WO2013003545A1

WO2013003545A1 - Tuning of fusel alcohol by-products during isobutanol production by recombinant microorganisms

Info

Publication number: WO2013003545A1
Application number: PCT/US2012/044563
Authority: WO
Inventors: Peter Meinhold; Matthew W. Peters; Andrew C. Hawkins; Reid M. FELDMAN; Jun Urano
Original assignee: Gevo, Inc.
Priority date: 2011-06-28
Filing date: 2012-06-28
Publication date: 2013-01-03

Abstract

The disclosure relates to recombinant microorganisms comprising biosynthetic pathways and methods of using said recombinant microorganisms to produce various beneficial metabolites. In various aspects, the recombinant microorganisms may further comprise one or more modifications resulting in the reduction or elimination of a 2-ketoisovalerate-derived by-product such as 3-methyl-1-butanol. The recombinant microorganisms may be microorganisms of the Saccharomyces dade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

Description

TUNING OF FUSEL ALCOHOL BY-PRODUCTS DURING ISOBUTANOL PRODUCTION BY RECOMBINANT WHCROORGAN!SW!S

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 81/502,171 , filed June 28, 201 1 , which is herein incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

[0002] Recombinant microorganisms and methods of producing such organisms are provided. Also provided are methods of producing beneficial metabolites including fuels, chemicals, and amino acids by contacting a suitable substrate with recombinant microorganisms and enzymatic preparations therefrom.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0003] The contents of the text file submitted electronically herewith are incorporated by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: GEVO_058_01 WO_SeqList_ST25.txt, date recorded: June 22, 2012, file size: 65 kilobytes).

BACKGROUND

[0004] The ability of microorganisms to convert sugars to beneficial metabolites including fuels, chemicals, and amino acids has been widely described in the literature in recent years. See, e.g., Alper, ef a/., 2009, Nature Microbiol. Rev. 7: 715-723 and McCourt, ef a/., 2006, Amino Acids 31 : 173-210. Recombinant engineering techniques have enabled the creation of microorganisms that express biosynthetic pathways capable of producing a number of useful products, such as valine, isoleucine, leucine, and panthothenic acid (vitamin B5). In addition, fuels such as isobutanoi have been produced recombinantiy in microorganisms expressing a heterologous metabolic pathway (See, e.g., WO/2007/050671 to Donaldson, et a/., and WO/2008/098227 to Liao, et a/.). Although engineered microorganisms represent potentially useful tools for the renewable production of fuels, chemicals, and amino acids, many of these microorganisms have fallen short of commercial relevance due to their low performance characteristics, including low- productivity, low titers, and low yields.

[0005] One of the reasons for the sub-optimal performance observed in many existing microorganisms is the undesirable conversion of pathway intermediates to unwanted by-products. The present inventors have identified one molecule, 3- methyl-1 -butanol that may be unwanted in the use of biosynthetic pathways used to produce fuels, chemicals, and amino acids. The accumulation of 3-methyl-1 -butanoi may negatively impact the synthesis and yield of desirable metabolites in a variety of fermentation reactions. The present invention, in part, results from the study of enzymes that form this molecule and shows that the suppression of one or more of these enzymes considerably reduces or eliminates the formation of 3-methy!-1 - butanoi, and concomitantly improves the yields and titers of beneficial metabolites derived from 2-ketoisovalerate.

[0006] On the other hand, the inventors also recognize instances in which the production of 3-methyl-1 -butanol may be beneficial. Thus, 3-methyl-1 -butanol may be produced alongside a beneficial metabolite, for instance, isobutanol, in a quantitated manner such that a mixture of products is isolated. Therefore, one may tune a recombinant microorganism to produce a blended product that is composed partially of 3-methyl-1 -butanol.

SU MA Y OF THE INVENTION

[0007] The present inventors have discovered that the occasionally unwanted byproduct 3-methy!-1 -butanol can accumulate during various fermentation processes, including fermentation of the biofuei candidate, isobutanol. The accumulation of this unwanted by-product may result from the undesirable conversion of 2- ketoisovalerate, an intermediate in a number of biosynthetic pathways. The conversion of 2-ketoisovalerate to this sometimes unwanted by-product can hinder the optimal productivity and yield of a desirable product. Therefore, the present inventors have developed methods for reducing the conversion of 2-ketoisovalerate to 3-methyl-1 -butanol during processes where 2-ketoisovaierate acts as a pathway intermediate. The accumulation of this unwanted by-product may also result from the undesirable conversion of leucine, an amino acid that may be present in a fermentation medium. The present inventors have developed methods for reducing the conversion of leucine to 3-methyl-1 -butanoi during processes where leucine is present in the fermentation medium. Also, however, the present inventors have recognized that, in certain situations, it may be desirable to produce a mixture of products that can comprise 3-methyi~1 -butanoL Therefore, the present inventors have developed methods for enhancing the conversion of 2-ketoisovalerate to 3- methyl-1 -butanol during processes where 2-ketoisovalerate acts as a pathway intermediate.

[0008] In a first aspect, the present invention relates to a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovalerate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 3-methyl-1 -butanol. In an embodiment according to this aspect, the present invention relates to a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovalerate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 2~isopropyimaiate; one or more enzymes catalyzing the conversion of 2-isopropyimaiate to 3-isopropyimalate; one or more enzymes catalyzing the conversion of 3-isopropylmalate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methyibutanal; and one or more enzymes catalyzing the conversion of 3-methy!butanal to 3-methyl- 1 -butanol.

[0009] In another aspect, the present invention relates to a recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 3- methyl-1 -butanoi. In an embodiment according to this aspect, the present invention relates to a recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovaierate to 2-isopropyimaiate; one or more enzymes catalyzing the conversion of 2-isopropylmaiate to 3- isopropy!ma!ate; one or more enzymes catalyzing the conversion of 3- isopropylmaiate to α-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproaie to 3-methylbutanal; and one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methyl-1 -butanoi.

[0010] In another aspect, the present invention relates to a recombinant microorganism comprising a biosynthetic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of leucine to 3-methy!-1 -butanol. In an embodiment according to this aspect, the present invention relates to a recombinant microorganism comprising a biosynthetic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression of one or more of the following: one or more enzymes catalyzing the conversion of leucine to a- ketoisocaproate; one or more enzymes catalyzing the conversion of a- ketoisocaproate to 3-methylbutanal; and one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methyi-1 -butanol,

[0011] In another aspect, the present invention relates to a recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of leucine to 3~methy!-1 ~ butanoi. In an embodiment according to this aspect, the present invention relates to a recombinant microorganism comprising an isobutanol producing metabolic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression of one or more of the following: one or more enzymes catalyzing the conversion of leucine to a-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methylbutanai; and one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methyl-1 -butano!.

[0012] In various embodiments described herein, the recombinant microorganisms are engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 3-methyl-1 - butanoi. In one embodiment, the enzyme is a 2-isopropylmalate synthase, catalyzing the conversion of 2-ketoisovaierate to 2-isopropyimalate. In a specific embodiment, the 2-isopropylmalate synthase is the S. cerevisiae Leu4 (SEQ ID NO: SEQ ID NO: 2) or the S. cerevisiae Leu9 (SEQ ID NO: SEQ ID NO: 4) or a homoiog or variant thereof. In one embodiment, the enzyme is an isopropyimalate isomerase, catalyzing the conversion of 2-isopropylmalate to 3-isopropy!malate. In a specific embodiment, the isopropyimalate isomerase is the S. cerevisiae Leu1 (SEQ ID NO: 8) or a homo!og or variant thereof. In one embodiment, the enzyme is a 3- isopropyimaiate dehydrogenase, catalyzing the conversion of 3-isopropylmalate to a- ketoisocaproate. In a specific embodiment, the 3-isopropylmalate dehydrogenase is the S. cerevisiae Leu2 (SEQ ID NO: 8) or a homolog or variant thereof. In one embodiment, the enzyme is a branched-chain amino acid transaminase, catalyzing the conversion of leucine to a-kefoisocaproate. In a specific embodiment, the branched-chain amino acid transaminase is the S. cerevisiae Bat1 (SEQ ID NO: 10) or the S. cerevisiae Bat2 (SEQ ID NO: 12) or a homoiog or variant thereof. In one embodiment, the enzyme is a keto-isocaproate decarboxylase, catalyzing the conversion of α-ketoisocaproate to 3-methylbutanai. !n a specific embodiment, the keto-isocaproate decarboxylase is the S. cerevisiae Aro10 (SEQ ID NO: 14) or the S. cerevisiae Thi3 (SEQ ID NO: 16) or a homoiog or variant thereof. In one embodiment, the enzyme is an alcohol dehydrogenase, catalyzing the conversion of 3-methylbutanal to 3-methyl-1 -butanoi. In a specific embodiment, the alcohol dehydrogenase is the S. cerevisiae Adh6 (SEQ ID NO: 18) or the S. cerevisiae Adh7 (SEQ ID NO: 20) or a homoiog or variant thereof.

[0013] In another specific embodiment described herein, the recombinant microorganisms are engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the initial steps in the conversion of 2- ketoisovalerate to 3-methyi-1 -butanoi. In one embodiment, the enzyme is a 2- isopropylmaiate synthase, catalyzing the conversion of 2-ketoisovalerate to 2~ isopropylmalate. In a specific embodiment, the 2-isopropylmalate synthase is the S, cerevisiae Leu4 (SEQ ID NO: 2) or the S. cerevisiae Leu9 (SEQ ID NO: 4) or a homoiog or variant thereof. In one embodiment, the enzyme is a branched-chain amino acid transaminase, catalyzing the conversion of leucine to a-ketoisocaproate. In a specific embodiment, the branched-chain amino acid transaminase is the S. cerevisiae Bat1 (SEQ ID NO: 10) or the S. cerevisiae Bat2 (SEQ ID NO: 12) or a homoiog or variant thereof.

[0014] In various embodiments described in the application, the recombinant microorganism comprises an isobutanoi producing metabolic pathway. In one embodiment, the isobutanoi producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanoi. In another embodiment, the isobutanoi producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, ail of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes.

[0015] In one embodiment, one or more of the isobutanol pathway genes encodes an enzyme that is localized to the cytosol. In one embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol. In another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least two isobutanol pathway enzymes localized in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least three isobutanol pathway enzymes localized in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least four isobutanol pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with five isobutanol pathway enzymes localized in the cytosol. In yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with all isobutanol pathway enzymes localized in the cytosol.

[0016] In various embodiments described herein, the isobutanol pathway genes may encode enzyme(s) selected from the group consisting of acetolactate synthase (ALS), ketoi-acid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2~ keto-acid decarboxylase, e.g., keto-isovalerate decarboxylase (KIVD), and alcohol dehydrogenase (ADH). In one embodiment, the KARI is an NADH-dependent KARI (NKR). In another embodiment, the ADH is an NADH-dependent ADH. In yet another embodiment, the KAR! is an NADH-dependent KAR! (NKR) and the ADH is an NADH-dependent ADH.

[0017] In various embodiments, the present invention also provides for a recombinant microorganism comprising a biosynthetic pathway which uses 2- ketoisovalerate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following and/or is substantially free of an enzyme catalyzing the conversion of one or more of the following: one or more enzymes catalyzing the conversion of 2~ ketoisovalerate to 2-isopropyimaiate; one or more enzymes catalyzing the conversion of 2-isopropylmalate to 3-isopropylmalate; one or more enzymes catalyzing the conversion of 3-isopropylmalate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of leucine to a-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methylbutanal; and one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methyl-1 - butanol.

[0018] In various aspects, the present invention also provides for a recombinant microorganism comprising an isobutanoi producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following and/or is substantially free of an enzyme catalyzing the conversion of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovaierate to 2-isopropyimaiate; one or more enzymes catalyzing the conversion of 2-isopropyimaiate to 3-isopropylmalate; one or more enzymes catalyzing the conversion of 3-isopropyimaiate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of leucine to α-ketoisocaproate; one or more enzymes catalyzing the conversion of α-ketoisocaproate to 3-methylbutanal; and one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methyl- 1 -butanoi.

[0019] In various aspects, the present invention also provides for a recombinant microorganism for the production of isobutanoi and 3-methyi-l -butanol, wherein said recombinant microorganism comprises an isobutanoi producing metabolic pathway and overexpresses one or more enzymes capable of converting 2-ketoisovaierate to 3-methyl-1 -butanoi. In certain embodiments, one or more enzymes catalyze the conversion of 3-methylbutanal to 3-methy!-1 -butanol. In certain embodiments, the enzyme is an alcohol dehydrogenase. In further embodiments, the alcohol dehydrogenase is NADH-dependent.

[0020] In various embodiments described herein, the recombinant microorganisms of the invention that comprise an isobutanol producing metabolic pathway may be further engineered to reduce or eliminate the expression or activity of one or more enzymes selected from a pyruvate decarboxylase (PDC), a glycerol- 3-phosphate dehydrogenase (GPD), a 3-keto acid reductase (3-KAR), or an aldehyde dehydrogenase (ALDH).

[0021] In various embodiments described herein, the recombinant microorganisms of the invention produce a 2-ketoisovaierate-derived product. In certain embodiments, the 2-ketoisovalerate-derived product is selected from isobutanol, valine, pantothenate, and coenzyme A. In these embodiments, production pathway enzymes may be overexpressed to yield a desired product.

[0022] In various embodiments described herein, the recombinant microorganism is a bacterium. In various embodiments, the recombinant microorganism may be selected from a genus of Citrobacter, Corynebacterium, Lactobacillus, Lactococcus, Salmonella, Enterobacter, Enterococcus, Erwinia, Pantoea, Morganella, Pectobacterium, Proteus, Serratia, Shigella, and Klebsiella. In one specific embodiment, the recombinant microorganism is a lactic acid bacterium such as, for example, a microorganism derived from the Lactobacillus or Lactococcus genus.

[0023] In various embodiments described herein, the recombinant microorganisms may be yeast microorganisms.

[0024] In some embodiments, the recombinant microorganisms may be yeast recombinant microorganisms of the Saccharomyces ciade.

[0025] In some embodiments, the recombinant microorganisms may be Saccharomyces sensu stricto microorganisms. In one embodiment, the Saccharomyces sensu stricto microorganism is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis, and hybrids thereof.

[0026] In some embodiments, the recombinant microorganisms may be Crabtree- negative recombinant yeast microorganisms. In one embodiment, the Crabtree- negative yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Hansenula, issatchenkia, or Candida. In additional embodiments, the Crabtree-negative yeast microorganism is selected from Saccharomyces kluyveri, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Pichia kudriavzevii, issatchenkia orientalis, Hansenula anomala, Candida utilis, and Kluyveromyces waltii.

[0027] In some embodiments, the recombinant microorganisms may be Crabtree- positive recombinant yeast microorganisms. In one embodiment, the Crabtree- positive yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Pichia, Candida, and Schizosaccharomyces. In additional embodiments, the Crabtree-positive yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelii, Kluyveromyces thermotolerans, Candida glabrata, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii, Pichia pastorius, and Schizosaccharomyces pombe.

[0028] In some embodiments, the recombinant microorganisms may be post- WGD (whole genome duplication) yeast recombinant microorganisms. In one embodiment, the post-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces or Candida. In additional embodiments, the post-WGD yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelii, and Candida glabrata.

[0029] In some embodiments, the recombinant microorganisms may be pre-WGD (whole genome duplication) yeast recombinant microorganisms. In one embodiment, the pre-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Debaryomyces, Hansenula, Issatchenkia, Pachysolen, Yarrowia and Schizosaccharomyces. In additional embodiments, the pre-WGD yeast is selected from the group consisting of Saccharomyces kluyveri, Kluyveromyces thermotolerans, Kiuyveromyces marxianus, Kiuyveromyces waltii, Kiuyveromyces lactis, Candida tropicaiis, Pichia pastoris, Pichia anomala, Pichia stipitis, Pichia kudriavzevii, Issatchenkia orientalis, Issatchenkia occidentaiis, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophiiis, Yarrowia iipolytica, and Schizosaccharomyces pombe.

[0030] In some embodiments, the recombinant microorganisms may be microorganisms that are non-fermenting yeast microorganisms, including, but not limited to those, classified into a genera selected from the group consisting of Tricosporon, Rhodotorula, Myxozyma, or Candida. In a specific embodiment, the non-fermenting yeast is C. xestobii.

[0031] In another aspect, the present invention provides methods of producing beneficial metabolites including fuels, chemicals, and amino acids using a recombinant microorganism as described herein. In one aspect, the method includes cultivating the recombinant microorganism in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the metabolite is produced. In one embodiment, the microorganism produces the metabolite from a carbon source at a yield of at least about 5 percent theoretical. In another embodiment, the microorganism produces the metabolite at a yield of at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 80 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5 percent theoretical. In one embodiment, the metabolite may be derived from a biosynthetic pathway which uses 2-ketoisovalerate as an intermediate, including, but not limited to, isobutanoi, valine, pantothenate, and coenzyme A pathways. In an exemplary embodiment, the metabolite is isobutanoi.

[0032] In one embodiment, the recombinant microorganism converts the carbon source to the beneficial metabolite under aerobic conditions. In another embodiment, the recombinant microorganism converts the carbon source to the beneficial metabolite under microaerobic conditions. In yet another embodiment, the recombinant microorganism converts the carbon source to the beneficial metabolite under anaerobic conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Illustrative embodiments of the invention are illustrated in the drawings, in which:

[0034] Figure 1 illustrates an exemplary embodiment of an isobutanoi pathway.

[0035] Figure 2 illustrates an exemplary embodiment of an NADH-dependent isobutanol pathway.

[0036] Figure 3 illustrates exemplary biosynthetic pathways utilizing 2- ketoisovalerate as an intermediate.

[0037] Figure 4 illustrates the conversion of 2-ketoisovalerate to 3-methyl-1 - butanol.

[0038] Figure 5A illustrates the production of 3-methyl-1 -butanol by an S. cerevissae parental strain (GEVO8014), and progeny strains in which Leu4 or Leu9 have been disrupted. The S. cerevisiae strains were grown in medium void of leucine.

[0039] Figure 5B illustrates the production of 3-methyl-1 -butanoi by an S. cerevisiae parental strain (GEVO8014), and progeny strains in which Leu4 or Leu9 have been disrupted. The S. cerevisiae strains were grown in medium supplemented with leucine.

DETAILED DESCRIPTION

[0040] The present inventors have discovered that the occasionally unwanted byproduct_., 3-methyi-l -butanol, can accumulate during various fermentation processes, including fermentation of the biofuei candidate, isobutanol. The accumulation of this unwanted by-product may result from the undesirable conversion of 2- ketoisovalerate, an intermediate in a number of biosynthetic pathways. The conversion of 2-ketoisovalerate to this sometimes unwanted by-product can hinder the optimal productivity and yield of a desirable product. Therefore, the present inventors have developed methods for reducing the conversion of 2-ketoisovalerate to 3-methyl-1 -butano! during processes where 2-ketoisovaierate acts as a pathway intermediate. The accumulation of this unwanted by-product may also result from the undesirable conversion of leucine, an amino acid that may be present in a fermentation medium. The present inventors have developed methods for reducing the conversion of leucine to 3-methyl-1 -butanoi during processes where leucine is present in the fermentation medium. Also, however, the present inventors have recognized that, in certain situations, it may be desirable to produce a mixture of products that can comprise 3-methyi-1 -butanoi. Therefore, the present inventors have developed methods for enhancing the conversion of 2-ketoisovalerate to 3- methyl-1 -butanoi during processes where 2-keioisovalerate acts as a pathway intermediate.

[0041] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the microorganism" includes reference to one or more microorganisms, and so forth.

[0042] Unless defined otherwise, ail technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

[0043] Any publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

[0044] The term "microorganism" includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria, and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista, among others. The terms "microbial ceils" and "microbes" are used interchangeably with the term microorganism.

[0045] The term "genus" is defined as a taxonomic group of related species according to the Taxonomic Outline of Bacteria and Archaea (Garrity, G.M., et a/. The Taxonomic Outline of Bacteria and Archaea. TOBA Release 7.7, March 2007. Michigan State University Board of Trustees, [http://www.taxonomicoutiine.org/]).

[0046] The term "species" is defined as a collection of closely related organisms with greater than 97% 16S ribosomai RNA sequence homology and greater than 70% genomic hybridization and sufficiently different from ail other organisms so as to be recognized as a distinct unit.

[0047] The terms "recombinant microorganism," "modified microorganism," and "recombinant host ceil" are used interchangeably herein and refer to microorganisms that have been genetically modified to express or to overexpress endogenous polynucleotides, to express heterologous polynucleotides, such as those included in a vector, in an integration construct, or which have an alteration in expression of an endogenous gene. By "alteration" it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the alteration. For example, the term "alter" can mean "inhibit," but the use of the word "alter" is not limited to this definition. It is understood that the terms "recombinant microorganism" and "recombinant host cell" refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0048] The term "expression" with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein results from transcription and translation of the open reading frame sequence. The level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the ceil, or the amount of the desired product encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantitated by qRT-PCR or by Northern hybridization (see Sambrook et a/., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that recognize and bind the protein. See Sambrook et a/., 1989, supra, [0049] The term "overexpression" refers to an elevated level (e.g., aberrant level) of mRNAs encoding for a protein(s), and/or to elevated levels of protein(s) in ceils as compared to similar corresponding unmodified ceils expressing basal levels of mRNAs or having basal levels of proteins. In particular embodiments, mRNA(s) or protein(s) may be overexpressed by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8- fold, 10-fold, 12-fold, 15-fold or more in microorganisms engineered to exhibit increased gene mRNA, protein, and/or activity. [0050] As used herein and as would be understood by one of ordinary skill in the art, "reduced activity and/or expression" of a protein such as an enzyme can mean either a reduced specific catalytic activity of the protein (e.g. reduced activity) and/or decreased concentrations of the protein in the cell (e.g. reduced expression). As would be understood by one or ordinary skill in the art, the reduced activity of a protein in a cell may result from decreased concentrations of the protein in the ceil.

[0051] The term "wild-type microorganism" describes a ceil that occurs in nature, i.e. , a cell that has not been genetically modified. A wild-type microorganism can be genetically modified to express or overexpress a first target enzyme. This microorganism can act as a parental microorganism in the generation of a microorganism modified to express or overexpress a second target enzyme. In turn, the microorganism modified to express or overexpress a first and a second target enzyme can be modified to express or overexpress a third target enzyme.

[0052] Accordingly, a "parental microorganism" functions as a reference cell for successive genetic modification events. Each modification event can be accomplished by introducing a nucleic acid molecule in to the reference cell. The introduction facilitates the expression or overexpression of a target enzyme. It is understood that the term "facilitates" encompasses the activation of endogenous polynucleotides encoding a target enzyme through genetic modification of e.g. , a promoter sequence in a parental microorganism. It is further understood that the term "facilitates" encompasses the introduction of heterologous polynucleotides encoding a target enzyme into a parental microorganism.

[0053] The term "engineer" refers to any manipulation of a microorganism that results in a detectable change in the microorganism, wherein the manipulation includes, but is not limited to, inserting a polynucleotide and/or polypeptide heterologous to the microorganism and mutating a polynucleotide and/or polypeptide native to the microorganism.

[0054] The term "mutation" as used herein indicates any modification of a nucleic acid and/or polypeptide which results in an altered nucleic acid or polypeptide. Mutations include, for example, point mutations, deletions, or insertions of single or multiple residues in a polynucleotide, which includes alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A genetic alteration may be a mutation of any type. For instance, the mutation may constitute a point mutation, a frame-shift mutation, a nonsense mutation, an insertion, or a deletion of part or ail of a gene. In addition, in some embodiments of the modified microorganism, a portion of the microorganism genome has been replaced with a heterologous polynucleotide. In some embodiments, the mutations are naturally-occurring. In other embodiments, the mutations are identified and/or enriched through artificial selection pressure. In still other embodiments, the mutations in the microorganism genome are the result of genetic engineering.

[0055] The term "biosynthetic pathway", also referred to as "metabolic pathway", refers to a set of anabolic or catabolic biochemical reactions for converting one chemical species into another. Gene products belong to the same "metabolic pathway" if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product.

[0056] As used herein, the term "isobutanol producing metabolic pathway" refers to an enzyme pathway which produces isobutanol from pyruvate.

[0057] The term "NADH-dependent" as used herein with reference to an enzyme, e.g., KARI and/or ADH, refers to an enzyme that catalyzes the reduction of a substrate coupled to the oxidation of NADH with a catalytic efficiency that is greater than the reduction of the same substrate coupled to the oxidation of NADPH at equal substrate and cofactor concentrations.

[0058] The term "exogenous" as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are not normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.

[0059] On the other hand, the term "endogenous" or "native" as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.

[0060] The term "heterologous" as used herein in the context of a modified host cell refers to various molecules, e.g. , polynucleotides, polypeptides, enzymes, etc., wherein at least one of the following is true: (a) the molecule(s) is/are foreign ("exogenous") to (i.e. , not naturally found in) the host cell; (b) the molecule(s) is/are naturally found in (e.g., is "endogenous to") a given host microorganism or host ceil but is either produced in an unnatural location or in an unnatural amount in the ceil; and/or (c) the molecule(s) differ(s) in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence(s) such that the molecule differing in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid as found endogenously is produced in an unnatural {e.g., greater than naturally found) amount in the ceil.

[0061] The term "feedstock" is defined as a raw material or mixture of raw materials supplied to a microorganism or fermentation process from which other products can be made. For example, a carbon source, such as biomass or the carbon compounds derived from biomass are a feedstock for a microorganism that produces a biofuel in a fermentation process. However, a feedstock may contain nutrients other than a carbon source.

[0062] The term "substrate" or "suitable substrate" refers to any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme. The term includes not only a single compound, but also combinations of compounds_., such as solutions, mixtures and other materials which contain at least one substrate, or derivatives thereof. Further, the term "substrate" encompasses not only compounds that provide a carbon source suitable for use as a starting material, such as any biomass derived sugar, but also intermediate and end product metabolites used in a pathway associated with a recombinant microorganism as described herein.

[0063] The term "fermentation" or "fermentation process" is defined as a process in which a microorganism is cultivated in a culture medium containing raw materials, such as feedstock and nutrients, wherein the microorganism converts raw materials, such as a feedstock, into products.

[0064] The term "volumetric productivity" or "production rate" is defined as the amount of product formed per volume of medium per unit of time. Volumetric productivity is reported in grams per liter per hour (g/L/h).

[0065] The term "specific productivity" or "specific production rate" is defined as the amount of product formed per volume of medium per unit of time per amount of ceils. Specific productivity is reported in grams or milligrams per liter per hour per OD (g/L/h/OD).

[0066] The term "yield" is defined as the amount of product obtained per unit weight of raw material and may be expressed as grams product per grams substrate (g/g). Yield may be expressed as a percentage of the theoretical yield. "Theoretical yield" is defined as the maximum amount of product that can be generated per a given amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to isobutanol is 0.41 g/g. As such, a yield of isobutanol from glucose of 0.39 g/g would be expressed as 95% of theoretical or 95% theoretical yield.

[0067] The term "titer" is defined as the strength of a solution or the concentration of a substance in solution. For example, the titer of a biofuel in a fermentation broth is described as grams of biofuel in solution per liter of fermentation broth (g/L).

[0068] "Aerobic conditions" are defined as conditions under which the oxygen concentration in the fermentation medium is sufficiently high for an aerobic or facultative anaerobic microorganism to use as a terminal electron acceptor.

[0069] In contrast, "anaerobic conditions" are defined as conditions under which the oxygen concentration in the fermentation medium is too low for the microorganism to use as a terminal electron acceptor. Anaerobic conditions may be achieved by sparging a fermentation medium with an inert gas such as nitrogen until oxygen is no longer available to the microorganism as a terminal electron acceptor. Alternatively, anaerobic conditions may be achieved by the microorganism consuming the available oxygen of the fermentation until oxygen is unavailable to the microorganism as a terminal electron acceptor. Methods for the production of isobutanol under anaerobic conditions are described in commonly owned and copending publication, US 2010/0143997, the disclosures of which are herein incorporated by reference in its entirety for all purposes.

[0070] "Aerobic metabolism" refers to a biochemical process in which oxygen is used as a terminal electron acceptor to make energy, typically in the form of ATP, from carbohydrates. Aerobic metabolism occurs e.g. via glycolysis and the TCA cycle, wherein a single glucose molecule is metabolized completely into carbon dioxide in the presence of oxygen.

[0071] In contrast, "anaerobic metabolism" refers to a biochemical process in which oxygen is not the final acceptor of electrons contained in NADH. Anaerobic metabolism can be divided into anaerobic respiration, in which compounds other than oxygen serve as the terminal electron acceptor, and substrate level phosphorylation, in which the electrons from NADH are utilized to generate a reduced product via a "fermentative pathway."

[0072] In "fermentative pathways," NAD(P)H donates its electrons to a molecule produced by the same metabolic pathway that produced the electrons carried in NAD(P)H. For example, in one of the fermentative pathways of certain yeast strains, NAD(P)H generated through glycolysis transfers its electrons to pyruvate, yielding ethanoi. Fermentative pathways are usually active under anaerobic conditions but may also occur under aerobic conditions, under conditions where NADH is not fully oxidized via the respiratory chain. For example, above certain glucose concentrations, Crabtree positive yeasts produce large amounts of ethanoi under aerobic conditions.

[0073] The term "byproduct" or "by-product" means an undesired product related to the production of an amino acid, amino acid precursor, chemical, chemical precursor, biofuel, or biofuel precursor.

[0074] The term "substantially free" when used in reference to the presence or absence of enzymatic activities (non-limiting examples include, 2-isopropylmalate synthase, isopropy!ma!ate isomerase, 3-isoproy!ma!ate dehydrogenase, branched- chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, GPD, etc.) in carbon pathways that compete with the desired metabolic pathway (e.g., an isobutanol-producing metabolic pathway) means the level of the enzyme activity is substantially less than that of the same enzyme activity in the wild-type host, wherein less than about 50% of the wild- type level is preferred and less than about 30% is more preferred. The activity may be less than about 20%, less than about 10%, less than about 5%, or less than about 1 % of wild-type activity. Microorganisms which are "substantially free" of a particular enzymatic activity (non-limiting examples include, 2-isopropylmalate synthase, isopropy!ma!ate isomerase, 3-isoproylmalate dehydrogenase, branched- chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, GPD, etc.) may be created through recombinant means or identified in nature.

[0075] The term "non-fermenting yeast" is a yeast species that fails to demonstrate anaerobic metabolism in which the electrons from NADH are utilized to generate a reduced product via a fermentative pathway such as the production of ethanoi and CO2 from glucose. Non-fermentative yeast can be identified by the "Durham Tube Test" (Barnett J.A., et a/. 2000, Yeasts Characteristics and Identification. 3^rd edition, p. 28-29. Cambridge University Press, Cambridge, UK) or by monitoring the production of fermentation productions such as ethanol and CO2 [0076] The term "polynucleotide" is used herein interchangeably with the term "nucleic acid" and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single stranded or double stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or antisense ribonucleic acid (RNA) of any length, including siRNA. The term "nucleotide" refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or a pyrimidine base and to a phosphate group, and that are the basic structural units of nucleic acids. The term "nucleoside" refers to a compound (as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. The term "nucleotide analog" or "nucleoside analog" refers, respectively, to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called a nucleotidic oligomer or oligonucleotide.

[0077] It is understood that the polynucleotides described herein include "genes" and that the nucleic acid molecules described herein include "vectors" or "plasmids." Accordingly, the term "gene," also called a "structural gene" refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non- transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. The transcribed region of the gene may include untranslated regions, including introns, 5'-untranslated region (UTR), and 3'-UTR, as well as the coding sequence.

[0078] The term "operon" refers to two or more genes which are transcribed as a single transcriptional unit from a common promoter. In some embodiments, the genes comprising the operon are contiguous genes. It is understood that transcription of an entire operon can be modified (i.e., increased, decreased, or eliminated) by modifying the common promoter. Alternatively, any gene or combination of genes in an operon can be modified to alter the function or activity of the encoded polypeptide. The modification can result in an increase in the activity of the encoded polypeptide. Further, the modification can impart new activities on the encoded polypeptide. Exemplary new activities include the use of alternative substrates and/or the ability to function in alternative environmental conditions.

[0079] A "vector" is any means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components. Vectors include viruses, bacteriophage, pro-viruses, piasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes), and the like, that are "episomes," that is, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine -conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that are not episomal in nature, or it can be an organism which comprises one or more of the above polynucleotide constructs such as an agrobacterium or a bacterium.

[0080] "Transformation" refers to the process by which a vector is introduced into a host ceil. Transformation (or transduction, or transfection), can be achieved by any one of a number of means including chemical transformation (e.g. lithium acetate transformation), eiectroporation, microinjection, bioHstics (or particle bombardment- mediated delivery), or agrobacterium mediated transformation.

[0081] The term "enzyme" as used herein refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide, but can include enzymes composed of a different molecule including polynucleotides.

[0082] The term "protein," "peptide," or "polypeptide" as used herein indicates an organic polymer composed of two or more amino acidic monomers and/or analogs thereof. As used herein, the term "amino acid" or "amino acidic monomer" refers to any natural and/or synthetic amino acids including glycine and both D or L optical isomers. The term "amino acid analog" refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, or with a different functional group. Accordingly, the term polypeptide includes amino acidic polymers of any length including full length proteins, and peptides as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called a protein oligomer or oligopeptide.

[0083] The term "homoiog," used with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural, or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PGR. Identity of cloned sequences as homologs can be confirmed using functional assays and/or by genomic mapping of the genes.

[0084] A protein has "homology" or is "homologous" to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences (thus, the term "homologous proteins" is defined to mean that the two proteins have similar amino acid sequences).

[0085] The term "analog" or "analogous" refers to nucleic acid or protein sequences or protein structures that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure.

Recombinant Microorganisms with Reduced By-Product Accumulation

[0086] Yeast ceils convert sugars to produce pyruvate, which is then utilized in a number of pathways of cellular metabolism, !n recent years, yeast cells have been engineered to produce a number of desirable products via pyruvate-d riven biosynthetic pathways. In many of these biosynthetic pathways, 2-ketoisovalerate is involved.

[0087] One route of 2-ketoisovalerate formation is a conversion from 2,3- dihydroxyisova!erate via a dihydroxyacid dehydratase in a pathway for the formation of isobutanol. In addition to the isobutanoi production pathway, a number of other biosynthetic pathways also use 2-ketoisovalerate as an intermediate, including, but not limited to, valine, pantothenate, and coenzyme A pathways. Engineered biosynthetic pathways for the synthesis of these beneficial 2-ketoisovalerate-derived metabolites are found in Table 1 and Figure 2,

Table 1. Biosynthetic Pathways Utilizing 2-Ketoisova!erate as an Intermediate

a - The contents of each of the references in this table are herein incorporated by reference in their entireties for all purposes.

[0088] Each of the biosynthetic pathways shares the common 2-ketoisovalerate intermediate (Figure 3) and therefore, the product yield from these biosynthetic pathways will in part depend upon the amount of 2-ketoisovalerate that is available to downstream enzymes of said biosynthetic pathways.

[0089] As described herein, the present inventors have characterized the enzymes responsible for the accumulation of 3-methyl-1 -butanoi. In some circumstances, these enzymatic activities may hinder the optimal productivity and yield of 2-ketoisovalerate-derived products, including, but not limited to, isobutanoi, valine, pantothenate, and coenzyme A. The present inventors have found that suppressing these newly-characterized enzymatic activities considerably reduces or eliminates the formation of 3-methyi-1 -butanol, and concomitantly improves the yields and titers of beneficial metabolites.

Reduced Accumulation of 3-Methyl-1 -Butanoi

[0090] As described herein, the present inventors have found that the sometimes unwanted by-product, 3-niethy!-1 -butanol, can accumulate during fermentation processes with microorganisms comprising a pathway involving a 2-ketoisovalerate intermediate. The conversion of 2-ketoisovalerate to 3-methy!-1 -butano! is illustrated in Figure 4.

[0091] The present inventors have found that the deletion of the pathway steps by which 3-methyi-1 -butanoi is produced may remove competition for 2-ketoisovalerate and may further removes competition in engineered biosynthetic pathways for reduced co-factors, which increases the NAD(P)H/NAD(P ratio such that flux through an engineered biosynthetic pathway (e.g., an isobutanoi producing metabolic pathway) may increase. As described herein, the activities of multiple enzymes are shown to be responsible for the formation of 3-methyl-1 -butanoi (Figure 4),

[0092] The present inventors describe herein multiple strategies for reducing the conversion of 2-ketoisovaierate to 3-methyl-1 -butano!, a process which is accompanied by an increase in the yield of desirable metabolites. As described herein, reducing the conversion of 2-ketoisovalerate to 3-methy!-1 -butano! enables the increased production of beneficial metabolites such as isobutanoi, valine, pantothenate, and coenzyme A, which are derived from biosynthetic pathways using 2-ketoisovalerate as an intermediate.

[0093] Accordingly, in one embodiment, the present invention relates to a recombinant microorganism comprising a biosynthetic pathway which uses 2- ketoisovalerate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 3-methyi-1 -butanoi.

[0094] In another embodiment, the present invention relates to a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovaierate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovaierate to 2-isopropyimalafe; one or more enzymes catalyzing the conversion of 2-isopropy!malate to 3-isopropy!ma!ate; one or more enzymes catalyzing the conversion of 3-isopropyimaIate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3- methylbutanal; and one or more enzymes catalyzing the conversion of 3- methylbutanal to 3-methyi~1 -butanoL

[0095] In another embodiment, the present invention relates to a recombinant microorganism comprising an isobutanoi producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 2-isopropyimaiate; one or more enzymes catalyzing the conversion of 2-isopropyimaiate to 3-isopropylmalate; one or more enzymes catalyzing the conversion of 3-isopropylmalate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methyibutana!; and one or more enzymes catalyzing the conversion of 3-methyibutana! to 3-methyl- 1 -butanol.

[0096] In another aspect, the present invention relates to a recombinant microorganism comprising a biosynthetic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of leucine to 3-methyi-1 -butanoI. In an embodiment according to this aspect, the present invention relates to a recombinant microorganism comprising a biosynthetic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression of one or more of the following: one or more enzymes catalyzing the conversion of leucine to a- ketoisocaproate; one or more enzymes catalyzing the conversion of a- ketoisocaproate to 3-methylbutanai; and one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methy!-1 -butanol.

[0097] In another aspect, the present invention relates to a recombinant microorganism comprising an isobutanoi producing metabolic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of leucine to 3-methy!-1 - butanoi. In an embodiment according to this aspect, the present invention relates to a recombinant microorganism comprising an isobutanoi producing metabolic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression of one or more of the following: one or more enzymes catalyzing the conversion of leucine to α-ketoisocaproate; one or more enzymes catalyzing the conversion of α-ketoisocaproate to 3-methylbutanal; and one or more enzymes catalyzing the conversion of 3-methy!butanai to 3-methyl-1 -butanol.

[0098] In another embodiment, the recombinant microorganisms are engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the initial steps in the conversion of 2-ketoisovalerate to 3-methyi-1 -butanoi. in one embodiment, the enzyme is a 2-isopropyimaiate synthase, catalyzing the conversion of 2-ketoisovaierate to 2-isopropyimaiate. In a specific embodiment, the 2- isopropylmaiate synthase is the S. cerevisiae Leu4 (SEQ ID NO: 2) or the S. cerevisiae Leu9 (SEQ ID NO: 4) or a homolog or variant thereof. in one embodiment, the enzyme is a branched-chain amino acid transaminase, catalyzing the conversion of leucine to a-ketoisocaproate. In a specific embodiment, the branched-chain amino acid transaminase is 8. cerevisiae Bail (SEQ ID NO: 10) or S. cerevisiae Bat2 (SEQ ID NO: 12) or a homolog or variant thereof. Homologs of Leu4, Leu9, Bat1 , and Bat2 are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, a Leu4, Leu9, Bat1 , and/or Bat2 polypeptide derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaiyomyces, Gibbereiia, Giomereila, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Peniciiiium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces,

Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces may be disrupted, deleted, or mutated.

Reduced Enzymatic Conversions Involving 2-Ketoisovaierate as an Intermediate [0099] In one embodiment, the invention is directed to a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovaierate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 3-methyl-1 -butano!.

[00100] In some embodiments, the enzyme catalyzing the conversion of 2~ ketoisovalerate to 2-isopropy!malate is a 2-isopropyimaiate synthase. In an exemplary embodiment the 2-isopropy!ma!ate synthase is the S. cerevisiae Leu4 (SEQ ID NO: 2) or S. cerevisiae Leu9 (SEQ ID NO: 4) or a homolog or variant thereof. Homologs of Leu4 and Leu9 are known to occur in yeast other than S. cerevisiae, Accordingly, in additional embodiments, a Leu4 and/or Leu9 polypeptide derived from a yeast selected from Ajeilomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibbereila, Giomereila, Grosmannia, issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosaiiorya, Neurospora, Paracoccidioides, Peniciliiurn, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces,

Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticiliium, Yarrowia or Zygosaccharomyces may be disrupted, deleted, or mutated.

[00101] As used herein, the term "2-isopropyimaiate synthase" refers to a polypeptide having an enzymatic activity that catalyzes the conversion of 2- ketoisovalerate to 2-isopropylmalate. Exemplary 2-isopropy!ma!ate synthases are found in a variety of microorganisms, e.g., S. cerevisiae (see above), £. coti LeuA and L !actis LeuA,

[00102] In some embodiments, the enzyme catalyzing the conversion of 2~ isopropyimalate to 3-isopropylmaiate is a isopropy!ma!ate isomerase. In an exemplary embodiment the isopropyimalate isomerase is the S. cerevisiae Leu1 (SEQ ID NO: 8) or a homolog or variant thereof. Homoiogs of Leu4 and Leu9 are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, a Leu4 and/or Leu9 polypeptide derived from a yeast selected from Ajeliomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaeiomium, Ciavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Lepiosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerelia, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Scierotinia, Sordaria, Taiaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticiliium, Yarrowia or Zygosaccharomyces may be disrupted, deleted, or mutated.

[00103] As used herein, the term "isopropyimalate isomerase" refers to one or more a polypeptides having an enzymatic activity that catalyzes the conversion of 2- isopropyimaiate to 3-isopropy!malate. Exemplary isopropyimalate isomerases are known as EC 4.2.1 .33 and are found in a variety of microorganisms, e.g., S. cerevisiae (see above), £. coti LeuC and LeuD and L, iactis LeuG and LeuD.

[00104] In some embodiments, the enzyme catalyzing the conversion of 3- isopropylmaiate to a-ketoisocaproate is an 3-isoproyimaiate dehydrogenase. In an exemplary embodiment the 3-isoproyimalate dehydrogenase is the S. cerevisiae Leu2 (SEQ ID NO: 8) or a homolog or variant thereof. Homoiogs of Leu2 are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, a Leu2 polypeptide derived from a yeast selected from Ajeliomyces, Arthroderma, Ashbya, Aspergillus, Botsyotinia, Candida, Chaeiomium, Ciavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Scierotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticiliium, Yarrowsa or Zygosaccharomyces may be disrupted, deleted, or mutated

[00105] As used herein, the term "3-isoproylmaiate dehydrogenase" reiers to a polypeptide having an enzymatic activity that catalyzes the conversion of 3~ isopropylmaiate to a-ketoisocaproate. Exemplary 3-isoproylmalate dehydrogenases are known as EC 1 .1 .1 .85 and are found in a variety of microorganisms, e.g. S. cerevssiae (see above) and E. cols LeuB and L lactis LeuB.

[00106] In some embodiments, the enzyme catalyzing the conversion of leucine to a-ketoisocaproate is a branched-chain amino acid transaminase. In an exemplary embodiment the branched-chain amino acid transaminase is S. cerevssiae Bat1 (SEQ ID NO: 10) or S. cerevssiae Bat2 (SEQ ID NO: 12) or a homo!og or variant thereof. Homologs of Bat1 and/or Bat2 are known to occur in yeast other than S. cerevssiae. Accordingly, in additional embodiments, a Bat1 and/or Bat2 polypeptide derived from a yeast selected from Ajeliomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Ciavispora, Coccidioides, Debaryomyces, Gibbereila, Giomereiia, Grosmannia, issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces,

Schizosaccharomyces, Scierotinia, Sordaria, Talarosvyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticiliium, Yarrowsa or Zygosaccharomyces may be disrupted, deleted, or mutated.

[00107] As used herein, the term "branched-chain amino acid transaminase" refers to a polypeptide having an enzymatic activity that catalyzes the conversion of leucine to a-ketoisocaproate. Exemplary branched-chain amino acid transaminases are known as EC 2.6.1 .42 and are found in a variety of microorganisms, e.g. S. cerevssiae (see above) and £. coii llvE and L lactis HvE.

[00108] In some embodiments, the enzyme catalyzing the conversion of a- ketoisocaproate to 3-methy!butanal is a keto-isocaproate decarboxylase. In an exemplary embodiment the keto-isocaproate decarboxylase is S. cerevssiae Aro10 (SEQ ID NO: 14), Ths3 (SEQ ID NO: 18) or homologs or variants thereof. Homologs of Aro10 and Thi3 are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, an Aro10 and/or Thi3 polypeptide derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberel!a, G!omere!la, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meye zyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Peniciiiium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Taiaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces may be disrupted, deleted, or mutated.

[00109] As used herein, the term "keto-isocaproate decarboxylase" refers to a polypeptide having an enzymatic activity that catalyzes the conversion of a- kefoisocaproate to 3-methy!butanai. Exemplary keto-isocaproate decarboxylases are known as EC 4.1 .1 .1 or EC 4.1 .1 .72 and are found in a variety of microorganisms, e.g. S. cerevisiae (see above) and L. lactis KivD and L. lactis KdcA.

[00110] In some embodiments, alteration of keto-isocaproate decarboxylase, e.g. reduction or deletion, is done so in a manner that ensures that the isobutanoi pathway genes for the enzymes converting 2-ketoisovaierate to isobutyraldehyde are not reduced or deleted. In some embodiments, the keto-isocaproate decarboxylase is altered such that its substrate specificity for a-ketoisocaproate or 2-ketoisovalerate is changed relative to a parental strain. In some embodiments, the enzyme is engineered to favor 2-ketoisovalerate over a-ketoisocaproate, for instance, when isobutanoi is being produced and 3-methyl-1 -butanoi is to be reduced.

[00111] In some embodiments, the enzyme catalyzing the conversion of 3- methylbutanal to 3-methyl-1 -butanoi is an alcohol dehydrogenase. In an exemplary embodiment, the alcohol dehydrogenase is the S. cerevisiae Adh6 (SEQ ID NO: 18), Adh7 (SEQ ID NO: 20) or homologs or variants thereof, as summarized below:

&cat [S j

S. cerevisiae Adh6 139'¹ 233°

5. cerevisiae Adh7 48^b 16(†

aLarroy, C, Biochem. J. (2002) 361:163-172; numbers are for 3-methyl-l-butanal (the contents of which are hereby incorporated by reference) °Larroy, C, Eur. J. Biochem. (2002) 289:5738-5745; numbers are for 3-methyi-l-bi!tanal (the contents of which are hereby incorporated by reference).

[00112] Horno!ogs of Adh8 and Adh7 are known to occur in yeast other than S, cerevisiae. Accordingly, in additional embodiments, an Adh6 and/or Adh7 polypeptide derived from a yeast selected from Aje!iomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Giomereila, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Scierotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticiiiium, Yarrowia or Zygosaccharomyces may be disrupted, deleted, or mutated.

[00113] As used herein, the term "alcohol dehydrogenase" refers to a polypeptide having an enzymatic activity that catalyzes the conversion of 3-methylbutanal to 3- methyM -butanoi. Exemplary alcohol dehydrogenases are known as EC 1 .1 .1 .1 or EC 1 .1 .1 .2 and are found in a variety of microorganisms, e.g. S. cerevisiae Adh6 (see above) and L. iactis AdhA.

[00114] In some embodiments, alteration of alcohol dehydrogenase, e.g. reduction or deletion, is done so in a manner that ensures that the isobutanol pathway genes for the enzymes converting isobutyraldehyde to isobutanol are not reduced or deleted. In some embodiments, the alcohol dehydrogenase is altered such that its substrate specificity for 3-methylbutanal or isobutyraldehyde is changed relative to a parental strain. In some embodiments, the enzyme is engineered to favor isobutyraldehyde over 3-methylbutanal, for instance, when isobutanol is being produced and 3-methyl-1 -butanoi is to be reduced.

[00115] In one embodiment, the invention is directed to a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovaierate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the initial steps in the conversion of 2-ketoisovalerate to 3-methyl-1 -butanol. In one embodiment, the enzyme is a 2-isopropylma!ate synthase, cataiyzing the conversion of 2-ketoisovalerate to 2-isopropylmalate. In a specific embodiment, the 2- isopropylmaiate synthase is the S. cerevisiae Leu4 (SEQ !D NO: 2) or the S. cerevisiae Leu9 (SEQ ID NO: 4) or a homoiog or variant thereof. In one embodiment, the enzyme is a branched-chain amino acid transaminase, catalyzing the conversion of leucine to a-ketoisocaproate. In a specific embodiment, the branched-chain amino acid transaminase is S. cerevisiae Bat1 (SEQ ID NO: 10) or S. cerevisiae Bat2 (SEQ ID NO: 12) or a homoiog or variant thereof.

[00116] In one embodiment, the recombinant microorganism of the invention includes a mutation in at least one gene encoding for at least any of the enzymes mentioned herein resulting in a reduction of enzymatic activity of a polypeptide encoded by said gene. In another embodiment, the recombinant microorganism includes a partial deletion of a gene encoding for at least any of the enzymes mentioned herein resulting in a reduction of enzymatic activity of a polypeptide encoded by the gene. In another embodiment, the recombinant microorganism comprises a complete deletion of a gene encoding for at least any of the enzymes mentioned herein resulting in a reduction of enzymatic activity of a polypeptide encoded by the gene. In yet another embodiment, the recombinant microorganism includes a modification of the regulatory region associated with the gene encoding for at least any of the enzymes mentioned herein resulting in a reduction of expression of an enzyme polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism comprises a modification of a transcriptional regulator resulting in a reduction of transcription of a gene encoding for any of the enzymes mentioned herein. In yet another embodiment, the recombinant microorganism comprises mutations in ail genes encoding for at least any of the enzymes mentioned herein resulting in a reduction of activity of a polypeptide encoded by the gene(s). As would be understood in the art, naturally occurring homo!ogs of any of the enzymes mentioned herein in yeast other than S. cerevisiae can similarly be inactivated using the methods of the present invention. Homoiogs of the enzymes mentioned herein and methods of identifying such homoiogs are described herein.

[00117] As is understood by those skilled in the art, there are several additional mechanisms available for reducing or disrupting the activity of a protein such as the enzymes described herein, including, but not limited to, the use of a regulated promoter, use of a weak constitutive promoter, disruption of one of the two copies of the gene in a diploid yeast, disruption of both copies of the gene in a diploid yeast, expression of an anti-sense nucleic acid, expression of an siRNA, over expression of a negative regulator of the endogenous promoter, alteration of the activity of an endogenous or heterologous gene, use of a heterologous gene with lower specific activity, and the like or combinations thereof.

[00118] As described herein, the recombinant microorganisms of the present invention are engineered to produce less 3-methyl-1 -butanol than an unmodified parental microorganism. In one embodiment, the recombinant microorganism produces 3-methyl-1 -butanol from a carbon source at a carbon yield of less than about 20 percent. In another embodiment, the microorganism produces 3-methyi-1 - butanoi from a carbon source at a carbon yield of less than about 10, less than about 5, less than about 2, less than about 1 , less than about 0.5, less than about 0.1 , or less than about 0.01 percent.

[00119] In one embodiment, the 3-methyl-1 -butanol derived from 2-ketoisovalerate is reduced by at least about 50% in a recombinant microorganism as compared to a parental microorganism that does not comprise a reduction or deletion of the activity or expression of one or more of the enzymes described herein. In another embodiment, the 3-methyl-1 -butanoi derived from 2-ketoisovaierate is reduced by at least about 60%, by at least about 65%, by at least about 70%, by at least about 75%, by at least about 80%, by at least about 85%, by at least about 90%, by at least about 95%, by at least about 99%, by at least about 99.9%, or by at least about 100% as compared to a parental microorganism that does not comprise a reduction or deletion of the activity or expression of one or more of the enzymes described herein.

[00120] In an additional embodiment, the yield of a desirable fermentation product is increased in the recombinant microorganisms comprising a reduction or elimination of the activity or expression of one or more of the enzymes described herein. In one embodiment, the yield of a desirable fermentation product is increased by at least about 0.1 % as compared to a parental microorganism that does not comprise a reduction or elimination of the activity or expression of one or more of the enzymes described herein. In another embodiment, the yield of a desirable fermentation product is increased by at least about 0.5%, by at least about 1 %, by at least about 5%, or by at least about 10% as compared to a parental microorganism that does not comprise a reduction or elimination of the activity or expression of one or more of the enzymes described herein. The desirable fermentation product is derived from any biosynthetic pathway in which 2- ketoisovalerate acts as an intermediate, including, but not limited to, isobutanol, valine, pantothenate, and coenzyme A pathways.

[00121] Methods for identifying additional enzymes catalyzing any of the reactions described herein are outlined as follows: endogenous yeast genes coding for potential proteins with the ability to undertake any of the reactions described herein are deleted from the genome of a yeast strain comprising a biosynthetic pathway in which 2-ketoisovalerate is an intermediate. These deletion strains are compared to the parent strain by fermentation and analysis of the fermentation broth for the presence and concentration of the 3-methyl-1 -butanol by-product. In S. cerevisiae, deletions that reduce the production of the 3-methyl-1 -butanoi by-product are combined by construction of strains carrying multiple deletions. Many of these deletion strains are available commercially (for example Open Biosystems YSC1054). These deletion strains are transformed with a piasmid pGV2435 from which the ALS gene (e.g., the B. subtiiis alsS) is expressed under the control of the CUP1 promoter. The transformants are cultivated in YPD medium containing 150 g/L glucose in shake flasks at 30°C, 75 rpm in a shaking incubator for 48 hours. After 48 hours samples from the shake flasks are analyzed by HPLC for the concentration of the 3~methyl-1 ~butanol by-product. As would be understood in the art, naturally occurring homoiogs of any of the enzymes described herein in yeast other than S. cerevisiae can similarly be inactivated using the methods of the present invention. These homoiogs and methods of identifying such homoiogs are described herein.

[00122] Another way to screen the deletion library is to incubate yeast cells with 2- ketoisovalerate or leucine and analyze the broth for the production of the 3-methyi-1 - butanol by-product.

[00123] An alternative approach for finding additional endogenous activity responsible for the production of the 3-methy!-1 -butano! by-product derived from 2- ketoisovalerate or from leucine is to analyze yeast strains that overexpress the genes suspected of encoding the enzyme responsible for production of the 3-methyl- 1 -butanol by-product. Such strains are commercially available for many of the candidate genes listed above (for example Open Biosystems YSC3870). The ORF overexpressing strains are processed in the same way as the deletion strains. They are transformed with a piasmid for ALS expression and screened for 3-methy!-1 - butanol by-product production levels. To narrow the list of possible genes causing the production of the 3-rnethyi-1 -butanol by-product, their expression can be analyzed in fermentation samples. Genes that are not expressed during a fermentation that produced the 3-methy!-1 -butanol by-product can be excluded from the list of possible targets. This analysis can be done by extraction of RNA from fermenter samples and submitting these samples to whole genome expression analysis, for example, by Roche NimbleGen.

[00124] As described herein, strains that naturally produce low levels of 3-methyl- 1 -butanol can also have applicability for producing increased levels of desirable fermentation products that are derived from biosynthetic pathways comprising a 2- ketoisovalerate intermediate. As would be understood by one skilled in the art equipped with the instant disclosure, strains that naturally produce low levels of 3- methyl-1 -butanol may inherently exhibit low or undetectable levels of endogenous enzyme activity, resulting in the reduced conversion of 2-ketoisovalerate to 3-methyl- 1 -butanol or leucine to 3-methyl-1 -butanol, a trait favorable for the production of a desirable fermentation product such as isobutanol. Described herein are several approaches for identifying a native host microorganism which is substantially free of activity of any of the enzymes described herein. For example, one approach to finding a host microorganism which exhibits inherently low or undetectable endogenous enzyme activity responsible for the production of 3-methyi-1 -butanol is to analyze yeast strains by incubating the yeast ceils with 2-ketoisovaierate or leucine and analyze the broth for the production of 3-methyl-1 -butanol.

Recombinant Microorganisms for the Production of 3-Methyi-1 -Butanol

[00125] The present invention also provides for, in some embodiments, the production of 3-methy!-1 -butanol as a product or a metabolic intermediate. In some embodiments, the recombinant microorganisms described herein are used to produce 3-methy!-1 -butanol. In some embodiments, a blend of isobutanol and 3- methyl-1 -butanoi is produced.

[00126] In one embodiment, one or more enzymes required for the production of 3- methyl-1 -butanol is overexpressed, as described herein, to yield increased amounts of 3-methy!-1 -butanol. In one embodiment, the enzyme to be overexpressed is a 2- isopropy!ma!ate synthase, catalyzing the conversion of 2-ketoisovalerate to 2- isopropyimaiate. In a specific embodiment, the 2-isopropylmalafe synthase is the S. cerevisiae Leu4 (SEQ ID NO: 2) or the S. cerevisiae Leu9 (SEO ID NO: 4) or a homoiog or variant thereof. In one embodiment, the enzyme to be overexpressed is a isopropylmalate isomerase, catalyzing the conversion of 2-isopropylmalate to 3- isopropylrnalate. In a specific embodiment, the isopropylmalate isomerase is the S, cerevisiae Leu1 (SEQ ID NO: 8) or a homoiog or variant thereof. In one embodiment, the enzyme to be overexpressed is a 3-isopropylmalate dehydrogenase, catalyzing the conversion of 3-isopropyimaiate to a-ketoisocaproate. In a specific embodiment, the 3-isopropylmalate dehydrogenase is the S. cerevisiae Leu2 (SEQ ID NO: 8) or a homoiog or variant thereof. In one embodiment, the enzyme to be overexpressed is a keto-isocaproate decarboxylase, catalyzing the conversion of a-ketoisocaproate to 3-methyibutanai. In a specific embodiment, the keto-isocaproate decarboxylase is the S. cerevisiae Aro10 (SEQ ID NO: 14) or the S. cerevisiae Thi3 (SEQ ID NO: 16) or homologs or variants thereof. In one embodiment, the enzyme is an alcohol dehydrogenase, catalyzing the conversion of 3-methylbutanal to 3-methyl-1 -butano!. In a specific embodiment, the alcohol dehydrogenase is the S. cerevisiae Adh6 (SEQ ID NO: 18) or the S. cerevisiae Adh7 (SEQ ID NO: 20) or homologs or variants thereof. In some embodiments, the overexpressed ADH may be engineered to require an NADH, as opposed to NADPH, cofactor.

[00127] In one embodiment, a blend of isobutanoi and 3-methyl-1 -butanol is produced that comprises a ratio of at least about 90:10 isobutanoi to 3~methyl-1 ~ butanoi. In another embodiment, a blend of isobutanoi and 3-methyi-1 -butanoi that comprises a ratio of at least about 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, or 10:90 isobutanoi to 3-methyl~1 -butanoi is produced.

Isobutanoi-Producing Yeast Microorganisms

[00128] A variety of microorganisms convert sugars to produce pyruvate, which is then utilized in a number of pathways of cellular metabolism. In recent years, microorganisms, including yeast, have been engineered to produce a number of desirable products via pyruvate-d riven biosynthetic pathways, including isobutanoi, an important commodity chemical and biofuei candidate (See, e.g., commonly owned and co-pending patent publications, US 2009/0228991 , US 2010/0143997, US 201 1/0020889, US 201 1/0076733, and WO 2010/075504). [00129] As described herein, the present invention relates to recombinant microorganisms for producing isobutanoi, wherein said recombinant microorganisms comprise an isobutanoi producing metabolic pathway. In one embodiment, the isobutanoi producing metabolic pathway to convert pyruvate to isobutanoi can be comprised of the following reactions:

1 . 2 pyruvate→ acetolactate + CO2

2. acetolactate + NAD(P)H ---> 2,3-dihydroxyisovalerate + NAD(P)⁺

3. 2,3-dihydroxyisovalerate→ alpha-ketoisovaierate

4. alpha-ketoisovaierate -→ isobutyraidehyde + CO2

5. isobutyraidehyde +NAD(P)H→ isobutanoi + NADP

[00130] In one embodiment, these reactions are carried out by the enzymes 1 ) Acetolactate synthase (ALS), 2) Ketol-acid reductoisomerase (KARI), 3) Dihydroxy- acid dehydratase (DHAD), 4) 2-keto-acid decarboxylase, e.g., Keto-isovalerate decarboxylase (KIVD), and 5) an Alcohol dehydrogenase (ADH) (Figure 1 ). in some embodiments, the recombinant microorganism may be engineered to overexpress one or more of these enzymes. In an exemplary embodiment, the recombinant microorganism is engineered to overexpress ail of these enzymes.

[00131] Alternative pathways for the production of isobutanoi in yeast have been described in WO/2007/050871 and in Dickinson ef a/., 1998, J Bioi Chem 273:25751 -6. These and other isobutanoi producing metabolic pathways are within the scope of the present application. In one embodiment, the isobutanoi producing metabolic pathway comprises five substrate to product reactions. In another embodiment, the isobutanoi producing metabolic pathway comprises six substrate to product reactions. In yet another embodiment, the isobutanoi producing metabolic pathway comprises seven substrate to product reactions.

[00132] In various embodiments described herein, the recombinant microorganism comprises an isobutanoi producing metabolic pathway. In one embodiment, the isobutanoi producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanoi. In another embodiment, the isobutanoi producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi. in yet another embodiment, the isobutanoi producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi. In yet another embodiment, the isobutano! producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi. In yet another embodiment, the isobutanoi producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi. In yet another embodiment, all of the isobutanoi producing metabolic pathway steps in the conversion of pyruvate to isobutanoi are converted by exogenously encoded enzymes.

[00133] In one embodiment, one or more of the isobutanoi pathway genes encodes an enzyme that is localized to the cytosol. In one embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least one isobutanoi pathway enzyme localized in the cytosol. In another embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least two isobutanoi pathway enzymes localized in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least three isobutanoi pathway enzymes localized in the cytosol. In yet another embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least four isobutanoi pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with five isobutanoi pathway enzymes localized in the cytosol. In yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with all isobutanoi pathway enzymes localized in the cytosol. Isobutanoi producing metabolic pathways in which one or more genes are localized to the cytosol are described in commonly owned and copending U.S. Publication No. 201 1/0078733, which is herein incorporated by reference in its entirety for all purposes.

[00134] As is understood in the art, a variety of organisms can serve as sources for the isobutanoi pathway enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. iactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including V. spp. stipitis, Toruiaspora pretonensis, Issatchenkia orientalis, Schizosaccharomyces spp., including S. pombe, Crypiococcus spp., Aspergillus spp., Neurospora spp., or Ustilago spp. Sources of genes from anaerobic fungi include, but not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp. Sources of prokaryotic enzymes that are useful include, but not limited to, Escherichia spp., Zymomonas spp., Staphylococcus spp., Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., Streptococcus spp., Salmonella spp., Siackia spp., Cryptobacterium spp., and Eggerthella spp.

[00135] In some embodiments, one or more of these enzymes can be encoded by native genes. Alternatively, one or more of these enzymes can be encoded by heterologous genes.

[00136] For example, acetolactate synthases capable of converting pyruvate to acetoiactate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including B, subiiiis (GenBank Accession No. Q04789.3), L lactis (GenBank Accession No. NP__267340.1 ), S. mutans (GenBank Accession No. NP_721805.1 ), K. pneumoniae (GenBank Accession No. ZPJ36014957.1 ), C. glutamicum (GenBank Accession No. P42483.1 ), E, cloacae (GenBank Accession No. YP_00361361 1 .1 ), M. maripaludis (GenBank Accession No. ABX01060.1 ), M. grisea (GenBank Accession No. AAB81248.1 ), T. stipitatus (GenBank Accession No. XP_002485976.1 ), or S. cerevisiae ILV2 (GenBank Accession No. NPJ313826.1 ). Additional acetoiactate synthases capable of converting pyruvate to acetolactate are described in commoniy owned and co-pending US Publication No. 201 1/0076733, which is herein incorporated by reference in its entirety. A review article characterizing the biosynthesis of acetoiactate from pyruvate via the activity of acetolactate synthases is provided by Chipman et a/., 1998, Biochimica et Biophysica Acta 1385: 401 -19, which is herein incorporated by reference in its entirety. Chipman et a/, provide an alignment and consensus for the sequences of a representative number of acetolactate synthases. Motifs shared in common between the majority of acetolactate synthases are disclosed in commonly owned and co-pending PCT Application No. PCT/US12/42824. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit acetoiactate synthase activity.

[00137] Ketol-acid reductoisomerases capable of converting acetoiactate to 2,3- dihydroxyisovalerate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including E. coli (GenBank Accession No. EGB30597.1 ), L. lactis (GenBank Accession No. YPJ3033S371 Q.1 ), S. exigua (GenBank Accession No. ZP_06160130.1 ), C. curiam (GenBank Accession No. YP_..003151266.1 ), Shewanella sp. (GenBank Accession No. YP__732498.1 ), V. fischeri (GenBank Accession No. YP__20591 1 .1 ), M. maripaludis (GenBank Accession No. YP_001097443.1 ), B. subtilis (GenBank Accession No. CAB14789), S. pombe (GenBank Accession No. NP__001018S45), B, thetaiotamicron (GenBank Accession No. NP__81 Q987), or S. cerevisiae ILV5 (GenBank Accession No. NP_013459.1 ). Additional ketoi-acid reductoisomerases capabie of converting acetoiac ate to 2,3- dihydroxyisovalerate are described in commonly owned and co-pending US Publication No. 201 1/0076733, which is herein incorporated by reference in its entirety. An alignment and consensus for the sequences of a representative number of ketoi-acid reductoisomerases is provided in commonly owned and co-pending US Publication No. 2010/0143997, which is herein incorporated by reference in its entirety. Motifs shared in common between the majority of ketol-acid reductoisomerases are disclosed in commonly owned an co-pending US Provisional Application No. 61/625,324, which is herein incorporated by reference in its entirety. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit ketoi-acid reductoisomerase activity.

[00138] Dihydroxy acid dehydratases capabie of converting 2,3- dihydroxyisova!erate to a-ketoisovaierate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including E. coli (GenBank Accession No. YP 026248.1 ), L iactis (GenBank Accession No. NP_267379.1 ), S. mutans (GenBank Accession No. NP__722414.1 ), M. stadtmanae (GenBank Accession No. YP__448586.1 ), M. iractuosa (GenBank Accession No. YP_004053736.1 ), Eubacterium SCB49 (GenBank Accession No. ZP_01890126.1 ), G, forsetti (GenBank Accession No. YP__862145.1 ), Y. lipolytica (GenBank Accession No. XP__502180.2), N. crassa (GenBank Accession No. XP_963045.1 ), or S. cerevisiae ILV3 (GenBank Accession No. NP__012550.1 ). Additional dihydroxy acid dehydratases capable of 2,3-dihydroxyisovalerate to a-ketoisovalerate are described in commonly owned and co-pending US Publication No. 201 1/0076733. Motifs shared in common between the majority of dihydroxy acid dehydratases are disclosed in commonly owned and co-pending PCT Application No. PCT/US12/42624. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit dihydroxy acid dehydratase activity. [00139] 2-keio-acid decarboxylases capable of converting a-ketoisovalerate to isobutyraldehyde may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including L laciis kivD (GenBank Accession No. YPJ3033S382Q.1 ), £. cloacae (GenBank Accession No. P23234.1 ), M. smegmatis (GenBank Accession No. A0R480.1 ), M. tuberculosis (GenBank Accession No. 053885.1 ), M. avium (GenBank Accession No. Q742Q2.1 , A. brasilense (GenBank Accession No. P51852.1 ), L lactis kdcA (GenBank Accession No. AAS49166.1 ), S. epidermidis (GenBank Accession No. NP 765765.1 ), M. caseoiyticus (GenBank Accession No. YP_002560734.1 ), B. megaterium (GenBank Accession No. YP_003561644.1 ), S. cerevisiae Aro10 (GenBank Accession No. NP 010668.1 ), or S. cerevisiae Thi3 (GenBank Accession No. CAA98646.1 ). Additional 2-keto-acid decarboxylases capable of converting a-ketoisovalerate to isobutyraldehyde are described in commonly owned and co-pending US Publication No. 201 1/0076733. Motifs shared in common between the majority of 2-keto-acid decarboxylases are disclosed in commonly owned and co-pending PCT Application No. PCT/US 12/42824. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit 2-keto-acid decarboxylase activity.

[00140] Alcohol dehydrogenases capable of converting isobutyraldehyde to isobutanol may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including L lactis (GenBank Accession No. YP_003354381 ), S. cereus (GenBank Accession No. YP 001374103.1 ), N. meningitidis (GenBank Accession No. CBA03985.1 ), S. sanguinis (GenBank Accession No. YP_001035842.1 ), L, brevis (GenBank Accession No. YP__794451 .1 ), B. thuringiensis (GenBank Accession No. ZP_04101989.1 ), P. acidiiactici (GenBank Accession No. ZP_06197454.1 ), B. subtilis (GenBank Accession No. EHA31 1 15.1 ), N. crassa (GenBank Accession No. CAB91241 .1 ) or S. cerevisiae Adh6 (GenBank Accession No. NP_014051 .1 ). Additional alcohol dehydrogenases capable of converting isobutyraldehyde to isobutanol are described in commonly owned and co-pending US Publication Nos. 201 1/0076733 and 201 1/0201072. Motifs shared in common between the majority of alcohol dehydrogenases are disclosed in commonly owned and co-pending PCT Application No. PCT/US 12/42624. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit alcohol dehydrogenase activity. [00141] In an exemplary embodiment, pathway steps 2 and 5 of the isobutanol pathway may be carried out by KARI and ADH enzymes that utilize NADH (rather than NADPH) as a cofactor. It has been found previously that utilization of NADH- dependent KARI (NKR) and ADH enzymes to catalyze pathway steps 2 and 5, respectively, surprisingly enables production of isobutanol at theoretical yield and/or under anaerobic conditions. See, e.g., commonly owned and co-pending patent publication US 2010/0143997. An example of an NADH-dependent isobutanol pathway is illustrated in Figure 2. Thus, in one embodiment, the recombinant microorganisms of the present invention may use an NKR to catalyze the conversion of acetoiactate to produce 2,3-dihydroxyisovalerate, In another embodiment, the recombinant microorganisms of the present invention may use an NADH-dependent ADH to catalyze the conversion of isobutyraidehyde to produce isobutanol. In yet another embodiment, the recombinant microorganisms of the present invention may use both an NKR to catalyze the conversion of acetoiactate to produce 2,3- dihydroxyisovalerate, and an NADH-dependent ADH to catalyze the conversion of isobutyraidehyde to produce isobutanol.

[00142] In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutanol. In one embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutyraidehyde. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to keto-isovaierate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to 2,3-dihydroxyisovalerate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to acetoiactate.

[00143] Furthermore, any of the genes encoding the foregoing enzymes (or any others mentioned herein (or any of the regulatory elements that control or modulate expression thereof)) may be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast.

[00144] In one embodiment, the invention is directed to a recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 2-isopropyimaiate; one or more enzymes catalyzing the conversion of 2-isopropyimaiate to 3-isopropylmalate; one or more enzymes catalyzing the conversion of 3-isopropylmalate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of leucine to a-ketoisocaproate; one or more enzymes catalyzing the conversion of α-ketoisocaproate to 3-methy!butanal; and one or more enzymes catalyzing the conversion of 3-methyibutana! to 3-methyl- -butanoi.

[00145] In some embodiments, the recombinant microorganisms comprising an isobutanol producing metabolic pathway are engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2- ketoisovalerate to 3-methyl-1 -butanoI. In one embodiment, the enzyme is a 2- isopropylmalate synthase, catalyzing the conversion of 2-ketoisovalerate to 2- isopropyimaiate. In a specific embodiment, the 2-isopropyimaiate synthase is the S. cerevisiae Leu4 (SEO ID NO: 2) or S. cerevisiae Leu9 (SEQ ID NO: 4) or a homoiog or variant thereof. In one embodiment, the enzyme is a isopropyimalate isomerase, catalyzing the conversion of 2-isopropyimaiate to 3-isopropylmalate. In a specific embodiment, the isopropyimalate isomerase is the S. cerevisiae Leu1 (SEQ ID NO: 8) or a homoiog or variant thereof. In one embodiment, the enzyme is an 3- isoproyimaiate dehydrogenase, catalyzing the conversion of 3-isopropylmalate to Q- ketoisocaproate. In a specific embodiment, the 3-isoproylmalate dehydrogenase is the S. cerevisiae Leu2 (SEQ ID NO: 8) or a homoiog or variant thereof. In one embodiment, the enzyme is a branched-chain amino acid transaminase, catalyzing the conversion of leucine to α-kefoisocaproate. In a specific embodiment, the branched-chain amino acid transaminase is S. cerevisiae Bat1 (SEQ ID NO: 10) or S. cerevisiae Bat2 (SEQ ID NO: 12) or a homoiog or variant thereof. In one embodiment, the enzyme is a keto-isocaproate decarboxylase, catalyzing the conversion of a-ketoisocaproate to 3-methy!butanai. In a specific embodiment, the keto-isocaproate decarboxylase is S. cerevisiae Aro10 (SEQ ID NO: 14), Ths3 (SEQ ID NO: 16), or homoiogs or variants thereof. In one embodiment, the enzyme is an alcohol dehydrogenase, catalyzing the conversion of 3-methylbutanal to 3-methyl-1 - butanoi. In a specific embodiment, the alcohol dehydrogenase is the S. cerevisiae Adh6 (SEQ ID NO: 18), Adh7 (SEQ ID NO: 20), or homoiogs or variants thereof. [00146] In various aspects, the present invention also provides for a recombinant microorganism comprising an isobutanoi producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following and/or substantially free of an enzyme catalyzing the conversion of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovaierate to 2-isopropy!malate; one or more enzymes catalyzing the conversion of 2-isopropyimaiate to 3-isopropylmalate; one or more enzymes catalyzing the conversion of 3-isopropyima!ate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of leucine to a-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methylbutana!; and one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methyl- 1 -butanoi.

[00147] In another embodiment, the recombinant microorganisms are engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the initial steps in the conversion of 2-ketoisovalerate to 3-methy!-1 -butanol. In one embodiment, the enzyme is a 2-isopropylmaiate synthase, catalyzing the conversion of 2-ketoisovaierate to 2-isopropylmalate, In a specific embodiment, the 2- isopropylmaiate synthase is the S. cerevisiae Leu4 (SEQ ID NO: 2) or the S. cerevisiae Leu9 (SEQ ID NO: 4) or a homolog or variant thereof. In one embodiment, the enzyme is a branched-chain amino acid transaminase, catalyzing the conversion of leucine to α-ketoisocaproate. In a specific embodiment, the branched-chain amino acid transaminase is S. cerevisiae Bat1 (SEQ ID NO: 10) or S. cerevisiae Bat.2 (SEQ ID NO: 12) or a homolog or variant thereof.

The Microorganism in General

[00148] As described herein, the recombinant microorganisms of the present invention can express a plurality of heterologous and/or native enzymes involved in pathways for the production of a beneficial metabolite such as isobutanoi,

[00149] As described herein, "engineered" or "modified" microorganisms are produced via the introduction of genetic material into a host or parental microorganism of choice and/or by modification of the expression of native genes, thereby modifying or altering the cellular physiology and biochemistry of the microorganism, Through the introduction of genetic material and/or the modification of the expression of native genes the parental microorganism acquires new properties, e.g., the ability to produce a new, or greater quantities of, an intracellular and/or extracellular metabolite. As described herein, the introduction of genetic material into and/or the modification of the expression of native genes in a parental microorganism results in a new or modified ability to produce beneficial metabolites, such as isobutanoi, valine, pantothenate, and coenzyme A, from a suitable carbon source. The genetic material introduced into and/or the genes modified for expression in the parental microorganism contains gene{s), or parts of genes, coding for one or more of the enzymes involved in a biosynthetic pathway for the production of one or more metabolites selected from isobutanoi, valine, pantothenate, and coenzyme A and may also include additional elements for the expression and/or regulation of expression of these genes, e.g., promoter sequences.

[00150] In addition to the introduction of a genetic material into a host or parental microorganism, an engineered or modified microorganism can also include the alteration, disruption, deletion or knocking-out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism. Through the alteration, disruption, deletion or knocking-out of a gene or polynucleotide, the microorganism acquires new or improved properties (e.g., the ability to produce a new metabolite or greater quantities of an intracellular metabolite, to improve the flux of a metabolite down a desired pathway, and/or to reduce the production of by-products).

[00151] Recombinant microorganisms provided herein may also produce metabolites in quantities not available in the parental microorganism. A "metabolite" refers to any substance produced by metabolism or a substance necessary for or taking part in a particular metabolic process. A metabolite can be an organic compound that is a starting material (e.g., glucose or pyruvate), an intermediate (e.g., 2-ketoisovaierate), or an end product (e.g., isobutanoi) of metabolism. Metabolites can be used to construct more complex molecules, or they can be broken down into simpler ones. Intermediate metabolites may be synthesized from other metabolites, perhaps used to make more complex substances, or broken down into simpler compounds, often with the release of chemical energy.

[00152] The disclosure identifies specific genes useful in the methods, compositions and organisms of the disclosure; however it will be recognized that absolute identify to such genes is not necessary. For example, changes in a particular gene or polynucleotide comprising a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically, such changes comprise conservative mutations and silent mutations. Such modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art.

[00153] Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or functionally equivalent polypeptides can also be used to done and express the polynucleotides encoding such enzymes.

[00154] As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low- usage codons. Codons can be substituted to reflect the preferred codon usage of the host, in a process sometimes called "codon optimization" or "controlling for species codon bias."

[00155] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (Murray et a/., 1989, Nuci Acids Res. 17: 477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and £. coli commonly use UAA as the stop codon (Daiphin et a/., 1996, Nuci Acids Res. 24: 218-8). Methodology for optimizing a nucleotide sequence for expression in a plant is provided, for example, in U.S. Pat. No. 6,015,891 , and the references cited therein.

[00156] Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds differing in their nucleotide sequences can be used to encode a given enzyme of the disclosure. The native DNA sequence encoding the biosynthetic enzymes described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide. Furthermore, the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.

[00157] In addition, homo!ogs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein.

[00158] As used herein, two proteins (or a region of the proteins) are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and nonhomologous sequences can be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 80%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[00159] When "homologous" is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson W.R., 1994, Methods in Mo! Biol 25: 365-89.

[00160] The following six groups each contain amino acids that are conservative substitutions for one another: 1 ) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Giutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Alanine (A), Valine (V), and 8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[00161] Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See commonly owned U.S. Pat. No. 8,017,375. A typical algorithm used comparing a molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST. When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms described in commonly owned U.S. Pat. No. 8,017,375.

[00162] It is understood that a range of microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of beneficial metabolites from biosynthetic pathways requiring the use of 2-ketoisovalerate as an intermediate. In various embodiments, microorganisms may be selected from yeast microorganisms. Yeast microorganisms for the production of a metabolite such as isobutanoi, valine, pantothenate, and coenzyme A may be selected based on certain characteristics.

[00163] One characteristic may include the property that the microorganism is selected to convert various carbon sources into beneficial metabolites such as isobutanoi, valine, pantothenate, and coenzyme A. The term "carbon source" generally refers to a substance suitable to be used as a source of carbon for prokaryotic or eukaryotic ceil growth. Examples of suitable carbon sources are described in commonly owned U.S. Pat. No. 8,017,375. Accordingly, in one embodiment, the recombinant microorganism herein disclosed can convert a variety of carbon sources to products, including but not limited to glucose, galactose, mannose, xylose, arabinose, lactose, sucrose, and mixtures thereof.

[00164] The recombinant microorganism may thus further include a pathway for the production of isobutanol from five-carbon (pentose) sugars including xylose. Most yeast species metabolize xylose via a complex route, in which xylose is first reduced to xylitol via a xylose reductase (XR) enzyme. The xylitol is then oxidized to xylulose via a xylitol dehydrogenase (XDH) enzyme. The xylulose is then phosphoryiated via a xyiulokinase (XK) enzyme. This pathway operates inefficiently in yeast species because it introduces a redox imbalance in the ceil. The xyiose-to- xylitol step uses primarily NADPH as a cofactor (generating NADP+), whereas the xylitol-to-xylulose step uses NAD+ as a cofactor (generating NADH). Other processes must operate to restore the redox imbalance within the cell. This often means that the organism cannot grow anaerobicaily on xylose or other pentose sugars. Accordingly, a yeast species that can efficiently ferment xylose and other pentose sugars into a desired fermentation product is therefore very desirable.

[00165] Thus, in one aspect, the recombinant microorganism is engineered to express a functional exogenous xylose isomerase. Exogenous xylose isomerases (XI) functional in yeast are known in the art. See, e.g., Rajgarhia et ai., U.S. Pat. No. 7,943,366, which is herein incorporated by reference in its entirety. In an embodiment according to this aspect, the exogenous XI gene is operatively linked to promoter and terminator sequences that are functional in the yeast cell. In a preferred embodiment, the recombinant microorganism further has a deletion or disruption of a native gene that encodes for an enzyme (e.g., XR and/or XDH) that catalyzes the conversion of xylose to xylitol. In a further preferred embodiment, the recombinant microorganism also contains a functional, exogenous xyiulokinase (XK) gene operatively linked to promoter and terminator sequences that are functional in the yeast ceil. In one embodiment, the xyiulokinase (XK) gene is overexpressed.

[00166] In one embodiment, the yeast microorganism has reduced or no pyruvate decarboxylase (PDC) activity. PDC catalyzes the decarboxylation of pyruvate to acetaldehyde, which is then reduced to ethanol by ADH via an oxidation of NADH to NAD+. Ethanol production is the main pathway to oxidize the NADH from glycolysis. Deletion, disruption, or mutation of this pathway increases the pyruvate and the reducing equivalents (NADH) available for a biosynthetic pathway which uses pyruvate as the starting material and/or as an intermediate. Accordingly, deletion, disruption, or mutation of one or more genes encoding for pyruvate decarboxylase and/or a positive transcriptional regulator thereof can further increase the yield of the desired pyruvate-derived metabolite {e.g., isobutanoi). In one embodiment, said pyruvate decarboxylase gene targeted for disruption, deletion, or mutation is selected from the group consisting of PDC1, PDC5, and PDC6, or homologs or variants thereof. In another embodiment, all three of PDC1, PDC5, and PDC6 are targeted for disruption, deletion, or mutation. In yet another embodiment, a positive transcriptional regulator of the PDC1, PDC5, and/or PDC6 is targeted for disruption, deletion or mutation. In one embodiment, said positive transcriptional regulator is PDC2, or homologs or variants thereof,

[00167] As is understood by those skilled in the art, there are several additional mechanisms available for reducing or disrupting the activity of a protein encoded by PDC1, PDC5, PDC6, and/or PDC2, including, but not limited to, the use of a regulated promoter, use of a weak constitutive promoter, disruption of one of the two copies of the gene in a diploid yeast, disruption of both copies of the gene in a diploid yeast, expression of an anti-sense nucleic acid, expression of an siRNA, over expression of a negative regulator of the endogenous promoter, alteration of the activity of an endogenous or heterologous gene, use of a heterologous gene with lower specific activity, the like or combinations thereof. Yeast strains with reduced PDC activity are described in commonly owned U.S. Pat. No. 8.017,375, as well as commonly owned and co-pending US Patent Publication No. 201 1/0183392,

[00168] In another embodiment, the microorganism has reduced giycerol-3- phosphate dehydrogenase (GPD) activity. GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD+. Glycerol is then produced from G3P by Glycerol-3- phosphatase (GPP). Glycerol production is a secondary pathway to oxidize excess NADH from glycolysis. Reduction or elimination of this pathway would increase the pyruvate and reducing equivalents (NADH) available for the production of a pyruvate-derived metabolite (e.g., isobutanoi). Thus, disruption, deletion, or mutation of the genes encoding for giycerol-3-phosphate dehydrogenases can further increase the yield of the desired metabolite (e.g., isobutanoi). Yeast strains with reduced GPD activity are described in commonly owned and co-pending US Patent Publication Nos. 201 1/0020889 and 201 1/0183392. [00169] In yet another embodiment, the microorganism has reduced 3-keto acid reductase (3-KAR) activity. 3-KARs catalyze the conversion of 3-keto acids {e.g., acetolactate) to 3-hydroxyacids (e.g., DH2MB). Yeast strains with reduced 3-KAR activity are described in commonly owned U.S. Pat. Nos. 8,133,715, 8,153,415, and 8,158,404, which are herein incorporated by reference in their entireties.

[00170] In yet another embodiment, the microorganism has reduced aldehyde dehydrogenase (ALDH) activity. Aldehyde dehydrogenases catalyze the conversion of aldehydes (e.g., isobutyraldehyde) to acid by-products (e.g., isobutyrate). Yeast strains with reduced ALDH activity are described in commonly owned U.S. Pat. Nos. 8,133,715, 8,153,415, and 8,158,404, which are herein incorporated by reference in their entireties.

[00171] In one embodiment, the yeast microorganisms may be selected from the "Saccharomyces Yeast C!ade", as described in commonly owned U.S. Pat. No. 8,017,375.

[00172] The term "Saccharomyces sensu stricto" taxonomy group is a cluster of yeast species that are highly related to S. cerevssiae (Rainier! et ai., 2003, J. Biosci Bioengin 96: 1 -9). Saccharomyces sesisu stricto yeast species include but are not limited to S. cerevssiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarurn, S. carocanis and hybrids derived from these species (Masneuf et ai., 1998, Yeast 7: 61 - 72).

[00173] An ancient whole genome duplication (WGD) event occurred during the evolution of the hemiascomycete yeast and was discovered using comparative genomic tools (Kellis et ai, 2004, Nature 428: 617-24; Dujon et ai, 2004, Nature 430:35-44; Langkjaer et a/., 2003, Nature 428: 848-52; Wolfe et ai, 1997, Nature 387: 708-13). Using this major evolutionary event, yeast can be divided into species that diverged from a common ancestor following the WGD event (termed "post- WGD yeast" herein) and species that diverged from the yeast lineage prior to the WGD event (termed "pre-WGD yeast" herein).

[00174] Accordingly, in one embodiment, the yeast microorganism may be selected from a post-WGD yeast genus, including but not limited to Saccharomyces and Candida. The favored post-WGD yeast species include: S. cerevisiae, S. uvarurn, S. bayanus, S. paradoxus, S. casie!li, and C. glabrata.

[00175] In another embodiment, the yeast microorganism may be selected from a pre-whole genome duplication (pre-WGD) yeast genus including but not limited to Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Yarrowia and, Schizosaccharomyces. Representative pre-WGD yeast species inciude: S. kluyveri, K. thermotolerans, K. marxianus, K. waltii, K. iactis, C. tmpicalis, P. pastoris, P, anoma!a, P. stipitis, I. orientalis, I. occidentalis, I. scutulata, D. hansenii, H. anomala, Y. lipolytica, and S. pombe.

[00176] A yeast microorganism may be either Crabtree-negative or Crabtree- positive as described in described in commonly owned U.S. Pat. No. 8,017,375. In one embodiment the yeast microorganism may be selected from yeast with a Crabtree-negative phenotype including but not limited to the following genera: Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida. Crabtree-negative species include but are not limited to: S. kluyveri, K. Iactis, K. marxianus, P. anomala, P. stipitis, I. orientaiis, I. occidentalis, i scutulata, H. anomala, and C. utiiis. In another embodiment, the yeast microorganism may be selected from yeast with a Crabtree-positive phenotype, including but not limited to Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Pichia and Schizosaccharomyces. Crabtree-positive yeast species include but are not limited to: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S. castelli, K, thermotolerans, C. glabrata, Z. basils^', Z. rouxii, D. hansenii, P, pastorius, and S. pombe.

[00177] Another characteristic may include the property that the microorganism is that it is non-fermenting. In other words, it cannot metabolize a carbon source anaerobicaliy while the yeast is able to metabolize a carbon source in the presence of oxygen. Nonfermenting yeast refers to both naturally occurring yeasts as well as genetically modified yeast. During anaerobic fermentation with fermentative yeast, the main pathway to oxidize the NADH from glycolysis is through the production of ethanoi. Ethanol is produced by alcohol dehydrogenase (ADH) via the reduction of acetaidehyde, which is generated from pyruvate by pyruvate decarboxylase (PDC). !n one embodiment, a fermentative yeast can be engineered to be non-fermentative by the reduction or elimination of the native PDC activity. Thus, most of the pyruvate produced by glycolysis is not consumed by PDC and is available for the isobutanoi pathway. Deletion of this pathway increases the pyruvate and the reducing equivalents available for the biosynthetic pathway. Fermentative pathways contribute to low yield and low productivity of pyruvate-derived metabolites such as isobutanol. Accordingly, deletion of one or more PDC genes may increase yield and productivity of a desired metabolite {e.g., isobutanol).

[00178] In some embodiments_., the recombinant microorganisms may be microorganisms that are non-fermenting yeast microorganisms, including, but not limited to those, classified into a genera selected from the group consisting of Tricosporon, Rhodotorula, Myxozyma, or Candida. In a specific embodiment, the non-fermenting yeast is C. xestobii.

[00179] In alternative embodiments, the recombinant microorganisms may be derived from bacterial microorganisms. In various embodiments the recombinant microorganism may be selected from a genus of Citrobacter, Corynebacterium, Lactobacillus, Lactococcus, Salmonella, Enterobacter, Enterococcus, Erwinia, Pantoea, Morganella, Pectobacterium, Proteus, Serratia, Shigella, and Klebsiella. In one specific embodiment, the recombinant microorganism is a Iactic acid bacteria such as, for example, a microorganism derived from the Lactobacillus or Lactococcus genus.

Methods in General

Identification of Homoiogs of the Enzymes Described Herein

[00180] Any method can be used to identify genes that encode for enzymes that are homologous to the genes described herein {e.g., 2-isopropyImalate synthase homoiogs, isopropyimaiate isomerase homoiogs, 3-isoproyimaiate dehydrogenase homoiogs, branched-chain amino acid transaminase homoiogs, keto-isocaproate decarboxylase homoiogs, and alcohol dehydrogenase homoiogs, etc.). Generally, genes that are homologous or similar to the 2-isopropyimalate synthases, isopropyimaiate isomerases, 3-isoproy!maiate dehydrogenases, branched-chain amino acid transaminases, keto-isocaproate decarboxylases, and alcohol dehydrogenases described herein may be identified by functional, structural, and/or genetic analysis. In most cases, homologous or similar genes and/or homologous or similar enzymes will have functional, structural, or genetic similarities.

[00181] Techniques known to those skilled in the art may be suitable to identify additional homologous genes and homologous enzymes. Generally, analogous genes and/or analogous enzymes can be identified by functional analysis and will have functional similarities. Techniques known to those skilled in the art may be suitable to identify analogous genes and analogous enzymes. For example, to identify homologous or analogous genes, proteins, or enzymes, techniques may include, but are not limited to, cloning a gene by PGR using primers based on a published sequence of a gene/enzyme or by degenerate PGR using degenerate primers designed to amplify a conserved region among dehydratase genes. Further, one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity. Techniques include examining a ceil or ceil culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity (e.g. as described herein or in Kiritani, K. Branched- Chain Amino Acids Methods Enzymoiogy, 1970), then isolating the enzyme with said activity through purification, determining the protein sequence of the enzyme through techniques such as Edman degradation, design of PGR primers to the likely nucleic acid sequence, amplification of said DNA sequence through PGR, and cloning of said nucleic acid sequence. To identify homologous or similar genes and/or homologous or similar enzymes, analogous genes and/or analogous enzymes or proteins, techniques also include comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC, The candidate gene or enzyme may be identified within the above mentioned databases in accordance with the teachings herein.

[00182] Additional homologs using a profile Hidden Markov Model (HMM) following the user guide which is available from: HMMER (Janelia Farm Research Campus, Ashburn, Va.). The output of the HMMER software program is a profile Hidden Markov Model (profile HMM) that characterizes the input sequences. As stated in the user guide, profile HMMs are statistical descriptions of the consensus of a multiple sequence alignment. They use position-specific scores for amino acids (or nucleotides) and position specific scores for opening and extending an insertion or deletion. Compared to other profile based methods, HMMs have a formal probabilistic basis. Profile HMMs for a large number of protein families are publicly available in the PFAM database (Janelia Farm Research Campus, Ashburn, Va.). Any homologous protein that matches the Profile HMM with an E value of < 10^"3 using hmmsearch program in the HMMER package is expected to be a functional homoiog. Accordingly, the present invention provides in some embodiments, microorganisms with the reduced expression and/or activity of 2-isopropylmalate synthase homologs, isopropylmalate isomerase homologs, 3-isoproylmalate dehydrogenase homologs, branched-chain amino acid transaminase homologs, keto-isocaproate decarboxylase homologs, and alcohol dehydrogenase homologs, wherein said homologs have an MM search profile E value of < 10^"3 using the hmmsearch program.

[00183] In various embodiments, the endogenous nucleic acid or polypeptide identified herein is the S. cerevisiae version of the nucleic acid or polypeptide {e.g., Leu4, Leu9, Leu1 , Leu2, Bat1 , Bat2, Aro10, Thi3, Adh8, Adh7, etc.). Any method can be used to identify genes that encode for the endogenous polypeptide of interest in a variety of yeast strains. Generally, genes that are homologous or similar to the endogenous polypeptide of interest can be identified by functional, structural, and/or genetic analysis. Homologous or similar polypeptides will generally have functional, structural, or genetic similarities.

[00184] The chromosomal location of the genes encoding endogenous S. cerevisiae polypeptides (e.g., Leu4, Leu9, Leu1 , Leu2, Bat1 , Bat2, Aro10, Thi3, Adh6, Adh7, etc.) may be syntenic to chromosomes in many related yeast [Byrne, K.P. and K. H. Wolfe (2005) "The Yeast Gene Order Browser: combining curated homology and syntenic context reveals gene fate in polyploid species." Genome Res. 15(10):1458-61 . Scannell, D. R., K, P. Byrne, J. L. Gordon, S. Wong, and K. H. Wolfe (2008) "Multiple rounds of speciation associated with reciprocal gene loss in polyploidy yeasts." Nature 440: 341 -5. Scannell, D. R., A. C. Frank, G. C. Conant, K. P. Byrne, M. Woolfit, and K. H. Wolfe (2007)" Independent sorting-out of thousands of duplicated gene pairs in two yeast species descended from a whole- genome duplication." Proc Nail Acad Sci U S A 104: 8397-402]. Using this syntenic relationship, species-specific versions of these genes are readily identified in a variety of yeast, including but not limited to, Ashbya gossypii, Candida glabrata, Kiuyveromyces lactis, Kiuyveromyces polyspora, Kiuyveromyces thermotolerans, Kiuyveromyces waitii, Saccharomyces kiuyveri, Saccharomyces casteiii, Saccharomyces bayanus, and Zygosaccharomyces rouxii.

[00185] As will be understood by one skilled in the art, this technique is therefore additionally suitable for the identification homologous {e.g., Leu4, Leu9, Leu1 , Leu2, Bati , Bat2, Aro10, Thi3, Adh8, Adh7, etc.) polypeptides in yeast other than S. cerevisiae. Genetic insertions and Deletions

[00186] Any method can be used to introduce a nucleic acid molecule into yeast and many such methods are well known. For example, transformation and electroporation are common methods for introducing nucleic acid into yeast cells. See, e.g., Gietz et ai., 1992, Nuc Acids Res. 27: 69-74; Ito et ai, 1983, J. Bacterioi. 153: 183-8; and Becker et al., 1991 , Methods in Enzymo!ogy 194: 182-7,

[00187] In an embodiment, the integration of a gene of interest into a DNA fragment or target gene of a yeast microorganism occurs according to the principle of homologous recombination. According to this embodiment, an integration cassette containing a module comprising at least one yeast marker gene and/or the gene to be integrated (internal module) is flanked on either side by DNA fragments homologous to those of the ends of the targeted integration site (recombinogenic sequences). After transforming the yeast with the cassette by appropriate methods, a homologous recombination between the recombinogenic sequences may result in the interna! module replacing the chromosomal region in between the two sites of the genome corresponding to the recombinogenic sequences of the integration cassette. (Orr-Weaver ef a/., 1981 , PNAS USA 78: 6354-58),

[00188] In an embodiment, the integration cassette for integration of a gene of interest into a yeast microorganism includes the heterologous gene under the control of an appropriate promoter and terminator together with the selectable marker flanked by recombinogenic sequences for integration of a heterologous gene into the yeast chromosome, In an embodiment, the heterologous gene includes an appropriate native gene desired to increase the copy number of a native gene(s). The selectable marker gene can be any marker gene used in yeast, including but not limited to, HIS3, TRP1, Leu2, URA3, bar, hie, hph, and kan. The recombinogenic sequences can be chosen at will, depending on the desired integration site suitable for the desired application.

[00189] In another embodiment, integration of a gene into the chromosome of the yeast microorganism may occur via random integration (Kooistra et ai., 2004, Yeast 21 : 781 -792).

[00190] Additionally, in an embodiment, certain introduced marker genes are removed from the genome using techniques well known to those skilled in the art. For example, URA3 marker loss can be obtained by plating URA3 containing cells in FOA (5~fluoro~orotic acid) containing medium and selecting for FOA resistant colonies (Boeke et al., 1984, MoL Gen. Genet 197: 345-47).

[00191] The exogenous nucleic acid molecule contained within a yeast cell of the disclosure can be maintained within that ceil in any form, For example, exogenous nucleic acid molecules can be integrated into the genome of the cell or maintained in an episoma! state that can stably be passed on ("inherited") to daughter cells. Such extra-chromosomal genetic elements (such as piasmids, mitochondrial genome, etc.) can additionally contain selection markers that ensure the presence of such genetic elements in daughter cells. Moreover, the yeast cells can be stably or transiently transformed, !n addition, the yeast cells described herein can contain a single copy, or multiple copies of a particular exogenous nucleic acid molecule as described above.

Reduction of Enzymatic Activity

[00192] Yeast microorganisms within the scope of the invention may have reduced enzymatic activity such as, for example, reduced 2-isopropylmalafe synthase, isopropylmalate isomerase, 3-isoproylmaiate dehydrogenase, branched-chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, or GPD activity. The term "reduced" as used herein with respect to a particular enzymatic activity refers to a lower level of enzymatic activity than that measured in a comparable yeast cell of the same species. The term reduced also refers to the elimination of enzymatic activity as compared to a comparable yeast ceil of the same species. Thus, for example, yeast cells lacking 2-isopropylmalate synthase, isopropylmalate isomerase, 3-isoproylmalate dehydrogenase, branched- chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, or GPD activity are considered to have reduced 2-isopropylmaiate synthase, isopropylmalate isomerase, 3-isoproylmalate dehydrogenase, branched-chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, or GPD activity since most, if not ail, comparable yeast strains have at least some 2-isopropylmalate synthase, isopropylmalate isomerase, 3-isoproylmalate dehydrogenase, branched- chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, or GPD activity. Such reduced enzymatic activities can be the result of lower enzyme concentration, lower specific activity of an enzyme, or a combination thereof. Many different methods can be used to make yeast having reduced enzymatic activity. For example, a yeast cell can be engineered to have a disrupted enzyme-encoding locus using common mutagenesis or knock-out technology. See, e.g., Methods in Yeast Genetics (1997 edition), Adams, et a!., Cold Spring Harbor Press (1998). In addition, certain poinf- mutation(s) can be introduced which results in an enzyme with reduced activity. Also included within the scope of this invention are yeast strains which when found in nature, are substantially free of one or more activities selected from 2- isopropyimaiate synthase, isopropylmalate isomerase, 3-isoproylmalate dehydrogenase, branched-chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, or GPD activity.

[00193] Alternatively, anfisense technology can be used to reduce enzymatic activity. For example, yeast can be engineered to contain a cDNA that encodes an antisense molecule that prevents an enzyme from being made. The term "antisense molecule" as used herein encompasses any nucleic acid molecule that contains sequences that correspond to the coding strand of an endogenous polypeptide. An antisense molecule also can have flanking sequences (e.g., regulatory sequences). Thus antisense molecules can be ribozymes or antisense oligonucleotides. A ribozyme can have any general structure including, without limitation, hairpin, hammerhead, or axhead structures, provided the molecule cleaves RNA.

[00194] Yeasts having a reduced enzymatic activity can be identified using many methods. For example, yeasts having reduced 2-isopropyimalate synthase, isopropyimaiate isomerase, 3-isoproylmaiate dehydrogenase, branched-chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, or GPD activity can be easily identified using common methods, which may include, for example, measuring for the formation of the by-products produced by such enzymes via liquid chromatography.

Overexpression of Heterologous Genes

[00195] Methods for overexpressing a polypeptide from a native or heterologous nucleic acid molecule are well known. Such methods include, without limitation, constructing a nucleic acid sequence such that a regulatory element promotes the expression of a nucleic acid sequence that encodes the desired polypeptide. Typically, regulatory elements are DNA sequences that regulate the expression of other DNA sequences at the level of transcription. Thus, regulatory elements include, without limitation, promoters, enhancers, and the like. For example, the exogenous genes can be under the control of an inducible promoter or a constitutive promoter, Moreover, methods for expressing a polypeptide from an exogenous nucleic acid molecule in yeast are well known. For example, nucleic acid constructs that are used for the expression of exogenous polypeptides within Kluyveromyces and Saccharomyces are well known (see, e.g., U.S. Pat. Nos. 4,859,596 and 4,943,529, for Kluyveromyces and, e.g., Gellissen et a/., Gene 190(1 ):87-97 (1997) for Saccharomyces). Yeast piasmids have a selectable marker and an origin of replication. In addition certain piasmids may also contain a centromeric sequence. These centromeric piasmids are generally a single or low copy plasmid. Piasmids without a centromeric sequence and utilizing either a 2 micron (S. cerevisiae) or 1 .8 micron (K. lactls) replication origin are high copy piasmids. The selectable marker can be either prototrophic, such as HIS3, TRP1, Leu2, URA3 or ADE2, or antibiotic resistance, such as, bar, ble, hph, or kan,

[00196] In another embodiment, heterologous control elements can be used to activate or repress expression of endogenous genes. Additionally, when expression is to be repressed or eliminated, the gene for the relevant enzyme, protein or RNA can be eliminated by known deletion techniques,

[00197] As described herein, any yeast within the scope of the disclosure can be identified by selection techniques specific to the particular enzyme being expressed, over-expressed or repressed. Methods of identifying the strains with the desired phenotype are well known to those skilled in the art. Such methods include, without limitation, PGR, RT-PCR, and nucleic acid hybridization techniques such as Northern and Southern analysis, altered growth capabilities on a particular substrate or in the presence of a particular substrate, a chemical compound, a selection agent and the like. In some cases, immunohistochemistry and biochemical techniques can be used to determine if a cell contains a particular nucleic acid by detecting the expression of the encoded polypeptide. For example, an antibody having specificity for an encoded enzyme can be used to determine whether or not a particular yeast ceil contains that encoded enzyme. Further, biochemical techniques can be used to determine if a ceil contains a particular nucleic acid molecule encoding an enzymatic polypeptide by detecting a product produced as a result of the expression of the enzymatic polypeptide. For example, transforming a cell with a vector encoding aceto!aciate synthase and detecting increased acetoiactate concentrations compared to a cell without the vector indicates that the vector is both present and that the gene product is active. Methods for detecting specific enzymatic activities or the presence of particular products are well known to those skilled in the art. For example, the presence of acetoiactate can be determined as described by Hugenho!tz and Starrenburg, 1992, Appl. Micro. Blot. 38:17-22.

Increase of Enzymatic Activity

[00198] Yeast microorganisms of the invention may be further engineered to have increased activity of enzymes (e.g., increased activity of enzymes involved in an isobutanoi producing metabolic pathway). The term "increased" as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured in a comparable yeast ceil of the same species. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Increased activities for enzymes involved in glycolysis or the isobutanoi pathway would result in increased productivity and yield of isobutanoi.

[00199] Methods to increase enzymatic activity are known to those skilled in the art. Such techniques may include increasing the expression of the enzyme by increased copy number and/or use of a strong promoter, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the KM for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231 ), ed. Arnold and Georgiou, Humana Press (2003).

Methods of Using Recombinant Microorganisms for High-Yield Fermentations

[00200] For a biocatalyst to produce a beneficial metabolite most economically, it is desirable to produce said metabolite at a high yield. Preferably, the only product produced is the desired metabolite, as extra products (i.e. by-products) lead to a reduction in the yield of the desired metabolite and an increase in capital and operating costs, particularly if the extra products have little or no value. These extra products also require additional capital and operating costs to separate these products from the desired metabolite.

[00201] In one embodiment, the present invention provides a method of producing a beneficial metabolite derived from a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovalerate as an intermediate in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the beneficial metabolite is produced. In one embodiment, the present invention provides a method of producing a isobutanol derived from a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovaierate as an intermediate in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the isobutanol is produced, in an exemplary embodiment, said recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of 2- ketoisovalerate to 2-isopropylma!ate. in one embodiment, the recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of 2-isopropylmalate to 3-isopropylmaiate. In another embodiment, the recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of 3- isopropylmaiate to a-ketoisocaproate. In yet another embodiment, the recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of leucine to a-ketoisocaproate. In yet another embodiment, the recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of a- ketoisocaproate to 3-methyibutanal. In yet another embodiment, the recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of 3-methylbutanal to 3-methy!-1 -butanol.

[00202] The beneficial metabolite may be derived from any biosynthetic pathway which uses 2-ketoisovalerate as intermediate, including, but not limited to, biosynthetic pathways for the production of isobutanol, valine, pantothenate, and coenzyme A. In a specific embodiment, the beneficial metabolite is isobutanol.

[00203] In certain situations the production of 3-methyl-1 -butanol may be desired. Further, it may be desired to produce a blend of 3-methyl-1 -butanol and a beneficial metabolite. Further still, it may be desired to produce a blend of 3-methyl-1 -butanol and isobutanol.

[00204] Accordingly, in some embodiments of the present invention, a method of producing isobutanol and 3-methyi-1 -butanol comprising cultivating a recombinant microorganism described above and herein in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the isobutanoi and 3-methyl-1 -butano! is produced, is provided.

[00205] In some embodiments, a blend product of isobutanoi and 3-methyl-1 - butanol is produced. In one embodiment, a blend of isobutanoi and 3-methyl-1 - butanoi is produced that comprises a ratio of at least about 90:10 isobutanoi to 3- methyl-1 -butanoi. In another embodiment, a blend of isobutanoi and 3-methyl-1 - butanoi that comprises a ratio of at least about 80:20, 70:30, 80:40, 50:50, 40:60, 30:70, 20:80, or 10:90 isobutanoi to 3-methyi is produced.

[00206] In a method to produce a beneficial metabolite from a carbon source, the yeast microorganism is cultured in an appropriate culture medium containing a carbon source. In certain embodiments, the method further includes isolating the beneficial metabolite from the culture medium. For example, isobutanoi may be isolated from the culture medium by any method known to those skilled in the art, such as distillation, pervaporation, or liquid-liquid extraction.

[00207] In one embodiment, the recombinant microorganism may produce the beneficial metabolite from a carbon source at a yield of at least 5 percent theoretical. In another embodiment, the microorganism may produce the beneficial metabolite from a carbon source at a yield of at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 85 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5% theoretical. In a specific embodiment, the beneficial metabolite is isobutanoi.

Distillers Dried Grains Comprising Spent Yeast Biocatalysts

[00208] In an economic fermentation process, as many of the products of the fermentation as possible, including the co-products that contain biocatalyst ceil material, should have value. Insoluble material produced during fermentations using grain feedstocks, like corn, is frequently sold as protein and vitamin rich animal feed called distillers dried grains (DDG). See, e.g., commonly owned and co-pending U.S. Publication No. 2009/0215137, which is herein incorporated by reference in its entirety for all purposes. As used herein, the term "DDG" generally refers to the solids remaining after a fermentation, usually consisting of unconsumed feedstock solids, remaining nutrients, protein, fiber, and oil, as well as spent yeast biocatalysts or cell debris therefrom that are recovered by further processing from the fermentation, usually by a solids separation step such as centrifugation.

[00209] Distillers dried grains may also include soluble residual material from the fermentation, or syrup, and are then referred to as "distillers dried grains and solubles" (DDGS). Use of DDG or DDGS as animal feed is an economical use of the spent biocatalyst following an industrial scale fermentation process.

[00210] Accordingly, in one aspect, the present invention provides an animal feed product comprised of DDG derived from a fermentation process for the production of a beneficial metabolite (e.g., isobutanol), wherein said DDG comprise a spent yeast biocatalyst of the present invention. In an exemplary embodiment, said spent yeast biocatalyst has been engineered to comprise an isobutanol producing metabolic pathway.

[00211] In certain additional embodiments, the DDG comprising a spent yeast biocatalyst of the present invention comprise at least one additional product selected from the group consisting of unconsumed feedstock solids, nutrients, proteins, fibers, and oils.

[00212] In another aspect, the present invention provides a method for producing DDG derived from a fermentation process using a yeast biocatalyst (e.g., a recombinant yeast microorganism of the present invention), said method comprising: (a) cultivating said yeast biocatalyst in a fermentation medium comprising at least one carbon source; (b) harvesting insoluble material derived from the fermentation process, said insoluble material comprising said yeast biocatalyst; and (c) drying said insoluble material comprising said yeast biocatalyst to produce the DDG.

[00213] In certain additional embodiments, the method further comprises step (d) of adding soluble residual material from the fermentation process to said DDG to produce DDGS. In some embodiments, said DDGS comprise at least one additional product selected from the group consisting of unconsumed feedstock solids, nutrients, proteins, fibers, and oils.

[00214] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of ail references, patents, and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference for ail purposes EXAMPLES

[00215] Media: Medium used is standard yeast medium (see, for example Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3rd ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press and Guthrie, C. and Fink, G.R. eds. Methods in Enzymoiogy Part B: Guide to Yeast Genetics and Molecular and Cell Biology 350:3-823 (2002)). YP medium contains 1 % (w/v) yeast extract, 2% (w/v) peptone. YPD is YP containing 2% glucose unless specified otherwise. YPE is YP containing 25 mL/L ethanoi. SC medium is 8.7 g/L Difco™ Yeast Nitrogen Base, 14g/L Sigma™ Synthetic Dropout Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), 0.076 g/L histidine, 0.078 g/L tryptophan, 0.380 g/L leucine, and 0.078 g/L uracil. SCD is SC containing 2% (w/v) glucose unless otherwise noted. Drop-out versions of SC and SCD media are made by omitting one or more of histidine (-H), tryptophan (-W), leucine (-L), or uracil (-U). Solid versions of the above described media contain 2% (w/v) agar.

[00216] Cloning techniques: Standard molecular biology methods for cloning and plasmid construction are generally used, unless otherwise noted (Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press). Cloning techniques included digestion with restriction enzymes, PGR to generate DNA fragments (KOD Hot Start Polymerase, Cat# 71086, Merck, Darmstadt, Germany), ligations of two DNA fragments using the DNA Ligation Kit (Mighty Mix Cat# TAK 6023, Clontech Laboratories, Madison, Wl), and bacterial transformations into competent E.coii cells (Xtreme Efficiency DH5a Competent Cells, Cat# ABP-CE-CC02098P, Allele Biotechnology, San Diego, CA). Plasmid DNA is purified from E. coil ceils using the Qiagen QIAprep Spin Miniprep Kit (Cat# 27106, Qiagen, Valencia, CA). DNA is purified from agarose gels using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, CA; Catalog #04002) according to manufacturer's protocols.

[00217] Colony PGR: Yeast colony PGR uses the FailSafe™ PGR System (EPICENTRE© Biotechnologies, Madison, Wl; Catalog #FS99250) according to manufacturer's protocols. A PGR cocktail containing 15 pL of Master Mix E buffer, 10.5 pL water, 2 pL of each primer at 10 pM concentration, 0.5 pL polymerase enzyme mix from the kit is added to a 0.2 mL PGR tube for each sample (30 pL each). For each candidate a sma!i amount of cells is added to the reaction tube using a sterile pipette tip. Presence of the positive PGR product is assessed using agarose gel electrophoresis.

[00218] SOE PGR: The PGR reactions are incubated in a thermocycler using the following PCR conditions: 1 cycle of 94°C x 2 min, 35 cycles of 94°C x 30 s, 53°C x 30 s, 72°C x 2 min and 1 cycle of 72°C x 10 min. A master mix is made such that each reaction contained the following: 3 pL MgSG₄ (25 mM), S pL 10X KOD buffer, 5 pL 50% DMSO, 5 pL dNTP mix (2 mM each), 1 pL KOD, 28 pL dH₂O, 1 .5 pL forward primer (10 μΜ), 1 .5 pL reverse primer (10 pM), 0.5 pL template (plasmid or genomic DNA).

[00219] Genomic DNA Isolation: The Zymo Research ZR Fungal/Bacterial DNA Kit (Zymo Research Orange, CA; Catalog #06005) is used for genomic DNA isolation according to manufacturer's protocols with the following modifications. Following resuspension of pellets, 200 pL is transferred to 2 separate ZR BashingBead™ Lysis Tubes (to maximize yield). Following lysis by bead beating, 400 pL of supernatant from each of the ZR BashingBead™ Lysis Tubes is transferred to 2 separate Zymo-Spin™ IV Spin Filters and centrifuged at 7,000 rpm for 1 min. Following the spin, 1 .2 rnL of Fungal/Bacterial DNA Binding Buffer is added to each filtrate. In 800 pi a!iquots, filtrate from both filters is transferred to a single Zymo-Spin™ HC Column in a collection tube and centrifuged at 10,000 x g for 1 min. For the eiution step, instead of eiuting in 100 pL of EB (elution buffer, Qiagen), 50 pL of EB is added, incubated 1 min then the columns are centrifuged for 1 min. This elution step is repeated for a final eiution volume of 100 pL.

[00220] S, cerevisiae Transformations, S. cerevisiae strains are grown in YPD containing 1 % ethanoi. Transformation-competent cells are prepared by resuspension of S. cerevisiae cells in 100 mM lithium acetate. Once the cells are prepared, a mixture of DNA (final volume of 15 pL with sterile water), 72 pL 50% PEG, 10 pL 1 M lithium acetate, and 3 pL of denatured salmon sperm DNA (10 mg/mL) is prepared for each transformation. In a 1 .5 rnL tube, 15 pL of the ceil suspension is added to the DNA mixture (100 pL), and the transformation suspension is vortexed for 5 short pulses. The transformation is incubated for 30 min at 30°C, followed by incubation for 22 min at 42°C. The cells are collected by centrifugation (18,000 x g, 10 seconds, 25°C). The cells are resuspended in 350 pL YPD and after an overnight recovery shaking at 30°C and 250 rpm, the cells are spread over YPD plates containing 0.2 g/L G418 seiective plates. Transformants are then single colony purified onto G418 selective plates.

Analytical Chemistry:

[00221] Gas Chromatography (GC). Analysis of volatile organic compounds, including isobutanol and 3-methy!-1 -butanol is performed on a Agilent 5890/6890/7890 gas chromatograph fitted with an Agilent 7673 Autosampier, a ZB- FFAP column (J&W; 30 m length, 0.32 mm ID, 0.25 μΜ film thickness) or equivalent connected to a flame ionization detector (FID). The temperature program is as follows: 200°C for the injector, 300°C for the detector, 100°C oven for 1 minute, 70^cC/minute gradient to 230°C, and then hold for 2.5 min. Analysis is performed using authentic standards (>99%, obtained from Sigma-Aldrich, and a 5-point calibration curve with 1 -pentanoi as the internal standard.

[00222] High Performance Liquid Chromatography (LC): Analysis of organic acid metabolites including glucose is performed on an Agilent 1200 or equivalent High Performance Liquid Chromatography system equipped with a Bio-Rad Micro- guard Cation H Cartridge and two Phenomenex Rezex RFQ-Fast Fruit H+ (8%), 100 x 7.8-mm columns in series, or equivalent. Organic acid metabolites are detected using an Agilent 1 100 or equivalent UV detector (210 rim) and a refractive index detector. The column temperature is 60°C. This method is isocratic with 0.0180 N H2SO4 in Miili-Q water as mobile phase. Flow is set to 1 .1 mL/min. Injection volume is 20 pL and run time is 16 min. Quantitation of organic acid metabolites is performed using a 5~point calibration curve with authentic standards (>99% or highest purity available).

Example 1 : Deletion of Enzymes Used in the Production of 3-methyl-1 -butanoi

[00223] This example describes elimination of genes encoding activities required for production of 3-methy!-l -butanol, including Leu4, Leu9, Leu2, Leu1, Bail, and

Bat2, from a yeast strain engineered for production of isobutanol.

[00224] Elimination of Leu4 from the S. cerevisiae strain GEV03991 (Table 2) is accomplished by transformation with a PGR product generated with primers designed to target and replace the Let 4-iocus with a genetic marker (URA3) flanked with a repeat sequence (T_Sc CYCI) that enables the strain to grow in medium lacking uracil. Primers to amplify the genetic marker sequence targeted for the Leu4-locus are designed with > 40 bp sequence homologous to the region immediately upstream and downstream of the open reading frame of Leu4, The PGR product is introduced into GEV03991 following transformation protocols described above,.

[00225] Following transformation, strains in which Leu4 has been eliminated are selected on agar plated containing defined medium lacking uracil. Replacement of Leu4 in the genome GEV03991 is confirmed with PGR with sets of primers designed to verify the absence of Leu4 and the presence of the genetic marker. A confirmed strain is spread onto plates containing 5-FOA to select for loss of the URA3-marker gene to recycle the marker, and the resulting strain is designated GEVO###2 (Table 2).

[00226] Elimination of Leu9 from the S. cerevisiae strain GEV03991 (Table 2) is accomplished by transformation with a PGR product generated with primers designed to target and replace the Leu Aocus with a genetic marker (URA3) flanked with a repeat sequence (TS_C_CYCI) that enables the strain to grow in medium lacking uracil. Primers to amplify the genetic marker sequence targeted for the Leu9-locus are designed with > 40 bp sequence homologous to the region immediately upstream and downstream of the open reading frame of Leu9. The PGR product is introduced into GEV03991 following transformation protocols described. Following transformation, strains in which Leu9 has been eliminated are selected on agar plated containing defined medium lacking uracil. Replacement of Leu9 in the genome GEV03991 is confirmed with PGR with sets of primers designed to verify the absence of Leu9 and the presence of the genetic marker. A confirmed strain is spread onto plates containing 5-FOA to select for loss of the URA3-marker gene to recycle the marker, and the resulting strain is designated GEVO###3 (Table 2).

[00227] Elimination of Leu2 from the S. cerevisiae strain GEV03991 (Table 2) is accomplished by transformation with a PGR product generated with primers designed to target and replace the Leu2-\ocus with a genetic marker (URA3) flanked with a repeat sequence (TS_C_CYCI) that enables the strain to grow in medium lacking uracil. Primers to amplify the genetic marker sequence targeted for the Let/2~locus are designed with > 40 bp sequence homologous to the region immediately upstream and downstream of the open reading frame of Leu2. The PGR product is introduced into GEV03991 following transformation protocols described. Following transformation, strains in which Leu2 has been eliminated are selected on agar plated containing defined medium lacking uracil. Replacement of Leu2 in the genome GEV03991 is confirmed with PGR with sets of primers designed to verify the absence of Leu2 and the presence of the genetic marker. A confirmed strain is spread onto plates containing 5-FOA to select for loss of the URA3-marker gene to recycle the marker, and the resulting strain is designated GEVO###4 (Table 2).

[00228] Elimination of Leu1 from the S, cerevisiae strain GEVO3991 (Table 2) is accomplished by transformation with a PGR product generated with primers designed to target and replace the Leuf-iocus with a genetic marker {URA3) flanked with a repeat sequence (T_SC_CYCI) that enables the strain to grow in medium lacking uracil. Primers to amplify the genetic marker sequence targeted for the Lewi-locus are designed with > 40 bp sequence homologous to the region immediately upstream and downstream of the open reading frame of Leu1. The PGR product is introduced into GEVO3991 following transformation protocols described. Following transformation, strains in which Leu1 has been eliminated are selected on agar plated containing defined medium lacking uracil. Replacement of Leu1 in the genome GEVO3991 is confirmed with PGR with sets of primers designed to verify the absence of Leu1 and the presence of the genetic marker. A confirmed strain is spread onto plates containing 5-FOA to select for loss of the URA3-marker gene to recycle the marker, and the resulting strain is designated GEVO###5 (Table 2).

[00229] Combination of deletions of Leu4, Leu9, Leu2, and Leu1 are accomplished by sequential deletions performed initially in GEV03991 (Table 2). The deletions are performed as described with the selectable marker (URA3) and subsequent selection of removal of the marker in the presence of 5-FOA. Upon confirmation of elimination of all four genes, the resulting strain is designated GEVO###8 (Table 2).

[00230] Elimination of Bail from the S. cerevisiae strain GEV03991 (Table 2) is accomplished by transformation with a PGR product generated with primers designed to target and replace the Baif-iocus with a genetic marker {URA3) flanked with a repeat sequence (T_{Sc C}YCI) that enables the strain to grow in medium lacking uracil. Primers to amplify the genetic marker sequence targeted for the Saff-locus are designed with > 40 bp sequence homologous to the region immediately upstream and downstream of the open reading frame of Sail The PCR product is introduced into GEV03991 following transformation protocols described. Following transformation, strains in which Bat1 has been eliminated are selected on agar plated containing defined medium lacking uracil. Replacement of Bat1 in the genome GEV03991 is confirmed with PCR with sets of primers designed to verify the absence of Bail and the presence of the genetic marker. A confirmed strain is spread onto plates containing 5-FOA to select for loss of the URA3-marker gene to recycle the marker, and the resulting strain is designated GEVO###7 (Table 2).

[00231] Elimination of Bat2 from the S. cerevisiae strain GEV03991 (Table 2) is accomplished by transformation with a PGR product generated with primers designed to target and replace the Bat2-\ocus with a genetic marker (URA3) flanked with a repeat sequence (T_SC_CYCI ) that enables the strain to grow in medium lacking uracil. Primers to amplify the genetic marker sequence targeted for the Saf2-locus are designed with > 40 bp sequence homologous to the region immediately upstream and downstream of the open reading frame of Bat2. The PGR product is introduced into GEV03991 following transformation protocols described. Following transformation, strains in which Bat2 has been eliminated are selected on agar plated containing defined medium lacking uracil. Replacement of Bai2 in the genome GEV03991 is confirmed with PGR with sets of primers designed to verify the absence of Bat2 and the presence of the genetic marker. A confirmed strain is spread onto plates containing 5-FOA to select for loss of the URA3-marker gene to recycle the marker, and the resulting strain is designated GEVO###8 (Table 2).

[00232] Combination of deletions of Bat1 and Bat2 are accomplished by sequential deletions performed initially in GEV03991 (Table 2). The deletions are performed as described with the selectable marker (URA3) and subsequent selection of removal of the marker in the presence of 5-FOA. Upon confirmation of elimination of both genes, the resulting strain is designated GEVO###9 (Table 2).

Table 2. Genotype of Strains Disclosed in Example 1 .

GEVO###4 leu1A::T_Sc_cYci GEV03991

GEVO###5 Leu4A::Tsc_cYci GEV03991

GEVO###6 leu9A::T_Sc_cYci Leu4A::T_Sc_cYci ieu2A::T_Sc_cYci leu1A::T_Sc_cYci

GEV03991

GEVO###7 bat1A::T_Sc_cYci GEV03991

GEVO###8 bat2A::T_Sc_cYci GEV03991

GEVO###9 bat1A::T_Sc_cYci bat2A::Tsc_cYci GEV03991

Example 2: Reduced 3MB Production in Engineered Microorganisms

[00233] A fermentation is performed to determine the amount of isobutanol and 3- methyl-1 -butanoi produced by strains in which a gene or set of genes encoding activities required for production of 3-methyl-1 -butanol production are eliminated

(GEVO###2-GEVO###9 in Table 2) as compared to the parent strain, GEV03991 .

For these fermentations, single isolate cell colonies are transferred to 500 rnL baffled flasks containing 80 mL of YPD containing appropriate medium and incubated at

30^CC under temperature and agitation conditions conducive to generate sufficient biomass. The flask cultures are transferred to individual fermenter vessels,

Fermenters are operated for an appropriate duration of time under conditions conducive to isobutanol production. Periodically, samples (1 .5 mL) from each fermenter are removed. A portion of each sample is used to determine cell density

(OD₆oo), and the remainder of each sample is transferred into 1 .5 mL tubes and centrifuged in a microcentrifuge for 10 min at 18,000xg. The supernatants are analyzed by LC and GC analysis to determine of the amount of glucose consumed and isobutanol and 3-methyi-1 -butanol produced.

[00234] In strains in which enzymes required for production of 3-methyl-1 -butanol are eliminated (GEVO###2-GEVO###9 in Table 2), the amount of this metabolite is generally reduced, and the yield of isobutanol is increased.

Example 3: Production of Tuned Mixtures of 3MB and Isobutanol

[00235] This example describes overexpression of genes encoding activities required for production of 3-methyl-1 -butanol, including Leu4, LeuQ, Leu2, Leu1,

Bat1, and Bat2, from a strain engineered for production of isobutanol.

[00236] One or more of the following genes are cloned into an appropriate vector that also contains a selectable marker (e.g. URA3) under a promoter that drives transcription to the desired levels: Leu4, Leu9, Leu2, Leu1, Bat1, and/or Bat2, The resulting vector(s) are transformed into GEV03991 (Table 2) as described.

[00237] A fermentation is performed to determine the amount of isobutanol and 3- methyl-1 -butanoi produced by strains in which a gene or set of genes encoding activities required for production of 3-methyl-1 -butanol production are overexpressed in the isobutano!-production strain, GEV03991 (Table 2). For these fermentations, single isolate cell colonies are transferred to 500 rnL baffled flasks containing 80 mL of YPD containing appropriate medium and incubated at 30°C under temperature and agitation conditions conducive to generate sufficient biomass. The flask cultures are transferred to individual fermenter vessels. Fermenters are operated for an appropriate duration of time under conditions conducive to isobutanol production. Periodically, samples (1 .5 mL) from each fermenter are removed. A portion of each sample is used to determine cell density (OD₆oo), and the remainder of each sample is transferred into 1 .5 mL tubes and centrifuged in a microcentrifuge for 10 min at 18,000xg. The supernatants are analyzed by gas chromatography (GC) analysis to determine of the amount of isobutanol and 3-methy!-1 -butanol produced.

[00238] In strains in which an enzyme or enzymes required for production of 3- methyl-1 ~butanoi are overexpressed (GEVO###2-GEVO###9 in Table 2), the ratio of 3-methyl-1 -butanoi to isobutanol produced will generally be increased.

Example 4: LEU4 and LEU9 Reductions

[00239] In S. cerevisiae, two genes encode 2-isopropylmalate synthases, LEU4 and LEU9. In this example, the effect of the presence or absence of leucine in the medium on 3-methyI-1 -butanoi production was determined in strains in which LEU4 or LEU9 were disrupted.

Table 3. Strains Disclosed in Example 4.

Strain

GEVO6014 (S. cerevisiae) ^™

GEVO6014 ieu4A::P sc TEFI :bie:T Sc CYC1

GEVO6014 /eu9A-;P Sc TEFI :ble:T sc CYC1

[00240] In this example, the GEVO6014A/et/4, GEVO6014A/ey9, and GEVO6014 strains were inoculated for overnight growth at 30°C/250 rpm. These strains harbor an engineered isobutanol producing pathway comprising the B, subtilis a!sS gene, an engineered variant of E. coii HvC (EcJ!vC__coScP2D1-A1 , described in commonly-owned US Patent No. 8,097,440), the L lactis IlvD gene, the L. lactis kivD gene, and an engineered variant of L lactis adhA gene (U__adhA^R£1 , described in commonly owned US Patent Publication No. 201 10201072). Cells were harvested approximately 48 hrs later when the cell density was roughly

Cells were transferred into 50 ml Falcon tubes_., centrifuged at 3000 rpm, the supernatant was decanted, and ceil pellets were then resuspended in 20 ml sterile water, Cells were rinsed in 20 ml sterile water twice, finally centrifuged and resuspended in an appropriate volume of SCD medium to obtain cell density at

SCD medium contained 6.7 g/L yeast nitrogen base, 14 g/L synthetic dropout media supplement, 0.076 g/L histidine, 0.076 g/L tryptophan, and 20 g/L dextrose. Next, 2.5 ml of cells were added to 50 ml fiat-bottom flasks, containing SCD medium with or without leucine (0.38 g/L) addition. Cells were incubated at 30°C/75 rpm (production phase, time zero). Optical density of cells was measured at indicated time points and supernatants were analyzed via HPLC and GC.

[00241] Results: GEVO6014A/ew4 strains produced about 50% less 3-methyl-1 - butanoi ("3MB") in medium void of leucine addition as compared to the parental strain (Figure 5A). Consistent with this observation, the 3MB yield of GEVO6G14A/ei 4 was decreased by about 50% in medium void of leucine addition as compared to the parental strain. In SCD + leucine medium, no change in 3MB production was observed (Figure SB). In contrast, GEVO6014A/et 9 strains produced slightly less 3MB in SCD + leucine medium as compared to the parental strain (Figure SB), while no differences were observed in 3MB production in medium void of leucine addition (Figure 5A). Consistent with these observations, the ratio of IBuOH/3MB production was significantly higher in GEVO6014A/ei 4 strains in medium void of leucine addition.

[00242] The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood there from as modifications will be obvious to those skilled in the art.

[00243] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

[00244] The disclosures_., including the claims, figures and/or drawings, of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entireties.

Claims

WHAT IS CLAI ED IS:

1 . A recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovaierate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 3- methyI-1 -butanoi.

2. The recombinant microorganism of claim 1 , wherein said enzyme is selected from one or more of the following:

(i) one or more enzymes catalyzing the conversion of 2-ketoisovaierate to

2- isopropylma!ate;

(ii) one or more enzymes catalyzing the conversion of 2-isopropylmalate to

3- isopropyimaiate;

(iii) one or more enzymes catalyzing the conversion of 3-isopropylmalate to a-ketoisocaproate;

(iv) one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methylbutanal; and

(v) one or more enzymes catalyzing the conversion of 3-methyibutanai to 3-methyl-1 -butanol.

3. The recombinant microorganism of claim 2, wherein said enzyme catalyzing the conversion of 2-ketoisovalerate to 2-isopropylmalate is a 2- isopropylmalate synthase.

4. The recombinant microorganism of claim 3, wherein said 2-isopropylmalate is encoded by Leu4,

5. The recombinant microorganism of claim 3, wherein said 2-isopropyimalate synthase is encoded by Leu9.

8, The recombinant microorganism of claim 2, wherein said enzyme catalyzing the conversion of 2-isopropylmalate to 3-isopropyimaiate is an isopropylmalate isomerase.

7. The recombinant microorganism of claim 6, wherein said isopropylmalate isomerase is encoded by Leu1 .

8. The recombinant microorganism of claim 2, wherein said enzyme catalyzing the conversion of 3-isopropyimaiate to a-ketoisocaproate is a 3-isoproyimaiate dehydrogenase.

9. The recombinant microorganism of claim 8, wherein said 3-isoproylmalate dehydrogenase is encoded by Leu2.

10. The recombinant microorganism of claim 2, wherein said enzyme catalyzing the conversion of α-ketoisocaproate to 3-methy!butanai is a keto-isocaproate decarboxylase.

1 1 . The recombinant microorganism of claim 10, wherein the keto-isocaproate decarboxylase is encoded by a gene selected from the group consisting of Aro10 and Thi3.

12. The recombinant microorganism of claim 2, wherein said enzyme catalyzing the conversion of 3-methyibutanal to 3-methyl-1 -butano! is an alcohol dehydrogenase.

13. The recombinant microorganism of claim 12, wherein the alcohol dehydrogenase is encoded by a gene selected from the group consisting of Adh6 and Adh7.

14. A recombinant microorganism comprising a biosynthetic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of leucine to 3-methyl-1 -butanol.

15. The recombinant microorganism of claim 14, wherein said enzyme is selected from one or more of the following:

(i) one or more enzymes catalyzing the conversion of leucine to a- ketoisocaproate;

(ii) one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methylbutanal; and

(iii) one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methyl-1 -butanoi.

16. The recombinant microorganism of claim 15, wherein said enzyme catalyzing the conversion of leucine to a-ketoisocaproate is a branched-chain amino acid transaminase.

17. The recombinant microorganism of claim 16, wherein the branched-chain amino acid transaminase is encoded by Bat1 .

18. The recombinant microorganism of claim 18, wherein the branched-chain amino acid transaminase is encoded by Bat2.

19. The recombinant microorganism of any of the preceding claims, wherein said recombinant microorganism produces a 2-ketoisovaierate-derived product.

20. The recombinant microorganism of claim 19, wherein said 2-ketoisovalerate- derived product is selected from isobutanoi, valine, pantothenate, and coenzyme A.

21 . The recombinant microorganism of any of the preceding claims, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of a 3-keto acid to a 3-hydroxyacid by-product.

22. The recombinant microorganism of claim 21 , wherein said enzyme catalyzes acetolactate to 2,3-dihydroxy-2-methylbutanoic acid (DH2MB).

23. The recombinant microorganism of claim 21 , wherein said enzyme is a 3-keto acid reductase.

24. The recombinant microorganism of any of the preceding claims, wherein said recombinant microorganism comprises a biosynthetic pathway of which an aldehyde is an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of said aldehyde to an acid by-product.

25. The recombinant microorganism of claim 24, wherein said enzyme catalyzing the conversion of an aldehyde to an acid by-product is an aldehyde dehydrogenase.

26. The recombinant microorganism of any of the preceding claims, wherein said recombinant microorganism is engineered to reduce or eliminate pyruvate decarboxylase (PDC) activity.

27. The recombinant microorganism of any of the preceding claims, wherein said recombinant microorganism is engineered to reduce or eliminate giycerol-3- phosphate dehydrogenase (GPD) activity.

28. The recombinant microorganism of any of the preceding claims, wherein said recombinant microorganism is a yeast microorganism.

29. The recombinant microorganism of claim 28, wherein said recombinant microorganism is a yeast microorganism of the Saccharomyces clade.

30. The recombinant microorganism of claim 29, wherein said recombinant microorganism is a Saccharomyces sensu stricto microorganism.

31 . The recombinant microorganism of claim 30, wherein said Saccharomyces sensu stricto microorganism is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S. mikatae, S, bayanus, S, uvarum, S. carocanis and hybrids thereof.

32. The recombinant microorganism of claim 28, wherein said recombinant microorganism is a Crab ree-negafive yeast microorganism.

33. The recombinant microorganism of claim 32, wherein said Crabfree-negative yeast microorganism is classified into a genus seiected from a group consisting of Saccharomyces, Kluyveromyces, Pichia, Hansenula, Issatchenkia and Candida,

34. The recombinant microorganism of claim 33, wherein said Crabfree-negative yeast microorganism is selected from the group consisting of Saccharomyces kiuyveri, Kluyveromyces iaciis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Pichia kudriavzevii, issatchenkia orientalis, Hansenula anomala, Candida utilis and Kluyveromyces waitii.

35. The recombinant microorganism of claim 28, wherein said recombinant microorganism is a Crabtree-positive yeast microorganism.

38. The recombinant microorganism of claim 35, wherein said Crabtree-positive yeast microorganism is classified into a genus seiected from a group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Pichia, Candida, and Schizosaccharomyces.

37. The recombinant microorganism of claim 36, wherein said Crabtree-positive yeast microorganism is seiected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces casteili, Kluyveromyces thermotolerans, Candida glabrata, Zygosaccharomyces bailli, Zygosaccharomyces rouxii, Debaryomyces hansenii, Pichia pastorius, and Schizosaccharomyces pombe.

38. The recombinant microorganism of claim 28, wherein said recombinant microorganism is a post-WGD (whole genome duplication) yeast microorganism.

39. The recombinant microorganism of claim 38, wherein said post-WGD yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces or Candida.

40. The recombinant microorganism of claim 39, wherein said post-WGD yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and Candida glabrata.

41 . The recombinant microorganism of claim 28, wherein said recombinant microorganism is a pre-WGD (whole genome duplication) yeast microorganism.

42. The recombinant microorganism of claim 41 , wherein said pre-WGD yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Debaryomyces, Hansenula, issatchenkia, Pachysolen, Yarrowia and Schizosaccharomyces.

43. The recombinant microorganism of claim 42, wherein said pre-WGD yeast microorganism is selected from the group consisting of Saccharomyces kluyveri, Kluyveromyces thermotoierans, Kluyveromyces marxianus, Kluyveromyces waitii, Kluyveromyces iactis, Candida tropicaiis, Pichia pastoris, Pichia anomala, Pichia stipitis, Pichia kudriavzevii, Issatchenkia orientaiis, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yarrowia iipolytica, and Schizosaccharomyces pombe.

44. A method of producing a beneficial metabolite derived from a 2- ketoisovaierate intermediate, comprising:

(a) providing a recombinant microorganism according to any of the preceding claims; (b) cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source, until a recoverable quantity of the beneficial metabolite is produced.

45. The method of ciaim 44, wherein said beneficial metabolite is selected from isobutanol, valine, pantothenate, and coenzyme A.

48. A recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following:

(i) one or more enzymes catalyzing the conversion of 2-ketoisovalerate to

2- isopropylmalate;

(ii) one or more enzymes catalyzing the conversion of 2-isopropylmalate to

3- isopropyimaiate;

(iv) one or more enzymes catalyzing the conversion of leucine to a- ketoisocaproate;

(v) one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methylbutanal; and

(vi) one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methyl-1 -butanoi.

47. The recombinant microorganism of claim 48, wherein said enzyme catalyzing the conversion of 2-ketoisovalerate to 2-isopropyimaiate is a 2- isopropyimalate synthase.

48. The recombinant microorganism of ciaim 47, wherein said 2-isopropylmalate synthase is encoded by Leu4.

49. The recombinant microorganism of claim 47, wherein said 2-isopropylmalate synthase is encoded by Leu9.

50. The recombinant microorganism of ciaim 46, wherein said enzyme catalyzing the conversion of 2-isopropylmalate to 3-isopropyimaiate is an isopropylmalate isomerase.

51 . The recombinant microorganism of claim 50, wherein said isopropylmalate isomerase is encoded by Leu1 .

52. The recombinant microorganism of claim 48, wherein said enzyme catalyzing the conversion of 3-isopropyimaiate to a-ketoisocaproate is a 3-isoproyimaiate dehydrogenase.

53. The recombinant microorganism of claim 52, wherein said 3-isoproylmalate dehydrogenase is encoded by Leu2.

54. The recombinant microorganism of claim 46, wherein said enzyme catalyzing the conversion of leucine to a-ketoisocaproate is a branched-chain amino acid transaminase.

55. The recombinant microorganism of claim 54, wherein the branched-chain amino acid transaminase is encoded by Bat1 .

58. The recombinant microorganism of ciaim 54, wherein the branched-chain amino acid transaminase is encoded by Bat2.

57. The recombinant microorganism of claim 48, wherein said enzyme catalyzing the conversion of a-ketoisocaproate to 3-methylbutanal is a keto-isocaproate decarboxylase.

58. The recombinant microorganism of ciaim 57, wherein the keto-isocaproate decarboxylase is encoded by a gene selected from the group consisting of Aro10 and Thi3.

59. The recombinant microorganism of claim 46, wherein said enzyme catalyzing the conversion of 3-methylbutanal to 3-methyl-1 -butanoi is an alcohol dehydrogenase.

80. The recombinant microorganism of claim 59, wherein the alcohol dehydrogenase is encoded by a gene selected from the group consisting of Adh8 and Adh7.

61 . The recombinant microorganism of any of the preceding claims, wherein said recombinant microorganism produces a 2-ketoisovaierate-derived product.

62. The recombinant microorganism of claim 61 , wherein said 2-ketoisovaierate- derived product is selected from isobutanoi, valine, pantothenate, and coenzyme A.

63. The recombinant microorganism of any of claims 46-62, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of a 3-keto acid to a 3-hydroxyacid by-product.

64. The recombinant microorganism of any of claim 63, wherein the enzyme converts acetolactate to 2,3-dihydroxy-2-methyibutanoic acid (DH2MB).

65. The recombinant microorganism of claim 64, wherein said enzyme catalyzing the conversion of a 3-keto acid to a 3-hydroxyacid by-product is a 3-keto acid reductase.

66. The recombinant microorganism of any of claims 46-85, wherein said recombinant microorganism comprises a biosynthetic pathway of which an aldehyde is an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of said aldehyde to an acid by-product.

87. The recombinant microorganism of claim 86, wherein said enzyme catalyzing the conversion of an aldehyde to an acid by-product is an aldehyde dehydrogenase.

88. The recombinant microorganism of any of claims 48-87, wherein said recombinant microorganism is engineered to reduce or eliminate pyruvate decarboxylase (PDC) activity.

89. The recombinant microorganism of any of claims 48-88, wherein said recombinant microorganism is engineered to reduce or eliminate giyceroi-3- phosphate dehydrogenase (GPD) activity.

70. The recombinant microorganism of any of claims 48-69, wherein said recombinant microorganism is a yeast microorganism.

71 . The recombinant microorganism of claim 70, wherein said recombinant microorganism is a yeast microorganism of the Saccharomyces clade.

72. The recombinant microorganism of claim 71 , wherein said recombinant microorganism is a Saccharomyces sensu stricto microorganism.

73. The recombinant microorganism of claim 72, wherein said Saccharomyces sensu stricto microorganism is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrids thereof.

74. The recombinant microorganism of claim 70, wherein said recombinant microorganism is a Crabtree-negative yeast microorganism.

75. The recombinant microorganism of claim 74, wherein said Crabtree-negative yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces, Kluyveromyces, Pichia, Hansenula, issatchenkia and Candida.

76. The recombinant microorganism of claim 75, wherein said Crabtree-negative yeast microorganism is selected from the group consisting of Saccharomyces kluyveri, Kluyveromyces lactis. Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Pichia kudriavzevii, Issatchenkia orientalis, Hansenula anomala, Candida utiiis and Kluyveromyces waitii.

77. The recombinant microorganism of claim 70, wherein said recombinant microorganism is a Crabtree-positive yeast microorganism.

78. The recombinant microorganism of claim 77, wherein said Crabtree-positive yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Pichia, Candida, and Schizosaccharomyces.

79. The recombinant microorganism of claim 78, wherein said Crabtree-positive yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelii, Kluyveromyces thermotolerans, Candida glabrata, Zygosaccharomyces bailli, Zygosaccharomyces rouxii, Debaryomyces hansenii, Pichia pastonus, and Schizosaccharomyces pombe.

80. The recombinant microorganism of claim 70, wherein said recombinant microorganism is a post-WGD (whole genome duplication) yeast microorganism.

81 . The recombinant microorganism of claim 80, wherein said post-WGD yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces or Candida.

82. The recombinant microorganism of claim 81 , wherein said post-WGD yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and Candida glabrata.

S3. The recombinant microorganism of claim 70, wherein said recombinant microorganism is a pre-WGD (whole genome duplication) yeast microorganism.

84. The recombinant microorganism of claim 83, wherein said pre-WGD yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces, Kiuyveromyces, Candida, Pichia, Debaryomyces, Hansenula, issatchenkia, Pachysolen, Yarrowia and Schizosaccharomyces,

85. The recombinant microorganism of claim 84, wherein said pre-WGD yeast microorganism is selected from the group consisting of Saccharomyces kluyveri, Kiuyveromyces thermotolerans, Kiuyveromyces marxianus, Kiuyveromyces waltii, Kiuyveromyces lactis, Candida tropicalis, Pichia pastoris, Pichia anomala, Pichia stipitis, Pichia kudriavzevii, Issatchenkia orientaiis, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yarrowia lipoiytica, and Schizosaccharomyces pom be.

86. A method of producing isobutanol, comprising:

(a) providing a recombinant microorganism according to any of claims 48-85;

(b) cultivating the recombinant microorganism in a culture medium containing a feedstock providing the carbon source, until a recoverable quantity of isobutanol is produced.

87. A recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovaierate as an intermediate, wherein said recombinant microorganism is:

(a) engineered to reduce or eliminate the expression or activity of one or more of the following: (i) one or more enzymes catalyzing the conversion of 2- keioisovalerate to 2-isopropylmalate;

(ii) one or more enzymes catalyzing the conversion of 2- isopropyimalate to 3-isopropyimalate;

(iii) one or more enzymes catalyzing the conversion of 3- isopropylmalate to

a-keto!socaproate

(vi) one or more enzymes catalyzing the conversion of leucine to a- ketoisocaproate;

(iv) one or more enzymes catalyzing the conversion of a- ketoisocaproate to 3-methyibutanal; and

(v) one or more enzymes catalyzing the conversion of 3- methylbutanai to 3-methyi-1 -butanol and/or is

(b) substantially free of an enzyme catalyzing the conversion of one or more of the following:

(i) one or more enzymes catalyzing the conversion of 2- ketoisovaierate to 2-isopropyimaiate;

(ii) one or more enzymes catalyzing the conversion of 2~ isopropylmalate to 3-isopropylmalate;

(iii) one or more enzymes catalyzing the conversion of 3- isopropyimalate to

a-ketoisocaproate

(v) one or more enzymes catalyzing the conversion of 3~ methylbutanai to 3-methy!-1 -butanol.

A recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein said recombinant microorganism is: (a) engineered to reduce or eliminate the expression or activity of one or more of the following:

(i) one or more enzymes catalyzing the conversion of 2- ketoisovalerate to 2-isopropylmalate;

(ii) one or more enzymes catalyzing the conversion of 2- isopropyimalate to 3-isopropyImaiate;

(iii) one or more enzymes catalyzing the conversion of 3- isopropylma!ate to

a-ketoisocaproate

(iv) one or more enzymes catalyzing the conversion of a- ketoisocaproate to 3-methy!butanal; and

(v) one or more enzymes catalyzing the conversion of 3- methylbutanal to 3-methyl-1 -butanol and/or is

(ii) one or more enzymes catalyzing the conversion of 2- isopropylmalate to 3-isopropylmalate;

(iii) one or more enzymes catalyzing the conversion of 3- isopropy!malate to

a-ketoisocaproate

(v) one or more enzymes catalyzing the conversion of 3- methylbutanai to 3-methy!-1 -butanol.

89. A recombinant microorganism for the production of isobutanol and 3-methyl-1 - butanoi, wherein said recombinant microorganism comprises an isobutanoi producing metabolic pathway and overexpresses one or more enzymes capabie of converting 2-ketoisova!erate to 3-methyl-1 -butanol.

90. The recombinant microorganism of claim 89, wherein said enzyme catalyzing the conversion of 2-ketoisovalerate to 3-methyl-1 -butanol is a 2~ isopropylma!ate synthase.

91 . The recombinant microorganism of ciaim 90, wherein said 2-isopropy!ma!ate synthase is encoded by Leu4.

92. The recombinant microorganism of ciaim 91 , wherein said 2-isopropylmalate synthase is encoded by Leu9.

93. The recombinant microorganism of claim 89, wherein said enzyme catalyzing the conversion of 2-ketoisovaierate to 3-methyl-1 -butanoi is an isopropy!ma!ate isomerase.

94. The recombinant microorganism of claim 93, wherein said isopropy!malate isomerase is encoded by Leu1 .

95. The recombinant microorganism of claim 89, wherein said enzyme catalyzing the conversion of 2-ketoisovalerate to 3-methyi-1 -butanoi is a 3- isoproy!ma!ate dehydrogenase.

96. The recombinant microorganism of claim 95, wherein said 3-isoproylmalate dehydrogenase is encoded by Leu2.

97. The recombinant microorganism of claim 89, wherein said enzyme catalyzing the conversion of a-ketoisocaproate to 3-methylbutanal is a keto-isocaproate decarboxylase.

98. The recombinant microorganism of ciaim 97, wherein the keto-isocaproate decarboxylase is encoded by a gene selected from the group consisting of Aro10 and Thi3.

99. The recombinant microorganism of claim 89, wherein said enzyme catalyzing the conversion of 3-methy!butanal to 3-methyl-1 -butanol is an alcohol dehydrogenase.

100. The recombinant microorganism of claim 99, wherein the alcohol dehydrogenase is encoded by a gene selected from the group consisting of Adh8 and Adh7.

101 . A method for the production of isobutano! and 3-methy!-1 -butanol, comprising:

(a) providing a recombinant microorganism according to any of claims 89-100;

(b) cultivating the recombinant microorganism in a culture medium containing a feedstock providing the carbon source, until a recoverable quantity of isobutanol and 3-methyl-1 -butano! is produced.