HIGH-PERFORMANCE DIHYDROXY ACID DEHYDRATASES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 61/536,916, filed September 20, 201 1 , and U.S. Provisional Application Serial No. 61/619,154, filed April 2, 2012, each of which is herein incorporated by reference in its entirety for all purposes.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0002] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: filename: GEVO_070_02WO_SeqList_ST25.txt, date recorded: September 14, 2012, file size: 601 kilobytes).

TECHNICAL FIELD

[0003] Recombinant microorganisms and methods of producing such microorganisms are provided. Also provided are methods of producing beneficial metabolites including fuels and chemicals by contacting a suitable substrate with the recombinant microorganisms of the application and enzymatic preparations therefrom.

BACKGROUND

[0004] Dihydroxyacid dehydratase (DHAD) is an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate ("DHIV") to a-ketoisovalerate and of 2,3- dihydroxy-3-methyl valerate to 2-keto-3-methylvalerate. This enzyme plays an important role in a variety of biosynthetic pathways, including pathways producing valine, isoleucine, leucine and pantothenic acid (vitamin B5). DHAD also catalyzes the conversion of 2,3-dihydroxyisovalerate to a-ketoisovalerate as part of isobutanol biosynthetic pathways disclosed in commonly owned U.S. Patent Nos. 8,017,375, 8,017,376, 8,097,440, 8,133,715, 8,153,415, 8,158,404, and 8,232,089. In addition, biosynthetic pathways for the production of 3-methyl-1 -butanol and 2-methyl-1 - butanol use DHAD to convert DHIV to α-ketoisovalerate and 2,3-dihydroxy-3- methylvalerate to 2-keto-3-methylvalerate, respectively (Atsumi et al., 2008, Nature [0005] DHAD is an essential enzyme in all of these biosynthetic pathways, hence, it is desirable that recombinant microorganisms engineered to produce the above- mentioned compounds exhibit optimal DHAD activity. The optimal level of DHAD activity will typically have to be at levels that are significantly higher than those found in non-engineered microorganisms in order to sustain commercially viable productivities, yields, and titers. The present application addresses this need by identifying several enzymes that exhibit high activity for the conversion of DHIV to a- ketoisovalerate and/or 2,3-dihydroxy-3-methylvalerate to 2-keto-3-methylvalerate. In particular, the present inventors have identified DHAD enzymes that exhibit high activity when expressed recombinantly in yeast and exhibit one or more of the following desirable characteristics: 1 ) activity equivalent to or greater than the DHAD derived from L. lactis which has previously been found to exhibit DHAD activity in yeast as well as function in a recombinant isobutanol biosynthetic pathway in yeast (See, e.g., U.S. Patent No. 8,232,089), (2) a K _M for the substrate, DHIV, that is lower than that of the L. lactis DHAD, and/or (3) less inhibition by isobutanol as compared to the L. lactis DHAD.

SUMMARY OF THE INVENTION

[0006] The present inventors have discovered a group of dihydroxyacid dehydratase (DHAD) enzymes with high level activity for the conversion of 2,3- dihydroxyisovalerate ("DHIV") to a-ketoisovalerate and/or the conversion of 2,3- dihydroxy-3-methyl valerate to 2-keto-3-methylvalerate in various biosynthetic pathways, including an engineered isobutanol biosynthetic pathway. The use of one or more of these DHAD enzymes can improve the production of various beneficial metabolites described herein, including isobutanol.

[0007] In a first aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 81 . In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Candidatus Korarchaeum cryptofilum OPF8.

[0008] In another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 82. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Eubacterium siraeum.

[0009] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 96. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Halorhabdus. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Halorhabdus utahensis.

[0010] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 98. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Methanosphaera. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Methanosphaera stadtmanae.

[0011] In another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 100. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Haladaptatus. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Haladaptatus paucihalophilus.

[0012] In another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 101 . In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Bacteroides. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Bacteroides fragilis.

[0013] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 107. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the uncultured gamma proteobacterium eBACHOT4E07.

[0014] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 108. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Ustilago. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Ustilago maydis.

[0015] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 109. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Rhodopirellula. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Rhodopirellula baltica.

[0016] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 1 10. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Leadbetterella. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Leadbetterella byssophila.

[0017] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 1 1 1 . In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the unidentified eubacterium SCB49.

[0018] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 1 12. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Marivirga. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Marivirga tractuosa.

[0019] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 1 13. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Polaribacter. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Polaribacter irgensii.

[0020] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to a polypeptide selected from SEQ ID NOs: 1 14-1 15. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Pedobacter. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Pedobacter saltans or Pedobacter heparinus.

[0021] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 1 16. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the class Flavobacteria. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Flavobacteria bacterium BAL38.

[0022] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 1 17. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Prochlorococcus. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Prochlorococcus marinus.

[0023] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 1 19. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Streptococcus. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Streptococcus gordonii.

[0024] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 120. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Microscilla. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Microscilla marina.

[0025] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 122. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Paenibacillus. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Paenibacillus sp. JDR-2.

[0026] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 128. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Acaryochloris. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Acaryochloris marina.

[0027] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 130. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Candidatus Koribacter versatilis Ellin345.

[0028] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 142. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Flavobacterium. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Flavobacterium psychrophilum.

[0029] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 149. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Arabidopsis. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Arabidopsis thaliana.

[0030] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to a polypeptide selected from SEQ ID NOs: 151 -153. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Neurospora. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Neurospora crassa.

[0031] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to a polypeptide selected from SEQ ID NOs: 154-156. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Oryza. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Oryza sativa.

[0032] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 163. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Methanobacterium. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Methanobacterium sp. SWAN-1 .

[0033] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 164. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Methanobrevibacter. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Methanobrevibacter smithii.

[0034] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 165. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Methanococcus. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Methanococcus aeolicus.

[0035] In yet another aspect, the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to SEQ ID NO: 166. In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Methanosarcina. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Methanosarcina mazei.

[0036] In various embodiments described herein, the recombinant microorganism may comprise a biosynthetic pathway which uses DHAD to catalyze a pathway step. The biosynthetic pathway which uses DHAD to catalyze a pathway step may be selected from a pathway for the biosynthesis of isobutanol, 3-methyl-1 -butanol, 2- methyl-1 -butanol, valine, isoleucine, leucine, and pantothenic acid. In one embodiment, the pathway for the biosynthesis of isobutanol, 3-methyl-1 -butanol, 2- methyl-1 -butanol, valine, isoleucine, leucine, and pantothenic acid comprises at least one polypeptide encoded by an exogenous gene.

[0037] In various embodiments described herein, the recombinant microorganism comprises an isobutanol producing metabolic pathway. In one embodiment, the isobutanol producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanol. In another embodiment, the isobutanol 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, all of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes. In an exemplary embodiment, at least one of the exogenously encoded enzymes is a polypeptide with dihydroxyacid dehydratase (DHAD) activity that is at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a polypeptide selected from SEQ ID NOs: 81 -82, 96, 98, 100-101 , 107-1 17, 1 19-120, 122, 128, 130, 142, 149, 154-156, and 163-166.

[0038] 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.

[0039] In various embodiments described herein, the isobutanol pathway genes may encode enzyme(s) selected from the group consisting of acetolactate synthase (ALS), ketol-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 KARI is an NADH-dependent KARI (NKR) and the ADH is an NADH-dependent ADH. In an exemplary embodiment, the dihydroxyacid dehydratase is a polypeptide which is at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a polypeptide selected from SEQ ID NOs: 81 -82, 96, 98, 100-101 , 107-1 17, 1 19-120, 122, 128, 130, 142, 149, 154-156, and 163-166.

[0040] 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).

[0041] In one embodiment, the recombinant microorganisms may be recombinant prokaryotic microorganisms. In another embodiment, the recombinant microorganisms may be recombinant eukaryotic microorganisms. In a further embodiment, the recombinant eukaryotic microorganisms may be recombinant yeast microorganisms.

[0042] In some embodiments, the recombinant yeast microorganisms may be members of the Saccharomyces clade, Saccharomyces sensu stricto microorganisms, 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.

[0043] In some embodiments, the recombinant microorganisms may be yeast recombinant microorganisms of the Saccharomyces clade.

[0044] In some embodiments, the recombinant microorganisms may be Saccharomyces sensu stricto microorganisms. In one embodiment, the Saccharomyces sensu stricto is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrids thereof.

[0045] 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, Issatchenkia, Hansenula, or Candida. In additional embodiments, the Crabtree-negative yeast microorganism is selected from Saccharomyces kluyveri, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Hansenula anomala, Candida utilis and Kluyveromyces waltii. [0046] 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, Kiuyveromyces, Zygosaccharomyces, Debaryomyces, Candida, Pichia 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 castelli, Kiuyveromyces thermotolerans, Candida glabrata, Z. bailli, Z. rouxii, Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces uvarum.

[0047] 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 castelli, and Candida glabrata.

[0048] 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, Kiuyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Pachysolen, Yarrowia and Schizosaccharomyces. In additional embodiments, the pre-WGD yeast 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, Issatchenkia orientalis, Issatchenkia occidentalis, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yarrowia lipolytica, and Schizosaccharomyces pombe.

[0049] 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. [0050] In another aspect, the present application provides methods of producing a beneficial metabolite derived from a recombinant microorganism. In one embodiment, the method includes cultivating a recombinant microorganism in a culture medium containing a feedstock providing a carbon source until a recoverable quantity of the beneficial metabolite is produced and optionally, recovering the metabolite. In an exemplary embodiment, said recombinant microorganism has been engineered to express a polypeptide with DHAD activity, wherein said polypeptide is at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a polypeptide selected from SEQ ID NOs: 81 -82, 96, 98, 100-101 , 107-1 17, 1 19-120, 122, 128, 130, 142, 149, 154- 156, and 163-166. The beneficial metabolite may be derived from any biosynthetic pathway which uses DHAD to catalyze a pathway step, including, but not limited to, biosynthetic pathways for the production of isobutanol, 3-methyl-1 -butanol, 2-methyl- 1 -butanol, valine, isoleucine, leucine, and pantothenic acid. In a specific embodiment, the beneficial metabolite is isobutanol.

[0051] In a method to produce a beneficial metabolite from a carbon source, the 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, a beneficial metabolite such as isobutanol 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

[0052] 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 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% theoretical. In a specific embodiment, the beneficial metabolite is isobutanol.

[0053] In one embodiment, the recombinant microorganism converts the carbon source to isobutanol under aerobic conditions. In another embodiment, the recombinant microorganism converts the carbon source to isobutanol under microaerobic conditions. In yet another embodiment, the recombinant microorganism converts the carbon source to isobutanol under anaerobic conditions.

BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 illustrates an isobutanol pathway.

[0055] Figure 2 illustrates an NADH-dependent isobutanol pathway.

[0056] Figure 3 illustrates the specific activity (U/mg) of DHADs as measured via in vitro assays. Values plotted for the DHADs are the average of technical triplicate assays performed on biological triplicate samples, with one standard deviation.

Each screening round contains the L. lactis DHAD comparison activity and rounds 3-

7 contain a GEVO6770 background DHAD activity control (empty vector). The top line represents the average of the L. lactis DHAD samples minus two standard deviations. The bottom line represents the average of the background control

(empty vector) samples plus two standard deviations.

[0057] Figure 4 illustrates the specific activity (U/mg) of DHADs as measured via in vitro assays. Values plotted for the DHADs are the average of technical triplicate assays performed on biological triplicate samples, with one standard deviation. Each screening round contains the L. lactis DHAD comparison activity and a GEVO6770 background DHAD activity control (empty vector). The top line represents the average of the L. lactis DHAD samples minus two standard deviations. The bottom line represents the average of the background control (empty vector) samples plus two standard deviations

[0058] Figure 5 illustrates the specific activity (U/mg) of DHADs as measured via in vitro assays in the presence of DHIV at concentrations of 0.4 mM (Low DHIV) and 10 mM (Std DHIV). Values plotted for the DHADs are the average of technical triplicate assays performed on biological triplicate samples, with one standard deviation. Each screening round contains the L. lactis DHAD comparison activity and the GEVO6770 background DHAD activity control (empty vector).

[0059] Figures 6A and 6B illustrate the specific activity (U/mg) of DHADs as measured via in vitro assays in the presence (iBuOH DHAD) and absence (Std DHAD) of 20 g/L isobutanol in the DHAD assay. Values plotted for the DHADs are the average of technical triplicate assays performed on biological triplicate samples, with one standard deviation.

[0060] Figure 7 illustrates the average of isobutanol volumetric titers of 48 hour shake flask fermentations with one standard deviation for various DHAD homologs. [0061] Figure 8 illustrates the average of isobutanol volumetric titers of 48 hour shake flask fermentations with one standard deviation for various DHAD homologs.

[0062] Figure 9 illustrates the specific activity (U/mg) of DHADs as measured via in vitro assays at 24 hours of fermentation. Error bars represent the standard deviation of biological triplicates from the fermentation and technical triplicates within the DHAD assay.

DETAILED DESCRIPTION

[0063] 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.

[0064] Unless defined otherwise, all 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.

[0065] 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.

[0066] 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. The terms "microbial cells" and "microbes" are used interchangeably with the term microorganism.

[0067] The term "prokaryotes" is art recognized and refers to cells which contain no nucleus or other cell organelles. The prokaryotes are generally classified in one of two domains, the Bacteria and the Archaea. The definitive difference between organisms of the Archaea and Bacteria domains is based on fundamental differences in the nucleotide base sequence in the 16S ribosomal RNA.

[0068] The term "Archaea" refers to a categorization of organisms of the division Mendosicutes, typically found in unusual environments and distinguished from the rest of the prokaryotes by several criteria, including the number of ribosomal proteins and the lack of muramic acid in cell walls. On the basis of ssrRNA analysis, the Archaea consist of two phylogenetically-distinct groups: Crenarchaeota and Euryarchaeota. On the basis of their physiology, the Archaea can be organized into three types: methanogens (prokaryotes that produce methane); extreme halophiles (prokaryotes that live at very high concentrations of salt (NaCI); and extreme (hyper) thermophiles (prokaryotes that live at very high temperatures). Besides the unifying archaeal features that distinguish them from Bacteria (i.e., no murein in cell wall, ester-linked membrane lipids, etc.), these prokaryotes exhibit unique structural or biochemical attributes which adapt them to their particular habitats. The Crenarchaeota consist mainly of hyperthermophilic sulfur-dependent prokaryotes and the Euryarchaeota contain the methanogens and extreme halophiles.

[0069] "Bacteria", or "eubacteria", refers to a domain of prokaryotic organisms. Bacteria include at least eleven distinct groups as follows: (1 ) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1 ) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic +non-photosynthetic Gram-negative bacteria (includes most "common" Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non- sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; (1 1 ) Thermotoga and Thermosipho thermophiles.

[0070] "Gram-negative bacteria" include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0071] "Gram positive bacteria" include cocci, nonsporulating rods, and sporulating rods. The genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0072] The term "genus" is defined as a taxonomic group of related species according to the Taxonomic Outline of Bacteria and Archaea (Garrity, G.M., Lilburn, T.G., Cole, J.R., Harrison, S.H., Euzeby, J., and Tindall, B.J. (2007) The Taxonomic Outline of Bacteria and Archaea. TOBA Release 7.7, March 2007. Michigan State University Board of Trustees.

[0073] The term "species" is defined as a collection of closely related organisms with greater than 97% 16S ribosomal RNA sequence homology and greater than 70% genomic hybridization and sufficiently different from all other organisms so as to be recognized as a distinct unit.

[0074] The terms "recombinant microorganism," "modified microorganism," and "recombinant host cell" 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.

[0075] 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 cell, 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 al., 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 al., 1989, supra.

[0076] 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 cells as compared to similar corresponding unmodified cells 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.

[0077] 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 cell.

[0078] The term "wild-type microorganism" describes a cell 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.

[0079] 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 in to a parental microorganism.

[0080] 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.

[0081] 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 all 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.

[0082] 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.

[0083] As used herein, the term "isobutanol producing metabolic pathway" refers to an enzyme pathway which produces isobutanol from pyruvate.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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 cell but is either produced in an unnatural location or in an unnatural amount in the cell; 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 cell.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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 gram per liter per hour (g/L/h).

[0092] 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 cells. Specific productivity is reported in gram (or milligram) per gram cell dry weight per hour (g/g h).

[0093] The term "yield" is defined as the amount of product obtained per unit weight of raw material and may be expressed as g product per g 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.

[0094] 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 g of biofuel in solution per liter of fermentation broth (g/L).

[0095] "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.

[0096] 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.

[0097] "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. [0098] 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."

[0099] 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 ethanol. 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 ethanol under aerobic conditions.

[00100] 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.

[00101] The term "substantially free" when used in reference to the presence or absence of a protein activity (3-KAR enzymatic activity, ALDH enzymatic activity, PDC enzymatic activity, GPD enzymatic activity, etc.) means the level of the protein is substantially less than that of the same protein 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 protein activity (3-KAR enzymatic activity, ALDH enzymatic activity, PDC enzymatic activity, GPD enzymatic activity, etc.) may be created through recombinant means or identified in nature.

[00102] The term "non-fermenting yeast" is a yeast species that fails to demonstrate an anaerobic metabolism in which the electrons from NADH are utilized to generate a reduced product via a fermentative pathway such as the production of ethanol and CO2 from glucose. Non-fermentative yeast can be identified by the "Durham Tube Test" (J .A. Barnett, R.W. Payne, and D. Yarrow. 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.

[00103] 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 nucleotidic oligomer or oligonucleotide.

[00104] 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.

[00105] 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.

[00106] 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, plasmids, 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.

[00107] "Transformation" refers to the process by which a vector is introduced into a host cell. Transformation (or transduction, or transfection), can be achieved by any one of a number of means including chemical transformation (e.g. lithium acetate transformation), electroporation, microinjection, biolistics (or particle bombardment- mediated delivery), or agrobacterium mediated transformation.

[00108] 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 or polypeptides, but can include enzymes composed of a different molecule including polynucleotides.

[00109] 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 polymer 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

[00110] The term "homolog," used with respect to an original polynucleotide or polypeptide of a first family or species, refers to distinct polynucleotides or polypeptides of a second family or species which are determined by functional, structural or genomic analyses to be a polynucleotide or polypeptide of the second family or species which corresponds to the original polynucleotide or polypeptide of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of a polynucleotide or polypeptide can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.

[00111] A polypeptide has "homology" or is "homologous" to a second polypeptide if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a polypeptide has homology to a second polypeptide if the two polypeptides have "similar" amino acid sequences. (Thus, the terms "homologous polypeptides" or "homologous proteins" are defined to mean that the two polypeptides have similar amino acid sequences).

[00112] The term "analog" or "analogous" refers to polynucleotide or polypeptide sequences 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.

Isobutanol Producing Recombinant Microorganisms

[00113] 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-driven biosynthetic pathways, including isobutanol, an important commodity chemical and biofuel candidate (See, e.g., commonly owned U.S. Patent Nos. 8,017,375, 8,017,376, 8,097,440, 8,133,715, 8,153,415, 8,158,404, and 8,232,089).

[00114] As described herein, the present invention relates to recombinant microorganisms for producing isobutanol, wherein said recombinant microorganisms comprise an isobutanol producing metabolic pathway. In one embodiment, the isobutanol producing metabolic pathway to convert pyruvate to isobutanol 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-ketoisovalerate

4. alpha-ketoisovalerate→ isobutyraldehyde + CO ₂

5. isobutyraldehyde +NAD(P)H→ isobutanol + NAD(P) ⁺

[00115] 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 all of these enzymes.

[00116] Alternative pathways for the production of isobutanol in yeast have been described in WO/2007/050671 and in Dickinson et a/., 1998, J Biol Chem 273: 25751 -6. These and other isobutanol producing metabolic pathways are within the scope of the present application. In one embodiment, the isobutanol producing metabolic pathway comprises five substrate to product reactions. In another embodiment, the isobutanol producing metabolic pathway comprises six substrate to product reactions. In yet another embodiment, the isobutanol producing metabolic pathway comprises seven substrate to product reactions.

[00117] In various embodiments described herein, the recombinant microorganism comprises an isobutanol producing metabolic pathway. In one embodiment, the isobutanol producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanol. In another embodiment, the isobutanol 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, all of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes.

[00118] 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. Isobutanol producing metabolic pathways in which one or more genes are localized to the cytosol are described in commonly owned U.S. Patent No. 8,232,089, which is herein incorporated by reference in its entirety for all purposes.

[00119] As is understood in the art, a variety of organisms can serve as sources for the isobutanol pathway enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including Y. spp. stipitis, Torulaspora pretoriensis, Issatchenkia orientalis, Schizosaccharomyces spp., including S. pombe, Cryptococcus 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., Slackia spp., Cryptobacterium spp., and Eggerthella spp. [00120] 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.

[00121] For example, acetolactate synthases capable of converting pyruvate to acetolactate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including B. subtilis (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. ZP_06014957.1 ), C. glutamicum (GenBank Accession No. P42463.1 ), 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. NP_013826.1 ). Additional acetolactate synthases capable of converting pyruvate to acetolactate are described in commonly 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 acetolactate from pyruvate via the activity of acetolactate synthases is provided by Chipman et ai, 1998, Biochimica et Biophysica Acta 1385: 401 -19, which is herein incorporated by reference in its entirety. Chipman et al. 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 include:

SGPG(A/C/V)(T/S)N (SEQ ID NO: 167),

GX(P/A)GX(V/A/T) (SEQ ID NO: 168),

GX(Q/G)(T/A)(L/M)G(Y/F/W)(A/G)X(P/G)(W/A)AX(G/T)(A/V) (SEQ ID NO: 169), and

GD(G/A)(G/S/C)F (SEQ ID NO: 170)

motifs at amino acid positions corresponding to the 163-169, 240-245, 521 -535, and 549-553 residues, respectively, of the S. cerevisiae ILV2. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit acetolactate synthase activity.

[00122] Ketol-acid reductoisomerases capable of converting acetolactate 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. YP_003353710.1 ), 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_001018845), B. thetaiotamicron (GenBank Accession No. NP_810987), or S. cerevisiae ILV5 (GenBank Accession No. NP_013459.1 ). Additional ketol-acid reductoisomerases capable of converting acetolactate to 2,3- dihydroxyisovalerate are described in commonly owned U.S. Patent No. 8,232,089, which is herein incorporated by reference in its entirety. An alignment and consensus for the sequences of a representative number of ketol-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 include:

G(Y/C/W)GXQ(G/A) (SEQ ID NO: 171 ),

(F/Y/L)(S/A)HG(F/L) (SEQ ID NO: 172),

V(V/I/F)(M/L/A)(A/C)PK (SEQ ID NO: 173),

D(L/I)XGE(Q/R)XXLXG (SEQ ID NO: 174), and

S(D/N/T)TA(E/Q/R)XG (SEQ ID NO: 175)

motifs at amino acid positions corresponding to the 89-94, 175-179, 194-200, 262- 272, and 459-465 residues, respectively, of the E. coli ketol-acid reductoisomerase encoded by ilvC. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit ketol-acid reductoisomerase activity.

[00123] To date, all known, naturally existing ketol-acid reductoisomerases are known to use NADPH as a cofactor. In certain embodiments, a ketol-acid reductoisomerase which has been engineered to used NADH as a cofactor may be utilized to mediate the conversion of acetolactate to 2,3-dihydroxyisovalerate. Engineered NADH-dependent KARI enzymes ("NKRs") and methods of generating such NKRs are disclosed in commonly owned and co-pending US Publication No. 2010/0143997.

[00124] In accordance with the invention, any number of mutations can be made to a KARI enzyme, and in a preferred aspect, multiple mutations can be made to a KARI enzyme to result in an increased ability to utilize NADH for the conversion of acetolactate to 2,3-dihydroxyisovalerate. Such mutations include point mutations, frame shift mutations, deletions, and insertions, with one or more {e.g., one, two, three, four, five or more, etc.) point mutations preferred.

[00125] Mutations may be introduced into naturally existing KARI enzymes to create NKRs using any methodology known to those skilled in the art. Mutations may be introduced randomly by, for example, conducting a PCR reaction in the presence of manganese as a divalent metal ion cofactor. Alternatively, oligonucleotide directed mutagenesis may be used to create the NKRs which allows for all possible classes of base pair changes at any determined site along the encoding DNA molecule. In general, this technique involves annealing an oligonucleotide complementary (except for one or more mismatches) to a single stranded nucleotide sequence coding for the KARI enzyme of interest. The mismatched oligonucleotide is then extended by DNA polymerase, generating a double-stranded DNA molecule which contains the desired change in sequence in one strand. The changes in sequence can, for example, result in the deletion, substitution, or insertion of an amino acid. The double-stranded polynucleotide can then be inserted into an appropriate expression vector, and a mutant or modified polypeptide can thus be produced. The above-described oligonucleotide directed mutagenesis can, for example, be carried out via PCR.

[00126] 2-keto-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. lactis kivD (GenBank Accession No. YP_003353820.1 ), E. cloacae (GenBank Accession No. P23234.1 ), M. smegmatis (GenBank Accession No. A0R480.1 ), M. tuberculosis (GenBank Accession No. 053865.1 ), 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. caseolyticus (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 include:

FG(V/I)(P/S)G(D/E)(Y/F) (SEQ ID NO: 176),

(T/V)T(F/Y)G(V/A)G(E/A)(L/F)(S/N) (SEQ ID NO: 177),

N(G/A)(L/I/V)AG(S/A)(Y/F)AE (SEQ ID NO: 178),

(V/I)(L/I/V)XI(V/T/S)G (SEQ ID NO: 179), and

GDG(S/A)(L/F/A)Q(L/M)T (SEQ ID NO: 180) motifs at amino acid positions corresponding to the 21 -27, 70-78, 81 -89, 93-98, and 428-435 residues, respectively, of the L. lactis 2-keto-acid decarboxylase encoded by kivD. An additional "HH"-motif found at amino acids 1 12-1 13 in the L. lactis 2- keto-acid decarboxylase encoded by kivD is characteristic of thiamin diphosphate- dependent decarboxylases, a class of enzymes of which 2-keto acid decarboxylases belong. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit 2-keto-acid decarboxylase activity.

[00127] 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 ), B. cereus (GenBank Accession No. YP_001374103.1 ), N. meningitidis (GenBank Accession No. CBA03965.1 ), 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. acidilactici (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 include:

C(H/G)(T/S)D(L/I)H (SEQ ID NO: 181 ),

GHEXXGXV (SEQ ID NO: 182),

(L/V)(Q/K/E)(V/I/K)G(D/Q)(R/H)(V/A) (SEQ ID NO: 183),

CXXCXXC (SEQ ID NO: 184),

(C/A)(A/G/D)(G/A)XT(T/V) (SEQ ID NO: 185), and

G(L/A/C)G(G/P)(L/I/V)G (SEQ ID NO: 186) motifs at amino acid positions corresponding to the 39-44, 59-66, 76-82, 91 -97, 147- 152, and 171 -176 residues, respectively, of the L. lactis alcohol dehydrogenase encoded by adhA. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit alcohol dehydrogenase activity.

[00128] 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 isobutyraldehyde. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to keto-isovalerate. 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 acetolactate.

[00129] 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.

[00130] 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. The present inventors have found 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. 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 acetolactate 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 isobutyraldehyde to produce isobutanol. In yet another embodiment, the recombinant microorganisms of the present invention may use both an NKR to catalyze the conversion of acetolactate to produce 2,3- dihydroxyisovalerate, and an NADH-dependent ADH to catalyze the conversion of isobutyraldehyde to produce isobutanol.

Metabolic Pathways with Improved DHAD Properties

[00131] A number of biosynthetic pathways use DHAD activity to catalyze a reaction step, including pathways for the production of isobutanol, 3-methyl-1 - butanol, 2-methyl-1 -butanol, valine, isoleucine, leucine, and pantothenic acid. The metabolic pathway utilizing use DHAD activity to catalyze a reaction step may naturally occur in a microorganism {e.g., a natural pathway for the production of valine or isobutanol) or arise from the introduction of one or more heterologous polynucleotides through genetic engineering.

[00132] As used herein, the terms "DHAD" or "DHAD enzyme" or "dihydroxyacid dehydratase" are used interchangeably herein to refer to an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to ketoisovalerate and/or the conversion of 2,3-dihydroxy-3-nnethylvalerate to 2-keto-3-methylvalerate.

[00133] As described above, biosynthetic pathways for the production of isobutanol, 3-methyl-1 -butanol, 2-methyl-1 -butanol, valine, isoleucine, leucine, and pantothenic acid rely upon an enzyme exhibiting DHAD activity to catalyze a reaction step. Therefore, the product yield from these biosynthetic pathways will in part depend upon the level of activity of the enzyme exhibiting DHAD activity. The optimal level of DHAD activity will typically have to be at levels that are significantly higher than those found in non-engineered microorganisms in order to sustain commercially viable productivities, yields, and titers. The present application has identified several DHAD enzymes that exhibit high activity for the conversion of DHIV to a- ketoisovalerate and/or the conversion of 2,3-dihydroxy-3-methylvalerate to 2-keto-3- methylvalerate. In particular, the present inventors have identified DHAD enzymes that exhibit high activity when expressed recombinantly in yeast and exhibit one or more of the following desirable characteristics: 1 ) activity equivalent to or greater than the DHAD derived from L. lactis which has previously been found to exhibit DHAD activity in yeast as well as function in a recombinant isobutanol biosynthetic pathway in yeast (See, e.g., U.S. Patent No. 8,232,089), (2) a K _M for the substrate, DHIV, that is lower than that of the L. lactis DHAD, and/or (3) less inhibition by isobutanol as compared to the L. lactis DHAD.

[00134] As will be understood by one skilled in the art equipped with the present disclosure, the enzymes exhibiting DHAD activity described herein would have utility in any DHAD-requiring biosynthetic pathway, including pathways for the production of isobutanol, 3-methyl-1 -butanol, 2-methyl-1 -butanol, valine, isoleucine, leucine, and pantothenic acid. Thus, in one aspect, the present application relates to polypeptides with favorable dihydroxyacid dehydratase (DHAD) activities. Specifically, these polypeptides include those described in SEQ ID NOs: 81 -82, 96, 98, 100-101 , 107-1 17, 1 19-120, 122, 128, 130, 142, 149, 154-156, and 163-166.

[00135] Accordingly, one aspect of the application is directed to an isolated nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to a polypeptide selected from SEQ ID NOs: 81 -82, 96, 98, 100-101 , 107-1 17, 1 19-120, 122, 128, 130, 142, 149, 154-156, and 163-166. Further within the scope of present application are polypeptides with dihydroxyacid dehydratase (DHAD) activity which are at least about 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a polypeptide selected from SEQ ID NOs: 81 -82, 96, 98, 100-101 , 107-1 17, 1 19-120, 122, 128, 130, 142, 149, 154-156, and 163-166.

[00136] In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Candidatus Korarchaeum cryptofilum OPF8. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 81 .

[00137] In another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Eubacterium siraeum. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 82.

[00138] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Halorhabdus. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Halorhabdus utahensis. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 96.

[00139] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Methanosphaera. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Methanosphaera stadtmanae. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 98.

[00140] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Haladaptatus. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Haladaptatus paucihalophilus. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 100.

[00141] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Bacteroides. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Bacteroides fragilis. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 101 .

[00142] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the uncultured gamma proteobacterium eBACHOT4E07. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 107.

[00143] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Ustilago. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Ustilago maydis. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 108.

[00144] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Rhodopirellula. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Rhodopirellula baltica. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 109.

[00145] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Leadbetterella. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Leadbetterella byssophila. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 1 10.

[00146] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the unidentified eubacterium SCB49. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 1 1 1 .

[00147] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Marivirga. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Marivirga tractuosa. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 1 12.

[00148] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Polaribacter. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Polaribacter irgensii. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 1 13.

[00149] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Pedobacter. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Pedobacter saltans or Pedobacter heparinus. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is selected from SEQ ID NOs: 1 14-1 15.

[00150] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the class Flavobacteria. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Flavobacteria bacterium BAL38. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 1 16.

[00151] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Prochlorococcus. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Prochlorococcus marinus. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 1 17.

[00152] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Streptococcus. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Streptococcus gordonii. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 1 19.

[00153] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Microscilla. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Microscilla marina. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 120.

[00154] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Paenibacillus. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Paenibacillus sp. JDR-2. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 122.

[00155] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Acaryochloris. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Acaryochloris marina. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 128.

[00156] In one embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Candidatus Koribacter versatilis Ellin345. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 130.

[00157] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Flavobacterium. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Flavobacterium psychrophilum. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 142.

[00158] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Arabidopsis. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Arabidopsis thaliana. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 149.

[00159] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Neurospora. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Neurospora crassa. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is selected from SEQ ID NOs: 151 - 153.

[00160] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Oryza. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Oryza sativa. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is selected from SEQ ID NOs: 154-156.

[00161] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Methanobacterium. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Methanobacterium sp. SWAN-1 . In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 163. [00162] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Methanobrevibacter. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Methanobrevibacter smithii. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 164.

[00163] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Methanococcus. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Methanococcus aeolicus. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 165.

[00164] In yet another embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from the genus Methanosarcina. In a specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity is derived from Methanosarcina mazei. In another specific embodiment, the polypeptide with dihydroxyacid dehydratase (DHAD) activity comprises SEQ ID NO: 166.

[00165] The invention also includes fragments of the disclosed polypeptides with dihydroxyacid dehydratase (DHAD) activity which comprise at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 amino acid residues and retain one or more activities associated with dihydroxyacid dehydratases. Such fragments may be obtained by deletion mutation, by recombinant techniques that are routine and well-known in the art, or by enzymatic digestion of the polypeptides of interest using any of a number of well-known proteolytic enzymes. The invention further includes nucleic acid molecules which encode the above described polypeptides and polypeptide fragments exhibiting dihydroxyacid dehydratase (DHAD) activity.

[00166] Another aspect of the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70% identical to a polypeptide selected from SEQ ID NOs: 81 -82, 96, 98, 100- 101 , 107-1 17, 1 19-120, 122, 128, 130, 142, 149, 154-156, and 163-166. Further within the scope of present application are recombinant microorganisms comprising at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a polypeptide selected from SEQ ID NOs: 81 -82, 96, 98, 100-101 , 107-1 17, 1 19-120, 122, 128, 130, 142, 149, 154-156, and 163-166.

The Microorganism in General

[00167] As described herein, the recombinant microorganisms of the present application can express a plurality of heterologous and/or native enzymes involved in pathways for the production of a beneficial metabolite such as isobutanol, 3-methyl-

1 - butanol, 2-methyl-1 -butanol, valine, isoleucine, leucine, and pantothenic acid.

[00168] Each of these metabolic pathways utilizes an enzyme with DHAD activity to convert (a) DHIV to a-ketoisovalerate and/or (b) 2,3-dihydroxy-3-methylvalerate to

2- keto-3-methylvalerate. The metabolic pathway may naturally occur in a microorganism or arise from the introduction of one or more heterologous polynucleotides through genetic engineering. In an exemplary embodiment, the recombinant microorganisms expressing the biosynthetic pathway requiring an enzyme with DHAD activity are yeast cells.

[00169] Engineered biosynthetic pathways for synthesis of valine, leucine, and isoleucine (See, e.g., WO/2001/021772, and McCourt et al., 2006, Amino Acids 31 : 173-210), pantothenic acid (See, e.g., WO/2001/021772), 3-methyl-1 -butanol (See, e.g., WO/2008/098227, Atsumi et al., 2008, Nature 451 : 86-89, and Connor et al., 2008, Appl. Environ. Microbiol. 74: 5769-5775), and 2-methyl-1 -butanol (See, e.g., WO/2008/098227, WO/2009/076480, and Atsumi et al., 2008, Nature 451 : 86-89).

[00170] 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 isobutanol, 3-methyl-1 -butanol, 2-methyl-1 -butanol, valine, isoleucine, leucine, and pantothenic acid 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 isobutanol, 3- methyl-1 -butanol, 2-methyl-1 -butanol, valine, isoleucine, leucine, and pantothenic acid and may also include additional elements for the expression and/or regulation of expression of these genes, e.g., promoter sequences.

[00171] 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).

[00172] 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-ketoisovalerate), or an end product {e.g., isobutanol) 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.

[00173] The disclosure identifies specific genes useful in the methods, compositions and organisms of the disclosure; however it will be recognized that absolute identity 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.

[00174] 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 clone and express the polynucleotides encoding such enzymes. [00175] 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."

[00176] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (Murray et al., 1989, Nucl 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 E. coli commonly use UAA as the stop codon (Dalphin et ai, 1996, Nucl Acids Res. 24: 216-8). 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.

[00177] 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.

[00178] In addition, homologs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein.

[00179] 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 60%, 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.

[00180] 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 Mol Biol 25: 365-89).

[00181] 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), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[00182] 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 DHAD activity. In various embodiments, microorganisms may be selected from yeast microorganisms. Yeast microorganisms for the production of a metabolite such as isobutanol are described in commonly-owned U.S. Patent Nos. 8,017,375, 8,017,376, 8,133,715, 8,153,415, 8,158,404, and 8,232,089. 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 lactic acid bacteria such as, for example, a microorganism derived from the Lactobacillus or Lactococcus genus.

[00183] 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., isobutanol). 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.

[00184] 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.

[00185] In another embodiment, the microorganism has reduced glycerol-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., isobutanol). Thus, disruption, deletion, or mutation of the genes encoding for glycerol-3-phosphate dehydrogenases can further increase the yield of the desired metabolite {e.g., isobutanol). 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.

[00186] 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. In a specific embodiment, the 3-KAR is the S. cerevisiae protein YMR226c or a homolog thereof.

[00187] 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. In a specific embodiment, the ALDH is the S. cerevisiae protein ALD6 or a homolog thereof. [00188] In yet another embodiment, the microorganism has increased activator of ferrous transport (AFT) activity. Increased AFT activity has been demonstrated to improve the activity of DHAD and concomitantly improve the production of the beneficial metabolites in recombinant microorganisms comprising a DHAD-requiring biosynthetic pathway. See, e.g., commonly owned U.S. Patent Nos. 8,017,376 and 8,071 ,358, each of which is herein incorporated by reference in its entirety for all purposes. The microorganisms of the present application may be engineered to have increased AFT activity via the overexpression of one or more AFT polynucleotides and/or via the expression of one or more polynucleotides encoding one or more constitutively active AFT polypeptides. In some embodiments, the AFT polynucleotide to be overexpressed is a polynucleotide encoding a constitutively active AFT polypeptide. In additional embodiments, the microorganisms of the present application may be engineered to have increased AFT activity via the deletion of one or more negative regulators of AFT, e.g., GRX3 and/or FRA2.

Methods in General

[00189] Any method can be used to identify genes that encode for enzymes that are homologous to the genes described herein {e.g., dihydroxyacid dehydratase homologs). Generally, genes that are homologous or similar to the dihydroxyacid dehydratases 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.

[00190] 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 not limited to, cloning a gene by PCR using primers based on a published sequence of a gene/enzyme or by degenerate PCR using degenerate primers designed to amplify a conserved region among ketol-acid reductoisomerase genes. Further, one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity. 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.

[00191] Methods for gene insertion, gene deletion, and gene overexpression may be found in commonly-owned U.S. Patent Nos. 8,017,375, 8,017,376, 8,071 ,358, 8,097,440, 8,133,175, 8,153,415, 8,158,404, and 8,232,089, each of which is herein incorporated by reference in its entirety for all purposes.

Methods of Using Recombinant Microorganisms for Production of Beneficial Metabolites

[00192] In one aspect, the present application provides methods of producing a desired metabolite using a recombinant described herein. In one embodiment, the recombinant microorganism comprises a biosynthetic pathway requiring an enzyme with dihydroxyacid dehydratase (DHAD) activity, wherein said recombinant microorganism comprises at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a polypeptide selected from SEQ ID NOs: 81 -82, 96, 98, 100-101 , 107-1 17, 1 19-120, 122, 128, 130, 142, 149, 154-156, and 163-166.

[00193] In an exemplary embodiment, the biosynthetic pathway is a pathway for the production of a beneficial metabolite selected from isobutanol, 3-methyl-1 - butanol, 2-methyl-1 -butanol, valine, isoleucine, leucine, and pantothenic acid. In a further exemplary embodiment, the beneficial metabolite is isobutanol.

[00194] In a method to produce a beneficial metabolite {e.g., isobutanol) from a carbon source, the recombinant microorganism is cultured in an appropriate culture medium containing a carbon source. In certain embodiments, the method further includes isolating the beneficial metabolite {e.g., isobutanol) from the culture medium. For example, a beneficial metabolite {e.g., isobutanol) 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. In certain exemplary embodiments, the beneficial metabolite is selected from isobutanol, 3-methyl-1 - butanol, 2-methyl-1 -butanol, valine, isoleucine, leucine, and pantothenic acid. In a further exemplary embodiment, the beneficial metabolite is isobutanol.

[00195] In one embodiment, the recombinant microorganism may produce the beneficial metabolite {e.g., isobutanol) from a carbon source at a yield of at least 5 percent theoretical. In another embodiment, the microorganism may produce the beneficial metabolite {e.g., isobutanol) 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 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 a specific embodiment, the beneficial metabolite is isobutanol.

Distillers Dried Grains Comprising Spent Yeast Biocatalvsts

[00196] In an economic fermentation process, as many of the products of the fermentation as possible, including the co-products that contain biocatalyst cell 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.

[00197] 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.

[00198] 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 at least one nucleic acid molecule encoding a polypeptide with dihydroxyacid dehydratase (DHAD) activity, wherein said polypeptide is at least about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a polypeptide selected from SEQ ID NOs: 81 -82, 96, 98, 100-101 , 107-1 17, 1 19-120, 122, 128, 130, 142, 149, 154- 156, and 163-166. [00199] 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.

[00200] 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.

[00201] 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.

[00202] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures and the Sequence Listings, are incorporated herein by reference for all purposes.

EXAMPLES

Example 1 : In Vitro Screening of DHAD Activity in Yeast

[00203] In this example, genes encoding DHAD homologs from a variety of diverse organisms were cloned onto CEN, low-copy number plasmids downstream of a S. cerevisiae PDC1 promoter to provide high expression of the DHAD homologs. A copy of AFT1 was further included on the plasmid. Plasmids containing the DHAD homologs were transformed into S. cerevisiae strain, GEVO6770, and DHAD activities were determined on the yeast transformant lysates at the end of the growth phase of fermentation cultures.

[00204] A listing of the genes screened in Example 1 are listed in Table 1.

Table 1. Investigated DHAD Genes. Organism of Derivation SEQ ID NO: SEQ ID NO:

Nucleic Acid Polypeptide

Uncultured archaeon GZfos28B8 from Eel River sediment 1 80

Candidatus Korarchaeum cryptofilum OPF8 2 81

Eubacterium siraeum DSM 15702 3 82

Desulfitobacterium hafniense Y51 4 83

Fusobacterium nucleatum subsp. polymorphum ATCC 10953 5 84

Dehalococcoides sp. CBDB1 6 85

Halothermothrix orenii H 168 7 86

Desulfovibrio salexigens DSM 2638 8 87

Campylobacter jejuni subsp. jejuni CF93-6 9 88

Chloroherpeton thalassium ATCC 351 10 10 89

Candidatus Desulforudis audaxviator MP104C 1 1 90

Congregibacter litoralis KT71 12 91

Candidatus Blochmannia floridanus 13 92

Yersinia aldovae ATCC 35236 14 93

Aeromonas salmonicida subsp. salmonicida A449 15 94

Neisseria meningitidis alpha275 16 95

Halorhabdus utahensis DSM 12940 17 96

Methanocorpusculum labreanum Z 18 97

Methanosphaera stadtmanae DSM 3091 19 98

Clostridiales bacterium 1_7_47FAA 20 99

Haladaptatus paucihalophilus DX253 21 100

Bacteroides fragilis NCTC 9343 22 101

Maritimibacter alkaliphilus HTCC2654 23 102

Bradyrhizobium japonicum USDA 1 10 24 103

Nitrosopumilus maritimus SCM1 25 104

Brevundimonas subvibrioides ATCC 15264 26 105

Candidatus Carsonella ruddii PV 27 106

Uncultured gamma proteobacterium eBACHOT4E07 from 28 107 surface bacterioplankton from Hawaii Ocean Time-Series

(HOT) Station ALOHA seawater

Ustilago maydis 521 29 108

Rhodopirellula baltica SH 1 30 109

Leadbetterella byssophila DSM 17132 31 1 10

Unidentified eubacterium SCB49 32 1 1 1

Marivirga tractuosa DSM 4126 33 1 12

Polaribacter irgensii 23-P 34 1 13

Pedobacter saltans DSM 12145 35 1 14

Pedobacter heparinus DSM 2366 36 1 15

Flavobacteria bacterium BAL38 37 1 16

Prochlorococcus marinus str. NATL1A 38 1 17

Gemmata obscuriglobus UQM 2246 39 1 18

Streptococcus gordonii str. Challis substr. CH 1 40 1 19

Microscilla marina ATCC 23134 41 120

Psychromonas ingrahamii 37 42 121

LIJlvD (Lactococcus lactis subsp. lactis IL1403) 79 158

[00205] Each round of fermentations contained a comparison strain expressing the L. lactis DHAD (SEQ ID NO: 158) derived from sub-species lactis IL1403 to account for variations in the DHAD assay between fermentation rounds and to provide a benchmark for identifying DHAD homologs that gave similar or higher DHAD activity than the L. lactis DHAD of SEQ ID NO: 158, which was previously identified as demonstrating high activity in the yeast cytosol (See commonly owned U.S. Patent No. 8,232,089). A cut-off of two standard deviations below the average L. lactis DHAD activity from each individual fermentation round was used to identify DHADs that gave similar or higher DHAD activity than the L. lactis DHAD in yeast lysates. In other words, if the DHAD activity was at least as high as two standard deviations below the average L. lactis DHAD activity, the DHAD was considered to possess similar or higher DHAD activity as compared to the L. lactis DHAD. DHAD homologues that gave average activities in the yeast lysates with single standard deviations that overlapped with or were higher than this cut-off value were considered to have similar or higher DHAD activity than the L. lactis DHAD in that fermentation round. Also, during analysis of the DHAD activities from each round of fermentations after the first two rounds (i.e., R1 and R2), a cell pellet from a strain transformed with a control plasmid not expressing a DHAD or AFT1 (pGV2437) was included to provide background DHAD activities from GEVO6770. A cut off of two standard deviations above these background DHAD activities in each fermentation round was used to identify DHADs that had higher than background levels of DHAD activity in the yeast lysates if they did not have DHAD activities similar to or higher than the L. lactis DHAD in that fermentation round. DHAD homologs that gave average activities in the yeast lysates with single standard deviations that were higher than this background DHAD activity cut-off value were considered to have higher DHAD activity than the background DHAD activity in that fermentation round.

[00206] Figure 3 shows the DHAD activity results from the first seven rounds of screening of DHAD homologs. Figure 4 shows the DHAD activity results from screening rounds 8-13.

[00207] Amongst the tested DHAD homologs, 29 clones resulted in DHAD activities that were considered similar to or higher than the L. lactis DHAD. These results are summarized in Table 2.

Table 2. DHAD Homolog Genes Resulting in DHAD Activity in Yeast Lysates Similar to or Higher Than the L. lactis IlvD Gene (SEQ ID NO: 158).

DHAD Gene Description

Huta_DHAD_coScB Halorhabdus utahensis DSM 12940 DHAD gene

Msta_DHAD_coScB Methanosphaera stadtmanae DSM 3091 DHAD gene

UnGaPr_DHAD_coScB Uncultured gamma proteobacterium eBACHOT4E07 DHAD gene Umay_DHAD_coScB Ustilago maydis 521 DHAD gene

Rbal_DHAD_coScB Rhodopirellula baltica SH 1 DHAD gene

Lbys_DHAD_coScB Leadbetterella byssophila DSM 17132 DHAD gene

UnEuBa_DHAD_coScB Unidentified eubacterium SCB49 DHAD gene

Mtra_DHAD_coScB Marivirga tractuosa DSM 4126 DHAD gene

Fpsy_DHAD_coScB Flavobacterium psychrophilum JIP02/86 DHAD gene

Psal_DHAD_coScB Pedobacter saltans DSM 12145 DHAD gene

Phep_DHAD_coScB Pedobacter heparinus DSM 2366 DHAD gene

Lactococcus lactis subsp. cremoris strain MG1363 and strain NZ9000

Llsbcre_DHAD_coScB DHAD gene

Sgor_DHAD_coScB Streptococcus gordonii str. Challis substr. CH1 DHAD gene

Paen_sp_DHAD_coScB Paenibacillus sp. JDR-2 DHAD gene

Smut_DHAD_coScB Streptococcus mutans IlvD DHAD gene (coScB version)

Ncra2_DHAD_coScB Neurospora crassa llvD2 DHAD gene (coScB version)

Candidatus Koribacter versatilis Ellin345 (aka Acidobacteria bacterium

Acid_ba_DHAD_coSc Ellin345) DHAD gene

Gfor_DHAD_coSc Gramella forsetti KT0803 DHAD gene

Lyng_sp_DHAD_coSc Lyngbya sp. PCC 8106 DHAD gene

Ncra1_DHAD_coSc Neurospora crassa ilvD1 DHAD gene

Ncra2_DHAD_coSc Neurospora crassa ilvD2 DHAD gene

Sery_DHAD_coSc Saccharopolyspora erythraea NRRL 2338 DHAD gene

Smut_DHAD_coSc Streptococcus mutans UA159 DHAD gene

Ylip_DHAD_coSc Yarrowia lipolytica CLIB122 DHAD gene

P i ro_s p_D H AD_coSc Piromyces sp. E2 DHAD gene

Ftul_DHAD_coSc Francisella tularensis subsp. tularensis WY96-3418 DHAD gene

Saccharomyces cerevisiae DHAD gene missing N-terminal 19 amino

Sc_ILV3AN19 acids

Saccharomyces cerevisiae DHAD gene missing N-terminal 20 amino

Sc_ILV3AN20 acids

Saccharomyces cerevisiae DHAD gene missing N-terminal 23 amino

Sc_ILV3AN23 acids

[00208] An additional 10 DHAD homologs were identified that gave DHAD activities in GEVO6770 lysates that were not considered similar to or higher than that of the L. lactis DHAD but were higher than the background DHAD activity from GEVO6770 (summarized in Table 3). Two other DHAD homologs from the first screening round may also fall into this category. Specifically, even though a background DHAD activity control was not included with the first two screening rounds, the DHAD activity in GEVO6770 lysates from these two clones (containing the Candidatus Korarchaeum cryptofilum and Eubacterium siraeum DHAD genes) were higher than the highest average background DHAD activity plus two standard deviations from any of the screening rounds (Table 3). Table 3. DHAD Homolog Genes Resulting in DHAD Activity in Yeast Lysates Above the Background Control DHAD Activity.

* Higher activity than highest average background DHAD activity plus two standard deviations from any of the screening rounds.

Example 2: Identification of DHADs with Lower K _M for DHIV and Less Inhibition By Isobutanol

[00209] DHAD enzymes with a lower K _M for DHIV are desired for use in a recombinant microorganism. Lower K _M for the substrate provides favorable kinetic properties to the DHAD enzyme and thus yield a more efficient enzyme for use in DHAD-requiring biosynthetic pathways.

[00210] Further, DHAD enzymes that are minimally inhibited by isobutanol are desired for use in a recombinant microorganism comprising an isobutanol producing metabolic pathway.

[00211] In this example, cell pellets from DHAD homologs identified in Example 1 above were re-analyzed in DHAD assays containing 2,3-dihydroxyisovalerate (DHIV) using a standard level of DHIV (10 mM final concentration), a lower level of DHIV (0.4 mM final concentration), or with 20 g/L isobutanol added to the assay containing the standard level of DHIV. The assays with the lower DHIV concentration were expected to identify DHADs where DHIV was still saturating for the particular DHAD homologue at a concentration that was no longer saturating for the L. lactis DHAD. These DHADs might therefore have a lower K _M for DHIV than the L. lactis DHAD. The assays with added isobutanol were performed to identify DHADs with tolerance to at least 20 g/L of isobutanol.

[00212] Figure 5 shows the DHAD activities from the nine DHAD homolog clones that demonstrated equivalent or better DHAD activity in GEVO6770 lysates with the lower DHIV concentration compared with the higher DHIV concentration (summarized in Table 4). This figure also shows that L. lactis DHAD activity was decreased with the lower DHIV concentration compared with the standard DHIV concentration, as was the endogenous S. cerevisiae Ilv3 DHAD activity for the background control lysates.

Table 4. DHAD Homologs Resulting in Equivalent DHAD Activity in Yeast Lysates with 0.4 mM DHIV Compared with 10 mM DHIV.

[00213] Figure 6A shows the DHAD homologs that gave equivalent DHAD activities in the presence or absence of 20 g/L isobutanol in the DHAD assay (summarized in Table 5). In contrast, Figure 6B shows the DHAD homologs that gave lower DHAD activities in the presence of 20 g/L isobutanol in the DHAD assay (summarized in Table 6).

Table 5. DHAD Homologs Resulting in Equivalent DHAD Activity in Yeast Lysates with 20 g/L Isobutanol in DHAD Assays Compared with No Isobutanol.

Ncra2_DHAD_coScB

Sery_DHAD_coSc Saccharopolyspora erythraea NRRL 2338 DHAD gene

Ylip_DHAD_coSc Yarrowia lipolytica CLIB122 DHAD gene

P i ro_s p_D H AD_coSc Piromyces sp. E2 DHAD gene

Fpsy_DHAD_coScB Flavobacterium psychrophilum JIP02/86 DHAD gene

Ftul_DHAD_coSc Francisella tularensis subsp. tularensis WY96-3418 DHAD gene

Sc_ILV3, Sc_ILV3AN19, Saccharomyces cerevisiae DHAD gene and versions missing N- Sc_ILV3AN20, Sc_ILV3AN23 terminal 19, 20 or 23 amino acids

Table 6. DHAD Homologs Resulting in Lower DHAD Activity in Yeast Lysates with 20 g/L Isobutanol in DHAD Assays Compared with No Isobutanol.

Example 3: Characterization of DHAD Homologs for Isobutanol Production

[00214] In this example, 27 of the DHAD homologs identified in example 1 as exhibiting DHAD activities that were similar to or higher than that of the L. lactis ilvD were chosen for further characterization. Specifically, these DHAD homologs were incorporated into GEVO7931 , an S. cerevisiae strain comprising the B. subtilis acetolactate synthase, an engineered NADH-dependent ketol acid reductoisomerase

(E. coli llvC ^P2D1"A1 ), an L. lactis keto-isovalerate decarboxylase (kivD), and an engineered L. lactis alcohol dehydrogenase (L. lactis AdhA ^RE1 ). GEVO7931 additionally contained two copies of the L. lactis IlvD gene integrated into its genome and therefore isobutanol production from this strain background involves DHAD activity from these gene copies as well as the DHAD gene present on the plasmid transformed into GEVO7931 .

[00215] Volumetric isobutanol titer results of the fermentations are presented in Figure 7. The genes encoding DHADs from Piromyces sp. strain E3 and uncultured gamma proteobacterium eBACHOT4E07 resulted in higher volumetric isobutanol titers and specific isobutanol titers, respectively than the L. lactis IlvD. Genes encoding DHADs from Marivirga tractuosa, Methanosphaera stadtmanae, unidentified Eubacterium SCB49, Streptococcus mutans, Flavobacterium psychrophilum, Yarrowia lipolytica, Paenibacillus sp. JDR-2, Francisella tularensis, Lyngbya sp. PCC 8106, Neurospora crassa llvD2, and S. cerevisiae (ΙΙν3ΔΝ20) resulted in volumetric isobutanol titers similar to the L. lactis IlvD gene. Example 4: Characterization of Additional DHAD Homologs

[00216] As shown in example 3, a strain carrying the Methanosphaera stadtmanae DHAD was shown to produce comparable isobutanol titers to a strain carrying the L. lactis DHAD. Additional DHADs with similarity to the Methanosphaera stadtmanae DHAD were investigated for isobutanol production.

[00217] In this example, the S. cerevisiae strain GEVO9914 was transformed with DHAD enzymes listed in Table 7 that exhibit sequence similarity to the Methanosphaera stadtmanae DHAD.

Table 7. Additional Tested DHADs Exhibiting Similarity to the Methanosphaera stadtmanae DHAD.

[00218] Table 8 demonstrates the growth (OD ₆ oo) of GEVO9914 transformants harboring the indicated DHAD. Strains comprising the Methanococcus aeolicus and the Methanococcus stadtmanae DHADs exhibited the lowest accumulation of biomass at 48 hours, while strains with the Methanobrevibacter smithii DHAD grew better than the other strains in the fermentation.

Table 8. OD ₆ oo at 48 Hours of Fermentation

[00219] Figure 8 shows the total isobutanol titers for GEVO9914 transformants harboring the indicated DHAD. All of the new DHAD expressing strains produced equal or more isobutanol at 48 hours than the Methanosphaera stadtmanae DHAD control, with the exception of the strains containing the Maeo DHAD. Of note, strains containing the Methanobrevibacter smithii DHAD had the highest total titers at 9.41 g/L in 48 hours, which was significantly more than the Methanosphaera stadtmanae DHAD control. [00220] Figure 9 shows the specific isobutanol titers for GEVO9914 transformants harboring the indicated DHAD. While the Methanosphaera stadtmanae DHAD control exhibited the highest specific isobutanol titer at 1 .15 g/L/OD, the strains harboring the new DHADs generally had similar specific titers in the range of 0.9 to 1 .1 g/L/OD.

[00221] 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.

[00222] 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.

[00223] 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.