Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/16—Butanols
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
  • C12Y202/01—Transketolases and transaldolases (2.2.1)
  • C12Y202/01006—Acetolactate synthase (2.2.1.6)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• 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.
• 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).
• 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.
• the first step of the isobutanol producing metabolic pathway is catalyzed by acetolactate synthase (ALS), which converts pyruvate to acetolactate.
• ALS acetolactate synthase
• 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.
• 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.
• 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.
• the polypeptide is derived from the genus Magnaporthe.
• the polypeptide is derived from Magnaporthe grisea or Magnaporthe oryzae.
• 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.
• the polypeptide is derived from the genus Phaeosphaeria. In a specific embodiment, the polypeptide is derived from Phaeosphaeria nodonim.
• 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.
• the polypeptide is derived from the genus Trichoderma. In a specific embodiment, the polypeptide is derived from Trichoderma atroviride.
• 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.
• the polypeptide is derived from the genus Talaromyces.
• the polypeptide is derived from Talaromyces stipitatus.
• 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.
• the polypeptide is derived from the genus Penicillium. In a specific embodiment, the polypeptide is derived from Penicillium mameffei.
• 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.
• the polypeptide is derived from the genus Bacillus.
• 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.
• the polypeptide is derived from the genus Listeria.
• 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.
• the polypeptide is derived from the genus Paenibacillus.
• 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.
• the polypeptide is derived from the genus Exiguobacterium.
• 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.
• the polypeptide is derived from the genus Gemella.
• 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.
• the polypeptide is derived from the genus Lactobacillus.
• 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.
• the polypeptide is derived from the genus Enterococcus.
• 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.
• the polypeptide is derived from the genus Streptococcus.
• 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.
• the polypeptide is derived from the genus Pediococcus.
• 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.
• the polypeptide is derived from the genus Staphylococcus.
• 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.
• the polypeptide is derived from the genus Oenococcus.
• 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.
• the polypeptide is derived from the genus Pectobacterium.
• 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.
• the polypeptide is derived from the genus Serratia.
• 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.
• the polypeptide is derived from the genus Dickeya.
• 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.
• the polypeptide is derived from the genus Proteus.
• 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.
• the polypeptide is derived from the genus Klebsiella.
• 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.
• the polypeptide is derived from the genus Yersinia.
• 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.
• the polypeptide is derived from the genus Vibrio.
• 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.
• the polypeptide is derived from the genus Enterobacter.
• 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.
• the polypeptide is derived from the genus Leuconostoc.
• 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.
• the polypeptide is derived from the genus Erwinia.
• 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.
• the polypeptide is derived from the genus Pantoea.
• 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.
• the polypeptide is derived from the genus Clostridium.
• 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.
• the polypeptide is derived from the genus Pseudomonas.
• 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.
• the polypeptide is derived from the genus Burkholderia
• 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.
• the polypeptide is derived from the genus Gardnemlla.
• 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.
• the polypeptide is derived from the genus Arcobacter.
• 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.
• the polypeptide is derived from the genus Marinobacter.
• 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.
• 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.
• the recombinant microorganism comprises an isobutanol producing metabolic pathway.
• 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.
• the isobutanol producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol.
• 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.
• all of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes.
• 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.
• ALS acetolactate synthase
• one or more of the isobutanol pathway genes encodes an enzyme that is localized to the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least two isobutanol pathway enzymes localized in the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least three isobutanol pathway enzymes localized in the cytosol.
• 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.
• 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).
• the KARI is an NADH-dependent KARI (NKR).
• the ADH is an NADH-dependent ADH.
• the KARI is an NADH-dependent KARI (NKR) and the ADH is an NADH-dependent ADH.
• 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.
• 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).
• PDC pyruvate decarboxylase
• GPD glycerol- 3-phosphate dehydrogenase
• 3-KAR 3-keto acid reductase
• ALDH aldehyde dehydrogenase
• 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.
• ALS acetolactate synthase
• 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.
• ALS acetolactate synthase
• the recombinant microorganisms may be recombinant prokaryotic microorganisms.
• the recombinant microorganisms may be recombinant eukaryotic microorganisms.
• the recombinant eukaryotic microorganisms may be recombinant yeast microorganisms.
• 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.
• the recombinant microorganisms may be yeast recombinant microorganisms of the Saccharomyces clade.
• the recombinant microorganisms may be Saccharomyces sensu stricto microorganisms.
• 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.
• the recombinant microorganisms may be Crabtree- negative recombinant yeast microorganisms.
• the Crabtree- negative yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Hansenula, or Candida.
• the Crabtree-negative yeast microorganism is selected from Saccharomyces reteyven, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Hansenula anomala, Candida utilis and Kluyveromyces waltii.
• the recombinant microorganisms may be Crabtree- positive recombinant yeast microorganisms.
• the Crabtree- positive yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Candida, Pichia and Schizosaccharomyces.
• 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.
• the recombinant microorganisms may be post- WGD (whole genome duplication) yeast recombinant microorganisms.
• the post-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces or Candida.
• the post-WGD yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and Candida glabrata.
• the recombinant microorganisms may be pre-WGD (whole genome duplication) yeast recombinant microorganisms.
• 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.
• 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.
• 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.
• the non-fermenting yeast is C. xestobii.
• the present application provides methods of producing a beneficial metabolite derived from a recombinant microorganism.
• 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.
• 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.
• the beneficial metabolite is isobutanol.
• the microorganism is cultured in an appropriate culture medium containing a carbon source.
• the method further includes isolating the beneficial metabolite from the culture medium.
• 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
• the recombinant microorganism may produce the beneficial metabolite from a carbon source at a yield of at least 5 percent theoretical.
• 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.
• the beneficial metabolite is isobutanol.
• 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.
• Figure 1 illustrates an isobutanol pathway.
• Figure 2 illustrates an NADH-dependent isobutanol pathway.
• Figure 3 shows exemplary metabolic pathways involving acetolactate as a metabolic intermediate.
• 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.
• Figure 5 illustrates the plasmid map of pGV1730.
• Figure 6 illustrates the plasmid map of pGV1773.
• Figure 7 illustrates the plasmid map of pGV1800.
• Figure 8 illustrates the plasmid map of pGV1801.
• Figure 9 illustrates the plasmid map of pGV1802.
• Figure 10 illustrates the plasmid map of pGV1803.
• Figure 11 illustrates the plasmid map of pGV1804.
• Figure 12 illustrates the plasmid map of pGV2044.
• Figure 13 illustrates the plasmid map of pGV2047.
• Figure 14 illustrates the plasmid map of pGV2082.
• Figure 15 illustrates the plasmid map of pGV2114.
• Figure 16 illustrates the plasmid map of pGV2115.
• Figure 17 illustrates the plasmid map of pGV2116.
• Figure 18 illustrates the plasmid map of pGV2117.
• Figure 19 illustrates the plasmid map of pGV2118.
• Figure 20 illustrates the plasmid map of pGV2119.
• 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:
• Ts_ALS Talaromyces stipitatus
• Figure 22 illustrates the data of Figure 21 , normalized for cell growth.
• 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).
• 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).
• 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).
• 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.
• microbial cells and “microbes” are used interchangeably with the term microorganism.
• 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.
• 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.
• the Archaea consist of two phylogenetically-distinct groups: Crenarchaeota and Euryarchaeota.
• 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).
• methanogens prokaryotes that produce methane
• extreme halophiles prokaryotes that live at very high concentrations of salt (NaCI)
• extreme (hyper) thermophiles prokaryotes that live at very high temperatures.
• 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.
• Bacteria 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
• 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.
• 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.
• 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.
• genomic hybridization 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.
• recombinant microorganism refers 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.
• 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.
• alter can mean “inhibit,” but the use of the word “alter” is not limited to this definition.
• 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.
• expression refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein.
• 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.
• 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.
• 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.
• 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.
• 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).
• the reduced activity of a protein in a cell may result from decreased concentrations of the protein in the cell.
• 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.
• 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.
• 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.
• 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.
• engine 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.
• mutation 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.
• the modified microorganism a portion of the microorganism genome has been replaced with a heterologous polynucleotide.
• the mutations are naturally-occurring.
• the mutations are identified and/or enriched through artificial selection pressure.
• the mutations in the microorganism genome are the result of genetic engineering.
• 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.
• isobutanol producing metabolic pathway refers to an enzyme pathway which produces isobutanol from pyruvate.
• NADH-dependent 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.
• exogenous 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.
• endogenous or “native” as used herein with reference to various molecules refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
• heterologous 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
• 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.
• 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.
• a feedstock may contain nutrients other than a carbon source.
• 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.
• 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.
• 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.
• 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).
• 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).
• 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.
• titer is defined as the strength of a solution or the concentration of a substance in solution.
• 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).
• “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.
• 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.
• 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.
• 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.”
• NAD(P)H donates its electrons to a molecule produced by the same metabolic pathway that produced the electrons carried in NAD(P)H.
• 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.
• 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.
• 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 may be created through recombinant means or identified in nature.
• 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 C0 2 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 CO 2 .
• 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.
• DNA single stranded or double stranded
• RNA ribonucleic acid
• 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.
• 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.
• 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.
• the polynucleotides described herein include “genes” and that the nucleic acid molecules described herein include “vectors” or “plasmids.”
• 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.
• operon refers to two or more genes which are transcribed as a single transcriptional unit from a common promoter.
• 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.
• 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.
• 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.
• 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.
• enzyme 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.
• polypeptide indicates an organic polymer composed of two or more amino acidic monomers and/or analogs thereof.
• amino acid or “amino acidic monomer” refers to any natural and/or synthetic amino acids including glycine and both D or L optical isomers.
• 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.
• 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
• 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.
• 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.
• a polypeptide has homology to a second polypeptide if the two polypeptides have "similar” amino acid sequences.
• homology to a second polypeptide if the two polypeptides have "similar” amino acid sequences.
• analogs 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.
• microorganisms convert sugars to produce pyruvate, which is then utilized in a number of pathways of cellular metabolism.
• microorganisms including yeast
• microorganisms 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).
• the present invention relates to recombinant microorganisms for producing isobutanol, wherein said recombinant microorganisms comprise an isobutanol producing metabolic pathway.
• the isobutanol producing metabolic pathway to convert pyruvate to isobutanol can be comprised of the following reactions:
• 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).
• the recombinant microorganism may be engineered to overexpress one or more of these enzymes.
• the recombinant microorganism is engineered to overexpress all of these enzymes.
• isobutanol producing metabolic pathway comprises five substrate to product reactions.
• the isobutanol producing metabolic pathway comprises six substrate to product reactions.
• the isobutanol producing metabolic pathway comprises seven substrate to product reactions.
• the recombinant microorganism comprises an isobutanol producing metabolic pathway.
• 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.
• the isobutanol producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol.
• 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.
• one or more of the isobutanol pathway genes encodes an enzyme that is localized to the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least two isobutanol pathway enzymes localized in the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least three isobutanol pathway enzymes localized in the cytosol.
• 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.
• 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.
• 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.
• one or more of these enzymes can be encoded by native genes.
• one or more of these enzymes can be encoded by heterologous genes.
• 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.
• ketol-acid reductoisomerases 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:
• V(V/I/F)(M/L/A)(A/C)PK (SEQ ID NO: 548),
• a protein harboring one or more of these amino acid motifs can generally be expected to exhibit ketol-acid reductoisomerase activity.
• ketol-acid reductoisomerases are known to use NADPH as a cofactor.
• 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.
• 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.
• 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.
• 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.
• 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.
• CDKXXPG (SEQ ID NO: 552)
• a protein harboring one or more of these amino acid motifs can generally be expected to exhibit dihydroxy acid dehydratase activity.
• 2-keto-acid decarboxylases capable of converting ⁇ -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.
• 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).
• 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:
• 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.
• a protein harboring one or more of these amino acid motifs can generally be expected to exhibit 2-keto-acid decarboxylase activity.
• 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.
• sources e.g., bacterial, yeast, Archaea, etc.
• L. lactis GenBank Accession No. YP_003354381
• B. cereus GenBank Accession No. YP_001374103.1
• GHEXXGXV (SEQ ID NO: 564), (L/V)(Q/K/E)(V/I/K)G(D/Q)(R/H)(V/A) (SEQ ID NO: 565),
• 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.
• a protein harboring one or more of these amino acid motifs can generally be expected to exhibit alcohol dehydrogenase activity.
• 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.
• any of the genes encoding the foregoing 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. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast.
• 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.
• KARI NADH-dependent KARI
• ADH enzymes 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.
• the recombinant microorganisms of the present invention may use an NKR to catalyze the conversion of acetolactate to produce 2,3-dihydroxyisovalerate.
• the recombinant microorganisms of the present invention may use an NADH-dependent ADH to catalyze the conversion of isobutyraldehyde to produce isobutanol.
• 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.
• the first step of the isobutanol producing metabolic pathway is catalyzed by acetolactate synthase (ALS), which converts pyruvate to acetolactate.
• ALS acetolactate synthase
• 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.
• AHAS acetohydroxyacid synthase
• ALS acetolactate synthase
• 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.
• ALSs are specific for the conversion of two pyruvate molecules to acetolactate. Therefore, ALSs are favored over AHASs.
• 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.
• BLAST searches of fungal sequence databases were performed and resulted in the identification of ALS homologs from several fungal species (including, for example, M.
• ALS homologs from M. grisea, P. nodomm, T. atmviride, T. stipitatus, P. mameffei, and Glomemlla graminicola will generally be expected to be cytosolically localized.
• 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.
• 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.
• the polypeptide is derived from the genus Magnaporthe.
• the polypeptide is derived from Magnaporthe grisea or Magnaporthe oryzae.
• 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.
• the polypeptide is derived from the genus Phaeosphaeria. In a specific embodiment, the polypeptide is derived from Phaeosphaeria nodorum.
• 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.
• the polypeptide is derived from the genus Trichoderma. In a specific embodiment, the polypeptide is derived from Trichoderma atroviride.
• 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.
• the polypeptide is derived from the genus Talaromyces.
• the polypeptide is derived from Talaromyces stipitatus.
• 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.
• the polypeptide is derived from the genus Penicillium. In a specific embodiment, the polypeptide is derived from Penicillium mameffei.
• 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.
• the polypeptide is derived from the genus Bacillus.
• 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.
• the polypeptide is derived from the genus Listeria.
• 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.
• the polypeptide is derived from the genus Paenibacillus.
• 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.
• the polypeptide is derived from the genus Exiguobacterium.
• 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.
• the polypeptide is derived from the genus Gemella.
• 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.
• the polypeptide is derived from the genus Lactobacillus.
• 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.
• the polypeptide is derived from the genus Enterococcus.
• 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.
• the polypeptide is derived from the genus Streptococcus.
• 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.
• the polypeptide is derived from the genus Pediococcus.
• 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.
• the polypeptide is derived from the genus Staphylococcus.
• 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.
• the polypeptide is derived from the genus Oenococcus.
• 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.
• the polypeptide is derived from the genus Pectobacterium.
• 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.
• the polypeptide is derived from the genus Serratia.
• 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.
• the polypeptide is derived from the genus Dickeya.
• 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.
• the polypeptide is derived from the genus Proteus.
• 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.
• the polypeptide is derived from the genus Klebsiella.
• 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.
• the polypeptide is derived from the genus Yersinia.
• 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.
• the polypeptide is derived from the genus Vibrio.
• 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.
• the polypeptide is derived from the genus Enterobacter.
• 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.
• the polypeptide is derived from the genus Leuconostoc.
• 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.
• the polypeptide is derived from the genus Erwinia.
• 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.
• the polypeptide is derived from the genus Pantoea.
• 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.
• the polypeptide is derived from the genus Clostridium.
• 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.
• the polypeptide is derived from the genus Pseudomonas.
• 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.
• the polypeptide is derived from the genus Burkholderia.
• 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.
• the polypeptide is derived from the genus Gardnerella.
• 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.
• the polypeptide is derived from the genus Arcobacter.
• 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.
• the polypeptide is derived from the genus Marinobacter.
• 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.
• 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.
• ALS acetolactate synthase
• 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.
• ALS acetolactate synthase
• 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.
• DHAD dihydroxyacid dehydratase
• 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).
• 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).
• the recombinant microorganism as discussed herein is a prokaryotic microorganism.
• the recombinant microorganism as discussed herein is a yeast microorganism.
• the recombinant microorganism as discussed herein is a yeast microorganism of the Saccharomyces clade.
• the recombinant microorganism as discussed herein is a Saccharomyces sensu stricto microorganism.
• 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.
• the recombinant microorganism as discussed herein is a Crabtree-negative yeast microorganism.
• the Crabtree- negative yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces, Kluyveromyces, Pichia, Hansenula, Issatchenkia and Candida.
• 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.
• the recombinant microorganism as discussed herein is a Crabtree-positive yeast microorganism.
• the Crabtree-positive yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces, Kluyvenomyces, Zygosacchanomyces, Debanyomyces, Pichia, Candida, and Schizosacchanomyces.
• 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.
• the recombinant microorganism as discussed herein is a post-WGD (whole genome duplication) yeast microorganism.
• the post-WGD yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces or Candida.
• 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.
• the recombinant microorganism as discussed herein is a pre-WGD (whole genome duplication) yeast microorganism.
• 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.
• 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.
• 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.
• ALS acetolactate synthase
• 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.
• ALS acetolactate synthase
• the enzymes exhibiting acetolactate synthase (ALS) activity described herein would have utility in any of the above-described pathways.
• 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.
• ALS acetolactate synthase
• 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.
• protein engineering techniques are used to decrease the Michaelis-Menten constant (KM) of the foregoing ALS enzymes.
• the KM for the substrate pyruvate is lowered to below 10, 9. 8, 7, 6, 5, 4, 2, 1 , or below 1 mM.
• protein engineering techniques are used to increase the specific activity of the foregoing ALS enzyme for the conversion of pyruvate to acetolactate.
• the specific activity is increase by 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000%.
• 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.
• 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 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.
• the parental microorganism acquires new properties, e.g., the ability to produce a new, or greater quantities of, an intracellular and/or extracellular metabolite.
• 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host 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.
• 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.
• 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.
• the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.
• homologs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein.
• two 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.
• 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).
• 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.
• microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of beneficial metabolites from biosynthetic pathways requiring DHAD activity.
• 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.
• the recombinant microorganisms may be derived from bacterial microorganisms.
• 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.
• the recombinant microorganism is a lactic acid bacteria such as, for example, a microorganism derived from the Lactobacillus or Lactococcus genus.
• 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.
• NADH reducing equivalents
• 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).
• 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.
• all three of PDC1, PDC5, and PDC6 are targeted for disruption, deletion, or mutation.
• a positive transcriptional regulator of the PDC1, PDC5, and/or PDC6 is targeted for disruption, deletion or mutation.
• said positive transcriptional regulator is PDC2, or homologs or variants thereof.
• 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+.
• DHAP dihydroxyacetone phosphate
• G3P glycerol-3-phosphate
• 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).
• NADH pyruvate and reducing equivalents
• 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).
• desired metabolite e.g., isobutanol
• 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.
• the 3-KAR is the S. cerevisiae protein YMR226c or a homolog thereof.
• 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.
• the ALDH is the S. cerevisiae protein ALD6 or a homolog thereof.
• genes that encode for enzymes that are homologous to the genes described herein e.g., acetolactate synthase homologs.
• 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.
• Techniques known to those skilled in the art may be suitable to identify additional homologous genes and homologous enzymes.
• 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.
• 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.
• one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity.
• 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.
• the present application provides methods of producing a desired metabolite using a recombinant described herein.
• 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.
• 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.
• the beneficial metabolite is isobutanol.
• a beneficial metabolite e.g., isobutanol
• the recombinant microorganism is cultured in an appropriate culture medium containing a carbon source.
• the method further includes isolating the beneficial metabolite (e.g., isobutanol) from the culture medium.
• a beneficial metabolite e.g., isobutanol
• 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.
• the beneficial metabolite is isobutanol.
• the recombinant microorganism may produce the beneficial metabolite (e.g., isobutanol) from a carbon source at a yield of at least 5 percent theoretical.
• 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.
• the beneficial metabolite is isobutanol. Distillers Dried Grains Comprising Spent Yeast Biocatalvsts
• 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.
• 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).
• DDGS soluble residual material from the fermentation, or syrup
• Use of DDG or DDGS as animal feed is an economical use of the spent biocatalyst following an industrial scale fermentation process.
• 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.
• a beneficial metabolite e.g., isobutanol
• 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.
• ALS acetolactate synthase
• 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.
• 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.
• a yeast biocatalyst e.g., a recombinant yeast microorganism of the present invention
• the method further comprises step (d) of adding soluble residual material from the fermentation process to said DDG to produce DDGS.
• said DDGS comprise at least one additional product selected from the group consisting of unconsumed feedstock solids, nutrients, proteins, fibers, and oils.
• 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).
• 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 FailSafeTM PCR System (EPICENTRE ® Biotechnologies, Madison, Wl; Catalog # FS99250).
• a PCR cocktail containing 15 ⁇ of Master Mix E buffer, 10.5 ⁇ water, 2 ⁇ of each primer at 10 ⁇ concentration, and 0.5 ⁇ polymerase enzyme mix from the kit was added to a 0.2 mL PCR tube for each sample (30 ⁇ 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.
• 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 .
• Transformation of integration plasmids was performed according to the SOP using standard techniques. Integration plasmids were digested with ⁇ rul, 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.
• 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)).
• LB plus 100 ⁇ g/ml ampicillin was used for pGV1730 based plasmids.
• SC-drop out media was used for plasmid selection and growth in S. cerevisiae strains.
• SCD-Trp media is 14 g/L SigmaTM Synthetic Dropout Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), 6.7 g/L DifcoTM 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.
• 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, CuSO 4 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.
• 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.
• ALS assays were performed in triplicate for each lysate, both with and without substrate.
• 100 ⁇ L of lysate (diluted 1:2 with lysis buffer) was mixed with 900 ⁇ L of buffer (50 mM potassium phosphate buffer pH 6.0, 1 mM MgSO 4 , 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 ⁇ L from each reaction was mixed with 25 ⁇ L 35% H2SO4 and incubated at 37°C for 30 min. Acetoin production was quantified by HPLC.
• 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 OD 600 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 OD 600 of 0.1 was obtained.
• Gevo2618 was constructed by transforming Gevo1187 with the integration plasmid pGV2114.
• the plasmid was first linearized with ⁇ , 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 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 ⁇ 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• ALS enzymes 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.
• AHAS FAD-dependent acetohydroxyacid synthase
• the large subunit of AHAS shares significant sequence homology with ALS, despite ALS being FAD-independent.
• 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.
• 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).
• lactis alsS is ⁇ .' 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.
• 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.
• 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.
• SEQ ID NO: 2 P. nodorum sequence
• ALS homologs were 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.
• Example 3 Isobutanol Production in Recombinant Microorganisms with Exemplary ALS Enzvmes
• Example 2 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.
• pGV2082 containing E. coli ilvC (Q110V), L. lactis ilvD, L. lactis kivD, and D. melanogaster ADH
• FIG. 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.
• 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 OD 600 , 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).
• 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.
• 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.
• 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.
• libraries (-1,000 - 10,000 mutants with one to two amino acid mutations on average) are created by error-prone PCR using MnCI 2 (0, 50, 100, and 150 ⁇ ) to induce mutations, Taq DNA polymerase (Roche), approximately 50 ng of plasmid DNA as template, and appropriate primers
• 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.
• 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.
• 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.
• 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.
• ALS assay 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.

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