood with the sash set at the proper height and blower turned on; CO was dispensed from a compressed gas cylinder and the regulator connected to a Schlenk line. The latter ensures that equal concentrations of CO were dispensed to each of several possible cuvettes or vials. The Schlenk line was set up containing an oxygen scrubber on the input side and an oil pressure release bubbler and vent on the other side. Assay cuvettes were both anaerobic and CO-containing. Threfore, the assay cuvettes were tightly sealed with a rubber stopper and reagents were added or removed using gas-tight needles and syringes. Secondly, small (~50 mL) cultures were grown with saturating CO in tightly stoppered serum bottles. As with the biochemical assays, the CO saturated microbial cultures were equilibrated in the fume hood using the Schlenk line setup. Both the biochemical assays and microbial cultures were in portable, sealed containers and in small volumes making for safe handling outside of the fume hood. The compressed CO tank was adjacent to the fume hood. Typically, a Schlenk line was used to dispense CO to cuvettes, each vented. Rubber stoppers on the cuvettes were pierced with 19 or 20 gage disposable syringe needles and were vented with the same. An oil bubbler was used with a CO tank and oxygen scrubber. The glass or quartz spectrophotometer cuvettes have a circular hole on top into which a Kontes 237 stopper sleeve, Sz7 774250-0007 was fitted. The CO detector unit was positioned proximal to the fume hood. B. Handling of CO in larger quantities fed to large-scale cultures. Fermentation cultures are fed either CO or a mixture of CO and H 2 to simulate syngas as a feedstock in fermentative production. Therefore, quantities of cells ranging from 1 liter to several liters can include the addition of CO gas to increase the dissolved concentration of CO in the medium. In these circumstances, fairly large and continuously administered quantities of CO gas are added to the cultures. At different points, the cultures are harvested or samples removed. Alternatively, cells are harvested with an integrated continuous flow centrifuge that is part of the fermenter. The fermentative processes are carried out under anaerobic conditions. In some cases, it is uneconomical to pump oxygen or air into fermenters to ensure adequate oxygen saturation to provide a respiratory environment. In addition, the reducing power generated during anaerobic fermentation may be needed in product formation rather than respiration. Furthermore, many of the enzymes for various pathways are oxygen-sensitive to varying degrees. Classic acetogens such as M. thermoacetica are obligate anaerobes and the enzymes in the Wood-Ljungdahl pathway are highly sensitive to irreversible inactivation by molecular oxygen. While there are oxygen-tolerant acetogens, the repertoire of enzymes in the Wood Ljungdahl pathway might be incompatible in the presence of oxygen because most are metallo-enzymes, key components are ferredoxins, and regulation can divert metabolism away from the Wood-Ljungdahl pathway to maximize energy acquisition. At the same time, cells in culture act as oxygen scavengers that moderate the need for extreme measures in the presence of large cell growth. C. Anaerobic chamber and conditions. Exemplary anaerobic chambers are available commercially (see, for example, Vacuum Atmospheres Company, Hawthorne CA; MBraun, Newburyport MA). Conditions included an 02 concentration of 1 ppm or less and 1 atm pure N 2 . In one example, 3 oxygen scrubbers/catalyst regenerators were used, and the chamber included an 02 electrode (such as Teledyne; City of Industry CA). Nearly all items and reagents were cycled four times in the airlock of the chamber prior to opening the inner chamber door. Reagents with a volume >5mL were sparged with pure N 2 prior to introduction into the chamber. Gloves are changed twice/yr and the catalyst containers were regenerated periodically when the chamber displays increasingly sluggish response to 238 changes in oxygen levels. The chamber's pressure was controlled through one-way valves activated by solenoids. This feature allowed setting the chamber pressure at a level higher than the surroundings to allow transfer of very small tubes through the purge valve. The anaerobic chambers achieved levels of 02 that were consistently very low and were needed for highly oxygen sensitive anaerobic conditions. However, growth and handling of cells does not usually require such precautions. In an alternative anaerobic chamber configuration, platinum or palladium can be used as a catalyst that requires some hydrogen gas in the mix. Instead of using solenoid valves, pressure release can be controlled by a bubbler. Instead of using instrument-based 02 monitoring, test strips can be used instead. D. Anaerobic microbiology. Small cultures were handled as described above for CO handling. In particular, serum or media bottles are fitted with thick rubber stoppers and aluminum crimps are employed to seal the bottle. Medium, such as Terrific Broth, is made in a conventional manner and dispensed to an appropriately sized serum bottle. The bottles are sparged with nitrogen for ~30 min of moderate bubbling. This removes most of the oxygen from the medium and, after this step, each bottle is capped with a rubber stopper (such as Bellco 20 mm septum stoppers; Bellco, Vineland, NJ) and crimp-sealed (Bellco 20 mm). Then the bottles of medium are autoclaved using a slow (liquid) exhaust cycle. At least sometimes a needle can be poked through the stopper to provide exhaust during autoclaving; the needle needs to be removed immediately upon removal from the autoclave. The sterile medium has the remaining medium components, for example buffer or antibiotics, added via syringe and needle. Prior to addition of reducing agents, the bottles are equilibrated for 30 60 minutes with nitrogen (or CO depending upon use). A reducing agent such as a 100 x 150 mM sodium sulfide, 200 mM cysteine-HCl is added. This is made by weighing the sodium sulfide into a dry beaker and the cysteine into a serum bottle, bringing both into the anaerobic chamber, dissolving the sodium sulfide into anaerobic water, then adding this to the cysteine in the serum bottle. The bottle is stoppered immediately as the sodium sulfide solution generates hydrogen sulfide gas upon contact with the cysteine. When injecting into the culture, a syringe filter is used to sterilize the solution. Other components are added through syringe needles, such as B12 (10 pM cyanocobalamin), nickel chloride (NiCl 2 , 20 microM final concentration from a 40 mM stock made in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture), and 239 ferrous ammonium sulfate (final concentration needed is 100 pM-made as 100-1000x stock solution in anaerobic water in the chamber and sterilized by autoclaving or by using a syringe filter upon injection into the culture). To facilitate faster growth under anaerobic conditions, the 1 liter bottles were inoculated with 50 mL of a preculture grown anaerobically. Induction of the pAl-lacO1 promoter in the vectors was performed by addition of isopropyl p-D-1 thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM and was carried out for about 3 hrs. Large cultures can be grown in larger bottles using continuous gas addition while bubbling. A rubber stopper with a metal bubbler is placed in the bottle after medium addition and sparged with nitrogen for 30 minutes or more prior to setting up the rest of the bottle. Each bottle is put together such that a sterile filter will sterilize the gas bubbled in and the hoses on the bottles are compressible with small C clamps. Medium and cells are stirred with magnetic stir bars. Once all medium components and cells are added, the bottles are incubated in an incubator in room air but with continuous nitrogen sparging into the bottles. EXAMPLE XI CO oxidation (CODH) Assay This example describes assay methods for measuring CO oxidation (CO dehydrogenase; CODH). The 7 gene CODH/ACS operon of Moorella thermoacetica was cloned into E. coli expression vectors. The intact ~10 kbp DNA fragment was cloned, and it is likely that some of the genes in this region are expressed from their own endogenous promoters and all contain endogenous ribosomal binding sites. These clones were assayed for CO oxidation, using an assay that quantitatively measures CODH activity. Antisera to the M. thermoacetica gene products was used for Western blots to estimate specific activity. M. thermoacetica is Gram positive, and ribosome binding site elements are expected to work well in E. coli. This activity, described below in more detail, was estimated to be ~1/50th of the M. thermoacetica specific activity. It is possible that CODH activity of recombinant E. coli cells could be limited by the fact that M. thermoacetica enzymes have temperature optima around 55C. Therefore, a mesophilic CODH/ACS pathway could be advantageous such as the close relative of Moorella that is mesophilic and does have an apparently intact CODH/ACS operon and a Wood-Ljungdahl pathway, Desulfitobacterium hafniense. Acetogens as 240 potential host organisms include, but are not limited to, Rhodospirillum rubrum, Moorella thermoacetica and Desulfitobacterium hafniense. CO oxidation is both the most sensitive and most robust of the CODH/ACS assays. It is likely that an E. coli-based syngas using system will ultimately need to be about as anaerobic as Clostridial (i.e., Moorella) systems, especially for maximal activity. Improvement in CODH should be possible but will ultimately be limited by the solubility of CO gas in water. Initially, each of the genes was cloned individually into expression vectors. Combined expression units for multiple subunits/i complex were generated. Expression in E. coli at the protein level was determined. Both combined M. thermoacetica CODH/ACS operons and individual expression clones were made. CO oxidation assay. This assay is one of the simpler, reliable, and more versatile assays of enzymatic activities within the Wood-Ljungdahl pathway and tests CODH (Seravalli et al., Biochemistry 43:3944-3955 (2004)). A typical activity of M. thermoacetica CODH specific activity is 500 U at 55'C or ~60U at 25'C. This assay employs reduction of methyl viologen in the presence of CO. This is measured at 578 nm in stoppered, anaerobic, glass cuvettes. In more detail, glass rubber stoppered cuvettes were prepared after first washing the cuvette four times in deionized water and one time with acetone. A small amount of vacuum grease was smeared on the top of the rubber gasket. The cuvette was gassed with CO, dried 10 min with a 22 Ga. needle plus an exhaust needle. A volume of 0.98 mlL of reaction buffer (50 mM Hepes, pH 8.5, 2mM dithiothreitol (DTT) was added using a 22 Ga. needle, with exhaust needled, and 100%CO. Methyl viologen (CH 3 viologen) stock was 1 M in water. Each assay used 20 microliters for 20 mM final concentration. When methyl viologen was added, an 18 Ga needle (partial) was used as a jacket to facilitate use of a Hamilton syringe to withdraw the CH 3 viologen. 4 -5 aliquots were drawn up and discarded to wash and gas equilibrate the syringe. A small amount of sodium dithionite (0.1 M stock) was added when making up the CH 3 viologen stock to slightly reduce the CH 3 viologen. The temperature was equilibrated to 55'C in a heated Olis spectrophotometer (Bogart GA). A blank reaction (CH 3 viologen + buffer) was run first to measure the base rate of CH 3 viologen reduction. Crude E. coli cell extracts of ACS90 and ACS91 (CODH-ACS operon of M. thermoacetica with and 241 without, respectively, the first coo C). 10 microliters of extract were added at a time, mixed and assayed. Reduced CH 3 viologen turns purple. The results of an assay are shown in Table I. Table I. Crude extract CO Oxidation Activities. ACS90 7.7 mg/ml ACS91 11.8 mg/ml Mta98 9.8 mg/ml Mta99 11.2 mg/ml Extract Vol QD/ U/ml U/mR ACS90 10 microliters 0.073 0.376 0.049 ACS91 10 microliters 0.096 0.494 0.042 Mta99 10 microliters 0.0031 0.016 0.0014 ACS90 10 microliters 0.099 0.51 0.066 Mta99 25 microlite rs 0.012 0.025 0.0022 ACS91 25 microliters 0.215 0.443 0.037 Mta98 25 microliters 0.019 0.039 0.004 ACS91 10 microliters 0.129 0.66 0.056 Averages ACS90 0.057 U/mg ACS91 0.045 U/mg Mta99 0.0018 U/mg Mta98/Mta99 are E. coli MG1655 strains that express methanol methyltransferase genes from M. thermoacetia and, therefore, are negative controls for the ACS90 ACS91 E. coli strains that contain M. thermoacetica CODH operons. If ~1% of the cellular protein is CODH, then these figures would be approximately 1 OOX less than the 500 U/mg activity of pure M thermoacetica CODH. Actual estimates based on Western blots are 0.5% of the cellular protein, so the activity is about 50X less than for M. thermoacetica CODH. Nevertheless, this experiment demonstrates CO oxidation activity in recombinant E. coli with a much smaller amount in the negative controls. The small amount of CO oxidation (CH 3 viologen reduction) seen in the negative controls indicates that E. coli may have a limited ability to reduce CH 3 viologen. To estimate the final concentrations of CODH and Mtr proteins, SDS-PAGE followed by Western blot analyses were performed on the same cell extracts used in the CO oxidation, ACS, methyltransferase, and corrinoid Fe-S assays. The antisera used were polyclonal to 242 purified M. thermoacetica CODH-ACS and Mtr proteins and were visualized using an alkaline phosphatase-linked goat-anti-rabbit secondary antibody. The Westerns were performed and results are shown in Figure 24. The amounts of CODH in ACS90 and ACS91 were estimated at 50 ng by comparison to the control lanes. Expression of CODH-ACS operon genes including 2 CODH subunits and the methyltransferase were confirmed via Western blot analysis. Therefore, the recombinant E. coli cells express multiple components of a 7 gene operon. In addition, both the methyltransferase and corrinoid iron sulfur protein were active in the same recombinant E. coli cells. These proteins are part of the same operon cloned into the same cells. The CO oxidation assays were repeated using extracts of Moorella thermoacetica cells for the positive controls. Though CODH activity in E. coli ACS90 and ACS91 was measurable, it was at about 130 - 150 X lower than the M. thermoacetica control. The results of the assay are shown in Figure 25. Briefly, cells (M. thermoacetica or E. coli with the CODH/ACS operon; ACS90 or ACS91 or empty vector: pZA33S) were grown and extracts prepared as described above. Assays were performed as described above at 55 0 C at various times on the day the extracts were prepared. Reduction of methylviologen was followed at 578 nm over a 120 sec time course. These results describe the CO oxidation (CODH) assay and results. Recombinant E. coli cells expressed CO oxidation activity as measured by the methyl viologen reduction assay. EXAMPLE X11 E. coli CO Tolerance Experiment and CO Concentration Assay (myoglobin assay) This example describes the tolerance of E. coli for high concentrations of CO. To test whether or not E. coli can grow anaerobically in the presence of saturating amounts of CO, cultures were set up in 120 ml serum bottles with 50 ml of Terrific Broth medium (plus reducing solution, NiCl 2 , Fe(II)NH 4

SO

4 , cyanocobalamin, IPTG, and chloramphenicol) as described above for anaerobic microbiology in small volumes. One half of these bottles were equilibrated with nitrogen gas for 30 min. and one half was equilibrated with CO gas for 30 min. An empty vector (pZA33) was used as a control, and cultures containing the pZA33 empty vector as well as both ACS90 and ACS91 were tested with both 243

N

2 and CO. All were inoculated and grown for 36 hrs with shaking (250 rpm) at 37'C. At the end of the 36 hour period, examination of the flasks showed high amounts of growth in all. The bulk of the observed growth occurred overnight with a long lag. Given that all cultures appeared to grow well in the presence of CO, the final CO concentrations were confirmed. This was performed using an assay of the spectral shift of myoglobin upon exposure to CO. Myoglobin reduced with sodium dithionite has an absorbance peak at 435 nm; this peak is shifted to 423 nm with CO. Due to the low wavelength and need to record a whole spectrum from 300 nm on upwards, quartz cuvettes must be used. CO concentration is measured against a standard curve and depends upon the Henry's Law constant for CO of maximum water solubility = 970 micromolar at 20C and 1 atm. For the myoglobin test of CO concentration, cuvettes were washed 1OX with water, 1X with acetone, and then stoppered as with the CODH assay. N 2 was blown into the cuvettes for ~10 min. A volume of 1 ml of anaerobic buffer (HEPES, pH 8.0, 2mM DTT) was added to the blank (not equilibrated with CO) with a Hamilton syringe. A volume of 10 microliter myoglobin (~1 mM-can be varied, just need a fairly large amount) and 1 microliter dithionite (20 mlM stock) were added. A CO standard curve was made using CO saturated buffer added at 1 microliter increments. Peak height and shift was recorded for each increment. The cultures tested were pZA33/CO, ACS90/CO, and ACS91/CO. Each of these was added in 1 microliter increments to the same cuvette. Midway through the experiment a second cuvette was set up and used. The results are shown in Table II. Table II. Carbon Monoxide Concentrations, 36 hrs. Strain and Growth Conditions Final CO concentration (micromolar) pZA33-CO 930 ACS90-CO 638 494 734 883 ave 687 SD 164 ACS91-CO 728 812 760 611 ave. 728 SD 85 244 The results shown in Table II indicate that the cultures grew whether or not a strain was cultured in the presence of CO or not. These results indicate that E. coli can tolerate exposure to CO under anaerobic conditions and that E. coli cells expressing the CODH-ACS operon can metabolize some of the CO. These results demonstrate that E. coli cells, whether expressing CODH/ACS or not, were able to grow in the presence of saturating amounts of CO. Furthermore, these grew equally well as the controls in nitrogen in place of CO. This experiment demonstrated that laboratory strains of E. coli are insensitive to CO at the levels achievable in a syngas project performed at normal atmospheric pressure. In addition, preliminary experiments indicated that the recombinant E. coli cells expressing CODH/ACS actually consumed some CO, probably by oxidation to carbon dioxide. Example XIII 3-Hydroxyacid decarboxylase enzymes for formation of 1,3-butadiene and 3-butene-1-ol 3-Hydroxyacid decarboxylase enzymes catalyze the ATP-driven decarboxylation of 3-hydroxyacids to alkene derivatives. 3-Hyroxyacid decarboxylase enzymes have recently been described that catalyze the formation of isobutylene, propylene and ethylene (WO 2010/001078 and Gogerty and Bobik, Appl. Environ. Microbiol., p. 8004-80 10, Vol. 76, No. 24 (2010)). We propose here a the application of similar enzymes to catalyze the conversion of 3-hydroxypent-4-enoate (3HP4) to 1,3-butadiene, shown in Figure 16 and 3,5 dihydroxypentanoate to 3-butene-1-ol , shown in Figure 17. The 3-butene-1-ol product can then be converted to butadiene via chemical dehydration or biological dehydration via a 3 butene- 1 -ol dehydratase enzyme. Conversion of 3-hydroxypent-4-enoate to butadiene is carried out by a 3 hydroxypent-4-enoate decarboxylase. Similarly, conversion of 3,5-dihydroxypentanoate to 3 butene-1-ol is carried out by a 3,5-dihydroxypentanoate decarboxylase. Such enzymes may share similarity to mevalonate pyrophosphate decarboxylase or diphosphomevalonate decarboxylase enzymes. One potential 3-hydroxypent-4-enoate decarboxylase is the Saccharomyces cerevisiae mevalonate diphosphate decarboxylase (ScMDD or ERG 19) which was shown to convert 3-hydroxy-3-methylbutyrate (3-HMB) to isobutene (Gogerty and Bobik, 2010, Appl. Environ. Microbiol., p. 8004-80 10, Vol. 76, No. 24). Two improved variants of the enzyme, ScMDD1 (1145F) and ScMDD2 (R74H), were demonstrated to 245 achieve 19-fold and 38-fold increases compared to the wild-type His-tagged enzyme. ERG19 and additional enzymes candidates are provided below. Enzyme GI Accession Number Organism ERG19 6324371 NP_014441.1 Saccharomyces cerevisiae S288C CAWG_01359 238879484 EEQ43122.1 Candida albicans WO-1 ANI_1_332184 145256805 XP_001401521.1 Aspergillus niger CBS 513.88 MVD 4505289 NP_002452.1 Homo sapiens Ahos_1490 332797171 YP_004458671.1 Acidianus hospitalis W1 SS02989 15899699 NP_344304.1 Sulfolobus solfataricus P2 UNLARM2_0386 255513677 EET89942.1 Candidatus Micrarchaeum acidiphilum ARMAN-2 mvaD 146329706 YP001209416.1 Dichelobacter nodosus VCS1703A MPTP_0700 332686202 YP_004455976.1 Melissococcus plutonius ATCC 35311 RKLH1l_3963 254513287 ZP_05125352.1 Rhodobacteraceae bacterium KLH 11 Example XIV Chemical dehydration of 3-butene-1-ol to butadiene Alcohols can be converted to olefins by reaction with a suitable dehydration catalyst under appropriate conditions. Typical dehydration catalysts that convert alcohols such as butanols and pentanols into olefins include various acid treated and untreated alumina (e.g., y-alumina) and silica catalysts and clays including zeolites (e.g., p-type zeolites, ZSM-5 or Y- 246 type zeolites, fluoride-treated p-zeolite catalysts, fluoride-treated clay catalysts, etc.), sulfonic acid resins (e.g., sulfonated styrenic resins such as Amberlyst@ 15), strong acids such as phosphoric acid and sulfuric acid, Lewis acids such boron trifluoride and aluminum trichloride, and many different types of metal salts including metal oxides (e.g., zirconium oxide or titanium dioxide) and metal chlorides (e.g., Latshaw B E, Dehydration of Isobutanol to Isobutylene in a Slurry Reactor, Department of Energy Topical Report, February 1994). Dehydration reactions can be carried out in both gas and liquid phases with both heterogeneous and homogeneous catalyst systems in many different reactor configurations. Typically, the catalysts used are stable to the water that is generated by the reaction. The water is usually removed from the reaction zone with the product. The resulting alkene(s) either exit the reactor in the gas or liquid phase (e.g., depending upon the reactor conditions) and are captured by a downstream purification process or are further converted in the reactor to other compounds (such as butadiene or isoprene) as described herein. The water generated by the dehydration reaction exits the reactor with unreacted alcohol and alkene product(s) and is separated by distillation or phase separation. Because water is generated in large quantities in the dehydration step, the dehydration catalysts used are generally tolerant to water and a process for removing the water from substrate and product may be part of any process that contains a dehydration step. For this reason, it is possible to use wet (i.e., up to about 95% or 98% water by weight) alcohol as a substrate for a dehydration reaction and remove this water with the water generated by the dehydration reaction (e.g., using a zeolite catalyst as described U.S. Pat. Nos. 4,698,452 and 4,873,392). Additionally, neutral alumina and zeolites will dehydrate alcohols to alkenes but generally at higher temperatures and pressures than the acidic versions of these catalysts. Dehydration of 3-buten-1-ol to butadiene is well known in the art (Gustav. Egloff and George. Hulla, Chem. Rev., 1945, 36 (1), pp 63-141). For example, 3-buten-1-ol is formed as an intermediate in the dehydration of 1,4-butanediol to 1,3-butadiene (Sato, et al, Catalysis Communications, 5 (8), 2004, p. 397-400).

247 Example XV Pathways to 2,4-pentadienoate, 3-butene-1-ol and 1,3-butadiene This example describes pathways to 2,4-pentadienoate, 3-butene-1-ol and 1,3 butadiene. Novel pathways to the intermediates 3HP4, 3,5-dihydroxypentanoate and 3 butene-1-ol are shown in Figures 18-21. Pathways to 3HP4 are shown in Figures 18, 19 and 20 (new). Pathways to 3,5-dihydroxypentanoate and 3-butene-1-ol are shown in Figures 19 and 21 (new). Additional pathways to the butadiene precursor 2,4-pentadienoate are shown in Figures 19, 20 and 21 (new). Several pathways to 2,4-pentadienoate are disclosed in earlier examples (see for example, Figures 4, 12, 13 14 and 15. Hydration of 2,4-pentadienoate yields 3-hydroxypent 4-enoate as shown in Figure 18 (new). 3-Hydroxypent-4-enoate can be subsequently decarboxylated to butadiene by a 3-hydroxyacid decarboxylase, also described above. Additional pathways from acrylyl-CoA and 3-HP-CoA to 2,4-pentadienoate are shown in Figure 19. Also shown here are pathways to 3HP4, 3-butene-1-ol and butadiene. Pathways to 3HP4 from acrylyl-CoA include: steps M/O/P, steps M/N/T Pathways to 3HP4 from 3-HP-CoA include: steps A/L/O/P, steps A/L/N/T Pathways to 3HP4 and 24PD from succinyl-CoA are shown in Figure 20. Succinyl CoA and acetyl-CoA are first joined by 3-oxoadipyl-CoA thiolase to form 3-oxoadipyl-CoA (Step A). In one pathway the 3-oxo group of 3-oxoadipyl-CoA is reduced to form 3 hydroxyadipyl-CoA (Step G). The CoA moiety is then converted to an acid group by a CoA hydrolase, synthetase or transferase. 3-Hydroxyadipate is then oxidized to form 3 hydroxyhex-4-enedioate (Step I). This product is then decarboxylated to form 3HP4. In an alternate route, 3-oxoadipyl-CoA is converted to 3-oxoadipate by a CoA transferase, synthetase or hydrolase in Step B. 3-Oxoadipate is then reduced to 3-hydroxyadipate (Step K), which is converted to 3HP4 as previously described. Alternately, 3-oxoadipate is converted to 2-fumarylacetate in Step C. The 3-oxo group of 2-fumarylacetate is then reduced to 3-hydroxyhex-4-enedioate, which is decarboxylated to 3HP4. In yet another embodiment, the 2-fumarylacetate is decarboxylated to form 3-oxopent-4-enoate (Step D), which is subsequently reduced to 3HP4 (Step E). 3HP4 can then be converted to butadiene in one step 248 by decarboxylation by a 3HP4 decarboxylase (Step M), or in two steps by dehydration to 2,4 pentadiene followed by decarboxylation (Steps F, N). Pathways to 3-butene-1-ol, butadiene and 2,4-pentadienoate from malonyl-CoA and acetyl-CoA are shown in Figure 21. In these pathways, malonyl-CoA and acetyl-CoA are joined by a thiolase to form 3-oxoglutaryl-CoA. This intermediate can then be converted to 3,5-dihydroxypentanoate by several alternate routes (Steps B/C/D, Steps B/G, Steps H/I/J, Steps H/L/D, K/J). Once formed, 3,5-dihydroxypentanoate can be decarboxylated by a 3 hydroxyacid decarboxylase to form 3-butene-1-ol (Step M). Subsequent dehydration by a 3 butene-1-ol dehydrogenase or a chemical catalyst yields butadiene (Step 0). Alternately, 3,5 dihydroxypentanoate can be dehydrated to 5-hydroxypent-2-enoate as shown in Step E. This intermediate can then be decarboxylated to 3-butene- 1 -ol (Step N) or further dehydrated to 2,4-pentadienoate (Step F). Enzymes for catalyzing the transformations shown in Figures 18-21 are categorized by EC number (Table 1) and described further below. Label Function Step 19 I,B,N,P 20 G,K,L,E 1.1.1.a Oxidoreductase (oxo to alcohol) 21 B, D, J, I,L 1.1.1.c Oxidoreductase (2 step, acyl-CoA to alcohol) 21 G,K 1.2.1 .b Oxidoreductase (acyl-CoA to aldehyde) 21 C,H 1.3.1.a Oxidoreductase (alkene to alkane) 20 1,C 19A,M 2.3.1 .b Beta-ketothiolase 20A,21A 19F,O,G,T,E, H 2.8.3.a Coenzyme-A transferase 20 B,H 19F,O,G,T,E, H 3.1.2.a Thiolester hydrolase (CoA specific) 20 B,H 19 U,Y,V,X 20 D,J,M,N 4.1.1.a Carboxy-lyase 21 M,N,P 19 S,K,L,R,D,C,J,Q,W 20 F 4.2.1.a Hydro-lyase 21 E,F,O 19F,O,G,T,E, H 6.2.1.a Acid-thiol ligase 20 B,H 249 Several reactions shown in Figures 19-21 are catalyzed by alcohol dehydrogenase enzymes. These reactions include Steps B, I, N and P of Figure 19, Steps E, G, K and L of Figure 20 and Steps B, D, I, J and L of Figure 21. Exemplary genes encoding enzymes that catalyze the reduction of an aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalently aldehyde reductase) include alrA encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al., Appl.Environ.Microbiol. 66:5231-5235 (2000)), yqhD andfucO from E. coli (Sulzenbacher et al., 342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicum which converts butyryaldehyde into butanol (Walter et al., 174:7149-7158 (1992)). YqhD catalyzes the reduction of a wide range of aldehydes using NADPH as the cofactor, with a preference for chain lengths longer than C(3) (Sulzenbacher et al., 342:489-502 (2004);Perez et al., J Biol.Chem. 283:7346-7353 (2008)). The adhA gene product from Zymomonas mobilisE has been demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita et al., AppI Microbiol Biotechnol 22:249-254 (1985)). Additional aldehyde reductase candidates are encoded by bdh in C. saccharoperbutylacetonicum and Cbei_1722, Cbei_2181 and Cbei_2421 in C. Beijerinckii. Additional aldehyde reductase gene candidates in Saccharomyces cerevisiae include the aldehyde reductases GRE3, ALD2-6 and HFD1, glyoxylate reductases GORI and YPL113C and glycerol dehydrogenase GCY1 (WO 2011/022651A1; Atsumi et al., Nature 451:86-89 (2008)). The enzyme candidates described previously for catalyzing the reduction of methylglyoxal to acetol or lactaldehyde are also suitable lactaldehyde reductase enzyme candidates. Protein GENBANK ID GI NUMBER ORGANISM airA BAB 12273.1 9967138 Acinetobacter sp. strain M-1 ADH2 NP_014032.1 6323961 Saccharomyces cerevisiae yqhD NP_417484.1 16130909 Escherichia coli fucO NP_417279.1 16130706 Escherichia coli bdh I NP_349892.1 15896543 Clostridium acetobutylicum bdh II NP_349891.1 15896542 Clostridium acetobutylicum adhA YP_162971.1 56552132 Zymomonas mobilis bdh BAF45463.1 124221917 Clostridium saccharoperbutylacetonicum Cbei_1722 YP_001308850 150016596 Clostridium beijerinckii Cbei_2181 YP_001309304 150017050 Clostridium beijerinckii 250 Cbei_2421 YP_001309535 150017281 Clostridium beijerinckii GRE3 P38715.1 731691 Saccharomyces cerevisiae ALD2 CAA89806.1 825575 Saccharomyces cerevisiae ALD3 NP_013892.1 6323821 Saccharomyces cerevisiae ALD4 NP_015019.1 6324950 Saccharomyces cerevisiae ALD5 NP_010996.2 330443526 Saccharomyces cerevisiae ALD6 ABX39192.1 160415767 Saccharomyces cerevisiae HFD] Q0445 8.1 2494079 Saccharomyces cerevisiae GOR] NP_014125.1 6324055 Saccharomyces cerevisiae YPL13C AAB68248.1 1163100 Saccharomyces cerevisiae GCY1 CAA99318.1 1420317 Saccharomyces cerevisiae Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC 1.1.1.61) also fall into this category. Such enzymes have been characterized in Ralstonia eutropha (Bravo et al., JForens Sci, 49:379-387 (2004)), Clostridium kluyveri (Wolff et al., Protein Expr.Purif 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al., JBiol Chem, 278:41552 41556 (2003)). The A. thaliana enzyme was cloned and characterized in yeast (Breitkreuz et al., JBiol.Chem. 278:41552-41556 (2003)). Yet another gene is the alcohol dehydrogenase adhI from Geobacillus thermoglucosidasius (Jeon et al., JBiotechnol 135:127-133 (2008)). PROTEIN GENBANK ID GI NUMBER ORGANISM 4hbd YP_726053.1 113867564 Ralstonia eutropha H16 4hbd L21902.1 146348486 Clostridium kluyveri DSM 555 4hbd Q94B07 75249805 Arabidopsis thaliana adhI AAR91477.1 40795502 Geobacillus thermoglucosidasius Another exemplary aldehyde reductase is methylmalonate semialdehyde reductase, also known as 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). This enzyme participates in valine, leucine and isoleucine degradation and has been identified in bacteria, eukaryotes, and mammals. The enzyme encoded by P84067 from Thermus thermophilus HB8 has been structurally characterized (Lokanath et al., JMoI Biol, 352:905-17 (2005)). The reversibility of the human 3-hydroxyisobutyrate dehydrogenase was demonstrated using isotopically labeled substrate (Manning et al., Biochem J, 231:481-4 (1985)). Additional genes encoding this enzyme include 3hidh in Homo sapiens (Hawes et al., Methods Enzymol, 324:218-228 251 (2000)) and Oryctolagus cuniculus (Hawes et al., supra; Chowdhury et al., Biosci.Biotechnol Biochem. 60:2043-2047 (1996)), mmsB in Pseudomonas aeruginosa and Pseudomonas putida, and dhat in Pseudomonas putida (Aberhart et al., J Chem.Soc.[Perkin 1] 6:1404-1406 (1979); Chowdhury et al., Biosci.Biotechnol Biochem. 60:2043-2047 (1996); Chowdhury et al., Biosci.Biotechnol Biochem. 67:438-441 (2003)). Several 3-hydroxyisobutyrate dehydrogenase enzymes have been characterized in the reductive direction, including mmsB from Pseudomonas aeruginosa (Gokam et al., US Patent 739676, (2008)) and mmsB from Pseudomonas putida. PROTEIN GENBANK ID GI NUMBER ORGANISM P84067 P84067 75345323 Thermus thermophilus 3hidh P31937.2 12643395 Homo sapiens 3hidh P32185.1 416872 Oryctolagus cuniculus mmsB NP_746775.1 26991350 Pseudomonas putida mmsB P2881 1.1 127211 Pseudomonas aeruginosa dhat Q59477.1 2842618 Pseudomonas putida There exist several exemplary alcohol dehydrogenases that reduce a ketone to a hydroxyl functional group. Two such enzymes from E. coli are encoded by malate dehydrogenase (mdh) and lactate dehydrogenase (ldhA). In addition, lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high activities on 2-ketoacids of various chain lengths includings lactate, 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (Steinbuchel et al., Eur.JBiochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta (Suda et al., Arch.Biochem.Biophys. 176:610-620 (1976); Suda et al., Biochem.Biophys.Res.Commun. 77:586-591 (1977)). An additional oxidoreductase is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the human heart which has been cloned and characterized (Marks et al., JBiol.Chem. 267:15459-15463 (1992)). Alcohol dehydrogenase enzymes of C. beijerinckii (Ismaiel et al., JBacteriol. 175:5097-5105 (1993)) and T. brockii (Lamed et al., Biochem.J 195:183-190 (1981); Peretz et al., Biochemistry. 28:6549-6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase catalyzes the reduction of MEK to 2-butanol. Exemplary MEK reductase enzymes can be found in Rhodococcus ruber (Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and Pyrococcusfuriosus (van der et al., Eur.JBiochem. 268:3062-3068 (2001)).

252 Protein GenBank Accession No. GI No. Organism mdh AAC76268.1 1789632 Escherichia coli ldhA NP_415898.1 16129341 Escherichia coli ldh YP_725182.1 113866693 Ralstonia eutropha bdh AAA58352.1 177198 Homo sapiens adh AAA23199.2 60592974 Clostridium beijerinckii NRRL B593 adh P14941.1 113443 Thermoanaerobacter brockii HTD4 sadh CAD36475 21615553 Rhodococcus ruber adhA AAC25556 3288810 Pyrococcusfuriosus A number of organisms can catalyze the reduction of 4-hydroxy-2-butanone to 1,3 butanediol, including those belonging to the genus Bacillus, Brevibacterium, Candida, and Klebsiella among others, as described by Matsuyama et al. ( (1995)). A mutated Rhodococcus phenylacetaldehyde reductase (Sar268) and a Leifonia alcohol dehydrogenase have also been shown to catalyze this transformation at high yields (Itoh et al., Appl.Microbiol Biotechnol. 75:1249-1256 (2007)). Alcohol dehydrogenase enzymes that reduce 3-oxoacyl-CoA substrates to their corresponding 3-hyroxyacyl-CoA product are also relevant to the pathways depicted in Figures 19-21. 3-Oxoacyl-CoA dehydrogenase enzymes (EC 1.1.1.35) convert 3-oxoacyl CoA molecules into 3-hydroxyacyl-CoA molecules and are often involved in fatty acid beta oxidation or phenylacetate catabolism. For example, subunits of two fatty acid oxidation complexes in E. coli, encoded byfadB andfadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et al., Methods Enzymol. 71 Pt C:403-411 (1981)). Given the proximity in E. coli ofpaaH to other genes in the phenylacetate degradation operon (Nogales et al., 153:357-365 (2007)) and the fact that paaH mutants cannot grow on phenylacetate (Ismail et al., Eur.JBiochem. 270:3047-3054 (2003)), it is expected that the E. colipaaH gene also encodes a 3-hydroxyacyl-CoA dehydrogenase. Acetoacetyl-CoA reductase participates in the acetyl-CoA fermentation pathway to butyrate in several species of Clostridia and has been studied in detail (Jones et al., Microbiol Rev. 50:484-524 (1986)). The enzyme from Clostridium acetobutylicum, encoded by hbd, has been cloned and functionally expressed in E. coli (Youngleson et al., JBacteriol. 171:6800-6807 (1989)). Yet other genes demonstrated to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA are phbB from Zoogloea ramigera (Ploux et al., Eur.JBiochem. 174:177-182 (1988)) and phaB from 253 Rhodobacter sphaeroides (Alber et al., Mol.Microbiol 61:297-309 (2006)). The former gene is NADPH-dependent, its nucleotide sequence has been determined (Peoples et al., Mol.Microbiol 3:349-357 (1989)) and the gene has been expressed in E. coli. Substrate specificity studies on the gene led to the conclusion that it could accept 3-oxopropionyl-CoA as a substrate besides acetoacetyl-CoA (Ploux et al., Eur.JBiochem. 174:177-182 (1988)). Additional genes include phaB in Paracoccus denitrificans, Hbd1 (C-terminal domain) and Hbd2 (N-terminal domain) in Clostridium kluyveri (Hillmer and Gottschalk, Biochim. Biophys. Acta 3334:12-23 (1974)) and HSD17B1O in Bos taurus (Wakil et al., JBiol.Chem. 207:631-638 (1954)). The enzyme from Paracoccus denitrificans has been functionally expressed and characterized in E. coli (Yabutani et al., FEMS Microbiol Lett. 133:85-90 (1995)). A number of similar enzymes have been found in other species of Clostridia and in Metallosphaera sedula (Berg et al., Science. 318:1782-1786 (2007)). The enzyme from Candida tropicalis is a component of the peroxisomal fatty acid beta-oxidation multifunctional enzyme type 2 (MFE-2). The dehydrogenase B domain of this protein is catalytically active on acetoacetyl-CoA. The domain has been functionally expressed in E. coli, a crystal structure is available, and the catalytic mechanism is well-understood (Ylianttila et al., Biochem Biophys Res Commun 324:25-30 (2004); Ylianttila et al., JMoI Biol 358:1286-1295 (2006)). Protein GENBANK ID GI NUMBER ORGANISM fadB P21177.2 119811 Escherichia coli fadJ P77399.1 3334437 Escherichia coli paaH NP_415913.1 16129356 Escherichia coli Hbd2 EDK34807.1 146348271 Clostridium kluyveri Hbdl EDK32512.1 146345976 Clostridium kluyveri HSD17BJO 002691.3 3183024 Bos taurus phbB P23238.1 130017 Zoogloea ramigera phaB YP_353825.1 77464321 Rhodobacter sphaeroides phaB BAA08358 675524 Paracoccus denitrificans Hbd NP_349314.1 15895965 Clostridium acetobutylicum Hbd AAM 14586.1 20162442 Clostridium beijerinckii Msed_1423 YP_001191505 146304189 Metallosphaera sedula Msed_0399 YP_001190500 146303184 Metallosphaera sedula Msed_0389 YP_001190490 146303174 Metallosphaera sedula 254 Msed_1993 YP_001192057 146304741 Metallosphaera sedula Fox2 Q02207 399508 Candida tropicalis Bifunctional oxidoreductases convert an acyl-CoA to its corresponding alcohol. Enzymes with this activity are required to convert 3-hydroxyglutaryl-CoA to 3,5 dihydroxypentanoate (Figure 21, Step G) and 3-oxoglutaryl-CoA to 5-hydroxy-3 oxopentanoate (Figure 21, Step K). Exemplary bifunctional oxidoreductases that convert an acyl-CoA to alcohol include those that transform substrates such as acetyl-CoA to ethanol (e.g., adhE from E. coli (Kessler et al., FEBS.Lett. 281:59-63 (1991))) and butyryl-CoA to butanol (e.g. adhE2 from C. acetobutylicum (Fontaine et al., JBacteriol. 184:821-830 (2002))). The C. acetobutylicum enzymes encoded by bdh I and bdh II (Walter, et al., J Bacteriol. 174:7149-7158 (1992)), reduce acetyl-CoA and butyryl-CoA to ethanol and butanol, respectively. In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxide the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al., JGen.Appl.Microbiol. 18:43-55 (1972); Koo et al., Biotechnol Lett, 27:505 510 (2005)). Another exemplary enzyme can convert malonyl-CoA to 3-HP. An NADPH dependent enzyme with this activity has characterized in Chloroflexus aurantiacus where it participates in the 3-hydroxypropionate cycle (Hugler et al., JBacteriol, 184:2404-2410 (2002); Strauss et al., Eur JBiochem, 215:633-643 (1993)). This enzyme, with a mass of 300 kDa, is highly substrate-specific and shows little sequence similarity to other known oxidoreductases (Hugler et al., supra). No enzymes in other organisms have been shown to catalyze this specific reaction; however there is bioinformatic evidence that other organisms may have similar pathways (Klatt et al., Env Microbiol, 9:2067-2078 (2007)). Enzyme candidates in other organisms including Roseiflexus castenholzii, Erythrobacter sp. NAP] and marine gamma proteobacterium HTCC2080 can be inferred by sequence similarity. Protein GenBank ID GI Number Organism adhE NP_415757.1 16129202 Escherichia coli adhE2 AAK09379.1 12958626 Clostridium acetobutylicum bdh I NP_349892.1 15896543 Clostridium acetobutylicum bdh 11 NP_349891.1 15896542 Clostridium acetobutylicum 255 Protein GenBank ID GI Number Organism adhE AAV66076.1 55818563 Leuconostoc mesenteroides mcr AAS20429.1 42561982 Chloroflexus aurantiacus Rcas_2929 YP_001433009.1 156742880 Roseiflexus castenholzii NAP]_02720 ZP_01039179.1 85708113 Erythrobacter sp. NAP] MGP2080_00535 ZP_01626393.1 119504313 marine gamma proteobacterium HTCC2080 Longer chain acyl-CoA molecules can be reduced to their corresponding alcohols by enzymes such as the jojoba (Simmondsia chinensis) FAR which encodes an alcohol-forming fatty acyl-CoA reductase. Its overexpression in E. coli resulted in FAR activity and the accumulation of fatty alcohol (Metz et al., Plant Physiol, 122:635-644 (2000)). Protein GenBank ID GI Number Organism FAR AAD38039.1 5020215 Simmondsia chinensis Another candidate for catalyzing these steps is 3-hydroxy-3-methylglutaryl-CoA reductase (or HMG-CoA reductase). This enzyme naturally reduces the CoA group in 3 hydroxy-3-methylglutaryl-CoA to an alcohol forming mevalonate. The hmgA gene of Sulfolobus solfataricus, encoding 3-hydroxy-3-methylglutaryl-CoA reductase, has been cloned, sequenced, and expressed in E. coli (Bochar et al., JBacteriol. 179:3632-3638 (1997)). S. cerevisiae also has two HMG-CoA reductases in it (Basson et al., Proc.Nat.Acad.Sci. U.S.A 83:5563-5567 (1986)). The gene has also been isolated from Arabidopsis thaliana and has been shown to complement the HMG-COA reductase activity in S. cerevisiae (Learned et al., Proc.Natl.Acad.Sci. U.S.A 86:2779-2783 (1989)). Protein GenBank ID GI Number Organism HMG1 CAA86503.1 587536 Saccharomyces cerevisiae HMG2 NP_013555 6323483 Saccharomyces cerevisiae HMG1 CAA70691.1 1694976 Arabidopsis thaliana hmgA AAC45370.1 2130564 Sulfolobus solfataricus 256 Acyl-CoA reductases in the 1.2.1 family reduce an acyl-CoA to its corresponding aldehyde. Such a conversion is required in steps C and H of Figure 21. Several acyl-CoA dehydrogenase enzymes have been described in the open literature and represent suitable candidates for these steps. These are described below. Exemplary acyl-CoA reductase enzymes include fatty acyl-CoA reductase, succinyl CoA reductase (EC 1.2.1.76), acetyl-CoA reductase and butyryl-CoA reductase. Exemplary fatty acyl-CoA reductase enzymes are encoded by acr1 of Acinetobacter calcoaceticus (Reiser, Journal ofBacteriology 179:2969-2975 (1997)) and Acinetobacter sp. M-1 (Ishige et al., Appl. Environ. Microbiol. 68:1192-1195 (2002)). Enzymes with succinyl-CoA reductase activity are encoded by sucD of Clostridium kluyveri (Sohling, J Bacteriol. 178:871-880 (1996)) and sucD of P. gingivalis (Takahashi, J Bacteriol 182:4704-4710 (2000)). Additional succinyl-CoA reductase enzymes participate in the 3-hydroxypropionate/4 hydroxybutyrate cycle of thermophilic archaea including Metallosphaera sedula (Berg et al., Science 318:1782-1786 (2007)) and Thermoproteus neutrophilus (Ramos-Vera et al., J Bacteriol., 191:4286-4297 (2009)). The M. sedula enzyme, encoded by Msed_0709, is strictly NADPH-dependent and also has malonyl-CoA reductase activity. The T. neutrophilus enzyme is active with both NADPH and NADH. The enzyme acylating acetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yet another as it has been demonstrated to oxidize and acylate acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde (Powlowski, J Bacteriol. 175:377-385 (1993)). In addition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to oxidize the branched chain compound isobutyraldehyde to isobutyryl-CoA (Kazahaya, J Gen. Apple. Microbiol. 18:43-55 (1972); and Koo et al., Biotechnol Lett. 27:505-510 (2005)). Butyraldehyde dehydrogenase catalyzes a similar reaction, conversion of butyryl-CoA to butyraldehyde, in solventogenic organisms such as Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci Biotechnol Biochem., 71:58 68 (2007)). Protein GenBank ID GI Number Organism acr1 YP_047869.1 50086359 Acinetobacter calcoaceticus acri AAC45217 1684886 Acinetobacter baylyi acri BAB85476.1 18857901 Acinetobacter sp. Strain M-1 MSED_0709 YP_001190808.1 146303492 Metallosphaera sedula 257 Protein GenBank ID GI Number Organism Tneu_0421 ACB39369.1 170934108 Thermoproteus neutrophilus sucD P38947.1 172046062 Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonas gingivalis bphG BAA03892.1 425213 Pseudomonas sp adhE AAV66076.1 55818563 Leuconostoc mesenteroides Bld AAP42563.1 31075383 Clostridium saccharoperbutylacetonicum An additional enzyme type that converts an acyl-CoA to its corresponding aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic semialdehyde. Malonyl CoA reductase is a key enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal bacteria (Berg, Science 318:1782-1786 (2007); and Thauer, Science 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor and has been characterized in Metallosphaera and Sulfolobus sp. (Alber et al., J Bacteriol. 188:855 1 8559 (2006); and Hugler, J Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded by Msed_0709 in Metallosphaera sedula (Alber et al., J Bacteriol. 188:8551-8559 (2006); and Berg, Science 318:1782-1786 (2007)). A gene encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and heterologously expressed in E. coli (Alber et al., J Bacteriol 188:8551-8559 (2006). This enzyme has also been shown to catalyze the conversion of methylmalonyl-CoA to its corresponding aldehyde (W02007141208 (2007)). Although the aldehyde dehydrogenase functionality of these enzymes is similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little sequence similarity. Both malonyl-CoA reductase enzyme candidates have high sequence similarity to aspartate semialdehyde dehydrogenase, an enzyme catalyzing the reduction and concurrent dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde. Additional gene candidates can be found by sequence homology to proteins in other organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius and have been listed below. Yet another candidate for CoA-acylating aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999). This enzyme has been reported to reduce acetyl-CoA and butyryl-CoA to their corresponding aldehydes. This gene is very similar to eutE that encodes acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth, Appl. Environ. Microbiol. 65:4973-4980 (1999).

258 Protein GenBank ID GI Number Organism Msed_0709 YP_001190808.1 146303492 Metallosphaera sedula Mcr NP_378167.1 15922498 Sulfolobus tokodaii asd-2 NP_343563.1 15898958 Sulfolobus solfataricus Saci_2370 YP_256941.1 70608071 Sulfolobus acidocaldarius Ald AAT66436 49473535 Clostridium beijerinckii eutE AAA80209 687645 Salmonella typhimurium eutE P77445 2498347 Escherichia coli 2-Enoate reductase enzymes, some of which are reversible, are known to catalyze the NAD(P)H-dependent reduction of a wide variety of a, -unsaturated carboxylic acids and aldehydes (Rohdich et al., 276:5779-5787 (2001)). These enzymes represent suitable candidates to carry out the transformations depicted by steps I and C of Figure 20. Several examples are provided below. In the recently published genome sequence of C. kluyveri, 9 coding sequences for enoate reductases were reported, out of which one has been characterized (Seedorf et al., Proc.Natl.Acad.Sci U.S.A 105:2128-2133 (2008)). The enr genes from both C. tyrobutyricum and M. thermoaceticum have been cloned and sequenced and show 59% identity to each other. The former gene is also found to have approximately 75% similarity to the characterized gene in C. kluyveri (Giesel et al., 135:51-57 (1983)). It has been reported based on these sequence results that enr is very similar to the dienoyl CoA reductase in E. coli (fadH) (Rohdich et al., JBiol.Chem. 276:5779-5787 (2001)). The C. thermoaceticum enr gene has also been expressed in a catalytically active form in E. coli (Rohdich et al., J Biol.Chem. 276:5779-5787 (2001)). This enzyme exhibits activity on a broad range of alpha, beta-unsaturated carbonyl compounds.

259 Protein GenBank ID GI Number Organism enr ACA54153.1 169405742 Clostridium botulinum A3 str enr CAA7 1086.1 2765041 Clostridium tyrobutyricum enr CAA76083.1 3402834 Clostridium kluyveri enr YP_430895.1 83590886 Moorella thermoacetica fadH NP_417552.1 16130976 Escherichia coli Another candidate 2-enoate reductase is maleylacetate reductase (MAR, EC 1.3.1.32), an enzyme catalyzing the reduction of 2-maleylacetate (4-oxohex-2-enedioate) to 3 oxoadipate. MAR enzymes naturally participate in aromatic degradation pathways (Kaschabek et al., JBacteriol. 175:6075-6081 (1993); Kaschabek et al., JBacteriol. 177:320 325 (1995); Camara et al., JBacteriol. (2009); Huang et al., Appl Environ.Microbiol 72:7238-7245 (2006)). The enzyme activity was identified and characterized in Pseudomonas sp. strain B13 (Kaschabek et al., 175:6075-6081 (1993); Kaschabek et al., 177:320-325 (1995)), and the coding gene was cloned and sequenced (Kasberg et al., JBacteriol. 179:3801-3803 (1997)). Additional MAR gene candidates include clcE gene from Pseudomonas sp. strain B13 (Kasberg et al., JBacteriol. 179:3801-3803 (1997)), macA gene from Rhodococcus opacus (Seibert et al., 180:3503-3508 (1998)), the macA gene from Ralstonia eutropha (also known as Cupriavidus necator) (Seibert et al., Microbiology 150:463-472 (2004)), tfdFII from Ralstonia eutropha (Seibert et al., JBacteriol. 175:6745 6754 (1993)) and NCglI112 in Corynebacterium glutamicum (Huang et al., Appl Environ.Microbiol 72:7238-7245 (2006)). A MAR in Pseudomonas reinekei MT1, encoded by ccaD, was recently identified (Camara et al., JBacteriol. (2009)).

260 Protein GenBank ID GI Number Organism clcE 3913241 030847.1 Pseudomonas sp. strain B13 macA 7387876 084992.1 Rhodococcus opacus macA 5916089 AAD55886 Cupriavidus necator tfdFII 1747424 AAC44727.1 Ralstonia eutropha JMP134 NCgll112 19552383 NP_600385 Corynebacterium glutamicum ccaD 134133940 ABO61029.1 Pseudomonas reinekei MT1 Step A of Figures 19-21 and Step M of Figure 19 require condensation of either 3 hydroxypropionyl-CoA, acrylyl-CoA, succinyl-CoA or malonyl-CoA with acetyl-CoA. Several D -ketothiolase enzymes have been described in the open literature and represent suitable candidates for these steps. These are described below. For example, 3-Oxoadipyl-CoA thiolase represents one type of beta-ketothiolase enzyme that is suitable for the aforementioned steps. 3-Oxoadipyl-CoA thiolase (EC 2.3.1.174) naturally converts beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA and is a key enzyme of the beta-ketoadipate pathway for aromatic compound degradation. The enzyme is widespread in soil bacteria and fungi including Pseudomonas putida (Harwood et al., JBacteriol. 176:6479-6488 (1994)) and Acinetobacter calcoaceticus (Doten et al., J Bacteriol. 169:3168-3174 (1987)). The gene products encoded bypcaF in Pseudomonas strain B13 (Kaschabek et al., JBacteriol. 184:207-215 (2002)), phaD in Pseudomonasputida U(Olivera et al., Proc.Natl.Acad.Sci U.S.A 95:6419-6424 (1998)), paaE in Pseudomonas fluorescens ST (Di et al., Arch.Microbiol 188:117-125 (2007)), and paaJ from E. coli (Nogales et al., Microbiology 153:357-365 (2007)) also catalyze this transformation. Several beta-ketothiolases exhibit significant and selective activities in the oxoadipyl-CoA forming direction including bkt from Pseudomonas putida, pcaF and bkt from Pseudomonas aeruginosa PA01, bkt from Burkholderia ambifaria AMMD, paaJ from E. coli, and phaD from P. putida.

261 Protein GI# GenBank Accession # Organism paaJ 16129358 NP_415915.1 Escherichia coli pcaF 17736947 AAL02407 Pseudomonas knackmussii (B 13) phaD 3253200 AAC24332.1 Pseudomonasputida pcaF 506695 AAA85138.1 Pseudomonas putida pcaF 141777 AAC37148.1 Acinetobacter calcoaceticus paaE 106636097 ABF82237.1 Pseudomonasfluorescens bkt 115360515 YP_777652.1 Burkholderia ambifaria AMMD bkt 9949744 AAG06977.1 Pseudomonas aeruginosa PA01 pcaF 9946065 AAG03617.1 Pseudomonas aeruginosa PA01 Glutaryl-CoA and acetyl-CoA are condensed to form 3-oxopimeloyl-CoA by oxopimeloyl-CoA:glutaryl-CoA acyltransferase, a beta-ketothiolase (EC 2.3.1.16). An enzyme catalyzing this transformation is found in Ralstonia eutropha (formerly known as Alcaligenes eutrophus), encoded by genes bktB and bktC (Slater et al., JBacteriol. 180:1979-1987 (1998); Haywood et al., 52:91-96 (1988)). The sequence of the BktB protein is known; however, the sequence of the BktC protein has not been reported. The pim operon of Rhodopseudomonas palustris also encodes a beta-ketothiolase, encoded by pimB, predicted to catalyze this transformation in the degradative direction during benzoyl-CoA degradation (Harrison et al., 151:727-736 (2005)). A beta-ketothiolase enzyme candidate in S. aciditrophicus was identified by sequence homology to bktB (430% identity, evalue = le 93). Protein GenBank ID GI Number Organism bktB YP_725948 11386745 Ralstonia eutropha pimB CAE29156 39650633 Rhodopseudomonas palustris syn_02642 YP_462685.1 85860483 Syntrophus aciditrophicus Beta-ketothiolase enzymes catalyzing the formation of beta-ketovalerate from acetyl CoA and propionyl-CoA may also be able to catalyze the condensation of acetyl-CoA with 3 hydroxypropionyl-CoA, acrylyl-CoA, succinyl-CoA, or malonyl-CoA. Zoogloea ramigera 262 possesses two ketothiolases that can form [ -ketovaleryl-CoA from propionyl-CoA and acetyl-CoA and R. eutropha has a [ -oxidation ketothiolase that is also capable of catalyzing this transformation (Gruys et al., US Patent 5,958,745 (1999)). The sequences of these genes or their translated proteins have not been reported, but several candidates in R. eutropha, Z. ramigera, or other organisms can be identified based on sequence homology to bktB from R. eutropha. These include: Protein GenBank ID GI Number Organism phaA YP_725941.1 113867452 Ralstonia eutropha h16_Al 713 YP_726205.1 113867716 Ralstonia eutropha pcaF YP_728366.1 116694155 Ralstonia eutropha h16_B1369 YP_840888.1 116695312 Ralstonia eutropha h16_AOl70 YP_724690.1 113866201 Ralstonia eutropha h16_A0462 YP_724980.1 113866491 Ralstonia eutropha h16_A1528 YP_726028.1 113867539 Ralstonia eutropha h16_B0381 YP_728545.1 116694334 Ralstonia eutropha h16_B0662 YP_728824.1 116694613 Ralstonia eutropha h16_B0759 YP_728921.1 116694710 Ralstonia eutropha h16_B0668 YP_728830.1 116694619 Ralstonia eutropha h16_A1720 YP_726212.1 113867723 Ralstonia eutropha h16_A1887 YP_726356.1 113867867 Ralstonia eutropha phbA P07097.4 135759 Zoogloea ramigera bktB YP_002005382.1 194289475 Cupriavidus taiwanensis Rmet_1362 YP_583514.1 94310304 Ralstonia metallidurans Bphy_0975 YP_001857210.1 186475740 Burkholderiaphymatum Additional candidates include beta-ketothiolases that are known to convert two molecules of acetyl-CoA into acetoacetyl-CoA (EC 2.1.3.9). Exemplary acetoacetyl-CoA thiolase enzymes include the gene products of atoB from E. coli (Martin et al., Nat.Biotechnol 21:796-802 (2003)), thiA and thiB from C. acetobutylicum (Hanai et al., Appl Environ Microbiol 73:7814-7818 (2007); Winzer et al., J.Mol.Microbiol Biotechnol 2:531 541 (2000)), and ERG1O from S. cerevisiae (Hiser et al., J.Biol.Chem. 269:31383-31389 (1994)).

263 Protein GenBank ID GI Number Organism atoB NP_416728 16130161 Escherichia coli thIA NP_349476.1 15896127 Clostridium acetobutylicum thIB NP_149242.1 15004782 Clostridium acetobutylicum ERG1O NP_015297 6325229 Saccharomyces cerevisiae Enzymes in the 2.8.3 family catalyze the reversible transfer of a CoA moiety from one molecule to another. Such a transformation is required by steps F, 0, G, T, H, and E of figure 19 and steps B and H of Figure 20. Several CoA transferase enzymes have been described in the open literature and represent suitable candidates for these steps. These are described below. Many transferases have broad specificity and thus can utilize CoA acceptors as diverse as acetate, succinate, propionate, butyrate, 2-methylacetoacetate, 3-ketohexanoate, 3 ketopentanoate, valerate, crotonate, 3-mercaptopropionate, propionate, vinylacetate, butyrate, among others. For example, an enzyme from Roseburia sp. A2-183 was shown to have butyryl-CoA: acetate: CoA transferase and propionyl-CoA: acetate: CoA transferase activity (Charrier et al., Microbiology 152, 179-185 (2006)). Close homologs can be found in, for example, Roseburia intestinalis L1-82, Roseburia inulinivorans DSM 16841, Eubacterium rectale ATCC 33656. Another enzyme with propionyl-CoA transferase activity can be found in Clostridium propionicum (Selmer et al., Eur JBiochem 269, 372-380 (2002)). This enzyme can use acetate, (R)-lactate, (S)-lactate, acrylate, and butyrate as the CoA acceptor (Selmer et al., Eur JBiochem 269, 372-380 (2002); Schweiger and Buckel, FEBS Letters, 171(1) 79-84 (1984)). Close homologs can be found in, for example, Clostridium novyi NT, Clostridium beijerinckii NCIMB 8052, and Clostridium botulinum C str. Eklund. YgfH encodes a propionyl CoA:succinate CoA transferase in E. coli (Haller et al., Biochemistry, 39(16) 4622-4629). Close homologs can be found in, for example, Citrobacteryoungae ATCC 29220, Salmonella enterica subsp. arizonae serovar, and Yersinia intermedia ATCC 29909. These proteins are identified below .

264 Protein GenBank ID GI Number Organism Ach1 AAX19660.1 60396828 Roseburia sp. A2-183 ROSINTL182_07121 ZP_04743841.2 257413684 Roseburia intestinalis L1-82 ROSEINA2194_0364 ZP_03755203.1 225377982 Roseburia inulinivorans 2 EUBREC_3075 YP_002938937.1 238925420 Eubacterium rectale A TCC 33656 Pct CAB77207.1 7242549 Clostridium propionicum NT01CX_2372 YP_878445.1 118444712 Clostridium novyi NT Cbei_4543 YP_001311608.1 150019354 Clostridium beijerinckii CBC_A0889 ZP_02621218.1 168186583 Clostridium botulinum C str. Eklund ygfH NP_417395.1 16130821 Escherichia coli CIT292_04485 ZP_03838384.1 227334728 Citrobacter youngae A TCC 29220 SARI_04582 YP_001573497.1 161506385 Salmonella enterica subsp. arizonae serovar yinte000l_14430 ZP_04635364.1 238791727 Yersinia intermedia A TCC 29909 An additional candidate enzyme is the two-unit enzyme encoded by pcaI and pcaJ in Pseudomonas, which has been shown to have 3-oxoadipyl-CoA/succinate transferase activity (Kaschabek et al., supra). Similar enzymes based on homology exist in Acinetobacter sp. ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)) and Streptomyces coelicolor. Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases are present in Helicobacterpylori (Corthesy-Theulaz et al., JBiol.Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al., Protein.Expr.Purif 53:396-403 (2007)). These proteins are identified below. Protein GenBank ID GI Number Organism pcal AAN69545.1 24985644 Pseudomonas putida pcaJ NP_746082.1 26990657 Pseudomonas putida pcal YP_046368.1 50084858 Acinetobacter sp. ADP1 pcaJ AAC37147.1 141776 Acinetobacter sp. ADP1 265 Protein GenBank ID GI Number Organism pcal NP_630776.1 21224997 Streptomyces coelicolor pcaJ NP_630775.1 21224996 Streptomyces coelicolor HPAG1_0676 YP_627417 108563101 Helicobacter pylori HPAG1_0677 YP_627418 108563102 Helicobacter pylori ScoA NP_391778 16080950 Bacillus subtilis ScoB NP_391777 16080949 Bacillus subtilis A CoA transferase that can utilize acetate as the CoA acceptor is acetoacetyl-CoA transferase, encoded by the E. coli atoA (alpha subunit) and atoD (beta subunit) genes (Vanderwinkel et al., Biochem.Biophys.Res Commun. 33:902-908 (1968); Korolev et al., Acta Crystallogr.D Biol Crystallogr. 58:2116-2121 (2002)). This enzyme has also been shown to transfer the CoA moiety to acetate from a variety of branched and linear acyl-CoA substrates, including isobutyrate (Matthies et al., App/ Environ Microbiol 58:1435-1439 (1992)), valerate (Vanderwinkel et al., supra) and butanoate (Vanderwinkel et al., supra). Similar enzymes exist in Corynebacterium glutamicum ATCC 13032 (Duncan et al., App/ Environ Microbiol 68:5186-5190 (2002)), Clostridium acetobutylicum (Cary et al., App/ Environ Microbiol 56:1576-1583 (1990)), and Clostridium saccharoperbutylacetonicum (Kosaka et al., Biosci.Biotechnol Biochem. 71:58-68 (2007)). These proteins are identified below. Protein GenBank ID GI Number Organism atoA P76459.1 2492994 Escherichia coli K12 atoD P76458.1 2492990 Escherichia coli K12 actA YP_226809.1 62391407 Corynebacterium glutamicum ATCC 13032 cg0592 YP_224801.1 62389399 Corynebacterium glutamicum ATCC 13032 ctfA NP_149326.1 15004866 Clostridium acetobutylicum ctfB NP_149327.1 15004867 Clostridium acetobutylicum ctfA AAP42564.1 31075384 Clostridium saccharoperbutylacetonicum ctfB AAP42565.1 31075385 Clostridium saccharoperbutylacetonicum 266 Additional exemplary transferase candidates are catalyzed by the gene products of cat], cat2, and cat3 of Clostridium kluyveri which have been shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA transferase activity, respectively (Seedorf et al., supra; Sohling et al., Eur.JBiochem. 212:121-127 (1993); Sohling et al., JBacteriol. 178:871-880 (1996)). Similar CoA transferase activities are also present in Trichomonas vaginalis (van Grinsven et al., JBiol.Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., JBiol.Chem. 279:45337-45346 (2004)). These proteins are identified below. Protein GenBank ID GI Number Organism catl P38946.1 729048 Clostridium kluyveri cat2 P38942.2 172046066 Clostridium kluyveri cat3 EDK35586.1 146349050 Clostridium kluyveri TVAG_395550 XP_001330176 123975034 Trichomonas vaginalis G3 Tb11.02.0290 XP_828352 71754875 Trypanosoma brucei The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobic bacterium Acidaminococcusfermentans reacts with diacid glutaconyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS Lett. 405:209-212 (1997)). The genes encoding this enzyme are gctA and gctB. This enzyme has reduced but detectable activity with other CoA derivatives including glutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckel et al., Eur.JBiochem. 118:315-321 (1981)). The enzyme has been cloned and expressed in E. coli (Mack et al., Eur.JBiochem. 226:41-51 (1994)). These proteins are identified below. Protein GenBank ID GI Number Organism gctA CAA57199.1 559392 Acidaminococcus fermentans gctB CAA57200.1 559393 Acidaminococcus fermentans Enzymes in the 3.1.2 family hydrolyze acyl-CoA molecules to their corresponding acids. Such a transformation is required by steps F, 0, G, T, H, and E of figure 19 and steps B and H of Figure 20. Several such enzymes have been described in the literature and represent suitable candidates for these steps.

267 For example, the enzyme encoded by acot12 from Rattus norvegicus brain (Robinson et al., Biochem.Biophys.Res.Commun. 71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and malonyl-CoA. The human dicarboxylic acid thioesterase, encoded by acot8, exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., JBiol.Chem. 280:38125-38132 (2005)). The closest E. coli homolog to this enzyme, tesB, can also hydrolyze a range of CoA thiolesters (Naggert et al., JBiol Chem 266:11044-11050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana R., Biochem Int 26:767-773 (1992)). Additional enzymes with hydrolase activity in E. coli include ybgC, paaI, and ybdB (Kuznetsova, et al., FEMS Microbiol Rev, 2005, 29(2):263-279; Song et al., JBiol Chem, 2006, 281(16):11028-38). Though its sequence has not been reported, the enzyme from the mitochondrion of the pea leaf has a broad substrate specificity, with demonstrated activity on acetyl-CoA, propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, and crotonyl-CoA (Zeiher et al., Plant.Physiol. 94:20-27 (1990)) The acetyl-CoA hydrolase, ACH1, from S. cerevisiae represents another candidate hydrolase (Buu et al., JBiol.Chem. 278:17203-17209 (2003)) . Protein GenBank Accession # GI# Organism acot12 NP_570103.1 18543355 Rattus norvegicus tesB NP_414986 16128437 Escherichia coli acot8 CAA15502 3191970 Homo sapiens acot8 NP_570112 51036669 Rattus norvegicus tesA NP_415027 16128478 Escherichia coli ybgC NP_415264 16128711 Escherichia coli paaI NP_415914 16129357 Escherichia coli ybdB NP_415129 16128580 Escherichia coli ACH1 NP_009538 6319456 Saccharomyces cerevisiae Yet another candidate hydrolase is the glutaconate CoA-transferase from Acidaminococcusfermentans. This enzyme was transformed by site-directed mutagenesis into an acyl-CoA hydrolase with activity on glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS.Lett. 405:209-212 (1997)).This suggests that the enzymes encoding succinyl-CoA:3-ketoacid-CoA transferases and acetoacetyl-CoA:acetyl-CoA transferases 268 may also serve as candidates for this reaction step but would require certain mutations to change their function. Protein GenBank Accession # GI# Organism gctA CAA57199 559392 Acidaminococcusfermentans gctB CAA57200 559393 Acidaminococcusfermentans Additional hydrolase enzymes include 3-hydroxyisobutyryl-CoA hydrolase which has been described to efficiently catalyze the conversion of 3-hydroxyisobutyryl-CoA to 3 hydroxyisobutyrate during valine degradation (Shimomura et al., JBiol Chem. 269:14248 14253 (1994)). Genes encoding this enzyme include hibch of Rattus norvegicus (Shimomura et al., Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomura et al., supra). Similar gene candidates can also be identified by sequence homology, including hibch of Saccharomyces cerevisiae and BC_2292 of Bacillus cereus. Protein GenBank Accession # GI# Organism hibch Q5XIE6.2 146324906 Rattus norvegicus hibch Q6NVY1.2 146324905 Homo sapiens hibch P28817.2 2506374 Saccharomyces cerevisiae BC_2292 AP09256 29895975 Bacillus cereus Decarboxylase enzymes in the EC class 4.1.1 are required to catalyze steps U,Y,V, and X of Figure 19, steps D,J,M, and N of figure 20, and steps M,N, and P of figure 21. Candidate decarboxylase enzymes have been described earlier in this application. The hydration of a double bond can be catalyzed by hydratase enzymes in the 4.2.1 family of enzymes. The removal of water to form a double bond is catalyzed by dehydratase enzymes in the 4.2.1 family of enzymes. Hydratase enzymes are sometimes reversible and also catalyze dehydration. Dehydratase enzymes are sometimes reversible and also catalyze hydration. The addition or removal of water from a given substrate is required by steps S, K, L, R, D, C, J, Q, and W in Figure 19, by step F in Figure 20, and by steps E, F, and 0 in Figure 21. Several hydratase and dehydratase enzymes have been described in the literature and represent suitable candidates for these steps.

269 For example, many dehydratase enzymes catalyze the alpha, beta-elimination of water which involves activation of the alpha-hydrogen by an electron-withdrawing carbonyl, carboxylate, or CoA-thiol ester group and removal of the hydroxyl group from the beta position (Buckel et al, JBacteriol, 117:1248-60 (1974); Martins et al, PNAS 101:15645-9 (2004)). Exemplary enzymes include 2-(hydroxymethyl)glutarate dehydratase (EC 4.2.1.-), fumarase (EC 4.2.1.2), 3-dehydroquinate dehydratase (EC 4.2.1.10), cyclohexanone hydratase (EC 4.2.1.-) and 2-keto-4-pentenoate dehydratase (EC 4.2.1.80), citramalate hydrolyase and dimethylmaleate hydratase. 2-(Hydroxymethyl)glutarate dehydratase is a [4Fe-4S] -containing enzyme that dehydrates 2-(hydroxymethyl)glutarate to 2-methylene-glutarate, studied for its role in nicontinate catabolism in Eubacterium barkeri (formerly Clostridium barkeri) (Alhapel et al., Proc Natl Acad Sci 103:12341-6 (2006)). Similar enzymes with high sequence homology are found in Bacteroides capillosus, Anaerotruncus colihominis, and Natranaerobius thermophilius. These enzymes are homologous to the alpha and beta subunits of [4Fe-4S] containing bacterial serine dehydratases (e.g., E. coli enzymes encoded by tdcG, sdhB, and sdaA). An enzyme with similar functionality in E. barkeri is dimethylmaleate hydratase, a reversible Fe2+-dependent and oxygen-sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3-dimethylmalate. This enzyme is encoded by dmdAB (Alhapel et al., Proc Natl Acad Sci USA 103:12341-6 (2006); Kollmann-Koch et al., Hoppe Seylers.Z.Physiol Chem. 365:847-857 (1984)). Protein GenBank ID GI Number Organism hmd ABC88407.1 86278275 Eubacterium barkeri BACCAP_02294 ZP_02036683.1 154498305 Bacteroides capillosus ANACOL_02527 ZP_02443222.1 167771169 Anaerotruncus colihominis NtherDRAFT 2368 ZP_02852366.1 169192667 Natranaerobius thermophilus dmdA ABC88408 86278276 Eubacterium barkeri dmdB ABC88409 86278277 Eubacterium barkeri Fumarate hydratase (EC 4.2.1.2) enzymes naturally catalyze the reversible hydration of fumarate to malate. Although the ability of fumarate hydratase to react with 3-oxobutanol as a substrate has not been described in the literature, a wealth of structural information is available for this enzyme and other researchers have successfully engineered the enzyme to 270 alter activity, inhibition and localization (Weaver, 61:1395-1401 (2005)). E. coli has three fumarases: FumA, FumB, and FumC that are regulated by growth conditions. FumB is oxygen sensitive and only active under anaerobic conditions. FumA is active under microanaerobic conditions, and FumC is the only active enzyme in aerobic growth (Tseng et al., JBacteriol, 183:461-467 (2001); Woods et al., 954:14-26 (1988); Guest et al., JGen Microbiol 131:2971-2984 (1985)). Additional enzyme candidates are found in Campylobacterjejuni (Smith et al., Int.JBiochem.Cell Biol 31:961-975 (1999)), Thermus thermophilus (Mizobata et al., Arch.Biochem.Biophys. 355:49-55 (1998)) and Rattus norvegicus (Kobayashi et al., J Biochem, 89:1923-1931 (1981)). Similar enzymes with high sequence homology includefum] from Arabidopsis thaliana and fumC from Corynebacterium glutamicum. The MmcBC fumarase from Pelotomaculum thermopropionicum is another class of fumarase with two subunits (Shimoyama et al., FEMS Microbiol Lett, 270:207-213 (2007)). Protein GenBank ID GI Number Organism fumA NP_416129.1 16129570 Escherichia coli fumB NP_418546.1 16131948 Escherichia coli fumC NP_416128.1 16129569 Escherichia coli fumC 069294 9789756 Campylobacterjejuni fumC P84127 75427690 Thermus thermophilus fumH P14408 120605 Rattus norvegicus fumI P93033 39931311 Arabidopsis thaliana fumC Q8NRN8 39931596 Corynebacterium glutamicum MmcB YP_001211906 147677691 Pelotomaculum thermopropionicum MmcC YP_001211907 147677692 Pelotomaculum thermopropionicum Dehydration of 4-hydroxy-2-oxovalerate to 2-oxopentenoate is catalyzed by 4 hydroxy-2-oxovalerate hydratase (EC 4.2.1.80). This enzyme participates in aromatic degradation pathways and is typically co-transcribed with a gene encoding an enzyme with 4 hydroxy-2-oxovalerate aldolase activity. Exemplary gene products are encoded by mhpD of E. coli (Ferrandez et al., JBacteriol. 179:2573-2581 (1997); Pollard et al., Eur JBiochem. 251:98-106 (1998)), todG and cmtF of Pseudomonas putida (Lau et al., Gene 146:7-13 (1994); Eaton, JBacteriol. 178:1351-1362 (1996)), cnbE of Comamonas sp. CNB-1 (Ma et al., Appl Environ Microbiol 73:4477-4483 (2007)) and mhpD of Burkholderia xenovorans 271 (Wang et al., FEBSJ272:966-974 (2005)). A closely related enzyme, 2-oxohepta-4-ene-1,7 dioate hydratase, participates in 4-hydroxyphenylacetic acid degradation, where it converts 2 oxo-hept-4-ene-1,7-dioate (OHED) to 2-oxo-4-hydroxy-hepta-1,7-dioate using magnesium as a cofactor (Burks et al., JAm.Chem.Soc. 120: (1998)). OHED hydratase enzyme candidates have been identified and characterized in E. coli C (Roper et al., Gene 156:47-51 (1995); Izumi et al., JMol.Biol. 370:899-911 (2007)) and E. coli W (Prieto et al., JBacteriol. 178:111-120 (1996)). Sequence comparison reveals homologs in a wide range of bacteria, plants and animals. Enzymes with highly similar sequences are contained in Klebsiella pneumonia (910% identity, eval = 2e- 138) and Salmonella enterica (910% identity, eval = 4e 138), among others. Protein GenBank Accession No. GI No. Organism mhpD AAC73453.2 87081722 Escherichia coli cmtF AAB62293.1 1263188 Pseudomonas putida todG AAA61942.1 485738 Pseudomonas putida cnbE YP_001967714.1 190572008 Comamonas sp. CNB-1 mhpD Q13VUO 123358582 Burkholderia xenovorans hpcG CAA57202.1 556840 Escherichia coli C hpaH CAA86044.1 757830 Escherichia coli W hpaH ABR80130.1 150958100 Klebsiella pneumoniae Sari_01896 ABX21779.1 160865156 Salmonella enterica Another enzyme candidate is citramalate hydrolyase (EC 4.2.1.34), an enzyme that naturally dehydrates 2-methylmalate to mesaconate. This enzyme has been studied in Methanocaldococcusjannaschii in the context of the pyruvate pathway to 2-oxobutanoate, where it has been shown to have a broad substrate specificity (Drevland et al., JBacteriol. 189:4391-4400 (2007)). This enzyme activity was also detected in Clostridium tetanomorphum, Morganella morganii, Citrobacter amalonaticus where it is thought to participate in glutamate degradation (Kato et al., Arch.Microbiol 168:457-463 (1997)). The M. jannaschii protein sequence does not bear significant homology to genes in these organisms.

272 Protein GenBank ID GI Number Organism leuD Q58673.1 3122345 Methanocaldococcusjannaschii Dimethylmaleate hydratase (EC 4.2.1.85) is a reversible Fe2+-dependent and oxygen sensitive enzyme in the aconitase family that hydrates dimethylmaeate to form (2R,3S)-2,3 dimethylmalate. This enzyme is encoded by dmdAB in Eubacterium barkeri (Alhapel et al., supra; Kollmann-Koch et al., Hoppe Seylers.Z.Physiol Chem. 365:847-857 (1984)). Protein GenBank ID GI Number Organism dmdA ABC88408 86278276 Eubacterium barkeri dmdB ABC88409.1 86278277 Eubacterium barkeri Oleate hydratases represent additional suitable candidates as suggested in WO2011076691. These are particularly useful for step W of Figure 19 and step 0 of Figure 21. Examples include the following proteins. Protein GenBank ID GI Number Organism OhyA ACT54545.1 254031735 Elizabethkingia meningoseptica HMPREF0841_1446 ZP_07461147.1 306827879 Streptococcus pyogenes A TCC 10782 P700755_13397 ZP_01252267.1 91215295 Psychroflexus torquis A TCC 700755 RPB_2430 YP_486046.1 86749550 Rhodopseudomonas palustris Enoyl-CoA hydratases (EC 4.2.1.17) catalyze the dehydration of a range of 3 hydroxyacyl-CoA substrates (Roberts et al., Arch.Microbiol 117:99-108 (1978); Agnihotri et al., Bioorg.Med.Chem. 11:9-20 (2003); Conrad et al., JBacteriol. 118:103-111 (1974)). The enoyl-CoA hydratase of Pseudomonas putida, encoded by ech, catalyzes the conversion of 3 hydroxybutyryl-CoA to crotonyl-CoA (Roberts et al., Arch.Microbiol 117:99-108 (1978)). This transformation is also catalyzed by the crt gene product of Clostridium acetobutylicum, the crt1 gene product of C. kluyveri, and other clostridial organisms Atsumi et al., Metab Eng 10:305-311 (2008); Boynton et al., JBacteriol. 178:3015-3024 (1996); Hillmer et al., FEBS 273 Lett. 21:351-354 (1972)). Additional enoyl-CoA hydratase candidates are phaA and phaB, of P. putida, and paaA and paaB from P.fluorescens (Olivera et al., Proc.Natl.Acad.Sci U.S.A 95:6419-6424 (1998)). The gene product ofpimF in Rhodopseudomonas palustris is predicted to encode an enoyl-CoA hydratase that participates in pimeloyl-CoA degradation (Harrison et al., Microbiology 151:727-736 (2005)). Lastly, a number of Escherichia coli genes have been shown to demonstrate enoyl-CoA hydratase functionality including maoC (Park et al., JBacteriol. 185:5391-5397 (2003)), paaF (Ismail et al., Eur.JBiochem. 270:3047-3054 (2003); Park et al., Appl.Biochem.Biotechnol 113-116:335-346 (2004); Park et al., Biotechnol Bioeng 86:681-686 (2004)) and paaG (Ismail et al., Eur.JBiochem. 270:3047-3054 (2003); Park and Lee, Appl.Biochem.Biotechnol 113-116:335-346 (2004); Park and Yup, Biotechnol Bioeng 86:681-686 (2004)). Protein GenBank Accession No. GI No. Organism ech NP_745498.1 26990073 Pseudomonas putida crt NP_349318.1 15895969 Clostridium acetobutylicum crt1 YP_001393856 153953091 Clostridium kluyveri phaA ABF82233.1 26990002 Pseudomonas putida phaB ABF82234.1 26990001 Pseudomonas putida paaA NP_745427.1 106636093 Pseudomonasfluorescens paaB NP_745426.1 106636094 Pseudomonasfluorescens maoC NP_415905.1 16129348 Escherichia coli paaF NP_415911.1 16129354 Escherichia coli paaG NP_415912.1 16129355 Escherichia coli Alternatively, the E. coli gene products offadA andfadB encode a multienzyme complex involved in fatty acid oxidation that exhibits enoyl-CoA hydratase activity (Yang et al., Biochemistry 30:6788-6795 (1991); Yang, JBacteriol. 173:7405-7406 (1991); Nakahigashi et al., Nucleic Acids Res. 18:4937 (1990)). Knocking out a negative regulator encoded byfadR can be utilized to activate thefadB gene product (Sato et al., JBiosci.Bioeng 103:38-44 (2007)). ThefadI andfadJ genes encode similar functions and are naturally expressed under anaerobic conditions (Campbell et al., Mol.Microbiol 47:793-805 (2003)). Protein GenBank ID GI Number Organism 274 fadA YP_026272.1 49176430 Escherichia coli fadB NP_418288.1 16131692 Escherichia coli fadI NP_416844.1 16130275 Escherichia coli fadJ NP_416843.1 16130274 Escherichia coli fadR NP_415705.1 16129150 Escherichia coli The conversion of acyl-CoA substrates to their acid products can be catalyzed by a CoA acid-thiol ligase or CoA synthetase in the 6.2.1 family of enzymes, several of which are reversible. Several reactions shown in Figures 19-20 are catalyzed by acid-thiol ligase enzymes. These reactions include Steps F, 0, G, T, H, and E of Figure 19 and Steps B and H of Figure 20. Several enzymes catalyzing CoA acid-thiol ligase or CoA synthetase activities have been described in the literature and represent suitable candidates for these steps. For example, ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that couples the conversion of acyl-CoA esters to their corresponding acids with the concomitant synthesis of ATP. ACD I from Archaeoglobusfulgidus, encoded by AF1211, was shown to operate on a variety of linear and branched-chain substrates including isobutyrate, isopentanoate, and fumarate (Musfeldt et al., JBacteriol. 184:636-644 (2002)). A second reversible ACD in Archaeoglobusfulgidus, encoded by AF1983, was also shown to have a broad substrate range with high activity on cyclic compounds phenylacetate and indoleacetate (Musfeldt and Schonheit, JBacteriol. 184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as a succinyl-CoA synthetase) accepts propionate, butyrate, and branched-chain acids (isovalerate and isobutyrate) as substrates, and was shown to operate in the forward and reverse directions (Brasen et al., Arch Microbiol 182:277-287 (2004)). The ACD encoded by PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized ACDs, reacting with acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al, supra). Directed evolution or engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism. The enzymes from A.fulgidus, H. marismortui and P. aerophilum have all been cloned, functionally expressed, and characterized in E. coli (Brasen and Schonheit, supra; Musfeldt and Schonheit, JBacteriol. 184:636-644 (2002)). An additional candidate is succinyl-CoA synthetase, encoded by sucCD of E. coli and LSCJ and LSC2 genes of Saccharomyces cerevisiae. These enzymes catalyze 275 the formation of succinyl-CoA from succinate with the concomitant consumption of one ATP in a reaction which is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)). The acyl CoA ligase from Pseudomonas putida has been demonstrated to work on several aliphatic substrates including acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds such as phenylacetic and phenoxyacetic acids (Fernandez-Valverde et al., Appl.Environ.Microbiol. 59:1149-1154 (1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) from Rhizobium leguminosarum could convert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-, cyclopropyl-, cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into their corresponding monothioesters (Pohl et al., JAm.Chem.Soc. 123:5822-5823 (2001)). Protein GenBank ID GI Number Organism AF1211 NP_070039.1 11498810 Archaeoglobusfulgidus AF1983 NP_070807.1 11499565 Archaeoglobusfulgidus Scs YP_135572.1 55377722 Haloarcula marismortui PAE3250 NP_560604.1 18313937 Pyrobaculum aerophilum str. IM2 sucC NP_415256.1 16128703 Escherichia coli sucD AAC73823.1 1786949 Escherichia coli LSCJ NP_014785 6324716 Saccharomyces cerevisiae LSC2 NP_011760 6321683 Saccharomyces cerevisiae paaF AAC24333.2 22711873 Pseudomonas putida matB AAC83455.1 3982573 Rhizobium leguminosarum Another candidate enzyme for these steps is 6-carboxyhexanoate-CoA ligase, also known as pimeloyl-CoA ligase (EC 6.2.1.14), which naturally activates pimelate to pimeloyl CoA during biotin biosynthesis in gram-positive bacteria. The enzyme from Pseudomonas mendocina, cloned into E. coli, was shown to accept the alternate substrates hexanedioate and nonanedioate (Binieda et al., Biochem.J340 ( Pt 3):793-801 (1999)). Other candidates are found in Bacillus subtilis (Bower et al., JBacteriol. 178:4122-4130 (1996)) and Lysinibacillus sphaericus (formerly Bacillus sphaericus) (Ploux et al., Biochem.J 287 ( Pt 3):685-690 (1992)).

276 Protein GenBank ID GI Number Organism bioW NP_390902.2 50812281 Bacillus subtilis bioW CAA10043.1 3850837 Pseudomonas mendocina bioW P22822.1 115012 Bacillus sphaericus Additional CoA-ligases include the rat dicarboxylate-CoA ligase for which the sequence is yet uncharacterized (Vamecq et al., Biochem.J230:683-693 (1985)), either of the two characterized phenylacetate-CoA ligases from P. chrysogenum (Lamas-Maceiras et al., Biochem.J395:147-155 (2006); Wang et al., 360:453-458 (2007)), the phenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco et al., JBiol Chem 265:7084-7090 (1990)) and the 6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower et al. J Bacteriol 178(14):4122-4130 (1996)). Acetoacetyl-CoA synthetases from Mus musculus (Hasegawa et al., Biochim Biophys Acta 1779:414-419 (2008)) and Homo sapiens (Ohgami et al., Biochem.Pharmacol. 65:989-994 (2003)) naturally catalyze the ATP-dependent conversion of acetoacetate into acetoacetyl-CoA. Protein Accession No. GI No. Organism phl CAJ15517.1 77019264 Penicillium chrysogenum ph/B ABS 19624.1 152002983 Penicillium chrysogenum paaF AAC24333.2 22711873 Pseudomonas putida bioW NP_390902.2 50812281 Bacillus subtilis AACS NP_084486.1 21313520 Mus musculus AACS NP_076417.2 31982927 Homo sapiens Like enzymes in other classes, certain enzymes in the EC class 6.2.1 have been determined to have broad substrate specificity. The acyl CoA ligase from Pseudomonas putida has been demonstrated to work on several aliphatic substrates including acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids and on aromatic compounds such as phenylacetic and phenoxyacetic acids (Femandez-Valverde et al., Applied and Environmental Microbiology 59:1149-1154 (1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) from Rhizobium trifolii could convert several diacids, namely, ethyl-, propyl-, allyl-, isopropyl-, dimethyl-, cyclopropyl-, cyclopropylmethylene-, 277 cyclobutyl-, and benzyl-malonate into their corresponding monothioesters (Pohl et al., JAm.Chem.Soc. 123:5822-5823 (2001)). Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank and GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.

Claims (243)

1. A non-naturally occurring microbial organism, comprising a microbial organism having a toluene pathway comprising at least one exogenous nucleic acid encoding a toluene pathway enzyme expressed in a sufficient amount to produce 5 toluene, said toluene pathway selected from (A) 1) one or both of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, and 3) phenylacetaldehyde decarbonylase; (B) 1) one or more of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, 3) one or more of phenylacetaldehyde dehydrogenase 10 and phenylacetaldehyde oxidase, and 4) phenylacetate decarboxylase; (C) 1) one or more of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate oxidase, and 3) phenylacetate decarboxylase; and (D) 1) phenylalanine aminotransferase and/or phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate oxidase and 3) phenylacetate decarboxylase. 15

2. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises two exogenous nucleic acids each encoding a toluene pathway enzyme.

3. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises three exogenous nucleic acids each encoding a toluene 20 pathway enzyme.

4. The non-naturally occurring microbial organism of claim 3, wherein said three exogenous nucleic acids encode 1) phenylalanine aminotransferase or phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, 3) phenylacetaldehyde decarbonylase. 25

5. The non-naturally occurring microbial organism of claim 1, wherein said microbial organism comprises four exogenous nucleic acids each encoding a toluene pathway enzyme.

6. The non-naturally occurring microbial organism of claim 5, wherein said four exogenous nucleic acids encode 1) phenylalanine aminotransferase or 279 phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, 3) phenylacetaldehyde dehydrogenase or oxidase, and 4) phenylacetate decarboxylase.

7. The non-naturally occurring microbial organism of claim 1, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 5

8. The non-naturally occurring microbial organism of claim 1, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

9. A method for producing toluene, comprising culturing a non-naturally occurring microbial organism having a toluene pathway, said pathway comprising at 10 least one exogenous nucleic acid encoding a toluene pathway enzyme expressed in a sufficient amount to produce toluene, under conditions and for a sufficient period of time to produce toluene, said toluene pathway selected from (A) 1) one or both of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, and 3) phenylacetaldehyde decarbonylase; (B) 1) one or 15 more of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, 3) one or more of phenylacetaldehyde dehydrogenase and phenylacetaldehyde oxidase, and 4) phenylacetate decarboxylase; (C) one or more of phenylalanine aminotransferase and phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate oxidase, and 3) phenylacetate decarboxylase; and (D) 20 1) phenylalanine aminotransferase and/or phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate oxidase and 3) phenylacetate decarboxylase.

10. The method of claim 9, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

11. The method of claim 9, wherein said microbial organism comprises two 25 exogenous nucleic acids each encoding a toluene pathway enzyme.

12. The method of claim 9, wherein said microbial organism comprises three exogenous nucleic acids each encoding a toluene pathway enzyme. 280

13. The method of claim 12, wherein said three exogenous nucleic acids encode 1) phenylalanine aminotransferase or phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, and 3) phenylacetaldehyde decarbonylase. 5

14. The method of claim 9, wherein said microbial organism comprises four exogenous nucleic acids each encoding a toluene pathway enzyme.

15. The method of claim 14, wherein said four exogenous nucleic acids encode 1) phenylalanine aminotransferase or phenylalanine oxidoreductase (deaminating), 2) phenylpyruvate decarboxylase, 3) phenylacetaldehyde dehydrogenase or oxidase, and 10 4) phenylacetate decarboxylase.

16. The method of claim 9, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

17. A non-naturally occurring microbial organism, comprising a microbial organism having a benzene pathway comprising at least one exogenous nucleic acid 15 encoding a benzene pathway enzyme expressed in a sufficient amount to produce benzene, said benzene pathway comprising a phenylalanine benzene-lyase.

18. The non-naturally occurring microbial organism of claim 17, wherein said at least one exogenous nucleic acid is said phenylalanine benzene-lyase.

19. The non-naturally occurring microbial organism of claim 17, wherein said 20 at least one exogenous nucleic acid is a heterologous nucleic acid.

20. The non-naturally occurring microbial organism of claim 17, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

21. A method for producing benzene, comprising culturing a non-naturally 25 occurring microbial organism having a benzene pathway, said pathway comprising at least one exogenous nucleic acid encoding a benzene pathway enzyme expressed in a sufficient amount to produce benzene, under conditions and for a sufficient period of 281 time to produce benzene, said benzene pathway comprising a phenylalanine benzene lyase.

22. The method of claim 21, wherein said at least one exogenous nucleic acid is said phenylalanine benzene-lyase. 5

23. The method of claim 21, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

24. The method of claim 21, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

25. A non-naturally occurring microbial organism, comprising a microbial 10 organism having a styrene pathway comprising at least one exogenous nucleic acid encoding a styrene pathway enzyme expressed in a sufficient amount to produce styrene, said styrene pathway selected from (A) 1) benzoyl-CoA acetyltransferase, 2) one or more of 3-oxo-3-phenylpropionyl-CoA synthetase, transferase, and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetopheone reductase, and 5) 1 -phenylethanol 15 dehydratase; or (B) 1) benzoyl-CoA acetyltransferase, 2) phosphotrans-3-oxo-3 phenylpropionylase, 3) benzoyl-acetate kinase , 4) benzoyl-acetate decarboxylase, 5) acetopheone reductase, and 6) 1 -phenylethanol dehydratase.

26. The non-naturally occurring microbial organism of claim 25, wherein said microbial organism comprises two exogenous nucleic acids each encoding a styrene 20 pathway enzyme.

27. The non-naturally occurring microbial organism of claim 25, wherein said microbial organism comprises three exogenous nucleic acids each encoding a styrene pathway enzyme.

28. The non-naturally occurring microbial organism of claim 25, wherein said 25 microbial organism comprises four exogenous nucleic acids each encoding a styrene pathway enzyme. 282

29. The non-naturally occurring microbial organism of claim 25, wherein said microbial organism comprises five exogenous nucleic acids each encoding a styrene pathway enzyme.

30. The non-naturally occurring microbial organism of claim 29, wherein said 5 five exogenous nucleic acids encode 1) benzoyl-CoA acetyltransferase, 2) one of 3 oxo-3-phenylpropionyl-CoA synthetase, transferase, and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetopheone reductase, and 5) 1 -phenylethanol dehydratase.

31. The non-naturally occurring microbial organism of claim 25, wherein said microbial organism comprises six exogenous nucleic acids each encoding a styrene 10 pathway enzyme.

32. The non-naturally occurring microbial organism of claim 31, wherein said six exogenous nucleic acids encode 1) benzoyl-CoA acetyltransferase, 2) phosphotrans 3-oxo-3-phenylpropionylase, 3) benzoyl-acetate kinase , 4) benzoyl-acetate decarboxylase, 5) acetopheone reductase, and 6) 1 -phenylethanol dehydratase. 15

33. The non-naturally occurring microbial organism of claim 25, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

34. The non-naturally occurring microbial organism of claim 25, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 20

35. A method for producing styrene, comprising culturing a non-naturally occurring microbial organism having a styrene pathway, said pathway comprising at least one exogenous nucleic acid encoding a styrene pathway enzyme expressed in a sufficient amount to produce styrene, under conditions and for a sufficient period of time to produce styrene, said styrene pathway selected from (A) 1) benzoyl-CoA 25 acetyltransferase, 2) one or more of 3-oxo-3-phenylpropionyl-CoA synthetase, transferase, and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetopheone reductase, and 5) 1-phenylethanol dehydratase; or (B) 1) benzoyl-CoA acetyltransferase, 2) 283 phosphotrans-3-oxo-3-phenylpropionylase, 3) benzoyl-acetate kinase , 4) benzoyl acetate decarboxylase, 5) acetopheone reductase, and 6) 1-phenylethanol dehydratase.

36. The method of claim 35, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 5

37. The method of claim 35, wherein said microbial organism comprises two exogenous nucleic acids each encoding a styrene pathway enzyme.

38. The method of claim 35, wherein said microbial organism comprises three exogenous nucleic acids each encoding a styrene pathway enzyme.

39. The method of claim 35, wherein said microbial organism comprises four 10 exogenous nucleic acids each encoding a styrene pathway enzyme.

40. The method of claim 35, wherein said microbial organism comprises five exogenous nucleic acids each encoding a styrene pathway enzyme.

41. The method of claim 40, wherein said five exogenous nucleic acids encode 1) benzoyl-CoA acetyltransferase, 2) one of 3-oxo-3-phenylpropionyl-CoA synthetase, 15 transferase, and hydrolase, 3) benzoyl-acetate decarboxylase, 4) acetopheone reductase, and 5) 1 -phenylethanol dehydratase.

42. The method of claim 35, wherein said microbial organism comprises six exogenous nucleic acids each encoding a styrene pathway enzyme.

43. The method of claim 42, wherein said six exogenous nucleic acids encode 20 1) benzoyl-CoA acetyltransferase, 2) phosphotrans-3-oxo-3-phenylpropionylase, 3) benzoyl-acetate kinase, 4) benzoyl-acetate decarboxylase, 5) acetopheone reductase, and 6) 1 -phenylethanol dehydratase.

44. The method of claim 35, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 25

45. A non-naturally occurring microbial organism, comprising a microbial organism having a 1,3-butadiene pathway comprising at least one exogenous nucleic 284 acid encoding a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene, said 1,3-butadiene pathway selected from (A) 1) trans, trans muconate decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (B) 1) cis, trans-muconate cis-decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (C) 1) 5 cis, trans-muconate trans-decarboxylase 2) cis-2,4-pentadienoate decarboxylase; (D) 1) cis, cis-muconate decarboxylase and 2) cis-2,4-pentadienoate decarboxylase; (E) cis 2,4-pentadienoate decarboxylase; and (F) trans-2,4-pentadienoate decarboxylase.

46. The non-naturally occurring microbial organism of claim 45, wherein said microbial organism comprises two exogenous nucleic acids each encoding a 1,3 10 butadiene pathway enzyme.

47. The non-naturally occurring microbial organism of claim 46, wherein said two exogenous nucleic acids encode a set selected from (A) 1) trans, trans-muconate decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (B) 1) cis, trans muconate cis-decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (C) 1) cis, 15 trans-muconate trans-decarboxylase 2) cis-2,4-pentadienoate decarboxylase; and (D) 1) cis, cis-muconate decarboxylase and 2) cis-2,4-pentadienoate decarboxylase.

48. The non-naturally occurring microbial organism of claim 45, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

49. The non-naturally occurring microbial organism of claim 45, wherein said 20 non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

50. A method for producing 1,3-butadiene, comprising culturing a non naturally occurring microbial organism having a 1,3-butadiene pathway, said pathway comprising at least one exogenous nucleic acid encoding a 1,3-butadiene pathway 25 enzyme expressed in a sufficient amount to produce 1,3-butadiene, under conditions and for a sufficient period of time to produce 1,3-butadiene, said 1,3-butadiene pathway selected from (A) 1) trans, trans-muconate decarboxylase and 2) trans-2,4 pentadienoate decarboxylase; (B) 1) cis, trans-muconate cis-decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (C) 1) cis, trans-muconate trans-decarboxylase 285 2) cis-2,4-pentadienoate decarboxylase; (D) 1) cis, cis-muconate decarboxylase and 2) cis-2,4-pentadienoate decarboxylase; (E) cis-2,4-pentadienoate decarboxylase; and (F) trans-2,4-pentadienoate decarboxylase.

51. The method of claim 50, wherein said non-naturally occurring microbial 5 organism is in a substantially anaerobic culture medium.

52. The method of claim 50, wherein said microbial organism comprises two exogenous nucleic acids each encoding a 1,3-butadiene pathway enzyme.

53. The method of claim 52, wherein said two exogenous nucleic acids encode a set selected from (A) 1) trans, trans-muconate decarboxylase and 2) trans-2,4 10 pentadienoate decarboxylase; (B) 1) cis, trans-muconate cis-decarboxylase and 2) trans-2,4-pentadienoate decarboxylase; (C) 1) cis, trans-muconate trans-decarboxylase 2) cis-2,4-pentadienoate decarboxylase; and (D) 1) cis, cis-muconate decarboxylase and 2) cis-2,4-pentadienoate decarboxylase.

54. The method of claim 50, wherein said at least one exogenous nucleic acid 15 is a heterologous nucleic acid.

55. A non-naturally occurring microbial organism, comprising a microbial organism having a (2-hydroxy-4-oxobutoxy)phosphonate pathway comprising at least one exogenous nucleic acid encoding a (2-hydroxy-4-oxobutoxy)phosphonate pathway enzyme expressed in a sufficient amount to produce (2-hydroxy-4 20 oxobutoxy)phosphonate, said (2-hydroxy-4-oxobutoxy)phosphonate pathway comprising erythrose-4-phosphate dehydratase and (2,4-dioxobutoxy)phosphonate reductase.

56. The non-naturally occurring microbial organism of claim 55, wherein said microbial organism comprises two exogenous nucleic acids each encoding a (2 25 hydroxy-4-oxobutoxy)phosphonate pathway enzyme. 286

57. The non-naturally occurring microbial organism of claim 56, wherein said two exogenous nucleic acids encode erythrose-4-phosphate dehydratase and (2,4 dioxobutoxy)phosphonate reductase.

58. The non-naturally occurring microbial organism of claim 55, wherein at 5 least one exogenous nucleic acid is a heterologous nucleic acid.

59. The non-naturally occurring microbial organism of claim 55, wherein said microbial organism is cultured anaerobically.

60. A non-naturally occurring microbial organism, comprising a microbial organism having a benzoate pathway comprising at least one exogenous nucleic acid 10 encoding a benzoate pathway enzyme expressed in a sufficient amount to produce benzoate, said benzoate pathway comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase. 15

61. The non-naturally occurring microbial organism of claim 60, wherein said microbial organism comprises two exogenous nucleic acids each encoding a benzoate pathway enzyme.

62. The non-naturally occurring microbial organism of claim 60, wherein said microbial organism comprises three exogenous nucleic acids each encoding a benzoate 20 pathway enzyme.

63. The non-naturally occurring microbial organism of claim 60, wherein said microbial organism comprises four exogenous nucleic acids each encoding a benzoate pathway enzyme.

64. The non-naturally occurring microbial organism of claim 60, wherein said 25 microbial organism comprises five exogenous nucleic acids each encoding a benzoate pathway enzyme. 287

65. The non-naturally occurring microbial organism of claim 60, wherein said microbial organism comprises six exogenous nucleic acids each encoding a benzoate pathway enzyme.

66. The non-naturally occurring microbial organism of claim 60, wherein said 5 microbial organism comprises seven exogenous nucleic acids each encoding a benzoate pathway enzyme.

67. The non-naturally occurring microbial organism of claim 60, wherein said microbial organism comprises eight exogenous nucleic acids each encoding a benzoate pathway enzyme. 10

68. The non-naturally occurring microbial organism of claim 67, wherein said eight exogenous nucleic acids encode 2-dehydro-3-deoxyphosphoheptonate synthase; 3 dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase. 15

69. The non-naturally occurring microbial organism of claim 60 further comprising a (2-hydroxy-4-oxobutoxy)phosphonate pathway comprising erythrose-4 phosphate dehydratase and (2,4-dioxobutoxy)phosphonate reductase.

70. The non-naturally occurring microbial organism of claim 60, wherein at least one exogenous nucleic acid is a heterologous nucleic acid. 20

71. The non-naturally occurring microbial organism of claim 60, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

72. A method for producing benzoate, comprising culturing the non-naturally occurring microbial organism of claim 60 under conditions and for a sufficient period of 25 time to produce benzoate.

73. The method of claim 72, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 288

74. The method of claim 72, wherein said microbial organism comprises eight exogenous nucleic acids each encoding a benzoate pathway enzyme.

75. The method of claim 74, wherein said eight exogenous nucleic acids encode 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3 5 dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3 phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase.

76. The method of claim 72, wherein said microbial organism further comprises a (2-hydroxy-4-oxobutoxy)phosphonate pathway comprising erythrose-4-phosphate 10 dehydratase and (2,4-dioxobutoxy)phosphonate reductase.

77. The method of claim 72, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

78. A non-naturally occurring microbial organism, comprising a microbial organism having a benzene pathway comprising at least one exogenous nucleic acid 15 encoding a benzene pathway enzyme expressed in a sufficient amount to produce benzene, said benzene pathway is selected from a set of pathway enzymes selected from: a) benzoate decarboxylase; b) benzoate reductase and benzaldehyde decarbonylase; c) benzoate kinase, (benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase; d) (benzoyl-CoA synthetase, transferase and/or 20 hydrolase), phosphotransbenzoylase, (benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase; and e) (benzoyl-CoA synthetase, transferase and/or hydrolase), benzoyl-CoA reductase and benzaldehyde decarbonylase, f) (benzoyl-CoA synthetase, transferase and/or hydrolase), benzoate decarboxylase, g) benzoyl-CoA reductase and benzaldehyde decarbonylase, h) phosphotransbenzoylase, 25 (benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase, i) phosphotransbenzoylase, benzoate kinase, benzoate decarboxylase, j) phosphotransbenzoylase, benzoate kinase, benzoate reductase, benzaldehyde decarbonylase; k) phosphotransbenzoylase, (benzoyloxy)phosphonate reductase, and 289 benzaldehyde decarbonylase; and 1) benzoyl-CoA reductase and benzaldehyde decarbonylase.

79. The non-naturally occurring microbial organism of claim 78, wherein said benzene pathway comprises benzoate decarboxylase. 5

80. The non-naturally occurring microbial organism of claim 78, wherein said benzene pathway comprises benzoate reductase and benzaldehyde decarbonylase

81. The non-naturally occurring microbial organism of claim 78, wherein said benzene pathway comprises benzoate kinase, (benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase. 10

82. The non-naturally occurring microbial organism of claim 78, wherein said benzene pathway comprises (benzoyl-CoA synthetase, transferase and/or hydrolase), phosphotransbenzoylase, (benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase.

83. The non-naturally occurring microbial organism of claim 78, wherein said 15 benzene pathway comprises (benzoyl-CoA synthetase, transferase and/or hydrolase), benzoyl-CoA reductase and benzaldehyde decarbonylase.

84. The non-naturally occurring microbial organism of claim 78, wherein said microbial organism comprises two exogenous nucleic acids each encoding a benzene pathway enzyme. 20

85. The non-naturally occurring microbial organism of claim 78, wherein said microbial organism comprises three exogenous nucleic acids each encoding a benzene pathway enzyme.

86. The non-naturally occurring microbial organism of claim 78, wherein said microbial organism comprises four exogenous nucleic acids each encoding a benzene 25 pathway enzyme. 290

87. The non-naturally occurring microbial organism of claim 78, further comprising a benzoate pathway comprising at least one exogenous nucleic acid encoding a benzoate pathway enzyme expressed in a sufficient amount to produce benzoate, said benzoate pathway comprising 2-dehydro-3-deoxyphosphoheptonate 5 synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase.

88. The non-naturally occurring microbial organism of claim 78, further comprising a (2-hydroxy-4-oxobutoxy)phosphonate pathway comprising erythrose-4 10 phosphate dehydratase and (2,4-dioxobutoxy)phosphonate reductase.

89. The non-naturally occurring microbial organism of claim 78, wherein at least one exogenous nucleic acid is a heterologous nucleic acid.

90. The non-naturally occurring microbial organism of claim 78, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture 15 medium.

91. A method for producing benzene, comprising culturing the non-naturally occurring microbial organism of claim 78 under conditions and for a sufficient period of time to produce benzene.

92. The method of claim 91, wherein said benzene pathway comprises benzoate 20 decarboxylase.

93. The method of claim 91, wherein said benzene pathway comprises benzoate reductase and benzaldehyde decarbonylase

94. The method of claim 91, wherein said benzene pathway comprises benzoate kinase, (benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase. 25

95. The method of claim 91, wherein said benzene pathway comprises (benzoyl-CoA synthetase, transferase and/or hydrolase), phosphotransbenzoylase, (benzoyloxy)phosphonate reductase, and benzaldehyde decarbonylase. 291

96. The method of claim 91, wherein said benzene pathway comprises (benzoyl-CoA synthetase, transferase and/or hydrolase), benzoyl-CoA reductase and benzaldehyde decarbonylase.

97. The method of claim 91, wherein said microbial organism comprises two 5 exogenous nucleic acids each encoding a benzene pathway enzyme.

98. The method of claim 91, wherein said microbial organism comprises three exogenous nucleic acids each encoding a benzene pathway enzyme.

99. The method of claim 91, wherein said microbial organism comprises four exogenous nucleic acids each encoding a benzene pathway enzyme. 10

100. The method of claim 91, further comprising a benzoate pathway comprising at least one exogenous nucleic acid encoding a benzoate pathway enzyme expressed in a sufficient amount to produce benzoate, said benzoate pathway comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3 15 phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase.

101. The method of claim 91, further comprising a (2-hydroxy-4 oxobutoxy)phosphonate pathway comprising erythrose-4-phosphate dehydratase and (2,4-dioxobutoxy)phosphonate reductase. 20

102. The method of claim 91, wherein at least one exogenous nucleic acid is a heterologous nucleic acid.

103. The method of claim 91, wherein said non-naturally occurring microbial organism is cultured in a substantially anaerobic culture medium.

104. A non-naturally occurring microbial organism, comprising a microbial 25 organism having a toluene pathway comprising at least one exogenous nucleic acid encoding a toluene pathway enzyme expressed in a sufficient amount to produce toluene, said toluene pathway is selected from a set of pathway enzymes selected from: 292 a) p-toluate decarboxylase; b) p-toluate reductase and p-methylbenzaldehyde decarbonylase; c) p-toluate kinase, (p-methylbenzoyloxy)phosphonate reductase, and p methylbenzaldehyde decarbonylase; d) (p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), phosphotrans-p-methylbenzoylase, (p 5 methylbenzoyloxy)phosphonate reductase, and p-methylbenzaldehyde decarbonylase; and e) (p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), p methylbenzoyl-CoA reductase and p-methylbenzaldehyde decarbonylase, f) (p methylbenzoyl-CoA synthetase, transferase and/or hydrolase), p-toluate decarboxylase, g) p-methylbenzoyl-CoA reductase and p-methylbenzaldehyde decarbonylase, h) 10 phosphotrans-p-methylbenzoylase, (p-methylbenzoyloxy)phosphonate reductase, and p methylbenzaldehyde decarbonylase, i) phosphotrans-p-methylbenzoylase, p-toluate kinase, p-toluate decarboxylase, j) phosphotrans-p-methylbenzoylase, p-toluate kinase, p-toluate reductase, p-methylbenzaldehyde decarbonylase; k) phosphotrans-p methylbenzoylase, (p-methylbenzoyloxy)phosphonate reductase (dephosphorylating), 15 and p-methylbenzaldehyde decarbonylase; and 1) p-methylbenzoyl-CoA reductase and p-methylbenzaldehyde decarbonylase.

105. The non-naturally occurring microbial organism of claim 104, wherein said toluene pathway comprises p-toluate decarboxylase.

106. The non-naturally occurring microbial organism of claim 104, wherein 20 said toluene pathway comprises p-toluate reductase and p-methylbenzaldehyde decarbonylase

107. The non-naturally occurring microbial organism of claim 104, wherein said toluene pathway comprises c) p-toluate kinase, (p-methylbenzoyloxy)phosphonate reductase, and p-methylbenzaldehyde decarbonylase. 25

108. The non-naturally occurring microbial organism of claim 104, wherein said toluene pathway comprises (p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), phosphotrans-p-methylbenzoylase, (p-methylbenzoyloxy)phosphonate reductase, and p-methylbenzaldehyde decarbonylase. 293

109. The non-naturally occurring microbial organism of claim 104, wherein said toluene pathway comprises (p-methylbenzoyl-CoA synthetase, transferase and/or hydrolase), p-methylbenzoyl-CoA reductase and p-methylbenzaldehyde decarbonylase.

110. The non-naturally occurring microbial organism of claim 104, wherein 5 said microbial organism comprises two exogenous nucleic acids each encoding a toluene pathway enzyme.

111. The non-naturally occurring microbial organism of claim 104, wherein said microbial organism comprises three exogenous nucleic acids each encoding a toluene pathway enzyme. 10

112. The non-naturally occurring microbial organism of claim 104, wherein said microbial organism comprises four exogenous nucleic acids each encoding a toluene pathway enzyme.

113. The non-naturally occurring microbial organism of claim 104, further comprising ap-toluate pathway comprising at least one exogenous nucleic acid 15 encoding a p-toluate pathway enzyme expressed in a sufficient amount to produce p toluate, said p-toluate pathway comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3-phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase. 20

114. The non-naturally occurring microbial organism of claim 104, further comprising a (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway comprising DXP synthase, DXP reductoisomerase, and 2ME4P dehydratase.

115. The non-naturally occurring microbial organism of claim 104, wherein at least one exogenous nucleic acid is a heterologous nucleic acid. 25

116. The non-naturally occurring microbial organism of claim 104, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 294

117. A method for producing toluene, comprising culturing the non-naturally occurring microbial organism of claim 104 under conditions and for a sufficient period of time to produce toluene.

118. The method of claim 117, wherein said toluene pathway comprises p 5 toluate decarboxylase.

119. The method of claim 117, wherein said toluene pathway comprises p toluate reductase and p-methylbenzaldehyde decarbonylase

120. The method of claim 117, wherein said toluene pathway comprises c)p toluate kinase, (p-methylbenzoyloxy)phosphonate reductase, and p-methylbenzaldehyde 10 decarbonylase.

121. The method of claim 117, wherein said toluene pathway comprises (p methylbenzoyl-CoA synthetase, transferase and/or hydrolase), phosphotrans-p methylbenzoylase, (p-methylbenzoyloxy)phosphonate reductase, and p methylbenzaldehyde decarbonylase. 15

122. The method of claim 117, wherein said toluene pathway comprises (p methylbenzoyl-CoA synthetase, transferase and/or hydrolase), p-methylbenzoyl-CoA reductase and p-methylbenzaldehyde decarbonylase.

123. The method of claim 117, wherein said microbial organism comprises two exogenous nucleic acids each encoding a toluene pathway enzyme. 20

124. The method of claim 117, wherein said microbial organism comprises three exogenous nucleic acids each encoding a toluene pathway enzyme.

125. The method of claim 117, wherein said microbial organism comprises four exogenous nucleic acids each encoding a toluene pathway enzyme.

126. The method of claim 117, further comprising a p-toluate pathway 25 comprising at least one exogenous nucleic acid encoding a p-toluate pathway enzyme expressed in a sufficient amount to produce p-toluate, said p-toluate pathway 295 comprising 2-dehydro-3-deoxyphosphoheptonate synthase; 3-dehydroquinate synthase; 3-dehydroquinate dehydratase; shikimate dehydrogenase; shikimate kinase; 3 phosphoshikimate-2-carboxyvinyltransferase; chorismate synthase; and chorismate lyase. 5

127. The method of claim 117, further comprising a (2-hydroxy-3-methyl-4 oxobutoxy)phosphonate pathway comprising DXP synthase, DXP reductoisomerase, and 2ME4P dehydratase.

128. The method of claim 117, wherein at least one exogenous nucleic acid is a heterologous nucleic acid. 10

129. The method of claim 117, wherein said non-naturally occurring microbial organism is cultured in a substantially anaerobic culture medium.

130. A non-naturally occurring microbial organism, comprising a microbial organism having a 2,4-pentadienoate pathway comprising at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient 15 amount to produce 2,4-pentadienoate, said 2,4-pentadienoate pathway having a set of enzymes selected from A) i) a 4-hydroxy-2-oxovalerate aldolase, ii) a 4-hydroxy-2 oxovalerate dehydratase, iii) a 2-oxopentenoate reductase, and iv) a 2 hydroxypentenoate dehydratase; B) i) an AKP deaminase, ii) an acetylacrylate reductase, and iii) a 4-hydroxypent-2-enoate dehydratase; C) i) an AKP 20 aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2-reductase, iii) a 2 hydroxy-4-oxopentanoate dehydratase, iv) an acetylacrylate reductase, and v) a 4 hydroxypent-2-enoate dehydratase; D) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-4-reductase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate 25 dehydratase; and E) i) an AKP reductase, ii) a 2-amino-4-hydroxypentanoate aminotransferase and/or dehydrogenase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase. 296

131. The non-naturally occurring microbial organism of claim 130, wherein said microbial organism comprises two exogenous nucleic acids each encoding a 2,4 pentadienoate pathway enzyme.

132. The non-naturally occurring microbial organism of claim 130, wherein 5 said microbial organism comprises three exogenous nucleic acids each encoding a 2,4 pentadienoate pathway enzyme.

133. The non-naturally occurring microbial organism of claim 132, wherein said three exogenous nucleic acids encode i) an AKP deaminase, ii) an acetylacrylate reductase, and iii) a 4-hydroxypent-2-enoate dehydratase. 10

134. The non-naturally occurring microbial organism of claim 130, wherein said microbial organism comprises four exogenous nucleic acids each encoding a 2,4 pentadienoate pathway enzyme.

135. The non-naturally occurring microbial organism of claim 134, wherein said four exogenous nucleic acids encode i) a 4-hydroxy-2-oxovalerate aldolase, ii) a 4 15 hydroxy-2-oxovalerate dehydratase, iii) a 2-oxopentenoate reductase, and iv) a 2 hydroxypentenoate dehydratase.

136. The non-naturally occurring microbial organism of claim 130, wherein said microbial organism comprises five exogenous nucleic acids each encoding a 2,4 pentadienoate pathway enzyme. 20

137. The non-naturally occurring microbial organism of claim 136, wherein said five exogenous nucleic acids encode i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2-reductase, iii) a 2-hydroxy-4-oxopentanoate dehydratase, iv) an acetylacrylate reductase, and v) a 4-hydroxypent-2-enoate dehydratase. 25

138. The non-naturally occurring microbial organism of claim 136, wherein said five exogenous nucleic acids encode i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-4-reductase, iii) a 4-hydroxy-2-oxovalerate 297 dehydratase, iv) a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase.

139. The non-naturally occurring microbial organism of claim 136, wherein said five exogenous nucleic acids encode i) an AKP reductase, ii) a 2-amino-4 5 hydroxypentanoate aminotransferase and/or dehydrogenase, iii) a 4-hydroxy-2 oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase.

140. The non-naturally occurring microbial organism of claim 130, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 10

141. The non-naturally occurring microbial organism of claim 130, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

142. The non-naturally occurring microbial organism of claim 130, further comprising a 2,4-pentadienoate decarboxylase expressed in a sufficient amount to 15 produce 1,3-butadiene by conversion of 2,4-pentadienoate to 1,3-butadiene.

143. The non-naturally occurring microbial organism of claim 130, further comprising at least one of an AKP thiolase, an omithine 4,5-aminomutase, a 2,4 diaminopentanoate 4-aminotransferase and a 2,4-diaminopentanoate 4-dehydrogenase.

144. A method for producing 2,4-pentadienoate, comprising culturing a non 20 naturally occurring microbial organism having a 2,4-pentadienoate pathway, said pathway comprising at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate, under conditions and for a sufficient period of time to produce 2,4-pentadienoate, said 2,4 pentadienoate pathway selected from A) i) a 4-hydroxy-2-oxovalerate aldolase, ii) a 4 25 hydroxy-2-oxovalerate dehydratase, iii) a 2-oxopentenoate reductase, and iv) a 2 hydroxypentenoate dehydratase; B) i) an AKP deaminase, ii) an acetylacrylate reductase, and iii) a 4-hydroxypent-2-enoate dehydratase; C) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2-reductase, iii) a 2- 298 hydroxy-4-oxopentanoate dehydratase, iv) an acetylacrylate reductase, and v) a 4 hydroxypent-2-enoate dehydratase; D) i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-4-reductase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate 5 dehydratase; and E) i) an AKP reductase, ii) a 2-amino-4-hydroxypentanoate aminotransferase and/or dehydrogenase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase.

145. The method of claim 144, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 10

146. The method of claim 144, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

147. The method of claim 144, wherein said microbial organism comprises two exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme.

148. The method of claim 144, wherein said microbial organism comprises 15 three exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme.

149. The method of claim 148, wherein said three exogenous nucleic acids encode i) an AKP deaminase, ii) an acetylacrylate reductase, and iii) a 4-hydroxypent-2 enoate dehydratase.

150. The method of claim 144, wherein said microbial organism comprises four 20 exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme.

151. The method of claim 150, wherein said four exogenous nucleic acids encode i) a 4-hydroxy-2-oxovalerate aldolase, ii) a 4-hydroxy-2-oxovalerate dehydratase, iii) a 2-oxopentenoate reductase, and iv) a 2-hydroxypentenoate dehydratase. 25

152. The method of claim 144, wherein said microbial organism comprises five exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. 299

153. The method of claim 152, wherein said five exogenous nucleic acids encode i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-2 reductase, iii) a 2-hydroxy-4-oxopentanoate dehydratase, iv) an acetylacrylate reductase, and v) a 4-hydroxypent-2-enoate dehydratase. 5

154. The method of claim 152, wherein said five exogenous nucleic acids encode i) an AKP aminotransferase and/or dehydrogenase, ii) a 2,4-dioxopentanoate-4 reductase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2-oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase.

155. The method of claim 152, wherein said five exogenous nucleic acids 10 encode i) an AKP reductase, ii) a 2-amino-4-hydroxypentanoate aminotransferase and/or dehydrogenase, iii) a 4-hydroxy-2-oxovalerate dehydratase, iv) a 2 oxopentenoate reductase, and v) a 2-hydroxypentenoate dehydratase.

156. The method of claim 144, further comprising a 2,4-pentadienoate decarboxylase expressed in a sufficient amount to convert 2,4-pentadienoate to 1,3 15 butadiene.

157. The method of claim 144, further comprising at least one of an AKP thiolase, an omithine 4,5-aminomutase, a 2,4-diaminopentanoate 4-aminotransferase and a 2,4-diaminopentanoate 4-dehydrogenase.

158. A non-naturally occurring microbial organism, comprising a microbial 20 organism having a 2,4-pentadienoate pathway comprising at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate, said 2,4-pentadienoate pathway having a set of enzymes selected from: 1) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5 25 hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5 hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 300 2) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5 hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, H. 5 hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, D. 5-hydroxypent 5 2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 3) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, C. 3,5 10 dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 4) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, 15 transferase and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4 dienoyl-CoA synthetase, transferase and/or hydrolase; 5) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5 dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. 20 pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 6) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5 dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5 dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, 25 transferase and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4 dienoyl-CoA synthetase, transferase and/or hydrolase; 7) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, G. 3,5-dihydroxypentanoyl-CoA 301 synthetase, transferase and/or hydrolase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 8) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA 5 acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 10 9) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl CoA synthetase, transferase and/or hydrolase; 10) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA 15 dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, D. 5 hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 20 11) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, 25 transferase and/or hydrolase; 12) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, , F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, 302 D. 5-hydroxypent-2-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase and/or hydrolase; 13) M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, R. 3-hydroxypent-4-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, 5 transferase and/or hydrolase; 14) M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, S. 3-hydroxypent-4 enoate dehydratase; and 15) M. acrylyl-CoA acetyltransferase, 0. 3-oxopent-4-enoyl-CoA synthetase, 10 transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, S. 3-hydroxypent-4 enoate dehydratase; 16) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5 hydroxypentanoyl-CoA dehydratase, N. 3-oxopent-4-enoyl-CoA reductase, R. 3 hydroxypent-4-enoyl-CoA dehydratase, E. pent-2,4-dienoyl-CoA synthetase, transferase 15 and/or hydrolase; 17) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5 hydroxypentanoyl-CoA dehydratase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3 hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, S. 3-hydroxypent-4 enoate dehydratase; 20 18) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5 hydroxypentanoyl-CoA dehydratase, 0. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, S. 3-hydroxypent-4-enoate dehydratase. 19) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5 25 hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, H. 5 hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, Q. 5-hydroxypent 2-enoate dehydratase; 303 20) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5 hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, Q. 5 hydroxypent-2-enoate dehydratase; 5 21) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5 hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase I. 3-oxo-5 hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, Q. 5 hydroxypent-2-enoate dehydratase; 22) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA 10 dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, Q. 5-hydroxypent-2-enoate dehydratase; 23) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl 15 CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, Q. 5-hydroxypent-2-enoate dehydratase; and 24) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl CoA dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or 20 hydrolase, Q. 5-hydroxypent-2-enoate dehydratase.

159. The non-naturally occurring microbial organism of claim 158, wherein said microbial organism comprises two, three, four, five, six, seven, or eight exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme.

160. The non-naturally occurring microbial organism of claim 158, wherein 25 said at least one exogenous nucleic acid is a heterologous nucleic acid. 304

161. The non-naturally occurring microbial organism of claim 158, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

162. The non-naturally occurring microbial organism of claim 158, further 5 comprising a 2,4-pentadiene decarboxylase to convert 2,4-pentadienoate to 1,3 butadiene.

163. A method for producing 2,4-pentadienoate, comprising culturing a non naturally occurring microbial organism according to claim 158, under conditions and for a sufficient period of time to produce 2,4-pentadienoate. 10

164. The method of claim 163, wherein said microbial organism comprises two, three, four, five, six, seven, or eight exogenous nucleic acids each encoding a 2,4 pentadienoate pathway enzyme.

165. The method of claim 163, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 15

166. The method of claim 163, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

167. A method for producing 1,3-butadiene, comprising culturing a non naturally occurring microbial organism according to claim 162, under conditions and for a sufficient period of time to produce 1,3-butadiene. 20

168. The method of claim 167, wherein said microbial organism comprises two, three, four, five, six, seven, or eight exogenous nucleic acids each encoding a 2,4 pentadienoate pathway enzyme.

169. The method of claim 167, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 25

170. The method of claim 167, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 305

171. A non-naturally occurring microbial organism, comprising a microbial organism having a 1,3-butadiene pathway comprising at least one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene, said 1,3-butadiene pathway having a set of enzymes selected 5 from: 1) M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3 hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, Y. 3-hydroxypent-4 enoate decarboxylase; 2) M. acrylyl-CoA acetyltransferase, 0. 3-oxopent-4-enoyl-CoA synthetase, 10 transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, Y. 3-hydroxypent-4 enoate decarboxylase; 3) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3-hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, Y. 3-hydroxypent-4-enoate decarboxylase; 15 4) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, 0. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4 enoate reductase, Y. 3-hydroxypent-4-enoate decarboxylase; 5) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5 hydroxypentanoyl-CoA dehydratase, N. 3-oxopent-4-enoyl-CoA reductase, T. 3 20 hydroxypent-4-enoyl-CoA transferase, synthetase or hydrolase, Y. 3-hydroxypent-4 enoate decarboxylase; 6) A. 3-hydroxypropanoyl-CoA acetyltransferase, L. 3-oxo-5 hydroxypentanoyl-CoA dehydratase, 0. 3-oxopent-4-enoyl-CoA synthetase, transferase and/or hydrolase, P. 3-oxopent-4-enoate reductase, Y. 3-hydroxypent-4-enoate 25 decarboxylase; 306

172. The non-naturally occurring microbial organism of claim 171, wherein said microbial organism comprises two, three, four, or five exogenous nucleic acids each encoding a 1,3-butadiene pathway enzyme.

173. The non-naturally occurring microbial organism of claim 171, wherein 5 said at least one exogenous nucleic acid is a heterologous nucleic acid.

174. The non-naturally occurring microbial organism of claim 171, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

175. A method for producing 1,3-butadiene, comprising culturing a non 10 naturally occurring microbial organism according to claim 171, under conditions and for a sufficient period of time to produce 1,3-butadiene.

176. The method of claim 175, wherein said microbial organism comprises two, three, four, or five exogenous nucleic acids each encoding a 1,3-butadiene pathway enzyme. 15

177. The method of claim 175, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

178. The method of claim 175, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

179. A non-naturally occurring microbial organism, comprising a microbial 20 organism having a 1,3-butadiene pathway comprising at least one exogenous nucleic acid encoding a 3-butene-1-ol pathway enzyme expressed in a sufficient amount to produce 3-butene-1-ol, said 3-butene-1-ol pathway having a set of enzymes selected from: 1) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl 25 CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate decarboxylase; 307 2) A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5 -hydroxypent-2-enoate decarboxylase; 3) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5 5 hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase; 4) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5 hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5 10 hydroxypent-2-enoate decarboxylase; 5) A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5 hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, H. 5 hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent 2-enoate decarboxylase; 15 6) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate decarboxylase; 7) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA 20 dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 8) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl 25 CoA synthetase, transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase; 308 9) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 5 10) M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl CoA dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate decarboxylase; 11) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA 10 acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate decarboxylase; 12) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or 15 hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 13) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5 dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, U. 3,5 20 dihydroxypentanoate decarboxylase; 14) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5 dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5 dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 25 15) K. 3-hydroxypropanoyl-CoA dehydratase, A. 3-hydroxypropanoyl-CoA acetyltransferase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5 dihydroxypentanoyl-CoA dehydratase, H. 5-hydroxypent-2-enoyl-CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate decarboxylase; 309 16) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, U. 3,5-dihydroxypentanoate decarboxylase; 5 17) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, F. 3-oxo-5-hydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, I. 3-oxo-5-hydroxypentanoate reductase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 18) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, 10 L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, U. 3,5-dihydroxypentanoate decarboxylase; 19) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA 15 reductase, G. 3,5-dihydroxypentanoyl-CoA synthetase, transferase and/or hydrolase, J. 3,5-dihydroxypentanoate dehydratase, V. 5-hydroxypent-2-enoate decarboxylase; 20) K. 3-hydroxypropanoyl-CoA dehydratase, M. acrylyl-CoA acetyltransferase, L. 3-oxo-5-hydroxypentanoyl-CoA dehydratase, B. 3-oxo-5-hydroxypentanoyl-CoA reductase, C. 3,5-dihydroxypentanoyl-CoA dehydratase, H. 5-hydroxypent-2-enoyl 20 CoA synthetase, transferase and/or hydrolase, V. 5-hydroxypent-2-enoate decarboxylase;

180. The non-naturally occurring microbial organism of claim 179, wherein said microbial organism comprises two, three, four, five, six, or seven, exogenous nucleic acids each encoding a 3-butene-1-ol pathway enzyme. 25

181. The non-naturally occurring microbial organism of claim 179, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 310

182. The non-naturally occurring microbial organism of claim 179, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

183. The non-naturally occurring microbial organism of claim 179, further 5 comprising a 3-butene-1-ol dehydratase to convert 3-butene-1-ol to 1,3-butadiene.

184. A method for producing 3-butene-1-ol, comprising culturing a non naturally occurring microbial organism according to claim 179, under conditions and for a sufficient period of time to produce 3-butene-1-ol.

185. The method of claim 184, wherein said microbial organism comprises two, 10 three, four, five, six, or seven exogenous nucleic acids each encoding a 3-butene-1-ol pathway enzyme.

186. The method of claim 184, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

187. The method of claim 184, wherein said non-naturally occurring microbial 15 organism is in a substantially anaerobic culture medium.

188. The method of claim 184 further comprising the chemical dehydration of 3 butene-1-ol to provide 1,3-butadiene.

189. A method for producing 1,3-butadiene, comprising culturing a non naturally occurring microbial organism according to claim 183, under conditions and 20 for a sufficient period of time to produce 1,3-butadiene.

190. The method of claim 189, wherein said microbial organism comprises two, three, four, five, six, seven, or eight exogenous nucleic acids each encoding a 1,3 butadiene pathway enzyme.

191. The method of claim 189, wherein said at least one exogenous nucleic acid 25 is a heterologous nucleic acid. 311

192. The method of claim 189, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

193. A non-naturally occurring microbial organism, comprising a microbial organism having a 1,3-butadiene pathway comprising at least one exogenous nucleic 5 acid encoding a 1,3-butadiene pathway enzyme expressed in a sufficient amount to produce 1,3-butadiene, said 1,3-butadiene pathway selected from: (A) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3 oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate 10 reductase; and a 3-hydroxypent-4-enoate decarboxylase; (B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate reductase; a 3 hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate decarboxylase; 15 (C) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase; (D) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA 20 transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3 oxoadipate dehydrogenase; a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate decarboxylase; (E) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, 25 a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase; 312 (F) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate 5 decarboxylase; (G) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA synthetase or a 3 hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4 enedioate decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase; and 10 (H) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA synthetase or a 3 hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4 enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4 pentadienoate decarboxylase. 15

194. The non-naturally occurring microbial organism of claim 193, wherein said microbial organism comprises two, three, four, five, six or seven exogenous nucleic acids each encoding a 1,3-butadiene pathway enzyme.

195. The non-naturally occurring microbial organism of claim 194, wherein said microbial organism comprises exogenous nucleic acids encoding each of the 20 enzymes selected from: (A) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3 oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate reductase; and a 3-hydroxypent-4-enoate decarboxylase; 25 (B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate reductase; a 3 hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate decarboxylase; 313 (C) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase; 5 (D) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3 oxoadipate dehydrogenase; a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate decarboxylase; 10 (E) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase; (F) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, 15 a 3-oxoadipyl-CoA synthetase or a 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4-pentadienoate decarboxylase; (G) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; 20 a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA synthetase or a 3 hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4 enedioate decarboxylase; and a 3-hydroxypent-4-enoate decarboxylase; and (H) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA synthetase or a 3 25 hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4 enedioate decarboxylase; a 3-hydroxypent-4-enoate dehydratase; and a 2,4 pentadienoate decarboxylase. 314

196. The non-naturally occurring microbial organism of claim 193, wherein said microbial organism further comprises: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least 5 one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least 10 one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected 15 from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof.

197. The non-naturally occurring microbial organism of claim 196, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate 20 dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.

198. The non-naturally occurring microbial organism of claim 196, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme 25 selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

199. The non-naturally occurring microbial orgnaism of claim 196, wherein said microbial organism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; 315 wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous 5 nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase.

200. The non-naturally occurring microbial organism of claim 193, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

201. The non-naturally occurring microbial organism of claim 193, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture 10 medium.

202. A method for producing 1,3-butadiene, comprising culturing a non naturally occurring microbial organism of any one of claims 193-201 under conditions and for a sufficient period of time to produce 1,3-butadiene.

203. A non-naturally occurring microbial organism, comprising a microbial 15 organism having a 2,4-pentadienoate pathway comprising at least one exogenous nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate, said 2,4-pentadienoate pathway selected from: (A) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3 20 oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate reductase; and a 3-hydroxypent-4-enoate dehydratase; (B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate 25 decarboxylase; and a 3-hydroxypent-4-enoate dehydratase; (C) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate 316 reductase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate dehydratase; and (D) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA synthetase or a 3 5 hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4 enedioate decarboxylase; and a 3-hydroxypent-4-enoate dehydratase.

204. The non-naturally occurring microbial organism of claim 203, wherein said microbial organism comprises two, three, four, five, or six exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. 10

205. The non-naturally occurring microbial organism of claim 204, wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes selected from: (A) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3 15 oxoadipate dehydrogenase; a 2-fumarylacetate decarboxylase; a 3-oxopent-4-enoate reductase; and a 3-hydroxypent-4-enoate dehydratase; (B) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate dehydrogenase; a 2-fumarylacetate reductase; a 3-hydroxyhex-4-enedioate 20 decarboxylase; and a 3-hydroxypent-4-enoate dehydratase; (C) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA transferase, a 3-oxoadipyl-CoA synthetase ora 3-oxoadipyl-CoA hydrolase; a 3-oxoadipate reductase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4-enedioate decarboxylase; and a 3-hydroxypent-4-enoate dehydratase; and 25 (D) a succinyl-CoA:acetyl-CoA acyltransferase; a 3-oxoadipyl-CoA reductase; a 3-hydroxyadipyl-CoA transferase, a 3-hydroxyadipyl-CoA synthetase or a 3- 317 hydroxyadipyl-CoA hydrolase; a 3-hydroxyadipate dehydrogenase; a 3-hydroxyhex-4 enedioate decarboxylase; and a 3-hydroxypent-4-enoate dehydratase.

206. The non-naturally occurring microbial organism of claim 203, wherein said microbial organism further comprises: 5 (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; 10 (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 15 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof.

207. The non-naturally occurring microbial organism of claim 206, wherein said microbial 20 organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. 25

208. The non-naturally occurring microbial organism of claim 206, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. 318

209. The non-naturally occurring microbial orgnaism of claim 206, wherein said microbial organism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids 5 encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase.

210. The non-naturally occurring microbial organism of claim 203, wherein 10 said at least one exogenous nucleic acid is a heterologous nucleic acid.

211. The non-naturally occurring microbial organism of claim 203, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

212. A method for producing 2,4-pentadienoate, comprising culturing a non 15 naturally occurring microbial organism of any one of claims 203-211 under conditions and for a sufficient period of time to produce 2,4-pentadienoate.

213. A non-naturally occurring microbial organism, comprising a microbial organism having a 1,3-butadiene pathway comprising at least one exogenous nucleic acid encoding a 1,3-butadiene pathway enzyme expressed in a sufficient amount to 20 produce 1,3-butadiene, said 1,3-butadiene pathway selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy 3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent 2-enoate dehydratase; and a 2,4-pentadiene decarboxylase; 25 (B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy 3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent 2-enoate decarboxylase; and a 3-butene-1-ol dehydratase; 319 (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy 3-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene 1 -ol dehydratase; 5 (D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4 pentadiene decarboxylase; (E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase 10 (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3 butene- 1 -ol dehydratase; (F) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 15 dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase; (G) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5 oxopentanoate reductase; a 3,5 -dihydroxypentanoate dehydratase; a 5-hydroxypent-2 enoate dehydratase; and a 2,4-pentadiene decarboxylase; 20 (H) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5 oxopentanoate reductase; a 3,5 -dihydroxypentanoate dehydratase; a 5-hydroxypent-2 enoate decarboxylase; and a 3-butene-1-ol dehydratase; (I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase 25 (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5 oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1 ol dehydratase; 320 (J) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5 hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase; 5 (K) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5 hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase; (L) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase 10 (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene- 1 -ol dehydratase; (M) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 15 dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4 pentadiene decarboxylase; (N) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3 20 butene-1-ol dehydratase; and (0) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase.

214. The non-naturally occurring microbial organism of claim 213, wherein 25 said microbial organism comprises two, three, four, five, six or seven exogenous nucleic acids each encoding a 1,3-butadiene pathway enzyme. 321

215. The non-naturally occurring microbial organism of claim 214, wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase 5 (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy 3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent 2-enoate dehydratase; and a 2,4-pentadiene decarboxylase; (B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy 10 3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5-hydroxypent 2-enoate decarboxylase; and a 3-butene-1-ol dehydratase; (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy 3-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene 15 1-ol dehydratase; (D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4 pentadiene decarboxylase; 20 (E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3 butene- 1 -ol dehydratase; (F) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase 25 (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase; 322 (G) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5 oxopentanoate reductase; a 3,5 -dihydroxypentanoate dehydratase; a 5-hydroxypent-2 enoate dehydratase; and a 2,4-pentadiene decarboxylase; 5 (H) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5 oxopentanoate reductase; a 3,5 -dihydroxypentanoate dehydratase; a 5-hydroxypent-2 enoate decarboxylase; and a 3-butene-1-ol dehydratase; (I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase 10 (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5 oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene-1 ol dehydratase; (J) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 15 hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5 hydroxypent-2-enoate dehydratase; and a 2,4-pentadiene decarboxylase; (K) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; a 5 20 hydroxypent-2-enoate decarboxylase; and a 3-butene-1-ol dehydratase; (L) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate decarboxylase; and a 3-butene- 1 -ol dehydratase; 25 (M) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate dehydratase; and a 2,4 pentadiene decarboxylase; 323 (N) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 dihydroxypentanoate dehydratase; a 5-hydroxypent-2-enoate decarboxylase; and a 3 butene-1-ol dehydratase; and 5 (0) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 dihydroxypentanoate decarboxylase; and a 3-butene-1-ol dehydratase.

216. The non-naturally occurring microbial organism of claim 213, wherein said microbial organism further comprises: 10 (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; 15 (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 20 hydrogenase; or (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof.

217. The non-naturally occurring microbial organism of claim 216, wherein said microbial 25 organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate 324 dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.

218. The non-naturally occurring microbial organism of claim 216, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme 5 selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

219. The non-naturally occurring microbial orgnaism of claim 216, wherein said microbial organism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; 10 wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase. 15

220. The non-naturally occurring microbial organism of claim 213, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

221. The non-naturally occurring microbial organism of claim 213, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 20

222. A method for producing 1,3-butadiene, comprising culturing a non naturally occurring microbial organism of any one of claims 213-221 under conditions and for a sufficient period of time to produce 1,3-butadiene.

223. A non-naturally occurring microbial organism, comprising a microbial organism having a 2,4-pentadienoate pathway comprising at least one exogenous 25 nucleic acid encoding a 2,4-pentadienoate pathway enzyme expressed in a sufficient amount to produce 2,4-pentadienoate, said 2,4-pentadienoate pathway selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy- 325 3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5 hydroxypent-2-enoate dehydratase; (B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 5 dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase; (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5 oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5 hydroxypent-2-enoate dehydratase; 10 (D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5 hydroxypent-2-enoate dehydratase; and (E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase 15 (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase.

224. The non-naturally occurring microbial organism of claim 223, wherein said microbial organism comprises two, three, four, five or six exogenous nucleic acids each encoding a 2,4-pentadienoate pathway enzyme. 20

225. The non-naturally occurring microbial organism of claim 224, wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy 25 3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5 hydroxypent-2-enoate dehydratase; 326 (B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase; (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase 5 (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5 oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5 hydroxypent-2-enoate dehydratase; (D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 10 hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5 hydroxypent-2-enoate dehydratase; and (E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate dehydratase. 15

226. The non-naturally occurring microbial organism of claim 223, wherein said microbial organism further comprises: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a 20 citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin 25 oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or 327 (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof.

227. The non-naturally occurring microbial organism of claim 226, wherein said microbial 5 organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. 10

228. The non-naturally occurring microbial organism of claim 226, wherein said microbial organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

229. The non-naturally occurring microbial orgnaism of claim 226, wherein said microbial 15 organism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or 20 wherein said microbial organism comprising (iii) comprises two exogenous nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase.

230. The non-naturally occurring microbial organism of claim 223, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

231. The non-naturally occurring microbial organism of claim 223, wherein 25 said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

232. A method for producing 2,4-pentadienoate, comprising culturing a non naturally occurring microbial organism of any one of claims 223-231 under conditions and for a sufficient period of time to produce 2,4-pentadienoate. 328

233. A non-naturally occurring microbial organism, comprising a microbial organism having a 3-butene-1-ol pathway comprising at least one exogenous nucleic acid encoding a 3-butene-1-ol pathway enzyme expressed in a sufficient amount to produce 3-butene-1-ol, said 3-butene-1-ol pathway selected from: 5 (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy 3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5 hydroxypent-2-enoate decarboxylase; (B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase 10 (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy 3-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; 15 (D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5 20 oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5 hydroxypent-2-enoate decarboxylase; (F) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5 oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; 25 (G) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3- 329 hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5 hydroxypent-2-enoate decarboxylase; (H) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 5 hydroxy-5-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; and (J) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase 10 (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); and a 3,5 dihydroxypentanoate decarboxylase.

234. The non-naturally occurring microbial organism of claim 233, wherein said microbial organism comprises two, three, four or five exogenous nucleic acids each encoding a 3-butene-1-ol pathway enzyme. 15

235. The non-naturally occurring microbial organism of claim 234, wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes selected from: (A) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy 20 3-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5 hydroxypent-2-enoate decarboxylase; (B) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (aldehyde reducing); a 5-hydroxy 3-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; 25 (C) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; a 3,5 dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; 330 (D) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (CoA reducing and alcohol forming); a 5-hydroxy-3-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (E) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase 5 (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5 oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5 hydroxypent-2-enoate decarboxylase; (F) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (aldehyde forming); a 3,5-dioxopentanoate reductase (ketone reducing); a 3-hydroxy-5 10 oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (G) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 hydroxy-5-oxopentanoate reductase; a 3,5-dihydroxypentanoate dehydratase; and a 5 hydroxypent-2-enoate decarboxylase; 15 (H) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (aldehyde forming); a 3 hydroxy-5-oxopentanoate reductase; and a 3,5-dihydroxypentanoate decarboxylase; (I) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); a 3,5 20 dihydroxypentanoate dehydratase; and a 5-hydroxypent-2-enoate decarboxylase; and (J) a malonyl-CoA:acetyl-CoA acyltransferase; a 3-oxoglutaryl-CoA reductase (ketone-reducing); a 3-hydroxyglutaryl-CoA reductase (alcohol forming); and a 3,5 dihydroxypentanoate decarboxylase.

236. The non-naturally occurring microbial organism of claim 233, wherein 25 said microbial organism further comprises: (i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least 331 one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; (ii) a reductive TCA pathway comprising at least one exogenous nucleic 5 acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase; or 10 (iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H 2 hydrogenase, and combinations thereof.

237. The non-naturally occurring microbial organism of claim 236, wherein said microbial organism comprising (i) further comprises an exogenous nucleic acid encoding an enzyme 15 selected from a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.

238. The non-naturally occurring microbial organism of claim 236, wherein said microbial 20 organism comprising (ii) further comprises an exogenous nucleic acid encoding an enzyme selected from an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof.

239. The non-naturally occurring microbial orgnaism of claim 236, wherein said microbial organism comprising (i) comprises four exogenous nucleic acids encoding an ATP-citrate lyase, 25 citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase; wherein said microbial organism comprising (ii) comprises five exogenous nucleic acids encoding a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2 hydrogenase; or wherein said microbial organism comprising (iii) comprises two exogenous 30 nucleic acids encoding a CO dehydrogenase and an H 2 hydrogenase. 332

240. The non-naturally occurring microbial organism of claim 233, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

241. The non-naturally occurring microbial organism of claim 233, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture 5 medium.

242. A method for producing 3-butene-1-ol, comprising culturing a non naturally occurring microbial organism of any one of claims 233-241 under conditions and for a sufficient period of time to produce 3-butene-1-ol.

243. A method for producing 1,3-butadiene comprising, culturing a non 10 naturally occurring microbial organism of any one of claims 233-241 under conditions and for a sufficient period of time to produce 3-butene- 1 -ol, and chemically converting said 3-butene-1-ol to 1,3-butadiene.