AU750057B2 - Modification of fatty acid metabolism in plants - Google Patents

Description

WO 99/45122 PCT/US99/04999 MODIFICATION OF FATTY ACID METABOLISM IN PLANTS Background Of The Invention The present invention is generally in the field oftransgenic plant systems for the production of polyhydroxyalkanoate materials, modification oftriglycerides and fatty acids, and methods for altering seed production in plants.

Methods for producing stable transgenic plants for agronomic crops have been developed over the last 15 years. Crops have been genetically modified for improvements in both input and output traits. In the former traits, tolerance to specific agrochemicals has been engineered into crops, and specific natural pesticides, such as the Bacillus thuringenesis toxin, have been expressed directly in the plant. There also has been significant progress in developing male sterility systems for the production of hybrid plants.

With respect to output traits, crops are being modified to increase the value of the product, generally the seed, grain, or fiber of the plant. Critical metabolic targets include the modification of starch, fatty acid, and oil biosynthetic pathways.

There is considerable commercial interest in producing microbial polyhydroxyalkanoate (PHA) biopolymers in plant crops. See, for example, U.S. Patent Nos. 5,245,023 and 5,250,430 to Peoples and Sinskey; U.S.

Patent No. 5,502,273 to Bright et al.; U.S. Patent No. 5,534,432 to Peoples and Sinskey; U.S. Patent No. 5,602,321 to John; U.S. Patent No. 5,610,041 to Somerville et al.; PCT WO 91/00917; PCT WO 92/19747; PCT WO 93/02187; PCT WO 93/02194; PCT WO 94/12014; Poirier et al., Science 256:520-23 (1992); van der Leij Witholt, Can. J. Microbiol.

41(supplement):222-38 (1995); Nawrath Poirier, The International Symposium on Bacterial Polyhydroxyalkanoates, (Eggink et al., eds.) Davos Switzerland (August 18-23, 1996); and Williams and Peoples, CHEMTECH 26: 38-44 (1996). PHAs are natural, thermoplastic polyesters and can be processed by traditional polymer techniques for use in an enormous variety WO 99/45122 PCT/US99/04999 of applications, including consumer packaging, disposable diaper linings and garbage bags, food and medical products.

Early studies on the production of polyhydroxybutyrate in the chloroplasts of the experimental plant system Arabidopsis thaliana resulted in the accumulation of up to 14% of the leaf dry weight as PHB (Nawrath et al., 1993). Arabidopsis, however, has no agronomic value. Moreover, in order to economically produce PHAs in agronomic crops, it is desirable to produce the PHAs in the seeds, so that the current infrastructure for harvesting and processing seeds can be utilized. The options for recovery of the PHAs from plant seeds (PCT WO 97/15681) and the end use applications (Williams Peoples, CHEMTECH 26:38-44 (1996)) are significantly affected by the polymer composition. Therefore, it would be advantageous to develop transgenic plant systems that produce PHA polymers having a well-defined composition.

Careful selection of the PHA biosynthetic enzymes on the basis of their substrate specificity allows for the production of PHA polymers of defined composition in transgenic systems Patent Nos. 5,229,279; 5,245,023; 5,250,430; 5,480,794; 5,512,669; 5,534,432; 5,661,026; and 5,663,063).

In bacteria, each PHA group is produced by a specific pathway. In the case of the short pendant group PHAs, three enzymes are involved: 3ketothiolase, acetoacetyl-CoA reductase, and PHA synthase. The homopolymer PHB, for example, is produced by the condensation of two molecules of acetyl-coenzyme A to give acetoacetyl-coenzyme A. The latter then is reduced to the chiral intermediate R-3-hydroxybutyryl-coenzyme A by the reductase, and subsequently polymerized by the PHA synthase enzyme. The PHA synthase notably has a relatively wide substrate specificity which allows it to polymerize C3-C5 hydroxy acid monomers including both 4-hydroxy and 5-hydroxy acid units. This biosynthetic pathway is found in a number of bacteria such as Alcaligenes eutrophus, A.

latus, Azotobacter vinlandii, and Zoogloea ramigera. Long pendant group PHAs are produced for example by many different Pseudomonas bacteria.

WO 99/45122 PCT/US99/04999 Their biosynthesis involve the P-oxidation of fatty acids and fatty acid synthesis as routes to the hydroxyacyl-coenzyme A monomeric units. The latter then are converted by PHA synthases which have substrate specificities favoring the larger C6-C14 monomeric units (Peoples Sinskey, 1990).

In the case of the PHB-co-HX copolymers which usually are produced from cells grown on fatty acids, a combination of these routes can be responsible for the formation of the different monomeric units. Indeed, analysis of the DNA locus encoding the PHA synthase gene inAeromonas caviae, which produces the copolymer PHB-co-3-hydroxyhexanoate, was used to identify a gene encoding a D-specific enoyl-CoA hydratase responsible for the production of the D-p-hydroxybutyryl-CoA and D-P hydroxyhexanoyl-CoA units (Fukui Doi, J Bacteriol. 179:4821-30 (1997); Fukui et. al., J. Bacteriol. 180:667-73 (1998)). Other sources of such hydratase genes and enzymes include Alcaligenes, Pseudomonas, and Rhodospirillum.

The enzymes PHA synthase, acetoacetyl-CoA reductase, and Pketothiolase, which produce the short pendant group PHAs in A. eutrophus, are coded by an operon comprising thephbC-phbA-phbB genes; Peoples et al., 1987; Peoples Sinskey, 1989). In the Pseudomonas organisms, the PHA synthases responsible for production of the long pendant group PHAs have been found to be encoded on thepha locus, specifically by thephaA andphaC genes Patent Nos. 5,245,023 and 5,250,430; Huisman et. al., J. Biol. Chem. 266:2191-98 (1991)). Since these earlier studies, a range of PHA biosynthetic genes have been isolated and characterized or identified from genome sequencing projects. Examples of known PHA biosynthetic genes are disclosed in the following references: Aeronomas caviae (Fukui Doi, 1997, J. Bacteriol. 179:4821-30); Alcaligenes eutrophus Patent Nos. 5,245,023; 5,250,430; 5,512,669; and 5,661,026; Peoples Sinskey, J.

Biol. Chem. 264:15298-03 (1989)); Acinetobacter (Schembri et. al., FEMS Microbiol. Lett. 118:145-52 (1994)); Chromatium vinosum (Liebergesell Steinbuchel, Eur. J. Biochem. 209:135-50 (1992)); Methylobacterium extorquens (Valentin Steinbuchel, Appl. Microbiol. Biotechnol. 39:309-17 WO 99/45122 PCT/US99/04999 (1993)); Nocardia corallina (GENBANK Accession No. AF019964; Hall et.

al., 1998, Can. J. Microbiol. 44:687-69); Paracoccus denitrificans (Ueda et al., 1 Bacteriol. 178:774-79 (1996); Yabutani et. al., FEAMSMicrobiol. Lett.

133:85-90 (1995)); Pseudomonas acidophila (Umeda et. al., 1998, Applied Biochemistry and Biotechnology, 70-72:341-52); Pseudomonas sp. 61-3 (Matsusaki et al., 1998, J Bacteriol. 180:6459-67); Nocardia corallina; Pseudomonas aeruginosa (Timm Steinbuchel, Eur. J. Biochem. 209:15-30 (1992)); P. oleovorans Patent Nos. 5,245,023 and 5,250,430; Huisman et. al., J. Biol. Chem. 266(4):2191-98 (1991); Rhizobium etli (Cevallos et. al., J Bacteriol. 178:1646-54 (1996)); R. meliloti (Tombolini et. al., Microbiology 141:2553-59 (1995)); Rhodococcus ruber (Pieper-Furst Steinbuchel, FEMS Microbiol. Lett. 75:73-79 (1992)); Rhodospirillum rubrum (Hustede et. al., FEMSMicrobiol. Let 93:285-90 (1992)); Rhodobacter sphaeroides (Hustede et. al., FEMSMicrobiol. Rev. 9:217-30 (1992); Biotechnol. Lett. 15:709-14 (1993); Synechocystis sp. (DNA Res.

3:109-36 (1996)); Thiocapsiae violacea (Appl. Microbiol. Biotechnol.

38:493-501 (1993)) and Zoogloea ramigera (Peoples et. al., J. Biol. Chem.

262:97-102 (1987); Peoples Sinskey, Molecular Microbiology 3:349-57 (1989)). The availability of these genes or their published DNA sequences should provide a range of options for producing PHAs.

PHA synthases suitable for producing PHB-co-HH copolymers comprising from 1-99% HH monomers are encoded by the Rhodococcus ruber, Rhodospirillum rubrum, Thiocapsiae violacea, and Aeromonas caviae PHA synthase genes. PHA synthases useful for incorporating 3hydroxyacids of 6-12 carbon atoms in addition to R-3-hydroxybutyrate i.e.

for producing biological polymers equivalent to the chemically synthesized copolymers described in PCT WO 95/20614, PCT WO 95/20615, and PCT WO 95/20621 have been identified in a number ofPseudomonas and other bacteria (Steinbiichel Wiese, Appl. Microbiol. Biotechnol. 37:691-97 (1992); Valentin et al., Appl. Microbiol. Biotechnol. 36:507-14 (1992); Valentin et al., Appl. Microbiol. Biotechnol. 40:710-16 (1994); Lee et al., Appl. Microbiol. Biotechnol. 42:901-09 (1995); Kato et al., Appl. Microbiol.

WO 99/45122 PCT/US99/04999 Biotechnol. 45:363-70 (1996); Abe et al., Int. J. Biol. Macromol. 16:115-19 (1994); Valentin et al., Appl. Microbiol. Biotechnol. 46:261-67 (1996)) and can readily be isolated as described in U.S. Patent Nos. 5,245,023 and 5,250,430. The PHA synthase from P. oleovorans Patent Nos.

5,245,023 and 5,250,430; Huisman et. al., J. Biol. Chem. 266(4): 2191-98 (1991)) is suitable for producing the long pendant group PHAs. Plant genes encoding 0-ketothiolase also have been identified (Vollack Bach, Plant Physiol. 111:1097-107 (1996)).

Despite this ability to modify monomer composition by selection of the syntheses and substrates, it is desirable to modify other features of polymer biosynthesis, such as that which involves fatty acid metabolism.

It is therefore an object of the present invention to provide a method and DNA constructs to introduce fatty acid oxidation enzyme systems for manipulating the cellular metabolism of plants.

It is another object of the present invention to provide methods for enhancing the production of PHAs in plants, preferably in the oilseeds thereof.

Summary Of The Invention Methods and systems to modify fatty acid biosynthesis and oxidation in plants to make new polymers are described. Two enzymes are essential: a hydratase such as D-specific enoyl-CoA hydratase, for example, the hydratase obtained from Aeromonas caviae, and a P-oxidation enzyme system. Some plants have a 3-oxidation enzyme system which is sufficient to modify polymer synthesis when the plants are engineered to express the hydratase.

Examples demonstrate production of polymer by expression of these enzymes in transgenic plants. Examples also demonstrate that modifications in fatty acid biosynthesis can be used to alter plant phenotypes, decreasing or eliminating seed production and increasing green plant biomass, as well as producing PHAs.

WO 99/45122 PCT/US99/04999 Brief Description of the Drawings Figure 1 is a schematic of fatty acid 0-oxidation routes to produce polyhydroxyalkanoate monomers.

Figure 2 is a schematic showing plasmid constructs pSBS2024 and pSBS2025.

Figures 3A and 3B are schematics showing plasmid constructs pCGmfl24 and Figures 4A and 4B are schematics showing plasmid constructs pmf1249 and pmfl254.

Figures 5A and 5B are schematics showing plasmid constructs pCGmf224 and pCGmf225.

Figures 6A and 6B are schematics showing plasmid constructs pCGmflP2S and pCGmf2P S.

Detailed Description Of The Invention Methods and DNA constructs for manipulating the cellular metabolism of plants by introducing fatty acid oxidation enzyme systems into the cytoplasm or plastids of developing oilseeds or green tissue are provided. Fatty acid oxidation systems typically comprise several enzyme activities including a 0-ketothiolase enzyme activity which utilizes a broad range of 1-ketoacyl-CoA substrates.

It surprisingly was found that expression of at least one of these transgenes from the bean phaseolin promoter results in male sterility.

Interestingly, these plants did not set seed, but instead produced higher than normal levels ofbiomass leafs, stems, stalks). Therefore the methods and constructs described herein also can be used to create male sterile plants, for example, for hybrid production or to increase the production of biomass of forage, such as alfalfa or tobacco. Plants generated using these methods and DNA constructs are useful for producing polyhydroxyalkanoate biopolymers or for producing novel oil compositions.

The methods described herein include the subsequent incorporation of additional transgenes, in particular encoding additional enzymes involved WO 99/45122 PCT/US99/04999 in fatty acid oxidation or polyhydroxyalkanoate biosynthesis. For polyhydroxyalkanoate biosynthesis, the methods include the incorporation of transgenes encoding enzymes, such as NADH and/or NADPH acetoacetyl- CoenzymeA reductases, PHB synthases, PHA synthases, acetoacetyl-CoA thiolase, hydroxyacyl-CoA epimerases, delta3-cis-delta2-trans enoyl-CoA isomerases, acyl-CoA dehydrogenase, acyl-CoA oxidase and enoyl-CoA hydratases by subsequent transformation of the transgenic plants produced using the methods and DNA constructs described herein or by traditional plant breeding methods.

I. Plant Expression Systems In a preferred embodiment, the fatty acid oxidation transgenes are expressed from a seed specific promoter, and the proteins are expressed in the cytoplasm of the developing oilseed. In an alternate preferred embodiment, fatty acid oxidation transgenes are expressed from a seed specific promoter and the expressed proteins are directed to the plastids using plastid targeting signals. In another preferred embodiment, the fatty acid oxidation transgenes are expressed directly from the plastid chromosome where they have been integrated by homologous recombination. The fatty acid oxidation transgenes may also be expressed throughout the entire plant tissue from a constitutive promoter. It is also useful to be able to control the expression of these transgenes by using promoters that can be activated following the application of an agrochemical or other active ingredient to the crop in the field. Additional control of the expression of these genes encompassed by the methods described herein include the use of recombinase technologies for targeted insertion of the transgenes into specific chromosomal sites in the plant chromosome or to regulate the expression of the transgenes.

The methods described herein involve a plant seed having a genome including a promoter operably linked to a first DNA sequence and a 3'untranslated region, wherein the first DNA sequence encodes a fatty acid oxidation polypeptide and optionally a promoter operably linked to a second DNA sequence and a 3'-untranslated region, wherein the second WO 99/45122 PCT/US99/04999 DNA sequence encodes a fatty acid oxidation polypeptide. Expression of the two transgenes provides the plant with a functional fatty acid B-oxidation system having at least P-ketothiolase, dehydrogenase and hydratase activities in the cytoplasm or plastids other than peroxisomes or glyoxisomes. The first and/or second DNA sequence may be isolated from bacteria, yeast, fungi, algae, plants, or animals. It is preferable that at least one of the DNA sequences encodes a polypeptide with at least two, and preferably three, enzyme activities.

Transformation Vectors DNA constructs useful in the methods described herein include transformation vectors capable of introducing transgenes into plants. Several plant transformation vector options are available, including those described in "Gene Transfer to Plants" (Potrykus, et al., eds.) Springer-Verlag Berlin Heidelberg New York (1995); "Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins" (Owen, et al., eds.) John Wiley Sons Ltd. England (1996); and "Methods in Plant Molecular Biology: A Laboratory Course Manual" (Maliga, et al. eds.) Cold Spring Laboratory Press, New York (1995). Plant transformation vectors generally include one or more coding sequences of interest under the transcriptional control of and 3' regulatory sequences, including a promoter, a transcription termination and/or polyadenylation signal, and a selectable or screenable marker gene. The usual requirements for 5' regulatory sequences include a promoter, a transcription termination and/or a polyadenylation signal. For the expression of two or more polypeptides from a single transcript, additional RNA processing signals and ribozyme sequences can be engineered into the construct Patent No. 5,519,164). This approach has the advantage of locating multiple transgenes in a single locus, which is advantageous in subsequent plant breeding efforts. An additional approach is to use a vector to specifically transform the plant plastid chromosome by homologous recombination Patent No. 5,545,818), in which case it is possible to take advantage of the prokaryotic nature of the plastid genome and insert a number of transgenes as an operon.

WO 99/45122 PCT/US99/04999 Promoters A large number of plant promoters are known and result in either constitutive, or environmentally or developmentally regulated expression of the gene of interest. Plant promoters can be selected to control the expression of the transgene in different plant tissues or organelles for all of which methods are known to those skilled in the art (Gasser Fraley, Science 244:1293-99 (1989)). The 5' end of the transgene may be engineered to include sequences encoding plastid or other subcellular organelle targeting peptides linked in-frame with the transgene. Suitable constitutive plant promoters include the cauliflower mosaic virus promoter (CaMV) and enhanced CaMV promoters (Odell et. al., Nature, 313: 810 (1985)), actin promoter (McElroy et al., Plant Cell 2:163-71 (1990)), AdhI promoter (Fromm et. al., Bio/Technology 8:833-39 (1990); Kyozuka et al., Mol. Gen. Genet. 228:40-48 (1991)), ubiquitin promoters, the Figwort mosaic virus promoter, mannopine synthase promoter, nopaline synthase promoter and octopine synthase promoter. Useful regulatable promoter systems include spinach nitrate-inducible promoter, heat shock promoters, small subunit ofribulose biphosphate carboxylase promoters and chemically inducible promoters Patent No. 5,364,780 to Hershey et al.).

In a preferred embodiment of the methods described herein, the transgenes are expressed only in the developing seeds. Promoters suitable for this purpose include the napin gene promoter Patent Nos. 5,420,034 and 5,608,152), the acetyl-CoA carboxylase promoter Patent Nos.

5,420,034 and 5,608,152), 2S albumin promoter, seed storage protein promoter, phaseolin promoter (Slightom et. al., Proc. Natl. Acad. Sci. USA 80:1897-1901 (1983)), oleosin promoter (Plant et. al., Plant Mol. Biol.

25:193-205 (1994); Rowley et al., Biochim. Biophys. Acta. 1345:1-4 (1997); U.S. Patent No. 5,650,554; and PCT WO 93/20216), zein promoter, glutelin promoter, starch synthase promoter, and starch branching enzyme promoter.

The transformation of suitable agronomic plant hosts using these vectors can be accomplished with a variety of methods and plant tissues.

WO 99/45122 PCTIUS99/04999 Representative plants useful in the methods disclosed herein include the Brassica family including napus, rappa, sp. carinata andjuncea; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards including Sinapis alba; and flax. Crops harvested as biomass, such as silage corn, alfalfa, or tobacco, also are useful with the methods disclosed herein.

Representative tissues for transformation using these vectors include protoplasts, cells, callus tissue, leaf discs, pollen, and meristems.

Representative transformation procedures include Agrobacterium-mediated transformation, biolistics, microinjection, electroporation, polyethylene glycol-mediated protoplast transformation, liposome-mediated transformation, and silicon fiber-mediated transformation Patent No.

5,464,765; "Gene Transfer to Plants" (Potrykus, et al., eds.) Springer-Verlag Berlin Heidelberg New York (1995); "Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins" (Owen, et al., eds.) John Wiley Sons Ltd. England (1996); and "Methods in Plant Molecular Biology: A Laboratory Course Manual" (Maliga, et al. eds.) Cold Spring Laboratory Press, New York (1995)).

H. Methods for Making and Screening for Transgenic Plants In order to generate transgenic plants using the constructs described herein, the following procedures can be used to obtain a transformed plant expressing the transgenes subsequent to transformation: select the plant cells that have been transformed on a selective medium; regenerate the plant cells that have been transformed to produce differentiated plants; select transformed plants expressing the transgene at such that the level of desired polypeptide is obtained in the desired tissue and cellular location.

For the specific crops useful for practicing the described methods, transformation procedures have been established, as described for example, in "Gene Transfer to Plants" (Potrykus, et al., eds.) Springer-Verlag Berlin Heidelberg New York (1995); "Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins" (Owen, et al., eds.) John Wiley Sons Ltd. England (1996); and "Methods in Plant Molecular Biology: A Laboratory Course Manual" (Maliga, et al. eds.) Cold Spring Laboratory WO 99/45122 PCT/US99/04999 Press, New York (1995).

Brassica napus can be transformed as described, for example, in U.S.

Patent Nos. 5,188,958 and 5,463,174. Other Brassica such as rappa, carinata andjuncea as well as Sinapis alba can be transformed as described by Moloney et. al., Plant Cell Reports 8:238-42 (1989). Soybean can be transformed by a number of reported procedures Patent Nos.

5,015,580; 5,015,944; 5,024,944; 5,322,783; 5,416,011; and 5,169,770).

Several transformation procedures have been reported for the production of transgenic maize plants including pollen transformation Patent No.

5,629,183), silicon fiber-mediated transformation Patent No.

5,464,765), electroporation of protoplasts Patent Nos. 5,231,019; 5,472,869; and 5,384,253) gene gun Patent Nos. 5,538,877 and 5,538,880 and Agrobacterium-mediated transformation (EP 0 604 662 Al; PCT WO 94/00977). The Agrobacterium-mediated procedure is particularly preferred, since single integration events of the transgene constructs are more readily obtained using this procedure, which greatly facilitates subsequent plant breeding. Cotton can be transformed by particle bombardment (U.S.

Patent Nos. 5,004,863 and 5,159,135). Sunflower can be transformed using a combination of particle bombardment and Agrobacterium infection (EP 0 486 233 A2; U.S. Patent No. 5,030,572). Flax can be transformed by either particle bombardment or Agrobacterium-mediated transformation.

Recombinase technologies include the cre-lox, FLP/FRT, and Gin systems.

Methods for utilizing these technologies are described for example in U.S.

Patent No. 5,527,695 to Hodges et al.; Dale Ow, Proc. Natl. Acad. Sci.

USA 88:10558-62 (1991); Medberry et. al., Nucleic Acids Res. 23:485-90 (1995).

Selectable Marker Genes Selectable marker genes useful in practicing the methods described herein include the neomycin phosphotransferase gene nptlI Patent Nos.

5,034,322 and 5,530,196), hygromycin resistance gene Patent No.

5,668,298), bar gene encoding resistance to phosphinothricin Patent No. 5,276,268). EP 0 530 129 Al describes a positive selection system WO 99/45122 PCT/US99/04999 which enables the transformed plants to outgrow the non-transformed lines by expressing a transgene encoding an enzyme that activates an inactive compound added to the growth media. Screenable marker genes useful in the methods herein include the 3-glucuronidase gene (Jefferson et. al., EMBO J 6:3901-07 (1987); U.S. Patent No. 5,268,463) and native or modified green fluorescent protein gene (Cubitt et. al., Trends Biochem Sci.

20:448-55 (1995); Pang et. al., Plant Physiol. 112:893-900 (1996)). Some of these markers have the added advantage of introducing a trait, such as herbicide resistance, into the plant of interest, thereby providing an additional agronomic value on the input side.

In a preferred embodiment of the methods described herein, more than one gene product is expressed in the plant. This expression can be achieved via a number of different methods, including introducing the encoding DNAs in a single transformation event where all necessary DNAs are on a single vector; introducing the encoding DNAs in a cotransformation event where all necessary DNAs are on separate vectors but introduced into plant cells simultaneously; introducing the encoding DNAs by independent transformation events successively into the plant cells i.e. transformation oftransgenic plant cells expressing one or more of the encoding DNAs with additional DNA constructs; and transformation of each of the required DNA constructs by separate transformation events, obtaining transgenic plants expressing the individual proteins and using traditional plant breeding methods to incorporate the entire pathway into a single plant.

III. P-Oxidation Enzyme Pathways Production of PHAs in the cytosol of plants requires the cytosolic localization of enzymes that are able to produce R-3-hydroxyacyl CoA thioesters as substrates for PHA synthases. Both eukaryotes and prokaryotes possess a P-oxidation pathway for fatty acid degradation that consists of a series of enzymes that convert fatty acyl CoA thioesters to acetyl CoA.

While these pathways proceed via intermediate 3-hydroxyacyl CoA, the stereochemistry of this intermediate varies among organisms. For example, WO 99/45122 PCT/US99/04999 the -oxidation pathways of bacteria and the peroxisomal pathway of higher eukaryotes degrade fatty acids to acetyl CoA via S-3-hydroxyacyl CoA (Schultz, "Oxidation of Fatty Acids" in Biochemistry ofLipids, Lipoproteins andMembranes (Vance et al., eds.) pp. 101-06 (Elsevier, Amsterdam 1991)).

In Escherichia coli, an epimerase activity encoded by the P-oxidation multifunctional enzyme complex is capable of converting S-3-hydroxyacyl CoA to R-3-hydroxyacyl CoA. Yeast possesses a peroxisomal localized fatty acid degradation pathway that proceeds via intermediate R-3hydroxyacyl CoA (Hiltunen, et al. J. Biol. Chem. 267: 6646-53 (1992); Filppula, et al. J. Biol. Chem. 270:27453-57 (1995)), such that no epimerase activity is required to produce PHAs.

Plants, like other higher eukaryotes, possesses a 1-oxidation pathway for fatty acid degradation localized subcellularly in the peroxisomes (Gerhardt, "Catabolism of Fatty Acids [a and 1 Oxidation]" in Lipid Metabolism in Plants (Moore, Jr., ed.) pp. 527-65 (CRC Press, Boca Raton, Florida 1993)). Production of PHAs in the cytosol of plants therefore necessitates the cytosolic expression of a P-oxidation pathway, for conversion of fatty acids to R-3-hydroxyacyl CoA thioesters of the correct chain length, as well as cytosolic expression of an appropriate PHA synthase, to polymerize R-3-hydroxyacyl CoA to polymer.

Fatty acids are synthesized as saturated acyl-ACP thioesters in the plastids of plants (Hartwood, "Plant Lipid Metabolism" in Plant Biochemistry (Dey et al., eds.) pp. 237-72 (Academic Press, San Diego 1997)). Prior to export from the plastid into the cytosol, the majority of fatty acids are desaturated via a A9 desaturase. The pool of newly synthesized fatty acids in most oilseed crops consists predominantly of oleic acid (cis 9octadecenoic acid), stearic acid (octadecanoic acid), and palmitic acid (hexadecanoic acid). However, some plants, such as coconut and palm kernel, synthesize shorter chain fatty acids (C8-14). The fatty acid is released from ACP via a thioesterase and subsequently converted to an acyl- CoA thioester via an acyl CoA synthetase located in the plastid membrane (Andrews, et al., "Fatty acid and lipid biosynthesis and degradation" in Plant WO 99/45122 PCT/US99/04999 Physiology, Biochemistry, and Molecular Biology (Dennis et al., eds.) pp.

345-46 (Longman Scientific Technical, Essex, England 1990); Harwood, "Plant Lipid Metabolism" in Plant Biochemistry (Dey et al., eds) p. 246 (Academic Press, San Diego 1997)).

The cytosolic conversion of the pool of newly synthesized acyl CoA thioesters via fatty acid degradation pathways and the conversion of intermediates from these series of reactions to R-3-hydroxyacyl-CoA substrates for PHA synthases can be achieved via the enzyme reactions outlined in Figure 1. The PHA synthase substrates are C4-C16 R-3hydroxyacyl CoAs. For saturated fatty acyl CoAs, conversion to R-3hydroxyacyl CoA thioesters using fatty acids degradation pathways necessitates the following sequence of reactions: conversion of the acyl CoA thioester to trans-2-enoyl-CoA (reaction hydration of trans-2-enoyl-CoA to R-3-hyddroxy acyl CoA (reaction 2a, e.g. yeast system operates through this route and the Aeromonas caviae D-specific hydratase yields C4-C7 R-3hydroxyacyl-CoAs), hydration of trans-2-enoyl-CoA to S-3-hydroxy acyl CoA (reaction 2b), and epimerization of S-3-hydroxyacyl CoA to R-3hydroxyacyl CoA (reaction 5, e.g. cucumber tetrafunctional protein, bacterial systems). If 3-hydroxyacyl CoA is not polymerized by PHA synthase forming PHA, it can proceed through the remainder of the P-oxidation pathway as follows: oxidation of 3-hydroxyacyl CoA to form P-keto acyl CoA (reaction 3) followed by thiolysis in the presence of CoA to yield acetyl CoA and a saturated acyl CoA thioester shorter by two carbon units (reaction The acyl CoA thioester produced in reaction 4 is free to re-enter the Poxidation pathway at reaction 1 and the acetyl-CoA produced can be converted to R-3-hydroxyacyl CoA by the action of 0-ketothiolase (reaction 7) and NADH or NADPH acetoacetyl-CoA reductase (reaction This latter route is useful for producing R-3-hydroxybutyryl-CoA, R-3hydroxyvaleryl-CoA and R-3-hydroxyhexanoyl-CoA. The R-3hydroxyacids of four to sixteen carbon atoms produced by this series of enzymatic reactions can be polymerized by PHA synthases expressed from a transgene, or transgenes in the case of the two subunit synthase enzymes, WO 99/45122 PCT/US99/04999 into PHA polymers.

For A9 unsaturated fatty acyl CoAs, a variation of the reaction sequences described is required. Three cycles of 1-oxidation, as detailed in Figure 1, will remove six carbon units yielding an unsaturated acyl CoA thioester with a cis double bond at position 3. Conversion of the cis double bond at position 3 to a trans double bond at position 2, catalyzed by A 3 -cis- A2-trans-enoyl CoA isomerase will allow the P-oxidation reaction sequences outlined in Figure 1 to proceed. This enzyme activity is present on the microbial 3-oxidation complexes and the plant tetrafunctional protein, but not on the yeastfoxl.

Acyl CoA thioesters also can be degraded to a P-keto acyl CoA and converted to R-3-hydroxyacyl CoA via a NADH or NADPH dependent reductase (reaction 6).

Multifunctional enzymes that encode S-specific hydratase, S-specific dehydrogenase, 0-ketothiolase, epimerase and A 3 -cis-A 2 -trans-enoyl CoA isomerase activities have been found in bacteria such as Escherichia coli (Spratt, et al., J. Bacteriol. 158:535-42 (1984)) and Pseudomonasfragi (Immure, et al., J. Biochem. 107:184-89 (1990)). The multifunctional enzyme complexes consist of two copies of each of two subunits such that catalytically active protein forms a heterotetramer. The hydratase, dehydrogenase, epimerase, and A 3 -cis-A 2 -trans-enoyl CoA isomerase activities are located on one subunit, whereas the thiolase is located on another subunit. The genes encoding the enzymes from organisms such as E.

coli (Spratt, et al., J. Bacteriol. 158:535-42 (1984); DiRusso, J. Bacteriol.

172:6459-68 (1990)) and P. fragi (Sato, et al., J. Biochem. 111:8-15 (1992)) have been isolated and sequenced and are suitable for practicing the methods described herein. Furthermore, the E. coli enzyme system has been subjected to site-directed mutagenesis analysis to identify amino acid residues critical to the individual enzyme activities (He Yang, Biochemistry 35:9625-30 (1996); Yang et. al., Biochemistry 34:6641-47 (1995); He Yang, Biochemistry 36:11044-49 (1997); He et. al., Biochemistry 36:261-68 (1997); Yang Elzinga, J. Biol. Chem. 268:6588- WO 99/45122 PCT/US99/04999 92 (1993)). These mutant genes also could be used in some embodiments of the methods described herein.

Mammals, such as rat, possess a trifunctional 1-oxidation enzyme in their peroxisomes that contains hydratase, dehydrogenase, and A 3 -cis-A 2 trans-enoyl CoA isomerase activities. The trifunctional enzyme from rat liver has been isolated and has been found to be monomeric with a molecular weight of 78 kDa (Palosaari, et al., J. Biol. Chem. 265:2446-49 (1990)).

Unlike the bacterial system, thiolase activity is not part of the multienzyme protein (Schultz, "Oxidation of Fatty Acids" in Biochemistry ofLipids, Lipoproteins and Membranes (Vance et al., eds) p. 95 (Elsevier, Amsterdam (1991)). Epimerization in rat occurs by the combined activities of two distinct hydratases, one which converts R-3-hydroxyacyl CoA to trans-2enoyl CoA, and another which converts trans-2-enoyl CoA to S-3hydroxyacyl CoA (Smeland, et al., Biochemical and Biophysical Research Communications 160:988-92 (1989)). Mammals also possess P-oxidation pathways in their mitochondria that degrade fatty acids to acetyl CoA via intermediate S-3-hydroxyacyl CoA (Schultz, "Oxidation of Fatty Acids" in Biochemistry ofLipids, Lipoproteins and Membranes (Vance et al., eds) p.

96 (Elsevier, Amsterdam (1991)). Genes encoding mitochondrial Poxidation activities have been isolated from several animals including a Rat mitochondrial long chain acyl CoA hydratase/3-hydroxy acyl CoA dehydrogenase (GENBANK Accession D16478) and a Rat mitochondrial thiolase (GENBANK Accession #s D13921, D00511).

Yeast possesses a multifunctional enzyme, Fox2, that differs from the 0-oxidation complexes of bacteria and higher eukaryotes in that it proceeds via a R-3-hydroxyacyl CoA intermediate instead of S-3-hydroxyacyl CoA (Hiltunen, et al., J. Biol. Chem. 267:6646-53 (1992)). Fox2 possesses Rspecific hydratase and R-specific dehydrogenase enzyme activities. This enzyme does not possess the A 3 -cis-A 2 -trans-enoyl CoA isomerase activity needed for degradation of A9-cis-hydroxyacyl CoAs to form R-3hydroxyacyl CoAs. The gene encodingfox2 from yeast has been isolated and sequenced and encodes a 900 amino acid protein. The DNA sequence of WO 99/45122 PCT/US99/04999 the structural gene and amino acid sequence of the encoded polypeptide is shown in SEQ ID NO:1 and SEQ ID NO:2.

Plants have a tetrafunctional protein similar to the yeast Fox2, but also encoding a A 3 -cis-A 2 -trans-enoyl CoA isomerase activity (Muller et., al., J. Biol. Chem. 269:20475-81 (1994)). The DNA sequence of the cDNA and amino acid sequence of the encoded polypeptide is shown in SEQ ID NO:3 and SEQ ID NO:4.

IV. Targeting of Enzymes to the Cytoplasm of Oil Seed Crops Engineering PHA production in the cytoplasm of plants requires directing the expression of P-oxidation to the cytosol of the plant. No targeting signals are present in the bacterial systems, such as faoAB. In fungi, yeast, plants, and mammals, P-oxidation occurs in subcellular organelles. Typically, the genes are expressed from the nuclear chromosome, and the polypeptides synthesized in the cytoplasm are directed to these organelles by the presence of specific amino acid sequences. To practice the methods described herein using genes isolated from eukaryotic sources, fatty acid oxidation enzymes from eukaryotic sources, such as yeast, fungi, plants, and mammals, the removal or modification of subcellular targeting signals is required to direct the enzymes to the cytosol.

It may be useful to add signals for directing proteins to the endoplasmic reticulum. Peptides useful in this process are well known in the art. The general approach is to modify the transgene by inserting a DNA sequence specifying an ER targeting peptide sequence to form a chimeric gene.

Eukaryotic acyl CoA dehydrogenases, as well as other mitrochondrial proteins, are targeted to the mitochondria via leader peptides on the Nterminus of the protein that are usually 20-60 amino acids long (Horwich, Current Opinion in Cell Biology, 2:625-33 (1990)). Despite the lack of an obvious consensus sequence for mitochondrial import leader peptides, mutagenesis of key residues in the leader sequence have been demonstrated to prevent the import of the mitochrondrial protein. For example, the import of Saccharomyces cerevisiae F -ATPase was prevented by mutagenesis of its leader sequence, resulting in the accumulation of the modified precursor WO 99/45122 PCT/US99/04999 protein in the cytoplasm (Bedwell, et al., Mol. Cell Biol. 9:1014-25 (1989)) Three eukaryotic peroxisomal targeting signals have been reported (Gould, et al., J Cell Biol. 108:1657-64 (1989); Brickner, et al., J. Plant Physiol., 113:1213-21 (1997)). The tripeptide targeting signal S/A/C- K/H/R-L occurs at the C-terminal end of many peroxisomal proteins (Gould, et al., J. Cell Biol. 108:1657-64 (1989)). Mutagenesis of this sequence has been shown to prevent import of proteins into peroxisomes. Some peroxisomal proteins do not contain the tripeptide at the C-terminal end of the protein. For these proteins, it has been suggested that targeting occurs via the tripeptide in an internal position within the protein sequence (Gould, et al., J Cell Biol. 108:1657-64 (1989)) or via an unknown, unrelated sequence (Brickner, et al., Plant Physiol. 113:1213-21 (1997)). The results of in vitro peroxisomal targeting experiments with fragments of acyl CoA oxidase from Candida tropicalis appear to support the latter theory and suggest that there are two separate targeting signals within the internal amino acid sequence of the polypeptide (Small, et al., The EMBO Journal 7:1167- 73 (1988)). In the aforementioned study, the targeting signals were localized to two regions of 118 amino acids in length, and neither of regions was found to contain the targeting signal S/A/C-K/H/R-L. A small number of peroxisomal proteins appear to contain an amino terminal leader sequence for import into peroxisomes (Brickner, et al., J. Plant Physiol. 113:1213-21 (1997)). These targeting signals can be deleted or altered by site directed mutagenesis.

V. Cultivation and Harvesting of Transgenic Plant The transgenic plants can be grown using standard cultivation techniques. The plant or plant part also can be harvested using standard equipment and methods. The PHAs can be recovered from the plant or plant part using known techniques such as solvent extraction in conjunction with traditional seed processing technologies, as described in PCT WO 97/15681, or can be used directly, for example, as animal feed, where it is unnecessary to extract the PHA from the plant biomass.

Several lines which did not produce seed, produced much higher WO 99/45122 PCT/US99/04999 levels of biomass. produced much higher levels of biomass. This phenotype therefore may be useful as a means to increase the amount of green biomass produced per acre for silage, forage, or other biomass crops. End uses include the more cost effective production of forage crops for animal feed or as energy crops for electric power generation. Other uses include increasing biomass levels in crops, such as alfalfa or tobacco, for subsequent recovery of industrial products, such as PHAs by extraction.

The compositions and methods of preparation and use thereof described herein are further described by the following non-limiting examples.

Example 1: Isolation and Characterization of the Pseudomonas putidafaoAB Genes and Fao Enzyme All DNA manipulations, including PCR, DNA sequencing E. coli transformation, and plasmids purification, were performed using standard procedures, as described, for example, by Sambrook et. al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York (1989)). The genes encodingfaoAB from Pseudomonasputida were isolated using a probe generated from P. putida genomic DNA by PCR (polymerase chain reaction) using primers 1 and 2 possessing homology to faoB from Pseudomonasfragi (Sato, et al., J. Biochem. 111:8-15 (1992)).

Primer 1: gat ggg ccg ctc caa ggg tgg 3' (SEQ ID Primer 2: 5' caa ccc gaa ggt gcc gcc att 3' (SEQ ID NO:6) A 1.1 kb DNA fragment was purified from the PCR reaction and used as a probe to screen a P. putida genomic library constructed in plasmid pBKCMV using the lambda ZAP expression system (Stratagene). Plasmid pMFX was selected from the positive clones and the DNA sequence of the insert containing thefaoAB genes and flanking sequences determined. This is shown in SEQ ID NO:7. A fragment containingfaoAB was subcloned with the native P. putida ribosome binding site intact into the expression WO 99/45122 PCT/US99/04999 vector pTRCN forming plasmid pMFX3 as follows. Plasmid pMFXI was digested with BsrG I. The resulting protruding ends were filled in with Klenow. Digestion with Hind III yielded a 3.39 kb blunt ended/Hind III fragment encoding FaoAB. The expression vector pTRCN was digested with Sma I/Hind III and ligated with thefaoAB fragment forming the 7.57 kb plasmid pMFX3.

Enzymes in the FaoAB multienzyme complex were assayed as follows. Hydratase activity was assayed by monitoring the conversion of NAD to NADH using the coupling enzyme L-0-hydroxyacyl CoA dehydrogenase as previously described, except that assays were run in the presence of CoA (Filppula, et al., J. Biol. Chem. 270:27453-57 (1995)).

Severe product inhibitation of the coupling enzyme was observed in the absence of CoA. The assay contained (1 mL final volume) 60 pM crotonyl CoA, 50 pM Tris-CI, pH 9, 50 p.g bovine serum albumin per mL, 50 mM KC1, 1 mM NAD, 7 p.g L-specific P-hydroxyacyl CoA dehydrogenase from porcine heart per mL, and 0.25 mM CoA. The assay was initiated with the addition of FaoAB to the assay mixture. A control assay was performed without substrate to determine the rate of consumption of NAD in the absence of the hydratase generated product, S-hydroxybutyryl CoA. One unit of activity is defined as the consumption of one p.Mol of NAD per min (6340= 6220 M"'cm'l).

Hydroxyacyl CoA dehydrogenase was assayed in the reverse direction with acetoacetyl CoA as the substrate by monitoring the conversion of NADH to NAD at 340 nm (Binstock, et al., Methods in Enzymology, 71:403 (1981)). The assay contained (1 mL final volume) 0.1 M KH 2

PO

4 pH 7, 0.2 mg bovine serum albumin per mL, 0.1 mM NADH, and 33 pM acetoacetyl CoA. The assay was initiated with the addition of FaoAB to the assay mixture. When necessary, enzyme samples were diluted in 0.1 M

KH

2

PO

4 pH 7, containing 1 mg bovine serum albumin per mL. A control assay was performed without substrate acetoacetyl CoA to detect the rate of consumption of NADH in the crude due to enzymes other than hydroxyacyl CoA dehydrogenase. One unit of activity is defined as the consumption of WO 99/45122 PCT/US99/04999 one gMol of NADH per minute (8340 6220 HydroxyacylCoA dehydrogenase was assayed in the forward direction with crotonyl CoA as a substrate by monitoring the conversion of NAD to NADH at 340 nm (Binstock, et al., Methods in Enzymology, 71:403 (1981)). The assay mixture contained (1 mL final volume) 0.1 M KH2PO 4 pH 8, 0.3 mg bovine serum albumin per mL, 2 mM P-mercaptoethanol, 0.25 mM CoA, 30 pM crotonyl CoA, and an aliquot of FaoAB. The reaction was preincubated for a couple of minutes to allow in situ formation of Shydroxybutyryl CoA. The assay then was initiated by the addition of NAD (0.45 mM). A control assay was performed without substrate to detect the rate of consumption of NAD due to enzymes other than hydroxyacyl CoA dehydrogenase. One unit of activity is defined as the consumption of one p.Mol of NAD per minute (8340 6220 Thiolase activity was determined by monitoring the decrease in absorption at 304 nm due to consumption of substrate acetoacetyl CoA as previously described with some modifications (Palmer, et al., J. Biol. Chem.

266:1-7 (1991)). The assay contained (final volume 1 mL) 62.4 mM Tris-Cl, pH 8.1, 4.8 mM MgC1 2 62.5 pLM CoA, and 62.5 pM acetoacetyl CoA. The assay was initiated with the addition of FaoAB to the assay mixture. A control sample without enzyme was performed for each assay to detect the rate of substrate degradation of pH 8.1 in the absence of enzyme. One unit of activity is defined as the consumption of one pMol of substrate acetoacetyl CoA per minute (8340= 16900 M'cm').

Epimerase activity was assayed as previously described (Binstock, et al., Methods in Enzymology, 71:403 (1981)) except that R-3-hydroxyacyl CoA thioesters were utilized instead of D,L-3-hydroxyacyl CoA mixtures.

The assay contained (final volume 1 mL) 30 pM R-3-hydroxyacyl CoA, 150 mM KH 2

PO

4 (pH 0.3 mg/mL BSA, 0.5 mM NAD, 0.1 mM CoA, and 7 p.g/mL L-specific 0-hydroxyacyl CoA dehydrogenase from porcine heart.

The assay was initiated with the addition of FaoAB.

For expression of FaoAB in DH5a/pMFX3, cultures were grown in 2xTY medium at 30 2xTY medium contains (per L) 16 g tryptone, 10 g WO 99/45122 PCT/US99/04999 yeast, and 5 g NaCl. A starter culture was grown overnight and used to inoculate inoculum) fresh medium (100 mL in a 250 mL Erlenmeyer flask for small scale growths; 1.5L in a 2.8L flask for large scale growths).

Cells were induced with 0.4 mM IPTG when the absorbance at 600 nm was in the range of 0.4 to 0.6. Cells were cultured an additional 4 h prior to harvest. Cells were lysed by sonication, and the insoluble matter was removed from the soluble proteins by centrifugation. Acyl CoA dehydrogenase activity was monitored in the reverse direction to ensure activity of the FaoA subunit (SEQ ID NO:31) and thiolase activity was assayed to determine activity of the Fao subunit. FaoAB in DH5o/pMFX3 contained dehydrogenase and thiolase activity values of 4.3 and 0.99 U/mg, respectively, which is significantly more than the 0.0074 and 0.0033 U/mg observed for dehydrogenase and thiolase, respectively, in control strain DHSopTRCN.

FaoAB was purified from DH5c/pMFX3 using a modified procedure previously described for the purification of FaoAB from Pseudomonasfragi (Imamura, et al., J. Biochem. 107:184-89 (1990)). Thiolase activity (assayed in the forward direction) and dehydrogenase activities (assayed in the reverse direction) were monitored throughout the purification. Three liters of DH5atpMFX3 cells (2 X 1.5 L aliquots in 2.8 L Erlenmeyer flasks) were grown in 2 x TY medium using the cell growth procedure previously described for preparing cells for enzyme activity analysis. Cells (15.8 g) were resuspended in 32 mL of 10 mM KH 2 PO4, pH 7, and lysed by sonication. Soluble proteins were removed from insoluble cells debris by centrifugation (18,000 RPM, 30 min., 4 The soluble extract was made in acetone and the precipitated protein was isolated by centrifugation and redissolved in 10 mM KH 2

PO

4 pH 7. The sample was adjusted to 33% saturation with (NH 4 2

SO

4 and the soluble and insoluble proteins were separated by centrifugation. The resulting supernatant was adjusted to 56% saturation with (NH4) 2

SO

4 and the insoluble pellet was isolated by centrifugation and dissolved in 10 mM KH 2

PO

4 pH 7. The sample was heated at 50 0 C for 30 min. and the soluble proteins were isolated by WO 99/45122 PCT/US99/04999 centrifugation and dialyzed in a 6,000 to 8,000 molecular weight cut off membrane in 10 mM KH 2

PO

4 pH 7 (2 X 3L; 20 The sample was loaded on a Toyo Jozo DEAE FPLC column (3 cm x 14 cm) that previously had been equilibrated in 10 mM KH 2

PO

4 pH 7. The protein was eluted with a linear gradient (100 mL by 100 mL; 0 to 500 mM NaCI in 10 KH 2 P0 4 pH 7) at a flow of 3 mL/min. FaoAB eluted between 300 and 325 mM NaC1. The sample was dialyzed in a 50,000 molecular weight cut off membrane in mM KH 2

PO

4 pH 7 (1 X 2L; 15h) prior to loading on a macro-prep hydroxylapatite 18/30 (Biorad) FPLC column (2 cm x 15 cm) that previously had been equilibrated in 10 mM KH 2 P0 4 pH 7. The protein was eluted with a linear gradient (250 mL by 250 mL; 10 to 500 mM KH 2

PO

4 pH 7) at a flow rate of 3 mL/min. FaoAB eluted between 70 and 130 mM KH 2 PO4.

The fractions containing activity were concentrated to 9 mL using a MILLIPORETM 100,000 molecular weight cutoff concentrator. The buffer was exchanged 3 times with 10 mM KH 2

PO

4 pH 7 containing 20% sucrose and frozen at -70 0 C. Enzyme activities of the hydroxylapatite purified fraction were assayed with a range of substrates. The results are shown in Table 1 below.

Table 1: Enzyme Substrates and Activities Enzyme Substrate Activity (U/mg) hydratase crotonyl CoA 8.8 dehydrogenase (forward) crotonyl CoA 0.46 dehydrogenase (reverse) acetoacetyl CoA 29 thiolase acetoacetyl CoA 9.9 epimerase R-3-hydroxyoctanyl CoA 0.022 epimerase R-3- hydroxyhexanyl CoA 0.0029 epimerase R-3- hydroxybutyryl CoA 0.000022 Example 2: Production of Antibodies to the FaoAB and FaoAB Polypeptides Following purification of the FaoAB protein as described in Example 1, a sample was separated by SDS-PAGE. The protein band corresponding to the FaoA (SEQ ID NO:31) and FaoB (SEQ ID NO:26) was excised and used to immunize New Zealand white rabbits with complete Freunds adjuvant. Boosts were performed using incomplete Freunds at three week WO 99/45122 PCT/US99/04999 intervals. Antibodies were recovered from serum by affinity chromatography on Protein A columns (Pharmacia) and tested against the antigen by Western blotting procedures. Control extracts of Brassica seeds were used to test for cross reactivity to plant proteins. No cross reactivity was detected.

Example 3: Construction of Plasmids for Expression of the Pseudomonasputidofao AB Genes in Transgenic Oilseeds Construction of pSBS2024 Oligonucleotide primers GVR471 '-CGGTACCCATTGTACTCCCAGTATCAT-3' (SEQ ID NO:8) and GVR472 TTTAAA TAGTAGAGTATTGAATATG-3' (SEQ ID NO:9) homologous to sequences flanking the 5' and 3' ends (underlined), respectively, of the bean phaseolin promoter (SEQ ID NO: 10; Slightom et al., 1983) were designed with the addition of KpnI (in italics, nucleotides 1-7 in SEQ ID NO:8) and SwaI (in italics, nucleotides 1-9 in SEQ ID NO:9) at the 5' ends of GVR471 and GVR472, respectively. These restriction sites were incorporated to facilitate cloning. The primers were used to amplify a 1.4 kb phaseolin promoter, which was cloned at the SmaI site in pUC19 by blunt ended ligation. The designated plasmid, pCPPI (see Figure 2) was cut with Sall and Swal and ligated to a SalI/SwaI phaseolin terminator (SEQ ID NO:27). The bean phaseolin terminator sequence encompassing the polyadenylation signals was amplified using the following PCR primers: GVR396: 5'-GATTTAAATGCAAGCTTAAATAAGTATGAACTAAAATGC-3' (SEQ ID NO:22) and GVR397: 5'-CGGTACCTTAGTTGGTAGGGTGCTA-3' (SEQ ID NO.23) and the 1.2Kb fragment (SEQ ID NO:27) cloned into Sall-Sal site ofpCCPl to obtain pSBS2024 (Figure The resulting plasmid which contains a unique HindIII site for cloning was called pSBS2024 (Figure 2).

WO 99/45122 PCT/US99/04999 Construction of pSBS2025 A soybean oleosin promoter fragment (SEQ ID NO: 11; Rowley et al., 1997) was simplified with primers that flank the DNA sequence.

Primer JA408 5' -TCTA GATACATCCATTTCTTAATATAATCCTCTTATTC-3' (SEQ ID NO: 12) contains sequences that are complementary to the 5' end (underlined).

Primer npl -CA TTTAAA TGGTTAAGGTGAAGGTAGGGCT-3' (SEQ ID NO:13) contains sequences homologous to the 3' end (underlined) of the promoter fragment. The restriction sites Xbal (in italics) and Swal (in italics) were incorporated at the 5' end of JA408 and npl, respectively, to facilitate cloning. The primers were used to amplify a 975 bp promoter fragment, which then was cloned into Small site of pUC 19 (see Figure The resulting plasmid, pCSPI, was cut with Sall and Swal and ligated to the soybean terminator (SEQ ID NO:28). The soybean oleosin terminator was amplified by PCR using the following primers: JA410: 5'-AAGCTTACGTGATGAGTATTAATGTGTTGTTATG-3' (SEQ ID NO:29) and JA411: 5'-TCTAGACAATTCATCAAATACAAATCACATTGCC-3' (SEQ ID and the 225 bp fragment cloned into the SalI-Swal site ofpCSPl to obtain plasmid pSBS2025 (Figure The designated plasmid, pSBS2025, carried a unique HindIII site for cloning (Figure 2).

WO 99/45122 PCT/US99/04999 Construction of Promoter-coding Sequence Fusions Two oligonucleotide primers were synthesized: np2 5'AAGCTTAAAATGATTTACGAAGGTAAAGCC-3' (SEQ IDNO:14) homologous to nucleotides 553 to 573 of the 5' flanking sequences, and np3 A TTGCTTCAGTTGAAGCGCTG-3' (SEQ ID NO: complementary to nucleotides 2700 to 2683 flanking the 3' end ofmfl (faoA, SEQ ID NO:24) of plasmid pmfx3. A HindIIl (in italics) site was introduced at the 5' end of primers np2 and np3 to facilitate cloning. In addition, a 3 bp AAA sequence (bold) was incorporated to obtain a more favorable sequence surrounding the plant translational initiation codon. Primers np2 and np3 were used to amplify the fragment and cloned into SmaI site of pUC 19. The resulting plasmid was called pCmfl (Figures 3A and 3B). Plasmid pBmf2 was constructed in a similar process (Figures 5A and 5B). In order to generate a HindIll (in italics) at 5' and 3' ends of the mf2 (faoB) gene (SEQ ID NO:25) for cloning, a second set of synthetic primers were designed.

Primers np4 5'-AAGCTTAAAATGAGCCTGAATCCAAGAGAC-3' (SEQ ID NO:16) complementary to 5' (nucleotides 2732-2752 bp) and -3' (SEQ ID NO: 17) homologous to 3' (nucleotides 3907-3886 bp) sequences of mf2 (faoB, SEQ ID NO:25) of plasmid pmfx3 were used in a PCR reaction to amplify the 1.17 kb DNA fragment. The resulting PCR product was cloned into the EcoRV site of pBluescript. The plasmid was referred to as pBmf2.

Both plasmids were individually cut with HindI and their inserts cloned in plasmids pSBS2024 and pSBS2025, which had previously been linearized with the same restriction enzyme. As a result, the following WO 99/45122 PCT/US99/04999 plasmids were generated: pmfl24 and pmfl25 (Figures 3A and 3B) and pmf224 and pmf225 (Figures 5A and 5B) containing the Fao genes (mfl and mf2) fused to either the phaseolin or soybean promoters. DNA sequence analysis confirmed the correct promoter-coding sequence-termination sequence fusions for pmfl24, pmfl25, pmf224, and pmf225.

Example 4: Assembly of Promoter-coding Sequence Fusions into Plant Transformation Vectors After obtaining plasmids pmfl24, pmfl25, pmf224, and pmf225, promoter-coding sequence fusions were independently cloned into the binary vectors, pCGN1559 (McBride and Summerfelt, 1990) containing the CaMV promoter driving the expression ofNPTII gene (conferring resistance to the antibiotic kanamycin) and pSBS2004 containing a parsley ubiquitin promoter driving the PPT gene, which confers resistance to the herbicide phosphinothricine. Binary vectors suitable for this purpose with a variety of selectable markers can be obtained from several sources.

The phaseolin-mf21 fusion cassette was released from the parent plasmid with XbaI and ligated with pCGN1559, which had been linearized with the same restriction enzyme. The resulting plasmid was designated pCGmfl24 (Figures 3A and 3B). Plasmid pCGmfl25 containing the soybean-mfl fusion was constructed in a similar way (Figures 3A and 3B), except that both pmfl25 and pCGN 1559 were cut with BamHI before ligation.

Construction ofpmfl249 an pmfl254 The plasmid pSBS2004 was linearized with BamHI fragment containing the soybean-mfl fusion. This plasmid was designated pmfl254 (Figures 4A and 4B). Similarly, the XbaI phaseolin-mfl fusion fragment was ligated to pSBS2004 which had been linearized with the same restriction enzyme. The resulting plasmid was designated pmfl249 (Figures 4A and 4B).

Construction of pCGmf224 and pCGmf225 The phaseolin-mf2 and soybean-mf2 fusions were constructed by WO 99/45122 PCT/US99/04999 excising the fusions from the vector by cutting with either BamHI or XbaI, and cloned into pCGN1559 which had been linearized with either restriction enzyme (Figures 5A and Construction of pCGmflP2S and pCGmf2P1S The two expression cassettes containing the promoter-coding sequence fusions were assembled on the same binary vector as follows: Plasmid pmfl24 containing the phaseolin-mfl fusion was cut with BamHI and cloned into the BamHI site of pCGN1559 to create pCGmfB124. This plasmid then was linearized with XbaI and ligated to the XbaI fragment of pmf225 containing the soybean-mf2 fusion. The final plasmid was designated pCGmflP2S (Figures 6A and 6B). Plasmid pCGmf2P1S was assembled in similar manner. The phaseolin-mf2 fusion was released from pmf224 by cutting with BamHI and cloned at the BamHI site of pCGN 1559.

The resulting plasmid, pCGmfB224, was linearized with XbaI and ligated to the XbaI fragment of pmfl25 containing the soybean-mfl fusion (Figures 6A and 6B).

Example 5: Transformation of Brassica Brassica seeds were surface sterilized in 10% commercial bleach (Javex, Colgate-Palmolive) for 30 min. with gentle shaking. The seeds were washed three times in sterile distilled water. Seeds were placed in germination medium comprising Murashige-Skoog (MS) salts and vitamins, 3% sucrose and 0.7% phytagar, pH 5.8 at a density of 20 per plate and maintained at 24 oC and a 16 h light 8 h dark photoperiod at a light intensity of 60-80 p/Em-2s" for four to five days.

Each of the constructs, pCGmfl24, pCGmfl25, pCGmf224, pCGmflP2S, and pCGmf2P1 S were introduced into Agrobacterium tumefacians strain EHA101 (Hood et al., J. Bacteriol. 168:1291-1301 (1986)) by electroporation. Prior to transformation of cotyledonary petioles, single colonies of strain EHA101 harboring each construct were grown in ml of minimal medium supplemented with 100 mg kanamycin per liter and 100 mg gentamycin per liter for 48 hr at 28 OC. One milliliter of bacterial WO 99/45122 PCT/US99/04999 suspension was pelletized by centrifugation for 1 min in a microfuge. The pellet was resuspended in 1 ml minimal medium.

For transformation, cotyledons were excised from 4 day old, or in some cases 5 day old, seedlings, so that they included approximately 2 mm of petiole at the base. Individual cotyledons with the cut surface of their petioles were immersed in diluted bacterial suspension for 1 s and immediately embedded to a depth of approximately 2 mm in co-cultivation medium, MS medium with 3% sucrose and 0.7% phytagar and enriched with 20 pM benzyladenine. The inoculated cotyledons were plated at a density of 10 per plate and incubated under the same growth conditions for 48 h. After co-cultivation, the cotyledons then were transferred to regeneration medium comprising MS medium supplemented with 3% sucrose, 20 uM benzyladenine, 0.7% phytagar, pH 5.8, 300 mg timentinin per liter, and 20 mg kanamycin sulfate per liter.

After two to three weeks, regenerant shoots obtained were cut and maintained on "shoot elongation" medium (MS medium containing, 3% sucrose, 300 mg timentin per liter, 0.7% phytagar, 300 mg timentinin per liter, and 20 mg kanamycin sulfate per liter, pH 5.8) in Magenta jars.

The elongated shoots were transferred to "rooting" medium comprising MS medium, 3% sucrose, 2 mg indole butyric acid per liter, 0.7% phytagar, and 500 mg carbenicillin per liter. After roots emerged, plantlets were transferred to potting mix (Redi Earth, W.R. Grace and The plants were maintained in a misting chamber (75% relative humidity) under the same growth conditions. Two to three weeks after growth, leaf samples were taken for neomycin phosphotransferase (NPTII) assays (Moloney et al., Plant Cell Reports 8:238-42 (1989)).

Seeds from the FaoA and FaoB transgenic lines can be analyzed for expression of the fatty acid oxidation polypeptides by western blotting using the anti-FaoA and anti-FaoB antibodies. The FaoB polypeptide (SEQ ID NO:26) is not functional in the absence of the FaoA gene product; however, the FaoAB gene product has enzyme activity.

Transgenic lines expressing the FaoA and FaoB complex are obtained WO 99/45122 PCT/US99/04999 by crossing the FaoA and FaoB transgenic lines expressing the individual polypeptides and seeds analyzed by western blotting and enzymes assays as described.

Example 6: Transformation ofB. napus cv. Westar and Analysis of Transgenic Lines Transformation The protocol used was adopted from a procedure described by Moloney et al. (1989). Seeds ofBrassica napus cv. Westar were surface sterilized in 10% commercial bleach (Javex, Colgate-Palmolive Canada Inc.) for 30 min with gentle shaking. The seeds were washed three times in sterile distilled water. Seeds were placed on germination medium comprising Murashige-Skoog (MS) salts and vitamins, 3% sucrose and 0.7% phytagar, pH 5.8 at a density of 20 per plate and maintained at 24 °C in a 16 h light/8 h dark photoperiod at a light intensity of 60-80 pEm- 2 s- for four to five days.

Each of the constructs, pCGmfl24, pCGmfl25, pCGmf224, pCGmf225, pCGmflP2S, and pCGmf2PlS were introduced into Agrobacterium tumefaciens strain EHA101 (Hood et al. 1986) by electroporation. Prior to transformation of cotyledonary petioles, single colonies of strain EHA101 harboring each construct were grown in 5 mL of minimal medium supplemented with 100 mg kanamycin per liter, and 100 mg gentamycin per liter for 48 h at 28 One milliliter of bacterial suspension was pelletized by centrifugation for 1 min in a microfuge. The pellet was resuspended in 1 mL minimal medium.

For transformation, cotyledons were excised from four-day-old, or in some cases five-day-old, seedlings so that they included approximately 2 mm of petiole at the base. Individual cotyledons with the cut surface of their petioles were immersed in diluted bacterial suspension for 1 s and immediately embedded to a depth of approximately 2 mm in co-cultivation medium, MS medium with 3% sucrose and 0.7% phytagar, enriched with ItM benzyladenine. The inoculated cotyledons were plated at a density of per plate and incubated under the same growth conditions for 48 h. After WO 99/45122 PCT/US99/04999 co-cultivation, the cotyledons then were transferred to regeneration medium, which comprised MS medium supplemented with 3% sucrose, 20 p.M benzyladenine, 0.7% phytagar, pH 5.8, 300 mg timentinin per liter, and mg kanamycin sulfate per liter.

After two to three weeks, regenerant shoots were obtained, cut, and maintained on "shoot elongation" medium (MS medium containing 3% sucrose, 300mg timentin per liter, 0.7% phytagar, and 20 mg kanamycin per liter, pH 5.8) in Magenta jars. The elongated shoots then were transferred to "rooting" medium, which comprised MS medium, 3% sucrose, 2 mg indole butyric acid per liter, 0.7% phytagar and 500 mg carbenicillin per liter. After roots emerged, the plantlets were transferred to potting mix (Redi Earth, W.R. Grace and Co. Canada Ltd.). The plants were maintained in a misting chamber (75% RH) under the same growth conditions. Two to three weeks after growth, leaf samples were taken for neomycin phosphotransferase (NPT II) assays (Moloney ef al. 1989). The results are shown in Table 2 below. The data show the number of plants that were confirmed to be transformed.

Table 2: NPT I Activity in Transformed Plants Constructs No. of NPTII NPTII No. of plants plants assayed confirmed confirmed transformed pCGmf24 47 27 23 33 37 28 18 18 pCGmf224 49 40 30 39 pCGmf225 52 37 28 34 pCGmflP2S 27 27 21 21 pCGmf2PIS

I

pCGmfl24 bean phaseolin regulatory sequences driving FaoA gene pCGmfl25 soybean oleosin regulatory sequences driving FaoA gene 3 pCGmf224 bean phaseolin regulatory sequences driving FaoB gene 4 pCGmf225 soybean oleosin regulatory sequences driving FaoB gene pCGmfl92S bean phaseolin and soybean oleosin regulatory sequences driving FaoA FaoB genes, respectively 6 pCGmf2P1S bean phaseolin and soybean oleosin regulatory sequences driving FaoB FaoA genes, respectively The fate of the transforming DNA was investigated for sixteen randomly selected transgenic lines. Southern DNA hybridization analysis showed that the FaoA and/or FaoB were integrated into the genomes of the WO 99/45122 PCT/US99/04999 transgenic lines tested.

Approximately 80% of the pmfl24 transgenic plants in which the FaoA gene is expressed from the strong bean phaseolin promoter were observed to be male sterile. Clearly high level expression of the FaoA gene from this promoter results in functional expression of the FaoA gene product which impairs seed and/or pollen development. This result was very unexpected, since it was not anticipated that the plant cells would be capable of carrying out the first step in the p-oxidation pathway in the cytosol. This result, however, provides additional applications for expressing p-oxidation genes in plants for male sterility for hybrid production or to prevent the production of seed. It was also note that in a side-by-side comparison with normal transgenic lines, the pmfl24 lines produced much higher levels of biomass, presumably due to the elimination of seed development. This phenotype therefore may be useful as a means to increase the amount of green biomass produced per acre for silage, forage, or other biomass crops.

Here, the use of an inducible promoter system or recombinase technology could be used to produce seed for planting. Seven of the sterile plants were successfully cross-pollinated with pollen from pmf225 transgenic lines and set seeds.

Northern analysis on RNA from seeds from pmf224 lines containing the phaseolin promoter-FaoB constructs showed a signal indicative of the expected 1.2 kb transcript in all the samples tested except the control.

Northern analysis on RNA from seeds from pmfl25 lines containing the weak soybean oleolsin promoter-FaoA constructs revealed a transcript of the expected size of 2.1 kb. Western blotting on 300-500 jig of protein from approximately 80% of seeds of pmfl25 plants where the FaoA gene is expressed from the relatively weak soybean oleosin promoter were inconclusive, although a weak signal was detected in one transgenic line.

Fatty Acid Analysis Given the unexpected results indicating a strong metabolic effect of expressing the FaoA gene from the strong bean phaseolin promoter in seeds, the fatty acid profile of the seeds from transgenic lines expressing the FaoA WO 99/45122 PCT/US99/04999 gene from the weak soybean oleosin promoter was analyzed. Seeds expressing only the FaoA gene or also expressing the FaoB gene from the bean phaseolin promoter were examined. The analysis was carried out as described in Millar et al., The Plant Cell 11:1889-902 (1998). Seed fatty acid methyl esters (FAMES) were prepared by placing ten seeds of B. napus in 15 x 45-mm screw capped glass tubes and heating at 80 °C in 0.75 mL of IN methanolic HCI reagent (Supelco, PA) and 10 giL of 1 mg 17:0 methyl ester (internal standard) per mL overnight. After cooling the samples, the FAMES were extracted with 0.3 mL hexane and 0.5 mL 0.9% NaCl by vortexing vigorously. The samples were allowed to stand to separate the phases, and 300 pL of the organic phase was drawn and analyzed on a Hewlett-Packard gas chromatograph.

Fatty acid profile analysis indicated the presence of an additional component or enhanced component in the lipid profile in all of the transgenic plants expressing the FaoA gene SEQ ID NO:24 which was absent from the control plants. This result again proves conclusively that the FaoA gene is being transcribed and translated and that the FaoA polypeptide SEQ ID NO: 27 is catalytically active. This peak also was observed in eleven additional transgenic plants harboring SoyP-FaoA, PhaP-FaoA-SoyP-FaoB, SoyP- FaoA-PhaP-FaoB genes and a sterile (PhaP-FaoA) plant cross-pollinated with SoyP-FaoB. These data clearly demonstrate functional expression of the FaoA gene and that even the very low levels of expression are sufficient to change the lipid profile of the seed. Adapting the methods described herein, one of skill in the art can express these genes at levels intermediate between that obtained with the phaseolin promoter and the soybean oleosin promoter using other promoters such as the Arabidopsis oleosin promoter, napin promoter, or cruciferin promoter, and can use inducible promoter systems or recombinase technologies to control when fatty acid oxidation transgenes are expressed.

Example 7: Yeast P-oxidation Multi-functional Enzyme Complex S. cerevisiae contains a P-oxidation pathway that proceeds via R- WO 99/45122 PCT/US99/04999 hydroxyacyl CoA rather than the S-3-hydroxyacyl CoA observed in bacteria and higher eukaryotes. Thefox2 gene from yeast encodes a hydratase that produces R-3-hydroxyacyl CoA from trans-2-enoyl-CoA and a dehydrogenase that utilizes R-3-hydroxyacyl-CoA to produce P-keto acyl CoAs.

Thefox2 gene (sequence shown in SEQ ID NO: 1) was isolated from S. cerevisiae genomic DNA by PCR in two pieces. Primers N-fox2b and Nbamfox2b were utilized to PCR a 1.1 kb SmaI/BamHI fragment encoding the N-terminal region of Fox2, and primers C-fox2 and C-bamfox2 were utilized to PCR a 1.6 kb BamHI/XbaI fragment encoding the C-terminal Fox2 region. The fullfox2 gene was reconstructed via subcloning in vector pTRCN.

N-fox2b fox2 tcc ccc ggg agg agg ttt tta tta tgc ctg gaa att tat cct tca aag ata gag tt (SEQ ID NO:18) N-bamfox2b fox2 aaggatccttgatgtcatttacaactacc (SEQ ID NO: 19) C-fox2 fox2 get cta gat agg gaa aga tgt atg taa g (SEQ ID C-bamfox2 fox2 tgacatcaaggatcctttt (SEQ ID NO:21) Thefoxl gene, however, does not possess a 0-ketothiolase activity and this activity must be supplied by a second transgene. Representative sources of such a gene include algae, bacteria, yeast, plants, and mammals. The bacterium Alcaligenes eutrophus possesses a broad specificity P-ketothiolase gene suitable for use in the methods described herein. It can be readily isolated using the acetoacetyl-CoA thiolase gene as a hybridization probe, as described in U.S. Patent 5,661,026 to Peoples et al. This enzyme also has been purified (Haywood et al., FEMSMicro. Lett. 52:91 (1988)), and the purified enzyme is useful for preparing antibodies or determining protein sequence information as a basis for the isolation of the gene.

WO 99/45122 PCT/US99/04999 Example 8: Plant f-Oxidation Gene The DNA sequence of the cDNA encoding P-oxidation tetrafunctional protein, shown in SEQ ID NO:4, can be isolated as described in Preisig-Muller et al., J. Biol. Chem. 269:20475-81 (1994). The equivalent gene can be isolated from other plant species including Arabidopsis, Brassica, soybean, sunflower, and corn using similar procedures or by screening genomic libraries, many of which are commercially available, for example from Clontech Laboratories Inc., Palo Alto, California, USA. A peroxisomal targeting sequence P-R-M was identified at the carboxy terminus of the protein. Constructs suitable for expressing in the plant cytosol can be prepared by PCR amplification of this gene using primers designed to delete this sequence.

EDITORIAL NOTE NO. 3071499 The sequence listing is numbered from page 1-25 The claims pages follow, starting from page number 36.

WO 99/45122 WO 9945122PCTIUS99/04999 SEQUENCE LISTING <110> Metabolix, Inc.

<120> Modification of Fatty Acid Metabolism in Plants <130> MBX 024 <140> <141> <150> 60/077,107 <151> 1998-03-06 <160> 31 <170> Patentln Ver. <210> <211> <212> <213> <220> <221> <222> <223> 1 2703

DNA

Sac charomyce s gene (2703) fox2 gene <400> 1 atgcctggaa ttaggtaagg ctaggtggca gagataaaaa gagaaaataa gctggaatat gtagatgttc tctcagaaat ggtcaagc ta aaggagggtg atgac agaaa cccttagtac gctgctggat gaccccaaga agggac aagc atcaccaaag tgcaacaaag tggtt tgcac gttgaagaaa atttatcctt caaagataga gttgttgtaa tcacgggcgc tggagggggc tgtatgcact ctttgggtgg aagccggagg ttgaaacggc taagggatgt atttgacagg ttggtagaat attattcagc ccaaatacaa acgtgttacc tctatttgac tctttggaca catatac tcc catttaacaa caaaaaaatt tcgtagtagt ggtacggtgc taaataaact agcttacgca ttcaggacat tatagctgtg tataaaagaa ttcatttgca tggctataag cattaacacc agctaaaatg cattaatgtt accacatatc acacgaaagt gctcagatgg tgaagcaatt aactcagcat acctcccaat tacgggtgca gaaggtagtt atatggtgaa agcagaggtg aactccaaag gcaaattacg ttcggcaggg aagatgacag c tatcgcgtg gcttcccctg ggcttagttg aattcaattg ttgaaacagt acgaaagtgt gagaggtctt ttaaataagt ccatatcaac gaacaaggct ggaggtggtc gtaaatgaca ggcacagcca caaaagtggt ctgcagactt actctgttaa ttgatgtact aacgtgagtt ctgcttggcc ccggtctatt gtttggcgga cgccattggc taggaccgga caaactccat c tggacaaat ggaaggaaat tctcggatta cagtgaaaat ttgggaagtc tcaaggatcc ttccagattc cgtcaatgat agtggtggat tgaaaatgga aattaacaac tgcatctgtg ttatatgcgc tggaaatttt aaccctcgcg tagatcacgt aaaaattgtt ttttgaactc tttcaatcca cacagactat taatgattta caagtcgctt tcatgcaatc tttttcagtt ccatgatgtg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 WO 99/45122 WO 9945122PCTIUS99/04999 gtcaccgaag t tggtcaata tggtttgctg ccaatattta tatggtaatt aaaactattg gcagaaacgg gcatctcaag ggaagaaggg tggcaaagaa gaaaatztgga gagtcttcta gatggattat acaagcaaag ttcgccgtca aacttcaatt atgccaagta ggtaaagccg atagcttata gtgagggatg gtaccagatt tctggcgatt acgccaattc tatggtccat c taaaggtta agaaacgtca taa ctcctctcat acgctggtat tcctgaaagt ccaaacaaaa ttggacaggc cac tggaagg Ct atgacaaa tctccccact ttattggcca gttccggtta accacatcac tggcaacctt tcaagtacac agcttaagta ttccatttat atgcaatgtt atggaac tc t ctttagttgt acgaaggatc ggaaaagagc ttgaggcgga tcaatccttt tgcatgggct atgaggagtt aagcttggaa ttgtattgga tatccaaact tttgcgtgac ccaccttttt gtctggattt caattatgcc tgccaagaga gactatattc tgttgttttg attattcgaa tgtttctatt tgatttcagt gcaagccgtg taccaaggat cacctacgag gcaagctact actgcatgga aaagacactt tggtggcttc gttcttcatc caagtttgct gatttctacg acatatcgat ttgtacatta gaaagtgaga gcaaggc tcg taacgccgct gcaataagta aaatcttttt tccacatttt attatcaata gc tgcaaaag ggaattattg tcggagaagg ttggcatctg gttggcggtg aaagagac ta cgcaacac ta caaaaagcgc tgtatcttgt aatgatccag gccacac tag gaacaatatt gctaaacctt gaaacttatg aggggcgcac gtccaaaatt aataaagatc cccacgctag ggtattagtg tttaccaatg gttgtcgttt gtaaaactat agtttcagag taaaaa tgaa cattgtcaaa ctacttctac ccgccatttt ttaatgttat aattatcaaa aagaac taca gttggtgtgg ttgaaccgga tcaacccgag actcttcaaa acaatttagg acttccaagt ctatggacaa ttaagctctg tacaagtact acattaaaac atgtacctcc ttgaagtgcc aagccgcatt ccaaagcagt cgaaagcatt ttgttttccc ttcaaacaat cgcaggcaaa agtagacatc agatgaggaa agcagtatgg ctcaggaatt aggattcagt cgctcctcat ccactttgat aaagtattct gcaaaccaga agaaattaaa c tccacagag ggagttggat acttggatgc tttgcccacg tttagtcgat cacgccgaca tgacaagaat taagaaac tc agaaaaggaa acatggaaag gtacaggtta taaatttcc t gtttgaacat aggtgatact tgatacgacc atctaaacta 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2703 <210> 2 <211> 900 <212> PRT <213> Saccharomyces <220> <221> PEPTIDE <222> (900) <223> fox2 encoded polypeptide <400> 2 Met Pro Gly Asn Leu Ser Phe Lys Asp Arg 1 5 10 Val Val Val Ile Thr Gly Ala Gly Gly Gly Leu Gly Lys Val Tyr 25 Ala Leu Ala Tyr Ala Ser Arg Gly Ala Lys Val Val Val Asn Asp 40 Leu Gly Gly Thr Leu Gly Gly Ser WO 99/45122 WO 9945122PCT/US99/04999 Gly His Ala Gly Giu Lys Leu Ile Thr Giu Tyr Lys 130 Gly Arg 145 Gly Gin Giu Thr Ile Ala His Ile 210 Tyr Leu 225 Ala Ala Ile Phe Lys Trp Gin His 290 Asn Gly Ile Asn Arg 115 Leu Ile Ala Leu Pro 195 Leu Thr Giy Asn Lys 275 Pro Ser Ile Ile Asn 100 Giu Ser Ile Asn Ala 180 Leu Lys His Phe Pro 260 Giu Tyr Lys Ala Giu Ala Phe Arg Asn Tyr 165 Lys Ala Gin Giu Phe 245 Asp Ile Gin Ala Ala 55 Val Ala 70 Thr Ala Gly Ile Ala Ser Ala Ala 135 Thr Ala 150 Ser Ala Giu Gly Arg Ser Leu Gly 215 Ser Thr 230 Gly Gin Asp Asn Ile Leu Val 120 Trp, Ser Ala Ala Arg 200 Pro Lys Leu Thr Tyr 280 Asp Leu Vai Val Tyr Lys Arg 105 Val Pro Pro Lys Lys 185 Met Glu Val Arg Tyr 265 Arg Tyr Asp Giu 90 Asp Asp Tyr Ala Met 170 Tyr Thr Lys Ser Trp 250 Thr Asp Asn Ser 75 Phe Vai Val Met Gly 155 Gly Asn Glu Ile Asn 235 Glu Pro Lys Asp Asp Val Gly Ser His Arg 140 Leu Leu Ile Asn Val 220 Ser Arg Glu Pro Leu 300 Glu Asn Arg Phe Leu 125 Ser Phe Val Asn Val 205 Pro Ile Ser Ala Phe 285 Ile Ile Giu Val Ala 110 Thr Gin Giy Gly Val 190 Leu Leu Phe Ser le 270 Asn Thr Lys Asn Asp Lys Gly Lys Asn Leu 175 Asn Pro Val Giu Gly 255 Leu Lys Lys Lys Gly Val Met Gly Phe Phe 160 Ala Ser Pro Leu Leu 240 Gin Asn Thr Ala WO 99/45122 WO 9945122PCTIUS99/04999 Lys 305 Cys Ser Asp Gly Pro 385 Leu Lys Phe Gly Gly 465 Lys Ile Lys Val Ile 545 Leu Lys Ala Lys 355 Gly Ile Asn Glu Leu 435 Ile Ala Ile Pro Leu 515 Leu Gin Pro Pro Asn 310 Val Val Val 325 Ile Trp Phe 340 Asp Pro Phe Thr Ala Ile Ile Gin Thr 390 Asn Ala Giy 405 Glu Trp Phe 420 Ser Lys Ala Ile Asn Thr Asn Tyr Ala 470 Ala Leu Giu 485 His Ala Giu 500 Ser Asn His Ala Ser Giu Leu Phe Giu 550 Giu Val Al a Ser Pro 375 Al a Ile Ala Val Thr 455 Ala Gly Thr Phe Giu 535 Val Gin Thr Arg Val 360 Asp Ile Leu Val Trp 440 Ser Ala Ala Ala Asp 520 Leu Gly Gly Gly Tyr 345 Val Ser Ser Arg Leu 425 Pro Thr Lys Lys Met 505 Ala Gin Val 315 Gly Ala Giu Asp Phe 395 Lys Val Phe Gly Ala 475 Gly Lys Gin Lys Gly Lys Ile Val 380 Gin Ser His Thr Ile 460 Ile Ile Thr Val Ile Giy Val Asn 365 Val Arg Phe Leu Lys 445 Tyr Leu Ser Giy 335 Val Leu Glu Asp Lys 415 Ser Lys Asn Phe Asn 495 Ser Leu Arg Ser Pro 525 Tyr Ser Gly Arg 540 Giy Gly Trp Cys Gly Gin Thr Arg WO 99/45122 WO 9945122PCT/US99/04999 Trp Giu Thr Ala Lys 625 Thr Val Leu His Giy 705 Gly Thr Ala Phe Giu 785 Gin Giu Ile Val 610 Tyr Ser Leu Ala Gly 690 Thr Lys Lys His Ala 770 Ala Arg Ile Asn 595 Gin Thr Lys Pro Met 675 Glu Leu Ala Lys Val 755 Val Glu Ser Lys 580 Pro Lys Thr Giu Thr 660 Asp Gin Lys Ala Leu 740 Pro Gin Ile Ser 565 Glu Ser Ala Lys Leu 645 Phe Asn Tyr Thr Leu 725 Ile Pro Asn Ser Gly Tyr Val Ser Asn Ser His Asp 630 Lys Ala Leu Phe Leu 710 Val Ala Glu Phe Thr 790 Trp, Thr Ser 615 cys Tyr Val Val Lys 695 Ala Val Tyr Lys Giu 775 Asn Asn His 585 Glu Giu 600 Ser Lys Ile Leu Thr Tyr Ile Pro 665 Asp Asn 680 Leu Cys Lys Pro Gly Gly Asn Giu 745 Giu Val 760 Val Pro Lys Asp Ile 570 Ile Ser Glu Tyr Giu 650 Phe Phe Thr Leu Phe 730 Gly Arg His Gin Lys Thr Ser Leu Asn 635 Asn Met Asn Pro Gin 715 Glu Ser Asp Gly Ala 795 Giu Thr Asp Met Asp 620 Leu Asp Gin Tyr Thr 700 Val Thr Phe Gly Lys 780 Al a Phe Ala 605 Asp Gly Pro Ala Aia 685 Met Leu Tyr Phe Lys 765 Val Ile Ser 590 Thr Gly Leu Asp Thr 670 Met Pro Asp Asp Ile 750 Arg Pro Giu 575 Arg Leu Leu Gly Phe 655 Ala Leu Ser Lys Ile 735 Arg Ala Asp Pro Asn Gin Phe Cys 640 Gin Thr Leu Asn Asn 720 Lys Gly Lys Phe Leu 800 Leu Tyr Arg Ser Gly Asp Phe Pro Leu His Ile Asp 810 Pro Thr Leu Ala Lys Ala 815 WO 99/45122 WO 9945122PCT/US99/04999 Val Lys Phe Ser Ala Lys 835 Pro 820 Thr Pro Ile Leu Gly Leu Cys Thr Leu Gly Ile 8 Giu Leu Lys Ala Leu Phe Glu His 840 Tyr Gly Pro Tyr Giu 845 Val Arg 850 Phe Thr Asn Val Phe Pro Gly Asp Leu Lys Val Lys Ala 865 Trp Lys Gin Gly Val Val Val Phe Thr Ile Asp Thr Arg Asn Vai Ile Leu Asp Asn Ala Ala 890 Val Lys Leu Ser Gin Ala 895 Lys Ser Lys Leu 900 <210> 3 <211> 2177 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> <220> <223> gene (2177) tetrafunctional beta-oxidation gene Description of Artificial Sequence <400> 3 atgggaagc a atcaccatca gatagttatg aagggaaaat ggggagcaac gcccgaaaac gccatggctt cagctcggaa tcaaaggccc ttggggttag gctcttgaaa ttagagtc tc cagtacccaa tctggccctc gatacttgca atgcaaaagg tcaaccctcc aacaagcctt tttctggtgg caaatgttag c tgcggttgc gtcatgctcg taattcctgg tagaaatgat tggatgccat tcctagagcg ttgctgaggc atcttaagca gtgc tggac t aaagcttaat aagaacggta agttaactcc gagaagagat ctttgatata aaacatatca agcgatagat aatatcaact ttttggagga gttgacgtca tgtccctccc gagaagacca taggaaaata tacaattgcc t tggaaggag tcatatcttc atggaggtgg ttgtcttttg gatgtgaagg actgcttttg attgaaatga ggacttgctt cctaccgctc acacaacggc aagccaatta gaagagttga tgggttcaca tttaacttag tgcattgatg gctgaagaat tttgcccagc gaactgatgg atgtgttatt caattgttgt gtgtactcca tcac tgatat tgggtggagg aattagggtt ttccacgtct aaggacaaga tcaacactgc gtcttcacag ctagagctca ctgttgaaac ttcagggact gttcaacaac agtagcaata cagcctgaga tacaggtgca aggaggaaag ttttgaagct gttggaggtt gcctgaactt tgttggtctc agctcattct acgtagatgg gactgacaag ggcaaagaaa gggtgtcgtc cctacattct taaggtacct WO 99/45122 WO 99/51 22PCT/US99/04999 ggagttactg ggattaatgg aaagaagtga agccgagtca aagggagttc gagaatgttt tgcatgcttg tctcgtgaca aagaatataa atgttttttc c aga ttgac a C ttgttggtt agaacttata actcgtaaag aagaaatata aagttacccg cgtgtccttg ggaatgggtt attacatcta gtggatactt ctaaacctcg ttgtccgtac atctgggttt gatc tggtat acgataagtt aaaaagggaa ttaactacga ctttgaagca ctactaatac ggattattgg agaaaacacc cctactctca gggctatttc ttggtgtggc aatcgatgct gtttc tatgt tcgagaaggc aaaaggacat ccgaaggcat tcccctccta ttcaaggttg agctgaaagg aatgtaagct caagcat ggtaccgaga agc tacagca cttgcaggct tatgactaat aagttttaaa acaaatcttt ttccacaata aacagc tgca.

agttgtcgtt ggctgcaatt caagtttgga agcagcaac t aattcctctg ctatgacaag taggaacagt tgtggagatg agcagtcaaa cggggaggac gaggaatggt gc tgttcagg catttcttta caaatcaaga ttgattctta ggcattgaca gagaaattcg gatgtagata tctgatcttg gac ttggagt caagtgattg ggaaattgta ttacttgcag atgccaatgg gccagtcagt atgcaagagg aaccgaaaag tctggtgttt atatttttcc gcagctgacc tcatgttctg cgaaacagta gtgcaactct gtcctgctca aagttgc tat gcaactatca gagtcagagc aaaagagtat tggtgataga aaaaatattg tgattggaga ttgatctgct caggttttgc aacatggggt gac cc ttc ag ttgttcaagc ataagaatgc ctgggccaaa cagttgatcc cagtggtgaa tggacattgc ggcggattct tggtggattt gagtgctccg catcatgccg tgtcggagga tgtggtactt aaacc tacaa ttctttactc ggctgttatt ccccccacat gagaataaag agatgttggg tgtcaacaga agatccctat gttgtgcgac ttttccagaa aggtgaatcc tccagagtta taagctcaca tgaagcatgt tggtgtaatg cttggatcaa ttcaagccct ggtggccatg ctattggaaa 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2177 <210> 4 <211> 725 <212> PRT <213> Cucum~ber <22 0> <221> <222> <223>

PEPTIDE

tetrafunctionai beta-oxidation protein <400> 4 Met Gly 1 Ser Asn Ala Lys Gly Arg Thr Val Met Giu Val Gly Thr Asp Gly Val Ala Ile Thr Ile Ile Pro Pro Val Asn Ser Leu Ser Phe Asp Val Leu Phe Ser Leu Arg Asp Ser Tyr Glu. Ala Leu Arg Arg Asp Asp Val Lys Ala Val Val Thr Gly Al a Lys Gly Lys Phe Ser Gly Gly Phe Asp Thr Ala Phe Gly Val Leu Gin Gly Gly WO 99/45122 WO 9945122PCTIUS99/04999 Gly Ile Al a Ser Ile 145 Ser Glu Leu Arg Ala 225 Gin Thr Glu le Leu 305 Gly Glu Phe Leu Thr 130 Pro Lys Ala Ile Pro 210 Glu Tyr Gly Phe Phe 290 Giy Leu Gin Giu Gly 115 Pro Gly Ala His Asn 195 Trp Ala Pro Val Gin 275 Phe Leu Pro Asn Ala Ala 100 Gly Gly Thr Ala Phe Gly Leu Glu 165 Ser Leu 180 Thr Ala Val His Arg Lys Asn Leu 245 Val Ser 260 Gly Leu Ala Gin Val Arg Asn Ile Ser Ile Glu Met Ile Thr Asp 90 Arg Leu Gin Gly 150 Met Gly Arg Ser Ile 230 Lys Gly Leu Arg Arg 310 Gly Lys Glu Leu 135 Thr Met Leu Arg Leu 215 Phe His Pro His Ser 295 Gin Ile Pro Val 120 Gly Gin Leu Val Trp 200 His Asn Thr Arg Ser 280 Thr Ile Ala 8 Ala 105 Al a Leu Arg Thr Asp 185 Ala Arg Leu Ile Ala 265 Asp Thr Lys Thr Val Ala Met Ala Pro Giu Leu Pro 155 Ser Lys 170 Ala Ile Leu Giu Thr Asp Ala Arg 235 Ala Cys 250 Gly Leu Thr Cys Lys Val Lys Val 315 Ala Leu 330 Ala Ile Cys His 125 Leu Gin 140 Arg Leu Pro Ile Val Pro Ile Leu 205 Lys Leu 220 Ala Gin le Asp Trp Lys Lys Ser 285 Pro Gly 300 Ala Ile Ile Leu Asp Gly 110 Ala Arg Leu Gly Val Gly Lys Gly 175 Pro Glu 190 Giu Arg Glu Ser Ala Lys Ala Val 255 Glu Ala 270 Leu Ile Val Thr Val Gly Ser Asn 335 Leu Ile Ile Leu 160 Gin Giu Arg Leu Lys 240 Giu Giu His Asp Gly 320 Tyr Met Gly Ser 325 WO 99/45122 WO 9945122PCT/US99/04999 His Val Asp Arg Thr Asn 370 Asn Tyr 385 Glu Asn Cys Pro Giu Leu Thr Ala 450 Lys Lys 465 Arg Met Gly Val Pro Met Ala Ala 530 Lys Ser 545 Ser Thr Pro Asn Val Val 355 Giu Giu Val Pro Ile 435 Ala Thr Phe Asp Gly 515 Thr Met Arg Pro Leu 340 Arg Lys Ser Ser His 420 Gly Gin Pro Phe Pro 500 Pro Ala Leu Lys Glu 580 Lys Ala Phe Phe Leu 405 Cys Giu Val Val Pro 485 Tyr Phe Ser Ile Giy 565 LeU Giu Asn Glu Lys 390 Lys Met Arg Ile Vai 470 Tyr Gin Arg Gin Pro 550 Phe Lys Val Asn Leu Gin 360 Lys Ser 375 Asp Val Gin Gin Leu Ala Ile Lys 440 Val Asp 455 Val Gly Ser Gin Ile Asp Leu Cys 520 Phe Val 535 Leu Met Tyr Val Lys Tyr 9 Asp 345 Ser Ile Asp Ile Thr 425 Ser Leu Asn Ala Arg 505 Asp Gin Gin Tyr Ile 585 Lys Arg Ser Met Phe 410 Asn Arg Leu Cys Aia 490 Ala Leu Ala Giu Asp 570 Giu Phe Vai Leu Vai 395 Ser Thr Asp Asp Thr 475 Ile Ile Vai Phe Asp 555 Lys Lys Leu Lys Leu 380 Ile Asp Ser Arg Val 460 Giy Leu Ser Gly Pro 540 Lys Asn Ala Gin Lys 365 Lys Giu Leu Thr Ile 445 Gly Phe Leu Lys Phe 525 Giu Asn Arg Arg Gly Ile Asn Met Vai Leu Val Ile 400 Lys Tyr 41i5 Asp Leu Gly His Asn Ile Val Asn 480 Giu His 495 Gly Met Val Ala Thr Tyr Giy Giu 560 Ala Gly 575 Ser Ser WO 99/45122 WO 9945122PCT/US99/04999 Gly Val Ser 595 Val Asp Pro Lys Leu Thr Lys Leu Pro Giu 605 Lys Asp Ile Val Glu 610 Met Ile Phe Phe Val Val Asn Glu Ala 620 Cys Arg Val Leu Ala 625 Glu Gly Ile Ala Lys Ala Ala Asp Asp Ile Ala Gly Met Gly Met Gly Phe 645 Pro Ser Tyr Arg Gly 650 Gly Leu Met Phe Trp Ala 655 Asp Ser Leu.

Lys Gin Tyr 675 Gly 660 Ser Asn Tyr Ile Ser Arg Leu Glu Glu Trp Ser 670 Ala Glu Arg Gly Gly Phe Phe Pro Cys Gly Tyr Leu.

685 Ala Val 690 Gin Gly Ala Thr Ser Ala Pro Gly Gly His Ala Lys Pro 700 Met Pro Leu Leu. Glu Met Ala His Phe Phe 710 Ser Pro Ala His Ile 715 Ile Val. Arg Thr <210> <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer 1 <400> gatgggccgc tccaagggtg g <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: WO 99/45122 WO 9945122PCTIUS99/04999 oligonucleotide primer- Primer 2 <400> 6 caacccgaag gtgccgccat t <210> 7 <211> 3907 <212> DNA <213> Pseudomonas putida <220> <221> gene <222> (1)..(3907) <223> faoAB gene sequence <400> 7 gatggcgttt ctggtcaatc tgccaagtcg ttcaagcaag tgcttgtggt ggacgataga cccatctttt gtatgtcatg ggcagcttag ggagatcagt atcgtcgagc ctgaacgagc atcgtcagga aacttcaagc aacgcgttcg ggcggcctgg ggcctgccgg cgcctgatcg gaagatgccc ggtgccctcg cagccgaagc gccaagggc t aagagcatcc ggctttgcca gatcaggaac gccgc cgtgC ggtacgccga gcctccaagt gaggccctca gtcgtcgagg ggccaggtga ctggccaagg ttactgaaaa tgttc tgtaa.

ccgctttgcg tgccagtgta caatcgctga aaatgactgg cggagaatgt acgccaagcc gcctgcttct tgatgattta tcaagttcga tgcgccaggc gtggcaagga tgcctgaggc aagacc tcga aaatgtgcct aagtcaagc t gctcggacaa tgaaagtggg acc tgatcaa tggaaaagct tcgtcgctgg agaaagccgc agctggccaa tcaagcgcaa tcggcgccgg tcc tgatgaa tgc ttggcaa acgccattcg ctgtggtcga aggacgatgc cgctcaagcg tttgcctccg cgacaaagcg ttctgctgcg gtcgccttgg catgaatgac aaagtcgcgt gtgcacaccc atacttccga gctgcagcga cgaaggtaaa cctcaagggt cgtcgatgcc cgtgttcatc cgaactggtc agtgccgacc ggcggccgac gggtatctac cgccatcgag ggccgtcgac gcgtgccatc caagctcaat ccaggccggc caacttcggt gacctctgtc ggccaaggcg catcatgggc ggacatccgc ccgcgtcgag cccgaccctg gaacccgaag gatcctcgct cccggaaaac gccatagaat gcggc tcggc cagaaaaggc ctcttcgcga cggcgatcac tttcgccgtg aaatcaaacg c tagcgcgc t tcggcgtgta gccatcacgg gagtccgtca atccgggccg gtcggcgccg gctggcaacc gttgccgcca taccgggtca ccgggctttg tggatcgccg gccgtggtcg agtggcgagc gccatcgagc ccgaactacc cgcgacaagg gccgagagcc catgacgaga ggcggtatcg gaggaagcca aagggccgcc tcctatggcg gtc aagcaag tccaacacct ttcgtcggca ctcctacggg ataaccctga caggcaggcc agcgcaaagc tgtcatcatg c tcgtgagca ggcgtatgaa gacgggtgtg cagttcagct ttaaggctct acaagttcaa atgcttcggt acatcaccga tggaagccaa tcaacggcat tgtccaccag gcggtaccgt ccggcaagga cccctgagct tggac tacaa agatgatggc cggccccggt ccctggaagt tgatcggctt tcgcccacga cctaccagtc ttcagctggg tgaccccggc atttcgccaa cggtac tggc ctaccatctc tgcacttctt ggcatccagg aggggtgggg gggttattca aataagccga ttttgtaccc tttgacaacg ttgagcgttt cctggggcaa tccatatcgt tgaaagtggc ccgccttacc caagggcgtg gttcgtcgac tcgcatcttc cgcgc tgggc cgccaggatc gcgcctgccg aaaccgtgcc gctgctggcc ggccaagcgc cttcgagact cgaagcgatc cgaagccgca gttcctcaac cgtgaagcag ggcggtcaaa tc tgaacgag caagatggcc tgtcgacatc ggaagtggaa catcaacctg caac ccggtg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 WO 99/45122 WO 9945122PCTIUS99/04999 cacatgatgc accaccgtgg ggctttttgg gccggtgtcg ccagcctact gccgaaggct gaggccaacc cgcggcaagc ttcgagcagc c ttgagaccg ggcctggtct tcgatcggtg taccacccga gcggtcaacg tgtcgacttc cgccgaagac cccgaaagaa gaacatcgcc cgtcagccgc gaccggtaac gatgcatggc gatgggcctg cc tgttcggc ggacgagatc cgacgaaacc caacccgaaa gtgcatgatc gatccgttcg atcgacccag gctcaacgaa caagatggat ttgctccggg gctgggtgtt cgtctga cgctggttga cctacgccaa tcaaccgcgt acttcgtgcg tgatggacgt tcccggatcg gcctgggcca cgaagaaggt gtgaagtcac tgcgttgcct acggcattgg tggccgaatt ccgccaagct agc tagagcg ggtcgcacgc atgtcggcgc gtcgaggacg cgcatggctt ctgtgcggct ggtgatgtgt gtagacccca actgcagaaa ttgcgttcgc atcccgatgc attcgcccgg ggcggtacgg gtcatgtccg atggcagtgg aaagccctca gccttcgctg gagaaggtta gcgcggattt gcgaccatgt agtgatccgt gaaaa tgggc gctgttcccg catcgacaag ggtcggcatc catgaaggac gaagaacggt cttcgatgcc tgacgaagac ggaagacggc tttccctccc cgtcgccctg gcgtgaaatg agagatttga caatgggccg acctgatcag tgatctgggg cgctgatgac cgtccatgag tcgtggtcgg acccgcacct tgctcggcaa accagctggc agggctacga aaaccaccct tcacggccgg gtcagcgtgc ccggtgtcga agcgtgcggg cgcaggccct acc tgcacgg ccggcaccct gcgtcggcct ggcgagaagt aagaacccga tactttggcg gtcatggaga gacaccggcc gagcgccgct aagggcttct accgtgc tcg atcatcaact atcgtcgaaa ttccgcggtg gccgatcagt gccaagaacg tatgagcctg ctccaagggt caagc tgc tg ctgcgtcaac cccgatcccg cgcgctgcac tggcgtggag gtccttgcat gatgcacggc ccacaaggcc cgagaacggc cgaaggcctg tacctcgtcg catggacc tc cccggcaatc cttgaccatg gcccgtgctg cggcgccatt gctcaacgtc gggccaaggt ccagtgacgt tcgtggtcaa gttttgccaa agttcggctg accacggccg cggcagtcga acgcctacga acgtgctcaa ggatgatggt ccgc tgccga gtgcgc tgcg atgccgacct gccagcgctt aatccaagag ggcatgcacc gaacgcaacg cagaccctgg cacacctctg acggccgccc cacatgggcc gccgccaagg atcacccgtg acggtcgaag ttcctgaagg gcatcgctca cagatcaccg ggtatccagc atgggctacg gccgatatcg aaagac ttga gctttgggcc atgaagcaaa atcaccactg ggcggtcgcc cgactgcccg gctggtcagc gccgatgggc tgacgtcatg cgcgttgtac aaccgacaag accgatcgtg cccgctgtgc agccgacatg ttacatcgac ggggccgctg cttcaactga acgtggtgat gcaacacccg gcaaggtcga agcagggc tg cggcgcagac aggcgatcat acgtcagcat cttccgggat agcagcagga gcaagttcaa tgttcgattt agcctgcgtt acggcgcctc cattggcggt gcccggtgcc acttcatcga aagtgc tcga acccgttcgg atggcggtac tc ttcgaacg 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3907 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer GVR471.

<400> 8 cggtacccat tgtactccca gtatcat <210> 9 WO 99/45122 WO 9945122PCT/US99/04999 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer GVR472 <400> 9 catttaaata gtagagtatt gaatatg <210> <211> 1558 <212> DNA <213> Bean Phaseolin <220> <221> promoter <222> (1)..(1558) <400> cggtacc cat tttttacctc ttgttacttt cctacaaatt ctatctttaa ttggtagaaa ataaacaaat caaatatttt taactccata atttttttct ttcaccaaac ttcccatttg taaagtaatt acttctaaaa gttgaatttg taaagtaaat acatttatgg aagtccgtaa atgggtcttg acgcaatcac atgtctaaat ctcaacccac attcattctc tgtcatccca tataaatacc tctactacta tgtactccca tatttaaagg aatttctcat tattatttgt tgtagtctaa gcataaagat tctttacctt tcaaccacgt atttttttat ttgaataaaa aatcatttgt acactacgga ttaataatag aattaattag tgactattga ataagtaatg tggactaatt ctagaattac cgcaagaaaa acaaccaac t gccatgcaaa acacaaacac ttccgccacc tgcccaaatc tctaatatca taatacccca gtatcattat ggttttccac aatc tttggt taaacatttt cattttcata ttattcttat aagaaggatt aaatctcata tcgac tgatc aaatccaatt ggtatttctg agtaactgaa ttactatatt atataattaa tttattattc tagtagagtg ttcatatatt agtgggt tgc agacaaagaa caaattagtc gcaacacgtg attgcctttt tcaatttctt tccatgcatg ctcacttctt acccaactca agtgaaagt t c taaaaattc tgaaattatc caaaccgcat ttgaaatata tcttcttcat tcccatttta ataataagtt ttaaagcaac atcattgtat aagcaagtca gatctgcttt caagatttca aatattactt tactatgttt ttagagtgtt tc ttattgct catggcactc caaagaaaaa actggctgat cttaacatgc tcttcatcat cacttcaaca ttccaaccac tcatcatcca tattcaatac ttggctctct tggtatcatt acgcttccgc aaaattttat taatttactt ataaatgttt tattttaaaa gtttcaaaag acccagtgac tttttttata tgttatgcaa tacatgcgag tatatcaaat ttttaatttt aaattgtttt accctaaacc tttacctttt tgtggtc ttt agacaaaaca caagatcgcc actttaaatg caccacaacc cacgtcaacc cttctctctt tc catc caga tactctacta cgccggtggt ctcactttac acacgatatc gaagtcccgt aattttagcg aatatacaat atatatttat taataaaatt acaactagcc caatgaaaat aattctataa acacatcttc actcaatatt aagtttaatt atagatagtt ataaactata cttggtatgt tggttcatgc gagagacaaa gcgtccatgt gctcacccat acctgtatat tgcatatgcg atataatacc gtactactac tttaaatg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1558 WO 99/45122 WO 9945122PCT[US99/04999 <210> 11 <211> 983 <212> DNA <213> Soybean oleosin <220> <221> promoter <222> (983) <400> 11 tctagataca tgtatggact tctgtcaaat gt taggtaat gttgagacat attaaaagta tgcaattaaa ttaaacttaa gattaatttt ttcatttact gataccacca aataaaaatg ttaatttttc caactcatca cctcccccat cttcacttgc aacttcacct tccatttctt atggc tcgag gcctattgtt ggacattaat tgactattgt aataataatc accagaagaa tgttatattt agtattgtct aatttatgta acaccaacac taaatagtac tgatgtactt tgcatcatgt cacactcccc agaacttagt taaccattta aatataatcc gaatccatac gttgaaataa gtaaagtgac gtgctatttc gctaaacatc ctaaatctac aaggaaaagg ttaatgagaa atgtgatttc caccaccacc attattctcc aacccagggc acgtgtcatc atctctctaa tctctgttgc atg tcttattcaa atagagacat agtaac ttat ttatagaatc aaaaagatc t atcaaaatat attaccatac acgaaactta ttaaagtttt aataagtgag tgcgaaac tg ttgcctgtca aaactgaaac atccagcaac cacacacata atcatcatca ~Attgcaattg tccccactcg gcatcaattt catatttgta gataaaatat taatcattag ttaagactat aaacttaaat attcactaat gtaaactccg tacgtatctc ttatttatgt atgttcctca tccacttttg cccccaacta tcttcattag cccagatctc tatacttgta aaaacaatca taacaaagat ttaataaaac attcaatttt ac atagagaa acaattgtat ttatgattat attgattgaa aattgtcctt gcccccagct tgcaaagccc ctatataact acaataattc tgttagccct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 983 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer JA408 <400> 12 tctagataca tccatttctt aatataatcc tcttattc <210> <211> <212> <213> <220> <223> 13

DNA

Artificial Sequence Description of Artificial Sequence: oligonucleotide primer- npl WO 99/45122 PCT/US99/04999 <400> 13 catttaaatg gttaaggtga aggtagggct <210> 14 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer np2 <400> 14 aagcttaaaa tgatttacga aggtaaagcc <210> <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer np3 <400> attgctttca gttgaagcgc tg 22 <210> 16 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer np4 <400> 16 aagcttaaaa tgagcctgaa tccaagagac <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer WO 99/45122 WO 9945122PCTIUS99/04999 <400> 17 aagctttcag acgcgttcga agacagtg 28 <210> 18 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer N-f ox2b <400> 18 tcccccggga ggaggttttt attatgcctg gaaatttatc cttcaaagat agagtt 56 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer N-banifox2b <400> 19 aaggatcctt gatgtcattt acaactacc 29 <210> <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer C-f ox2 <400> gctctagata gggaaagatg tatgtaag 28 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer C-bamfox2 WO 99/45122 PTU9/49 PCTIUS99/04999 <400> 21 tgacatcaag gatcctttt <210> <211> <212> <213> 22 39

DNA

Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide Primer- Primer GVR396 <400> 22 gatttaaatg caagcttaaa taagtatgaa ctaaaatgc <210> 23 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer GVR397 <400> 23 cggtacctta gttggtaggg tgcta <210> 24 <211> 2162 <212> DNA <213> Pseudomonas putida <220> <221> gene <222> (1)..(2162) <223> FaoA gene sequence <400> 24 aagcttaaat tcgagctcaa acgagc tgcg tcaggagtgg tcaagc tgcc cgttcgaaga gcctggaaat tgccggaagt tgatcggctc gatttacgaa gttcgacctc ccaggccgtc caaggacgtg tgaggccgaa cctcgaagtg gtgcc tggcg caagctgggt ggacaacgcc ggtaaagcca aagggtgagt gatgccatcc ttcatcgtcg ctggtcgctg ccgaccgttg gccgac tacc atctacccgg atcgagtgga tcacggttaa ccgtcaacaa gggccgatgc gcgccgacat gcaacctgga ccgccatcaa gggtcatgtc gctttggcgg tcgccgccgg ggc tc ttgaa gttcaaccgc ttcggtcaag caccgagttc agccaatcgc cggcatcgcg caccagcgcc taccgtgcgc caaggaaaac agtggcatcg c ttaccctga ggcgtgatcg gtcgacaact atcttcaacg c tgggcggcg aggatcggcc ctgccgcgcc cgtgccgaag WO 99/45122 WO 99/5 122PCT/US99/04999 atgccctgaa agtgggggcc CCCtcgacct gatcaagcgt cgaagc tgga aaagc tcaag agggcttcgt cgctggccag gcatccagaa agccgccaac ttgccaagct ggccaagacc aggaactcaa gcgcaaggcc ccgtgctcgg cgccggcatc cgccgatcct gatgaaggac ccaagttgct tggcaaccgc ccctcaacgc cattcgcccg tcgaggctgt ggtcgagaac aggtgaagga cgatgcgatc ccaaggcgct caagcgcccg tgatgccgct ggttgaagtg ccgtggccta cgccaagaaa ttttggtcaa ccgcgtgctg gtgtcgactt cgtgcgcatc cctacttgat ggacgtggtc aaggcttccc ggatcgcatg ccaaccgcct gggccagaag gcaagccgaa gaaggtcttc agcagcgtga agtcactgac agaccgtgcg ttgcctggaa tggtctacgg cattggtttc tcggtgtggc cgaattcgtc acccgaccgc caagctgcgt tt gtcgacgccg gccatcagtg ctcaatgcca gccggcccga ttcggtcgcg tctgtcgccg aaggcgcatg atgggcggcg atccgcgagg gtcgagaagg accctgtcct ccgaaggtca ctcgcttcca gaaaacttcg atccgtggcg atgggcaaga ttcccgtact gacaaggtca ggcatcgaca aaggacgagc aacggtaagg gatgccaccg gaagacatca gacggcatcg CCtcccttcc gccctggccg gaaa tggcca tggtcgc ccc gcgagctgga tcgagcagat ac tac ccggc acaaggccct agagcctgat acgagatcgc gtatcgccta aagccattca gccgcctgac atggcgatti agcaagcggt acacctctac tcggcatgca agaagtccag acccgatcgt ttggcggttt tggagaagtt ccggccacca gccgctcggC gcttctacgc tgctcgacgt tcaactggat tcgaaaccgc gcggtggtgc atcagtatgc agaacggcca tgagctgctg ctacaaggcc gatggccttc cccggtcgaa ggaagtcgaa cggc ttgttc ccacgacgtg ccagtcggcg gc tgggtc tg cccggccaag cgccaatgtc ac tggcggaa catctccatc cttcttcaac tgacgtggcg ggtcaacgac tgcc aagc tg cggc tggccg cggccgtgac agtcgacgcg ctacgaaacc gc tcaaaccg gatggtcccg tgccgaagcc gctgcgttac cgacctgggg gcgcttcttc c tggccggtg aagcgccagc gagactgcca gcgatcaaga gccgcaggc t c tcaacgatc aagcaggccg gtcaaaggta aacgaggcc t atggccgagg gacatcgtcg gtggaaggcc aacc tgc tgg ccggtgcaca gtcgccacca tgcccgggct gtcagcgccg atgggcccag gtcatggccg ttgtacgagg gacaagcgcg atcgtgttcg ctgtgccttg gacatgggcc atcgactcga ccgctgtacc aac tgaaagc 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2162 <210> <211> 1190 <212> DNA <213> Pseudomonas putida <220> <221> gene <222> (1190) <223> FaoB gene sequence <400> aagcttaaat tgggccgctc tgatcagcaa tc tggggc tg tgatgacccc ccatgagcgc tggtcggtgg cgcacctgtc gagcctgaat caagggtggc gctgctggaa cgtcaaccag gatcccgcac gctgcacacg cgtggagcac cttgcatgcc ccaagagacg atgcaccgca cgcaacggca accctggagc acctctgcgg gccgcccagg atgggccacg gccaaggctt tggtgattgt acacccgcgc aggtcgaccc agggc tggaa cgcagaccgt cgatcatgac tcagcatgat ccgggatgat cgacttcggt cgaagacatg gaaagaagtc catcgcccgc cagccgcctg cggtaacggt gcatggcgta gggcctgact cgcacgccaa tcggcgcacc gaggacgtga atggc ttcgc tgcggctcgt gatgtgttcg gaccccaacc gcagaaatgc WO 99/45122 WO 9945122PCTJIUS99/04999 tcggcaagat agctggccca gctacgacga ccaccctcga cggccggtac agcgtgccat gtgtcgaccc gtgcgggctt aggccctgcc tgcacggcgg gcaccctgct tcggcc tggg gcacggcatc caaggccacg gaacggcttc aggcctggca C tcgtcgcag ggacctcggt ggcaatcatg gacCa tggc C cgtgctgaaa cgccattgct caacgtcatg ccaaggtatc acccgtgagc gtcgaaggca ctgaaggtgt tcgctcaagc atcaccgacg atccagccat ggctacggcc gatatcgact gacttgaaag ttgggccacc aagcaaaatg accactgtct agcaggacc t agttcaagga tCgatttcga ctgcgttcaa gcgcctcgtg tggcggtgat cggtgccatc tcatcgagct tgctcgacaa cgttcggttg gcggtacgct tcgaacgcgt gttcggcttg cgttcgcacc 540 cgagatcatc ccgatgcagg 600 cgaaaccatt cccgaaaggc catgatcgtc ccgttcgatg gacccagaaa caacgaagcc gatggatgag C tccggggcg gggtgttgcg ctgaaagctt cgcccggaaa ggtacggtc a atgtccggtc gcagtggccg gccctcaagc ttcgctgcgc aaggttaacc cggatttccg accatgtgcg 660 720 780 840 900 960 1020 1080 1140 1190 <210> 26 <211> 391 <212> PRT <213> Pseudomonas putida <220> <221> <222> <223>

PEPTIDE

.(391) FaoB amino acid sequence <400> 26 Met Ser Leu Asn Pro Arg Asp Val Val 1 5 Ile 10 Val Asp Phe Gly Arg Thr Pro Met Gly Asp Met Ser Ser Lys Gly Gly His Arg Asn Thr Arg Ala Glu Asn Gly Lys Ala His Leu Ile Ser Lys Leu Leu Glu Val Asp so Pro Lys Giu Val Asp Val Ile Trp Gly Cys Val Asn Gin Thr Leu Glu Gin Gly Trp Asn Ile Ala Arg Ala Ser Leu Met Pro Ile Pro His Ser Ala Ala Gin Thr Val Ser Arg Leu Cys Gly Ser Ser Met Ala Leu His Thr Ala Ala Gin Ala Ile Met Thr Gly Asn Gly Asp Val Phe Val Val 115 Gly Val Giu His Met 125 Gly His Val WO 99/45122 WO 9945122PCT/US99/04999 Ser Al a 145 Met His Ile Asp Ser 225 Thr Gly Ser Val Asp 305 Pro Asn Gly Met 130 Lys His Gin Ile Phe 210 Leu Ser Gin Met Pro 290 le Val Leu Ala Met Al a Gly Leu Pro 195 Asp Lys Ser Arg Ala 275 Ser Asp Leu His Arg 355 His Ser Ile Ala 180 Met Glu Pro Gin Ala 260 Val Gly Gly Thr 165 His Gin Thr Al a Ile 245 Met Al a Val Met 150 Arg Lys Asp 135 Met Giu Ala Gly Tyr Ile Arg 215 Phe Asn 230 Thr Asp Asp Leu Gly Val Lys Ala 295 Giu Leu 310 Leu Lys Ala Ile Pro Asn Gly Leu Gin Gin Thr Val 185 Asp Glu 200 Pro Glu Pro Lys Gly Ala Gly Ile 265 Asp Pro 280 Leu Lys Asn Glu Val Leu Ala Leu 345 Leu Leu 360 Met Cys Pro Thr Asp 170 Giu Asn Thr Gly Ser 250 Gin Ala Arg Ala Asp 330 Gly Asn Val His Ala 155 Leu Gly Gly Thr Gly 235 Cys Pro Ile Ala Phe 315 Lys His Val Gly Leu 140 Glu Phe Lys Phe Leu 220 Thr Met Leu Met Gly 300 Al a Met Pro Met Leu 380 Ser Met Gly Phe Leu 205 Giu Val Ile Al a Gly 285 Leu Ala Asp Phe Lys 365 Gly Leu His Leu Gly Leu Arg 175 Lys Asp 190 Lvs Val Thr Gin Phe Ile Lys Asp 325 Gly Gly Giy Leu Ala Thr Ala Gly 240 Val Met Ser 255 Val Ile Arg 270 Tyr Gly Pro Thr Met Ala Gin Ala Leu 320 Giu Lys Val 335 Giy Cys Ser 350 Gin Asn Gly Gin Gly Ile Ser Gly Thr Val Ala Thr Gly Thr Leu Gly WO 99/45122 WO 9945122PCT/US99/04999 Thr Thr Val Phe Glu Arg Val 385 390 <210> 27 <211> 1244 <212> DNA <213> Bean Phaseolin <220> <221> terminator <222> (1244) <400> 27 gatttaaatg gagagcatgg tcttctatga gttctatgat tgcttcaaat agcattgtga tctccattta taacaattat attatactta tttagttgat gatcatcctt tgagttggtt aatatatgta ataaatctat acatatttga.

tttt tatcgg tacaaccaac tttttaattt ccttttagca tcagcaaaga aaccccaaaa caagcttaaa aatattgtat ataaacaaag aaatttcctc agtacaaaaa acgagacata tatattatat aaagagagaa tccacttatt atgtatatga aaagtgggtc tgataaaata tataaattta acaatcgttt c tttttggtt caaggaaata ttccacagga.

gggttgtctt gtagagcaat ataaataaaa acaagtttcc taagtatgaa ccgaccatgt gatgttatga ttattattat caaatgtgta agtgttaaga attacccact gtttgtatcc taatgtcttt aagggtac ta tatttaattt ttgaaggatt ttataatata agccttgctg atttaacaaa aaattaaatt aggtcaggtc gtttgctgca ggttgaccgt taaaatgaga tagcacccta ctaaaatgca aacagtataa tatattaaca aaatcatc tg ctataagact agacataaca tatgtattat atttatatat ataaggtttg tttgaactct tattgcttct taaaataata acatttatct gacgactctc ttattattta aggagggaca ggggacaaca taatttatgc gtgcttagct cacttcaggg ccaactaagg tgtaggtgta taac tgagc t ctctatctat aatcgtgacg ttctaaacaa attataatgg attaggatgt tatatactac atccatgata cttactctgt tacagataaa ataaataata ataaaaaagt aattatttaa acac tatatg atggtgtgtc aaaaaacagg agtaaaacac tcttttattt atgtttcaac tacc agagctcatg ccatctcact gcaccttatt gc ttatggaa ttctaacttt aagaagtttg taaggagaca ccatttatat tttctaatat ataaaggttg aaaaaaatta aataacatat aaatattgtc acgagagtaa aaattttttt ccaatcctta caagggaaat tacacataac tattttttta ccttatacaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1244 <210> 28 <211> 225 <212> DNA <213> Soybean oleosin <220> <221> terminator <222> <400> 28 aagcttacgt gatgagtatt aatgtgttgt tatgaactta tgatgttggt ttatgtgggg aaataaatga tgtatgtacc tcttcttgcc tatgtagtag gtttgggtgt tttgttgtct 120 WO 99/45122 WO 9945122PCT[US99/04999 agctttgctt atttagtaat tagtagaagg gatgttcgtt cgtgtctcat aaaaaggggt 180 actaccactc tggcaatgtg atttgtattt gatgaattgt ctaga 225 <210> 29 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer JA410 <400> 29 aagcttacgt gatgagtatt aatgtgttgt tatg 34 <210> <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: oligonucleotide primer- Primer JA411 <400> tctagacaat tcatcaaata caaatcacat tgcc 34 <210> 31 <211> 715 <212> PRT <213> Pseudomonas putida <220> <221> PEPTIDE <222> <223> FaoA amino acid sequence <400> 31 Met Ile Tyr Glu Gly Lys Ala Ile Thr Val Lys Ala Leu. Glu Ser Gly 1 5 10 Ile Val Giu Leu Lys Phe Asp Leu Lys Gly Glu Ser Val Asn Lys Phe 25 Asn Arg Leu Thr Leu. Asn Glu Leu Arg Gin Ala Val Asp Ala Ile Arg 40 Ala Asp Ala Ser Val Lys Gly Val Ile Val Arg Ser Gly Lys Asp Val Phe Pro Asn Ile Val Ile 145 Ser Giu Leu Glu Leu 225 Val Lys Val Ser WO 99/45122 s0 Ile Val G Glu Ala G3 Ala Phe G] Ala Leu G] 115 Met Ser T1 130 Tyr Pro GI Asp Asn A] Asp Ala Lt Leu Leu A] 195 Leu Asp T 210 Asn Ala 11 Ala Gly G] Ser Ile G] 26 Glu Ala A] 275 Leu Ile G] 290 Ly Lu Lu '0 -n rn PCTIUS99/04999 Ala Leu Asp Gly Ser Phe Ile 165 Lys Gly Lys Glu Ala 245 Lys Gly Leu Asp 70 Val Leu Gly Ala Gly 150 Glu Val Ala Ala Gin 230 Gly Al a Phe Phe Glu Phe Asn Pro 105 Met Gly Val Al a Val 185 Leu Gin Al a Tyr Phe 265 Leu Leu Thr Cys Leu Arg Ala 170 Asp Ile Pro Phe Pro 250 Gly Ala Val 75 Giu Val Leu Pro Leu 155 Gly Ala Lys Lys Glu 235 Ala Arg Lys Asp Ala Ala Ala Glu 140 Pro Lys Val Arg Leu 220 Thr Pro Asp Thr Leu 300 Asn Asn Ala Ala 125 Val Arg Glu Val Ala 205 Glu Ala Val Lys Ser 285 Lys Leu Phe Gly Arg Gly Gly 160 Ala Glu Gly Lys Phe 240 Ile Glu Glu Ala Asp Gin Glu Lys Ala His Asp Glu Ile Ala Lys la Hs Ap Gi lieAla Asp Val Lys'Gin Ala Ala Val Leu WO 99/45122 WO 9945122PCT/US99/04999 Gly Ala Gly Ile Gly Gly Arg Thr 385 Val Gly Ser Gly Ile 465 Tyr Gly Lys Glu Gly 545 Thr Pro Ile 340 Leu Leu 370 Leu Val Gin Ile Met 450 Arg Al a Phe Leu Lys 530 Ile Asn Glu 355 Thr Pro Ser Tyr Glu Asn Val Lys 420 Asn Leu 435 His Phe Giy Giu Lys Lys Leu Val 500 Vai Ser 515 Phe Giy Asp Thr Met Gly 325 Leu Met Ala Ser Ala Lys Gly Asp 390 Pro Lys 405 Asp Asp Leu Ala Phe Asn Lys Ser 470 Met Gly 485 Asn Arg Ala Gly Trp Pro Gly His 550 Gly Lys Lys Met 375 Phe Val Al a Lys Pro 455 Ser Lys Vai Val Met 535 His Gly Asp Leu 360 Al a Ala Lys Ile Ala 440 Val Asp Asn Leu Asp 520 Gly Gly Arg Ile Ile 345 Leu Glu Asn Gin Leu 425 Leu His Val Pro Phe 505 Phe Pro Arg Arg Ala 330 Arg Gly Ala Val Al a 410 Ala Lys Met Ala Ile 490 Pro Val Al a Asp Ser Tyr Glu Asn Leu Asp 395 Val Ser Arg Met Val 475 Val Tyr Arg Tyr Val 555 Ala Glu Arg Asn 380 Ile Leu Asn Pro Pro 460 Ala Val Phe Ile Leu 540 Met Al a Val 365 Al a Val Ala Thr Glu 445 Leu Thr Asn Gly Asp 525 Met Al a Gin Ser Ala Val 335 Gin Lys Arg Glu Val 415 Thr Phe Glu Val Cys 495 Phe Val Val Gly Lys Leu Gly Pro Ala 400 Glu Ile Val Val Ala 480 Pro Ala Met Val Phe 560 Tyr Pro Asp Arg Met Lys Asp Glu Val Asp Ala Leu WO 99/45122 WO 9945122PCT/US99/04999 Glu Glu Leu Glu 625 Arg Gly Arg Gin Ala Asn Thr Asn Asp 610 Asp 595 Val Ile Arg 580 Lys Leu Ile Leu Arg Lys Asn Asp 645 Gly Ser Leu Gin Lys Lys Pro 600 Ile Val Cys Leu Glu Leu Tyr Tyr 690 Val Ile 675 Al a Met Val Phe Val 680 Leu Asn 585 Lys Phe Val Glu Pro 665 Ala Tyr Lys Val Gin Leu 635 Ala Phe Phe Pro Gly Phe Arg 620 Cys Ala Arg Val Thr 700 Phe Asp 605 Giu Leu Glu Gly Ala 685 Ala Tyr Ala Tyr 590 Ala Thr Val Val Thr Asp Giu Thr Val 640 Ala Asp Met 655 Gly Ala Leu 670 Leu Ala Asp Lys Leu Arg Giu Met Ala Lys Asn Gly Gin Arg Phe Phe Asn

Claims (44)

1. A method for manipulating the metabolism of a plant, comprising introducing into a plant cell at least one gene encoding a fatty acid oxidation enzyme which is active either in the cytosol or plastids of the plant, wherein the gene encodes an enzyme selected from the group consisting of enzymes converting acyl CoA thioester to trans-2-enoyl-CoA, hydrating trans-2-enoyl-CoA to R-3-hydroxyacyl CoA, hydrating trans-2- enoyl-CoA to S-3-hydroxyacyl CoA, epimerizing S-3-hydroxyacyl CoA to R-3-hydroxyacyl CoA, oxidizing 3-hydroxyacyl CoA to form p-keto acyl CoA, and thiolyzing P-keto acyl CoA to yield acetyl CoA.

2. A method according to claim 1 wherein the fatty acid oxidation enzymes are :i* expressed from genes selected from the group consisting of bacterial, yeast, fungal, plant, and mammalian. S.

3. A method according to claim 1 or claim 2 wherein the fatty acid oxidation enzymes are expressed from genes from bacteria selected from the group consisting of Escherichia, Pseudomonads, Alcaligenes, and Coryneform.

4. A method according to claim 3 wherein the genes are Pseudomonas putida faoAB.

5. A method according to any one of claims 1 to 4 further comprising expressing genes of bacterial, fungal, yeast, plant or animal origin encoding enzymes selected from the group consisting of polyhydroxyalkanoate synthases, acetoacetyl-CoA reductases, P ketoacyl-CoA thiolases, and enoyl-CoA hydratases, wherein the enzymes encoded by these genes are directed to the cytosol or plastids other than the peroxisomes or glyoxisomes, or mitochondria of the plant.

6. A DNA construct for use in a method of manipulating the metabolism of a plant cell comprising, in phase, a promoter region functional in a plant; a structural DNA sequence encoding at least one fatty acid oxidation enzyme selected from the group consisting of enzymes converting acyl CoA thioester to trans-2- enoyl-CoA, hydrating trans-2-enoyl-CoA to R-3-hydroxyacyl CoA, hydrating trans-2-enoyl- SI36 IT 36 CoA to S-3-hydroxyacyl CoA, epimerizing S-3-hydroxyacyl CoA to R-3-hydroxyacyl CoA, oxidizing 3-hydroxyacyl CoA to form P-keto acyl CoA, and thiolyzing P-keto acyl CoA to yield acetyl CoA; and a 3'nontranslated region of a gene naturally expressed in a plant, wherein the nontranslated region encodes a signal sequence for polyadenylation of mRNA.

7. A DNA construct according to claim 6 wherein the promoter is a seed specific promoter.

8. A DNA construct according to claim 7 wherein the seed specific promoter is selected from the group consisting of napin promoter, phaseolin promoter, oleosin promoter, 2S albumin promoter, zein promoter, 1-conglycinin promoter, acyl-carrier protein promoter, and fatty acid desaturase promoter. 15

9. A DNA construct according to claim 7 or claim 8 wherein the promoter is a constitutive promoter. *e A DNA construct according to any one of claims 6 to 8 wherein the promoter is selected from the group consisting of CaMV 35S promoter, enhanced CaMV 35S promoter, and ubiquitin promoter.

S*

11. A method for enhancing the biological production of polyhydroxyalkanoates in a transgenic plant, comprising expressing genes encoding heterologous fatty acid oxidation enzymes selected from the group consisting of enzymes converting acyl CoA thioester to trans-2-enoyl-CoA, hydrating trans-2-enoyl-CoA to R-3-hydroxyacyl CoA, hydrating trans-2- enoyl-CoA to S-3-hydroxyacyl CoA, epimerizing S-3-hydroxyacyl CoA to R-3-hydroxyacyl CoA, oxidizing 3-hydroxyacyl CoA to form 3-keto acyl CoA, and thiolyzing p-keto acyl CoA to yield acetyl CoA, in the cytosol or plastids other than the peroxisomes, glyoxisomes or mitochondria of the plant.

12. A method according to claim 11 wherein the transgenic plant is selected from the group consisting of Brassica, maize, soybean, cottonseed, sunflower, palm, coconut, safflower, peanut, mustards, flax, tobacco, and alfalfa.

13. A method according to claims 11 or 12 wherein the R-3-hydroxyacyl CoAs are polymerised by a polyhydroxyalkanoate synthase to form polyhydroxyalkanoates.

14. A transgenic plant or part thereof comprising heterologous genes encoding fatty acid oxidation enzymes selected from the group consisting of enzymes converting acyl CoA thioester to trans-2-enoyl-CoA, hydrating trans-2- enoyl-CoA to R-3-hydroxyacyl CoA, hydrating trans-2-enoyl-CoA to S-3-hydroxyacyl CoA, epimerizing S-3-hydroxyacyl CoA to R-3-hydroxyacyl CoA, oxidizing 3-hydroxyacyl CoA to form P-keto acyl CoA, and thiolyzing p-keto acyl CoA to yield acetyl CoA, in the cytosol or plastids other than the peroxisomes, glyoxisomes or mitochondria of the plant.

A plant or part thereof according to claim 14 wherein the fatty acid oxidation enzymes are expressed from genes selected from the group consisting of bacterial, yeast, •fungal, plant, and mammalian. 1

16. A plant or part thereof according to claim 14 or claim 15 wherein the fatty acid oxidation enzymes are expressed from genes from bacteria selected from the group consisting of Escherichia, Pseudomonas, Alcaligenes, and Coryneform.

17. A plant or part thereof according to any one of claims 14 to 16 wherein the S*o. genes are Pseudomonas putida faoAB.

18. A plant or part thereof according to any one of claims 14 to 17 further comprising genes encoding enzymes selected from the group consisting of 25 polyhydroxyalkanoate synthases, acetoacetyl-CoA reductases, P-ketoacyl- CoA thiolases, Sand enoyl-CoA hydratases.

19. A plant or part thereof according to any one of claims 14 to 18 wherein the plant is selected from the group consisting of Brassica, maize, soybean, cottonseed, sunflower, palm, coconut, safflower, peanut, mustards, flax, tobacco, and alfalfa.

A plant or part thereof according to any one of claims 14 to 19 comprising a DNA construct comprising, in phase, a promoter region functional in a plant; a structural DNA sequence encoding at least one fatty acid oxidation enzyme activity selected from the group consisting of enzymes converting acyl CoA thioester to trans- 2-enoyl-CoA, hydrating trans-2-enoyl-CoA to R-3-hydroxyacyl CoA, hydrating trans-2-enoyl- CoA to S-3-hydroxyacyl CoA, epimerizing S-3-hydroxyacyl CoA to R-3-hydroxyacyl CoA, oxidizing 3-hydroxyacyl CoA to form p-keto acyl CoA, and thiolyzing p-keto acyl CoA to yield acetyl CoA; and a 3'nontranslated region of a gene naturally expressed in a plant, wherein the nontranslated region encodes a signal sequence for polyadenylation of mRNA.

21. A plant or part thereof according to claim 20 wherein the promoter is a seed specific promoter.

22. A plant or part thereof according to claim 21 wherein the seed specific promoter is selected from the group consisting of napin promoter, phaseolin promoter, 15 oleosin promoter, 2S albumin promoter, zein promoter, P-conglycinin promoter, acyl-carrier protein promoter, and fatty acid desaturase promoter.

23. A plant or part thereof according to any one of claims 20 to 22 wherein the promoter is a constitutive promoter.

24. A plant or part thereof according to any one of claims 20 to 23 wherein the promoter is selected from the group consisting of CaMV 35S promoter, enhanced CaMV promoter, and ubiquitin promoter S 25

25. A method of preventing or suppressing seed production in a plant, comprising oo expressing heterologous genes encoding fatty acid oxidation enzymes selected from the group consisting of enzymes converting acyl CoA thioester to trans-2-enoyl-CoA, hydrating trans-2-enoyl-CoA to R-3-hydroxyacyl CoA, hydrating trans-2-enoyl-CoA to S-3- hydroxyacyl CoA, epimerizing S-3-hydroxyacyl CoA to R-3-hydroxyacyl CoA, oxidizing 3- hydroxyacyl CoA to form p-keto acyl CoA, and thiolyzing p-keto acyl CoA to yield acetyl CoA, in the cytosol or plastids other than the peroxisomes, glyoxisomes or mitochondria of the plant.

26. A method for manipulating the metabolism of a plant, comprising introducing 35'k into a plant cell a gene encoding an enzyme, which enzyme is a hydratase.

27. A method according to claim 26 wherein the enzyme is an enoyl-CoA hydratase.

28. A method according to claims 26 or 27 wherein the gene encoding the enzyme is engineered to be expressed and active in the plant cell excluding the peroxisomes.

29. A method according to claim 28 wherein the gene encoding the enzyme is engineered to be expressed and active in the plant cell cytosol and/or plastids.

A method according to any one of claims 26 to 29 wherein the enzyme hydrates trans-2-enoyl-CoA to 3-hydroxyacyl CoA.

31. A method according to claim 30 wherein the 3-hydroxyacyl CoA takes the R and/or S forms wherein in the latter case, the S form is subsequently transformed to the R form.

32. A method according to any one of claims 26 to 31 wherein the 3-hydroxyacyl CoAs are polymerised by a polyhydroxyalkanoate synthase to form polyhydroxyalkanoates.

33. A method according to any one of claims 26 to 32 wherein the gene expressing the hydratase is selected from genes from the group consisting of bacterial, yeast, fungal, plant and mammalian. 2

34. A method according to any one of claims 26 to 33 wherein the gene expressing the hydratase is selected from genes from bacteria from the group of Escherichia, Pseudomonads, Alcaligenes and Coryneform.

35. A method according to any one of claims 26 to 34 wherein the gene expressing the hydratase is from Pseudomonas putida.

36. Polyhydroxyalkanoates obtainable by a method according to claim 13 or claim 32. IA PP 'y

37. A plant obtainable by a method according to any one of claims 26 to

38. A DNA construct for use in a method of manipulating the metabolism of a plant cell comprising, in phase, a promoter region functional in a plant; a structural DNA sequence encoding an enzyme capable of hydrating trans- 2-enoyl-CoA to R-3-hydroxyacyl CoA and/or hydrating trans-2-enoyl-CoA to S-3-hydroxyacyl CoA, a 3'nontranslated region of a gene naturally expressed in a plant, wherein the nontranslated region encodes a signal sequence for polyadenylation of mRNA.

39. A transgenic plant or part thereof comprising heterologous genes encoding an enzyme capable of hydrating trans-2-enoyl-CoA to R-3-hydroxyacyl CoA and/or hydrating trans-2-enoyl-CoA to S-3-hydroxyacyl CoA in the cytosol or plastids other than the peroxisomes, glyoxisomes or mitochondria of the plant.

40. A method for enhancing the biological production of polyhydroxyalkanoates in a transgenic plant, comprising expressing genes encoding an enzyme capable of hydrating trans-2-enoyl-CoA to R-3-hydroxyacyl CoA and/or hydrating trans-2-enoyl-CoA to S-3- hydroxyacyl CoA, in the cytosol or plastids other than the peroxisomes, glyoxisomes or mitochondria of the plant.

41. A method of preventing or suppressing seed production in a plant, comprising expressing a gene encoding a hydratase, in the cytosol or plastids other than the 25 peroxisomes, glyoxisomes or mitochondria of the plant.

42. A method for manipulating the metabolism of a plant substantially as hereinbefore described with reference to any one of the examples 1 to 8

43. A plant substantially as hereinbefore described with reference to any one of the examples 1 to 8.

44. A DNA construct substantially as hereinbefore described with reference to any one of the examples 1 to 8. A method for enhancing the biological production of polyhydroxyalkanoates in a transgenic plant substantially as hereinbefore described with reference to any one of the examples 1 to 8. DATED THIS TWENTIETH DAY OF MAY 2002 METABOLIX, INC BY PIZZEYS PATENT AND TRADE MARK ATTORNEYS 9 *o