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WO2013009818A2 - Cétoacide réductoisomérases de haute performance - Google Patents

Cétoacide réductoisomérases de haute performance Download PDF

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Publication number
WO2013009818A2
WO2013009818A2 PCT/US2012/046185 US2012046185W WO2013009818A2 WO 2013009818 A2 WO2013009818 A2 WO 2013009818A2 US 2012046185 W US2012046185 W US 2012046185W WO 2013009818 A2 WO2013009818 A2 WO 2013009818A2
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WIPO (PCT)
Prior art keywords
kari
seq
derived
recombinant microorganism
mutant
Prior art date
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PCT/US2012/046185
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English (en)
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WO2013009818A3 (fr
Inventor
Peter Meinhold
Doug Lies
Stephanie Porter-Scheinman
Christopher Smith
Christopher Snow
Sabine Bastian
Sebastian Schoof
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Gevo, Inc.
California Institute Of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from US13/303,884 external-priority patent/US20120190089A1/en
Application filed by Gevo, Inc., California Institute Of Technology filed Critical Gevo, Inc.
Priority to US14/131,984 priority Critical patent/US20140295513A1/en
Publication of WO2013009818A2 publication Critical patent/WO2013009818A2/fr
Publication of WO2013009818A3 publication Critical patent/WO2013009818A3/fr

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    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01086—Ketol-acid reductoisomerase (1.1.1.86)
    • 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 of the invention and enzymatic preparations therefrom.
  • Isobutanoi also 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 a/., WO/2008/098227 to Liao et a/. , and WO/2009/103533 to Festei et a/.).
  • 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.
  • KARl ketol-acid reductoisomerase
  • KARl enzyme Another important feature of a KARl enzyme is the ability to use NADH as a cofactor for the conversion of acetoiactate to 2,3-dihydroxyisovaierate.
  • NADH NADH-dependent alcohol dehydrogenase
  • isobutanoi can be produced at theoretical yield and/or under anaerobic conditions. See, e.g. , commonly owned and co-pending US Publication No. US 2010/0143997.
  • NADH-dependence is an important feature of a KAR! enzyme, the present inventors have identified several beneficial mutations which can be made to the KARl enzymes identified herein to switch the cofactor specificity of the enzymes from NADPH to NADH.
  • the present inventors have discovered a group of KARI enzymes with high level activity in the isobutanoi pathway.
  • the use of one or more of these KAR! enzymes, or NADH-dependent variants thereof, can improve production of the isobutanoi.
  • the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 80% identical to SEQ !D NO: 2.
  • KARI ketol-acid reductoisomerase
  • the KARI is derived from the genus Shewanella.
  • the KARI is derived from Shewanella sp. strain MR-4.
  • the KARI is encoded by SEQ ID NO: 1 .
  • the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 80% identical to SEQ ID NO: 4.
  • KARI ketol-acid reductoisomerase
  • the KARI is derived from the genus Vibrio.
  • the KARI is derived from Vibrio fischeri.
  • the KARI is encoded by SEQ ID NO: 3.
  • the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 80% identical to SEQ ID NO: 6.
  • KARI ketol-acid reductoisomerase
  • the KARI is derived from the genus Gramella.
  • the KARI is derived from Gramella forsetii.
  • the KARI is encoded by SEQ ID NO: 5.
  • the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 80% identical to SEQ ID NO: 8.
  • KARI ketol-acid reductoisomerase
  • the KARI is derived from the genus Cytophaga.
  • the KARI is derived from Cytophaga hutchinsonii.
  • the KARI is encoded by SEQ ID NO: 7.
  • the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 80% identical to SEQ ID NO: 10.
  • the KARI is derived from a genus selected from Lactococcus and Streptococcus.
  • the KARI is derived from Lactococcus lactis, Streptococcus equinus, or Streptococcus infantahus.
  • the KARI is encoded by SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, or SEQ ID NO: 25.
  • the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 80% identical to SEQ ID NO: 28.
  • the KARI is derived from the genus Methanococcus.
  • the KAR! is derived from Methanococcus maripaiudis, Methanococcus vannielii, or Methanococcus voltae.
  • the KARI is encoded by SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
  • the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 80% identical to SEQ ID NO: 40.
  • the KARI is derived from a genus selected from Zymomonas, Erythrobacter, Sphingomonas, Sphingobium, and Novosphingobium.
  • the KARI is derived from Zymomonas mobilis, Erythrobacter litoralis, Sphingomonas wittichii, Sphingobium japonicum, Sphingobium chlorophenolicum, or Novosphingobium nitrogenifigens,
  • the KARI is encoded by SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , or SEQ ID NO: 53.
  • the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 80% identical to SEQ ID NO: 58.
  • KARI ketol-acid reductoisomerase
  • the KARI is derived from the genus Bacteroides.
  • the KARI is derived from Bacteroides thetaiotaomicron.
  • the KARI is encoded by SEQ ID NO: 55.
  • the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 80% identical to SEQ ID NO: 58.
  • the KARI is derived from the genus Schizosaccharomyces. !n a specific embodiment, the KARI is derived from Schizosaccharomyces pombe or Schizosaccharomyces japonicus. In another specific embodiment, the KARI is encoded by SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61 .
  • the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 99% identical to SEQ ID NO: 84.
  • the KARI is derived from the genus Salmonella.
  • the KARI is derived from Salmonella entehca.
  • the KARI is encoded by SEQ ID NO: 83.
  • the KARI may be modified to be NADH-dependent. Accordingly, the present application further relates to NADH-dependent ketol-acid reductoisomerases (NKRs) derived from a KARI that is at least about 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58.
  • the present application relates to a recombinant microorganism comprising a NKR derived from a KAR! that is at least about 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58.
  • the present application further relates to NADH- dependent ketoi-acid reductoisomerases (NKRs) derived from a KAR! that is at least about 99% identical to SEQ ID NO: 84.
  • NRRs NADH- dependent ketoi-acid reductoisomerases
  • the present application relates to a recombinant microorganism comprising a NKR derived from a KARI that is at least about 99% identical to SEQ ID NO: 84.
  • the present application also relates to mutated ketol-acid reductoisomerase (KAR!) enzymes that utilize NADH rather than NADPH.
  • KAR ketol-acid reductoisomerase
  • KARIs include enzymes having one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the Shewanella sp, KARI (SEQ ID NO: 2); (b) arginine 76 of the Shewanelia sp. KARI (SEQ ID NO: 2); (c) serine 78 of the Shewanella sp. KAR!; and (d) glutamine 1 10 of the Shewanella sp. KARI (SEQ ID NO: 2).
  • the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 71 of the Shewanella sp. KARI (SEQ ID NO: 2). in another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 78 of the Shewane!a sp. KARl (SEQ ID NO: 2). In yet another embodiment, the KARl enzyme contains a modification or mutation at the amino acid corresponding to position 78 of the Shewanella sp. KARl (SEQ ID NO: 2). In yet another embodiment, the KARl enzyme contains a modification or mutation at the amino acid corresponding to position 1 10 of the Shewanella sp. KARl (SEO ID NO: 2).
  • the KARl enzyme contains two or more modifications or mutations at the amino acids corresponding to the positions described above, !n another embodiment, the KARl enzyme contains three or more modifications or mutations at the amino acids corresponding to the positions described above. In yet another embodiment, the KARl enzyme contains four modifications or mutations at the amino acids corresponding to the positions described above. Further included within the scope of the application are KARl enzymes, other than the Shewanella sp. KARl (SEQ ID NO: 2), which contain modifications or mutations corresponding to those set out above. In an exemplary embodiment, the modified KAR! is derived from a KAR! that is at least about 80% identical to SEQ ID NO: 2.
  • Additional mutated ketol-acid reductoisomerase (KARl) enzymes that utilize NADH rather than NADPH include enzymes having one or more modifications or mutations at positions corresponding to amino acids selected from: (a) valine 48 of the L lactis KARl (SEQ ID NO: 10); (b) arginine 49 of the L lactis KARl (SEQ ID NO: 10); (c) lysine 52 of the L lactis KARl (SEQ ID NO: 10); (d) serine 53 of the L. lactis KARl (SEQ ID NO: 10); (e) glutamic acid 59 of the L.
  • KARl ketol-acid reductoisomerase
  • lactis KARl (SEQ ID NO: 10): (f); leucine 85 of the L lactis KARl (SEQ ID NO: 10); (g) isoleucine 89 of the L lactis KARl (SEQ ID NO: 10); (h) lysine 1 18 of the L. lactis KARl (SEQ ID NO: 10); (i) threonine 182 of the L lactis KARl (SEQ ID NO: 10); and (j) glutamic acid 320 of the L. lactis KARl (SEQ ID NO: 10).
  • the KARl enzyme contains a modification or mutation at the amino acid corresponding to position 48 of the L. lactis KARl (SEQ ID NO: 10). In another embodiment, the KARl enzyme contains a modification or mutation at the amino acid corresponding to position 49 of the L lactis KARl (SEQ ID NO: 10). In yet another embodiment, the KARl enzyme contains a modification or mutation at the amino acid corresponding to position 52 of the L lactis KARl (SEQ ID NO: 10). In yet another embodiment, the KARl enzyme contains a modification or mutation at the amino acid corresponding to position 53 of the L. lactis KARl (SEQ ID NO: 10). In yet another embodiment, the KAR!
  • the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 59 of the L. lactis KARI (SEQ ID NO: 10). In yet another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 85 of the L. !actis KARI (SEQ ID NO: 10). In yet another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 89 of the L iactis KARI (SEO ID NO: 10). In yet another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 1 18 of the L lactis KARI (SEQ ID NO: 10).
  • the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 182 of the L lactis KARI (SEQ ID NO: 10). In yet another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 320 of the L iactis KARI (SEQ ID NO: 10). In one embodiment, the KARI enzyme contains two or more modifications or mutations at the amino acids corresponding to the positions described above, in another embodiment, the KARI enzyme contains three or more modifications or mutations at the amino acids corresponding to the positions described above. In yet another embodiment, the KARI enzyme contains four or more modifications or mutations at the amino acids corresponding to the positions described above.
  • the KARI enzyme contains five or more modifications or mutations at the amino acids corresponding to the positions described above. In yet another embodiment, the KARI enzyme contains six or more modifications or mutations at the amino acids corresponding to the positions described above. In yet another embodiment, the KARI enzyme contains seven or more modifications or mutations at the amino acids corresponding to the positions described above. In yet another embodiment, the KARI enzyme contains eight or more modifications or mutations at the amino acids corresponding to the positions described above. In yet another embodiment, the KARI enzyme contains nine or more modifications or mutations at the amino acids corresponding to the positions described above. In yet another embodiment, the KARI enzyme contains ten modifications or mutations at the amino acids corresponding to the positions described above.
  • KARI enzymes other than the L lactis KARI (SEQ ID NO: 10), which contain modifications or mutations corresponding to those set out above.
  • the modified KARI is derived from a KARI that is at least about 80% identical to SEQ ID NO: 10.
  • Additional mutated ketoi-acid reductoisomerase (KAR!) enzymes that utilize NADH rather than NADPH include enzymes having one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the S, enterica KARI (SEQ ID NO: 64); (b) arginine 78 of the S, enterica KARI (SEQ ID NO: 64); (c) serine 78 of the S. enterica KARI (SEQ ID NO: 64); (d) glutamine 1 10 of the S. enterica KARI (SEQ ID NO: 64); (e) aspartic acid 146 of the S. enterica KARI (SEQ ID NO: 64); (f) glycine 185 of the S. enterica KARI (SEQ ID NO: 64); and (g) lysine 433 of the S. enterica KARI (SEQ ID NO: 64),
  • the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 71 of the S, enterica KARI (SEQ ID NO: 64). In another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 76 of the S. enterica KARI (SEQ ID NO: 64), In yet another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 78 of the S. enterica KARI (SEQ ID NO: 64). In yet another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 1 10 of the S. enterica KARI (SEQ ID NO: 64).
  • the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 146 of the S. enterica KARI (SEQ ID NO: 64). In yet another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 185 of the S. enterica KARI (SEQ ID NO: 64). In yet another embodiment, the KARI enzyme contains a modification or mutation at the amino acid corresponding to position 433 of the S. enterica KARI (SEQ ID NO: 64). In one embodiment, the KARI enzyme contains two or more modifications or mutations at the amino acids corresponding to the positions described above. In another embodiment, the KARI enzyme contains three or more modifications or mutations at the amino acids corresponding to the positions described above.
  • the KARI enzyme contains four or more modifications or mutations at the amino acids corresponding to the positions described above. In yet another embodiment, the KARI enzyme contains five or more modifications or mutations at the amino acids corresponding to the positions described above. In yet another embodiment, the KARI enzyme contains six or more modifications or mutations at the amino acids corresponding to the positions described above. In yet another embodiment, the KARI enzyme contains seven modifications or mutations at the amino acids corresponding to the positions described above. Further included within the scope of the application are KARI enzymes, other than the S. entehca KARI (SEQ ID NO: 84), which contain modifications or mutations corresponding to those set out above. In an exemplary embodiment, the modified KARI is derived from a KARI that is at least about 99% identical to SEQ ID NO: 64.
  • the modified or mutated KARI may exhibit an increased catalytic efficiency with NADH as compared to the wild- type KARI.
  • the KAR! has at least about a 5% increased catalytic efficiency with NADH as compared to the wild-type KARI.
  • the KARI has at least about a 25%, at least about a 50%, at least about a 75%, at least about a 100%, at least about a 500%, at least about 1000%, or at least about a 10000% increased catalytic efficiency with NADH as compared to the wild-type KAR!.
  • the modified or mutated KARI may exhibit a decreased Michaelis Menten constant (KM) for NADH as compared to the wild-type KARI.
  • the KARI has at least about a 5% decreased K for NADH as compared to the wild-type KARI.
  • the KARI has at least about a 25%, at least about a 50%, at least about a 75%, at least about a 90%, at least about a 95%, or at least about a 97.5% decreased K for NADH as compared to the wild-type KARI.
  • the modified or mutated KARI may exhibit an increased catalytic constant (k cai ) with NADH as compared to the wild-type KARL
  • the KARI has at least about a 5% increased k ca t with NADH as compared to the wild-type KARI.
  • the KARI has at least about a 25%, at least about a 50%, at least about a 75%, at least about 100%, at least about 200%, or at least about a 500% increased k cat with NADH as compared to the wild-type KARI.
  • the modified or mutated KARI may exhibit an increased Michaelis Menten constant (KM) for NADPH as compared to the wild-type KARI.
  • the KARI has at least about a 5% increased KM for NADPH as compared to the wild-type KARL
  • the KARI has at least about a 25%, at least about a 50%, at least about a 100%, at least about a 500%, at least about a 1000%, or at least about a 5000% increased KM for NADPH as compared to the wild-type KARI.
  • the modified or mutated KARI may exhibit a decreased catalytic constant (k ca i) with NADPH as compared to the wild-type KARI.
  • the KARI has at least about a 5% decreased kcai with NADPH as compared to the wild-type KARL
  • the KARI has at least about a 25%, at least about a 50%, or at least about a 75%, at least about 90% decreased k ca i with NADPH as compared to the wild-type KARI.
  • the catalytic efficiency of the modified or mutated KARI with NADH is increased with respect to the catalytic efficiency with NADPH of the wild-type KARI.
  • the catalytic efficiency of said KARI with NADH is at least about 10% of the catalytic efficiency with NADPH of the wild-type KARI.
  • the catalytic efficiency of said KARI with NADH is at least about 25%, at least about 50%, or at least about 75% of the catalytic efficiency with NADPH of the wild-type KARL
  • the modified or mutated KARI preferentially utilizes NADH rather than NADPH.
  • the application is directed to NADH-dependent KARI enzymes having a catalytic efficiency with NADH that is greater than the catalytic efficiency with NADPH.
  • the catalytic efficiency of the NADH-dependent KARI is at least about 2-fold greater with NADH than with NADPH.
  • the catalytic efficiency of the NADH-dependent KARI is at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or at least about 500-fold greater with NADH than with NADPH.
  • the application is directed to modified or mutated KARI enzymes that demonstrate a switch in cofactor specificity from NADPH to NADH.
  • the modified or mutated KARI has at least about a 2:1 ratio of catalytic efficiency (k ca t K ) with NADH over k ca t with NADPH.
  • the modified or mutated KARI has at least about a 10:1 ratio of catalytic efficiency (k ca t/K M ) with NADH over catalytic efficiency (k cat /K M ) with NADPH.
  • the KARI exhibits at least about a 1 :10 ratio of K for NADH over K M for NADPH.
  • the application is directed to modified or mutated KARI enzymes that have been codon optimized for expression in certain desirable host organisms, such as yeast and E. coli.
  • the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a KARI enzyme.
  • the nucleic acid molecule encodes a KARI that is at least about 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ !D NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58.
  • the nucleic acid molecule encodes a KARI this is at least about 99% identical to SEQ ID NO: 64.
  • the nucleic acid molecule encodes an NADH-dependent ketoi-acid reductoisomerase (NKR) derived from a KARI that is at least about 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58.
  • KRR NADH-dependent ketoi-acid reductoisomerase
  • the nucleic acid molecule encodes an NADH-dependent ketoi-acid reductoisomerase (NKR) derived from a KARI that is at least about 80% identical to SEQ ID NO: 2, wherein said NKR has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the Shewanella sp. KARI (SEQ ID NO: 2); (b) arginine 78 of the Shewane!la sp. KARI (SEQ ID NO: 2); (c) serine 78 of the Shewanella sp. KARI; and (d) glutamine 1 10 of the Shewanella sp. KARI (SEQ ID NO: 2).
  • NRR NADH- dependent ketoi-acid reductoisomerase
  • the nucleic acid molecule encodes an NADH-dependent ketoi-acid reductoisomerase (NKR) derived from a KARI that is at least about 80% identical to SEQ ID NO: 10, wherein said NKR has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) valine 48 of the L. lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI (SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO: 10); (d) serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e) glutamic acid 59 of the L.
  • NRR NADH-dependent ketoi-acid reductoisomerase
  • lactis KARI (SEQ ID NO: 10): (f); leucine 85 of the L. lactis KARI (SEQ ID NO: 10); (g) isoleucine 89 of the L lactis KARI (SEQ ID NO: 10); (h) lysine 1 18 of the L lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L lactis KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L. lactis KARI (SEQ ID NO: 10).
  • the nucleic acid molecule encodes an NADH-dependent ketoi-acid reductoisomerase (NKR) derived from a KARI that is at least about 99% identical to SEQ ID NO: 84.
  • the nucleic acid molecule encodes an NADH-dependent ketoi-acid reductoisomerase (NKR) derived from a KARI that is at least about 99% identical to SEQ ID NO: 64, wherein said NKR has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the S. enferica KARI (SEQ ID NO: 84); (b) arginine 78 of the S. enterica KAR!
  • SEQ ID NO: 64 (SEQ ID NO: 64); (c) serine 78 of the S. enterica KARI (SEQ ID NO: 84); (d) giutamine 1 10 of the S. enterica KARI (SEQ ID NO: 84); (e) aspartic acid 148 of the S. enterica KARI (SEQ ID NO: 64); (f) glycine 185 of the S. enterica KARI (SEQ ID NO: 84); and (g) lysine 433 of the S, enterica KARI (SEQ ID NO: 64),
  • the recombinant microorganism comprises an isobutanoi producing metabolic pathway.
  • the isobutanoi producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanoi.
  • the isobutanoi producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi.
  • the isobutanoi producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi.
  • the isobutanoi producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi. In yet another embodiment, the isobutanoi producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi. In yet another embodiment, ail of the isobutanoi producing metabolic pathway steps in the conversion of pyruvate to isobutanoi are converted by exogenousiy encoded enzymes.
  • At least one of the exogenousiy encoded enzymes is a KARI that is at least about 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58.
  • at least one of the exogenousiy encoded enzymes is a KARI that is at least about 99% identical to SEQ ID NO: 64.
  • at least one of the exogenousiy encoded enzymes is a KARI enzyme has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the Shewane!la sp.
  • At least one of the exogenously encoded enzymes is a KARl enzyme has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) valine 48 of the L Iactis KARl (SEQ ID NO: 10); (b) arginine 49 of the L.
  • iactis KARl (SEQ ID NO: 10); (c) lysine 52 of the L. iactis KARl (SEQ ID NO: 10); (d) serine 53 of the L. Iactis KARl (SEQ ID NO: 10); (e) glutamic acid 59 of the L. iactis KARl (SEQ ID NO: 10): (f); leucine 85 of the L iactis KARl (SEQ ID NO: 10); (g) isoleucine 89 of the L.
  • At least one of the exogenously encoded enzymes is a KARl enzyme has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the S. enterica KARl (SEQ ID NO: 64); (b) arginine 76 of the S.
  • enterica KARl (SEQ ID NO: 64); (c) serine 78 of the S. enterica KARl (SEQ ID NO: 84); (d) g!utamine 1 10 of the S. enterica KARl (SEQ ID NO: 64); (e) aspartic acid 146 of the S. enterica KARl (SEQ ID NO: 64); (f) glycine 185 of the S, enterica KARl (SEQ ID NO: 64); and (g) lysine 433 of the S. enterica KARl (SEQ ID NO: 84).
  • 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 isobutano! 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), ke oi-acid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2- keto-acid decarboxylase, e.g., keto-isova!erate 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 KARI is at least about 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ !D NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58. In another exemplary embodiment, the KARI is at least about 99% identical to SEQ ID NO: 64. In yet another exemplary embodiment, the KARI comprises one or more modifications or mutations at positions corresponding to amino acids selected from:
  • the KARI comprises one or more modifications or mutations at positions corresponding to amino acids selected from: (a) valine 48 of the L lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L lactis KARI (SEQ ID NO: 10); (c) lysine 52 of the L lactis KARI (SEQ ID NO: 10); (d) serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e) glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10): (f); leucine 85 of the L lactis KARI (SEQ ID NO: 10); (g) isoleucine 89 of the L.
  • lactis KARI (SEQ ID NO: 10); (h) lysine 1 18 of the L. lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L lactis KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L lactis KARI (SEQ ID NO: 10).
  • the KARI comprises one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the 8. enterica KARI (SEQ ID NO: 64);
  • 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 recombinant microorganisms may be recombinant yeast microorganisms.
  • the recombinant yeast microorganisms may be members of the Saccharomyces ciade, 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 kluyveri, Kluyveromyces iactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Hansenula anomala, Candida utilis and Kluyveromyces waitii.
  • 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 castelti, Kluyveromyces thermotolerans, Candida giabrata, Z, bailli, Z, rouxii, Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces
  • 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 casteili, and Candida glabrata.
  • the recombinant microorganisms may be pre- VGD (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, Yar wia and Schizosaccharomyces.
  • the pre-WGD yeast is selected from the group consisting of Saccharomyces kluyveri, Kluyveromyces thermotolerans, Kluyveromyces marxianus, Kluyveromyces waltii, Kluyveromyces lactis, Candida tropicalis, Pichia pastoris, Pichia anomala, Pichia stipitis, issatchenkia orientaiis, issatchenkia occidentalis, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yarrowia lipoiytica, 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 invention provides methods of producing isobutanoi using a recombinant microorganism as described herein.
  • the method includes cultivating the recombinant microorganism in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of isobutanoi is produced and optionally, recovering the isobutanoi.
  • the microorganism produces isobutanoi from a carbon source at a yield of at least about 5 percent theoretical.
  • the microorganism produces isobutanoi at a yield of at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 80 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5 percent theoretical.
  • 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 exemplary embodiment of an isobutanol pathway.
  • Figure 2 illustrates an exemplary embodiment of an NADH-dependent isobutanol pathway.
  • 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 ceils and “microbes” are used interchangeably with the term microorganism.
  • prokaryotes is art recognized and refers to ceils 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 168 ribosomai 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 ribosomai 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 haiophiles (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 haiophiles 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 hyperthermophiiic sulfur-dependent prokaryotes and the Euryarchaeota contain the methanogens and extreme haiophiles.
  • 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, Fiavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non
  • Gram-negative bacteria include cocci, nonenteric rods, and enteric rods.
  • the genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasieurella, Brucella, Yersinia, Franciselia, Haemophilus, Bordetelia, 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, nonsporuiating rods, and sporuiating 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., Li!burn, T.G., Cole, J.R., Harrison, S.H., Euzeby, J., and Tinda!l, 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 ribosomai RNA sequence homology and greater than 70% genomic hybridization and sufficiently different from ail other organisms so as to be recognized as a distinct unit.
  • 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 ceil” 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 ceil, 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 a/., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
  • Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that recognize and bind the protein. See Sambrook et ai, 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 ceils as compared to similar corresponding unmodified ceils 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- foid, 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 ceil (e.g. reduced expression).
  • reduced activity of a protein in a cell may result from decreased concentrations of the protein in the ceil.
  • wild-type microorganism describes a cell that occurs in nature, i.e. a ceil 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.
  • mutant indicates any modification of a nucleic acid and/or polypeptide which results in an altered nucleic acid or polypeptide. Mutations include, for example, point mutations, deletions, or insertions of single or multiple residues in a polynucleotide, which includes alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences.
  • a genetic alteration may be a mutation of any type. For instance, the mutation may constitute a point mutation, a frame-shift mutation, a nonsense mutation, an insertion, or a deletion of part or ail of a gene.
  • 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 cataboiic 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 molecu!e(s) is/are naturally found in (e.g., is "endogenous to") a given host microorganism or host ceil but is either produced in an unnatural location or in an unnatural amount in the cell; and/or (c) the molecu!e(s) differ(s) in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence(s) such that the molecule differing in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid as found endogenously is produced in an unnatural (e.
  • 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 ceils. 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 isobutanoi 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 biofuei 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 ethanoi.
  • Fermentative pathways are usually active under anaerobic conditions but may also occur under aerobic conditions, under conditions where NADH is not fully oxidized via the respiratory chain. For example, above certain glucose concentrations, Crabtree positive yeasts produce large amounts of 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, biofuei precursor, higher alcohol, or higher alcohol 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 CO2 from glucose.
  • Non-fermentative yeast can be identified by the "Durham Tube Test” (J.A. Barnett, R.W. Payne, and D. Yarrow. 2000. Yeasts Characteristics and Identification. 3 rd edition, p. 28-29. Cambridge University Press, Cambridge, UK) or by monitoring the production of fermentation productions such as ethanol and CO2.
  • 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.
  • 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 poiy-lysine -conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-corijugated 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 agrobaeterium or a bacterium.
  • Transformation refers to the process by which a vector is introduced into a host ceil. Transformation (or transduction, or transfection), can be achieved by any one of a number of means including chemical transformation (e.g. lithium acetate transformation), eiectroporation, microinjection, bioiistics (or particle bombardment- mediated delivery), or agrobaeterium 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, homoiogs will have functional, structural or genomic similarities. Techniques are known by which homoiogs of a polynucleotide or polypeptide can readily be cloned using genetic probes and PGR. 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 or “analogous” refers to polynucleotide or polypeptide sequences that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure. jsobutanol Producing Recombinant Microorganisms
  • 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 biofuei candidate (See, e.g., commonly owned and co-pending patent publications, US 2009/0228991 , US 2010/0143997, US 201 1/0020889, US 201 1/0076733, and WO 2010/075504).
  • the present invention relates to recombinant microorganisms for producing isobutanol, wherein said recombinant microorganisms comprise an isobutanol producing metabolic pathway, !n one embodiment, 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-isova!erate 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 isobutanoi producing metaboiic pathway with at least three isobutanoi pathway enzymes localized in the cytosol.
  • the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least four isobutanoi pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with five isobutanoi pathway enzymes localized in the cytosol. In yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with all isobutanoi pathway enzymes localized in the cytosol. Isobutanoi producing metabolic pathways in which one or more genes are localized to the cytosol are described in commonly owned and copending U.S. Application Serial No. 12/855,278, which is herein incorporated by reference in its entirety for all purposes.
  • isobutanoi pathway enzymes including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including V. 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., Cryptobacte um spp., and Eggerthelia 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.
  • acetolactate synthases capable of converting pyruvate to acetoiactate may be derived from a variety of sources ⁇ e.g., bacterial, yeast, Archaea, etc.), including B. subtilis (GenBank Accession No. Q04789.3), L iaciis (GenBank Accession No. NP__267340.1 ), S. mutans (GenBank Accession No. NP__721805.1 ), K. pneumoniae (GenBank Accession No. ZP_06014957.1 ), C. giutamicum (GenBank Accession No. P42463.1 ), E, cloacae (GenBank Accession No.
  • YP_00361361 1 .1 M. maripa!udis (GenBank Accession No. ABX01060.1 ), M. grisea (GenBank Accession No. AAB81248.1 ), T. stipitatus (GenBank Accession No. XPJ302485976.1 ), or S. cerevisiae !LV2 (GenBank Accession No. NPJ313828.1 ).
  • Additional acetoiactate synthases capable of converting pyruvate to acetolactate are described in commonly owned and co-pending US Publication No. 201 1/0078733, which is herein incorporated by reference in its entirety.
  • Chipman et ai provide an alignment and consensus for the sequences of a representative number of acetoiactate synthases. Motifs shared in common between the majority of acetoiactate synthases include:
  • a protein harboring one or more of these amino acid motifs can generally be expected to exhibit acetoiactate synthase activity.
  • Dihydroxy acid dehydratases capable of converting 2,3- dihydroxyisovalerate to a-ketoisovaierate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including £. coii (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. YPJ3040S3736.1 ), Eubacterium SCB49 (GenBank Accession No.
  • ZP__01890126,1 G. forsetti (GenBank Accession No. YP__862145.1 ), Y. lipolytica (GenBank Accession No. XP_ 502180.2), N. crassa (GenBank Accession No. XP ... 963045.1 ), or S. cerevisiae ILV3 (GenBank Accession No. NP__012550.1 ).
  • Additional dihydroxy acid dehydratases capable of 2,3-dihydroxyisovaierate to a-ketoisovalerate are described in commonly owned and co-pending US Publication No. 201 1/0076733. Motifs shared in common between the majority of dihydroxy acid dehydratases include: SLXSRXX!A (SEQ ID NO: 69),
  • CDKXXPG (SEG ID NO: 70),
  • GGSTN SEG ID NO: 72
  • 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 a-ketoisovaierate to isobutyraldehyde may be derived from a variety of sources ⁇ e.g., bacterial, yeast, Archaea, etc.), including L. iactis kivD (GenBank Accession No. YPJ30335382Q.1 ), E. cloacae (GenBank Accession No. P23234.1 ), M. smegmatis (GenBank Accession No. A0R480.1 ), M. tuberculosis (GenBank Accession No. 053885.1 ), M. avium (GenBank Accession No. Q742Q2.1 , A.
  • brasilense (GenBank Accession No. P51852.1 ), L. lactis kdcA (GenBank Accession No. AAS49186.1 ), S. epidermidis (GenBank Accession No. NP_765765.1 ), M. caseolyticus (GenBank Accession No. YP 002560734.1 ), B. megatehum (GenBank Accession No. YP_ 003561644.1 ), S. cerevisiae ARO10 (GenBank Accession No. NP__010868.1 ), 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. 201 1 /0078733. 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: 80) motifs at amino acid positions corresponding to the 21 -27, 70-78, 81 -89, 93-98, and 428-435 residues, respectively, of the L iactis 2-keto-acid decarboxylase encoded by ivD,
  • 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. YPJ301035842.1 ), L brevis (GenBank Accession No. YP 794451 .1 ), B, thuringiensis (GenBank Accession No.
  • 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-isovaierate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to 2,3-dihydroxyisovalerate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to 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,
  • the present application provides several KARl enzymes that give high performance when expressed in yeast in the context of isobutanoi production. Accordingly, this application describes methods of increasing isobutanoi production through the use of recombinant microorganisms comprising KARl enzymes with improved properties for the production of isobutanoi.
  • KARl ketoi-acid reductoisomerase
  • KARIs which are at least about 80%
  • SEQ ID NO: 2 SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO;
  • the KARl is derived from the genus Shewanella. In a specific embodiment, the KARl is derived from Shewanella sp. strain MR-4. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 1 . In another embodiment, the KARl is derived from the genus Vibrio. In a specific embodiment, the KARl is derived from Vibrio fischeri. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 3. In yet another embodiment, the KARl is derived from the genus Grameila. In a specific embodiment, the KARl is derived from Grameila forsetii.
  • the isolated nucleic acid molecule is comprised of SEQ ID NO: 5.
  • the KARl is derived from the genus Cytophaga.
  • the KARl is derived from Cytophaga hutchinsonii.
  • the isolated nucleic acid molecule is comprised of SEQ ID NO: 7.
  • the KARl is derived from a genus selected from Lactococcus and Streptococcus.
  • the KARI is derived from Lactococcus lactis, Streptococcus equinus, or Streptococcus infantarius.
  • the KARI is encoded by SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEO ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, or SEQ ID NO: 25.
  • the KARI is derived from the genus Methanococcus.
  • the KARI is derived from Methanococcus maripaludis, Methanococcus vannielii, or Methanococcus lakeae.
  • the KARI is encoded by SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
  • the KARI is derived from a genus selected from Zymomonas, Erythrobacter, Sphingomonas, Sphmgobium, and Novosphingobium.
  • the KARI is derived from Zymomonas mobiiis, Erythrobacter litoralis, Sphingomonas wittichii, Sphmgobium japonicum, Sphmgobium chlorophenolicum, or Novosphingobium niirogenifigens.
  • the KARI is encoded by SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , or SEQ ID NO: 53.
  • the KARI is derived from the genus Bacteroides.
  • the KARI is derived from Bacteroides thetaiotaomicron.
  • the KARI is encoded by SEQ ID NO: 55.
  • the KARI is derived from the genus Schizosaccharomyces.
  • the KARI is derived from Schizosaccharomyces pombe or Schizosaccharomyces japonicus. In another specific embodiment, the KARI is encoded by SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61 .
  • KARI enzymes that have been modified to be NADH-dependent. Accordingly, the present application further relates to NADH-dependent ketol-acid reductoisomerases (NKRs) derived from a KARI that is at least about 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58.
  • NRRs NADH-dependent ketol-acid reductoisomerases
  • KARI ketol-acid reductoisomerase
  • SEQ ID NO: 64 an isolated nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 99% identical to SEQ ID NO: 64.
  • the KARI is derived from the genus Salmonella.
  • the KARI is derived from Salmonella enterica.
  • the KARI is encoded by SEQ ID NO: 63.
  • the present application further relates to NADH-dependent ketol- acid reductoisomerases (NKRs) derived from a KARI that is at least about 99% identical to SEQ ID NO: 64.
  • the invention also includes fragments of the disclosed KARI enzymes 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 KARI enzymes. 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 KARI enzyme(s) of interest using any of a number of well-known proteolytic enzymes.
  • the invention further includes nucleic acid molecules which encode the above described KARI enzymes and KARI enzyme fragments.
  • KARI ketol-acid reductoisomerase
  • KARI ketol-acid reductoisomerase
  • the KARI is derived from the genus Shewanella. In a specific embodiment, the KARI is derived from Shewanella sp. strain MR-4. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 1 . In another embodiment, the KARI is derived from the genus Vibrio. In a specific embodiment, the KARI is derived from Vibrio fischeri. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 3. In yet another embodiment, the KARI is derived from the genus Grameiia. In a specific embodiment, the KARI is derived from Grameiia forsetii.
  • the isolated nucleic acid molecule is comprised of SEQ ID NO: 5.
  • the KARI is derived from the genus Cytophaga.
  • the KARI is derived from Cytophaga hutchinsonii.
  • the isolated nucleic acid molecule is comprised of SEQ ID NO: 7.
  • the KARI is derived from a genus selected from Lactococcus and Streptococcus.
  • the KARI is derived from Lactococcus lactis, Streptococcus equinus, or Streptococcus infantarius.
  • the KARI is encoded by SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEO ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, or SEQ ID NO: 25.
  • the KARI is derived from the genus Methanococcus.
  • the KARI is derived from Methanococcus maripaludis, Methanococcus vannielii, or Methanococcus lakeae.
  • the KARI is encoded by SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
  • the KARI is derived from a genus selected from Zymomonas, Erythrobacter, Sphingomonas, Sphmgobium, and Novosphingobium.
  • the KARI is derived from Zymomonas mobiiis, Erythrobacter litoralis, Sphingomonas wittichii, Sphmgobium japonicum, Sphmgobium chlorophenolicum, or Novosphingobium niirogenifigens.
  • the KARI is encoded by SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , or SEQ ID NO: 53.
  • the KARI is derived from the genus Bacteroides.
  • the KARI is derived from Bacteroides thetaiotaomicron.
  • the KARI is encoded by SEQ ID NO: 55.
  • the KARI is derived from the genus Schizosaccharomyces.
  • the KARI is derived from Schizosaccharomyces pombe or Schizosaccharomyces japonicus. In another specific embodiment, the KARI is encoded by SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61 .
  • KARI ketol-acid reductoisomerase
  • SEQ ID NO: 64 a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 99% identical to SEQ ID NO: 64.
  • the KARI is derived from the genus Salmonella.
  • the KARI is derived from Saimonella enterica,
  • the KARI is encoded by SEQ ID NO: 63.
  • pathway steps 2 and 5 of the isobutanoi pathway may be carried out by KARI and ADH enzymes that utilize NADH (rather than NADPH) as a cofactor. It has been found previously that utilization of NADH-dependent KARI (NKR) and ADH enzymes to catalyze pathway steps 2 and 5, respectively, surprisingly enables production of isobutanoi at theoretical yield and/or under anaerobic conditions. See, e.g., commonly owned and co-pending patent publication US 2010/0143997. An example of an NADH-dependent isobutanoi pathway is illustrated in Figure 2.
  • the recombinant microorganisms of the present invention may use an NKR to catalyze the conversion of acetoiactate to produce 2,3-dihydroxyisovalerate.
  • the recombinant microorganisms of the present invention may use an NADH-dependent ADH to catalyze the conversion of isobufyraidehyde to produce isobutanoi.
  • the recombinant microorganisms of the present invention may use both an NKR to catalyze the conversion of acetoiactate to produce 2,3- dihydroxyisovalerate, and an NADH-dependent ADH to catalyze the conversion of isobufyraidehyde to produce isobutanoi.
  • the NKR is derived from a KARI that is at least about 80% identical to SEQ ID NO: 2.
  • the NKR is a KARI enzyme that has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the Shewanella sp. KARI (SEQ ID NO: 2); (b) arginine 76 of the Shewanella sp. KARI (SEQ ID NO: 2); (c) serine 78 of the Shewanella sp. KARI; and (d) glutamine 1 10 of the Shewanella sp. KARI (SEQ ID NO: 2).
  • the application is directed to KARI enzymes wherein the alanine corresponding to position 71 of the Shewanella sp. KARI (SEQ ID NO: 2) is replaced with an amino acid selected from serine, threonine, asparagine, or glutamine.
  • the application is directed to KARI enzymes wherein the arginine corresponding to position 78 of the Shewanella sp. KARI (SEQ ID NO: 2) is replaced with aspartic acid or glutamic acid.
  • the application is directed to KARI enzymes wherein the serine corresponding to position 78 of the Shewanella sp. KARI (SEQ ID NO: 2) is replaced with aspartic acid or glutamic acid.
  • the application is directed to KARI enzymes wherein the glutamine corresponding to position 1 10 of the Shewanella sp. KARI (SEQ ID NO: 2) is replaced with valine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, or tyrosine.
  • the application relates to a KARI enzyme having four modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the Shewanella sp. KARI (SEQ ID NO: 2); (b) arginine 76 of the Shewanella sp. KARI (SEQ ID NO: 2); (c) serine 78 of the Shewanella sp. KARI; and (d) glutamine 1 10 of the Shewanella sp, KARI (SEQ ID NO: 2).
  • the NKR is derived from a KARI that is at least about 80% identical to SEQ ID NO: 10.
  • the NKR is a KARI enzyme that has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) valine 48 of the L. lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI (SEQ ID NO: 10); (c) lysine 52 of the L.
  • lactis KARI (SEQ ID NO: 10); (d) serine 53 of the L lactis KARI (SEQ ID NO: 10); (e) glutamic acid 59 of the L lactis KARI (SEQ ID NO: 10): (f); leucine 85 of the L. lactis KARI (SEQ ID NO: 10); (g) isoleucine 89 of the L lactis KARI (SEQ ID NO: 10); (h) lysine 1 18 of the L lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L lactis KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L lactis KARI (SEQ ID NO: 10).
  • the application is directed to KARI enzymes wherein the valine corresponding to position 48 of the L lactis KARI (SEQ ID NO: 2) is replaced with leucine or proline.
  • the application is directed to KARI enzymes wherein the arginine corresponding to position 49 of the L, lactis KAR! (SEQ ID NO: 2) is replaced with valine, leucine, serine, or proline.
  • the application is directed to KARI enzymes wherein the lysine corresponding to position 52 of the L lactis KARI (SEQ ID NO: 2) is replaced with leucine, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, valine, aspartic acid, or glutamic acid.
  • the application is directed to KARI enzymes wherein the serine corresponding to position 53 of the L lactis KARI (SEQ ID NO: 2) is replaced with aspartic acid or glutamic acid.
  • the application is directed to KARI enzymes wherein the glutamic acid corresponding to position 59 of the L lactis KARI (SEQ !D NO: 2) is replaced with lysine, arginine, or histidine. !n yet another specific embodiment, the application is directed to KARI enzymes wherein the leucine corresponding to position 85 of the L lactis KARI (SEQ ID NO: 2) is replaced with threonine or alanine. In yet another specific embodiment, the application is directed to KARI enzymes wherein the isoleucine corresponding to position 89 of the L lactis KARI (SEQ ID NO: 2) is replaced with alanine.
  • the application is directed to KARI enzymes wherein the lysine corresponding to position 1 18 of the L !actis KARI (SEQ !D NO: 2) is replaced with glutamic acid or aspartic acid.
  • the application is directed to KARI enzymes wherein the threonine corresponding to position 182 of the L, lactis KARI (SEQ ID NO: 2) is replaced with serine, asparagine, or giutarnine.
  • the application is directed to KARI enzymes wherein the glutamic acid corresponding to position 320 of the L. lactis KARI (SEQ ID NO: 2) is replaced with lysine, arginine, or histidine.
  • the application relates to a KAR! enzyme having seven modifications or mutations at positions corresponding to amino acids selected from: (a) valine 48 of the L. lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI (SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO: 10); (d) serine 53 of the L lactis KARI (SEQ ID NO: 10); (e) glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10): (f) threonine 182 of the L. lactis KARI (SEQ ID NO: 10); and (g) glutamic acid 320 of the L. lactis KARI (SEQ ID NO: 10).
  • the NKR is derived from a KARI that is at least about 99% identical to SEQ ID NO: 64.
  • the NKR is a KARI enzyme that has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the S, enterica KARI (SEQ ID NO: 64); (b) arginine 76 of the S. enterica KAR! (SEQ ID NO: 84); (c) serine 78 of the S. enterica KARI (SEQ ID NO: 64); (d) giutarnine 1 10 of the S, enterica KARI (SEQ !D NO: 64); (e) aspartic acid 146 of the S. enterica KARI (SEQ ID NO: 64); (f) glycine 185 of the S. enterica KARI (SEQ ID NO: 64); and (g) lysine 433 of the S. enterica KARI (SEQ ID NO: 64).
  • the application is directed to KARI enzymes wherein the alanine corresponding to position 71 of the S. enterica KARI (SEQ ID NO: 64) is replaced with an amino acid selected from serine, threonine, asparagine, or giutarnine.
  • the application is directed to KARI enzymes wherein the arginine corresponding to position 76 of the S. enterica KARI (SEQ ID NO: 64) is replaced with aspartic acid or glutamic acid.
  • the application is directed to KARI enzymes wherein the serine corresponding to position 78 of the S. enterica KARI (SEQ ID NO: 64) is replaced with aspartic acid or glutamic acid.
  • the application is directed to KARI enzymes wherein the giutarnine corresponding to position 1 10 of the S. enterica KARI (SEQ ID NO: 84) is replaced with valine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, or tyrosine, !n yet another specific embodiment, the application is directed to KARl enzymes wherein the aspartic acid corresponding to position 146 of the S. enterica KARl (SEQ ID NO: 84) is replaced with glycine, cysteine, or proline. In yet another specific embodiment, the application is directed to KARl enzymes wherein the glycine corresponding to position 185 of the S.
  • enterica KARl (SEQ ID NO: 84) is replaced with arginine, histidine, or lysine.
  • the application is directed to KARl enzymes wherein the lysine corresponding to position 433 of the S. enterica KARl (SEQ ID NO: 64) is replaced with glutamic acid or aspartic acid.
  • the application relates to a KARl enzyme having seven modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the S. enterica KARl (SEQ ID NO: 64); (b) arginine 78 of the S. enterica KARl (SEQ ID NO: 84); (c) serine 78 of the S. enterica KARl (SEQ ID NO: 64); (d) giutamine 1 10 of the S. enterica KARl (SEQ ID NO: 64); (e) aspartic acid 148 of the S. enterica KARl (SEQ ID NO: 84); (f) glycine 185 of the S. enterica KARl (SEQ ID NO: 64); and (g) lysine 433 of the S. enterica KARl (SEQ ID NO: 64).
  • KARl enzymes other than the Shewanella sp. KARl (SEQ ID NO: 2), the L lactis KARl (SEQ ID NO: 10), or the 8. enterica KARl (SEQ ID NO: 64) which contain modifications or mutations corresponding to those set out above.
  • SEQ ID NO: 2 the Shewanella sp. KARl (SEQ ID NO: 2), the L lactis KARl (SEQ ID NO: 10), or the 8. enterica KARl (SEQ ID NO: 64) which contain modifications or mutations corresponding to those set out above.
  • the nucleotide sequences for several KARl enzymes are known.
  • a representative listing of KARl enzymes capable of being modified are disclosed in commonly owned and co-pending US Publication No. US 2010/0143997.
  • KARl enzyme identified herein e.g., the Shewanella sp, KARl, the L lactis KARl, or the S. enterica KARl
  • KARl enzymes identified herein may be readily identified for other KARl enzymes by one of skill in the art.
  • one with skill in the art can make one or a number of modifications which would result in an increased ability to utilize NADH, particularly for the conversion of acetolactate to 2,3- dihydroxyisovaierate, in any KARl enzyme of interest.
  • Residues to be modified in accordance with the present application may include those described in Examples 4- 5.
  • the application also includes fragments of the modified KARI enzymes 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 KARI enzymes.
  • 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 KARI enzyme(s) of interest using any of a number of well-known proteolytic enzymes.
  • the invention further includes nucleic acid molecules which encode the above described mutant KAR! enzymes and KAR! enzyme fragments.
  • KARI ketol-acid reductoisomerase
  • Yet another aspect of the application relates to a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) valine 48 of the L. lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI (SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARI (SEQ ID NO: 10); (d) serine 53 of the L. lactis KARI (SEQ ID NO: 10); (e) glutamic acid 59 of the L.
  • KARI ketol-acid reductoisomerase
  • lactis KARI (SEQ ID NO: 10): (f); leucine 85 of the L. lactis KARI (SEQ ID NO: 10); (g) isoleucine 89 of the L. lactis KARI (SEQ ID NO: 10); (h) lysine 1 18 of the L. lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L lactis KAR! (SEQ ID NO: 10); and (j) glutamic acid 320 of the L. lactis KARI (SEQ ID NO: 10). Further included within the scope of the application are recombinant microorganisms comprising a KARI enzyme, other than the L, lactis KARI (SEQ ID NO: 10), which contains modifications or mutations at positions corresponding to those set out above.
  • KARI ketol-acid reductoisomerase
  • SEQ ID NO: 64 a recombinant microorganism comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the S. enterica KAR! (SEQ ID NO: 64); (b) arginine 76 of the S. enterica KARI (SEQ ID NO: 64); (c) serine 78 of the S. enterica KARI (SEQ ID NO: 64); (d) glutamine 1 10 of the S. enterica KARI (SEQ ID NO: 64); (e) aspartic acid 146 of the S.
  • KARI ketol-acid reductoisomerase
  • enterica KARI SEQ ID NO: 64
  • glycine 185 of the S. enterica KARI SEQ ID NO: 64
  • glysine 433 of the S. enterica KARI SEQ ID NO: 64
  • recombinant microorganisms comprising a KARI enzyme, other than the S. enterica KARI (SEQ ID NO: 64), which contains modifications or mutations at positions corresponding to those set out above,
  • KARI ketol-acid reductoisomerase
  • KARI ketol-acid reductoisomerase
  • said KARI is at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a KARI having one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the Shewanella sp. KARI (SEQ ID NO: 2); (b) arginine 76 of the Shewanella sp. KARI (SEQ ID NO: 2); (c) serine 78 of the Shewanella sp.
  • KARI ketol-acid reductoisomerase
  • KARI ketol-acid reductoisomerase
  • KARI ketol-acid reductoisomerase
  • lactis KARI (SEQ ID NO: 10); (d) serine 53 of the L lactis KARI (SEQ ID NO: 10); (e) glutamic acid 59 of the L. lactis KARI (SEQ ID NO: 10): (f); leucine 85 of the L lactis KARI (SEQ ID NO: 10); (g) isoleucine 89 of the L lactis KARI (SEQ ID NO: 10); (h) lysine 1 18 of the L. lactis KARI (SEQ ID NO: 10); (i) threonine 182 of the L !actis KARI (SEQ ID NO: 10); and (j) glutamic acid 320 of the L, iactis KARI (SEQ ID NO: 10).
  • recombinant microorganisms comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI), wherein said KARI is at least about 99% identical to SEQ ID NO: 64
  • recombinant microorganisms comprising at least one nucleic acid molecule encoding a ketol-acid reductoisomerase (KARI)
  • KARI ketol-acid reductoisomerase
  • said KARI is at least about 99% identical to a KARI having one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the S, enterica KARI (SEQ ID NO: 84); (b) arginine 78 of the S.
  • enterica KARI SEQ ID NO: 64
  • serine 78 of the S. enterica KARI SEQ ID NO: 64
  • glutamine 1 10 of the S. enterica KARI SEQ ID NO: 64
  • glutamine 1 10 of the S. enterica KARI SEQ ID NO: 64
  • aspartic acid 146 of the S. enterica KARI SEQ ID NO: 84
  • glycine 185 of the S. enterica KARI SEQ ID NO: 64
  • lysine 433 of the S. enterica KARI SEQ ID NO: 64.
  • any number of mutations can be made to the KARI enzymes, and in a preferred aspect, multiple mutations can be made 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 the KARI enzymes of the present application to create NKRs using any methodology known to those skilled in the art. Mutations may be introduced randomly by, for example, conducting a PGR 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 ail 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 PGR.
  • the NADH-dependent activity of the modified or mutated KARI enzyme is increased.
  • the catalytic efficiency of the modified or mutated KARI enzyme is improved for the cofactor NADH.
  • the catalytic efficiency of the modified or mutated KARI enzyme is improved by at least about 5% as compared to the wild-type or parental KARI for NADH. More preferably the catalytic efficiency of the modified or mutated KAR! enzyme is improved by at least about 15% as compared to the wild-type or parental KARI for NADH. More preferably, the catalytic efficiency of the modified or mutated KARI enzyme is improved by at least about 25% as compared to the wild-type or parental KARI for NADH.
  • the catalytic efficiency of the modified or mutated KARI enzyme is improved by at least about 50% as compared to the wild-type or parental KARI for NADH. More preferably, the catalytic efficiency of the modified or mutated KARI enzyme is improved by at least about 75% as compared to the wild-type or parental KARI for NADH. More preferably, the catalytic efficiency of the modified or mutated KARI enzyme is improved by at least about 100% as compared to the wild- type or parental KARI for NADH. More preferably, the catalytic efficiency of the modified or mutated KAR! enzyme is improved by at least about 300% as compared to the wild-type or parental KARI for NADH.
  • the catalytic efficiency of the modified or mutated KARI enzyme is improved by at least about 500% as compared to the wild-type or parental KARI for NADH. More preferably, the catalytic efficiency of the modified or mutated KARI enzyme is improved by at least about 1000% as compared to the wild-type or parental KARI for NADH. More preferably, the catalytic efficiency of the modified or mutated KARI enzyme is improved by at least about 5000% as compared to the wild-type or parental KARI for NADH.
  • the catalytic efficiency of the modified or mutated KARI enzyme with NADH is increased with respect to the catalytic efficiency of the wild-type or parental enzyme with NADPH.
  • the catalytic efficiency of the modified or mutated KARI enzyme is at least about 10% of the catalytic efficiency of the wild-type or parental KARI enzyme for NADPH. More preferably, the catalytic efficiency of the modified or mutated KARI enzyme is at least about 25% of the catalytic efficiency of the wild-type or parental KARI enzyme for NADPH.
  • the catalytic efficiency of the modified or mutated KARI enzyme is at least about 50% of the catalytic efficiency of the wild-type or parental KARI enzyme for NADPH, More preferably, the catalytic efficiency of the modified or mutated KARI enzyme is at least about 75%, 85%, 95% of the catalytic efficiency of the wild-type or parental KARI enzyme for NADPH.
  • the K M of the KARI enzyme for NADH is decreased relative to the wild-type or parental enzyme.
  • a change in K M is evidenced by at least a 5% or greater increase or decrease in KM compared to the wild-type KARI enzyme.
  • modified or mutated KARI enzymes of the present invention may show greater than 10 times decreased K M for NADH compared to the wild-type or parental KARI enzyme. In certain embodiments, modified or mutated KARI enzymes of the present invention may show greater than 30 times decreased KM for NADH compared to the wild-type or parental KARI enzyme.
  • the k cat of the KARI enzyme with NADH is increased relative to the wild-type or parental enzyme.
  • a change in k ca t is evidenced by at least a 5% or greater increase or decrease in KM compared to the wild-type KARI enzyme.
  • modified or mutated KARI enzymes of the present invention may show greater than 50% increased k ca t for NADH compared to the wild-type or parental KARI enzyme.
  • modified or mutated KARI enzymes of the present invention may show greater than 100% increased k ca i for NADH compared to the wild-type or parental KARI enzyme.
  • modified or mutated KARI enzymes of the present invention may show greater than 200% increased k ca t for NADH compared to the wild-type or parental KARI enzyme.
  • KAR! enzymes to catalyze a reaction step, including pathways for the production of isoieucine, leucine, valine, pantothenate, coenzyme
  • each of these biosynthetic pathways uses a KARI enzyme to catalyze a reaction step. Therefore, the product yield from these biosynthetic pathways will in part depend upon the activity of KARI.
  • the KARI enzymes described herein would have utility in any of the above-described pathways.
  • the present application relates to a recombinant microorganism comprising a KARi-requiring biosynthetic pathway, wherein said recombinant microorganism comprises at least one nucleic acid molecule encoding a KARI that is at least about 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58.
  • the KARI is derived from the genus Shewanella.
  • the KARI is derived from Shewanella sp. strain MR-4. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 1 . In another embodiment, the KARI is derived from the genus Vibrio. In a specific embodiment, the KARI is derived from Vibrio fischeri. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 3. In yet another embodiment, the KARI is derived from the genus Gramella. In a specific embodiment, the KARI is derived from Gramella forsetii. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 5.
  • the KARI is derived from the genus Cytophaga. In a specific embodiment, the KARI is derived from Cytophaga hutchinsonii. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 7, In yet another embodiment, the KAR! is derived from a genus selected from Lactococcus and Streptococcus. In a specific embodiment, the KARI is derived from Lactococcus lactis, Streptococcus equinus, or Streptococcus infantarius.
  • the KARI is encoded by SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, or SEQ ID NO: 25.
  • the KARI is derived from the genus Methanococcus.
  • the KARI is derived from Methanococcus maripaludis, Methanococcus vannielii, or Methanococcus voltae.
  • the KARI is encoded by SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
  • the KARI is derived from a genus selected from Zymomonas, Erythrohacter, Sphingomonas, Sphingobium, and Novosphingobium.
  • the KARI is derived from Zymomonas mobilis, Erythrohacter litoralis, Sphingomonas wittichii, Sphingobium japonicum, Sphingobium chloropheno!icum, or Novosphingobium nitrogenifigens,
  • the KARI is encoded by SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , or SEQ ID NO: 53.
  • the KARI is derived from the genus Bacteroides, In a specific embodiment, the KARI is derived from Bacteroides thetaiotaomicron. In another specific embodiment, the KARI is encoded by SEQ ID NO: 55. In yet another embodiment, the KARI is derived from the genus Schizosaccharomyces. In a specific embodiment, the KARI is derived from Schizosaccharomyces pombe or Schizosaccharomyces japonicus. In another specific embodiment, the KARI is encoded by SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61 .
  • the KARI has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the Shewanella sp. KARI (SEQ ID NO: 2); (b) arginine 76 of the Shewanella sp. KARI (SEQ !D NO: 2); (c) serine 78 of the Shewanella sp. KARI; and
  • the KARI has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) valine 48 of the L lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI (SEQ ID NO: 10); (c) lysine 52 of the L, lactis KARI (SEQ ID NO: 10); (d) serine 53 of the L, lactis KARI (SEQ ID NO: 10);
  • the present application relates to a recombinant microorganism comprising a KARI-requiring biosynthetic pathway, wherein said recombinant microorganism comprises at least one nucleic acid molecule encoding a KARI that is at least about 99% identical to SEQ ID NO: 84.
  • the KARI is derived from the genus Salmonella.
  • the KARI is derived from Salmonella enterica.
  • the KAR! is encoded by SEQ ID NO: 63.
  • the KARI has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the S.
  • enterica KARI (SEQ ID NO: 64); (b) arginine 76 of the S. enterica KARI (SEQ !D NO: 64); (c) serine 78 of the S. enterica KARI (SEQ !D NO: 64); (d) giutamine 1 10 of the S. enterica KARI (SEQ ID NO: 64); (e) aspartic acid 146 of the S. enterica KARI (SEQ ID NO: 64); (f) glycine 185 of the S. enterica KARI (SEQ ID NO: 64); and (g) lysine 433 of the S. enterica KARI (SEQ ID NO: 84).
  • KARI-requiring biosynthetic pathway refers to any metabolic pathway which utilizes KARI to convert acetoiactate to 2,3- dihydroxyisovaierate or 2-aceto-2-hydroxy-butanoate to 2,3-dihydroxy-3- methylvalerate.
  • KARI-requiring biosynthetic pathways include, but are not limited to, isobutanol, isoieucine, leucine, valine, pantothenate, coenzyme A, 1 - butanoi, 2-methyl-1 -butanol, 3-methyI-1 -butanoi, 3-methyl-1 -pentanoi, 4 ⁇ methyl-1 - pentanol, 4-methyl-l -hexanoi, and 5-methyl-1 -heptanol metabolic pathways.
  • the metabolic pathway may naturally occur in a microorganism (e.g., a natural pathway for the production of valine) or arise from the introduction of one or more heterologous polynucleotides through genetic engineering.
  • the recombinant microorganisms expressing the KARI-requiring biosynthetic pathway are yeast cells.
  • the recombinant microorganisms of the present invention can express a plurality of heterologous and/or native enzymes involved in pathways for the production of a beneficial metabolite such as isobutanol.
  • 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 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 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 so taking part in a particular metabolic process.
  • a metabolite can be an organic compound that is a starting material (e.g., glucose or pyruvate), an intermediate (e.g., 2-ketoisovaierate), or an end product ⁇ e.g., isobutano! 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.
  • 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.
  • 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.
  • Sequence homology for polypeptides is typically measured using sequence analysis software. See commonly owned and co-pending application US 2009/0226991 .
  • a typical algorithm used comparing a molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST. When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms described in commonly owned U.S. Pat. No. 8,017,375.
  • microorganisms can be modified to include an isobutanol producing metabolic pathway suitable for the production of isobutanol.
  • the microorganisms may be selected from yeast microorganisms.
  • yeast microorganisms for the production of isobutanol may be selected based on certain characteristics:
  • One characteristic may include the property that the microorganism is selected to convert various carbon sources into isobutanol.
  • carbon source generally refers to a substance suitable to be used as a source of carbon for prokaryotic or eukaryotic cell growth. Examples of suitable carbon sources are described in commonly owned U.S. Pat. No. 8,017,375. Accordingly, in one embodiment, the recombinant microorganism herein disclosed can convert a variety of carbon sources to products, including but not limited to glucose, galactose, mannose, xylose, arabinose, lactose, sucrose, CO;?, and mixtures thereof.
  • the recombinant microorganism may thus further include a pathway for the production of isobutanol from five-carbon (pentose) sugars including xylose.
  • Most yeast species metabolize xylose via a complex route, in which xylose is first reduced to xylitol via a xylose reductase (XR) enzyme. The xylitol is then oxidized to xylulose via a xylitol dehydrogenase (XDH) enzyme. The xylulose is then phosphoryiated via a xyiulokinase (XK) enzyme.
  • XR xylose reductase
  • XDH xylitol dehydrogenase
  • XK xyiulokinase
  • This pathway operates inefficiently in yeast species because it introduces a redox imbalance in the ceil.
  • the xyiose-io- xyiitoi step uses primarily NADPH as a cofactor (generating NADP+), whereas the xyiitoi-to-xyiulose step uses NAD* as a cofactor (generating NADH).
  • NADPH a cofactor
  • Other processes must operate to restore the redox imbalance within the cell. This often means that the organism cannot grow anaerobically on xylose or other pentose sugars. Accordingly, a yeast species that can efficiently ferment xylose and other pentose sugars into a desired fermentation product is therefore very desirable.
  • the recombinant microorganism is engineered to express a functional exogenous xylose isomerase.
  • Exogenous xylose isomerases (X!) functional in yeast are known in the art. See, e.g., Rajgarhia et al., U.S. Pat. No. 7,943,368, which is herein incorporated by reference in its entirety.
  • the exogenous XI gene is operatively linked to promoter and terminator sequences that are functional in the yeast cell.
  • the recombinant microorganism further has a deletion or disruption of a native gene that encodes for an enzyme (e.g., XR and/or XDH) that catalyzes the conversion of xylose to xylitol.
  • the recombinant microorganism also contains a functional, exogenous xyiulokinase (XK) gene operatively linked to promoter and terminator sequences that are functional in the yeast ceil.
  • XK xyiuiokinase
  • the yeast microorganism has reduced or no pyruvate decarboxylase (PDC) activity.
  • PDC catalyzes the decarboxylation of pyruvate to acetaldehyde, which is then reduced to ethanoi by ADH via an oxidation of NADH to NAD+.
  • Ethanoi 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., isobutanoi).
  • 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 glyceroi-3- phosphate dehydrogenase (GPD) activity.
  • GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD+.
  • Glycerol is then produced from G3P by Glycerol-3- phosphatase (GPP).
  • Glycerol production Is a secondary pathway to oxidize excess NADH from glycolysis. Reduction or elimination of this pathway would increase the pyruvate and reducing equivalents (NADH) available for the production of a pyruvate-derived metabolite (e.g., isobutanol).
  • NADH pyruvate and reducing equivalents
  • disruption, deletion, or mutation of the genes encoding for giyceroi-3-phosphate dehydrogenases can further increase the yield of the desired metabolite (e.g., isobutanol).
  • desired metabolite e.g., isobutanol.
  • Yeast strains with reduced GPD activity are described in commonly owned and co-pending US Patent Publication Nos. 201 1/0020889 and 201 1/0183392.
  • the microorganism has reduced 3-keto acid reductase (3-KAR) activity.
  • 3-KARs catalyze the conversion of 3-keto acids (e.g., acetoiactate) to 3-hydroxyacids (e.g., DH2 B).
  • 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 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 yeast microorganisms may be selected from the "Saccharomyces Yeast C!ade", as described in commonly owned U.S. Pat. No. 8,017,375.
  • Saccharomyces sensu stricto yeast species include but are not limited to S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S, carocanis and hybrids derived from these species (Masneuf et a!., 1998, Yeast 7: 61 - 72).
  • the yeast microorganism may be selected from a post-WGD yeast genus, including but not limited to Saccharomyces and Candida.
  • the favored post-WGD yeast species include: S. cerevisiae, S. uvarum, S, bayanus, S. paradoxus, S, castelli, and C. g!abrata,
  • the yeast microorganism may be selected from a pre-whole genome duplication (pre-WGD) yeast genus including but not limited to Saccharomyces, Kluyveromyces . , Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Yarrowia and, Schizosaccharomyces,
  • pre-WGD yeast species include: S. kluyveri, K. thermotolerans, K. marxianus, K. waltii, K, lactis, C. tropicalis, P. pastoris, P. anomala, P. stipitis, I. orientalis, I. occidentals, I. scutulata, D. hansenii, H, anomala, Y. lipolytica, and S. pombe.
  • a yeast microorganism may be either Crabtree-negative or Crabtree- positive as described in described in commonly owned U.S. Pat. No. 8,017,375.
  • the yeast microorganism may be selected from yeast with a Crabtree-negative phenotype including but not limited to the following genera: Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida.
  • Crabtree-negative species include but are not limited to: S. kluyveri, K, lactis, K. marxianus, P. anomala, P. stipitis, I. orientalis, I, occidentaiis, I. scutulata, H.
  • the yeast microorganism may be selected from yeast with a Crabtree-positive phenotype, including but not limited to Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Pichia and Schizosaccharomyces.
  • Crabtree-positive yeast species include but are not limited to: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S. castelli, K. thermotolerans, C. glabrata. Z, basils, Z. rouxii, D. hansenii, P, pastohus, and S. pombe,
  • Another characteristic may include the property that the microorganism is that it is non-fermenting. In other words, it cannot metabolize a carbon source anaerobically while the yeast is able to metabolize a carbon source in the presence of oxygen.
  • Nonfermenting yeast refers to both naturally occurring yeasts as well as genetically modified yeast.
  • Ethanol is produced by alcohol dehydrogenase (ADH) via the reduction of aceta!dehyde, which is generated from pyruvate by pyruvate decarboxylase (PDC).
  • ADH alcohol dehydrogenase
  • PDC pyruvate decarboxylase
  • a fermentative yeast can be engineered to be non-fermentative by the reduction or elimination of the native PDC activity.
  • most of the pyruvate produced by glycolysis is not consumed by PDC and is available for the isobutanol pathway. Deletion of this pathway increases the pyruvate and the reducing equivalents available for the biosynthetic pathway.
  • Fermentative pathways contribute to low yield and low productivity of pyruvate-derived metabolites such as isobutanol. Accordingly, deletion of one or more PDC genes may increase yield and productivity of a desired metabolite (e.g., isobutanol).
  • 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, Rhodotoru!a, Myxozyma, or Candida,
  • the non-fermenting yeast is C. xesiobii.
  • genes that encode for enzymes that are homologous to the genes described herein e.g., KARI homologs.
  • genes that are homologous or similar to the KAR!s 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.
  • Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity (e.g. as described herein or in Kiritani, K. Branched-Chain Amino Acids Methods Enzymology, 1970), then isolating the enzyme with said activity through purification, determining the protein sequence of the enzyme through techniques such as Edman degradation, design of PGR primers to the likely nucleic acid sequence, amplification of said DNA sequence through PGR, and cloning of said nucleic acid sequence.
  • analogous genes and/or analogous enzymes or proteins techniques also include comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC.
  • the candidate gene or enzyme may be identified within the above mentioned databases in accordance with the teachings herein.
  • Any method can be used to introduce a nucleic acid molecule into yeast and many such methods are well known.
  • transformation and electroporation are common methods for introducing nucleic acid into yeast cells. See, e.g., Gietz et a!., 1992, Nuc Acids Res. 27: 69-74; Ito et ai., 1983, J. Bacteriol. 153: 183-8; and Becker et ai., 1991 , Methods in Enzymology 194: 182-7.
  • the integration of a gene of interest into a DNA fragment or target gene of a yeast microorganism occurs according to the principle of homologous recombination.
  • an integration cassette containing a module comprising at least one yeast marker gene and/or the gene to be integrated is flanked on either side by DNA fragments homologous to those of the ends of the targeted integration site (recombinogenic sequences).
  • recombinogenic sequences DNA fragments homologous to those of the ends of the targeted integration site
  • the integration cassette for integration of a gene of interest into a yeast microorganism includes the heterologous gene under the control of an appropriate promoter and terminator together with the selectable marker flanked by recombinogenic sequences for integration of a heterologous gene into the yeast chromosome.
  • the heterologous gene includes an appropriate native gene desired to increase the copy number of a native gene(s).
  • the selectable marker gene can be any marker gene used in yeast, including but not limited to, HIS3, TRP1, LEU2, URA3, bar, ble, hph, and kan.
  • the recombinogenic sequences can be chosen at will, depending on the desired integration site suitable for the desired application.
  • integration of a gene into the chromosome of the yeast microorganism may occur via random integration (Kooistra et a/., 2004, Yeast 21 : 781 -792).
  • URA3 marker loss can be obtained by plating URA3 containing cells in FOA (5-fluoro-orotic acid) containing medium and selecting for FOA resistant colonies (Boeke et al., 1984, Moi. Gen. Genet 197: 345-47).
  • exogenous nucleic acid molecule contained within a yeast cell of the disclosure can be maintained within that ceil in any form.
  • exogenous nucleic acid molecules can be integrated into the genome of the ceil or maintained in an episomai state that can stably be passed on ("inherited") to daughter cells.
  • extra-chromosomal genetic elements such as piasmids, mitochondrial genome, etc.
  • the yeast cells can be stably or transiently transformed.
  • the yeast cells described herein can contain a single copy, or multiple copies of a particular exogenous nucleic acid molecule as described above.
  • Yeast microorganisms within the scope of the invention may have reduced enzymatic activity such as reduced PDC, GPD, ALDH, or 3-KAR activity.
  • reduced as used herein with respect to a particular polypeptide activity refers to a lower level of polypeptide activity than that measured in a comparable yeast cell of the same species.
  • reduced also refers to the elimination of polypeptide activity as compared to a comparable yeast cell of the same species.
  • yeast ceils lacking activity for an endogenous PDC, GPD, ALDH, or 3-KAR are considered to have reduced activity for PDC, GPD, ALDH, or 3-KAR since most, if not al!, comparable yeast strains have at least some activity for PDC, GPD, ALDH, or 3- KAR.
  • Such reduced PDC, GPD, ALDH, or 3-KAR activities can be the result of lower PDC, GPD, ALDH, or 3-KAR concentration (e.g., via reduced expression), lower specific activity of the PDC, GPD, ALDH, or 3-KAR, or a combination thereof.
  • Many different methods can be used to make yeast having reduced PDC, GPD, ALDH, or 3-KAR activity.
  • a yeast cell can be engineered to have a disrupted PDC-, GPD-, ALDH-, or 3-KAR-encoding locus using common mutagenesis or knock-out technology. See, e.g., Methods in Yeast Genetics (1997 edition), Adams, Gottschiing, Kaiser, and Stems, Cold Spring Harbor Press (1998).
  • a yeast ceil can be engineered to partially or completely remove the coding sequence for a particular PDC, GPD, ALDH, or 3-KAR.
  • the promoter sequence and/or associated regulatory elements can be mutated, disrupted, or deleted to reduce the expression of a PDC, GPD, ALDH, or 3-KAR.
  • yeast strains which when found in nature, are substantially free of one or more PDC, GPD, ALDH, or 3-KAR activities.
  • antisense technology can be used to reduce PDC, GPD, ALDH, or 3-KAR activity.
  • yeasts can be engineered to contain a cDNA that encodes an antisense molecule that prevents a PDC, GPD, ALDH, or 3-KAR from being made.
  • antisense moiecuie encompasses any nucleic acid molecule that contains sequences that correspond to the coding strand of an endogenous polypeptide.
  • An antisense molecule also can have flanking sequences (e.g., regulatory sequences).
  • antisense molecules can be ribozymes or antisense oligonucleotides.
  • a ribozyme can have any genera! structure including, without limitation, hairpin, hammerhead, or axhead structures, provided the moiecuie cleaves RNA.
  • Methods for overexpressing a polypeptide from a native or heterologous nucleic acid moiecuie are well known. Such methods include, without limitation, constructing a nucleic acid sequence such that a regulatory element promotes the expression of a nucleic acid sequence that encodes the desired polypeptide.
  • regulatory elements are DNA sequences that regulate the expression of other DNA sequences at the level of transcription.
  • regulatory elements include, without limitation, promoters, enhancers, and the like.
  • the exogenous genes can be under the control of an inducible promoter or a constitutive promoter.
  • methods for expressing a polypeptide from an exogenous nucleic acid molecule in yeast are well known.
  • nucleic acid constructs that are used for the expression of exogenous polypeptides within Kluyveromyces and Saccharomyces are well known (see, e.g., U.S. Pat. Nos. 4,859,598 and 4,943,529, for Kluyveromyces and, e.g., Gellissen et al., Gene 190(1 ):87-97 (1997) for Saccharomyces).
  • Yeast piasmids have a selectable marker and an origin of replication.
  • certain piasmids may also contain a centromeric sequence. These centromeric piasmids are generally a single or low copy plasmid.
  • Piasmids without a centromeric sequence and utilizing either a 2 micron (S. cerevisiae) or 1 .6 micron (K. lactis) replication origin are high copy piasmids.
  • the selectable marker can be either prototrophic, such as HIS3, TRP1, LEU2, URA3 or ADE2, or antibiotic resistance, such as, bar, ble, hph, or kan.
  • heterologous control elements can be used to activate or repress expression of endogenous genes. Additionally, when expression is to be repressed or eliminated, the gene for the relevant enzyme, protein or RNA can be eliminated by known deletion techniques.
  • any yeast within the scope of the disclosure can be identified by selection techniques specific to the particular polypeptide (e.g. an isobutanol pathway enzyme) being expressed, over-expressed or repressed.
  • Methods of identifying the strains with the desired phenotype are well known to those skilled in the art. Such methods include, without limitation, PGR, RT-PCR, and nucleic acid hybridization techniques such as Northern and Southern analysis, altered growth capabilities on a particular substrate or in the presence of a particular substrate, a chemical compound, a selection agent and the like.
  • immunohistochemistry and biochemical techniques can be used to determine if a ceil contains a particular nucleic acid by detecting the expression of the encoded polypeptide.
  • an antibody having specificity for an encoded enzyme can be used to determine whether or not a particular yeast cell contains that encoded enzyme.
  • biochemical techniques can be used to determine if a cell contains a particular nucleic acid molecule encoding an enzymatic polypeptide by detecting a product produced as a result of the expression of the enzymatic polypeptide. For example, transforming a ceil with a vector encoding acetolactate synthase and detecting increased acetolactate concentrations compared to a cell without the vector indicates that the vector is both present and that the gene product is active. Methods for detecting specific enzymatic activities or the presence of particular products are well known to those skilled in the art. For example, the presence of acetolactate can be determined as described by Hugenholtz and Starrenburg, 1992, Appi. Micro, Biot. 38:17-22.
  • Yeast microorganisms of the invention may be further engineered to have increased activity of enzymes (e.g., increased activity of enzymes involved in an isobutanoi producing metabolic pathway).
  • increased activity of enzymes e.g., increased activity of enzymes involved in an isobutanoi producing metabolic pathway.
  • the term "increased” as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured in a comparable yeast cell of the same species. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Increased activities for enzymes involved in glycolysis or the isobutanoi pathway would result in increased productivity and yield of isobutanoi.
  • Methods to increase enzymatic activity are known to those skilled in the art. Such techniques may include increasing the expression of the enzyme by increased copy number and/or use of a strong promoter, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the K M for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231 ), ed. Arnold and Georgiou, Humana Press (2003).
  • the present application provides methods of producing a desired metabolite using a recombinant described herein.
  • the recombinant microorganism comprises a KARI-requiring biosynthetic pathway, wherein said recombinant microorganism comprises at least one nucleic acid molecule encoding a KARI that is at least about 80% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEO ID NO: 8, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58.
  • the KARI is derived from the genus Shewanella.
  • the KARI is derived from Shewanella sp. strain MR-4.
  • the isolated nucleic acid molecule is comprised of SEQ ID NO: 1 .
  • the KARI is derived from the genus Vibrio. In a specific embodiment, the KARI is derived from Vibrio fischeri. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 3. In yet another embodiment, the KARI is derived from the genus Gramella. In a specific embodiment, the KARI is derived from Gramelia forsetii. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 5. In yet another embodiment, the KARI is derived from the genus Cytophaga, In a specific embodiment, the KARI is derived from Cytophaga hutchinsonii. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 7.
  • the KARI is derived from a genus selected from Lactococcus and Streptococcus.
  • the KARI is derived from Lactococcus iactis, Streptococcus equinus, or Streptococcus infantarius.
  • the KARI is encoded by SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, or SEQ ID NO: 25.
  • the KARI is derived from the genus Methanococcus.
  • the KARI is derived from Methanococcus maripaludis, Methanococcus vannielii, or Methanococcus voltae.
  • the KARI is encoded by SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
  • the KARI is derived from a genus selected from Zymomonas, Erythrobacter, Sphingomonas, Sphingobium, and Novosphingobium.
  • the KARI is derived from Zymomonas mobilis, Erythrobacter iiioralis, Sphingomonas witiichii, Sphingobium japonicum, Sphingobium chlorophenolicum, or Novosphingobium nitrogenifigens.
  • the KARI is encoded by SEQ ID NO: 39, SEQ ID NO: 41 , SEQ !D NO: 43, SEQ !D NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , or SEQ ID NO: 53.
  • the KARI is derived from the genus Bacteroides.
  • the KARI is derived from Bacteroides thetaiotaomicron, In another specific embodiment, the KARI is encoded by SEQ ID NO: 55. In yet another embodiment, the KARI is derived from the genus Schizosaccharomyces. In a specific embodiment, the KARI is derived from Schizosaccharomyces pombe or Schizosaccharomyces japonicus. In another specific embodiment, the KARI is encoded by SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61 .
  • the KARI has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the Shewanella sp, KARI (SEQ ID NO: 2); (b) arginine 76 of the Shewanelia sp. KARI (SEQ ID NO: 2); (c) serine 78 of the Shewanella sp. KARI; and
  • the KARI has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) valine 48 of the L lactis KARI (SEQ ID NO: 10); (b) arginine 49 of the L. lactis KARI (SEQ ID NO: 10); (c) iysine 52 of the L. lactis KARI (SEQ ID NO: 10); (d) serine 53 of the L lactis KARI (SEQ ID NO: 10);
  • the recombinant microorganism comprises a KARI-requiring biosynfhetic pathway, wherein said recombinant microorganism comprises at least one nucleic acid molecule encoding a KARI that is at least about 99% identical to SEQ ID NO: 84.
  • the KARI is derived from the genus Salmonella.
  • the KARI is derived from Salmonella enterica.
  • the KARI is encoded by SEQ ID NO: 83.
  • the KARI has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the S.
  • enterica KARI SEQ ID NO: 64
  • arginine 76 of the S. enterica KARI SEQ ID NO: 64
  • serine 78 of the S. enterica KARI SEQ ID NO: 64
  • glutamine 1 10 of the S. enterica KARI SEQ ID NO: 64
  • glutamine 1 10 of the S. enterica KARI SEQ ID NO: 64
  • aspartic acid 148 of the S. enterica KARI SEQ ID NO: 64
  • glycine 185 of the S. enterica KARI SEQ ID NO: 64
  • lysine 433 of the S. enterica KARI SEQ ID NO: 64.
  • the KARI-requiring biosynthetic pathway is a pathway for the production of a metabolite selected from isobutanol, isoleucine, leucine, valine, pantothenate, coenzyme A, 1 -butanoi, 2-methyi-1 -butanoi, 3-methyi- 1 -butano!, 3-methy!-1 -pentanol, 4-methyl-1 -pentanol, 4-methyl-l -hexanoL and 5- methyl-1 -heptanoi.
  • 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, isoleucine, leucine, valine, pantothenate, coenzyme A, 1 -butanoi, 2-methyi-1 -butanoi, 3-methyi- 1 -butanoi, 3-methyi-1 -pentanoi, 4-methyl-1 -pentanoi, 4-methyi-1 -hexano!, and 5- methyl-1 -heptanol.
  • 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 80 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5% theoretical.
  • the beneficial metabolite is isobutanol.
  • 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.
  • said spent yeast biocatalyst has been engineered to comprise at least one nucleic acid molecule encoding a KARI that is at least about 80% identical to SEQ ID NO: 2, SEG ID NO: 4, SEG !D NO: 6, SEG ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 40, or SEQ ID NO: 58.
  • the KARI is derived from the genus Shewanella. In a specific embodiment, the KARI is derived from Shewanella sp. strain MR-4. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 1 . In another embodiment, the KARI is derived from the genus Vibrio, In a specific embodiment, the KARI is derived from Vibrio fischeri. In another specific embodiment, the isolated nucleic acid molecule is comprised of SEQ ID NO: 3. In yet another embodiment, the KARI is derived from the genus Grarnelia. In a specific embodiment, the KARI is derived from Gramella forsetii.
  • the isolated nucleic acid molecule is comprised of SEQ ID NO: 5.
  • the KARI is derived from the genus Cytophaga.
  • the KARI is derived from Cytophaga hutchinsonii.
  • the isolated nucleic acid molecule is comprised of SEQ ID NO: 7.
  • the KARI is derived from a genus selected from Lactococcus and Streptococcus.
  • the KARl is derived from Lactococcus lactis, Streptococcus equinus, or Streptococcus infaniarius.
  • the KARl is encoded by SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, or SEQ ID NO: 25.
  • the KARl is derived from the genus Methanococcus.
  • the KARl is derived from Methanococcus maripaludis, Methanococcus vannielii, or Methanococcus voltae.
  • the KARl is encoded by SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37.
  • the KARl is derived from a genus selected from Zyrnomonas, Erythrobacter, Sphingomonas, Sphingobiurn, and Novosphingobium.
  • the KARl is derived from Zyrnomonas mobilis, Erythrobacter litoralis, Sphingomonas wittichii, Sphingobiurn japonicum, Sphingobiurn chlorophenolicum, or Novosphingobium nitrogenifigens.
  • the KARl is encoded by SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , or SEQ ID NO: 53.
  • the KARl is derived from the genus Bacteroides.
  • the KARl is derived from Bacteroides thetaiotaomicmn.
  • the KARl is encoded by SEQ ID NO: 55.
  • the KARl is derived from the genus Schizosaccharomyces.
  • the KARl is derived from Schizosaccharomyces pombe or Schizosaccharomyces japonicus.
  • the KARl is encoded by SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 81 .
  • the KARl has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the Shewanella sp. KARl (SEQ ID NO: 2); (b) arginine 76 of the Shewanella sp. KARl (SEQ ID NO: 2); (c) serine 78 of the Shewanella sp. KARl; and
  • the KARl has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) valine 48 of the L. lactis KARl (SEQ ID NO: 10); (b) arginine 49 of the L lactis KARl (SEQ ID NO: 10); (c) lysine 52 of the L. lactis KARl (SEQ ID NO: 10); (d) serine 53 of the L lactis KARl (SEQ ID NO: 10);
  • said spent yeast biocataiyst has been engineered to comprise at least one nucleic acid molecule encoding a KARI that is at least about 99% identical to SEQ ID NO: 64.
  • the KARI is derived from the genus Salmonella.
  • the KARI is derived from Salmonella enterica.
  • the KARI is encoded by SEQ ID NO: 83.
  • the KAR! has one or more modifications or mutations at positions corresponding to amino acids selected from: (a) alanine 71 of the S, enterica KARI (SEQ ID NO: 84); (b) arginine 78 of the S.
  • enterica KARI SEQ ID NO: 64
  • serine 78 of the 8. enterica KARI SEQ ID NO: 64
  • glutamine 1 10 of the S. enterica KARI SEQ ID NO: 64
  • glutamine 1 10 of the S. enterica KARI SEQ ID NO: 64
  • aspartic acid 146 of the S. enterica KARI SEQ ID NO: 64
  • glycine 185 of the S. enterica KARI SEQ ID NO: 64
  • lysine 433 of the S. enterica KARI SEQ ID NO: 64.
  • the DDG comprising a spent yeast biocataiyst 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 biocataiyst (e.g., a recombinant yeast microorganism of the present invention), said method comprising: (a) cultivating said yeast biocataiyst 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 biocataiyst; and (c) drying said insoluble material comprising said yeast biocataiyst to produce the DDG.
  • a yeast biocataiyst 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.
  • KAR! genes were individually expressed from a yeast promoter in conjunction with other components of an isobutanoi production pathway in yeast such that KARI was the limiting enzyme in the pathway and the amount of isobutanoi produced during a fermentation was dependent on the KAR! activity level.
  • KARIs were identified and grouped by bioinformatic and phylogenetic methods based on the amino acid sequence. Individual KARIs were chosen for the above analysis to provide a representative sample of broadly diverse clades. KARI genes were designed and synthesized based on the primary amino acid sequence of the chosen KARI, with codon optimization of the genes for expression in S. cerevisiae, These genes were cloned downstream of the Sc__PDC1 'J0 ° promoter in pGV3009 to replace the Ec lvC__coSc P D1 ⁇ A 1 - hlsb gene present in the piasmid.
  • Shake Flask Fermentations Shake flask fermentations using GEV03956 carrying these individual piasmids were performed together in experiments with GEV03958 carrying pGV3022 (derived from pGV3009 but containing the E. coli ilvC. coSc gene expressed from the Sc__PDC1 '35G promoter) and GEV03956 carrying pGV3012 (equivalent to pGV3009 lacking the Sc__PDCT promoter and KARI gene) for comparison of isobutanoi production. The shake flask fermentations were performed as foliows.
  • the strains were grown overnight in 3 mL of YPD medium containing 1 % v/v ethanol and 0.1 g/L G418 at 30°C and 250 rpm. The OD 6 oo of these cultures was determined after overnight growth and the appropriate amount of culture was added to 50 mL of YP medium containing 5% w/v glucose, 1 % v/v ethanol, 200 mM MES, pH 6.5, and 0.1 g/L G418 to obtain an OD 600 of 0.1 in 250 mL baffled flasks with sleeve caps. Cultures were incubated at 30°C and 250 rpm overnight.
  • the fermentation cultures were incubated at 30°C and 250 rpm in non-baffled 250 mL flasks with vented screw cap tops. After 24, 48 and 72 hours of incubation, 1 .5 mL of culture was removed into 1 .5 mL microcentrifuge tubes from each culture. ODeoo values were determined from the samples and the remainder of each sample was centrifuged for 10 min at 14,000 rpm in a microcentrifuge and 1 mL of the supernatant was removed to be submitted for gas chromatographic analysis.
  • Table 4 shows the results of 48 hr and 72 hr isobutanoi fermentation timepoints.
  • Each of these identified KARIs share the property of being long-form KARIs, Long-form KARIs are found in plants, algae, and some bacteria, while short- form KARIs are found in fungi and bacteria.
  • the amino acid sequences of these high-performing bacterial long form KARIs were aligned with the sequences of 103 other KARIs representing broad biological diversity of KARIs chosen from the bioinformatic and phyiogenetic analysis above and were used to generate a phylogenetic tree in Clone Manager using the "Align Multiple Sequences" feature to perform a Multi-Way alignment of the amino acid sequences using the BLOSUM62 scoring matrix and otherwise default parameters.
  • the Shewaneila sp. KARI sequence amino acid sequence (SEQ ID NO: 2) was used to identify the 500 closest protein sequences in the GenBank database using the biastp algorithm on the non-redundant protein sequence database using the default parameters.
  • the COBALT Multiple Alignment link from the BLAST results page was used to perform a multiple alignment of the 500 closest protein sequences plus the Shewaneila sp. KARI sequence (SEQ ID NO: 2) using the default parameters.
  • This alignment was downloaded as a "Fasta plus gaps” file and opened with the Clone Manager "Align Multiple Sequences" feature to perform a Multi-Way alignment of the amino acid sequences using the BLOSUM62 scoring matrix and otherwise default parameters to generate a phylogenetic tree of the sequences.
  • KARI sequences representing the major clades of this tree were chosen and used to generate a representative subset phylogenetic tree.
  • the resulting subset phylogenetic tree showed a clade of proteins sequences containing the Shewaneila sp., Vibrio fischeri, Gramelia forsetii, Cytophaga hutchinsonii, and £. coli KARIs and KARIs that were closely related to those sequences (see Fig.
  • KARI was individually expressed from a yeast promoter in conjunction with other components of an isobutanol production pathway in yeast such that KARI was the limiting enzyme in the pathway and the amount of isobutanol produced during a fermentation was dependent on the KARI activity level.
  • KARIs were identified and grouped by bioinformatic and phylogenetic methods based on the amino acid sequence. Individual KARIs were chosen for the above analysis to provide a representative sample of broadly diverse clades. KARI genes were designed and synthesized based on the primary amino acid sequence of the chosen KARI, with codon optimization of the genes for expression in S. cerevisiae. These genes were cloned downstream of the Sc__PDC1 ⁇ j50 promoter in pGV3009 to replace the Ec_llvG_coSc h2lJ1 ⁇ A 1 ⁇ iSb gene present in the plasm id.
  • the strains were grown overnight in 3 mL of YPD medium containing 1 % v/v ethanol and 0.1 g/L G418 at 30°C and 250 rpm.
  • the OD 6 oo of these cultures was determined after overnight growth and the appropriate amount of culture was added to 50 mL of YP medium containing 5% w/v glucose, 1 % v/v ethanol, 200 mM MES, pH 6.5, and 0.1 g/L G418 to obtain an OD 6 oo of 0.1 in 250 mL baffled flasks with sleeve caps. Cultures were incubated at 30°C and 250 rpm overnight.
  • the fermentation cultures were incubated at 30°C and 250 rpm in non-baffled 250 mL flasks with vented screw cap tops. After 24, 48 and 72 hours of incubation, 1 .5 mL of culture was removed into 1 .5 mL microcentrifuge tubes from each culture. OD 6 oo values were determined from the samples and the remainder of each sample was centrifuged for 10 min at 14,000 rpm in a microcentrifuge and 1 mL of the supernatant was removed to be submitted for gas chromatographic analysis.
  • KARIs share the property of being short-form KARIs. Short-form KARIs are found in fungi and bacteria, while long-form KARIs are found in plants, algae, and some bacteria. An additional 21 short-form KARIs tested did not meet the yeast isobutanoi fermentation criteria in the above experiments.
  • Lactococcus lactis, Methanococcus maripaiudis, and Zymomonas mobilis KARIs were also identified as performing as well or better than the E. coii KARI in shake flask fermentations when expressed from a high copy number plasm id. Genes encoding these KARIs were cloned downstream of a Sc_TDH3 promoter to replace the Ec ivC_coSc p ⁇ u1'A 1JI/st' gene present in that plasmid.
  • the ODeoo of these cultures was determined after overnight growth and the appropriate amount of culture was added to 50 mL of YP medium containing 5% w/v glucose, 1 % v/v ethanoi, 200 mM MES, pH 6.5, and 0.1 g/L G418 to obtain an OD 6 oo of 0.1 in 250 mL baffled flasks with sleeve caps. Cultures were incubated at 30°C and 250 rpm overnight. The OD 6 oo of these cultures was determined after overnight growth and the appropriate amount of culture to total 250 ODs was added to 50 mL Falcon tubes and centrifuged at 2700 x g for 5 minutes.
  • the L Iactis, M. maripaludis, Z, mobilis, B. thetaiotaomicron, and S. pom be KARI amino acid sequences were used to identify the closest protein sequences in the GenBank database using the blastp algorithm on the non- redundant protein sequence database using the default parameters.
  • Table 8 discloses KARI sequences that have > 80% amino acid identity with the L, iactis, M, maripaludis, Z. mobilis, or S. pombe KARIs.
  • a phyiogenetic tree thai discloses the database identification numbers of 50 KAR! sequences that have >78% amino acid identity with the B. thetaiotaomicron KARI and their phyiogenetic relationship with the B. thetaiotaomicron KARI was generated (see Fig, 2 of U.S. Provisional Application No. 506,584, which is herein incorporated by reference).
  • thetaiotaomicron genome sequence project NCBI reference sequence NC 004863
  • the methionine codon for amino acid position 13 of the annotated protein (nucleotide positions 2600124-2600122 from NCB! reference sequence NC 004663).
  • the start codon in B. thetaiotaomicron is at the annotated position (nucleotides 2600180-2600158 from NCBI reference sequence NC 004863) or at the methionine codon for amino acid position 13 of the annotated protein (nucleotide positions 2800124-2600122 from NCBI reference sequence NC_004663).
  • thetaiotaomicron KARI lacking the N-terminal 12 amino acids of the annotated protein may function as well or better for isobutano! production in yeast compared with performance of the B. thetaiotaomicron KARL Such a protein would have the sequence of SEQ ID NO: 88.
  • Example 3 Cof a ctor Switch of the L !aciis KARI
  • the purpose of this example is to demonstrate how the cofactor specificity of the L, lactis KARI can be switched from NADPH to NADH,
  • the L lactis KARI is NADPH-dependent. To enable the enzyme's use in the production of isobutanoi at theoretical yield and/or under anaerobic conditions, the enzyme's cofactor usage was switched from NADPH to NADH.
  • CAAGCC SEQ ID NO: 1 15
  • CAAGCC SEQ ID NO: 1 16
  • AAGCC (SEQ ID NO: 1 17)
  • CAAGCC SEQ ID NO: 1 18
  • LI recomb 2b rev GGCTTGATATCCTCCTCGTATGCGGATTGTTGTGYCTCATCTG
  • LI recomb 3LS rev TTCCTTAGCTTTATCAAAAGATAGTCCGTG (SEQ ID NO: 128)
  • LI recomb 3LD rev TTCCTTAGCTTTATCAAAATCTAGTCCGTG (SEQ ID NO: 130)
  • nucleotide S (Strong - G or C), M (Amino - A or C), K (Keto - G or T), B (Not A - C, G, or T), W (Weak - A or T), V (Not T - A, C, or G)
  • L lactis KARI variants in £. coli: The expression of L. lactis KARI variants was conducted in 0.25-L Erlenrneyer flasks filled with 50 mL LB amp (Luria Bertani Broth, Research Products International Corp, supplemented with 100 pg/mL ampicillin) inoculated with overnight culture to an initial OD 6 oo of 0.1 . After growing the expression cultures at 37°C with shaking at 250 rpm for 4 h, the cultivation temperature was dropped to 25°C, and KARI expression was induced with IPTG to a final concentration of 0.5 mM. After 24 h at 25°C and shaking at 250 rpm, the ceils were pelleted at 5,300g for 10 min and then frozen at - 20 C C until further use.
  • L lactis KARI was purified over a 5- mL histrap column.
  • L. lactis KARI variants were purified over 1 -mL histrap columns.
  • a buffer exchange was performed on the purified Bs AlsS before the synthesis to remove as much glycerol as possible. This was done using a microcon filter with a 50 kDa nominal molecular weight cutoff membrane to filter 0.5 mL of the purified enzyme until only 50 pL were left on top of the membrane. 450 pL of 20 mM KP0 4 pH 7.0, 1 mM MgCI 2 , and 0.05 mM TPP were then added to the membrane and filtered again; this process was repeated three times. The final acetolactate concentration was determined by liquid chromatography and was -200 mM.
  • KARI assay in 1 -mL scale to measure NADPH and NADH KM values L lactis KARI activity or activities of its variants were assayed kineticaliy by monitoring the decrease in NADPH or NADH concentration by measuring the change in absorbance at 340 nm.
  • An assay buffer was prepared containing 100 mM potassium phosphate pH 7.0, 1 mM DTT, 2.5 mM (SJ-2-acetoiactate, and 10 mM MgCI 2 (final concentrations in the 1 -mL assay, accounting for dilution with enzyme and cofactor). Fifty pL purified enzyme and 930 pL of the assay buffer were placed into a 1 -mL cuvette.
  • the reaction was initiated by addition of 20 pL NADPH or NADH (200 ⁇ final concentration) for a general activity assay. Michae!is-Menten constants of the cofactors were determined with varying concentrations of NADPH (500 - 12 ⁇ final) or NADH (200 - 6 pM final).
  • the fragments served as templates in the assembly PCR using commercial T7 forward and reverse primers as flanking primers.
  • the purified assembly product (Zymo clean up) was restriction digested with Nde ⁇ and Xho ⁇ , ligated into pET22b(+), and electro-competent BL21 (D3) ceils (Lucigen) were transformed.
  • library 2 An assembly product of the recombination library described above was used to introduce S53D via SOE PCR using primer pairs 33 - 40 (Table 1 1 ).
  • the forward and reverse primers were mixed manually as described above.
  • the resulting fragments were gel purified and used as templates for the assembly PCR with flanking T7 primers.
  • the resulting assembly PCR product was treated as described above.
  • Double NNK library (K52NNKS53NNK) (Generation 3): The double NNK library was constructed via SOE PCR using construct pETU1 G2 as template and primers # 1 and 2, and 41 and 42. The construction of the library was as described above (site-saturation library). 2,800 colonies were picked for screening.
  • Cells were harvested at 5,300g and 4°C and then stored at -20°C.
  • the plates always contained four wild-type or parent L. iactis KARI colonies . , three BL21 (DE3) colonies carrying pET22b( ⁇ ) to control for background reactions in cell lysates, and one well that contained only media to make sure the plates were free of contaminations.
  • Results The residues chosen to test by site-saturation mutagenesis were Y25, V48, R49, G51 , S53, L85, and I89 of the L Iactis KARI (SEQ ID NO: 2).
  • Site- saturation libraries were constructed as described in the materials and method section. After successful transformation of BL21 (DE3) cells, 88 individual clones per library were chosen. The libraries were screened with NADH (not NADPH) as cofactor. Screening results are summarized in Table 12.
  • Recombination Libraries Generation 2: A recombination library introducing all mutations found at each site (Table 12) was constructed and also allowing for the wild-type residues as well using pGV3281 as template and primers #19-40. in addition, the S53D mutation was tested in the context of the recombination library. However, given previous experiences with this mutation (low expression levels, no switch in cofactor specificity, and loss of activity) the recombination library was constructed first, which only contained the mutations found in Generation 1 . This was deemed library 1 . Next, another round of SOE PGR was used to introduce S or D at position 53 (library 2). Both libraries were separately iigated and transformed.
  • variants 1A9, 1 C2, 1 G2, and 1 G5 were expressed, purified, and characterized (Tabie 14). Mutation L85A is noteworthy. In ail three cases (1A9, 1 C2, and 1 G5), the NADPH K M value was either cut in half (1A9) or below 1 ⁇ (1 C2 and 1 G5) and thus beneath the measureable threshold. These variants also showed the lowest NADH K M values with 99, 1 15, and 1 12 ⁇ . Mutation L85A could be conceived as being generally activating.
  • Generation 3 double NNK library at positions K52 and S53: Given the data presented in Table 14, the choice of the parent for the next round was between 1A9 and 1 G2. Both had a mutation at position R49 (L and P); in addition, 1A9 carried the L85A mutation described above; 1 G2 had a mutation at position 48 (V48L). Even though the NADH K M value and catalytic efficiency of 1 G2 were not as favorable as 1A9's, 1 G2 was chosen as parent because, due to the lack of L85A, its NADPH activities were parent-like. Thus, its improvements stem from increased NADH activity only.
  • a library with 1 G2 as parent was generated using primers # 41 and 42 and the commercial T7 primers (# 1 and 2). Approximately 2,800 individual colonies were screened for both NADH and NADPH consumption. The introduction of two negative charges, K52E and S53D, gives the highest NADH/NADPH ratio in the screen (variant 4H8). However, when the order of the residues is reversed, K52D and S53E (variant 3H5), the protein has potential folding issues. The introduction of one negative charge in combination with L, P, S, or K results in at least four-fold ratio improvements compared to parent 1 G2. However, when K52 is mutated to P and S53 is still able to bind phosphate, no beneficial effects on the ratio were observed (variant 2G8).
  • Li__NKR Gen6b"h!S retained mutations E59K, T182S, and E320K and had an almost 40-fold improved KM value for 2S-AL.
  • the catalytic efficiency of this enzyme in the presence of NADH was 14.8-fold increased compared to its parent, L!_NKR &en3 ⁇ hlS .
  • Li__NKR Gen6b"hiS showed a complete switch of cofactor preference.
  • the characterization data is summarized in Table 17.
  • Sh__sp__NKR DD and Sh__sp_ KR 6E6 were synthesized by GenScript USA Inc. (Piscataway, NJ 08854 USA) with flanking Ndel and Xhoi sites.
  • the genes were isolated by restriction enzyme digestion with Ndel and Xhoi for 1 hour at 37 C C.
  • the expression vector, pGV3195 was also digested with Ndel and Xhoi for 1 hour at 37 C C. The fragments were ligated using T4 DNA iigase from New England Biolabs (Ipswich, MA USA).
  • the ligated DNAs were transformed into chemically competent E, coii DH5cs ceils, incubated for 1 h at 37°C in SOC medium, and plated to LB am p agar plates (Luria Bertani Broth, Research Products International Corp, supplemented with 100 pg/mL ampici!lin) to yield single colonies. After confirming the correct sequence, £. coii BL21 (DE3) cells were transformed with the correct plasmids for expression.
  • Pfu turbo polymerase (Stratagene) was used as the polymerase in the following PGR program: 95 °C for 2 min; 95 °C for 30 s, 55 °C for 30 s, 72 °C for 8 min (repeat 15 times); 72 °C for 10 min.
  • the reaction mixtures were digested with Dpnl for 1 h at 37 C C.
  • chemically competent E. coii XL1 -Goid cells were transformed with 3 pL of the un-cleaned PGR mixtures and the cells were allowed to recover in SOC medium at 37 °C with shaking at 250 rpm for 1 h.
  • the recovery allowed the cells to close the nick-containing DNA produced during the PGR and thus to generate circularized plasmids.
  • Sh_sp__KARj and its NKR variants were expressed in 200-mL cultures and purified over a -mL histrap HP column. The K , k ca t, and specific acitivity values were measured as described above, and the results are summarized in Table 21 .
  • variants Sh_sp_NKR S78D , Sh__sp__NKR R76DS78D , and 8b__sp_ NKR 6E6 can be defined as being NADH-dependent KARIs (NKR) in terms of their catalytic efficiencies.
  • Se2__ KARI and its NKR variants were expressed in 200-mL cultures and purified over a 1 -mL histrap HP column. The K M , k C at, and specific acitivity values were measured as described above, and the results are summarized in Table 22.
  • variants Se2 NKR DD and Se2__NKR 6E6 can be defined as being NADH-dependent KARIs (NKR). Additional mutations at positions D146, G185, and K433 are generally expected to further improve activity of the Se2 KARI (data not shown).

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Abstract

La présente invention a pour objet des microorganismes de recombinaison comprenant au moins une molécule d'acide nucléique codant une cétoacide réductoisomérase (KARI) ou son variant dépendant de NADH modifié, ladite KARI étant identique à au moins environ 80 % à SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, SEQ ID NO : 8, SEQ ID NO : 10, SEQ ID NO : 28, SEQ ID NO : 40, ou SEQ ID NO : 58. La présente invention concerne également des microorganismes de recombinaison comprenant au moins une molécule d'acide nucléique codant une cétoacide réductoisomérase (KARI) ou son variant dépendant de NADH modifié, ladite KARI étant identique à au moins environ 99 % à SEQ ID NO : 64. Selon divers aspects de l'invention, les microorganismes de recombinaison peuvent comprendre une voie métabolique de production d'isobutanol et peuvent être utilisés dans des procédés de fabrication d'isobutanol.
PCT/US2012/046185 2011-07-11 2012-07-11 Cétoacide réductoisomérases de haute performance WO2013009818A2 (fr)

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AU2017247607B2 (en) * 2016-04-06 2021-08-19 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against AML and other cancers
WO2021227500A1 (fr) * 2020-05-13 2021-11-18 安徽华恒生物科技股份有限公司 Micro-organisme recombinant pour la production de l-valine, procédé de construction s'y rapportant et application associée

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US20080148432A1 (en) * 2005-12-21 2008-06-19 Mark Scott Abad Transgenic plants with enhanced agronomic traits
US8945899B2 (en) * 2007-12-20 2015-02-03 Butamax Advanced Biofuels Llc Ketol-acid reductoisomerase using NADH
WO2010051527A2 (fr) * 2008-10-31 2010-05-06 Gevo, Inc. Micro-organismes manipulés capables de produire des composés cibles en conditions anaérobies

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AU2017247607B2 (en) * 2016-04-06 2021-08-19 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against AML and other cancers
WO2021227500A1 (fr) * 2020-05-13 2021-11-18 安徽华恒生物科技股份有限公司 Micro-organisme recombinant pour la production de l-valine, procédé de construction s'y rapportant et application associée

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