WO2014039060A1 - Acetolactate synthases for improved metabolite production - Google Patents

Description

ACETOLACTATE SYNTHASES FOR IMPROVED METABOLITE PRODUCTION

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 61/545,024, filed October 7, 2011, 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: GEVO_071_01WO_SeqList_ST25.txt, date recorded: October 3, 2012, file size: 2,517 kilobytes)

ACKNOWLEDGMENT OF GOVERNMENTAL SUPPORT

[0003] This invention was made with government support under Contract No. IIP-

0823122, awarded by the National Science Foundation.

TECHNICAL FIELD

[0004] 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 and enzymatic preparations there from.

BACKGROUND

[0005] The ability of microorganisms to convert sugars to beneficial metabolites including fuels, chemicals, and amino acids has been widely described in the literature in recent years. See, e.g., Alper et al., 2009, Nature Microbiol. Rev. 7: 715- 723 and McCourt et al., 2006, Amino Acids 31: 173-210. Recombinant engineering techniques have enabled the creation of microorganisms that express biosynthetic pathways capable of producing a number of useful products, including the commodity chemical, isobutanol.

[0006] Isobutanol, a promising biofuel candidate, has been produced in recombinant microorganisms expressing a heterologous, five-step metabolic pathway (See, e.g., WO/2007/050671 to Donaldson et al., WO/2008/098227 to Liao et al., WO/2009/103533 to Festel et al, and U.S. Publication No. 2010/0143997). However, the microorganisms produced to date have fallen short of commercial relevance due to their low performance characteristics, including, for example low productivities, low titers, and low yields.

[0007] The first step of the isobutanol producing metabolic pathway is catalyzed by acetolactate synthase (ALS), which converts pyruvate to acetolactate. Because ALS is an essential enzyme in the isobutanol production pathway, it is desirable that recombinant microorganisms engineered to produce isobutanol exhibit optimal ALS activity. The present application addresses this need by identifying several enzymes that exhibit activity for the conversion of pyruvate to acetolactate within an isobutanol production pathway. Accordingly, this application describes methods of increasing isobutanol production through the use of recombinant microorganisms comprising enzymes with improved properties for the production of isobutanol.

SUMMARY OF THE INVENTION

[0008] The present application describes a group of enzymes with activity for the conversion of pyruvate to acetolactate in the isobutanol pathway. The use of one or more of these enzymes can improve production of isobutanol in recombinant microorganisms expressing an engineered isobutanol producing metabolic pathway.

[0009] One aspect of the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein the polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 1. In one embodiment, the polypeptide is derived from the genus Magnaporthe. In a specific embodiment, the polypeptide is derived from Magnaporthe grisea or Magnaporthe oryzae.

[0010] Another aspect of the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 2. In one embodiment, the polypeptide is derived from the genus Phaeosphaeria. In a specific embodiment, the polypeptide is derived from Phaeosphaeria nodonim. [0011] Another aspect of the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 3. In one embodiment, the polypeptide is derived from the genus Trichoderma. In a specific embodiment, the polypeptide is derived from Trichoderma atroviride.

[0012] In yet another aspect, of the invention there is provided a microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 4. In one embodiment, the polypeptide is derived from the genus Talaromyces. In a specific embodiment, the polypeptide is derived from Talaromyces stipitatus.

[0013] In another aspect of the invention there is provided a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 5. In one embodiment, the polypeptide is derived from the genus Penicillium. In a specific embodiment, the polypeptide is derived from Penicillium mameffei.

[0014] In another aspect of the invention there is provided a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 12-49, 51-55, 57-63, 65- 75, 77-81, 83-87, 101-103, 106-107, 135, 372, and 506. In one embodiment, the polypeptide is derived from the genus Bacillus.

[0015] In still another aspect, the present invention provides for a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 50, 56, 64, 76, 82, 88-97, 100, 105, 336, and 351. In one embodiment, the polypeptide is derived from the genus Listeria.

[0016] In still another aspect, the present invention provides for a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 98 and 99. In one embodiment, the polypeptide is derived from the genus Paenibacillus.

[0017] In another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 104 and 379. In one embodiment, the polypeptide is derived from the genus Exiguobacterium.

[0018] In another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 108. In one embodiment, the polypeptide is derived from the genus Gemella.

[0019] In yet another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 110, 122, 127, 136-139, 143, 154, 158-160, 181, 184, 190-192, 211-214, 230, 232-234, 253, 254, 256, 260, 266, 271 , 279, 296, and 338. In one embodiment, the polypeptide is derived from the genus Lactobacillus.

[0020] In yet another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 109, 111, 113-121, 123- 126, 128-134 and 345. In one embodiment, the polypeptide is derived from the genus Enterococcus.

[0021] In another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 140-142, 144-152, 156, 157, 161-163, 165, 166, 168, 170, 172, 173, 175, 177, 187, and 331. In one embodiment, the polypeptide is derived from the genus Streptococcus.

[0022] In another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 153 and 155. In one embodiment, the polypeptide is derived from the genus Pediococcus.

[0023] In still another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 164, 167, 171, 176, 178, 179, 180, 182, 183, 189, 193, 194-210, 215-219, 222, and 337. In one embodiment, the polypeptide is derived from the genus Staphylococcus.

[0024] In another aspect of the invention there is provided a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 221, 224, 225, 231, and 445. In one embodiment, the polypeptide is derived from the genus Oenococcus.

[0025] In still another aspect of the invention there is provided a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 235, 237, 239, 242, and 246. In one embodiment, the polypeptide is derived from the genus Pectobacterium.

[0026] In yet another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 240, 244, 249, and 251. In one embodiment, the polypeptide is derived from the genus Serratia.

[0027] In another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 248, 250, 255, and 258. In one embodiment, the polypeptide is derived from the genus Dickeya.

[0028] In still another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 263, 264, and 288. In one embodiment, the polypeptide is derived from the genus Proteus.

[0029] In another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 269, 273, 275, 283, 284, 286, 291, and 292. In one embodiment, the polypeptide is derived from the genus Klebsiella.

[0030] In another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 245, 247, 257, 268, 287, 294, and 295. In one embodiment, the polypeptide is derived from the genus Yersinia.

[0031] In yet another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 228, 298, 299, 301, 303, 312, 315-317, 319-324, 326, 327, 332, and 341. In one embodiment, the polypeptide is derived from the genus Vibrio.

[0032] In yet another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 252, 259, 267, 278, and 289. In one embodiment, the polypeptide is derived from the genus Enterobacter.

[0033] In another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 265, 272, 274, 280-282, 285, 302, and 304. In one embodiment, the polypeptide is derived from the genus Leuconostoc.

[0034] In a further aspect, the present invention provides a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 305, 308, 309, 311, 313, and 325. In one embodiment, the polypeptide is derived from the genus Erwinia.

[0035] In yet another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 293, 306, 310, 314, and 318. In one embodiment, the polypeptide is derived from the genus Pantoea.

[0036] In a further aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 348, 352-354, and 366. In one embodiment, the polypeptide is derived from the genus Clostridium.

[0037] In another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 431, 432, 436, 438, 441, 444, 448, 451, and 455. In one embodiment, the polypeptide is derived from the genus Pseudomonas.

[0038] In another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 409, 415, 423, 429, 433, 434, 437, 439, 440, 443, 447, 452, 456-463, 466, 468, 483, 485, 488, 489, 494, 498, 502, and 517. In one embodiment, the polypeptide is derived from the genus Burkholderia

[0039] In yet another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 474, 479, 487, 490, and 492. In one embodiment, the polypeptide is derived from the genus Gardnemlla.

[0040] In yet another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 470 and 481. In one embodiment, the polypeptide is derived from the genus Arcobacter.

[0041] In another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 480, 496, 504, and 519. In one embodiment, the polypeptide is derived from the genus Marinobacter.

[0042] In various embodiments described herein, the recombinant microorganism may comprise a biosynthetic pathway which uses ALS to catalyze a pathway step. The biosynthetic pathway which uses ALS to catalyze a pathway step may be selected from a pathway for the biosynthesis of isobutanol, 2-butanol, 1-butanol, 2- butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. In one embodiment, the pathway for the biosynthesis of isobutanol, 2-butanol, 1-butanol, 2- butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, or coenzyme A comprises at least one polypeptide encoded by an exogenous gene.

[0043] 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 acetolactate synthase (ALS) activity that is at least about 65%, 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: 1-535.

[0044] 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.

[0045] 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 acetolactate synthase is a polypeptide which is at least about 65%, 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: 1-535. [0046] 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).

[0047] In another aspect, the invention is directed to an isolated nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65%, 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: 1-535. In various embodiments, the invention comprises recombinant microorganisms comprising an isolated nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65%, 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: 1- 535.

[0048] 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.

[0049] 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.

[0050] In some embodiments, the recombinant microorganisms may be yeast recombinant microorganisms of the Saccharomyces clade.

[0051] 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. [0052] 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 kluyven, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Hansenula anomala, Candida utilis and Kluyveromyces waltii.

[0053] In some embodiments, the recombinant microorganisms may be Crabtree- positive recombinant yeast microorganisms. In one embodiment, the Crabtree- positive yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, 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, Kluyveromyces thermotolerans, Candida glabrata, Z. bailli, Z. rouxii, Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces uvarum.

[0054] 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.

[0055] In some embodiments, the recombinant microorganisms may be pre-WGD (whole genome duplication) yeast recombinant microorganisms. In one embodiment, the pre-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Pachysolen, Yarrowia and Schizosaccharomyces. In additional embodiments, the pre-WGD yeast is selected from the group consisting of Saccharomyces kluyven, Kluyveromyces thermotolerans, Kluyveromyces marxianus, Kluyveromyces waltii, Kluyveromyces lactis, Candida tropicalis, Pichia pastoris, Pichia anomala, Pichia stipitis, Issatchenkia orientalis, Issatchenkia occidentalis, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yarrowia lipolytica, and Schizosaccharomyces pombe.

[0056] 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.

[0057] 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 ALS activity, wherein said polypeptide is at least about 65%, 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: 1-535. The beneficial metabolite may be derived from any biosynthetic pathway which uses ALS to catalyze a pathway step, including, but not limited to, biosynthetic pathways for the production of isobutanol, 2-butanol, 1-butanol, 2- butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. In a specific embodiment, the beneficial metabolite is isobutanol.

[0058] 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

[0059] 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.

[0060] 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

[0061] Figure 1 illustrates an isobutanol pathway.

[0062] Figure 2 illustrates an NADH-dependent isobutanol pathway.

[0063] Figure 3 shows exemplary metabolic pathways involving acetolactate as a metabolic intermediate.

[0064] Figure 4 illustrates a sequence comparison, predicted localization and sequence identity to B. subtilis alsS of various identified fungal ALS. The FAD- binding sequence identified by Le and Chao, infra, and the analogous sequences in the ALS homologs are underlined.

[0065] Figure 5 illustrates the plasmid map of pGV1730.

[0066] Figure 6 illustrates the plasmid map of pGV1773.

[0067] Figure 7 illustrates the plasmid map of pGV1800.

[0068] Figure 8 illustrates the plasmid map of pGV1801.

[0069] Figure 9 illustrates the plasmid map of pGV1802.

[0070] Figure 10 illustrates the plasmid map of pGV1803.

[0071] Figure 11 illustrates the plasmid map of pGV1804.

[0072] Figure 12 illustrates the plasmid map of pGV2044.

[0073] Figure 13 illustrates the plasmid map of pGV2047.

[0074] Figure 14 illustrates the plasmid map of pGV2082. [0075] Figure 15 illustrates the plasmid map of pGV2114.

[0076] Figure 16 illustrates the plasmid map of pGV2115.

[0077] Figure 17 illustrates the plasmid map of pGV2116.

[0078] Figure 18 illustrates the plasmid map of pGV2117.

[0079] Figure 19 illustrates the plasmid map of pGV2118.

[0080] Figure 20 illustrates the plasmid map of pGV2119.

[0081] Figure 21 is a graph showing isobutanol produced by strains containing

ALS homologs (g/L). The strains compared are 1187 (no ALS control), 2280 (B. subtilis alsS: Bs_AlsS), 2618 (B. subtilis alsS, codon optimized for S. cerevisiae:

Bs_AlsS_coSc), 2621 (Trichoderma atroviride (SEQ ID NO: 3): Ta_ALS), and 2622

(Talaromyces stipitatus (SEQ ID NO: 4): Ts_ALS).

[0082] Figure 22 illustrates the data of Figure 21 , normalized for cell growth.

[0083] Figure 23 is a graph showing total adjusted isobutanol produced by strains containing ALS homologs. The strains compared are 1187 (no ALS), 2280 (Bs_AlsS), 2618 (Bs_AlsS_coSc), 2621 {Ta_ALS), 2622 (Ts_ALS) and 2623 (Pm_ALS).

[0084] Figure 24 is a graph showing total adjusted isobutanol produced by strains containing ALS homologs. The strains compared are 1187 (no ALS), 2280 (Bs_AlsS), 2618 (Bs_AlsS_coSc), 2621 (Ta_ALS), 2622 (Ts_ALS) and 2623 (Pm_ALS).

[0085] Figure 25 is a graph showing total adjusted specific isobutanol produced by strains containing ALS homologs. The strains compared are 1187 (no ALS), 2280 (Bs_AlsS), 2618 (Bs_AlsS_coSc), 2621 (Ta_ALS), 2622 (Ts_ALS) and 2623 (Pm_ALS).

DETAILED DESCRIPTION

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] "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; (11) Thermotoga and Thermosipho thermophiles.

[0093] "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.

[0094] "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.

[0095] The term "genus" is defined as a taxonomic group of related species according to the Taxonomic Outline of Bacteria and Archaea (Garrity, G.M., Lilbum, 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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. [00100] 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.

[00101] 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.

[00102] 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.

[00103] 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.

[00104] 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.

[00105] 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.

[00106] As used herein, the term "isobutanol producing metabolic pathway" refers to an enzyme pathway which produces isobutanol from pyruvate.

[00107] 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.

[00108] 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.

[00109] 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.

[00110] 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) drffer(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.

[00111] 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.

[00112] 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.

[00113] 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.

[00114] 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).

[00115] 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).

[00116] 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.

[00117] 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).

[00118] "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.

[00119] 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.

[00120] "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.

[00121] 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."

[00122] 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.

[00123] 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.

[00124] 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.

[00125] 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 C0₂ 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.

[00126] 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.

[00127] 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.

[00128] 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.

[00129] 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.

[00130] "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.

[00131] 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.

[00132] 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

[00133] 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.

[00134] 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).

[00135] 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-Producina Microorganisms

[00136] 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).

[00137] 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 + CO₂

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

[00138] 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.

[00139] 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.

[00140] 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.

[00141] 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.

[00142] 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. polymorphs, 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.

[00143] 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.

[00144] For example, 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_205911.1), 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: 546),

(F/Y/L)(S/A)HG(F/L) (SEQ ID NO: 547),

V(V/I/F)(M/L/A)(A/C)PK (SEQ ID NO: 548),

D(UI)XGE(Q/R)XXLXG (SEQ ID NO: 549), and S(D/N/T)TA(E/Q/R)XG (SEQ ID NO: 550)

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.

[00145] 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.

[00146] 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.

[00147] 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. [00148] Dihydroxy acid dehydratases capable of converting 2,3- dihydroxyisovalerate to a-ketoisovalerate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.). including E. coli (GenBank Accession No. YP_026248.1), L. lactis (GenBank Accession No. NP_267379.1 ), S. mutans (GenBank Accession No. NP_722414.1), M. stadtmanae (GenBank Accession No. YP_448586.1), M. tractuosa (GenBank Accession No. YP_004053736.1), Eubacterium SCB49 (GenBank Accession No. ZP_01890126.1), G. forsetti (GenBank Accession No. YP_862145.1), Y. lipolytica (GenBank Accession No. XP_502180.2), N. crassa (GenBank Accession No. XP_963045.1), or S. cerevisiae ILV3 (GenBank Accession No. NP_012550.1). Additional dihydroxy acid dehydratases capable of 2,3-dihydroxyisovalerate to a-ketoisovalerate are described in commonly owned and co-pending US Publication No. 2011/0076733. Motifs shared in common between the majority of dihydroxy acid dehydratases include:

SLXSRXXIA (SEQ ID NO: 551),

CDKXXPG (SEQ ID NO: 552),

GXCXGXXTAN (SEQ ID NO: 553),

GGSTN (SEQ ID NO: 554),

GPXGXPGMRXE (SEQ ID NO: 555),

ALXTDGRXSG (SEQ ID NO: 556), and

GHXXPEA (SEQ ID NO: 557)

motifs at amino acid positions corresponding to the 93-101, 122-128, 193-202, 276- 280, 482-491, 509-518, and 526-532 residues, respectively, of the £. co// dihydroxy acid dehydratase encoded by ilvD. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit dihydroxy acid dehydratase activity.

[00149] 2-keto-acid decarboxylases capable of converting α-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. 2011/0076733. 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: 558),

(T/V)T(F/Y)G(V/A)G(E/A)(L/F)(S/N) (SEQ ID NO: 559),

N(G/A)(L/I/V)AG(S/A)(Y/F)AE (SEQ ID NO: 560),

(V/I)(L/I/V)XI(V/T/S)G (SEQ ID NO: 561), and

GDG(S/A)(L/F/A)Q(L/M)T (SEQ ID NO: 562) 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 112-113 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.

[00150] 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. brews (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. EHA31115.1), 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. 2011/0076733 and 2011/0201072. Motifs shared in common between the majority of alcohol dehydrogenases include:

C(H/G)(T/S)D(L/I)H (SEQ ID NO: 563),

GHEXXGXV (SEQ ID NO: 564), (L/V)(Q/K/E)(V/I/K)G(D/Q)(R/H)(V/A) (SEQ ID NO: 565),

CXXCXXC (SEQ ID NO: 566),

(C/A)(A/G/D)(G/A)XT(T/V) (SEQ ID NO: 567), and

G(L/A/C)G(G/P)(L/I/V)G (SEQ ID NO: 568) 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.

[00151] 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.

[00152] 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.

[00153] 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.

Isobutanol-Producina Metabolic Pathways with Improved ALS Properties

[00154] The first step of the isobutanol producing metabolic pathway is catalyzed by acetolactate synthase (ALS), which converts pyruvate to acetolactate. The present application relates to the identification of several enzymes that exhibit activity for the conversion of pyruvate to acetolactate within an isobutanol production pathway. Accordingly, this application describes methods of increasing isobutanol production through the use of recombinant microorganisms comprising enzymes with improved properties for the production of isobutanol.

[00155] The conversion of two pyruvate molecules to acetolactate can be carried out by either an acetohydroxyacid synthase (AHAS) or an acetolactate synthase (ALS). AHASs are involved in biosynthesis of branched chain amino acids in the mitochondria of yeasts. They are FAD-dependent and are feedback inhibited by branched chain amino acids. ALSs are catabolic and are involved in the conversion of pyruvate to acetoin. ALSs are FAD-independent and not feedback inhibited by branched chain amino acids. In addition, ALSs are specific for the conversion of two pyruvate molecules to acetolactate. Therefore, ALSs are favored over AHASs. In addition, in the case of yeast, AHASs are normally mitochondrial, therefore a fungal ALS that is cytosolic is favored. Sequence analysis has shown that there is a conserved sequence 'RFDDR' found in AHASs that is not conserved among ALSs (Le and Choi (2005) Bull. Korean Chem. Soc. 26:916-920). This sequence is likely involved in FAD-binding by AHASs and thus could be used to distinguish between the FAD-dependent AHASs and the FAD-independent ALSs. Using this region to distinguish between AHASs and ALSs BLAST searches of fungal sequence databases were performed and resulted in the identification of ALS homologs from several fungal species (including, for example, M. grisea, P. nodomm, T. atmviride, T. stipitatus, P. mameffei, and Glomemlla graminicola). Of these sequences, the ALS homologs from M. grisea, P. nodomm, T. stipitatus, and T. atmviride will generally be expected to be cytosolically localized. Further, to identify further ALS enzymes, additional BLAST searches, not restricted to fungal sequences, were conducted using B. subtilis alsS and the ALS of Talaromyces stipitatus (SEQ ID NO: 4) as search queries.

[00156] Accordingly, one aspect of the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein the polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 1. In one embodiment, the polypeptide is derived from the genus Magnaporthe. In a specific embodiment, the polypeptide is derived from Magnaporthe grisea or Magnaporthe oryzae.

[00157] Another aspect of the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 2. In one embodiment, the polypeptide is derived from the genus Phaeosphaeria. In a specific embodiment, the polypeptide is derived from Phaeosphaeria nodorum.

[00158] Another aspect of the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 3. In one embodiment, the polypeptide is derived from the genus Trichoderma. In a specific embodiment, the polypeptide is derived from Trichoderma atroviride.

[00159] In yet another aspect, of the invention there is provided a microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 4. In one embodiment, the polypeptide is derived from the genus Talaromyces. In a specific embodiment, the polypeptide is derived from Talaromyces stipitatus.

[00160] In another aspect of the invention there is provided a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 5. In one embodiment, the polypeptide is derived from the genus Penicillium. In a specific embodiment, the polypeptide is derived from Penicillium mameffei.

[00161] In another aspect of the invention there is provided a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 12-49, 51-55, 57-63, 65- 75, 77-81, 83-87, 101-103, 106-107, 135, 372, and 506. In one embodiment, the polypeptide is derived from the genus Bacillus.

[00162] In still another aspect, the present invention provides for a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 50, 56, 64, 76, 82, 88-97, 100, 105, 336, and 351. In one embodiment, the polypeptide is derived from the genus Listeria.

[00163] In still another aspect, the present invention provides for a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 98 and 99. In one embodiment, the polypeptide is derived from the genus Paenibacillus.

[00164] In another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 104 and 379. In one embodiment, the polypeptide is derived from the genus Exiguobacterium.

[00165] In another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 108. In one embodiment, the polypeptide is derived from the genus Gemella.

[00166] In yet another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs:, 110, 122, 127, 136-139, 143, 154, 158-160, 181, 184, 190-192, 211-214, 230, 232-234, 253, 254, 256, 260, 266, 271 , 279, 296, and 338. In one embodiment, the polypeptide is derived from the genus Lactobacillus.

[00167] In yet another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 109, 111, 113-121, 123- 126, 128-134 and 345. In one embodiment, the polypeptide is derived from the genus Enterococcus.

[00168] In another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 140-142, 144-152, 156, 157, 161-163, 165, 166, 168, 170, 172, 173, 175, 177, 187, and 331. In one embodiment, the polypeptide is derived from the genus Streptococcus.

[00169] In another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 153 and 155. In one embodiment, the polypeptide is derived from the genus Pediococcus.

[00170] In still another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 164, 167, 171, 176, 178, 179, 180, 182, 183, 189, 193, 194-210, 215-219, 222, and 337. In one embodiment, the polypeptide is derived from the genus Staphylococcus.

[00171] In another aspect of the invention there is provided a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 221, 224, 225, 231, and 445. In one embodiment, the polypeptide is derived from the genus Oenococcus.

[00172] In still another aspect of the invention there is provided a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 235, 237, 239, 242, and 246. In one embodiment, the polypeptide is derived from the genus Pectobacterium.

[00173] In yet another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 240, 244, 249, and 251. In one embodiment, the polypeptide is derived from the genus Serratia.

[00174] In another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 248, 250, 255, and 258. In one embodiment, the polypeptide is derived from the genus Dickeya.

[00175] In still another aspect, the invention is directed to a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 263, 264, and 288. In one embodiment, the polypeptide is derived from the genus Proteus.

[00176] In another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 269, 273, 275, 283, 284, 286, 291, and 292. In one embodiment, the polypeptide is derived from the genus Klebsiella.

[00177] In another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 245, 247, 257, 268, 287, 294, and 295. In one embodiment, the polypeptide is derived from the genus Yersinia.

[00178] In yet another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 228, 298, 299, 301, 303, 312, 315-317, 319-324, 326, 327, 332, and 341. In one embodiment, the polypeptide is derived from the genus Vibrio.

[00179] In yet another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 252, 259, 267, 278, and 289. In one embodiment, the polypeptide is derived from the genus Enterobacter.

[00180] In another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 265, 272, 274, 280-282, 285, 302, and 304. In one embodiment, the polypeptide is derived from the genus Leuconostoc.

[00181] In a further aspect, the present invention provides a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 305, 308, 309, 311, 313, and 325. In one embodiment, the polypeptide is derived from the genus Erwinia.

[00182] In yet another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 293, 306, 310, 314, and 318. In one embodiment, the polypeptide is derived from the genus Pantoea.

[00183] In a further aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 348, 352-354, and 366. In one embodiment, the polypeptide is derived from the genus Clostridium.

[00184] In another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 431, 432, 436, 438, 441, 444, 448, 451, and 455. In one embodiment, the polypeptide is derived from the genus Pseudomonas.

[00185] In another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 409, 415, 423, 429, 433, 434, 437, 439, 440, 443, 447, 452, 456-463, 466, 468, 483, 485, 488, 489, 494, 498, 502, and 517. In one embodiment, the polypeptide is derived from the genus Burkholderia.

[00186] In yet another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 474, 479, 487, 490, and 492. In one embodiment, the polypeptide is derived from the genus Gardnerella.

[00187] In yet another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 470 and 481. In one embodiment, the polypeptide is derived from the genus Arcobacter.

[00188] In another aspect, the present invention includes a recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 480, 496, 504, and 519. In one embodiment, the polypeptide is derived from the genus Marinobacter.

[00189] The invention also includes fragments of the disclosed polypeptides with acetolactate synthase (ALS) 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 acetolactate synthases. 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 acetolactate synthase (ALS) activity. [00190] In another aspect, the invention is directed to an isolated nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 1-535.

[00191] In further aspects, the invention comprises recombinant microorganisms comprising an isolated nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 1-535. 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 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: 1-535.

[00192] In one embodiment, recombinant microorganism as discussed herein comprises a metabolic pathway for the production of a metabolite selected from isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1- pentanol, and coenzyme A (see Figure 3).

[00193] In one embodiment, the recombinant microorganism as discussed herein is a prokaryotic microorganism. In one embodiment, the recombinant microorganism as discussed herein is a yeast microorganism. In a specific embodiment, the recombinant microorganism as discussed herein is a yeast microorganism of the Saccharomyces clade. In another specific embodiment, the recombinant microorganism as discussed herein is a Saccharomyces sensu stricto microorganism. In a further specific embodiment the Saccharomyces sensu stricto microorganism is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrids thereof.

[00194] In one embodiment, the recombinant microorganism as discussed herein is a Crabtree-negative yeast microorganism. In a specific embodiment, the Crabtree- negative yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces, Kluyveromyces, Pichia, Hansenula, Issatchenkia and Candida. In further specific embodiments, the Crabtree-negative yeast microorganism is selected from the group consisting of Saccharomyces kluyveri, Kluyvenomyces lactis, Kluyvenomyces marxianus, Pichia anomala, Pichia stipitis, Pichia kudriavzevii, Issatchenkia orientalis, Hansenula anomala, Candida utilis and Kluyvenomyces waltii.

[00195] In another embodiment, the recombinant microorganism as discussed herein is a Crabtree-positive yeast microorganism. In a specific embodiment, the Crabtree-positive yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces, Kluyvenomyces, Zygosacchanomyces, Debanyomyces, Pichia, Candida, and Schizosacchanomyces. In further specific embodiments, the Crabtree-positive yeast microorganism is selected from the group consisting of Saccharomyces cenevisiae, Saccharomyces uvanum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, Kluyvenomyces thermotolerans, Candida glabnata, Zygosacchanomyces bailli, Zygosacchanomyces rouxii, Debanyomyces hansenii, Pichia pastorius, and Schizosacchanomyces pombe.

[00196] In another embodiment, the recombinant microorganism as discussed herein is a post-WGD (whole genome duplication) yeast microorganism. In a specific embodiment, the post-WGD yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces or Candida. In further specific embodiments, the post-WGD yeast microorganism is selected from the group consisting of Saccharomyces cenevisiae, Saccharomyces uvanum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and Candida glabnata.

[00197] In another embodiment, the recombinant microorganism as discussed herein is a pre-WGD (whole genome duplication) yeast microorganism. In a specific embodiment, the pre-WGD yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces, Kluyvenomyces, Candida, Pichia, Debanyomyces, Hansenula, Issatchenkia, Pachysolen, Yanrowia and Schizosacchanomyces. In further specific embodiments, the pre-WGD yeast microorganism is selected from the group consisting of Saccharomyces kluyveri, Kluyvenomyces thermotolerans, Kluyvenomyces marxianus, Kluyvenomyces waltii, Kluyvenomyces lactis, Candida tropicalis, Pichia pastohs, Pichia anomala, Pichia stipitis, Pichia kudriavzevii, Issatchenkia orientalis, Debanyomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yanrowia lipolytica, and Schizosacchanomyces pombe. Recombinant Microorganisms Comprising ALSs

[00198] In addition to isobutanol producing metabolic pathways, a number of biosynthetic pathways use enzymes exhibiting acetolactate synthase (ALS) activity to catalyze a reaction step, including pathways for the production of isobutanol, 2- butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. A representative list of the engineered biosynthetic pathways utilizing enzymes exhibiting acetolactate synthase (ALS) activity are described in Table 1.

[00199] As described above, each of these biosynthetic pathways uses an enzyme exhibiting acetolactate synthase (ALS) activity to catalyze a reaction step. Therefore, the product yield from these biosynthetic pathways will in part depend upon the activity of the enzyme exhibiting acetolactate synthase (ALS) activity. [00200] As will be understood by one skilled in the art equipped with the present disclosure, the enzymes exhibiting acetolactate synthase (ALS) activity described herein would have utility in any of the above-described pathways. Thus, in an additional aspect, the present application relates to a recombinant microorganism comprising a biosynthetic pathway requiring an enzyme with acetolactate synthase (ALS) activity, wherein said recombinant microorganism comprises at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65%, 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 1-535.

[00201] In various embodiments the invention provides for optimization of ALS enzymes. Any of the genes encoding the foregoing ALS enzymes 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 (see, e.g. Arnold, Frances H.; Georgiou, George (Eds.); Directed Enzyme Evolution Screening and Selection Method; Series: Methods in Molecular Biology; 2003, Vol. 230; Humana Press, incorporated by reference in its entirety; see, e.g. Arnold, Frances H.; Georgiou, George (Eds.); Directed Evolution Library Creation; Series: Methods in Molecular Biology, 2003, Vol. 231 ; Humana Press, incorporated by reference in its entirety). Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast.

[00202] In one embodiment, protein engineering techniques are used to decrease the Michaelis-Menten constant (KM) of the foregoing ALS enzymes. In one embodiment, the KM for the substrate pyruvate is lowered to below 10, 9. 8, 7, 6, 5, 4, 2, 1 , or below 1 mM.

[00203] In one embodiment, protein engineering techniques are used to increase the specific activity of the foregoing ALS enzyme for the conversion of pyruvate to acetolactate. In one embodiment, the specific activity is increase by 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000%.

The Microorganism in General

[00204] 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, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. Engineered pathways for the production of these beneficial metabolites are described above in Table 1.

[00205] 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, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl- 1-butanol, 4-methyl- l-pentanol, and coenzyme A from a suitable carbon source. The genetic material introduced into and/or the genes modified for expression in the parental microorganism contains gene(s), or parts of genes, coding for one or more of the enzymes involved in a biosynthetic pathway for the production of isobutanol, 2- butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A and may also include additional elements for the expression and/or regulation of expression of these genes, e.g., promoter sequences.

[00206] 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). [00207] 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.

[00208] 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.

[00209] 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 enzyme.

[00210] 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."

[00211] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (Murray et a/., 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 a/., 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.

[00212] 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.

[00213] In addition, homologs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein.

[00214] 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.

[00215] 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).

[00216] 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).

[00217] 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.

[00218] 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.

[00219] 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. 2011/0183392

[00220] 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. 2011/0020889 and 2011/0183392.

[00221] 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.

[00222] 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. Methods in General

[00223] Any method can be used to identify genes that encode for enzymes that are homologous to the genes described herein (e.g., acetolactate synthase homologs). Generally, genes that are homologous or similar to the acetolactate synthases 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.

[00224] 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.

[00225] 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 Usino Recombinant Microorganisms for Production of Beneficial Metabolites

[00226] 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 acetolactate synthase (ALS) activity, wherein said recombinant microorganism comprises at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65%, 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: 1-535.

[00227] In an exemplary embodiment, the biosynthetic pathway is a pathway for the production of a beneficial metabolite selected from isobutanol, 2-butanol, 1- butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. In a further exemplary embodiment, the beneficial metabolite is isobutanol.

[00228] 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, 2-butanol, 1- butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. In a further exemplary embodiment, the beneficial metabolite is isobutanol.

[00229] 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

[00230] 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 com, 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.

[00231] 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.

[00232] 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 acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65%, 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: 1-535.

[00233] 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.

[00234] 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.

[00235] 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.

[00236] 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

Materials and Methods

Strains. Plasmids and Primer Sequences

Molecular Biology

[00237] Standard molecular biology methods for cloning and plasmid construction were used, unless otherwise noted (Sambrook, supra).

[00238] Cloning techniques included digestion with restriction enzymes, gel purification of DNA fragments (Zymoclean Gel DNA Recovery Kit, Catalog # D4002, Zymo Research Corp, Orange, CA), ligations of two DNA fragments (Roche Rapid Ligation Kit, Catalog # 11 635 379 001 , Roche Diagnostics, Mannheim, Germany), Klenow treatment of fragments to give blunt ends (NEB DNA Polymerase I, Large (Klenow), Catalog # M0210S, Ipswich, MA), and bacterial transformations into chemically competent E. coli cells made at GEVO (TOP10). Plasmid DNA was purified from E. coli cells using the Qiagen QIAprep Spin Miniprep Kit (Catalog # 27106, Qiagen, Valencia, CA).

[00239] PCR was performed on an Eppendorf Mastercycler (Catalog # 71086, Novagen, Madison, Wl). The following PCR program was followed for all primer sets unless otherwise noted: 94^'C for 2 min then 40 cycles of (94^'C 30 sec, 54^*C 30 sec, 72 C 1 min) then 72 C for 10 min. Yeast colony PCR used the FailSafe™ PCR System (EPICENTRE^® Biotechnologies, Madison, Wl; Catalog # FS99250). A PCR cocktail containing 15 μΙ of Master Mix E buffer, 10.5 μΙ water, 2 μΙ of each primer at 10 μΜ concentration, and 0.5 μΙ polymerase enzyme mix from the kit was added to a 0.2 mL PCR tube for each sample (30 μΙ each). For each candidate a small amount of cells was added to the reaction tube using a sterile P10 pipette tip. Presence of the positive PCR product was assessed using agarose gel electrophoresis. The following primer pairs were used to verify integration (Table 3): primers 1432 and 1433 for the 5-end of all integrations, primers 1435 and 2233 for the 3'-end of pGV2114 integrations, primers 1435 and 2234 for the 3'-end of the pGV2115 integrations, primers 1435 and 2235 for the 3'-end of the pGV2116 integrations, primers 1435 and 2236 for the 3'-end of the pGV2117 integrations, primers 1435 and 2237 for the 3'-end of the pGV2118 integrations, and primers 1435 and 2238 for the 3-ends of the pGV2119 integrations .

[00240] Transformation of integration plasmids was performed according to the SOP using standard techniques. Integration plasmids were digested with Νrul, checked by gel electrophoresis for complete digestion and used directly from digestion. Integrative transformants were selected by plating the transformed cells on SCD-Trp agar medium. Once the transformants were single colony purified they were maintained on SCD-Trp plates. After transformants were screened by PCR for proper integration (as described above), each strain (2618-2623; Table 2) was transformed with the plasmid pGV2082 (Table 4). Transformants were plated to YPD plates containing 0.2 g/L G418.

[00241] Media used was standard E. coli and yeast media (see, e.g., Sambrook, supra and Guthrie, C. and Fink, G.R. eds. Methods in Enzymology Part B: Guide to Yeast Genetics and Molecular and Cell Biology 350:3-623 (2002)). For plasmid selection in E. coli, LB plus 100 μg/ml ampicillin was used for pGV1730 based plasmids. For plasmid selection and growth in S. cerevisiae strains, SC-drop out media was used. SCD-Trp media is 14 g/L Sigma™ Synthetic Dropout Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), 6.7 g/L Difco™ Yeast Nitrogen Base without amino acids. 0.076 g/L histidine, 0.076 g/L uracil, 0.380 g/L leucine, and 20 g/L glucose. YP media contains 1% (w/v) yeast extract, 2% (w/v) peptone. YPD is YP containing 2% (w/v) glucose.

ALS assays

[00242] Yeast cell extracts were made by growing cells in 4mL of YPD overnight at 30°C. The next day 1 mL of overnight culture was added to 50 ml of YPD in a 250 mL baffled flask and grown at 30°C shaking at 250 rpm. For cultures that were induced with copper, CuSO₄ was added to a final concentration of 1 mM when the cultures had reached a density of approximately 0.8 OD. The cultures were incubated an additional 2 hr (approximately 6 hr total) until an OD of 1-2 was reached. For cultures that were not induced with copper, cultures were grown for the same 6 hours to an OD of 1-2. To prepare cells lor assays, 50 ml of cells were collected by centrifugation at 2700 x g. After removal of the media, cells were resuspended in sterile dH₂0, centrifuged at 2700 x g and the remaining media was carefully removed. The cell pellets were weighed (empty tubes were preweighed) and then frozen at -80^°C until use. Cells were thawed on ice and resuspended in lysis buffer (250 mM KPO₄ pH 7.5, 10 mM MgCI₂ and 1 mM DTT) such that the result was a 20% cell suspension by mass. 1000 μΙ of glass beads (0.5 mm diameter) were added to a 1.5 ml Eppendorf tube and 875 μΙ of cell suspension was added. Yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, PA), mixing 6 X 1 min each at full speed with 1 min icing steps between each mixing step. The tubes were centrifuged for 10 min at 23,500x g at 4°C and the supernatant was removed. Extracts were held on ice until assayed.

[00243] Yeast extract protein concentration was determined using the BioRad Bradford Protein Assay Reagent Kit (Cat# 500-0006, BioRad Laboratories, Hercules, CA) and using BSA for the standard curve. Yeast cell extracts were diluted in lysis buffer to give a standard dilution which was then corrected for the calculations.

[00244] All ALS assays were performed in triplicate for each lysate, both with and without substrate. To assay each lysate, 100 μL of lysate (diluted 1:2 with lysis buffer) was mixed with 900 μL of buffer (50 mM potassium phosphate buffer pH 6.0, 1 mM MgSO₄, 1 mM thiamin-pyrophosphate, 110 mM pyruvate), and incubated for 15 min at 30°C. Buffers were prepared at room temperature. A no substrate control (buffer without pyruvate) and a no lysate control (lysis buffer instead of lysate) were also included. After incubation, 175 μL from each reaction was mixed with 25 μL 35% H2SO4 and incubated at 37°C for 30 min. Acetoin production was quantified by HPLC.

Fermentations

[00245] Strains with integrated ALS genes expressed from the CUP1 promoter were transformed with pGV2082 as described above. Strains were patched onto YPD plates containing 0.2 mg/mL G418. The following morning, cells were removed from the plate with a sterile toothpick and resuspended in 4mL of YPD with 0.2 mg/mL G418. The OD₆₀₀ was determined for each culture. Cells were added to 50 mL YP with 5% dextrose and 0.2 mg/mL G418 such that a final OD₆₀₀ of 0.1 was obtained. 1 mL of media was removed and the OD₆₀₀ for this undiluted sample was determined, leftover media was stored at 4°C to act as media blank for the analytical tests and as the t= 0 sample for the fermentation. At t=24 h, 2mL of media was removed and 25 μί used at a 1:40 dilution to determine OD₆₀₀- The remaining culture was centrifuged in a microcentrifuge at maximum speed for 10 min and a 1:10 dilution read on the YSI biochemical analyzer (YSI Instruments, Yellow Springs, OH, USA). 50% glucose containing 0.2 mg/mL G418 was added to a final concentration of 100 g/L glucose. At t=48 h, 2mL of media was removed and 25μί used at a 1 :40 dilution to determine OD₆₀₀. The remaining culture was centrifuged in a microcentrifuge at maximum speed for 10 min and a 1:10 dilution read on the YSI biochemical analyzer (YSI Instruments, Yellow Springs, OH, USA). 50% glucose plus water (with 0.2 mg/mL G418) were added to give a final concentration of glucose of 100g/L; the water was added to keep the volume of the fermentation media the same in every flask. At t=72 h, 2mL of media was removed and 25 L used at a 1 :40 dilution to determine OD₆₀₀- The remaining culture was centrifuged in a microcentrifuge at maximum speed for 10 min and a 1:10 dilution read on the YSI biochemical analyzer (YSI Instruments, Yellow Springs, OH, USA).

Yeast strain construction (Table 2)

[00246] Gevo2618 was constructed by transforming Gevo1187 with the integration plasmid pGV2114. The plasmid was first linearized with ΝπΛ, which cuts such that the linear plasmid will integrate into the PDC1 locus, and the DNA was transformed using the standard yeast transformation protocol described above. Twelve transformants were single colony purified. Correct integration was verified with colony PCR using primers described in Table 3.

[00247] Gevo2619 was constructed by transforming Gevol 187 with the integration plasmid pGV2115. The plasmid was first linearized with Nru\, which cuts such that the linear plasmid will integrate into the PDC1 locus, and the DNA was transformed using the standard yeast transformation protocol described above. Twelve transformants were single colony purified. Correct integration was verified with colony PCR using primers described in Table 3. [00248] Gevo2620 was constructed by transforming Gevol 187 with the integration plasmid pGV2116. The plasmid was first linearized with Nrul, which cuts such that the linear plasmid will integrate into the PDC1 locus, and the DNA was transformed using the standard yeast transformation protocol described above. Twelve transformants were single colony purified. Correct integration was verified with colony PCR using primers described in Table 3.

[00249] Gevo2621 was constructed by transforming Gevol 187 with the integration plasmids pGV2117. The plasmid was first linearized with Nrul, which cuts such that the linear plasmid will integrate into the PDC1 locus, and the DNA was transformed using the standard yeast transformation protocol described above. Twelve transformants were single colony purified. Correct integration was verified with colony PCR using primers described in Table 3.

[00250] Gevo2622 was constructed by transforming Gevol 187 with the integration plasmids pGV2118. The plasmid was first linearized with Nrul, which cuts such that the linear plasmid will integrate into the PDC1 locus, and the DNA was transformed using the standard yeast transformation protocol described above. Twelve transformants were single colony purified. Correct integration was verified with colony PCR using primers described in Table 3.

[00251] Gevo2623 was constructed by transforming Gevol 187 with the integration plasmids pGV2119. The plasmid was first linearized with Nrul, which cuts such that the linear plasmid will integrate into the PDC1 locus, and the DNA was transformed using the standard yeast transformation protocol described above. Twelve transformants were single colony purified. Correct integration was verified with colony PCR using primers described in Table 3.

[00252] Each ALS-containing strain was transformed with pGV2082, as described above. Control strains Gevo2280 (B. subtilis alsS) and Gevol 187 (no ALS) were also transformed with pGV2082. Transformants were single colony purified and maintained on YPD plates with 0.2 mg/mL G418.

Plasmid Construction (Table )

[00253] Construction of plasmid pGV2082 containing E. coli ilvC, codon optimized for S. cerevisiae (Q110V) (Ec-ilvCcoSc_Q110V), L. lactis ilvD (UJtvD), L. lactis (LI_kivD), and D. melanogaster ADH (Dm_ADH): The plasmid pGV2044 (Figure 12) carries the genes Ec-ilvCcoSc_Q110V, Bs_AlsS, UJlvD and Dm_ADH. The plasmid pGV2082 was created from pGV2044 by replacing the B. subtilis AlsS with LI_ KivD as follows: the U_KivD gene and associated PGK1 promoter were removed from pGV2047 (Figure 13) by digestion with Ανή\ and Nco\. The 2530 bp fragment was purified by gel electrophoresis and the fragment was prepared using the Zymoclean kit described above. Plasmid pGV2044 was digested with EcoR\ and Sbft to remove the B. subtilis AlsS gene and associated CUP1 promoter and the 11275 bp vector fragment was gel purified. The vector and insert were treated with Klenow fragment to produce blunt ends. The pGV2044 vector fragment and the PpGKi'.LI_kivD insert were ligated using standard methods in an approximately 5:1 insert:vector molar ratio and transformed into TOP10 chemically competent E. coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis.

[00254] Construction of plasmid pGV2114 containing B. subtilis AlsS_coSc driven by the CUP1 promoter: pGV1730 was digested with BamHl and Sa/I and the vector fragment of 4.9 kb was gel purified by agarose gel electrophoresis. pGV1773 was digested with BamHl and Sa/I and the 1.7 Kb fragment containing the B. subtilis AlsS_coSc was gel purified by agarose gel electrophoresis. The pGV1730 vector fragment was ligated to the pGV1773 insert fragment using the Roche rapid ligation kit in a ratio of 5:1 insert to vector ratio and transformed into TOP10 chemically competent E. coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis.

[00255] Construction of plasmid pGV2115 containing Mg_ALS driven by the CUP- 1 promoter: pGV1730 was digested with BamHl plus Sa/I and the vector fragment of 4.9 kb was gel purified by agarose gel electrophoresis. pGV1800 was digested with BamHl and Sa/I and the 1.8 kb fragment containing the Mg_ALS was gel purified by agarose gel electrophoresis. The pGV1730 vector fragment was ligated to the pGV1800 insert fragment using the Roche rapid ligation kit in a ratio of 5:1 insert to vector and transformed into TOP-10 chemically competent E.coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis consisting of digestion of potential clones.

[00256] Construction of plasmid pGV2116 containing Pn_ALS driven by the CUP-1 promoter: pGV1730 was digested with BamHl and Sa/I and the vector fragment of 4.9 kb was gel purified by agarose gel electrophoresis. pGV1801 was digested with BamHl and Sa/I and the 1.8 kb fragment containing Pn_ALS gel purified by agarose gel electrophoresis. The pGV1730 vector fragment was ligated to the pGV1801 insert fragment using the Roche rapid ligation kit in a ratio of 5:1 insert to vector and transformed into TOP-10 chemically competent E.coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis.

[00257] Construction of plasmid pGV2117 containing Ta_ALS driven by the CUP-1 promoter: pGV1730 was digested with BamHl and Sa/I and the vector fragment of 4.9 kb was gel purified by agarose gel electrophoresis. pGV1802 was digested with BamHl and Sa/I and the 1.8 kb fragment containing the Ta_ALS was gel purified by agarose gel electrophoresis. The pGV1730 vector fragment was ligated to the pGV1802 insert fragment using the Roche rapid ligation kit in a ration of 5:1 insert to vector ratio and transformed into TOP-10 chemically competent E.coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis.

[00258] Construction of plasmid pGV2118 containing Ts_ALS driven by the CUP-1 promoter: pGV1730 was digested with BamHI and Sail and the vector fragment of 4.9 kb was gel purified by agarose gel electrophoresis. pGV1803 was digested with BamHI and Sail and the 1.8 kb fragment containing the Ts_ALS gel purified by agarose gel electrophoresis. The pGV1730 vector fragment was ligated to the pGV1803 insert fragment using the Roche rapid ligation kit in a ration of 5:1 insert to vector ratio and transformed into TOP-10 chemically competent E.coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis.

[00259] Construction of plasmid pGV2119 containing Pm_ALS driven by the CUP- 1 promoter: pGV1730 was digested with BamHI and Sail and the vector fragment of 4.9 kb was gel purified by agarose gel electrophoresis. pGV1804 was digested with BamHI and Sail and the 1.8 kb fragment containing the Pm_ALS gel purified by agarose gel electrophoresis. The pGV1730 vector fragment was ligated to the pGV1804 insert fragment using the Roche rapid ligation kit in a ratio of 5:1 insert to vector ratio and transformed into TOP-10 chemically competent E.coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis.

[00260] The 5' end of the Als homologs in pGV 2114-2119 were sequenced using OGV2095 and Laragen sequencing services. All clones had the expected 5' sequence.

Example 1 : Identification of Sequences of ALS Enzvmes

[00261] As described above, identification of ALS enzymes from various host organisms, including, for example, yeast, is complicated by the existence of the two- subunit (small regulatory and large catalytic) FAD-dependent acetohydroxyacid synthase (AHAS) enzymes involved in branched-chain amino acid biosynthesis. The large subunit of AHAS shares significant sequence homology with ALS, despite ALS being FAD-independent. Accordingly, the present inventors sought to overcome this complication and distinguish ALS genes from AHAS genes of the basis of sequence. In doing so, the present inventors have identified numerous ALS enzymes (SEQ ID NOs: 1-535), including genes from fungal sources.

[00262] BLAST searches were performed using the BLAST utility found at the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Initially a non-redundant database confined to sequences for fungi (taxid: 4751) was searched. A broader search followed. For alignments, sequences were transferred to VectorNTI (Invitrogen) and aligned using the alignX utility (Invitrogen).

[00263] On the basis of a distinguishing characteristic between AHAS and ALS, i.e. that the former is FAD-dependent while the latter is not, focus was placed upon a short region in AHAS enzymes that is highly conserved ('RFDDR') and is reported to be involved in FAD binding (Le and Choi, supra). This region is not conserved among the known ALS enzymes. Figure 4 shows the comparison of this region between the AHAS large catalytic subunit, ilvl, from E. coli and the B. subtilis alsS and the L. lactis alsS. While the E. coli ilvl has the conserved 'RFDDR' sequence, this region in the B. subtilis alsS and the L. lactis alsS is ΌΡΙΕΥ.' This lack of sequence conservation in the ALSs supports the idea that this sequence can be used to distinguish the anabolic AHAS and the catabolic ALSs in sequence searching and identification. Additionally, it was found that ALS's may contain the specific TPP-binding domain designated by GenBank as cd02010.

[00264] The protein sequence of the B. subtilis alsS was used to perform BLASTp searches for ALSs. The search was initially confined to fungi (taxid:4751) databases to select for only ALSs that are from fungal origins. This initial BLASTp search identified sequences from two fungal plant pathogens, Magnaprthe grisea (SEQ ID NO: 1) and Phaeosphaeria nodomm (SEQ ID NO: 2). Examination of the 'FAD binding' region of these sequences showed that the M. grisea sequence is 'DEIEV and the P. nodorum sequence is 'DPVEV (Figure 4). Therefore, these sequences do not possess the conserved 'RFDDR' of AHAS enzymes. Rather, they possess sequences that are more similar to the 'DPIEV found in ALSs. Comparison of these sequences using Vector NTI showed that the M. grisea and the P. nodomm sequences exhibit 43.1% and 41.9% identity, respectively, to the B. subtilis alsS (Figure 4). Accordingly, the inventors projected that these sequences are fungal ALSs.

[00265] To identify additional fungal ALS genes, the M. grisea sequence (SEQ ID NO: 1) was used to carry out additional BLAST (tblastn) searches of fungal sequence databases. This search identified additional four potential ALSs from the fungi Trichoderma atroviride (SEQ ID NO: 3), Talaromyces stipitatus (SEQ ID NO: 4), Penicillium mameffei (SEQ ID NO: 5), and Glomerella graminicola (SEQ ID NO: 333). These ALSs sequences were also identified when the P. nodorum sequence (SEQ ID NO: 2) was used for tblastn searches, and no additional sequences were found. Full sequences were available for all these potential fungal ALSs, except for the sequence from G. graminicola, where an estimated 80 amino acids at the N- terminal could not be found. The 'FAD binding' region of these sequences (Figure 4) did not carry the conserved 'RFDDR' expected for AHAS enzymes. These sequences are more similar to ALSs. In addition these sequences all showed between 41-42% identity to the B. subtilis alsS when aligned using Vector NTI (Figure 4).

[00266] These initial BLAST searches identified a total of six potential fungal ALSs. These sequences were further analyzed using PSORT (http://wolfpsort.org/) to predict intracellular localization (Figure 4). Four of the six sequences (from M. grisea, P. nodorum, T. stipitatus, and P. mameffei) are predicted to be cytoplasmic. The sequence from T. atrovihdes was predicted to be cytoskeletal and the sequence from G. graminicola was not analyzed as the N-terminal region was not found.

[00267] To identify further ALS enzymes, additional BLAST searches, not restricted to fungal sequences, were conducted using B. subtilis alsS and the ALS of Talaromyces stipitatus (SEQ ID NO: 4) as search queries. Many of these sequences are included in SEQ ID NOs: 6-332 and 334-535.

Example 2: Characterization of Acetolactate Synthase Activity in Recombinant Microorganisms with Exemplary ALS Enzvmes

[00268] The inventors tested five of the ALS homologs as well as a version of the B. subtilis alsS gene that were codon optimized for yeast for acetolactate synthase activity. Each of the six ALS genes was placed under control of the CUP1 promoter and integrated into the PDC1 locus of Gevo1187 (Table 2). ALS activity was measured in lysates from the strains and some of the strains were tested in a fermentation for isobutanol production.

[00269] In a preliminary experiment to measure ALS activity, cells were grown and cell lysates were generated as described supra. Cell lysates from 1187 (no ALS), 2280 (B. subtilis alsS: Bs_alsS), 2618 (B. subtilis alsS, codon optimized for S. cerevisiae: Bs_alsS_coSc), 2619 {Magnaprthe grisea (SEQ ID NO: 1): Mg_ALS), 2620 (Phaeosphaeria nodorum (SEQ ID NO: 2): Pn_ALS), 2621 (Trichoderma atroviride (SEQ ID NO:3: Ta_ALS), 2622 (Talaromyces stipitatus (SEQ ID NO: 4): Ts_ALS), and 2623 (Penicillium mameffei (SEQ ID NO: 5): Pm_ALS) were tested for ALS activity as described above (Table 2). Strains containing the Bs_alsS, Bs_alsS_coSc, Ta_ALS, Ts_ALS, and Pm_ALS had the highest ALS activities above background and thus were selected to be tested for isobutanol production in a shake flask fermentation.

Example 3: Isobutanol Production in Recombinant Microorganisms with Exemplary ALS Enzvmes

[00270] The strains of Example 2 were transformed with a four-pathway component plasmid pGV2082 (containing E. coli ilvC (Q110V), L. lactis ilvD, L. lactis kivD, and D. melanogaster ADH) and tested for isobutanol production in shake flask fermentation experiments.

[00271] A first fermentation was performed with ALS-containing strains with a four- component pathway plasmid, pGV2082: Figure 21 shows fermentations of 1187, 2280, 2618, 2621, 2622 and transformed with pGV2082 which were carried out as described above. 2618 {Bs_AlsS_coSc), 2621 (Ta_ALS), and 2622 (Ts_ALS) were selected because they showed the highest ALS activity in ALS assays. In this experiment, strains containing the ALS homologs Ta_ALS and Ts_ALS produced more isobutanol than the strain containing the Bs_Als. The strain containing the Bs_Als_coSc produced the most isobutanol, yielding twice more isobutanol than the strain with the Bs_AlsS. The strain with the Ta_ALS produced about 50% more isobutanol than those with either the Bs_AlsS or Ts_ALS (Figure 21). Since the strain with the Bs_AlsS_coSc grew to the lowest OD₆₀₀, its specific isobutanol production was 120% higher than strain with the Bs_AlsS (Figure 22). Fermentation 2: ALS-containing strains with pGV2082: A second fermentation was performed with the strains 1187 (No ALS), 2280 (Bs_AlsS), 2618 (Bs_AlsS_coSc), 2621 (Ta_ALS), 2622 (Ts_ALS), and 2623 (Pm_ALS). In this fermentation the samples were incubated as previously (Figures 21, 22). The same pattern of isobutanol production was seen in this fermentation as above. Gevo2618 (Bs_AlsScoSc) produced the highest amounts of isobutanol (3.9 g/L and 0.27 g/L/OD), Gevo2621 {Ta_ALS) produced moderate levels (2.4 g/L and 0.17 g/L/OD), as did Gevo2622 (Ts_ALS) (2.0 g/L and 0.14 g/L/OD). Gevo2280 {Bs_AlsS) produced less than Gevo2618 (1.3 g/L and 0.09 g/L/OD). Gevo2623 (Pm_ALS) showed isobutanol production that was slightly above the no ALS control strain (Gevo1187) at 0.4 g/L and 0.03 g/L/OD) (Figures 23-25).

Example 4: Improvement of Acetolactate Synthase Activity in Recombinant Microorganisms with Exemplary ALS Enzvmes

[00272] A selected gene sequence encoding for an ALS enzyme disclosed herein is subjected to a protein engineering protocol comprising iterative rounds of (1) constructing a polynucleotide mutant library encoding mutant ALS enzymes, followed by (2) screening for decreased K_M and/or increased specific activity, and (3) constructing a next polynucleotide mutant library encoding mutant ALS enzymes.

[00273] The ALS encoding polynucleotide is cloned into an appropriate expression system for E. coli. Cloning of a codon-optimized polynucleotide and its adequate expression is accomplished via gene synthesis supplied from a commercial supplier using standard techniques. The gene is synthesized with a N-terminal six-histidine tag to enable affinity based protein purification and subsequent kinetic characterization. The gene may also be synthesized with a C-terminal six-histidine tag for the same purpose. Once obtained using standard methodology, the gene is cloned into an expression plasmid using standard techniques.

[00274] Libraries (-1,000 - 10,000 mutants with one to two amino acid mutations on average) are created by error-prone PCR using the Genemorph kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol.

[00275] Alternatively, libraries (-1,000 - 10,000 mutants with one to two amino acid mutations on average) are created by error-prone PCR using MnCI₂ (0, 50, 100, and 150 μΜ) to induce mutations, Taq DNA polymerase (Roche), approximately 50 ng of plasmid DNA as template, and appropriate primers

[00276] Alternatively, mutations are introduced into the ALS encoding polynucleotide by PCR overlap extension mutagenesis using a high-fidelity DNA polymerase. Amino acids to be mutated are identified based on the ALS protein structure or a homology model. For example, active site residues not implied in the catalytic mechanisms are targeted for mutagenesis. The oligonucleotide primers for each saturation mutagenesis library contain all possible combinations of bases, NNK (N = A, T, G, or C; K= T or G), at the codon that is mutated. For each mutation, two separate PCRs are performed, each using a perfectly complementary primer at the end of the sequence and a mutagenic primer. The resulting two overlapping fragments that contain the mutations are then annealed during a second PCR to amplify the complete mutated gene.

[00277] Alternatively, beneficial mutations that are found by screening an error prone PCR library and/or a saturation mutagenesis library are recombined in a combinatorial fashion using overlap extension PCR with degenerate primers or a mixture of primers.

[00278] After the library of ALS mutant genes is cloned into the expression plasmid using standard techniques, the resulting plasmids are transformed into an E. coli expression host using standard techniques and plated onto selective agar plates. Individual colonies are transferred into multi-titer plates containing a growth medium appropriate for gene expression from this plasmid.

[00279] Following ALS expression, the cells are lysed and the supernatant is subjected to an ALS assay, substantially as described above. Improved variants are isolated from this library based on increased activity under assay condition A where the substrate concentration is saturating and/or under assay condition B where the substrate concentration is significantly lower than the KM- Improved variants identified under assay condition A and/or B are confirmed via standard biochemical techniques.

[00280] 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.

[00281] 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.

[00282] The disclosures, including the claims, figures and/or drawings, of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 1.

2. The recombinant microorganism of claim 1, wherein said polypeptide is derived from the genus Magnaporthe.

3. The recombinant microorganism of claim 2, wherein said polypeptide is derived from Magnaporthe grisea or Magnaporthe oryzae.

4. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 2.

5. The recombinant microorganism of claim 4, wherein said polypeptide is derived from the genus Phaeosphaena.

6. The recombinant microorganism of claim 5, wherein said polypeptide is derived from Phaeosphaena nodorum.

7. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 3.

8. The recombinant microorganism of claim 7, wherein said polypeptide is derived from the genus Trichoderma.

9. The recombinant microorganism of claim 8, wherein said polypeptide is derived from Trichoderma atroviride.

10. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 4.

11. The recombinant microorganism of claim 10, wherein said polypeptide is derived from the genus Talaromyces.

12. The recombinant microorganism of claim 11, wherein said polypeptide is derived from Talaromyces stipitatus.

13. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 5.

14. The recombinant microorganism of claim 13, wherein said polypeptide is derived from the genus Penicillium.

15. The recombinant microorganism of claim 14, wherein said polypeptide is derived from the genus Penicillium mameffei.

16. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 12-49, 51-55, 57-63, 65-75, 77-81, 83-87, 101-103, 106-107, 135, 372, and 506.

17. The recombinant microorganism of claim 16, wherein said polypeptide is derived from the genus Bacillus.

18. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 50, 56, 64, 76, 82, 88-97, 100, 105, 336, and 351.

19. The recombinant microorganism of claim 18, wherein said polypeptide is derived from the genus Listeria.

20. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 98 and 99.

21. The recombinant microorganism of claim 20, wherein said polypeptide is derived from the genus Paenibacillus.

22. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 104 and 379.

23. The recombinant microorganism of claim 22, wherein said polypeptide is derived from the genus Exiguobacterium.

24. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide of SEQ ID NO: 108.

25. The recombinant microorganism of claim 24, wherein said polypeptide is derived from the genus Gemella.

26. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 110, 122, 127, 136-139, 143, 154, 158-160, 181, 184, 190-192, 211-214, 230, 232-234, 253, 254, 256, 260, 266, 271, 279, 296, and 338.

27. The recombinant microorganism of claim 26, wherein said polypeptide is derived from the genus Lactobacillus.

28. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 109, 111, 113-121, 123-126, 128-134 and 345.

29. The recombinant microorganism of claim 28, wherein said polypeptide is derived from the genus Enterococcus.

30. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 140-142, 144-152, 156, 157, 161-163, 165, 166, 168, 170, 172, 173, 175, 177, 187, and 331.

31. The recombinant microorganism of claim 30, wherein said polypeptide is derived from the genus Streptococcus.

32. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 153 and 155.

33. The recombinant microorganism of claim 32, wherein said polypeptide is derived from the genus Pediococcus.

34. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 164, 167, 171, 176, 178, 179, 180, 182, 183, 189, 193, 194- 210, 215-219, 222, and 337.

35. The recombinant microorganism of claim 34, wherein said polypeptide is derived from the genus Staphylococcus.

36. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 221, 224, 225, 231, and 445.

37. The recombinant microorganism of claim 36, wherein said polypeptide is derived from the genus Oenococcus.

38. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 235, 237, 239, 242, and 246.

39. The recombinant microorganism of claim 38, wherein said polypeptide is derived from the genus Pectobacterium.

40. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 240, 244, 249, and 251.

41. The recombinant microorganism of claim 40, wherein said polypeptide is derived from the genus Seiratia.

42. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 248, 250, 255, and 258.

43. The recombinant microorganism of claim 42, wherein said polypeptide is derived from the genus Dickeya.

44. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 263, 264, and 288.

45. The recombinant microorganism of claim 44, wherein said polypeptide is derived from the genus Proteus.

46. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 269, 273, 275, 283, 284, 286, 291, and 292.

47. The recombinant microorganism of claim 46, wherein said polypeptide is derived from the genus Klebsiella.

48. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 245, 247, 257, 268, 287, 294, and 295.

49. The recombinant microorganism of claim 48, wherein said polypeptide is derived from the genus Yersinia.

50. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 228, 298, 299, 301, 303, 312, 315-317, 319-324, 326, 327, 332, and 341.

51. The recombinant microorganism of claim 50, wherein said polypeptide is derived from the genus Vibrio.

52. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 252, 259, 267, 278, and 289.

53. The recombinant microorganism of claim 52, wherein said polypeptide is derived from the genus Enterobacter.

54. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 265, 272, 274, 280-282, 285, 302, and 304.

55. The recombinant microorganism of claim 54, wherein said polypeptide is derived from the genus Leuconostoc.

56. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 305, 308, 309, 311 , 313, and 325.

57. The recombinant microorganism of claim 56, wherein said polypeptide is derived from the genus Erwinia.

58. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 293, 306, 310, 314, and 318.

59. The recombinant microorganism of claim 58, wherein said polypeptide is derived from the genus Pantoea.

60. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 348, 352-354, and 366.

61. The recombinant microorganism of claim 60, wherein said polypeptide is derived from the genus Clostridium.

62. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 431 , 432, 436, 438, 441 , 444, 448, 451 , and 455.

63. The recombinant microorganism of claim 62, wherein said polypeptide is derived from the genus Pseudomonas.

64. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 409, 415, 423, 429, 433, 434, 437, 439, 440, 443, 447, 452, 456-463, 466, 468, 483, 485, 488, 489, 494, 498, 502, and 517.

65. The recombinant microorganism of claim 64, wherein said polypeptide is derived from the genus Burkholderia

66. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 474, 479, 487, 490, and 492.

67. The recombinant microorganism of claim 66, wherein said polypeptide is derived from the genus Gardnerella.

68. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 470 and 481.

69. The recombinant microorganism of claim 68, wherein said polypeptide is derived from the genus Arcobacter.

70. A recombinant microorganism comprising at least one nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 480, 496, 504, and 519.

71. The recombinant microorganism of claim 70, wherein said polypeptide is derived from the genus Marinobacter.

72. The recombinant microorganism of any of the preceding claims, wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway comprising one or more isobutanol metabolic pathway enzymes selected from ketol-acid reductoisomerase, dihydroxy acid dehydratase, 2- keto acid decarboxylase, and alcohol dehydrogenase.

73. The recombinant microorganism of claim 72, wherein said ketol-acid reductoisomerase is an NADH-dependent ketol-acid reductoisomerase (NKR).

74. The recombinant microorganism of claim 72, wherein said alcohol dehydrogenase is an NADH-dependent alcohol dehydrogenase.

75. The recombinant microorganism of any of the preceding claims, wherein said recombinant microorganism is engineered to reduce the expression and/or activity of one or more enzymes selected from a pyruvate decarboxylase, a glycerol-3-phosphate dehydrogenase, a 3-keto acid reductase, and an aldehyde dehydrogenase.

76. The recombinant microorganism of any of claims 1-71, wherein said recombinant microorganism comprises a metabolic pathway for the production of a metabolite selected from isobutanol, 2-butanol, 1-butanol, 2- butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl- 1-butanol, 4-methyl-1-pentanol, and coenzyme A.

77. The recombinant microorganism of any of the preceding claims, wherein said recombinant microorganism is a prokaryotic microorganism.

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

79. A method of producing isobutanol, comprising:

(a) providing a recombinant microorganism of any of claims 1-75 or 77-78;

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

80. An isolated nucleic acid molecule encoding a polypeptide with acetolactate synthase (ALS) activity, wherein said polypeptide is at least about 65% identical to a polypeptide selected from SEQ ID NOs: 1-535.

81. A recombinant microorganism comprising the isolated nucleic acid of claim 80.