EP2446043A1 - Organismes de levure pour la production d'isobutanol - Google Patents

Organismes de levure pour la production d'isobutanol

Info

Publication number
EP2446043A1
EP2446043A1 EP10792562A EP10792562A EP2446043A1 EP 2446043 A1 EP2446043 A1 EP 2446043A1 EP 10792562 A EP10792562 A EP 10792562A EP 10792562 A EP10792562 A EP 10792562A EP 2446043 A1 EP2446043 A1 EP 2446043A1
Authority
EP
European Patent Office
Prior art keywords
isobutanol
microorganism
yeast
nadh
recombinant microorganism
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP10792562A
Other languages
German (de)
English (en)
Other versions
EP2446043A4 (fr
Inventor
Reid M. Renny Feldman
Uvini Gunawardena
Jun Urano
Peter Meinhold
Aristos Aristidou
Catherine Asleson Dundon
Christopher Smith
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Gevo Inc
Original Assignee
Gevo Inc
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.)
Filing date
Publication date
Priority claimed from US12/696,645 external-priority patent/US20110183392A1/en
Application filed by Gevo Inc filed Critical Gevo Inc
Publication of EP2446043A1 publication Critical patent/EP2446043A1/fr
Publication of EP2446043A4 publication Critical patent/EP2446043A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • 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

  • Metabolically engineered microorganisms and methods of producing such organisms are provided. Also provided are methods of producing metabolites that are biofuels by contacting a suitable substrate with metabolically engineered microorganisms and enzymatic preparations there from.
  • Biofuels have a long history ranging back to the beginning of the 20th century. As early as 1900, Rudolf Diesel demonstrated at the World Exhibition in Paris, France, an engine running on peanut oil. Soon thereafter, Henry Ford demonstrated his Model T running on ethanol derived from corn. Petroleum-derived fuels displaced biofuels in the 1930s and 1940s due to increased supply, and efficiency at a lower cost.
  • Ethanol is the most abundant fermentatively produced fuel today but has several drawbacks when compared to gasoline. Butanol, in comparison, has several advantages over ethanol as a fuel: it can be made from the same feedstocks as ethanol but, unlike ethanol, it is compatible with gasoline at any ratio and can also be used as a pure fuel in existing combustion engines without modifications. Unlike ethanol, butanol does not absorb water and can thus be stored and distributed in the existing petrochemical infrastructure. Due to its higher energy content which is close to that of gasoline, the fuel economy (miles per gallon) is better than that of ethanol.
  • butanol-gasoiine blends have lower vapor pressure than ethanol-gasoline blends, which is important in reducing evaporative hydrocarbon emissions.
  • lsobutanol has the same advantages as butanol with the additional advantage of having a higher octane number due to its branched carbon chain, lsobutanol is also useful as a commodity chemical and is also a precursor to isobutylene and isobutylene-derived fuels and chemicals, lsobutanol has been produced recombinantly in yeast microorganisms expressing a heterologous metabolic pathway (See, e.g., WO/2007/050671 to Donaldson et a/., and WO/2008/098227 to Liao et a/.).
  • yeast microorganisms fall short of commercial relevance due to their low performance characteristics, including low productivity, low titer, low yield, and the requirement for oxygen during the fermentation process.
  • One of the primary reasons for the sub-optimal performance observed in existing isobutanol-producing microorganisms is the undesirable conversion of pathway intermediates to unwanted by-products.
  • the present inventors have addressed this need by providing recombinant microorganisms with reduced pyruvate decarboxylase (PDC) activity and reduced glycerol-3-phosphate dehydrogenase (GPD) activity.
  • PDC pyruvate decarboxylase
  • GPD glycerol-3-phosphate dehydrogenase
  • the present inventors have observed that by combining the expression of a cytosolically localized acetolactate synthase enzyme with reduced pyruvate decarboxylase (PDC) activity and/or reduced glycerol-3-phosphate dehydrogenase (GPD) activity, an unexpectedly high flux from pyruvate to acetolactate can be achieved.
  • the invention provides yeast cells that are engineered to exhibit an efficient conversion of pyruvate to acetolactate in the cytoplasm due to suppression of competing metabolic pathways.
  • the present invention has utility for the production of any acetolactate-derived product, including, but not limited to, isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3- butanediol, valine, leucine, and 3-methyl-1-butanol.
  • the invention provides a recombinant microorganism, such as a yeast cell, comprising a cytosolically-localized polypeptide having acetolactate synthase activity wherein the yeast cell is substantially free of an enzyme having pyruvate decarboxylase (PDC) activity and/or glycerol-3-phosphate dehydrogenase (GPD) activity, and wherein the cell converts pyruvate to acetolactate.
  • PDC pyruvate decarboxylase
  • GPD glycerol-3-phosphate dehydrogenase
  • the present invention provides recombinant microorganisms engineered to include reduced pyruvate decarboxylase (PDC) activity as compared to a parental microorganism.
  • PDC activity is eliminated.
  • PDC catalyzes the decarboxylation of pyruvate to acetaldehyde, which is reduced to ethanol by alcohol dehydrogenases via the oxidation of NADH to NAD+.
  • the recombinant microorganism includes a mutation in at least one PDC gene resulting in a reduction of PDC activity of a polypeptide encoded by said gene.
  • the recombinant microorganism includes a partial deletion of a PDC gene resulting in a reduction of PDC activity of a polypeptide encoded by said gene. In another embodiment, the recombinant microorganism comprises a complete deletion of a PDC gene resulting in a reduction of PDC activity of a polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism includes a modification of the regulatory region associated with at least one PDC gene resulting in a reduction of PDC activity of a polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism comprises a modification of the transcriptional regulator resulting in a reduction of PDC gene transcription.
  • the recombinant microorganism comprises mutations in all PDC genes resulting in a reduction of PDC activity of the polypeptides encoded by said genes.
  • the recombinant microorganism includes partial deletions of all PDC genes resulting in a reduction of PDC activity of the polypeptides encoded by said genes.
  • the recombinant microorganism comprises a deletion of all PDC genes resulting in the elimination of PDC activity of the polypeptides encoded by said genes.
  • the present invention provides recombinant microorganisms engineered to exhibit reduced glycerol-3-phosphate dehydrogenase (GPD) activity as compared to a parental microorganism.
  • GPD activity is eliminated.
  • GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD + .
  • Glycerol is produced from G3P by Glycerol-3-phosphatase (GPP).
  • the recombinant microorganism includes a mutation in at least one GPD gene resulting in a reduction of GPD activity of a polypeptide encoded by said gene.
  • the recombinant microorganism includes a partial deletion of a GPD gene resulting in a reduction of GPD activity of a polypeptide encoded by the gene. In another embodiment, the recombinant microorganism comprises a complete deletion of a GPD gene resulting in a reduction of GPD activity of a polypeptide encoded by the gene. In yet another embodiment, the recombinant microorganism includes a modification of the regulatory region associated with at least one GPD gene resulting in a reduction of GPD activity of a polypeptide encoded by said gene. In yet another embodiment, the recombinant microorganism comprises a modification of the transcriptional regulator resulting in a reduction of GPD gene transcription.
  • the recombinant microorganism includes partial deletions of all GPD genes resulting in a reduction of GPD activity of the polypeptides encoded by said genes.
  • the recombinant microorganism comprises mutations in all GPD genes resulting in a reduction of GPD activity of the polypeptides encoded by said genes.
  • the recombinant microorganism comprises a deletion of all GPD genes resulting in the elimination of GPDs activity of the polypeptides encoded by said genes.
  • the present invention provides a recombinant microorganism engineered to exhibit reduced pyruvate decarboxylase (PDC) activity and reduced glycerol-3-phosphate dehydrogenase (GPD) activity as compared to a parental microorganism.
  • PDC pyruvate decarboxylase
  • GPD glycerol-3-phosphate dehydrogenase
  • the present invention provides recombinant microorganisms engineered to exhibit reduced pyruvate dehydrogenase (PDH) activity as compared to a parental microorganism.
  • the recombinant microorganism is engineered to have reduced pyruvate decarboxylase (PDC) activity and reduced pyruvate dehydrogenase (PDH) activity.
  • the recombinant microorganism is engineered to have reduced glycerol-3-phosphate dehydrogenase (GPD) activity and reduced pyruvate dehydrogenase (PDH) activity.
  • the recombinant microorganism is engineered to have reduced pyruvate decarboxylase (PDC) activity, reduced glycerol-3-phosphate dehydrogenase (GPD) activity, and reduced pyruvate dehydrogenase (PDH) activity.
  • PDC pyruvate decarboxylase
  • GPD glycerol-3-phosphate dehydrogenase
  • PH reduced pyruvate dehydrogenase
  • the present invention provides recombinant microorganisms, including, but not limited to those, that comprise an isobutanol producing metabolic pathway.
  • the recombinant microorganisms can be engineered to express an isobutanol producing metabolic pathway comprising at least one exogenous gene that catalyzes a step in the conversion of pyruvate to isobutanol.
  • the recombinant microorganism may be engineered to express an isobutanol producing metabolic pathway comprising at least two exogenous genes.
  • the recombinant microorganism may be engineered to express an isobutanol producing metabolic pathway comprising at least three exogenous genes. In another embodiment, the recombinant microorganism may be engineered to express an isobutanol producing metabolic pathway comprising at least four exogenous genes. In another embodiment, the recombinant microorganism may be engineered to express an isobutanol producing metabolic pathway comprising five exogenous genes.
  • isobutanol producing metabolic pathway comprises at least one exogenous gene that catalyzes a step in the conversion of pyruvate to isobutanol.
  • the exogenous gene encodes a polypeptide selected from the group consisting of acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2- keto-acid decarboxylase (KIVD), and alcohol dehydrogenase (ADH).
  • the exogenous gene encodes an acetolactate synthase (ALS).
  • the acetolactate synthase is a cytosolically-locaiized acetolactate synthase.
  • the cytosolically-locaiized acetolactate synthase is encoded by the Lactococcus lactis gene alsS.
  • the cytosolically-locaiized acetolactate synthase is encoded by the Bacillus subtilis gene alsS.
  • the recombinant microorganism comprises an isobutanol producing metabolic pathway comprising genes encoding an NADH- dependent KARI and an NADH-dependent ADH.
  • the KARI and/or the ADH show at least a 10-fold higher catalytic efficiency using NADH as the cofactor as compared to the wild-type E.coli KARI HvC and a native E.coli ADH yqhD, respectively.
  • the KARI and/or the ADH have been modified or mutated to be NADH-dependent.
  • the KARI and/or the ADH has been identified in nature with increased activity using NADH as a cofactor as compared to the wild-type E.coli KARI HvC and a native E.coli ADH yqhD, respectively.
  • the invention provides a recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein said recombinant microorganism comprises a reduction in pyruvate decarboxylase (PDC) activity as compared to a parental microorganism.
  • said recombinant microorganism comprises a reduction in glycerol-3-phosphate dehydrogenase (GPD) activity as compared to a parental microorganism.
  • PDC pyruvate decarboxylase
  • GPD glycerol-3-phosphate dehydrogenase
  • the recombinant microorganism comprises a reduction in pyruvate decarboxylase (PDC) activity and glycerol-3-phosphate dehydrogenase (GPD) activity as compared to a parental microorganism.
  • the recombinant microorganism comprises a reduction in pyruvate decarboxylase (PDC) activity, a reduction in glycerol-3-phosphate dehydrogenase (GPD) activity, and a reduction in pyruvate dehydrogenase (PDH) activity as compared to a parental microorganism.
  • the present invention provides recombinant microorganisms that comprise a pathway for the fermentation of isobutanol from a pentose sugar.
  • the pentose sugar is xylose.
  • the recombinant microorganism is engineered to express a functional xylose isomerase (Xl).
  • the recombinant microorganism further comprises a deletion or disruption of a native gene encoding for an enzyme that catalyzes the conversion of xylose to xylitol.
  • the native gene is xylose reductase (XR).
  • the native gene is xylitol dehydrogenase (XDH).
  • both native genes are deleted or disrupted.
  • the recombinant microorganism further engineered to express xylulose kinase which catalyzes the conversion of xylulose to xylu!ose-5-phosphate.
  • the microorganisms of the present invention are engineered to grow on glucose independently of C2-compounds at a growth rate substantially equivalent to the growth rate of a parental microorganism without altered PDC activity.
  • the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Saccharomyces sensu stricto microorganisms, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.
  • the recombinant microorganisms may be yeast recombinant microorganisms of the Saccharomyces clade.
  • the recombinant microorganisms may be Saccharomyces sensu stricto microorganisms.
  • Saccharomyces sensu stricto is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrids thereof.
  • the recombinant microorganisms may be Crabtree- negative recombinant yeast microorganisms.
  • the Crabtree- negative yeast microorganism is classified into a genera selected from the group consisting of Kluyveromyces, Pichia, Hansenula, or Candida.
  • the Crabtree-negative yeast microorganism is selected from Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, P. kudriavzevii, Hansenula anomala, Candida utilis and Kluyveromyces waltii.
  • the recombinant microorganisms may be Crabtree- positive recombinant yeast microorganisms.
  • the Crabtree- positive yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Candida, Pichia and Schizosaccharomyces.
  • the Crabtree-positive yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, Saccharomyces kluyveri, Kluyveromyces thermotolerans, Candida glabrata, Z. bailli, Z. rouxii, Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces uvarum.
  • the recombinant microorganisms may be post- WGD (whole genome duplication) yeast recombinant microorganisms.
  • the post-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces or Candida.
  • the post-WGD yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and Candida glabrata.
  • the recombinant microorganisms may be pre-WGD (whole genome duplication) yeast recombinant microorganisms.
  • the pre-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Debaryomyces, Hansenula, Pachysolen, Yarrowia 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, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yarrowia lipolytica, and Schizosaccharomyces pombe.
  • the recombinant microorganisms may be microorganisms that are non-fermenting yeast microorganisms, including, but not limited to those, classified into a genera selected from the group consisting of Tricosporon, Rhodotorula, or Myxozyma.
  • the present invention provides methods of producing isobutanol using a recombinant microorganism of the invention.
  • the method includes cultivating the recombinant microorganism in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the isobutanol is produced and optionally, recovering the isobutanol.
  • the microorganism is selected to produce isobutanol from a carbon source at a yield of at least about 5 percent theoretical.
  • the microorganism is selected to produce isobutanol at a yield of at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, or at least about 95 percent theoretical.
  • the microorganism is selected to produce isobutanol from a carbon source at a specific productivity of at least about 0.7 mg/L/hr per OD. In another embodiment, the microorganism is selected to produce isobutanol from a carbon source at a specific productivity of at least about 1 mg/L/hr per OD, at least about 10 mg/L/hr per OD, at least about 50 mg/L/hr per OD, at least about 100 mg/L/hr per OD, at least about 250 mg/L/hr per OD, or at least about 500 mg/L/hr per OD.
  • Figure 1 illustrates an exemplary embodiment of an isobutanol pathway.
  • Figure 2 illustrates production of pyruvate via glycolysis, together with an isobutanol pathway which converts pyruvate to isobutanol and a PDC pathway which converts pyruvate to acetaldehyde and carbon dioxide.
  • Figure 3 illustrates an isobutanol pathway receiving additional pyruvate to form isobutano! at higher yield due to the deletion or reduction of the PDC pathway.
  • Figure 4 illustrates an isobutanol pathway receiving additional pyruvate to form isobutanol at higher yield due to deletion or reduction of the PDC pathway and the deletion or reduction of the GPD pathway.
  • Figure 5 illustrates the carbon source composition and feeding rate over time during chemostat evolution of the S. cerevisiae Pdc-minus strain GEVO1584.
  • This graph shows how the acetate was decreased over a period of 480 hours from
  • dilution rate can be calculated by dividing the feeding rate by 200 ml.
  • Figure 6 illustrates growth of evolved Pdc-minus mutant strain GEVO1863 in YPD compared to the parental strain, GEVO1187.
  • Figure 7 illustrates that the evolved PCD mutant, GEVO1863, does not produce ethanol in YPD medium, unlike the parental strain GEVO1187.
  • Figure 8 illustrates a schematic map of plasmid pGV1503.
  • Figure 9 illustrates a schematic map of plasmid pGV1537.
  • Figure 10 illustrates a schematic map of plasmid pGV1429.
  • Figure 11 illustrates a schematic map of plasmid pGV1430.
  • Figure 12 illustrates a schematic map of plasmid pGV1431.
  • Figure 13 illustrates a schematic map of plasmid pGV1472.
  • Figure 14 illustrates a schematic map of plasmid pGV1473.
  • Figure 15 illustrates a schematic map of plasmid pGV1475.
  • Figure 16 illustrates a schematic map of plasmid pGV1254.
  • Figure 17 illustrates a schematic map of plasmid pGV1295.
  • Figure 18 illustrates a schematic map of plasmid pGV1390.
  • Figure 19 illustrates a schematic map of plasmid pGV1438.
  • Figure 20 illustrates a schematic map of plasmid pGV1590.
  • Figure 21 illustrates a schematic map of plasmid pGV1726.
  • Figure 22 illustrates a schematic map of plasmid pGV1727.
  • Figure 23 illustrates a schematic map of plasmid pGV1056.
  • Figure 24 illustrates a schematic map of plasmid pGV1062.
  • Figure 25 illustrates a schematic map of plasmid pGV1102.
  • Figure 26 illustrates a schematic map of plasmid pGV1103.
  • Figure 27 illustrates a schematic map of plasmid pGV1104.
  • Figure 28 illustrates a schematic map of plasmid pGV1106.
  • Figure 29 illustrates a schematic map of plasmid pGV1649.
  • Figure 30 illustrates a schematic map of plasmid pGV1664.
  • Figure 31 illustrates a schematic map of plasmid pGV1672.
  • Figure 32 illustrates a schematic map of plasmid pGV1673.
  • Figure 33 illustrates a schematic map of plasmid pGV1677.
  • Figure 34 illustrates a schematic map of plasmid pGV1679.
  • Figure 35 illustrates a schematic map of plasmid pGV1683.
  • Figure 36 illustrates a schematic map of plasmid pGV1565.
  • Figure 37 illustrates a schematic map of plasmid pGV1568.
  • Figure 38 illustrates a schematic map of plasmid pGV2082.
  • Figure 39 illustrates a schematic map of plasmid pGV2114.
  • Figure 40 illustrates a schematic map of plasmid pGV2117.
  • Figure 41 illustrates a schematic map of plasmid pGV2118.
  • microorganism includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista.
  • microbial cells and “microbes” are used interchangeably with the term microorganism.
  • Bacteria or “eubacteria” refers to a domain of prokaryotic organisms.
  • Bacteria include at least 11 distinct groups as follows: (1 ) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1 ) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic +non-photosynthetic Gram-negative bacteria (includes most "common" Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non- sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; (11 ) Thermoto
  • Gram-negative bacteria include cocci, nonenteric rods, and enteric rods.
  • the genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.
  • Gram positive bacteria include cocci, nonsporulating rods, and sporulating rods.
  • the genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.
  • the term "genus” is defined as a taxonomic group of related species according to the Taxonomic Outline of Bacteria and Archaea (Garrity, G. M., Lilburn, T.G., Cole, J. R., Harrison, S. H., Euzeby, J., and Tindall, B.J. (2007) The Taxonomic Outline of Bacteria and Archaea. TOBA Release 7.7, March 2007. Michigan State University Board of Trustees, [http://www.taxonomicoutline.org/]).
  • genomic hybridization is defined as a collection of closely related organisms with greater than 97% 16S ribosomal RNA sequence homology and greater than 70% genomic hybridization and sufficiently different from all other organisms so as to be recognized as a distinct unit.
  • recombinant microorganism refers to microorganisms that have been genetically modified to express or over-express endogenous polynucleotides, or to express heterologous polynucleotides, such as those included in a vector, 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.
  • expression with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein.
  • expression of a protein results from transcription and translation of the open reading frame sequence.
  • the level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the cell, or the amount of the desired product encoded by the selected sequence.
  • mRNA transcribed from a selected sequence can be quantitated by PCR or by northern hybridization (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
  • Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that are recognize and bind reacting the protein. See Sambrook et al., 1989, supra.
  • the polynucleotide generally encodes a target enzyme involved in a metabolic pathway for producing a desired metabolite. It is understood that the terms "recombinant microorganism” and “recombinant host cell” refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent ceil, but are still included within the scope of the term as used herein.
  • wild-type microorganism describes a cell that occurs in nature, i.e. a cell that has not been genetically modified.
  • a wild-type microorganism can be genetically modified to express or overexpress a first target enzyme.
  • This microorganism can act as a parental microorganism in the generation of a microorganism modified to express or overexpress a second target enzyme.
  • the microorganism modified to express or overexpress a first and a second target enzyme can be modified to express or overexpress a third target enzyme.
  • a "parental microorganism" functions as a reference cell for successive genetic modification events.
  • Each modification event can be accomplished by introducing a nucleic acid molecule in to the reference cell.
  • the introduction facilitates the expression or overexpression of a target enzyme.
  • the term “facilitates” encompasses the activation of endogenous polynucleotides encoding a target enzyme through genetic modification of e.g., a promoter sequence in a parental microorganism. It is further understood that the term “facilitates” encompasses the introduction of heterologous polynucleotides encoding a target enzyme in to a parental microorganism
  • engine refers to any manipulation of a microorganism that result 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.
  • metabolism engineered or “metabolic engineering” involves rational pathway design and assembly of biosynthetic genes, genes associated with operons, and control elements of such polynucleotides, for the production of a desired metabolite.
  • Methodically engineered can further include optimization of metabolic flux by regulation and optimization of transcription, translation, protein stability and protein functionality using genetic engineering and appropriate culture condition including the reduction of, disruption, or knocking out of, a competing metabolic pathway that competes with an intermediate leading to a desired pathway.
  • the terms "metabolically engineered microorganism” and “modified microorganism” are used interchangeably herein and refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. 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.
  • mutation indicates any modification of a nucleic acid and/or polypeptide which results in an altered nucleic acid or polypeptide. Mutations include, for example, point mutations, deletions, or insertions of single or multiple residues in a polynucleotide, which includes alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences.
  • a genetic alteration may be a mutation of any type. For instance, the mutation may constitute a point mutation, a frame-shift mutation, an insertion, or a deletion of part or all of a gene.
  • a portion of the microorganism genome has been replaced with a heterologous polynucleotide.
  • the mutations are naturally- occurring.
  • the mutations are the results of artificial selection pressure.
  • the mutations in the microorganism genome are the result of genetic engineering.
  • biosynthetic pathway also referred to as “metabolic pathway” refers to a set of anabolic or catabolic biochemical reactions for converting one chemical species into another.
  • Gene products belong to the same “metabolic pathway” if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product.
  • heterologous indicates molecules that are expressed in an organism other than the organism from which they originated or are found in nature, independently of the level of expression that can be lower, equal or higher than the level of expression of the molecule in the native microorganism.
  • native or “endogenous” as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicates molecules that are expressed in the organism in which they originated or are found in nature, independently of the level of expression that can be lower equal or higher than the level of expression of the molecule in the native microorganism. It is understood that expression of native enzymes or polynucleotides may be modified in recombinant microorganisms.
  • 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 metabolically engineered microorganism as described herein.
  • C2-compound as used as a carbon source for engineered yeast microorganisms with mutations in all pyruvate decarboxylase (PDC) genes resulting in a reduction of pyruvate decarboxylase activity of said genes refers to organic compounds comprised of two carbon atoms, including but not limited to ethanol and acetate.
  • 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.
  • cell dry weight or “CDW” refers to the weight of the microorganism after the water contained in the microorganism has been removed using methods known to one skilled in the art. CDW is reported in grams.
  • biofuel refers to a fuel in which all carbon contained within the fuel is derived from biomass and is biochemically converted, at least in part, in to a fuel by a microorganism.
  • a biofuel is further defined as a non-ethanol compound which contains less than 0.5 oxygen atoms per carbon atom.
  • a biofuel is a fuel in its own right, but may be blended with petroleum-derived fuels to generate a fuel.
  • a biofuel may be used as a replacement for petrochemically-derived gasoline, diesel fuel, or jet fuel.
  • volumetric productivity or “production rate” is defined as the amount of product formed per volume of medium per unit of time. Volumetric productivity is reported in gram per liter per hour (g/L/h).
  • specific productivity or “specific production rate” is defined as the amount of product formed per volume of medium per unit of time per amount of cells. Volumetric productivity is reported in gram or milligram per liter per hour per
  • yield is defined as the amount of product obtained per unit weight of raw material and may be expressed as g product per g substrate (g/g).
  • Yield may be expressed as a percentage of the theoretical yield. "Theoretical yield” is defined as the maximum amount of product that can be generated per a given amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to isobutanol is 0.41 g/g. As such, a yield of isobutanol from glucose of 0.39 g/g would be expressed as 95% of theoretical or 95% theoretical yield.
  • titer is defined as the strength of a solution or the concentration of a substance in solution.
  • concentration of a substance in solution For example, the titer of a biofuel in a fermentation broth is described as g of biofuel in solution per liter of fermentation broth (g/L).
  • a "facultative anaerobic organism” or a “facultative anaerobic microorganism” is defined as an organism that can grow in either the presence or in the absence of oxygen.
  • a "strictly anaerobic organism” or a “strictly anaerobic microorganism” is defined as an organism that cannot grow in the presence of oxygen and which does not survive exposure to any concentration of oxygen.
  • an "anaerobic organism” or an “anaerobic microorganism” is defined as an organism that cannot grow in the presence of oxygen.
  • “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.
  • 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 acetaldehyde, yielding ethanol.
  • Fermentative pathways are usually active under anaerobic conditions but may also occur under aerobic conditions, under conditions where NADH is not fully oxidized via the respiratory chain. For example, above certain glucose concentrations, Crabtree-positive yeasts produce large amounts of ethanol under aerobic conditions.
  • byproduct or "by-product” means an undesired product related to the production of a biofuel or biofuel precursor. Byproducts are generally disposed as waste, adding cost to a production process.
  • substantially free when used in reference to the presence or absence of enzymatic activities (PDC, GPD, PDH, etc.) in carbon pathways that compete with the desired metabolic pathway (e.g. an isobutanol-producing metabolic pathway) means the level of the enzyme is substantially less than that of the same enzyme 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.
  • 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 CO 2 from glucose.
  • Non-fermentative yeast can be identified by the "Durham Tube Test” (J.A. Barnett, R.W. Payne, and D. Yarrow. 2000. Yeasts Characteristics and Identification. 3 rd edition, p. 28-29. Cambridge University Press, Cambridge, UK.) or by monitoring the production of fermentation productions such as ethanol and CO 2 .
  • polynucleotide is used herein interchangeably with the term “nucleic acid” and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single stranded or double stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or antisense ribonucleic acid (RNA) of any length, including siRNA.
  • DNA single stranded or double stranded
  • RNA ribonucleic acid
  • nucleotide refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or a pyrimidine base and to a phosphate group, and that are the basic structural units of nucleic acids.
  • nucleoside refers to a compound (as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids.
  • nucleotide analog or “nucleoside analog” refers, respectively, to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called nucleotidic oligomer or oligonucleotide.
  • the polynucleotides described herein include “genes” and that the nucleic acid molecules described herein include “vectors” or “plasmids.”
  • the term “gene”, also called a “structural gene” refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non- transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed.
  • the transcribed region of the gene may include untranslated regions, including introns, 5'-untranslated region (UTR), and 3'-UTR, as well as the coding sequence.
  • operon refers to two or more genes which are transcribed as a single transcriptional unit from a common promoter.
  • the genes comprising the operon are contiguous genes. It is understood that transcription of an entire operon can be modified (i.e., increased, decreased, or eliminated) by modifying the common promoter.
  • any gene or combination of genes in an operon can be modified to alter the function or activity of the encoded polypeptide.
  • the modification can result in an increase in the activity of the encoded polypeptide.
  • the modification can impart new activities on the encoded polypeptide. Exemplary new activities include the use of alternative substrates and/or the ability to function in alternative environmental conditions.
  • a "vector” is any means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components.
  • Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes), and the like, that are "episomes,” that is, that replicate autonomously or can integrate into a chromosome of a host cell.
  • a vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine -conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that are not episomal in nature, or it can be an organism which comprises one or more of the above polynucleotide constructs such as an agrobacterium or a bacterium.
  • Transformation refers to the process by which a vector is introduced into a host cell.
  • Transformation can be achieved by any one of a number of means including chemical transformation (e.g. lithium acetate transformation), electroporation, microinjection, biolistics (or particle bombardment- mediated delivery), or agrobacterium mediated transformation.
  • chemical transformation e.g. lithium acetate transformation
  • electroporation e.g. electroporation
  • microinjection e.g. electroporation
  • biolistics e.g. electroporation
  • microinjection e.g. lithium acetate transformation
  • biolistics or particle bombardment- mediated delivery
  • agrobacterium mediated transformation e.g. electroporation, microinjection, biolistics (or particle bombardment- mediated delivery), or agrobacterium mediated transformation.
  • enzyme refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide, but can include enzymes composed of a different molecule including polynucle
  • 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 enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.
  • a protein has "homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein.
  • a protein has homology to a second protein if the two proteins have "similar” amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences).
  • analogs refers to nucleic acid or protein sequences or protein structures that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure.
  • reduced activity and/or expression of an endogenous protein such an enzyme can mean either a reduced specific catalytic activity of the protein (e.g. reduced activity) and/or decreased concentrations of the protein in the cell (e.g. reduced expression), while “deleted activity and/or expression” of an endogenous protein such an enzyme can mean either no or negligible specific catalytic activity of the enzyme (e.g. deleted activity)and/or no or negligible concentrations of the enzyme in the cell (e.g. deleted expression).
  • reduced pyruvate decarboxylase activity means either a decreased concentration of the pyruvate decarboxylase enzyme in the cell or reduced or no specific catalytic activity of the pyruvate decarboxylase enzyme.
  • reduced glycerol-3-phosphate dehydrogenase activity means either a decreased concentration of the glycerol-3-phosphate dehydrogenase enzyme in the cell or reduced or no specific catalytic activity of the glycerol-3- phosphate dehydrogenase enzyme.
  • reduced pyruvate dehydrogenase activity means either a decreased concentration of the pyruvate dehydrogenase enzyme in the cell or reduced or no specific catalytic activity of the pyruvate dehydrogenase enzyme.
  • reduced xylose reductase activity means either a decreased concentration of the xylose reductase enzyme in the cell or reduced or no specific catalytic activity of the xylose reductase enzyme.
  • reduced xylitol dehydrogenase activity means either a decreased concentration of xylitol dehydrogenase enzyme in the cell or reduced or no specific catalytic activity of the xylitol dehydrogenase enzyme.
  • Yeast cells produce pyruvate from sugars, which is then utilized in a number of pathways of cellular metabolism. Yeast cells can be engineered to produce a number of desirable products with the initial biosynthetic pathway step being conversion of endogenous pyruvate to acetolactate.
  • the present inventors have observed that by combining the expression of a cytosolically localized acetolactate synthase enzyme with reduced pyruvate decarboxylase (PDC) activity and/or reduced glycerol-3-phosphate dehydrogenase (GPD) activity, an unexpectedly high flux from pyruvate to acetolactate can be achieved.
  • the invention provides yeast cells that are engineered to exhibit an efficient conversion of pyruvate to acetolactate in the cytoplasm due to suppression of competing metabolic pathways.
  • the present invention has utility for the production of any acetolactate-derived product, including, but not limited to, isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, valine, leucine, and 3-methyl-1-butanol.
  • the present invention provides recombinant microorganisms that comprise an isobutanol producing metabolic pathway.
  • Recombinant microorganisms provided herein can express a plurality of heterologous and/or native target enzymes involved in pathways for the production isobutanol from a suitable carbon source.
  • metabolically "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 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 isobutanol.
  • 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 alteration, disruption, deletion or knocking-out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism.
  • alteration, disruption, deletion or knocking-out of a gene or polynucleotide the microorganism acquires new or improved properties (e.g., the ability to produce a new metabolite or greater quantities of an intracellular metabolite, improve the flux of a metabolite down a desired pathway, and/or reduce the production of byproducts).
  • Recombinant microorganisms provided herein may also produce metabolites in quantities not available in the parental microorganism.
  • a "metabolite” refers to any substance produced by metabolism or a substance necessary for or taking part in a particular metabolic process.
  • a metabolite can be an organic compound that is a starting material (e.g., glucose or pyruvate), an intermediate (e.g., 2-ketoisovalerate), or an end product (e.g., isobutanol) of metabolism.
  • Metabolites can be used to construct more complex molecules, or they can be broken down into simpler ones.
  • Intermediate metabolites may be synthesized from other metabolites, perhaps used to make more complex substances, or broken down into simpler compounds, often with the release of chemical energy.
  • Exemplary metabolites include glucose, pyruvate, and isobutanol.
  • the metabolite isobutanol can be produced by a recombinant microorganism metabolically engineered to express or over-express a metabolic pathway that converts pyruvate to isobutanol.
  • An exemplary metabolic pathway that converts pyruvate to isobutanol may be comprised of an acetohydroxy acid synthase (ALS), a ketolacid reductoisomerase (KARI), a dihyroxy-acid dehydratase (DHAD), a 2-keto- acid decarboxylase (KIVD), and an alcohol dehydrogenase (ADH).
  • ALS acetohydroxy acid synthase
  • KARI ketolacid reductoisomerase
  • DHAD dihyroxy-acid dehydratase
  • KIVD 2-keto- acid decarboxylase
  • ADH alcohol dehydrogenase
  • recombinant microorganisms that produce isobutanol and in some aspects may include the elevated expression of target enzymes such as ALS, KARl, DHAD, KIVD, and ADH.
  • 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 mutation 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 they 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.
  • 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.
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).
  • R group side chain
  • a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see, e.g., Pearson W.R. Using the FASTA program to search protein and DNA sequence databases, Methods in Molecular Biology, 1994, 25:365-89, hereby incorporated herein by reference).
  • the following six groups each contain amino acids that are conservative substitutions for one another: 1 ) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) lsoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  • Sequence homology for polypeptides is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap” and "Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutant protein thereof. See, e.g., GCG Version 6.1.
  • 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 (Altschul, S. F., et al. (1990) "Basic local alignment search tool.” J. MoI. Biol. 215:403-410; Gish, W. and States, DJ. (1993) "Identification of protein coding regions by database similarity search.” Nature Genet. 3:266-272; Madden, T. L., et al. (1996) "Applications of network BLAST server” Meth. Enzymol. 266:131- 141 ; Aitschul, S. F., et al.
  • Typical parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.
  • polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1.
  • FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, W. R. (1990) "Rapid and Sensitive Sequence Comparison with FASTP and FASTA" Meth. Enzymol. 183:63-98).
  • a percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1 , hereby incorporated herein by reference.
  • the disclosure provides metabolically engineered microorganisms comprising a biochemical pathway for the production of isobutanol from a suitable substrate at a high yield.
  • a metabolically engineered microorganism of the disclosure comprises one or more recombinant polynucleotides within the genome of the organism or external to the genome within the organism.
  • the microorganism can comprise a reduction, disruption or knockout of a gene found in the wild-type organism and/or introduction of a heterologous polynucleotide and/or expression or overexpression of an endogenous polynucleotide.
  • the disclosure provides a recombinant microorganism comprising elevated expression of at least one target enzyme as compared to a parental microorganism or encodes an enzyme not found in the parental organism.
  • the microorganism comprises a reduction, disruption or knockout of at least one gene encoding an enzyme that competes with a metabolite necessary for the production of isobutanol.
  • the recombinant microorganism produces at least one metabolite involved in a biosynthetic pathway for the production of isobutanol.
  • the recombinant microorganisms comprises at least one recombinant metabolic pathway that comprises a target enzyme and may further include a reduction in activity or expression of an enzyme in a competitive biosynthetic pathway.
  • the pathway acts to modify a substrate or metabolic intermediate in the production of isobutanol.
  • the target enzyme is encoded by, and expressed from, a polynucleotide derived from a suitable biological source.
  • the polynucleotide comprises a gene derived from a prokaryotic or eukaryotic source and recombinantly engineered into the microorganism of the disclosure.
  • the polynucleotide comprises a gene that is native to the host organism.
  • microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of isobutanol.
  • 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.
  • Carbon sources include, but are not limited to, biomass hydrolysates, starch, sucrose, cellulose, hemicellulose, xylose, and lignin, as well as monomeric components of these substrates.
  • Carbon sources can comprise various organic compounds in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, etc.
  • biomass hydrolysates refers primarily to the stems, leaves, and starch-containing portions of green plants, and is mainly comprised of starch, lignin, cellulose, hemicellulose, and/or pectin.
  • Biomass can be decomposed by either chemical or enzymatic treatment to the monomeric sugars and phenols of which it is composed (Wyman, CE. 2003 Biotechnological Progress 19:254-62). This resulting material, called biomass hydrolysate, is neutralized and treated to remove trace amounts of organic material that may adversely affect the biocatalyst, and is then used as a feed stock for fermentations using a biocatalyst.
  • the recombinant microorganism herein disclosed can convert a variety of carbon sources to products, including but not limited to glucose, galactose, mannose, xylose, arabinose, lactose, sucrose, and mixtures thereof.
  • the recombinant microorganism may thus further include a pathway for the fermentation 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 phosphorylated via a xylulokinase (XK) enzyme.
  • XR xylose reductase
  • XDH xylitol dehydrogenase
  • XK xylulokinase
  • This pathway operates inefficiently in yeast species because it introduces a redox imbalance in the cell.
  • the xylose-to- xylitol step uses NADH as a cofactor
  • the xylitol-to-xylulose step uses NADPH as 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 sugar. Accordingly, a yeast species that can efficiently ferment xylose and other pentose sugars into a desired fermentation product is therefore very desirable.
  • the recombinant is engineered to express a functional exogenous xylose isomerase.
  • Exogenous xylose isomerases functional in yeast are known in the art. See, e.g., Rajgarhia et al, US20060234364, which is herein incorporated by reference in its entirety.
  • the exogenous xylose isomerase gene is operatively linked to promoter and terminator sequences that are functional in the yeast cell.
  • the recombinant microorganism 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.
  • an enzyme e.g. XR and/or XDH
  • the recombinant microorganism is engineered to exhibit reduced xylose reductase (XR) activity.
  • XDH xylitol dehydrogenase
  • the recombinant microorganism also contains a functional, exogenous xylulokinase (XK) gene operatively linked to promoter and terminator sequences that are functional in the yeast cell.
  • XK xylulokinase
  • the microorganism has reduced or no pyruvate decarboxylase (PDC) activity.
  • PDC catalyzes the decarboxylation of pyruvate to acetaldehyde, which is then reduced to ethanol by ADH via an oxidation of NADH to NAD+.
  • Ethanol production is the main pathway to oxidize the NADH from glycolysis. Deletion of this pathway increases the pyruvate and the reducing equivalents (NADH) available for the isobutanol pathway. Accordingly, deletion of PDC genes further increases the yield of isobutanol.
  • the microorganism has reduced or no glycerol-3- phosphate dehydrogenase (GPD) activity.
  • GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD+.
  • Glycerol is then produced from G3P by Glycerol-3- phosphatase (GPP).
  • Glycerol production is a secondary pathway to oxidize excess NADH from glycolysis. Reduction or elimination of this pathway increases the pyruvate and reducing equivalents (NADH) available for the isobutanol pathway.
  • NADH pyruvate and reducing equivalents
  • deletion of GPD genes further increases the yield of isobutanol.
  • the microorganism has reduced or no PDC activity and reduced or no GPD activity.
  • Another characteristic may include the property that the wild-type or parental microorganism 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.
  • Non-fermenting yeast refers to both naturally occurring yeasts as well as genetically modified yeast.
  • Ethanol is produced by alcohol dehydrogenase (ADH) via the reduction of acetaldehyde, 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 isobutanol pathway.
  • Fermentative pathways contribute to low yield and low productivity of isobutanol. Accordingly, deletion of PDC may increase yield and productivity of isobutanol.
  • a third characteristic may include the property that the biocatalyst is selected to convert various carbon sources into isobutanol.
  • the yeast microorganisms may be selected from the "Saccharomyces Yeast Clade", defined as an ascomycetous yeast taxonomic class by Kurtzman and Robnett in 1998 ("Identification and phylogeny of ascomycetous yeast from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences.”
  • Antonie van Leeuwenhoek 73: 331-371 See Figure 2 of Leeuwenhoek reference). They were able to determine the relatedness of approximately 500 yeast species by comparing the nucleotide sequence of the D1/D2 domain at the 5' end of the gene encoding the large ribosomal subunit 26S.
  • Saccharomyces sensu stricto taxonomy group is a cluster of yeast species that are highly related to S. cerevisiae (Rainieri, S. et al 2003. Saccharomyces Sensu Stricto: Systematics, Genetic Diversity and Evolution. J. Biosci Bioengin 96(1 )1-9. Saccharomyces sensu stricto yeast species include but are not limited to S. cerevisiae, S. cerevisiae, S.
  • yeast can be divided into species that diverged from a common ancestor following the WGD event (termed “post-WGD yeast” herein) and species that diverged from the yeast lineage prior to the WGD event (termed “pre- WGD yeast” herein).
  • 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. glabrata.
  • the yeast microorganism may be selected from a pre-whole genome duplication (pre-WGD) yeast genus including but not limited to Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Yarrowia and, Schizosaccharomyces.
  • Representative pre-WGD yeast species include: S. kluyveh, K. thermotolerans, K. marxianus, K. waltii, K. lactis, C. tropicalis, P. pastoris, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I. scutulata, D. hansenii, H. anomala, Y. lipolytica, and S. pombe.
  • a yeast microorganism may be either Crabtree-negative or Crabtree- positive.
  • a yeast cell having a Crabtree-negative phenotype is any yeast cell that does not exhibit the Crabtree effect.
  • the term "Crabtree-negative” refers to both naturally occurring and genetically modified organisms. Briefly, the Crabtree effect is defined as the inhibition of oxygen consumption by a microorganism when cultured under aerobic conditions due to the presence of a high concentration of glucose (e.g., 50 g-glucose L "1 ).
  • the yeast microorganism may be selected from yeast with a Crabtree-negative phenotype including but not limited to the following genera: Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida.
  • Crabtree-negative species include but are not limited to: K. lactis, K. marxianus, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I.
  • the yeast microorganism may be selected from a 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, S. kluyveri, K. thermotolerans, C. glabrata, Z. bailli, Z. rouxii, D. hansenii, P. pastorius, and S. 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, or Myxozyma.
  • a yeast microorganism is engineered to convert a carbon source, such as glucose, to pyruvate by glycolysis and the pyruvate is converted to isobutanol via an engineered isobutanol pathway (See, e.g.,
  • the engineered isobutanol pathway to convert pyruvate to isobutanol can be comprised of the following reactions:
  • the yeast microorganism is engineered to overexpress these enzymes.
  • these enzymes can be encoded by native genes.
  • these enzymes can be encoded by heterologous genes.
  • ALS can be encoded by the alsS gene of B, subtilis, alsS of L. lactis, or the HvK gene of K. pneumonia.
  • KARI can be encoded by the HvC genes of E. coli, C. glutamicum, M. maripaludis, or Piromyces sp E2.
  • DHAD can be encoded by the HvD genes of E. coli, C. glutamicum, or L. lactis.
  • KIVD can be encoded by the kivD gene of L. lactis.
  • ADH can be encoded by ADH2,
  • pathway steps 2 and 5 may be carried out by KARI and ADH enzymes that utilize NADH (rather than NADPH) as a co-factor.
  • NADH rather than NADPH
  • Such enzymes are described in commonly owned and co-pending applications US 12/610,784 and PCT/US09/62952 (published as WO/2010/051527), which are herein incorporated by reference in their entireties for all purposes.
  • the present inventors have found that utilization of NADH-dependent KARI and ADH enzymes to catalyze pathway steps 2 and 5, respectively, surprisingly enables production of isobutanol under anaerobic conditions.
  • the recombinant microorganisms of the present invention may use an NADH-dependent KARI to catalyze the conversion of acetolactate (+NADH) to produce 2, 3- dihydroxyisovalerate.
  • the recombinant microorganisms of the present invention may use an NADH-dependent ADH to catalyze the conversion of isobutyraldehyde (+NADH) to produce isobutanol.
  • the recombinant microorganisms of the present invention may use both an NADH-dependent KARI to catalyze the conversion of acetolactate (+NADH) to produce 2, 3- dihydroxyisovalerate, and an NADH-dependent ADH to catalyze the conversion of isobutyraldehyde (+NADH) to produce isobutanol.
  • the yeast microorganism of the invention may be engineered to have increased ability to convert pyruvate to isobutanol.
  • the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutyraldehyde.
  • the yeast microorganism may be engineered to have increased ability to convert pyruvate to keto-isovalerate.
  • the yeast microorganism may be engineered to have increased ability to convert pyruvate to 2, 3-dihydroxyisovalerate.
  • 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.
  • genes encoding these enzymes can be identified from other fungal and bacterial species and can be expressed for the modulation of this pathway. A variety of organisms could serve as sources for these enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S.
  • 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 coli, Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcu s spp., Enterobacter spp., and Salmonella spp.
  • any method can be used to identify genes that encode for enzymes with pyruvate decarboxylase (PDC) activity.
  • PDC catalyzes the decarboxylation of pyruvate to form acetaldehyde.
  • homologous or similar PDC genes and/or homologous or similar PDC enzymes can be identified by functional, structural, and/or genetic analysis. In most cases, homologous or similar PDC genes and/or homologous or similar PDC enzymes will have functional, structural, or genetic similarities. Techniques known to those skilled in the art may be suitable to identify 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 PDC 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 PDC genes.
  • degenerate PCR using degenerate primers designed to amplify a conserved region among PDC genes.
  • one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity.
  • Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity, then isolating the enzyme with said activity through purification, determining the protein sequence of the enzyme through techniques such as Edman degradation, design of PCR primers to the likely nucleic acid sequence, amplification of said DNA sequence through PCR, and cloning of said nucleic acid sequence.
  • 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.
  • PDC activity can be determined phenotypically. For example, ethanol production under fermentative conditions can be assessed. A lack of ethanol production may be indicative of a yeast microorganism with no PDC activity.
  • yeast pyruvate decarboxylase genes that may be targeted for disruption may be found in US Pat. No. 7,326,550.
  • Target genes for disruption include, but are not limited, to PDC1 (GenBank Accession No. CAA97573.1 ), PDC5 (GenBank Accession No. CAA97705.1 ), and PDC6 (GenBank Accession No. CAA97089.1 ) from S. cerevisiae, as well as genes encoding pyruvate decarboxylases from K. lactis (GenBank Accession No.
  • target genes such as those encoding pyruvate decarboxylase proteins having at least about 50-55%, 55%-60%, 60-65%, 65%-70%, 75-80%, 80-85%, 85%-90%, 90%-95%, or at least about 98% sequence identity to the S. cerevisiae pyruvate decarboxylases may be identified in the literature and in bio informatics databases well known to the skilled person.
  • GPD glycerol-3-phosphate dehydrogenase
  • DHAP dihydroxyacetone phosphate
  • G3P glycerol-3-phosphate
  • homologous or similar GPD genes and/or homologous or similar GPD enzymes can be identified by functional, structural, and/or genetic analysis. In most cases, homologous or similar GPD genes and/or homologous or similar GPD enzymes will have functional, structural, or genetic similarities. Techniques known to those skilled in the art may be suitable to identify 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. For example, to identify homologous or analogous genes, proteins, or enzymes, techniques may include, but not limited to, cloning a GPD 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 GPD genes. Further, one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity.
  • Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity, then isolating the enzyme with said activity through purification, determining the protein sequence of the enzyme through techniques such as Edman degradation, design of PCR primers to the likely nucleic acid sequence, amplification of said DNA sequence through PCR, and cloning of said nucleic acid sequence.
  • 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.
  • GPD activity can be determined phenotypically. For example, glycerol production under fermentative conditions can be assessed. A lack of glycerol production may be indicative of a yeast microorganism with no GPD activity. Examples of yeast glycerol-3-phosphate dehydrogenase genes that may be targeted for disruption may be found in US 2009/0053782. Other target genes, such as those encoding glycerol-3-phosphate dehydrogenase proteins having at least about 50-
  • S. cerevisiae glycerol-3-phosphate dehydrogenases may be identified in the literature and in bioinformatics databases well known to the skilled person.
  • 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/., Nucleic Acids Res. 27:69-74 (1992); lto et a/., J. Bactehol. 153:163-168 (1983); and Becker and Guarente, Methods in Enzymology 194:182- 187 (1991).
  • 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.
  • integration of a gene into the chromosome of the yeast microorganism may occur via random integration (Kooistra, R., Hooykaas, P.J.J. , Steensma, H.Y. 2004. Yeast 21 : 781-792).
  • certain introduced marker genes are removed from the genome using techniques well known to those skilled in the art.
  • 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, J. et al, 1984, MoI. Gen. Genet, 197, 345-47).
  • FOA fluoro-orotic acid
  • the exogenous nucleic acid molecule contained within a yeast cell of the disclosure can be maintained within that cell in any form.
  • exogenous nucleic acid molecules can be integrated into the genome of the cell or maintained in an episomal state that can stably be passed on ("inherited") to daughter cells.
  • extra-chromosomal genetic elements can additionally contain selection markers that ensure the presence of such genetic elements in daughter cells.
  • 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 pyruvate decarboxylase activity.
  • reduced refers to a lower level of enzymatic activity than that measured in a comparable yeast cell of the same species.
  • reduced also refers to the elimination of enzymatic activity than that measured in a comparable yeast cell of the same species.
  • yeast cells lacking pyruvate decarboxylase activity are considered to have reduced pyruvate decarboxylase activity since most, if not all, comparable yeast strains have at least some pyruvate decarboxylase activity.
  • Such reduced enzymatic activities can be the result of lower enzyme concentration, lower specific activity of an enzyme, or a combination thereof.
  • Many different methods can be used to make yeast having reduced enzymatic activity.
  • a yeast cell can be engineered to have a disrupted enzyme-encoding locus using common mutagenesis or knock-out technology. See, e.g., Methods in Yeast Genetics (1997 edition), Adams, Gottschling, Kaiser, and Stems, Cold Spring Harbor Press (1998).
  • certain point-mutation(s) can be introduced which results in an enzyme with reduced activity.
  • antisense technology can be used to reduce enzymatic activity.
  • yeast can be engineered to contain a cDNA that encodes an antisense molecule that prevents an enzyme from being made.
  • antisense molecule 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 general structure including, without limitation, hairpin, hammerhead, or axhead structures, provided the molecule cleaves RNA.
  • yeast having a reduced enzymatic activity can be identified using many methods. For example, yeast having reduced pyruvate decarboxylase activity can be easily identified using common methods, which may include, for example, measuring ethanol formation via gas chromatography.
  • Methods for overexpressing a polypeptide from a native or heterologous nucleic acid molecule are well known. Such methods include, without limitation, constructing a nucleic acid sequence such that a regulatory element promotes the expression of a nucleic acid sequence that encodes the desired polypeptide.
  • 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. 4,859,596 and 4,943,529, for Kluyveromyces and, e.g., Gellissen et ai, Gene 190(1 ):87-97 (1997) for Saccharomyces).
  • Yeast plasmids have a selectable marker and an origin of replication.
  • certain plasmids may also contain a centromeric sequence. These centromeric plasmids are generally a single or low copy plasmid.
  • Plasmids without a centromeric sequence and utilizing either a 2 micron (S. cerevisiae) or 1.6 micron (K. lactis) replication origin are high copy plasmids.
  • 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 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, PCR, RT-PCR, and nucleic acid hybridization techniques such as Northern and Southern analysis, altered growth capabilities on a particular substrate or in the presence of a particular substrate, a chemical compound, a selection agent and the like. In some cases, immunohistochemistry and biochemical techniques can be used to determine if a cell contains a particular nucleic acid by detecting the expression of the encoded polypeptide.
  • 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 cell 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, Appl. Microbiol. Biotechnol. 38:17-22 (1992). Increase of enzymatic activity
  • Yeast microorganisms of the invention may be further engineered to have increased activity of enzymes.
  • 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 isobutanol pathway would result in increased productivity and yield of isobutanol.
  • Methods to increase enzymatic activity are known to those skilled in the art.
  • Such techniques may include increasing the expression of the enzyme by increased copy number and/or use of a strong promoter, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the Km for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231 ), ed. Arnold and Georgiou, Humana Press (2003).
  • the biocatalyst herein disclosed can convert various carbon sources into isobutanol.
  • the term "carbon source” generally refers to a substance suitable to be used as a source of carbon for prokaryotic or eukaryotic cell growth.
  • Carbon sources include, but are not limited to, biomass hydrolysates, starch, sucrose, cellulose, hemicellulose, xylose, and lignin, as well as monomeric components of these substrates.
  • Carbon sources can comprise various organic compounds in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, etc.
  • photosynthetic organisms can additionally produce a carbon source as a product of photosynthesis.
  • carbon sources may be selected from biomass hydrolysates and glucose.
  • C2-compound as used as a carbon source for engineered yeast microorganisms with mutations in all pyruvate decarboxylase (PDC) genes resulting in a reduction of pyruvate decarboxylase activity of said genes refers to organic compounds comprised of two carbon atoms, including but not limited to ethanol and acetate
  • 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.
  • traditional carbohydrates refers to sugars and starches generated from specialized plants, such as sugar cane, corn, and wheat. Frequently, these specialized plants concentrate sugars and starches in portions of the plant, such as grains, that are harvested and processed to extract the sugars and starches.
  • Traditional carbohydrates are used as food and also to a lesser extent as carbon sources for fermentation processes to generate biofuels, such as and chemicals
  • biomass refers primarily to the stems, leaves, and starch-containing portions of green plants, and is mainly comprised of starch, lignin, cellulose, hemicellulose, and/or pectin. Biomass can be decomposed by either chemical or enzymatic treatment to the monomeric sugars and phenols of which it is composed (Wyman, CE. 2003 Biotechnological Progress 19:254-62). This resulting material, called biomass hydrolysate, is neutralized and treated to remove trace amounts of organic material that may adversely affect the biocatalyst, and is then used as a feed stock for fermentations using a biocatalyst.
  • starch refers to a polymer of glucose readily hydrolyzed by digestive enzymes. Starch is usually concentrated in specialized portions of plants, such as potatoes, corn kernels, rice grains, wheat grains, and sugar cane stems.
  • lignin refers to a polymer material, mainly composed of linked phenolic monomeric compounds, such as p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, which forms the basis of structural rigidity in plants and is frequently referred to as the woody portion of plants. Lignin is also considered to be the non-carbohydrate portion of the cell wall of plants.
  • cellulose refers is a long-chain polymer polysaccharide carbohydrate of beta-glucose of formula (C6H10O5)n, usually found in plant cell wails in combination with lignin and any hemicellulose.
  • hemicellulose refers to a class of plant cell-wall polysaccharides that can be any of several heteropolymers. These include xylane, xyloglucan, arabinoxylan, arabinogalactan, glucuronoxylan, glucomannan and galactomannan.
  • Monomeric components of hemicellulose include, but are not limited to: D-galactose, L-galactose, D-mannose, L-rhamnose, L-fucose, D-xylose, L- arabinose, and D-glucuronic acid.
  • This class of polysaccharides is found in almost all cell walls along with cellulose. Hemicellulose is lower in weight than cellulose and cannot be extracted by hot water or chelating agents, but can be extracted by aqueous alkali.
  • Polymeric chains of hemicellulose bind pectin and cellulose in a network of cross-linked fibers forming the cell walls of most plant cells.
  • isobutanol For a biocatalyst to produce isobutanol most economically, it is desired to produce a high yield. Preferably, the only product produced is isobutanol. Extra products lead to a reduction in product yield and an increase in capital and operating costs, particularly if the extra products have little or no value. Extra products also require additional capital and operating costs to separate these products from isobutanol.
  • the microorganism may convert one or more carbon sources derived from biomass into isobutanol with a yield of greater than 5% of theoretical.
  • the yield is greater than 10%.
  • the yield is greater than 50% of theoretical.
  • the yield is greater than 60% of theoretical.
  • the yield is greater than 70% of theoretical.
  • the yield is greater than 80% of theoretical.
  • the yield is greater than 85% of theoretical.
  • the yield is greater than 90% of theoretical.
  • the yield is greater than 95% of theoretical.
  • the yield is greater than 97.5% of theoretical.
  • the microorganism converts glucose, which can be derived from biomass into isobutanol with a yield of greater than 5% of theoretical.
  • the yield is greater than 10% of theoretical. In one embodiment, the yield is greater than 50% of theoretical. In one embodiment the yield is greater than 60% of theoretical. In another embodiment, the yield is greater than 70% of theoretical. In yet another embodiment, the yield is greater than 80% of theoretical. In yet another embodiment, the yield is greater than 85% of theoretical. In yet another embodiment the yield is greater than 90% of theoretical. In yet another embodiment, the yield is greater than 95% of theoretical. In still another embodiment, the yield is greater than 97.5% of theoretical
  • Microorganism expressing a cytosolically localized acetolactate synthase (ALS) [00207]
  • yeasts such as S. cerevisiae
  • the native acetolactate synthase encoded in S. cerevisiae by the ILV2 gene, is naturally expressed in the yeast mitochondria.
  • expression of heterologous, acetolactate synthases such as the B. subtilis alsS and the L. lactis alsS in yeast occurs in the yeast cytosol (i.e. cytosolically-localized).
  • cytosolic expression of acetolactate synthase is achieved by transforming a yeast with a gene encoding an acetolactate synthase protein (EC 2.2.1.6).
  • ALS homologs that could be cytosolically expressed and localized in yeast are predicted to lack a mitochondrial targeting sequence as analyzed using mitoprot (Claras et al., 1996, Eur. J. Biochem 241 : 779-86). Such cytosolically localized ALS proteins can be used as the first step in the isobutanol pathway.
  • ALS homologs include, but are not limited to, the following: the Serratia marcescens ALS (GenBank Accession No. ADH43113.1 ) (probability of mitochondrial localization 0.07), the Enterococcus faecalis ALS (GenBank Accession No.
  • NP_814940 probability of mitochondrial localization 0.21
  • Leuconostoc mesenteroides GenBank Accession No. YP_818010.1
  • Staphylococcus aureus ALS GenBank Accession No. YP_417545
  • Burkholderia cenocepacia ALS GenBank Accession No.
  • YP_624435 (probability of mitochondrial localization 0.15), Trichoderma atroviride ALS (SEQ ID NO: 77) probability of mitochondrial localization 0.19), Talaromyces stipitatus ALS (SEQ ID NO: 78) (probability of mitochondrial localization 0.19), and Magnaporthe grisea ALS (GenBank Accession No. EDJ99221 ) (probability of mitochondrial localization 0.02).
  • an ALS enzyme that is predicted to be mitochondria I Iy localized may be mutated or modified to remove or modify an N-terminal mitochondrial targeting sequence (MTS) to remove or eliminate its ability to target the ALS enzyme to the mitochondria. Removal of the MTS can increase cytosolic localization of the ALS and/or increase the cytosolic activity of the ALS as compared to the parental ALS.
  • MTS N-terminal mitochondrial targeting sequence
  • yeasts Methods for gene expression in yeasts are known in the art (See, e.g., Methods in Enzymology, 2004, Vo1 194, Guide to Yeast Genetics and Molecular and Cell Biology).
  • the expression of heterologous, prokaryotic genes in yeast typically requires a promoter, operably linked to a coding region of interest, and a transcriptional terminator.
  • a number of yeast promoters can be used in constructing expression cassettes for genes encoding an acetolactate synthase, including, but not limited to constitutive promoters FBA, GPD1, ADH1, and GPM, and the inducible promoters GALI, GAL10, and CUPI.
  • Suitable transcriptional terminators include, but are not limited to FBA, GPD, GPM, ERG10, GAL1, CYC1, and ADH1.
  • Microorganism characterized by production of isobutanol from pyruvate via an overexpressed isobutanol pathway and a Pdc-minus phenotype [00211]
  • yeast the conversion of pyruvate to acetaldehyde is a major drain on the pyruvate pool ( Figure 2), and, hence, a major source of competition with the isobutanol pathway.
  • This reaction is catalyzed by the pyruvate decarboxylase (PDC) enzyme.
  • PDC pyruvate decarboxylase
  • Reduction of this enzymatic activity in the yeast microorganism results in an increased availability of pyruvate and reducing equivalents to the isobutanol pathway and may improve isobutanol production and yield in a yeast microorganism that expresses a pyruvate-dependent isobutanol pathway ( Figure 3).
  • Reduction of PDC activity can be accomplished by 1) mutation or deletion of a positive transcriptional regulator for the structural genes encoding for PDC or 2) mutation or deletion of all PDC genes in a given organism.
  • transcriptional regulator can specify a protein or nucleic acid that works in trans to increase or to decrease the transcription of a different locus in the genome.
  • the PDC2 gene which encodes for a positive transcriptional regulator of PDC1,5,6 genes can be deleted; a S. cerevisiae in which the PDC2 gene is deleted is reported to have only -10% of wildtype PDC activity (Hohmann, MoI Gen Genet, 241 :657-666 (1993)).
  • all structural genes for PDC e.g. in S. cerevisiae, PDC1, PDC5, and PDC6, or in K. lactis, PDC1 are deleted.
  • Crabtree-positive yeast strains such as S. cerevisiae strain that contains disruptions in all three of the PDC alleles no longer produce ethanol by fermentation.
  • acetyl-CoA a downstream product of the reaction catalyzed by PDC, acetyl-CoA, is needed for anabolic production of necessary molecules. Therefore, the Pdc- mutant is unable to grow solely on glucose, and requires a two-carbon carbon source, either ethanol or acetate, to synthesize acetyl-CoA.
  • such a Crabtree-positive yeast strain may be evolved to generate variants of the PDC mutant yeast that do not have the requirement for a two-carbon molecule and has a growth rate similar to wild type on glucose.
  • Any method, including chemostat evolution or serial dilution may be utilized to generate variants of strains with deletion of three PDC alleles that can grow on glucose as the sole carbon source at a rate similar to wild type (van Maris et al., Directed Evolution of Pyruvate Decarboxylase-Negative Saccharomyces cerevisiae, Yielding a C2-lndependent, Glucose-Tolerant, and Pyruvate-Hyperproducing Yeast, Applied and Environmental Microbiology, 2004, 70(1 ), 159-166).
  • Microorganism characterized by production of isobutanol from pyruvate via an overexpressed isobutanol pathway and a PDC-minus GPD-minus phenotvpe [00215] Another pathway for NADH oxidation is through the production of glycerol. Dihydroxyacetone-phosphate, an intermediate of glycolysis is reduced to glycerol 3- phosphate by glycerol 3-phosphate dehydrogenase (GPD). Glycerol 3-phosphatase (GPP) converts glycerol 3-phosphate to glycerol. This pathway consumes carbon from glucose as well as reducing equivalents (NADH) resulting in less pyruvate and reducing equivalents available for the isobutanol pathway. These pathways contribute to low yield and low productivity of isobutanol. Accordingly, deletions of PDC and GPD would increase yield and productivity of isobutanol. As exemplified in
  • a yeast microorganism may include a recombinant microorganism having an engineered pathway to convert a carbon source, such as glucose, to isobutanol.
  • Yield of isobutanol can be increased also by reduction of the glycerol 3- phosphate dehydrogenase (GPD, EC1.1.1.8) activity, which is involved in the production of glycerol (Fig. 2).
  • This enzyme catalyzes the reduction of the glycolysis intermediate, dihydroxyacetone-phosphate, to glycerol 3-phosphate.
  • an NADH is oxidized to NAD+. Therefore, glycerol production would be a drain on the reducing equivalent (NADH) as well as on the carbon from glucose.
  • NADH reducing equivalent
  • PDH Pyruvate dehydrogenase
  • Branched-chain amino acid transaminase EC 2.6.1.42 interconverts valine ⁇ keto-isovalerate in the cytosol, and may therefore reduce or limit available keto-isovalerate to isobutanol pathway.
  • 3- methyl-2-oxobutanoate hydroxymethyltransferase (EC 2.1.2.11 ) directs the isobutanol pathway intermediate, keto-isovalerate, to the coenzyme A synthesis pathway.
  • Alphaisopropylmalate isomerase (EC 4.1.3.12) directs the isobutanol pathway intermediate, keto-isovalerate, to the synthesis of leucine. Therefore, all of these enzymatic activities represent possible additional targets for disruption, deletion, or both.
  • Microorganism characterized by production of isobutanol from pyruvate via an overexpressed balanced isobutanol pathway and a PDC-minus GPD-minus phenotype
  • Adh ⁇ p or Adh7p which are NADPH-dependent alcohol dehydrogenases.
  • the additional NADPH can be obtained from the pentose phosphate pathway, but this results in a reduced yield as only 5 moles of pyruvate is generated from 3 moles of glucose, while glycolysis generates 6 moles of pyruvate from 3 moles of glucose.
  • glycolysis can be engineered to generate NADPH instead of NADH. This is accomplished by replacing the endogenous NAD+-dependent glyceraldehydes 3- phosphate dehydrogenase (GAPDH, EC 1.2.1.12) with an NADP+-dependent GAPDH (EC 1.2.1.13).
  • GAPDH endogenous NAD+-dependent glyceraldehydes 3- phosphate dehydrogenase
  • NADP+-dependent GAPDH EC 1.2.1.13
  • Such NADP+-dependent GAPDHs have been identified in bacteria (i.e. gapB in B. subtilis), yeast (GDP1 in K. lactis) and plants. (Fillinger et ai, J Biol Chem.
  • an NADP + -dependent GAPDH is co-expressed with the endogenous NAD + -dependent GAPDH. This may allow the production of both NADPH and NADH from glycolysis and balance the consumption of 1 mole of NADPH and 1 mole of NADH by an isobutanol pathway utilizing an NADH-dependent alcohoi dehydrogenase.
  • the NADPH-dependent KARI enzyme in the pathway is engineered to use NADH.
  • HvC E. coli KARI
  • a KARI from Methanococcus species can be used.
  • These KARI enzymes have been reported to be able to utilize NADH with roughly 60% the activity with NADPH (Xing ⁇ t al., Journal of Bacteriology 1990).
  • the use of these NADH-utilizing HvC in combination with an NADH-dependent alcohol dehydrogenase also balances the NADH/NADPH imbalance.
  • any of the genes encoding the foregoing enzymes may be subject to directed evolution using methods known to those of skill in the art. Such action allows those of skill in the art to optimize the enzymes for expression and activity in yeast.
  • genes encoding these enzymes can be identified from other fungal and bacterial species and can be expressed for the modulation of this pathway.
  • a variety of organisms could serve as sources for these enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K, thermotolerans, K, lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including Y.
  • 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 coli, Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., and Salmonella spp.
  • the engineered metabolic pathway may be balanced with respect to NADH and NADPH as compared to a native or unmodified metabolic isobutanol pathway from a corresponding parental microorganism, wherein the native or unmodified metabolic pathway is not balanced with respect to NADH and NADPH.
  • the ideal production microorganism produces a desirable product at close to theoretical yield.
  • the ideal isobutanol producing organism produces isobutanol according to the following equation: 1 glucose -> isobutanol + 2 CO 2 + H 2 O.
  • NADPH is produced only sparingly by the cell - the reduced cofactor nicotinamide adenine dinucleotide (NADH) is the preferred equivalent. Respiration is required to produce NADPH in the large quantities required to support high-level production of isobutanol.
  • NADH reduced cofactor nicotinamide adenine dinucleotide
  • the decreased yield and the loss of productivity upon O 2 limitation indicate that the strain uses one or more metabolic pathways to generate the NADPH needed to support isobutanol production.
  • the production of isobutanol from glucose results in an imbalance between the cofactors reduced during glycolysis and the cofactors oxidized during the conversion of pyruvate to isobutanol.
  • glycolysis produces two moles of NADH
  • the isobutanol pathway consumes two moles of NADPH. This leads to a deficit of two moles of NADPH and overproduction of two moles of NADH per isobutanol molecule produced, a state described henceforth as cofactor imbalance.
  • cofactor balance or “balanced with respect to cofactor usage” refer to a recombinant microorganism comprising a metabolic pathway converting a carbon source to a fermentation product and a modification that leads to the regeneration of all redox cofactors within the recombinant microorganism producing said fermentation product from a carbon source and wherein the re-oxidation or re- reduction of said redox cofactors does not require the pentose phosphate pathway, the TCA cycle or the generation of additional fermentation products.
  • cofactor balance or “balanced with respect to cofactor usage” can refer to an advantageous modification that leads to the regeneration of all redox cofactors within the recombinant microorganism producing a fermentation product from a carbon source and wherein said re-oxidation or re- reduction of all redox cofactors does not require the production of byproducts or co- products.
  • cofactor balance or “balanced with respect to cofactor usage” can refer to an advantageous modification that leads to the regeneration of all redox cofactors within the recombinant microorganism producing a fermentation product from a carbon source under anaerobic conditions and wherein the production of additional fermentation products is not required for re- oxidation or re-reduction of redox cofactors.
  • the terms "cofactor balance” or “balanced with respect to cofactor usage” can refer to an advantageous modification that leads to the regeneration of all redox cofactors within the recombinant microorganism producing a fermentation product from a carbon source and wherein said modification increases production of said fermentation product under anaerobic conditions compared to the parental or wild type microorganism and wherein additional fermentation products are not required for the regeneration of said redox cofactors.
  • the cell has several options for resolving a cofactor imbalance. One is to change the relative fluxes going from glucose through glycolysis and through the pentose phosphate pathway (PPP).
  • Another result of the flux through the PPP is that one additional molecule of CO 2 is lost per molecule of glucose consumed, which limits the yield of isobutanol that can be achieved under aerobic conditions.
  • Another way the cell can generate NADPH is via the TCA cycle. Flux through the TCA cycle results in carbon loss through CO 2 and in production of NADH in addition to the NADPH required for the isobutanol pathway. The NADH would have to be utilized for energy production through respiration under aerobic conditions (and without an alternate electron acceptor) or for the production of byproducts.
  • the TCA cycle likely is not functional under anaerobic conditions and is therefore unsuitable for the production of stoichiometric amounts of NADPH in an anaerobic isobutanol process.
  • Feedstock cost is the major component of the overall operating cost, regardless of the nature of the feedstock and its current market price. From an economical perspective, this is important because the cost of isobutanol is dependent on the cost of the biomass-derived sugars. An increase in feedstock cost results in an increase in isobutanol cost. Thus, it is desirable to utilize NADH-dependent enzymes for the conversion of pyruvate to isobutanol.
  • An enzyme is "NADH-dependent" if it 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.
  • a microorganism is provided in which cofactor usage is balanced during the production of a fermentation product.
  • a microorganism is provided in which cofactor usage is balanced during the production of isobutanol, in this case, production of isobutanol from pyruvate utilizes the same cofactor that is produced during glycolysis.
  • a microorganism in which cofactor usage is balanced during the production of a fermentation product and the microorganism produces the fermentation product at a higher yield compared to a modified microorganism in which the cofactor usage in not balanced.
  • a microorganism is provided in which cofactor usage is balanced during the production of isobutanol and the microorganism produces isobutanol at a higher yield compared to a modified microorganism in which the cofactor usage in not balanced.
  • a modified microorganism in which cofactor usage is balanced during the production of a fermentation product may allow the microorganism to produce said fermentation product under anaerobic conditions at higher rates, and yields as compared to a modified microorganism in which the cofactor usage in not balanced during production of a fermentation product.
  • a modified microorganism in which cofactor usage is balanced during the production of isobutanol may allow the microorganism to produce isobutanol under anaerobic conditions at higher rates, and yields as compared to a modified microorganism in which the cofactor usage is not balanced during production of isobutanol.
  • One compound to be produced by the recombinant microorganism according to the present invention is isobutanol.
  • the present invention is not limited to isobutanol.
  • the invention may be applicable to any metabolic pathway that is imbalanced with respect to cofactor usage.
  • One skilled in the art is able to identify pathways that are imbalanced with respect to cofactor usage and apply this invention to provide recombinant microorganisms in which the same pathway is balanced with respect to cofactor usage.
  • One skilled in the art will recognize that the identified pathways may be of longer or shorter length, contain more or fewer genes or proteins, and require more or fewer cofactors than the exemplary isobutanol pathway.
  • certain embodiments of the present invention may be adapted to convert NADPH to NADH.
  • a metabolic pathway that converts pyruvate to a target product that consumes either two moles of NADPH or one mole of NADH and one mole of NADPH leads to cofactor imbalance.
  • One example of such a metabolic pathway is the isobutanol metabolic pathway described by Atsumi et a/. (Atsumi et a/., 2008, Nature 451 : 86-9), which converts two moles of pyruvate to one mole of isobutanol.
  • two enzymes are dependent upon NADPH: (1 ) KARI and (2) ADH, encoded by the E. coli HvC and E. coli yqhD, respectively.
  • the present invention provides a recombinant microorganism in which the NADPH-dependent enzymes KARI and
  • ADH are replaced with enzymes that preferentially depend on NADH (i.e. KARI and
  • ADH enzymes that are NADH-dependent are NADH-dependent.
  • the present invention in another embodiment provides recombinant microorganisms wherein the NADH-dependent
  • such enzymes may be identified in nature.
  • such enzymes may be generated by protein engineering techniques including but not limited to directed evolution or site-directed mutagenesis.
  • the two NADPH-dependent enzymes within an isobutanol biosynthetic pathway that converts pyruvate to isobutanol may be replaced with ones that utilize NADH.
  • These two enzymes may be KARI and an alcohol dehydrogenase (ADH).
  • NADH-dependent enzymes that catalyze the same reaction as the NADH-dependent enzymes are overexpressed. These two enzymes may be KARI and an alcohol dehydrogenase.
  • NADH-dependent KARI and ADH enzymes are identified in nature.
  • the NADPH-dependent KARI and ADH enzymes may be engineered using protein engineering techniques including but not limited to directed evolution and site-directed mutagenesis.
  • NADH-dependent isobutyraldehyde dehydrogenases or ketol-acid reductoisomerases (1 ) increase the NADH-dependent activity of an NADPH-dependent enzyme that is active towards the substrate of interest and/or (2) increase the activity of an NADH-dependent enzyme that is not sufficiently active towards the substrate of interest.
  • NADH-dependent isobutyraldehyde dehydrogenases or ketol-acid reductoisomerases (1 ) increase the NADH-dependent activity of an NADPH-dependent enzyme that is active towards the substrate of interest and/or (2) increase the activity of an NADH-dependent enzyme that is not sufficiently active towards the substrate of interest.
  • ketol-acid reductoisomerase (KARI) enzyme of the isobutanol biosynthetic pathway as disclosed by Atsumi et al. (Atsumi et al.,
  • NADPH acetolactate
  • NADH cell - nicotinamide adenine dinucleotide
  • KARI enzymes that preferentially utilize NADH rather than NADPH are desirable.
  • KARI enzymes for the conversion of acetolactate to 2, 3-dihydroxyisovalerate.
  • KARI enzymes convert acetolactate to 2, 3-dihydroxyisovalerate as part of the biosynthetic pathway for the production of 3-methyl-1-butanol (Atsumi et al., 2008, Nature 45: 86-9).
  • KARI or "KARI enzyme” or “ketol-acid reductoisomerase” are used interchangeably herein to refer to an enzyme that catalyzes the conversion of acetolactate to 2, 3-dihydroxyisovalerate and/or the conversion of 2-aceto-2-hydroxy-butyrate to 2, 3-dihydroxy-3-methylvalerate.
  • Enzymes for use in the compositions and methods of the invention include any enzyme having the ability to convert acetolactate to 2, 3-dihydroxyisovalerate and/or the ability to convert 2-aceto-2-hydroxy-butyrate to 2, 3-dihydroxy-3- methylvalerate.
  • Such enzymes include, but are not limited to, the E. coli HvC gene product and the S.
  • KARI sequences are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos: NP_418222 and NC_000913, Saccharomyces cerevisiae (GenBank Nos: NP_013459 and NC_001144, Methanococcus maripaludis (GenBank Nos: CAF30210 and BX957220, and Bacillus subtilis (GenBank Nos: CAB14789 and Z99118) and the KARI enzymes from Piromyces sp (GenBank No: CAA76356), Buchnera aphidicola (GenBank No: AAF13807), Spinacia oleracea (GenBank Nos: Q01292 and CAA40356J, Oryza sativa (GenBank No: NP_001056384J Chlamydomonas reinhardtii (GenBank No: XPJ)01702649), Neurospora crassa (
  • modified KARI enzymes may be obtained by recombinant or genetic engineering techniques that are routine and well-known in the art.
  • Mutant KARI enzymes can, for example, be obtained by mutating the gene or genes encoding the KARI enzyme of interest by site-directed or random mutagenesis. Such mutations may include point mutations, deletion mutations and insertional mutations. For example, one or more point mutations (e.g., substitution of one or more amino acids with one or more different amino acids) may be used to construct mutant KARI enzymes of the invention.
  • Ketol-acid reductoisomerase catalyzes the reduction of acetolactate to 2, 3-dihydroxyisovalerate.
  • the two-step reaction involves an alkyl migration and a ketone reduction that occurs at a single active site on the enzyme without dissociation of any reaction intermediates.
  • the enzyme is NADPH- dependent.
  • the cofactor specificity may be expanded or switched so that it will utilize both cofactors and preferentially NADH during the production of isobutanol.
  • a study published in 1997 (Rane, M.J. and K.C. Calvo, Archives of Biochemistry and Biophysics, 1997. 338: p. 83-89) describes a supposed cofactor-switched KARI quadruplet variant of the E.
  • 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, or four, etc.) point mutations preferred.
  • Mutations may be introduced into the KARI enzymes of the present invention using any methodology known to those skilled in the art. Mutations may be introduced randomly by, for example, conducting a PCR reaction in the presence of manganese as a divalent metal ion cofactor. Alternatively, oligonucleotide directed mutagenesis may be used to create the mutant KARI enzymes which allows for all possible classes of base pair changes at any determined site along the encoding DNA molecule. In general, this technique involves annealing an oligonucleotide complementary (except for one or more mismatches) to a single stranded nucleotide sequence coding for the KARI enzyme of interest.
  • the mismatched oligonucleotide is then extended by DNA polymerase, generating a double-stranded DNA molecule which contains the desired change in sequence in one strand.
  • the changes in sequence can, for example, result in the deletion, substitution, or insertion of an amino acid.
  • the double-stranded polynucleotide can then be inserted into an appropriate expression vector, and a mutant or modified polypeptide can thus be produced.
  • the above-described oligonucleotide directed mutagenesis can, for example, be carried out via PCR.
  • the invention further includes homologous KARI enzymes which are 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical at the amino acid level to a wild-type KARI enzyme (e.g., encoded by the EcJIvC gene or S. cerevisiae ilv5 gene) and exhibit an increased ability to utilize NADH for the conversion of acetolactate to 2,3-dihydroxyisovalerate.
  • a wild-type KARI enzyme e.g., encoded by the EcJIvC gene or S. cerevisiae ilv5 gene
  • KARI enzymes which are 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical at the amino acid level to a KARI enzyme comprising the amino acid sequence set out in SEQ ID NO: 56 and exhibit an increased ability to utilize NADH for the conversion of acetolactate to 2,3-dihydroxyisovalerate.
  • the invention also includes nucleic acid molecules which encode the above described KARI enzymes.
  • the invention also includes fragments of 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 mutant KARI enzymes and KARI enzyme fragments.
  • a protein or protein fragment having an amino acid sequence at least, for example, 50% "identical" to a reference amino acid sequence it is intended that the amino acid sequence of the protein is identical to the reference sequence except that the protein sequence may include up to 50 amino acid alterations per each 100 amino acids of the amino acid sequence of the reference protein.
  • up to 50% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 50% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • alterations of the reference sequence may occur at the amino (N-) and/or carboxy (C-) terminal positions of the reference amino acid sequence and/or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence and/or in one or more contiguous groups within the reference sequence.
  • whether a given amino acid sequence is, for example, at least 50% identical to the amino acid sequence of a reference protein can be determined conventionally using known computer programs such as those described above for nucleic acid sequence identity determinations, or using the CLUSTAL W program (Thompson, J. D., et ai, Nucleic Acids Res. 22:4673 4680 (1994)).
  • amino acid substitutions are made at one or more of the above identified positions (i.e., amino acid positions equivalent or corresponding to A71 , R76, S78, or Q110 of E. coli HvC).
  • the amino acids at these positions may be substituted with any other amino acid including Ala, Asn, Arg, Asp, Cys, GIn, GIu, GIy, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and VaI.
  • a specific example of a KARI enzyme which exhibits an increased ability to utilize NADH includes an E.
  • coli NvC KARI enzyme in which (1 ) the alanine at position 71 has been replaced with a serine, (2) the arginine at position 76 has been replaced with an aspartic acid, (3) the serine at position 78 has been replaced with an aspartic acid, and/or (4) the glutamine at position 110 has been replaced with valine (as described in commonly owned and co-pending applications US 12/610,784 and PCT/US09/62952 (published as WO/2010/051527).
  • Polypeptides having the ability to convert acetolactate to 2,3- dihydroxyisovalerate and/or 2-aceto-2-hydroxy-butyrate to 2,3-dihydroxy-3- methylvalerate for use in the invention may be isolated from their natural prokaryotic or eukaryotic sources according to standard procedures for isolating and purifying natural proteins that are well-known to one of ordinary skill in the art (see, e.g., Houts, G. E., et al., J. Virol. 29:517 (1979)).
  • polypeptides having the ability to convert acetolactate to 2,3-dihydroxyisovalerate and/or 2-aceto-2-hydroxy- butyrate to 2,3-dihydroxy-3-methylvalerate may be prepared by recombinant DNA techniques that are famiiiar to one of ordinary skill in the art (see, e.g., Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988); Soltis, D. A., and Skalka, A. M., Proc. Natl. Acad. Sci. USA 85:3372 3376 (1988)).
  • one or more mutations may be made in any KARI enzyme of interest in order to increase the ability of the enzyme to utilize NADH, or confer other properties described herein upon the enzyme, in accordance with the invention.
  • Such mutations include point mutations, frame shift mutations, deletions and insertions.
  • one or more point mutations, resulting in one or more amino acid substitutions are used to produce KARI enzymes having an enhanced or increased ability to utilize NADH, particularly to facilitate the conversion of acetolactate to 2,3-dihydroxyisovalerate and/or the conversion of 2-aceto-2- hydroxy-butyrate to 2,3-dihydroxy-3-methylvalerate.
  • one or more mutations at positions equivalent or corresponding to position A71 e.g., A71S
  • R76 e.g., R76D
  • S78 e.g. S78D
  • Q110 e.g. Q110V
  • D146 e.g. D146G
  • G185 e.g. G185R
  • K433 e.g. K433E
  • the corresponding positions of the KARI enzymes identified herein may be readily identified for other KARI enzymes by one of skill in the art.
  • the modified or mutated KARI enzymes have from 1 to 4 amino acid substitutions in amino acid regions involved in cofactor specificity as compared to the wild-type KARI enzyme proteins.
  • modified or mutated KARI enzymes have additional amino acid substitutions at other positions as compared to the respective wild-type KARI enzymes.
  • modified or mutated KARI enzymes may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40 different residues in other positions as compared to the respective wild-type KARI enzymes.
  • the number of additional positions that may have amino acid substitutions will depend on the wild-type KARI enzyme used to generate the variants. Thus, in some instances, up to 50 different positions may have amino acid substitutions.
  • the nucleotide sequences for several KARI enzymes are known.
  • the sequences of KARI enzymes are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank No: NP_418222), Saccharomyces cerevisiae (GenBank Nos: NP_013459, Mef/ianococci/s ma ⁇ paludis (GenBank No: YP_001097443), Bacillus subtilis (GenBank Nos: CAB 14789), and the KARI enzymes from Piromyces sp (GenBank No: CAA76356), Buchnera aphidicola (GenBank No: AAF13807), Spinacia oleracea (GenBank Nos: Q01292 and CAA40356J, Oryza sativa (GenBank No: NP_001056384) Chlamydomonas reinhardtii (GenBank No: XP_001702649)
  • 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 KARI 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 KARI 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. [00275] In a preferred embodiment, 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. More preferably, 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 K M 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.
  • modified or mutated KARI enzymes of the present invention may show greater than 30 times decreased KM for NADH compared to the w ⁇ d-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 cat is evidenced by at least a 5% or greater increase or decrease in K M compared to the wild-type KARI enzyme.
  • modified or mutated KARl enzymes of the present invention may show greater than 50% increased k cat 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 ⁇ 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 200% increased k Gat for NADH compared to the wild-type or parental KARI enzyme.
  • the cofactor specificity of the modified or mutated KARI enzyme is altered such that there is a cofactor switch from NADPH to NADH.
  • these modified or mutated KARI enzymes will have an increase in NADH-dependent activity and a substantially simultaneous decrease in NADPH dependent activity.
  • the methods of the present invention can be used to change the cofactor preference from NADPH to NADH.
  • Cofactor specificity is a measure of the specificity of an enzyme for one cofactor over another.
  • the methods of the present invention may be used to alter the cofactor preference of the target enzyme, such that the preference for the less favored cofactor is increased by 20%, 50%, 100%, 300%, 500%, 1000%, up to 2000%.
  • reductase enzymes have been described that favor NADPH over NADH (see WO/2002/022526; WO/2002/029019; Mittl et al., 1994, Protein Sd., 3: 1504-14; Banta et ai, (2002) Protein Eng., 15: 131-140; all of which are hereby incorporated by reference in their entirety).
  • the overall activity of the target protein is often limited.
  • target proteins that prefer NADPH as a cofactor it would be desirable to alter the cofactor specificity of the target protein (e.g. a KARI enzyme) to a cofactor that is more readily available, such as NADH.
  • the cofactor specificity of the KARl enzyme is switched.
  • switched herein is meant, that the cofactor preference (in terms of catalytic efficiency (k cat /K M ) of the KARI enzyme is changed to another cofactor
  • activity in terms of catalytic efficiency ( ⁇ /K M ) with the cofactor preferred by the wild-type KARI enzyme is reduced, while the activity with the less preferred cofactor is increased. This can be achieved, for example by increasing the k ca t for less preferred cofactor over the preferred cofactor or by decreasing K M for the less preferred cofactor over the preferred cofactor or both.
  • the KARI enzyme is modified or a mutated to become NADH-dependent.
  • NADH-dependent refers to the property of an enzyme to preferentially use NADH as the redox cofactor.
  • An NADH-dependent enzyme has a higher catalytic efficiency (k cat /K ⁇ v ⁇ ) with the cofactor NADH than with the cofactor NADPH as determined by in vitro enzyme activity assays.
  • NADPH-dependent refers to the property of an enzyme to preferentially use NADPH as the redox cofactor.
  • An NADPH dependent enzyme has a higher catalytic efficiency (kc at /K M ) with the cofactor NADPH than with the cofactor NADH as determined by in vitro enzyme activity assays.
  • the catalytic efficiency of the KARI enzyme for NADH is enhanced relative to the catalytic efficiency with NADPH.
  • the term "catalytic efficiency" describes the ratio of the rate constant k cat over the Michaelis- Menten constant KM.
  • the invention is directed to a modified or mutated KARI enzyme that exhibits at least about a 1 :10 ratio of catalytic efficiency (IWKM) with NADH over catalytic efficiency with NADPH.
  • the modified or mutated KARI enzyme exhibits at least about a 1 :1 ratio of catalytic efficiency ( ⁇ /K M ) with NADH over catalytic efficiency with NADPH.
  • the modified or mutated KARI enzyme exhibits at least about a 10:1 ratio of catalytic efficiency (IWKM) with NADH over catalytic efficiency with NADPH. In yet another embodiment, the modified or mutated KARI enzyme exhibits at least about a 100:1 ratio of catalytic efficiency (IWKM) with NADH over catalytic efficiency with NADPH. In an exemplary embodiment, the modified or mutated KARI enzyme exhibits at least about a 100:1 ratio of catalytic efficiency (IWKM) with NADH over catalytic efficiency with NADPH.
  • the K M of the KARI enzyme for NADH is decreased relative to the K M of the KARI enzyme for NADPH.
  • the invention is directed to a modified or mutated KARI enzyme that exhibits at least about a 10:1 ratio of K M for NADH over K M for NADPH.
  • the invention is directed to a modified or mutated KARI enzyme that exhibits at least about a 1 :1 ratio of K M for NADH over K M for NADPH.
  • the invention is directed to a modified or mutated KARI enzyme that exhibits at least about a 1 :10 ratio of K M for NADH over K M for NADPH.
  • the invention is directed to a modified or mutated KARI enzyme that exhibits at least about a 1 :20, 1 :100, 1 :1000 ratio of K M for NADH over K M for NADPH.
  • the k cat of the KARI enzyme with NADH is increased relative to k cat with NADPH.
  • modified or mutated KARI enzymes of the present invention may show greater than 0.8:1 ratio of k c at with NADH over k cat with NADPH.
  • modified or mutated KARI enzymes of the present invention may show greater than 1 :1 ratio of k cat with NADH over k cat with NADPH.
  • modified or mutated KARI enzymes of the present invention may show greater than 10:1 ratio of k cat with NADH over kcat with NADPH. In certain embodiments, modified or mutated KARI enzymes of the present invention may show greater than 100:1 ratio of k cat with NADH over k cat with NADPH.
  • E coli KARI (PDB ID NO. 1YRL) with rice KARI (PDB ID NO. 3FR8) as a representative of the shorter loop group
  • Ser165, Lys166, and Ser167 are sites of useful mutations in the E. coli context corresponded reasonably well with specific residues in the context of the shorter loop: Ser165, Lys166, and Ser167.
  • Ser165 of (corresponding to A71 in E. coli) therefore may be substituted with aspartate.
  • a charge reversal at position K166 (corresponding to position R76D) may yield the same result.
  • Ser167 may correspond to Ser78 and a mutation to aspartate corresponds to a beneficial mutation at Q110, and thus can be transferable in the aligned KARIs.
  • ADH2 gene is expected to be functionally expressed from pSA55 and required for catalyzing the final step of the isobutanol biosynthetic pathway, namely the conversion of isobutyraldehyde to isobutanol.
  • no isobutanol should be produced with the plasmid combination lacking ADH2 as adhE is deleted in JCL260.
  • the results of a fermentation using a strain without overexpression of any gene encoding an enzyme with ADH activity for the conversion of isobutyraldehyde to isobutanol showed that overexpression of an ADH enzyme is not required for isobutanol production in E. coli.
  • isobutyraldehyde reduction activity of an NADH-dependent enzyme with little native activity towards this substrate may be increased.
  • This approach has the advantages that (1) several specialized enzymes exist in nature that are highly active under fermentative conditions, (2) the binding sites of several of these enzymes are known, (3) mutational studies indicate that substrate specificity can easily be altered to achieve high activity on a new substrate.
  • Several alcohol dehydrogenase enzymes may be suitable candidates for conversion into an NADH-dependent isobutyraldehyde dehydrogenase: S.
  • ADHII are NADH-dependent enzymes responsible for the conversion of acetaldehyde to ethanol under anaerobic conditions. These enzymes are highly active.
  • the substrate specificity for these enzymes has been analyzed (Leskovac et al., 2002, Ferns Yeast Research, 2: 481- 94; Rellos et al., 1997, Protein Expression and Purification, 9: 89-90), the amino acid residues comprising the substrate binding pocket are known (Leskovac et al., 2002, Ferns Yeast Research, 2: 481-94; Rellos et al., 1997, Protein Expression and Purification, 9: 89-90), and attempts to alter the substrate specificity by mutation have revealed that the substrate specificity can be altered (Rellos et al., 1997, Protein Expression and Purification, 9: 89-90; Green et al., 1993, J.
  • Ec_YqhD and S. cerevisiae ADH7 are NADPH-dependent enzymes whose physiological functions are not as well understood. Ec_YqhD has been implicated in the protection of the cell from peroxide-derived aldehydes (Perez et al., 2008, J. Biol. Chem. 283: 7346-53). The substrate specificity of both enzymes is understood, and amino acids lining the substrate binding pocket are known (Perez et al., 2008, J. Biol. Chem. 283: 7346-53). Based on the known amino acid residues implicated in substrate binding (S. cere visiae ADH 1 , Z. mobilis ADHII) or the cofactor binding site ( ⁇ c_yqhD), sites with the highest likelihood of affecting desired enzyme features such as substrate specificity or cofactor specificity may be mutated to generate the desired function.
  • NNK libraries at each of the residues that interact with the cofactor. These libraries can be screened for activity in the presence of NADPH and NADH in order to identify which single mutations contribute to increased activity on NADH and altered specificity for NADH over NADPH. Combinations of mutations at aforementioned residues can be investigated by any method known in the art. For example, a combinatorial library of mutants may be designed based on the results of the saturation mutagenesis studies. For example, a combinatorial library of mutants may be designed including only those mutations that do not lead to decrease in NADH-dependent activity.
  • Another approach to increase the NADH-dependent activity of the enzyme is to perform saturation mutagenesis of a first amino acid that interacts with the cofactor, then isolate the mutant with the highest activity using NADH as the cofactor, then perform saturation mutagenesis of a second amino acid that interacts with the cofactor, and so on.
  • a limited number of amino acids that interact with the cofactor may be targeted for randomization simultaneously and then be screened for improved activity with NADH as the cofactor.
  • the selected, best mutant can then be subjected to the same procedure again and this approach may be repeated iteratively until the desired result is achieved.
  • Another approach is to use random oligonucleotide mutagenesis to generate diversity by incorporating random mutations, encoded on a synthetic oligonucleotide, into the cofactor binding region of the enzyme.
  • the number of mutations in individual enzymes within the population may be controlled by varying the length of the target sequence and the degree of randomization during synthesis of the oligonucleotides.
  • the advantages of this more defined approach are that all possible amino acid mutations and also coupled mutations can be found.
  • directed evolution via error-prone PCR may be used to obtain further improvements. Error-prone PCR mutagenesis of the first domain containing the cofactor binding pocket may be performed followed by screening for ADH activity with NADH and/or increased specificity for NADH over NADPH as the cofactor.
  • alcohol dehydrogenase enzymes that are not known to catalyze the reduction of isobutyraldehyde to isobutanol were identified that catalyze this reaction.
  • an alcohol dehydrogenase may be encoded by an NADH-dependent 1 ,3-propanediol dehydrogenase.
  • such an alcohol dehydrogenase may be encoded by an NADH-dependent 1 ,2-propanediol dehydrogenase.
  • Preferred enzymes of this disclosure include enzymes listed in Table 1 of co-pending and commonly owned US 12/610,784 and PCT/US09/62952 (published as WO/2010/051527). These enzymes exhibit NADH- dependent isobutyraldehyde reduction activity, measured as Unit per minute per mg of crude cell lysate (U min "1 mg "1 ) that is approximately six-fold to seven-fold greater than the corresponding NADPH-dependent isobutyraldehyde reduction activity. [00298] In addition to exhibiting increased activity with NADH as the cofactor as compared to the NADPH, alcohol dehydrogenases of the present invention may further be more active as compared to the native E. coli alcohol dehydrogenase Ec_YqhD.
  • alcohol dehydrogenases of the present invention may exhibit increased activity and/or decreased K M values with NADH as the cofactor as compared to Ec_YqhD with NADPH as the cofactor.
  • Exemplary enzymes that exhibit greater NADH-dependent alcohol dehydrogenase activity than the NADPH-dependent alcohol dehydrogenase activity are listed include the Drosophila melanogaster ADH, the L lactis adhA, K. pneumoniae dhaT, and E. coli fucO (see Table 1 of US 12/610,784).
  • Alcohol dehydrogenases of the present disclosure may also be utilized in metabolically-modified microorganisms that include recombinant biochemical pathways useful for producing additional alcohols such as 2-methyl-1-butanol, 3- methyl-1-butanol, 2-phenylethanol, 1-propanol, or 1-butanol via conversion of a suitable substrate by a modified microorganism.
  • these alcohols can be 1-propanol, 1-butanol, 2- methyl-1-butanol, 3-methyl-1-butanol or 2-phenylethanol and are generally produced from a metabolite comprising a 2-keto acid.
  • the 2-keto acid includes 2-ketobutyrate, 2-ketovaierate, 2-keto-3-methylvalerate, 2-keto-4-methyl- pentanoate, or phenylpyruvate.
  • the 2-ketoacid is converted to the respective aldehyde by a 2-ketoacid decarboxylase.
  • 2-ketobutyrate is converted to 1-propanal
  • 2-ketovalerate is converted to 1-butanal
  • 2-keto-3-methylvalerate is converted to 2-methyM-butanol
  • 2-keto-4-methyl-pentanoate is converted to 3- methyl-1-butanal
  • phenylpyruvate is converted to phenylethanal by a 2-ketoacid decarboxylase.
  • the recombinant microorganism includes elevated expression or activity of a 2-keto-acid decarboxylase, as compared to a parental microorganism.
  • the 2-keto-acid decarboxylase may be encoded by kivD from Lactococcus lactis, or homologs thereof.
  • the 2-keto-acid decarboxylase can be encoded by a polynucleotide derived from a gene selected from kivD from L lactis, or homologs thereof.
  • a microorganism in which the cofactor dependent final step for the conversion of the aldehyde to the respective alcohol is catalyzed by an NADH-dependent alcohol dehydrogenase.
  • NADH-dependent alcohol dehydrogenases are disclosed that catalyze the reduction aldehydes to alcohols, for example, of 1-propanal to I propanol, 1-butanal to 1-butanol, 2-methyl-1-butanal to 2-methyl-1-butanol, 3-methyl-1-butanal to 3- methyl-1-butanol, or phenylethanal to phenylethanol.
  • such an alcohol dehydrogenase may be encoded by the Drosophila melanogaster alcohol dehydrogenase Dm_Adh or homologs thereof.
  • such an alcohol dehydrogenase may be encoded by the Lactococcus lactis alcohol dehydrogenase (LI__AdhA) or homologs thereof.
  • LI__AdhA Lactococcus lactis alcohol dehydrogenase
  • such an alcohol dehydrogenase may be encoded by an NADH-dependent 1 ,3-propanediol dehydrogenase.
  • such an alcohol dehydrogenase may be encoded by an NADH-dependent
  • a method of producing an alcohol includes providing a recombinant microorganism provided herein; culturing the microorganism of in the presence of a suitable substrate or metabolic intermediate and under conditions suitable for the conversion of the substrate to an alcohol; and detecting the production of the alcohol.
  • the alcohol is selected from 1-propanol, 1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol, and 2-phenylethanol.
  • the substrate or metabolic intermediate includes a 2-keto acid-derived aldehyde, such as 1-propanal, 1-butanal, 2-methy!-1-butanal, 3-methyl-1-butanal, or phenylethanal.
  • the present invention is directed to recombinant host cells (i.e. metabolically "engineered” or “modified” microorganisms) comprising NADH-dependent KARI and/or ADH enzymes of the invention.
  • Recombinant microorganisms provided herein can express a plurality of additional heterologous and/or native target enzymes involved in pathways for the production of beneficial metabolites such as isobutanol from a suitable carbon source.
  • metabolically "engineered” or “modified” microorganisms are produced via the introduction of genetic material (i.e.
  • a NADH-dependent KARI and/or ADH enzymes into a host or parental microorganism of choice, 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 metabolite.
  • the introduction of genetic material and/or the modification of the expression of native genes into a parental microorganism results in a new or modified ability to produce beneficial metabolites such as isobutanol.
  • 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.
  • Recombinant microorganisms provided herein may also produce metabolites in quantities not available in the parental microorganism.
  • a "metabolite” refers to any substance produced by metabolism or a substance necessary for or taking part in a particular metabolic process.
  • a metabolite can be an organic compound that is a starting material (e.g., glucose or pyruvate), an intermediate (e.g., 2-ketoisovalerate), or an end product (e.g., 1-propanol, 1-butanol, isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol) 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.
  • Exemplary metabolites include glucose, pyruvate, 1-propanol, 1-butanol, isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol.
  • the metabolite 1-propanol can be produced by a recombinant microorganism engineered to express or over-express a metabolic pathway that converts pyruvate to 1-propanol.
  • An exemplary metabolic pathway that converts pyruvate to 1-propanol has been described in WO/2008/098227 and by Atsumi et al. (Atsumi et al., 2008, Nature 451 (7174): 86-9), the disclosures of which are herein incorporated by reference in their entireties.
  • metabolic pathway comprises a KARI and/or an ADH enzyme of the present invention.
  • the metabolite 1-butanol can be produced by a recombinant microorganism engineered to express or over-express a metabolic pathway that converts pyruvate to 3-methyl-1-butanol.
  • An exemplary metabolic pathway that converts pyruvate to 3-methyl-1-butanol has been described in WO/2008/098227 and by Atsumi et al. (Atsumi et al., 2008, Nature 451(7174): 86-9), the disclosures of which are herein incorporated by reference in their entireties.
  • metabolic pathway comprises a KARI and/or an ADH enzyme of the present invention.
  • the metabolite isobutanol can be produced by a recombinant microorganism engineered to express or over-express a metabolic pathway that converts pyruvate to isobutanol.
  • An exemplary metabolic pathway that converts pyruvate to isobutanol may be comprised of a acetohydroxy acid synthase (ALS) enzyme encoded by, for example, alsS from B. subtilis, a ketolacid reductoisomerase (KARI) of the present invention, a dihydroxy-acid dehydratase (DHAD), encoded by, for example HvD from E. coli or L. lactis, a 2-keto-acid decarboxylase (KiVD) encoded by, for example kivd from L. lactis, and an alcohol dehydrogenase (ADH) of the present invention.
  • ALS acetohydroxy acid synthase
  • alsS from B. subtilis
  • KARI ketolacid reductoi
  • the metabolite 3-methyl-1-butanol can be produced by a recombinant microorganism engineered to express or over-express a metabolic pathway that converts pyruvate to 3-methyl-1-butanol.
  • An exemplary metabolic pathway that converts pyruvate to 3-methyl-1 -butanol has been described in WO/2008/098227 and by Atsumi et al. (Atsumi et aL, 2008, Nature 451(7174): 86-9), the disclosures of which are herein incorporated by reference in their entireties.
  • metabolic pathway comprises a KARI and/or an ADH enzyme of the present invention.
  • the metabolite 2-methyl-1 -butanol can be produced by a recombinant microorganism engineered to express or over-express a metabolic pathway that converts pyruvate to 2-methyl-1 -butanol.
  • An exemplary metabolic pathway that converts pyruvate to 2-methyl-1 -butanol has been described in WO/2008/098227 and by Atsumi et al. (Atsumi et al., 2008, Nature 451 : 86-9), the disclosures of which are herein incorporated by reference in their entireties.
  • metabolic pathway comprises a KARI and/or an ADH enzyme of the present invention.
  • 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 mutation and silent mutations.
  • modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art.
  • homologs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein.
  • yeast microorganism exhibits a higher capacity to produce the intermediates of the isobutanoi pathway including, but not limited to, acetolactate, 2,3- dihydroxyisovalerate, keto-isovalerate, and isobutyraldehyde.
  • the yeast microorganism is cultured in an appropriate culture medium containing a carbon source.
  • Another exemplary embodiment provides a method for producing isobutanoi comprising a recombinant yeast microorganism of the invention in a suitable culture medium containing a carbon source that can be converted to isobutanoi by the yeast microorganism of the invention.
  • the method further includes isolating isobutanoi from the culture medium.
  • isobutanoi may be isolated from the culture medium by any method known to those skilled in the art, such as distillation, pervaporation, or liquid-liquid extraction.
  • Sample preparation Samples (2 mL) from the fermentation broth were stored at -20 0 C for later substrate and product analysis. Prior to analysis, samples were thawed and then centrifuged at 14,000 x g for 10 min. The supernatant was filtered through a 0.2 ⁇ m filter. Analysis of substrates and products was performed using authentic standards (>99%, obtained from Sigma-Aldrich), and a 5-point calibration curve (with 1-pentanol as an internal standard for analysis by gas chromatography).
  • optical density and cell dry weight The optical density of the yeast cultures was determined at 600 nm using a DU 800 spectrophotometer (Beckman-Coulter, Fullerton, CA, USA). Samples were diluted as necessary to yield an optical density of between 0.1 and 0.8. The cell dry weight was determined by centrifuging 50 mL of culture prior to decanting the supernatant. The cell pellet was washed once with 50 mL of milliQ H 2 O, centrifuged and the pellet was washed again with 25 mL of milliQ H 2 O. The cell pellet was then dried at 8O 0 C for at least 72 hours.
  • the cell dry weight was calculated by subtracting the weight of the centrifuge tube from the weight of the centrifuge tube containing the dried cell pellet.
  • Gas Chromatography Analysis of ethanol and isobutanol was performed on a HP 5890 gas chromatograph fitted with a DB-FFAP column (Agilent Technologies; 30 m length, 0.32 mm ID, 0.25 ⁇ M film thickness) or equivalent connected to a flame ionization detector (FID).
  • the temperature program was as follows: 200 0 C for the injector, 300 0 C for the detector, 100°C oven for 1 minute, 70°C/minute gradient to 235°C, and then hold for 2.5 min.
  • High Performance Liquid Chromatography Analysis of glucose and organic acids was performed on a HP-1100 High Performance Liquid Chromatography system equipped with an Aminex HPX-87H Ion Exclusion column (Bio-Rad, 300x7.8mm) or equivalent and an H + cation guard column (Bio-Rad) or equivalent.
  • Organic acids were detected using an HP-1100 UV detector (210nm, 8nm 360nm reference) while glucose was detected using an HP-1100 refractive index detector.
  • the column temperature was 60 0 C. This method was lsocratic with 0.008N sulfuric acid in water as mobile phase. Flow was set at 0.6 mL/min. Injection size was 20 ⁇ L and the run time was 30 minutes.
  • Anaerobic batch fermentations Anaerobic batch cultivations were performed at 30 0 C in stoppered 100 mL serum bottles. A total of 2OmL of synthetic medium with an initial glucose concentration of 20 g-glucose L "1 was used (Kaiser et al., Methods in Yeast Genetics, a Cold Spring Harbor Laboratory Manual (1994)). 2mL samples are taken at 24 and 48 hours. The fermentation is ended after 48 hours or when all glucose is consumed. Samples are processed and analyzed by Gas Chromatography and/or High Performance Liquid Chromatography as described above.
  • Yeast transformations - K. lactis Transformations were performed by electropo ration according to Kooistra et al., Yeast 21 :781-792 (2004).
  • Lithium Acetate transformations of S. cerevisiae strains were transformed by the Lithium Acetate method (Gietz et al., Nucleic Acids Res. 27:69-74 (1992). Cells were collected from overnight cultures grown in 50 mL of defined (SC) ethanol media at an OD ⁇ oo of approximately 0.8 to 1.0 by centrifugation at 2700 rcf for 2 minutes at room temperature.
  • SC defined
  • the cell pellet was resuspended in 50 mL sterile water, collected by centrifugation (2700 rcf; 2 min; room temp.), and resuspended in 25 ml_ sterile water.
  • the cells were collected by centrifugation (2700 rcf; 2 min; room temp.) and resuspended in 1 mL 100 mM lithium acetate.
  • the cell suspension was transferred to a sterile 1.5 mL tube and collected by centrifugation at full speed for 10 seconds.
  • the cells were resuspended in 100 mM lithium acetate with a volume four times the volume of the cell pellet (e.g. 400 ⁇ L for 100 ⁇ L cell pellet).
  • Yeast colony PCR Yeast cells were taken from agar medium and transferred to 30 ⁇ l 0.2% SDS and heated for 4mins at 90 0 C. The cells were spun down and 1 ⁇ l of the supernatant was used for PCR using standard Taq (NEB).
  • Molecular biology Standard molecular biology methods for cloning and plasmid construction were generally used, unless otherwise noted (Sambrook & Russell).
  • Media Media:
  • YP contains 1 % (w/v) yeast extract, 2% (w/v) peptone.
  • YPD is YP containing 2% (w/v) glucose
  • YPE is YP containing 2% (w/v) Ethanol.
  • SC+Complete 20 g/L glucose, 14g/L SigmaTM Synthetic Dropout Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), and 6.7 g/L DifcoTM Yeast Nitrogen Base. 0.076 g/L histidine, 0.076 g/L tryptophan, 0.380 g/L leucine, and 0.076 g/L uracil.
  • SC-HWUL 20 g/L glucose, 14g/L SigmaTM Synthetic Dropout Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), and 6.7 g/L -DifcoTM Yeast Nitrogen Base
  • SC-WLU 20 g/L glucose, 14g/L SigmaTM Synthetic Dropout Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), 6.7 g/L DifcoTM Yeast Nitrogen Base without amino acids, and
  • SC- HWU 20 g/L glucose, 14g/L SigmaTM Synthetic Dropout Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), 6.7 g/L DifcoTM Yeast Nitrogen Base without amino acids, and
  • Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), 6.7 g/L DifcoTM Yeast Nitrogen Base, and 0.380 g/L leucine.
  • Solid versions of the above described media contain 2% (w/v) agar.
  • Table 1 details the genotype of strains disclosed herein:
  • strains Gevol537 and Gevol538 were originally designated GG570 (derived from strain 12- 3D)and was obtained from Paul van Heusden from the University of Leiden, the Netherlands. For complete references for both strains, see: Flikweert, M.T. et al v (1996) Yeast 12:247-257.
  • Example 1 Construction and confirmation of PDC deletion in K. lactis [00337] The purpose of this Example is to describe how a PDC-deletion variant of a member of the Saccharomyces clade, Crabtree-negative yeast, pre-WGD yeast K. lactis was constructed and confirmed.
  • Plasmid pGV1537 (SEQ ID NO: 1 ) was constructed by the following series of steps. All PCR reactions carried out to generate pGV1537 used KOD polymerase (Novagen, Inc., Gibbstown, NJ) and Standard reaction conditions according to the manufacturer. A first round of two PCR reactions was carried out, wherein one PCR reaction contained primers 1006 and 1016 and used approximately 100ng of genomic DNA from K. lactis strain GEVO1287 as a template. The other first-round PCR reaction contained primers 1017 and 1009 and approximately 100ng of genomic DNA from K. lactis strain GEVO1287 as a template.
  • the two resulting PCR products (approximately 530bp and 630bp in size, respectively) were gel purified using a Zymo Research Gel DNA Extraction kit (Zymo Research, Orange, CA) according to manufacturer's instructions and eluted into 10 ⁇ L of water. Two (2) microliters of each eluted PCR product were then used as a template for a final round of KOD polymerase-catalyzed PCR, which also included primers 1006 plus 1009. The resulting product was purified (Zymo Research DNA Clean & Concentrate kit, Zymo Research, Orange, CA), digested to completion with the enzymes Mfe ⁇ and AatW, and the resulting product gel purified and eluted as described above.
  • Zymo Research Gel DNA Extraction kit Zymo Research, Orange, CA
  • This DNA was ligated into the vector pGV1503 ( Figure 8), which had been digested with EcoRI plus AatW, treated with calf alkaline phosphatase, and gel purified as described above. Colonies arising from transformation of the ligated DNA were screened by restriction digest analysis and confirmed by DNA sequencing reactions using primers 838, 1010, and 1019. Correct recombinant DNA resulting from the ligation and subsequent analysis was named pGV1537 ( Figure 9).
  • Colonies arising from the transformation were further selected by patching colonies onto YPD plates and then replica plating onto YPD containing 5 ⁇ M (final concentration) of the respiratory inhibitor Antimycin A, as Pdc- variants of K.lactis are unable to grow on glucose in the presence of Antimycin A (Bianchi, M., et al., (1996). "The petite negative yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity". Molecular Microbiology 19(1 ):27-36) and can therefore be identified by this method.
  • the suspension was pelleted by centrifugation (30sec, 16,000xg) and 1 ⁇ L of the supernatant was used as template in 50 ⁇ l_ PCR reactions.
  • the reactions contained Triton X-100 at a final concentration of 1.5% and DMSO at a final concentration of 5%.
  • the various primer sets used, and the expected amplicon sizes expected, are indicated in Table EX1-1.
  • Example 2 Construction and confirmation of PDC deletion in S. cerevisiae
  • the purpose of this Example is to describe how a PDC deletion variant of a member of the Saccharomyces sensu stricto yeast group, the Saccharomyces yeast clade, a Crabtree-positive yeast, and a post-WGD yeast, S. cerevisiae was constructed and confirmed.
  • the cells were resuspended in 5ml_ water to which was added O. ⁇ mL of a 1mg/mL solution (freshly made) of Zymolyase-T (in water) as well as 10 ⁇ L of ⁇ -mercaptoethanol.
  • the cell suspension was incubated overnight at 30 0 C in a shaker at 50 rpm.
  • Five mL of 1.5% Triton X-100 were added and the mixture was incubated on ice for 15 minutes.
  • the solution was sonicated three times for 30 seconds per cycle at 50% power, with 2 minutes rest on ice in between sonication cycles.
  • the suspension was centrifuged (1200 x g, 5 minutes) and washed twice with 5 mL of water.
  • the final cell pellet was resuspended in 1mL water and cells were plated to YP+2% EtOH. [00344] Following this procedure, the separate individual spores, were plated onto solid medium to obtain colonies, all of genotype HO pdc1::Tn5ble pdc5::Tn5ble pdc6:APT1 HIS3 LEU2 TRP1 URA3 and of unknown mating type. Some fraction of the cells were (homozygous) diploid due to the HO+ gene status and resultant mating type switching and re-mating to form diploids.
  • the genotype of the mating type locus of the putative Pdc-minus colonies was confirmed by PCR using Taq DNA polymerase (New England BioLabs, Ipswich, MA) under standard conditions using primers specific for the MAT a locus (primers #489 and #491 ) or MAT ⁇ locus (primers #490 and #491 ). Colonies that generated a single PCR product with one of the two possible primer sets primer set and no product when tested with the other were putative haploid Pdc-minus strains. To confirm the mating type, such strains were crossed to Gevo1187 and Gevo1188 (CEN. PK).
  • Resulting diploid progeny were selected on medium containing glucose (to select for the presence of PDC+ genes introduced by CEN. PK background) and also lacking at least one of the following nutrients: histidine, leucine, tryptophan, or uracil (to select for the appropriate prototrophy as provided by the wild-type allele of the corresponding gene from the Gevo1537 or GEVO1538 background.
  • Diploid cells were sporulated and germinated on agar plates containing YP + 2% ethanol (to permit growth of Pdc-minus isolates). To identify Pdc-minus candidates, viable colonies were streaked on to YPD agar plates and colonies that were inviable on glucose were isolated.
  • Pdc-minus strains were streaked to SC+Ethanol plates lacking one of: leucine, histidine, tryptophan, or uracil, to determine presence of auxotrophic mutations within these sfrains.
  • One Pdc-minus strain, GEVO1581 was auxotrophic for histidine, uracil, and tryptophan, and thus carried three of the makers (his3, ura3, and trp1).
  • Another Pdc-minus strain, GEVO1715 was auxotrophic for uracil and leucine and thus carried the two markers, ura3 and Ieu2.
  • GEVO1581 and GEVO1715 were screened by RFLP analysis to verify the presence of the ho allele.
  • a 447 bp portion of the HO locus was amplified by PCR that contained the codon that is altered in the ho allele (H475L) using primers 1375 and 1376.
  • This mutation introduces an AIuI restriction site, and consequently, digestion with Alu ⁇ (New England BioLabs, Ipswich, MA) yielded either a 447 bp fragment (HO) or a 122 bp fragment plus a 325 bp fragment (ho).
  • GEVO1581 was HO and GEVO1715 was ho.
  • Example 3 Other Pdc-minus S. cerevisiae strains.
  • S. cerevisiae engineered to be deficient in PDC activity have been previously described: (Flikweert, MT. , van der Zanden, L., Janssen, W.M.T.M, Steensma, H.Y., van Dijken J. P., Pronk JT. (1996) Yeast 12(3):247-57).Such strains may be obtained from these sources.
  • Example 4 Chemostat evolution of S. cerevisiae PDC triple-mutant [00351] This example demonstrates that a PDC deletion variant of a member Saccharomyces sensu stricto yeast group, the Saccharomyces clade yeast, Crabtree-positive, post-WGD yeast, S. cerevisiae, can be evolved so that it does not have the requirement for a two-carbon molecule and has a growth rate similar to the parental strain on glucose.
  • a DasGip fermentor vessel was sterilized and filled with 200ml of YNB (Yeast Nitrogen Base; containing per liter of distilled water: 6.7 g YNB without amino acids from Difco, the following were added per liter of medium: 0.076 g histidine, 0.076 g tryptophan, 0.380 g leucine, and/or 0.076 g uracil; medium was adjusted pH to 5 by adding a few drops of HCL or KOH) and contained 2% w/v ethanol.
  • the vessel was installed and all probes were calibrated according to DasGip instructions.
  • the vessel was also attached to an off-gas analyzer of the DasGip system, as well as to a mass spectrometer.
  • the amount of carbon dioxide (XC02) and oxygen (X 02) levels in the off-gas were used to assess the metabolic state of the cells.
  • the ethanol levels were monitored to ensure that there was no contamination, either from other yeast cells or from potential revertants of the mutant strain since the S. cerevisiae PDC triple-mutant (GEVO1584) does not produce ethanol.
  • the minimum pH in the vessel was set to 5, and a base control was set up to pump in potassium hydroxide into the vessel when the pH dropped below 5.
  • GEVO1584 was inoculated into 10 ml of YNB medium with 2% w/v ethanol as the carbon source. The culture was incubated at 30° C overnight with shaking.
  • the overnight culture was used to inoculate the DasGip vessel. Initially, the vessel was run in batch mode, to build up a high cell density. When about 3 g CDW/L of cell biomass was reached, the vessel was switched to chemostat mode and the dilution of the culture began.
  • the medium pumped into the vessel was YNB with 7.125 g/L glucose and 0.375 g/L of acetate (5% carbon equivalent).
  • the initial dilution rate was set to 0.1 h '1 , but as the cell density started dropping, the dilution rate was decreased to .025 h "1 to avoid washout.
  • GEVO1584 was mating type a.
  • the combined acetate going into the vessel was 0.375 g/L. Then, over a period of 3 weeks, the rate of pump A was slowly decreased and the rate of pump C was increased by the same amount so that the combined rate of feeding was always 20 mL/h. When the rate of pump A dropped below 3 mL/h the culture started to slowly wash out. To avoid complete washout the dilution rate was decreased to 0.075 h "1 from 0.1 h "1 ( Figure 5). At this dilution rate, the rate of pump A was finally reduced to 0, and the evolved strain was able to grow on glucose only. Over the period of about five weeks, a sample was occasionally removed, either from the vessel directly or from the effluent line.
  • Samples were analyzed for glucose, acetate, and pyruvate using HPLC, and were plated on YNB with glucose, YNB with ethanol, and YNB (w/o uracil) plus glucose or ethano! as negative control. Strains isolated from the chemostat did not grow on the YNB plates without uracil. OD 6 oo was taken regularly to make sure the chemostat did not wash out. Freezer stocks of samples of the culture were made regularly for future characterization of the strains.
  • YNB yeast extract, peptone, dextrose
  • YPE yeast extract, peptone, ethanol
  • the growth characterization was performed in either snap-cap test tubes or 48-well plates (7.5ml). The snap-cap test tubes were not closed completely so that air would vent in/out of the tubes, and the 48-well plates were covered with an air permeable membrane to allow for oxygen transfer.
  • YPD or YPE agar plates were used with the antibiotics G418 and Phleomycin.
  • the PDC triple mutant strain (GEVO1584) has both G418 and Phleomycin resistance markers, so the progeny of that strain were able to grow on the antibiotics.
  • Single colonies isolated from each chemostat sample were studied for growth rates.
  • a single colony isolated from the 35-day chemostat population was selected because of high growth rates on glucose as a sole carbon source, was resistant to both G418 and Phleomycin, and grew without the need for ethanol or acetate.
  • the single colony was further evolved through 24 successive serial transfers in test tubes on YPD at 30 0 C, 250 rpm shaking.
  • the resulting strain, GEVO1863, grew similarly to the wild-type yeast parent on glucose (Figure 6), did not produce ethanol ( Figure 7), and did not require ethanol or acetate for growth.
  • This example demonstrates isobutanol production in a member of the Saccharomyces clade, Crabtree-negative, pre-WGD yeast, K lactis.
  • the isobutanol production pathway was cloned in a K. lactis vector-based expression system: a Sac ⁇ - Mlu ⁇ fragment containing the TEF1 promoter. Lactococcus lactis alsS and part of the CYC1 terminator sequence was cloned into the same sites of the K. lactis expression plasmid, pGV1430 ( Figure 11 ), to generate pGV1472 ( Figure 13, SEQ ID NO: 2).
  • a Sad - MIuI fragment containing the TEF1 promoter, E. coll UvD, TDH3 promoter, E. coli HvC, and part of the CYC1 terminator was cloned into the same sites of the K. lactis expression plasmid, pGV1429 ( Figure 10), to generate pGV1473 ( Figure 14, SEQ ID NO: 3).
  • a BssHII - Not ⁇ fragment containing the TEF1 promoter, L lactis kivD, TDH3 promoter and S. cerevisiae ADH7. ScAdh7 was cloned into the K. lactis expression plasmid, pGV1431 ( Figure12), to obtain pGV1475 ( Figure15, SEQ ID NO: 4).
  • K. lactis strain GEVO1287 was transformed with the above plasmids, pGV1472, pGV1473, and pGV1475 (Table EX5-1 ) to express the isobutanol pathway.
  • K. lactis GEVO1287 was also transformed with empty vectors pGV1430, pGV1429, and pGV1431 (Table EX5-1 ).
  • Table EX5-1 K. lactis clones expressing an isobutanol pathway
  • Transformed cells were grown overnight and transferred to 10OmL fermentation bottles using 2OmL SC-WLU medium. Two mL samples were taken at 24 and 48 hours for GC analysis. At each time point, 2mL of a 20% glucose was added after removing samples for GC analysis. At 48 hours the fermentation was ended. GC samples were processed as described. Results are shown in Table EX5- 2 Up to 0.25 g/L isobutanol was produced in K. lactis transformed with an isobutanol pathway whereas the control strain without the pathway only produced 0.022 g/L in 48hours.
  • Example 6 lsobutanol production in Pdc plus S. cerevisiae
  • This example demonstrates isobutanol production in a member of Saccharomyces sensu stricto group, Saccharomyces clade, Crabtree-positive, post- WGD yeast, S. cerevisiae.
  • Plasmids pGV1254 ( Figure 16; SEQ ID NO: 10), pGV1295 ( Figure 17; SEQ ID NO: 11 ) pGV1390 ( Figure 18; SEQ ID NO: 12), and pGV1438 ( Figure 19; SEQ ID NO: 13) were high copy S. cerevisiae plasmids that together expressed the five genes of the isobutanol pathway (TABLE EX6-1 ).
  • pGV1390 was generated by cloning a Sail - BamH ⁇ fragment containing the L.
  • lactis alsS (SEQ ID NO: 5) into the high copy S. cerevisiae expression plasmid, pGV1387, where the L. lactis alsS would be expressed under the CUP1 promoter.
  • pGV1295 was generated by cloning a Sa/I - BamH ⁇ fragment containing the E. coli HvC (SEQ ID NO: 6) into the high copy S. cerevisiae expression plasmid, pGV1266, where the E. coli HvC would be expressed using the TDH3 promoter.
  • pGV1438 was generated by cloning a Sa/I - BamH ⁇ fragment containing the E. coli HvD (SEQ ID NO: 7) into the high copy S.
  • pGV1254 was made by cloning an EcoRI (filled in by Klenow polymerase treatment) - Xho ⁇ fragment containing the TDH3 promoter and S. cerevisiae ADH2 from pGV1241 into the BamH ⁇ (filled in by Klenow) and Xho ⁇ sites of pGV1186.
  • pGV1186 was made by cloning a Sa/I - BamH ⁇ fragment containing the L. lactis kivD (SEQ ID NO: 8) into a high copy S.
  • pGV1241 was made by cloning a Sa/I - BamHI fragment containing the S. cerevisiae ADH2 (SEQ ID NO: 9) into a high copy S. cerevisiae expression plasmid, pGV1106, where the S. cerevisiae ADH2 would be expressed using the TDH3 promoter.
  • GEVO1187 was transformed with plasmids as shown in Table EX6-1.
  • Table EX6-1 As a defective isobutanol pathway control, cells were transformed with pGV1056 ( Figure 23, empty vector control) instead of pGV1390. The transformants were plated onto appropriate selection plates. Single colonies from the transformation were isolated and tested for isobutanol production by fermentation. TABLE EX6-1
  • pGV1726 ( Figure 21 , SEQ ID NO: 15) is a yeast integration plasmid carrying the TRP1 marker and expressing B. subtilis alsS using the CUP1 promoter.
  • pGV1727 ( Figure 22, SEQ ID NO: 16) is a yeast integration plasmid carrying the LEU2 marker and expressing E. coli HvD under the TEF1 promoter and E. coli HvC under the TDH3 promoter.
  • Neither pGV1726 or pGV1727 carry a yeast replication origin.
  • lactis strain GEVO1742 by random integrations of the pathway genes.
  • GEVO1742 was transformed with the Acc65 ⁇ - ⁇ /goMIV fragment of pGV1590 containing the L. lactis kivd and S. cerevisiae ADH7 but without the yeast replication origin, to generate GEVO1794.
  • the presence of both L. lactis kivd and S. cerevisiae ADH7 was confirmed by colony PCR using primer sets 1334 + 1335 and 1338 + 1339, respectively.
  • GEVO1794 was transformed with pGV1727, a yeast integration plasmid carrying E. coli HvD (under the TEF1 promoter) and E.
  • GEVO1818 was confirmed by colony PCR for the presence of E. coli HvD and E. coli HvC using primer sets 1330 + 1331 and 1325 + 1328, respectively.
  • GEVO1818 was then transformed with pGV1726, a yeast integration plasmid carrying B. subtilis alsS (under the CUP1 promoter), that had been linearized by digesting with Ahdl to generate GEVO1829.
  • the presence of B. subtilis alsS was confirmed by colony PCR using primers 1321 + 1324.
  • Aerobic fermentations were carried out to test isobutanol production by the Pdc-minus strain carrying the isobutanol pathway, GEVO1829.
  • the Pdc-minus strain without the isobutanol pathway, GEVO1742 was used as a control.
  • These strains were cultured in YPD overnight at 30 0 C, 250 rpm, then diluted into 20 ml_ fresh YPD in a 125 ml_ flask and grown at 30 0 C, 250 rpm. 2 mL samples were taken at 24 and 48 hours, cells pelleted for 5 minutes at 14,000 x g and the supernatant was analyzed for isobutanol by GC.
  • Example 8A lsobutanol production in Pdc-minus S. cerevisiae GEVO1581 [00369] This example demonstrates isobutanol production in a Pdc-minus member of the Saccharomyces sensu stricto group, Saccharomyces clade yeast, Crabtree- positive yeast, post-WGD yeast, S. cerevisiae.
  • Strain GEVO1581 with the three genes encoding PDC activity deleted (pdd ⁇ , pdc ⁇ A, and pdc ⁇ A) was used to produce isobutanol.
  • Isobutanol pathway enzymes were encoded by genes cloned into three plasmids.
  • pGV1103 Figure 26, SEQ ID NO: 20
  • pGV1104 Figure 27, SEQ ID NO: 21
  • pGV1106 Figure 28, SEQ ID NO: 22
  • subtilis alsS gene express using the CUP1 promoter, was encoded on either a low copy CEN plasmid, pGV1673 ( Figure 32, SEQ ID NO: 26) or a high copy plasmid, pGV1649 ( Figure 29, SEQ ID NO: 23). Both of these plasmids used TRP1 as a marker gene. E. coli HvD (expressed using the TEF1 promoter) and E. coli HvC (expressed using the TDH3 promoter) were expressed off of the high copy plasmid pGV1677 ( Figure 33, SEQ ID NO: 27). This plasmid utilized HIS3 as a marker gene. L. lactis kivD (expressed using the TEF1 promoter) and S.
  • Fermentation experiments were carried out with GEVO1581 transformed with piasmids according to Table EX8-1 to determine the amount of isobutanol produced (titer) and the percentage of isobutanol to consumed glucose (yield).
  • Fermentations with Transformants of GEVO1581 Using cells grown in 3 ml. defined (SC-Ethanol) medium, 20 ml_ cultures were inoculated with transformants of GEVO1581 (3 independent colonies per transformation set) to an OD 6O o of approximately 0.1. The cultures were incubated at 30 0 C at 250 RPM in 125 mL metal cap flasks until they reached an OD 6 oo of approximately 1.
  • Table EX8A-3 48 hour time point data for fermentation in glucose, shown as an average of three replicates
  • Example 8B Isobutanol production in Pdc-minus S. cerevisiae GEVO1584
  • This example demonstrates isobutanol production in a Pdc-minus member of the Saccharomyces sensu stricto group, Saccharomyces clade, Crabtree-positive yeast, WGD yeast, S. cerevisiae.
  • GEVO1581 is a diploid strain, thus, a second backcross of a Pdc-minus yeast into the CEN. PK background was performed, yielding a Pdc-minus haploid strain GEVO1584 with the required auxotrophic markers for plasmid propagation.
  • Transformations of GEVO1584 The following combinations of plasmids were transformed into GEVO1584 (Table EX8B-1) using lithium acetate transformation (described in General Methods) followed by selection on appropriate minimal media.
  • pGV1672 ( Figure 31 , SEQ ID NO: 25), pGV1056 ( Figure 23, SEQ ID NO: 17), and pGV1062 ( Figure 24, SEQ ID NO: 18) were empty low copy CEN expression vectors that carry as marker genes, TRP1, HIS3, and URA3.
  • pGV1103 ( Figure 26, SEQ (D NO: 20), pGV1104 ( Figure 27, SEQ ID NO: 21) and pGV1102 ( Figure 25, SEQ ID NO: 19) were empty high copy expression vectors that carry as marker genes, URA3, HIS3 and TRP1, respectively.
  • the isobutanol pathway was expressed off of low copy CEN plasmids pGV1673 ( Figure 32, SEQ ID NO: 26), pGV1679 ( Figure 34, SEQ ID NO: 28) and pGV1683 ( Figure35, SEQ ID NO: 29).
  • pGV1673 carried the B. subtilis alsS under the CUP1 promoter and utilized the TRP1 marker gene.
  • pGV1679 carried the E. coli HvD and E. coli HvC genes expressed using the TEF1 and TDH3 promoters, respectively, and utilized the HIS3 marker gene.
  • pGV1683 carried the L. lactis kivd and the S.
  • pGV1649 carried the B. subtilis alsS under the CUP1 promoter and utilized the TRP 1 marker gene.
  • pGV1677 carried the E. coli HvD and E. coli HvC genes expressed using the TEF1 and TDH3 promoters, respectively, and utilized the HIS3 marker gene.
  • pGV1664 carried the L lactis kivd and the S. cerevisiae ADH7 genes expressed using the TEF1 and TDH3 promoters, respectively, and utilized the URA3 marker gene.
  • the cells were collected by centrifugation at 3000 RPM for 3 minutes and resuspended in 20 mL SC+Glucose-HWU media in 125 mL metal cap flasks. 250 ⁇ L of 100% ethano! was added to each culture to bring the concentration of ethanol to 1 %. A 2 mL aliquot was removed, the OD ⁇ oo was measured using 100 ⁇ L, and the remaining aliquot was centrifuged to pellet cells (14,000 xg, 5 min) and the supernatants were stored at -2O 0 C. The cultures were incubated at 125 rpm at 3O 0 C.
  • All Pdc-minus yeast consumed approximately 10 g/L of glucose and less than 2 g/L of ethanol after 48 hours. All strains accumulated ⁇ 1.5 g/L pyruvate, except for those carrying the isobutanol pathway on 2 ⁇ plasmids ( ⁇ 0.5 g/L). The accumulation of pyruvate and failure of the yeast to produce ethanol from glucose is confirmation that all lacked PDC activity. After 48 hours, the Pdc-minus yeast with the isobutanol pathway encoded on 2 ⁇ plasmids generated 0.248 ⁇ 0.032 g/L isobutanol at a theoretical yield of 6.36% of the consumed glucose (Table EX8B- 2).
  • the PDC-minus K. lactis strain GEVO1488, is engineered to delete GPD1 gene of K. lactis.
  • This PDC-minus GPD-minus strain is transformed with pGV1565 and pGV1568 ( Figure 36 and Figure 37). These transformants are then subjected to anaerobic batch fermentation and samples analyzed as described. As shown in Table EX9-1 , the additional deletion of GPD1 is expected to result in a significant increase in isobutanol yield.
  • Example 10 High-yield isobutanol fermentation using Crabtree-negative PDC-minus and GPD-minus K. lactis with balanced isobutanol pathway
  • Yield is further increased by the use of a pathway in which there is a balanced usage of NADH and NADPH. This balance is accomplished by the use of an engineered ilvC which is able to utilize NADH and the NADH-dependent alcohol dehydrogenase, Adh2. These constructs are used to express the isobutanol pathway in a PDC-minus and GPD-minus K. lactis. This strain is subjected to anaerobic batch fermentation as described above and samples are analyzed for isobutanol. As shown in Table EX9-1 , the yield of isobutanol using this pathway in a PDC-minus K. lactis is expected to result in a significant increase in yield.
  • Example 11 High-yield isobutanol fermentation using Crabtree-negative PDC-minus and GPD-minus K. lactis with balanced isobutanol pathway
  • An alternative route to balancing the NADH and NADPH usage is to overexpress an NADP + -dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in addition to the endogenous NAD + -dependent GAPDH, such that both NADH and NADPH are generated from glycolysis.
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • the isobutanol pathway can utilize an NADPH-dependent KARI enzyme and the NADH-dependent Adh2p.
  • PDC-minus and GPD-minus K. lactis is transformed with a construct expressing a NADP+-dependent GAPDH and an isobutanol pathway using Adh2.
  • This strain is subjected to anaerobic batch fermentation as described above and samples analyzed for isobutanol. As shown in Table EX9-1 , introduction of this NADP + -dependent GAPDH is expected to result in a significant increase in productivity of isobutanol.
  • Example 12 High-yield isobutanol fermentation using Crabtree-negative PDC-minus and GPD-minus K, lactis with balanced isobutanol pathway
  • Example 13 High-yield isobutanol fermentation using Crabtree-positive PDC-minus and GPD-minus S. cerevisiae
  • the PDC-minus S. cerevisiae strain is engineered to delete both GPD1 and GPD2.
  • This PDC-minus GPD-minus strain is transformed with plasmids expressing the isobutanol pathway in S. cerevisiae. These transformants are then subjected to anaerobic batch fermentation and samples analyzed as described. As is seen in Table EX13-1 , the additional deletions of GPD1 and GPD2 is expected to result in a significant increase in isobutanol yield.
  • Example 14 High-yield isobutanol fermentation using Crabtree-positive PDC-minus and GPD-minus S. cerevisiae with balanced isobutanol pathway [00384] Yield is further increased by the use of a pathway in which there is balanced usage of NADH and NADPH usage. This balance is accomplished by the use of an engineered KARI which is able to utilize NADH and the NADH-dependent alcohol dehydrogenase, Adh2p. These constructs are used to express the isobutanol pathway in a PDC-minus and GPD-minus S. cerevisiae. This strain is subjected to anaerobic batch fermentation as described above and samples are analyzed for isobutanol. As shown in Table EX13-1 , the yield of isobutanol using this pathway in a PDC-minus S. cerevisiae is expected to result in a significant increase in yield.
  • Example 15 High-yield isobutanol fermentation using Crabtree-positive PDC-minus GPD-minus S. cerevisiae with balanced isobutanol pathway
  • GAPDH NADP + -dependent glyceraldehydes 3-phosphate dehydrogenase
  • NADH and NADPH are generated from glycolysis.
  • the isobutanol pathway can utilize an NADPH-dependent KARI enzyme and the NADH-dependent Adh2.
  • PDC-minus and GPD-minus S. cerevisiae is transformed with a construct expressing a NADP + -dependent GAPDH and an isobutanol pathway using Adh2.
  • This strain is subjected to anaerobic batch fermentation as described above and samples analyzed for isobutanol. As shown in Table EX13-1, introduction of this NADP + -dependent GAPDH is expected to result in a significant increase in productivity of isobutanol.
  • Example 16 High-yield isobutanol fermentation using Crabtree-positive PDC-minus S. cerevisiae with balanced isobutanol pathway
  • Yet another alternative route to balancing the NADH and NADPH usage is to replace the endogenous NAD + -dependent GAPDH with an NADP + -dependent GAPDH in a PDC-minus and GPD-minus S. cerevisiae.
  • This strain is transformed with the isobutanol pathway and subjected to anaerobic batch fermentation as described above and samples analyzed for isobutanol.
  • introduction of this NADP + -dependent GAPDH is expected to result in a significant increase in productivity of isobutanol.
  • Table EX13-1 Isobutanol productivity in S. cerevisiae strains after 48 hours. (Listed numbers for the pdc-minus strains are expected numbers).
  • Example 17 High-yield isobutanol fermentation using Crabtree-negative PDC-minus GPD-minus evolved K. lactis with balanced isobutanol pathway [00387]
  • the yield for isobutanol may be increased by further engineering yeast microorganism to reduce production of minor byproducts. Isobutanol may be produced at a yield of about 90% theoretical.
  • Example 18 High-yield isobutanol fermentation using Crabtree-positive PDC-minus GPD-minus evolved S. cerevisiae with balanced isobutanol pathway [00388]
  • the yield for isobutanol may be increased by further engineering a yeast microorganism to reduce production of minor byproducts. Isobutanol may be produced at a yield of about 90% theoretical.
  • Sample preparation Samples were prepared from various timepoints for analysis by liquid chromatography and gas chromatography. 2 ml_ of media was removed and centrifuged at 14,000 x g for 10 min. The supernatant was removed and stored at 4 0 C until analysis.
  • optical density The optical density of the yeast cultures was determined at 600 nm using a DU 800 spectrophotometer (Beckman-Coulter,
  • the column and Rl temperatures were 6O 0 C. This method was lsocratic with 0.018N sulfuric acid in water as mobile phase. Flow was set at 1.1 mL/min. Injection size was 20 ⁇ L and the run time was 15 minutes
  • Lithium Acetate transformations of S. cerevisiae strains were transformed by the Lithium Acetate method (Gietz et al., Nucleic Acids Res. 27:69-74 (1992).
  • Cells were collected from overnight cultures grown in 50 mL of defined (SC) ethanol media at an OD 6 oo of approximately 0.8 to 1.0 by centrifugation at 2700 rcf for 2 minutes at room temperature.
  • the cell pellet was resuspended in 50 mL sterile water, collected by centrifugation (2700 rcf; 2 min; room temp.), and resuspended in 25 mL sterile water.
  • the cells were collected by centrifugation (2700 rcf; 2 min; room temp.) and resuspended in 1 mL 100 mM lithium acetate.
  • the cell suspension was transferred to a sterile 1.5 mL tube and collected by centrifugation at full speed for 10 seconds.
  • the cells were resuspended in 100 mM lithium acetate with a volume four times the volume of the cell pellet (e.g. 400 ⁇ L for 100 ⁇ L cell pellet).
  • Yeast colony PCR Yeast cells were taken from agar medium and transferred to 30 ⁇ l 0.2% SDS and heated for 4mins at 90 0 C. The cells were spun down and 1 ⁇ l of the supernatant was used for PCR using standard Taq (NEB).
  • YP contains 1 % (w/v) yeast extract, 2% (w/v) peptone.
  • YPD is YP containing 2% (w/v) glucose
  • YPE is YP containing 2% (w/v)
  • YPD80 medium is YP containing 80 g/L glucose, 0.2 g/L G418 antibiotic, 20 ⁇ M CuSO 4 , and 1% ethanol.
  • SC+Complete 20 g/L glucose, 14g/L SigmaTM Synthetic Dropout Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), and 6.7 g/L DifcoTM Yeast Nitrogen Base. 0.076 g/L histidine,
  • Solid versions of the above described media contain 2% (w/v) agar.
  • GEVO2962 is a modified yeast biocatalyst that contains genes within the chromosome of the biocatalyst which encode a pathway of enzymes that convert pyruvate into isobutanol.
  • GEVO2962 is GEVO2843 transformed with pGV2227 (SEQ ID NO: 57), which is a high copy yeast expression plasmid used to overexpress EcJlvC_Q110V ⁇ Escherichia coli HvC containing a Q to V mutation at position 110), LI _ilvD (Lactococcus lactis HvD), Z./_kivD2 (Lactococcus lactis kivD), and U__AdhA (Lactococcus lactis adhA).
  • the strain GEVO2843 is PDC-deficient and able to grow in high glucose media without addition of C2-compounds.
  • GEVO2843 has integrated into the PDC1 locus the Bs_alsS1_coSc (Bacillus subtilis alsS; SEQ ID NO: 58) and Li_kivD2_ coEc (Lactococcus lactis kivD, SEQ ID NO: 59) genes under the CUP1 and PGK1 promoters, respectively.
  • This strain also has the LI_kivD2_coEc (Lactococcus lactis kivD, SEQ ID NO: 59) and Dm_ ADH (Drosophila melanogaster ADH, SEQ ID NO: 60) under the TEF1 and TDH3 promoters, respectively, integrated into the PDC6 locus.
  • this strain has the Ec_ilvC_coSc__Q110V (SEQ ID NO: 61 ) and Sc_ILV3 ⁇ N20 (SEQ ID NO: 62) under the TDH3 and TEF1 promoters, respectively, integrated at the PDC5 locus.
  • the biocatalyst GEVO2962 was contacted with glucose in a medium suitable for growth of the biocatalyst, at about 30 0 C, the biocatalyst produced isobutanol from the glucose.
  • a 24-hour starter culture was started in a 50 mL conical tube with GEVO2962 cells from a frozen glycerol stock. The tube contained a 5 mL volume of YPD80 medium (Difco) at a starting OD 6 oo of about 1.0.
  • the starter culture was grown for approximately 24 hrs in a 3O 0 C shaker at 250 rpm. The entire contents of the starter culture were then transferred to a flask seed culture.
  • the flask seed culture was 1 L of YPD80 medium in a 2.8 L baffled Fembach flask. The seed flask culture was grown for approximately 24 hrs in a 3O 0 C shaker at 250 rpm. [00405] A portion of the flask seed culture was transferred to a 2 L DasGip seed fermenter containing about 750 ml_ of YPD80 medium to achieve a 1 OD ⁇ oo initial cell concentration.
  • the fermenter vessel was attached to a computer control system to monitor and control pH at 5.0 through addition of base, temperature at 3O 0 C, oxygen transfer rate (OTR), and agitation. The vessel was agitated, with a fixed agitation of 1000 rpm and 2 sL/h air flow overlay.
  • the dissolved oxygen was approximately zero throughout this experiment with an OTR of about 4-8 mM/h.
  • Continuous measurement of the fermentor vessel off-gas by mass spectrometer analysis was performed for oxygen, isobutanol, ethanol, and carbon dioxide throughout the experiment. Samples were aseptically removed from the fermenter vessel throughout the experiment and used to measure OD 60O , glucose concentration, and isobutanol concentration in the broth. [00406] At about 48 h intervals throughout the 470 h experiment, the fermenter whole broth was removed from the fermenter, cells were separated from the broth using centrifugation at about 2O 0 C and 4000 x g in 500 mL centrifuge bottles.
  • the cell pellets were resuspended in fresh YPD medium that contained 80 g/L glucose, 0.2 g/L G418 antibiotic, 20 ⁇ M CuSO 4 , and 1 % ethanol and returned to the fermenter.
  • fresh YPD medium that contained 80 g/L glucose, 0.2 g/L G418 antibiotic, 20 ⁇ M CuSO 4 , and 1 % ethanol and returned to the fermenter.
  • about 1 L of a flask culture of GEVO2962 at about 7 OD ⁇ oo was concentrated to 50-100 mL by centrifugation and then added to the fermenter vessel aseptically.
  • the fermenter vessel was attached by tubing to a smaller 400 mL fermenter vessel that served as a flash tank and operated in a recirculation loop with the fermenter.
  • Whole fermentation broth was recirculated between the flash tank and fermenter at a rate of about 10-30 mL per min.
  • the volume in the flash tank was approximately 100 ml. and the hydraulic retention time in the flash tank was about 3- 10 minutes.
  • Heat and vacuum were applied to the flash tank.
  • the vacuum level applied to the flash tank was initially set at about 60 mBar and the flash tank was set at approximately 36 0 C. Generally, the vacuum ranged from 50-65 mBar and the flash tank temperature ranged from 35 0 C to 37 0 C throughout the experiment.
  • Vapor from the heated flash tank was condensed into a collection vessel as distillate. Whole fermentation broth was continuously returned from the flash tank back to the fermentation vessel. When the concentration of isobutanol in the broth dropped below 1.5 g/L, the flash recycle system was turned off. The flash recycle was turned back on when the broth concentration of isobutanol reached above 2.5 g/L. [00408] The distillate recovered in the experiment was strongly enriched for isobutanol. Isobutanol formed an azeotrope with water and lead to a two phase distillate: an isobutanol rich top phase and an isobutanol lean bottom phase. Distillate samples were analyzed by GC for isobutanol concentration.
  • Isobutanol production reached a maximum at around 470 hrs with a total effective titer of about 111 g/L.
  • the isobutanol production rate was about 0.24 g/L/h on average over the course of the experiment.
  • the percent theoretical yield of isobutanol was approximately 36% at the end of the experiment.
  • Example 20 Isobutanol Production in Pdc- Gpd- Yeast
  • This example demonstrates isobutanol production at greater than 70% yield in a Pdc-minus, Gpd-minus member of the Saccharomyces sensu stricto group, Saccharomyces clade yeast, Crabtree-positive yeast, post-WGD yeast, S. cerevisiae.
  • the modified yeast biocatalyst, GEVO3158 encodes a heterologous pathway of enzymes that convert pyruvate into isobutanol. Genes for the pathway are located on the chromosome and on a single plasmid.
  • GEVO3158 is GEVO3124 transformed with pGV2247 (SEQ ID NO: 63), which is a high copy yeast expression plasmid used to overexpress Ec_ilvC_Q11 OV (Escherichia col i HvC containing a Q to V mutation at position 110), LIJIvD ⁇ Lactococcus lactis HvD), LI_kivD (Lactococcus lactis kivD), and LljadhA (Lactococcus lactis adhA).
  • the strain GEVO3124 is both GPD-deficient, PDC-deficient and able to grow in high glucose media without addition of C2-compounds.
  • GEVO3124 has integrated into the PDC1 locus the Bs_alsS1_coSc (Bacillus subtilis alsS; SEQ ID NO: 58J and LI_kivD2_ ⁇ Ec ⁇ Lactococcus lactis kivD, SEQ ID NO: 59) genes under the CUP1 and PGK1 promoters, respectively.
  • Bs_alsS1_coSc Bacillus subtilis alsS; SEQ ID NO: 58J and LI_kivD2_ ⁇ Ec ⁇ Lactococcus lactis kivD, SEQ ID NO: 59
  • This strain also has the Ec_ilvC_coSc_P2D1 -A1 (Escherichia coli HvC variant; SEQ ID NO: 64), LIJIvD_coSc, and LI_adhA (Lactococcus lactis adhA, SEQ ID NO: 66) under the TDH3, TEF1 and ENO2 promoters, respectively, integrated into the PDC6 locus.
  • this strain has the Ec JlvC_coSc_Q11 OV (Escherichia coli HvC containing a Q to V mutation at position 110; SEQ ID NO: 61) and Sc_ILV3 ⁇ N20 (SEQ ID NO: 62) under the TDH3 and TEF1 promoters, respectively, integrated at the PDC5 locus.
  • Ec JlvC_coSc_Q11 OV Escherichia coli HvC containing a Q to V mutation at position 110; SEQ ID NO: 61
  • Sc_ILV3 ⁇ N20 SEQ ID NO: 62
  • GEVO3124 was generated by deletion of GPD2 using Hph as marker in strain GEVO3059. Deletion of GPD2 was carried out by transforming a hygromycin resistance marker, Hph, flanked by the GPD2 5' and 3' targeting sequences. This gpd2::Hph disruption cassette was generated by multiple rounds of SOE PCR.
  • the GPD2 5' targeting sequence was amplified from pGV2164 (SEQ ID NO: 69), the CCW12 promoter was amplified from pGV1954 (SEQ ID NO: 70), the Hph ORF was amplified from pGV2074 (SEQ ID NO: 71 ), and the GPD2 3' targeting sequence was also amplified.
  • the GPD2 5' targeting sequence and the CCW12 promoter were stitched together by SOE-PCR, and the Hph ORF and GPD2 3' targeting sequence were stitched together by SOE-PCR. Lastly, these two SOE-PCR products were stitched together in another round of SOE-PCR.
  • GEVO3059 was then transformed into GEVO3059 recovered overnight in YPD+1 %EtOH+G418 or YPD+1 %EtOH+G418+1 g/L glycerol, and selected on YPD+G418+Hygro+1 g/L glycerol or YPD+G418+Hygro+10 g/L glycerol plates. Twelve colonies were re-streaked for singles and colony PCRs were performed in single colony isolates to test for correct 5' and 3' junctions and the loss of GPD2. [00413] GEVO3059 was generated by deletion of GPDI using KI_URA3 as marker in strain GEVO2994.
  • GPD1 Deletion of GPD1 was carried out by a bipartite integration scheme using the KI_URA3 marker.
  • the 5' bipartite fragment contained the GPD1J5' targeting sequence- T KLURA3 _s h o r t-P F B A i-KI_URA3_3' truncated and was generated by PCR with pGV2359 (SEQ ID NO: 72) as template.
  • the 3' bipartite fragment contained KI_URA3_5' truncated- T KI _URA 3 -GPD1_3' targeting sequence and was generated by PCR with pGV2157 (SEQ ID NO: 73) as template.
  • GEVO2994 was generated by integrating the Ec_ilvC_coSc_P2D1 -A1 (Escherichia coli HvC variant; SEQ ID NO: 64), LIJIvD_coSc, (Lactococcus lactis HvD, SEQ ID NO: 65) and LI_adhA (Lactococcus lactis adhA, SEQ ID NO: 66) under the TDH3, TEF1 and ENO2 promoters, respectively, into the PDC6 locus of GEVO2843.
  • This integration replaced the LI_kivD2_coEc (Lactococcus lactis kivD, SEQ ID NO: 59) and the Dm_ADH (Drosophila melanogaster ADH, SEQ ID NO: 60) that were present at the PDC6 locus in GEVO2843.
  • GEVO2843 was generated by integrating the Bs_alsS1_coSc (Bacillus subtilis alsS; SEQ ID NO: 58) and LI_kivD2__coEc (Lactococcus lactis kivD, SEQ ID NO: 59) into the PDC1 locus of GEVO2712
  • the flask seed culture was 80 ml_ of YPD80 medium in a 500 mL baffled Erlenmeyer flask.
  • the seed flask culture was grown for approximately 24 hrs in a 3O 0 C shaker at 250 rpm.
  • a portion of the flask seed culture was transferred to a 2 L DasGip fermenter containing about 750 mL of YPD80 medium to achieve a 0.5 OD 6 oo initial cell concentration.
  • the fermenter vessel was attached to a computer control system to monitor and control pH at 6.0 through addition of base, temperature at 3O 0 C, oxygen transfer rate (OTR), and agitation.
  • OTR oxygen transfer rate
  • the vessel was agitated, with a fixed agitation of 700 rpm to maintain an OTR of about 10 mM/h using a 5 sL/h air overlay until the OD 6 oo was about 8-10. After continuing growth for approximately 20 hrs, the OTR was decreased to approximately 0.2-0.7 mM/h by reducing agitation to a fixed 250-350 rpm and continued 5 sL/h airflow overlay.
  • Measurement of the fermentor vessel off-gas by mass spectrometer was included for ethanol, isobutanol, carbon dioxide, and oxygen. Continuous measurement of off-gas concentrations of carbon dioxide and oxygen were also measured by a DasGip off-gas analyzer throughout the experiment. Samples were aseptically removed from the fermenter vessel throughout the experiment and used to measure OD 6 oo. glucose concentration by HPLC, and isobutanol concentration in the broth by GC.
  • Isobutanol production reached a maximum at around 7 days with a titer of about 10 g/L.
  • Yield of the fermentation calculated when the titer of isobutanol was between 3.7 g/L and 10 g/L, was approximately 74% maximum theoretical.
  • Yield of the fermentation calculated when the titer of isobutanol was between 0 g/L and 10 g/L, was approximately 52% maximum theoretical.
  • Yield of the fermentation calculated when the titer of isobutanol was between 0.5 g/L and 10 g/L, was approximately 61 % maximum theoretical.
  • This example demonstrates isobutanol production at greater than 70% yield in a Pdc-minus, Gpd-minus member of the Saccharomyces sensu stricto group, Saccharomyces clade yeast, Crabtree-positive yeast, post-WGD yeast, S. cerevisiae.
  • the modified yeast biocatalyst encodes a heterologous pathway of enzymes that convert pyruvate into isobutanol. Genes for the pathway are located on the chromosome and on a single plasmid.
  • GEVO3159 is GEVO3124 transformed with pGV2082 (SEQ ID NO: 67), which is a high copy yeast expression plasmid used to overexpress EcJlvC_Q110V (Escherichia coli HvC containing a Q to V mutation at position 110), LMIvD (Lactococcus lactis HvD), LI_kivD2 (Lactococcus lactis kivD), and Dm_ADH (Drosophila melanogaster ADH).
  • pGV2082 SEQ ID NO: 67
  • the strain GEVO3124 is both GPD-deficient, PDC-deficient and able to grow in high glucose media without addition of C2-compounds.
  • GEVO3124 has integrated into the PDC1 locus the Bs_alsS1_coSc (Bacillus subtilis alsS; SEQ ID NO: 58) and LI_kivD2_coEc (Lactococcus lactis kivD, SEQ ID NO: 59) genes under the CUP1 and PGK1 promoters, respectively.
  • This strain also has the EcJlvC_ ⁇ Sc_P2D1-A1 (Escherichia coli HvC variant; SEQ ID NO: 64), LI_ilvD_coSc (Lactococcus lactis HvD, SEQ ID NO: 65), and LI_adhA (Lactococcus lactis adhA, SEQ ID NO: 66) under the TDH3, TEF1 and ENO2 promoters, respectively, integrated into the PDC6 locus.
  • EcJlvC_ ⁇ Sc_P2D1-A1 Escherichia coli HvC variant; SEQ ID NO: 64
  • LI_ilvD_coSc Lactococcus lactis HvD, SEQ ID NO: 65
  • LI_adhA Lactococcus lactis adhA
  • this strain has the Ec_ilvC_coSc__Q11 OV (Escherichia coli HvC containing a Q to V mutation at position 110; SEQ ID NO: 61) and Sc_ILV3 ⁇ N20 (SEQ ID NO: 62) under the TDH3 and TEF1 promoters, respectively, integrated at the PDC5 locus.
  • Ec_ilvC_coSc__Q11 OV Escherichia coli HvC containing a Q to V mutation at position 110; SEQ ID NO: 61
  • Sc_ILV3 ⁇ N20 SEQ ID NO: 62
  • GEVO3124 was generated by deletion of GPD2 using Hph as marker in strain GEVO3059. Deletion of GPD2 was carried out by transforming a hygromyc/A? res/stance marker, Hph, flanked by the GPD2 5' and 3' targeting sequences. This gpd2::Hph disruption cassette was generated by multiple rounds of SOE-PCR.
  • the GPD2 5' targeting sequence was amplified from pGV2164 (SEQ ID NO: 69), the CCW12 promoter was amplified from pGV1954 (SEQ ID NO: 70), the HPH ORF was amplified from pGV2074 (SEQ ID NO: 71 ), and the GPD2 3' targeting sequence was also amplified.
  • the GPD2 5' targeting sequence and the CCW12 promoter were stitched together by SOE-PCR, and the Hph ORF and GPD2 3' targeting sequence were also stitched together by SOE-PCR. Lastly, these two SOE-PCR products were stitched together in another round of SOE-PCR.
  • GEVO3059 was then transformed into GEVO3059 recovered overnight in YPD+1 %EtOH+G418 or YPD+1 %EtOH+G418+1 g/L glycerol, and selected on YPD+G418+Hygro+1 g/L glycerol or YPD+G418+Hygro+10 g/L glycerol plates. Twelve colonies were re-streaked for singles and colony PCRs were performed in single colony isolates to test for correct 5' and 3' junctions and the loss of GPD2. [00422] GEVO3059 was generated by deletion of GPD1 using KI_URA3 as marker in strain GEVO2994.
  • GPD1 Deletion of GPD1 was carried out by a bipartite integration scheme using the KI_URA3 marker.
  • the 5' bipartite fragment contained the GPDIJi targeting sequence-T K ⁇ _ ⁇ RA3 _ shor rP FBA i-KI_URA3_3' truncated and was generated by PCR with pGV2359 (SEQ ID NO: 72) as template.
  • the 3' bipartite fragment contained KI_URA3_5' Xrunca ⁇ e ⁇ -T K ⁇ _ ⁇ RA3 -GPD1 _3' targeting sequence and was generated by PCR with pGV2175 (SEQ ID NO: 73) as template.
  • GEVO2994 was generated by integrating the EcJlvC_coSc_P2D1-A1 (Escherichia coli HvC variant; SEQ ID NO: 64), LI_ilvD_coSc (Lactococcus lactis HvD,
  • GEVO2843 was generated by integrating the Bs_alsS1_coSc (Bacillus subtilis alsS; SEQ ID NO: 58 and LI_kivD2_coEc (Lactococcus lactis kivD, SEQ ID NO: 59) into the PDC1 locus of GEVO2712.
  • the biocatalyst GEVO3159 was contacted with glucose in a medium suitable for growth of the biocatalyst, at about 30 0 C, the biocatalyst produced isobutanol from the glucose.
  • a 24-hour starter culture was started in a 50 mL conical tube with GEVO3159 cells from a frozen glycerol stock. The tube contained a 5 mL volume of YPD80 at a starting OD 6 oo of about 1.0.
  • the starter culture was grown for approximately 24 hrs in a 3O 0 C shaker at 250 rpm. The entire contents of the starter culture were then transferred to a flask seed culture.
  • the flask seed culture was 80 mL of YPD80 in a 500 mL baffled Erlenmeyer flask. The seed flask culture was grown for approximately 24 hrs in a 3O 0 C shaker at 250 rpm. [00425] A portion of the flask seed culture was transferred to a 2 L DasGip fermenter containing about 750 mL of YPD80 medium to achieve a 0.5 OD 6 oo initial cell concentration. The fermenter vessel was attached to a computer control system to monitor and control pH at 6.0 through addition of base, temperature at 30 0 C, oxygen transfer rate (OTR), and agitation.
  • OTR oxygen transfer rate
  • the vessel was agitated, with a fixed agitation of 700 rpm to maintain an OTR of about 10 mlvl/h using a 5 sL/h air overlay until the OD 6 oo was about 8-10. After continuing growth for approximately 20 hrs, the OTR was decreased to approximately 0.2-0.7 mlvl/h by reducing agitation to a fixed 250-350 rpm and continued 5 sL/h airflow overlay.
  • Measurement of the fermentor vessel off-gas by mass spectrometer was included for ethanol, isobutanol, carbon dioxide, and oxygen. Continuous measurement of off-gas concentrations of carbon dioxide and oxygen were also measured by a DasGip off-gas analyzer throughout the experiment. Samples were aseptically removed from the fermenter vessel throughout the experiment and used to measure OD 6 oo. glucose concentration by HPLC, and isobutanol concentration in the broth by GC.
  • Isobutanol production reached a maximum at around 5 days with a titer of about 8.5 g/L.
  • Yield of the fermentation calculated when the titer of isobutanol was between 3.2 g/L and 8.5 g/L, was approximately 75% maximum theoretical.
  • Yield of the fermentation calculated when the titer of isobutanol was between 0 g/L and 8.5 g/L, was approximately 48% maximum theoretical.
  • Yield of the fermentation calculated when the titer of isobutanol was between 1 g/L and 8.5 g/L, was approximately 58% maximum theoretical.
  • Example 22 Isobutanol Production in Pdc- Gpd-, Co-Factor Balanced Yeast
  • This example demonstrates isobutanol production at greater than 70% yield in a Pdc-minus, Gpd-minus member of the Saccharomyces sensu stricto group, Saccharomyces clade yeast, Crabtree-positive yeast, post-WGD yeast, S. cerevisiae, expressing an NADH-dependent isobutanol biosynthetic pathway.
  • the recombinant yeast microorganism, GEVO3160 encodes a heterologous biosynthetic pathway that converts pyruvate into isobutanol.
  • GEVO3160 is GEVO3128 transformed with pGV2247(SEQ ID NO: 63), which is a high copy yeast expression plasmid used to overexpress Ec_ilvC_P2D1 -A1 (Escherichia coli HvC variant), LIJIvD (Lactococcus lactis HvD), U_kivD (Lactococcus lactis kivD), and LI_adhA (Lactococcus lactis adhA).
  • the strain GEVO3128 is both GPD-deficient, PDC-deficient and able to grow in high glucose media without addition of C2- compounds.
  • GEVO3128 has integrated into the PDC1 locus the Bs_alsS1_coSc (Bacillus subtilis alsS; SEQ ID NO: 58) and U_kivD2_coEc (Lactococcus lactis kivD, SEQ ID NO: 59) genes under the CUP1 and PGK1 promoters, respectively.
  • This strain also has the Ec_ilvC_coSc_P2D1 -A1 (Escherichia coli HvC variant; SEQ ID NO: 64), LIJIvD_coSc (Lactococcus lactis HvD, SEQ ID NO: 65), and LI__adhA (Lactococcus lactis adhA, SEQ ID NO: 66) under the TDH3, TEF1 and ENO2 promoters, respectively, integrated into the PDC6 locus.
  • Ec_ilvC_coSc_P2D1 -A1 Escherichia coli HvC variant; SEQ ID NO: 64
  • LIJIvD_coSc Lactococcus lactis HvD, SEQ ID NO: 65
  • LI_adhA Lactococcus lactis adhA
  • this strain has the Ec_ilvC_coSc__Q11 OV (Escherichia coli HvC containing a Q to V mutation at position 110; SEQ ID NO: 61) and Sc_ILV3 ⁇ N20 (SEQ ID NO: 62) under the TDH3 and TEF1 promoters, respectively, integrated at the PDC5 locus.
  • Ec_ilvC_coSc__Q11 OV Escherichia coli HvC containing a Q to V mutation at position 110; SEQ ID NO: 61
  • Sc_ILV3 ⁇ N20 SEQ ID NO: 62
  • GEVO3128 was generated by deletion of GPD1 using Hph as marker in strain GEVO3061.
  • deletion of GPD1 was pursued in the gpd2::KI_URA3 deletion strains GEVO3061
  • Deletion of GPD1 was carried out by transforming a hygromycin resistance marker, Hph, flanked by the GPD1 5' and 3' targeting sequences.
  • This gpd1::Hph disruption cassette was generated by multiple rounds of SOE PCR.
  • the GPD1 5' targeting sequence was amplified from pGV2163 (SEQ ID NO: 74), the CCW12 promoter was amplified from pGV1954 (SEQ ID NO: 70), the Hph ORF was amplified from pGV2074 (SEQ ID NO: 71 ), and the GPD1 3' targeting sequence was amplified by PCR.
  • the GPD1 5' targeting sequence and the CCW12 promoter were stitched together by SOE-PCR, and the Hph ORF and GPDI 3' targeting sequence were also stitched together by SOE-PCR.
  • these two SOE-PCR products were stitched together in another round of SOE-PCR.
  • the resulting product was then transformed into GEVO3061 , recovered overnight in YPD+1%EtOH+G418 or YPD+1%EtOH+G418+1 g/L glycerol, and selected on YPD+G418+Hygro+1 g/L glycerol or YPD+G418+Hygro+10 g/L glycerol plates
  • GEVO3061 was generated by deletion of GPD2 using KI_URA3 as marker in strain GEVO2994. Deletion of GPD2 was carried out by a bipartite integration scheme using the KIJJRA3 marker.
  • the 5' bipartite fragment contained the GPD2J5' targeting sequence- T K ⁇ _ ⁇ RA3 _ shO r t -P FBA i-KI_URA3_3' truncated and was generated by PCR with pGV2360 (SEQ ID NO: 75) as template.
  • the 3' bipartite fragment contained KIJJRA3J5' truncated- T K IJJRA 3 -GPD2_3' targeting sequence and was generated by PCR with pGV2381(SEQ ID NO: 76) as a template.
  • the 3' truncated and 5' truncated KIJURA3 overlapped by 347 bp to allow for recombination between the KI_URA3 sequences and reconstitution of a functional KI_URA3 gene.
  • the 5' and 3' bipartite fragments were co-transformed into GEVO2994 and selected on SCD-U+1g/L glycerol plates.
  • GEVO2994 was generated by integrating the Ec_ilvC__coSc__P2D1-A1 (Escherichia coli HvC variant; SEQ ID NO: 64), LI_ilvD_coSc (Lactococcus lactis HvD, SEQ ID NO: 65), and LI_adhA (Lactococcus lactis adhA, SEQ ID NO: 66) under the TDH3, TEF1 and ENO2 promoters, respectively, into the PDC6 locus of GEVO2843.
  • Ec_ilvC__coSc__P2D1-A1 Escherichia coli HvC variant; SEQ ID NO: 64
  • LI_ilvD_coSc Lactococcus lactis HvD, SEQ ID NO: 65
  • LI_adhA Lactococcus lactis adhA
  • GEVO2843 was generated by integrating the Bs_alsS1 __coSc (Bacillus subtilis alsS; SEQ ID NO: 58) and U_kivD2_coEc (Lactococcus lactis kivD, SEQ ID NO: 59) into the PDCI locus of GEVO2712.
  • the biocatalyst GEVO3160 was contacted with glucose in a medium suitable for growth of the biocatalyst, at about 3O 0 C, the biocatalyst produced isobutanol from the glucose.
  • a 24-hour starter culture was started in a 50 mL conical tube with GEVO3160 cells from a frozen glycerol stock. The tube contained a 5 mL volume of YPD80 medium at a starting OD 600 of about 1.0.
  • the starter culture was grown for approximately 24 hrs in a 3O 0 C shaker at 250 rpm. The entire contents of the starter culture were then transferred to a flask seed culture.
  • the flask seed culture was 80 mL of YPD80 medium in a 500 mL baffled Erlenmeyer flask. The seed flask culture was grown for approximately 24 hrs in a 3O 0 C shaker at 250 rpm. [00433] A portion of the flask seed culture was transferred to a 2 L DasGip fermenter containing about 750 mL of YPD80 medium to achieve a 0.5 OD 6 oo initial cell density. The fermenter vessel was attached to a computer control system to monitor and control pH at 6.0 through addition of base, temperature at 30 0 C, oxygen transfer rate (OTR), and agitation.
  • OTR oxygen transfer rate
  • the vessel was agitated, with a fixed agitation of 700 rpm to maintain an OTR of about 10 mlWh using a 5 sL/h air overlay until the OD 6O o was about 8-10. After continuing growth for approximately 20 hrs, the OTR was decreased to approximately 0.2-0.4 mM/h by reducing agitation to a fixed 200 rpm and continued 5 sL/h airflow overlay.
  • Measurement of the fermentor vessel off- gas by mass spectrometer was included for ethanol, isobutanol, carbon dioxide, and oxygen. Continuous measurement of off-gas concentrations of carbon dioxide and oxygen were also measured by a DasGip off-gas analyzer throughout the experiment.
  • This example demonstrates isobutanol production at greater than 70% yield in a Pdc-minus, Gpd-minus member of the Saccharomyces sensu stricto group, Saccharomyces clade yeast, Crabtree-positive yeast, post-WGD yeast, S. cerevisiae, expressing an NADH-dependent isobutanol biosynthetic pathway.
  • the recombinant yeast microorganism, GEVO3160 encodes a heterologous biosynthetic pathway that converts pyruvate into isobutanol. Genes for the pathway are located on the chromosome and on a single plasmid.
  • GEVO3160 is GEVO3128 transformed with pGV2247 (SEQ ID NO: 63), which is a high copy yeast expression plasmid used to overexpress Ec_ilvC_P2D1-A1 (Escherichia coli HvC variant), LIJIvD (Lactococcus lactis UvD), LIJkivD (Lactococcus lactis kivD), and LI_adhA (Lactococcus lactis adhA).
  • the strain GEVO3128 is both GPD-deficient, PDC-deficient and able to grow in high glucose media without addition of C2- compounds.
  • GEVO3128 has integrated into the PDC1 locus the Bs_alsS1_coSc (Bacillus subtilis alsS; SEQ ID NO: 58) and LI_kivD2_coEc (Lactococcus lactis kivD, SEQ ID NO: 59) genes under the CUP1 and PGK1 promoters, respectively.
  • This strain also has the Ec_ilvC_coSc_P2D1-A1 (Escherichia coli HvC variant; SEQ ID NO: 64), LI_ilvD_coSc (Lactococcus lactis HvD, SEQ ID NO: 65), and LI_adhA (Lactococcus lactis adhA, SEQ ID NO: 66) under the TDH3, TEF1 and ENO2 promoters, respectively, integrated into the PDC6 locus.
  • Ec_ilvC_coSc_P2D1-A1 Escherichia coli HvC variant; SEQ ID NO: 64
  • LI_ilvD_coSc Lactococcus lactis HvD, SEQ ID NO: 65
  • LI_adhA Lactococcus lactis adhA
  • this strain has the Ec_ilvC_coSc_Q110V (Escherichia coli HvC containing a Q to V mutation at position 110; SEQ ID NO: 61 ) and Sc_ILV3 ⁇ N20 (SEQ ID NO: 62) under the TDH3 and TEF1 promoters, respectively, integrated at the PDC5 locus.
  • GEVO3128 was generated by deletion of GPDI using Hph as marker in strain GEVO3061.
  • deletion of GPD1 was pursued in the gpd2::KI_URA3 deletion strains GEVO3061
  • Deletion of GPD1 was carried out by transforming a hygromycin resistance marker, Hph, flanked by the GPD1 5' and 3' targeting sequences.
  • This gpd1::Hph disruption cassette was generated by multiple rounds of SOE PCR.
  • the GPD1 5' targeting sequence was amplified from pGV2163 (SEQ ID NO: 74), the CCW12 promoter was amplified from pGV1954 (SEQ ID NO: 70), the Hph ORF was amplified from pGV2074 (SEQ ID NO: 71 ), and the GPD1 3' targeting sequence was amplified by PCR.
  • the GPDI 5' targeting sequence and the CCW12 promoter were stitched together by SOE-PCR, and the Hph ORF and GPD1 3' targeting sequence were also stitched together by SOE-PCR.
  • these two SOE-PCR products were stitched together in another round of SOE-PCR.
  • the resulting product was then transformed into GEVO3061 , recovered overnight in YPD+1 %EtOH+G418 or YPD+1 %EtOH+G418+1 g/L glycerol, and selected on YPD+G418+Hygro+1 g/L glycerol or YPD+G418+Hygro+10 g/L glycerol plates
  • GEVO3061 was generated by deletion of GPD2 using KI_URA3 as marker in strain GEVO2994. Deletion of GPD2 was carried out by a bipartite integration scheme using the KI URA3 marker.
  • the 5' bipartite fragment contained the GPD2J> targeting sequence-T KL uR A3 _s h or t -P FBA i-KI__URA3_3' truncated and was generated by PCR with pGV2360 (SEQ ID NO: 75) as template.
  • the 3' bipartite fragment contained KI_URA3_5' truncated-T K ⁇ j j RA3 -GPD2_3' targeting sequence and was generated by PCR with pGV2381 (SEQ ID NO: 76) as template.
  • the 5' and 3' bipartite fragments were co-transformed into GEVO2994 and selected on SCD-U+1g/L glycerol plates.
  • GEVO2994 was generated by integrating the EcJlvC_coSc__P2D1 -A1 (Escherichia coli HvC variant; SEQ ID NO: 64), LI_ilvD_coSc (Lactococcus lactis HvD, SEQ ID NO: 65), and LI_adhA (Lactococcus lactis adhA, SEQ ID NO: 66) under the TDH3, TEF1 and ENO2 promoters, respectively, into the PDC6 locus of GEVO2843.
  • GEVO2843 was generated by integrating the Bs_alsS1_coSc (Bacillus subtilis alsS; SEQ ID NO: 58) and U_kivD2_coEc (Lactococcus lactis kivD, SEQ ID NO: 59) into the PDC1 locus of GEVO2712.
  • the biocatalyst GEVO3160 was contacted with glucose in a medium suitable for growth of the biocatalyst, at about 3O 0 C, the biocatalyst produced isobutanol from the glucose.
  • a 24-hour starter culture was started in a 50 ml_ conical tube with GEVO3160 cells from a frozen glycerol stock. The tube contained a 5 mL volume of YPD80 medium at a starting OD 60O of about 1.0.
  • the starter culture was grown for approximately 24 hrs in a 3O 0 C shaker at 250 rpm. The entire contents of the starter culture were then transferred to a flask seed culture.
  • the flask seed culture was 80 mL of YPD80 medium in a 500 mL baffled Erlenmeyer flask. The seed flask culture was grown for approximately 24 hrs in a 3O 0 C shaker at 250 rpm. [00441] A portion of the fiask seed culture was transferred to a 2 L DasGip fermenter containing about 750 mL of YPD80 medium to achieve a 0.5 OD 6 oo initial cell concentration. The fermenter vessel was attached to a computer control system to monitor and control pH at 6.0 through addition of base, temperature at 3O 0 C, oxygen transfer rate (OTR), and agitation.
  • OTR oxygen transfer rate
  • the vessel was agitated, with a fixed agitation of 700 rpm to maintain an OTR of about 10 mM/h using a 5 sL/h air overlay until the OD 6 oo was about 8-10. After continuing growth for approximately 20 hrs, the OTR was decreased to approximately 0.3-0.8 mM/h by reducing agitation to a fixed 180-350 rpm and continued 5 sL/h airflow overlay.
  • Measurement of the fermentor vessel off-gas by mass spectrometer was included for ethanol, isobutanol, carbon dioxide, and oxygen. Continuous measurement of off-gas concentrations of carbon dioxide and oxygen were also measured by a DasGip off-gas analyzer throughout the experiment. Samples were aseptically removed from the fermenter vessel throughout the experiment and used to measure OD 6 oo, glucose concentration by HPLC 1 and isobutanol concentration in the broth by GC.
  • Isobutanol production reached a maximum at around 7 days with a titer of about 14 g/L.
  • Yield of the fermentation calculated when the titer of isobutanol was between 7.2 g/L and 12.6 g/L, was approximately 71 % maximum theoretical.
  • Yield of the fermentation calculated when the titer of isobutanol was between 0 g/L and 14 g/L, was approximately 52% maximum theoretical.
  • Yield of the fermentation calculated when the titer of isobutanol was between 0.5 g/L and 14 g/L, was approximately 60% maximum theoretical.
  • GEVO3647 contains P ADH rBs_alsS1_coSc ⁇ Bacillus subtilis alsS; SEQ ID NO: 58) with two copies of the Lactococcus lactis kivD gene (SEQ ID NO: 59) integrated at the PDC1 locus.
  • the strain is a transformation product of the parent strain GEVO3532 with plasmid pGV2563 (SEQ ID NO: 68).
  • Medium used for the fermentation was YP + 80 g/L glucose + 1 % v/v Ethanol + 0.2 g/L G418.
  • the medium was filter sterilized using a 1L bottle top Corning PES 0.22 ⁇ m filter (431174).
  • Medium was pH adjusted 6.0 in the fermenter vessels using 6N KOH.
  • Table EX 15-1 outlines medium components per liter of Di-
  • Fermenter vessels were sterilized, along with the appropriate dissolved oxygen probes and. pH probes, for 60 minutes at 121 0 C. pH probes were calibrated prior to sterilization however, dissolved oxygen probes were calibrated post sterilization in order to achieve complete polarization prior to calibration.
  • Table EX15-1 outlines the process control parameters used during the fermentation. Note that pH was controlled using 6N KOH and 2N H 2 SO 4 .
  • the fermentation was run for 84.3 h. Vessels were sampled every 6-10 h or 3 times daily. Sterile 5 ml_ syringes were used to collect 3 mL of fermenter broth via a sterile sample port. The sample was placed in a 2 mL microfuge tube and a portion was used to measure cell density (OD 6 oo) on a Genesys 10 spectrophotometer (Thermo Scientific). The remaining sample was filtered through a 0.22 ⁇ m Corning filter. The supernatant from each vessel was refrigerated in a 96- well deep well plate, and stored at 4 0 C prior to gas and liquid chromatography analysis.
  • Example 25 Cytosolic ALS homoloqs that support isobutanol production
  • This example demonstrates isobutanol production using expression of cytosolically localized ALS genes in the presence of the rest of the isobutanol pathway.
  • the ALS genes were integrated into the PDC1 locus of S. cerevisiae strain GEVO1187 and isobutanol production was achieved by expression from plasmid of the other genes in the isobutanol pathway.
  • Isobutanol production in strains carrying the ALS genes from T. atroviride ⁇ Ta_ALS) and T. stipitatus (Ts_ALS) was compared to isobutanol production in strains carrying the ALS gene from B. subtilis (either Bs_alsS2 or Bs_alsS1_coSc).
  • Strains, and plasmids are listed in Tables EX16-1 and EX16-2, respectively.
  • Cloning techniques included digestion with restriction enzymes, gel purification of DNA fragments (using the Zymoclean Gel DNA Recovery Kit, Cat# D4002, Zymo Research Corp, Orange, CA), ligations of two DNA fragments (using the Roche rapid ligation kit, Cat# 11 635 379 001 , Roche Diagnostics, Mannheim, Germany), Klenow treatment of fragments to give blunt ends (using the NEB DNA Polymerase I, Large (Klenow), cat# M0210S, Ipswich, MA), and bacterial transformations into chemically competent E. coli cells made at GEVO (TOP10). Plasmid DNA was purified from E.
  • coli cells using the Qiagen QIAprep Spin Miniprep Kit (Cat# 27106, Qiagen, Valencia, CA). [00457] PCR was performed on an Eppendorf Mastercycler (Cat# 71086, Novagen, Madison Wl). The following PCR program was followed for all primer sets unless otherwise noted: 94 0 C for 2 min then 40 cycles of (94 C 30 sec, 54 ° C 30 sec, 72 ° C 1 min) then 72 ° C for 10 min. Yeast colony PCR used the FailSafeTM PCR System EPICENTRE ® Biotechnologies, Madison, Wl; Catalog #FS99250).
  • a PCR cocktail containing 15 ⁇ l of Master Mix E buffer, 10.5 ⁇ l water, 2 ⁇ l of each primer at 10 ⁇ M concentration, 0.5 ⁇ l polymerase enzyme mix from the kit was added to a 0.2 mL PCR tube for each sample (30 ⁇ l each).
  • a small amount of cells was added to the reaction tube using a sterile P10 pipette tip. Presence of the positive PCR product was assessed using agarose gel electrophoresis. The following primer pairs were used.
  • Primers 1432 and 1433 for the 5'-ends of all integrations 800 bp band
  • primers 1435 and 2233 for the 3'-ends of pGV2114 integrations 1.1 Kb band
  • primers 1435 and 2234 for the 3'-end of the pGV2115 integrations 1.1 Kb band
  • primers 1435 and 2236 for the 3'-ends of the pGV2117 integrations 1.1 Kb band
  • primers 1435 and 2237 for the 3'-ends of the pGV2118 integrations (1.1 Kb band).
  • Transformation of integration plasmids was performed according to the lithium acetate protocol described above. Integration plasmids were digested with Nru ⁇ , checked by gel electrophoresis for complete digestion and used directly from digestion. Integrative transformants were selected by plating the transformed cells on SCD-T rp agar medium. Once the transformants were single colony purified they were maintained on SCD-Trp plates. Once transformants were screened by PCR as described above for proper integration, each strain was transformed with the plasmid pGV2082. Transformants were plated to YPD plates containing 0.2 g/L G418.
  • SCD-Trp 20 g/L glucose, 14g/L SigmaTM Synthetic Dropout Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), and 6.7 g/L DifcoTM Yeast Nitrogen Base. 0.076 g/L histidine, 0.380 g/L leucine, and 0.076 g/L uracil.
  • Strains with integrated ALS genes expressed from the CUP1 promoter were transformed with pGV2082 (which carries the other 4 isobutanol pathway genes Ec_ilvC_coScQ11 OV (SEQ ID NO: 61 ), LIJIvD (SEQ ID NO: 65), LI_kivd2__coEc (SEQ ID NO: 59), and Dm__ADH (SEQ ID NO: 60)).
  • Strains were patched onto YPD plates containing 0.2 mg/mL G418. The following morning cells were removed from the plate with a sterile toothpick and resuspended in 4 ml_ of YPD with 0.2 mg/mL G418.
  • GEVO2280 was constructed by transforming GEVO1187 with the integration plasmid pGV1730.
  • the plasmid pGV1730 was first linearized with NwI, which cuts such that the linear plasmid will integrate into the PDC1 locus, and the DNA was transformed using the standard yeast transformation protocol. Transformants were selected by plating to SCGal-Trp plates. Individual integrants were verified using colony PCR with primers 1432 and 1433 to detect proper integration at the 5'-end (803 bp fragment) and primers 1220 and 1435 to detect proper integration at the 3'-end (772 bp).
  • GEVO2618 was constructed by transforming GEVO1187 with the integration plasmid pGV2114.
  • the plasmid was first linearized with Nru ⁇ , which cuts such that the linear plasmid will integrate into the PDC1 locus, and the DNA was transformed using the standard yeast transformation protocol described above. Correct integration was verified with colony PCR using primers 1432 and 1433 to check the 5'-end of the integration (800 bp band) and primers 1435 and 2233 for the 3'-end of pGV2114 integration (1 ,100 bp band).
  • GEVO2621 was constructed by transforming GEVO1187 with the integration plasmid pGV2117.
  • the plasmid was first linearized with Nru ⁇ , which cuts such that the linear plasmid will integrate into the PDC1 locus, and the DNA was transformed using the standard yeast transformation protocol described above, integration was verified with colony PCR using primers 1432 and 1433 to check the 5'-end of the integration (800 bp band) and primers 1435 and 2236 for the 3'-end of pGV2117 integration (1 ,100 bp band).
  • GEVO2622 was constructed by transforming GEVO1187 with the integration plasmids pGV2118.
  • the plasmid was first linearized with Nru ⁇ , which cuts such that the linear plasmid will integrate into the PDC1 locus, and the DNA was transformed using the standard yeast transformation protocol described above. Twelve transformants were single colony purified. Correct integration was verified with colony PCR using primers 1432 and 1433 to check the 5'-end of the integration (800 bp band) and primers 1435 and 2237 for the 3'-end of pGV2118 integration (1 ,100 bp band).
  • Each ALS-containing strain was transformed with the 4 component pathway plasmid, pGV2082 (SEQ ID NO: 67), as described above.
  • Control strains GEVO2280 (Bs_alsS2) and GEVO1187 (no ALS) were also transformed with pGV2082.
  • Transformants were single colony purified and maintained on YPD plates with 0.2 mg/ml_ G418.
  • the plasmid pGV2044 carries the genes Ec_ilvC__coSc Q110V , Bs_AlsS2, LI_ilvD__coSc and Dm_ADH.
  • the plasmid pGV2082 was created from pGV2044 by replacing the Bs_AlsS2 with LI_kivD2_coEc as follows: the LI_kivD2_coEc gene and associated PGK1 promoter were removed from pGV2047 by digestion with AvrW and Nco ⁇ . The 2530 bp fragment was purified by gel electrophoresis and the fragment was prepared using the Zymoclean kit described above.
  • Plasmid pGV2044 was digested with EcoR ⁇ and Sbf ⁇ to remove the Bs_AlsS2 gene and associated CUP1 promoter and the 11275 bp vector fragment was gel purified. The vector and insert were treated with Klenow fragment to produce blunt ends. The pGV2044 vector fragment and the P PG ⁇ i:LI_kivD2_coEc insert were ligated using standard methods in an approximately 5:1 insertvector molar ratio and transformed into TOP10 chemically competent E. coli ceils.
  • Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis consisting of digestion of potential clones with the following enzymes: EcoRV to give correct fragments of 6.3 and 7.5 kb, EcoPN plus Nru ⁇ to give correct fragments of 2.9, 3.4, and 7.5 kb), EcoR ⁇ plus Ncol to give correct fragments of 2.5 and 11.2 Kb.
  • pGV1730 was digested with BamH ⁇ and Sa/I and the vector fragment of 4.9 kb was gel purified by agarose gel electrophoresis.
  • pGV1773 was digested with BamH ⁇ and Sa/I and the 1.7 Kb fragment containing the Bs_AlsS_coSc was gel purified by agarose gel electrophoresis.
  • the pGV1730 vector fragment was ligated to the pGV1773 insert fragment using the Roche rapid ligation kit in a ratio of 5:1 insert to vector ratio and transformed into TOP10 chemically competent E. coli cells.
  • Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis consisting of digestion of potential clones with Seal plus Pst) to give correct fragments of 2.7, 1.7, 1.4 and 0.9 Kb, AfIW to give correct fragments of 1.5 and 5.1 kb, ⁇ /ael plus Sftvl to give correct fragments of 1.4, 5.2 kb.
  • Construction of pGV2117 pGV1730 was digested with BamH ⁇ and Sa/I and the vector fragment of 4.9 kb was gel purified by agarose gel electrophoresis.
  • pGV1802 was digested with BamH ⁇ and Sa/I and the 1.8 kb fragment containing the Ta_ALS was gel purified by agarose gel electrophoresis.
  • Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis consisting of digestion of potential clones with BamH ⁇ plus Stu ⁇ to give correct fragments of 1.4 and 5.2 kb, Sa/I plus Pst ⁇ to give correct fragments of 0.7 and 5.9 kb, and Ahd ⁇ to give correct fragments of 1.9 and 4.7 kb.
  • Construction of pGV2118 pGV1730 was digested with BamH ⁇ and Sa/i and the vector fragment of 4.9 kb was gel purified by agarose gel electrophoresis.
  • pGV1803 was digested with BamH ⁇ and Sail and the 1.8 kb fragment containing the Ts_ALS gel purified by agarose gel electrophoresis.
  • the pGV1730 vector fragment was ligated to the pGV1803 insert fragment using the Roche rapid ligation kit in a ration of 5:1 insert to vector ratio and transformed into TOP-10 chemically competent E.coli cells. Plasmid DNA was isolated and correct clones were confirmed using restriction enzyme analysis consisting of digestion of potential clones with ⁇ /ael to give correct bands of 2.9 and 3.7 kb, EcoFA/ to give correct bands of 0.7 and 5.9 kb, and Hpa ⁇ plus Sacl to give correct bands of 1.9 and 4.7 kb.

Abstract

La présente invention porte sur des microorganismes recombinants comprenant une voie métabolique de production d'isobutanol et des procédés d'utilisation desdits microorganismes recombinants pour produire de l'isobutanol. Dans divers aspects de l'invention, les microorganismes recombinants peuvent comprendre une modification conduisant à la réduction de l'activité pyruvate décarboxylase et/ou de l'activité glycérol-3-phosphate déshydrogénase. Dans divers modes de réalisation décrits ici, les microorganismes recombinants peuvent être des microorganismes de clade des Saccharomyces, des microorganismes de levure-positive Crabtree-négative, des microorganismes de levure Crabtree, des microorganismes de levure post-WGD (duplication de génome entier), des microorganismes de levure pré-WGD (duplication de génome entier) et des microorganismes de levure non fermentante.
EP10792562A 2009-06-22 2010-06-22 Organismes de levure pour la production d'isobutanol Withdrawn EP2446043A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US21917309P 2009-06-22 2009-06-22
US12/696,645 US20110183392A1 (en) 2007-12-23 2010-01-29 Yeast organism producing isobutanol at a high yield
PCT/US2010/039447 WO2010151525A1 (fr) 2009-06-22 2010-06-22 Organismes de levure pour la production d'isobutanol

Publications (2)

Publication Number Publication Date
EP2446043A1 true EP2446043A1 (fr) 2012-05-02
EP2446043A4 EP2446043A4 (fr) 2013-02-13

Family

ID=45876515

Family Applications (1)

Application Number Title Priority Date Filing Date
EP10792562A Withdrawn EP2446043A4 (fr) 2009-06-22 2010-06-22 Organismes de levure pour la production d'isobutanol

Country Status (2)

Country Link
EP (1) EP2446043A4 (fr)
WO (1) WO2010151525A1 (fr)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8956850B2 (en) 2008-06-05 2015-02-17 Butamax Advanced Biofuels Llc Enhanced pyruvate to acetolactate conversion in yeast
US8273558B2 (en) 2005-10-26 2012-09-25 Butamax(Tm) Advanced Biofuels Llc Fermentive production of four carbon alcohols
US8455239B2 (en) 2007-12-23 2013-06-04 Gevo, Inc. Yeast organism producing isobutanol at a high yield
BR112012016042A2 (pt) 2009-12-29 2020-09-15 Butamax Advanced Biofuels Llc "celula hospedeira microbiana recombinante, metodo para a produção de isobutanol, método para a produção de 2-butanol e método para a produção de 14-butanol
GB2501143B (en) * 2010-02-12 2014-03-26 Gevo Inc Yeast microorganisms genetically engineered to improve isobutanol biosynthesis
EP2721165A2 (fr) * 2011-06-17 2014-04-23 Butamax Advanced Biofuels LLC Hydrolysats lignocellulosiques comme charges d'alimentation pour la fermentation de l'isobutanol
EP2583957A1 (fr) 2011-10-18 2013-04-24 LANXESS Deutschland GmbH Butène linéaire à partir d'isobutanol
EP2783007B9 (fr) 2011-11-21 2018-03-28 Metabolic Explorer Souches de micro-organismes pour la production de 2,3-butanediol
US9689004B2 (en) 2012-03-23 2017-06-27 Butamax Advanced Biofuels Llc Acetate supplemention of medium for butanologens
KR101553383B1 (ko) 2012-11-06 2015-09-30 한국생명공학연구원 글리세롤 또는 폐글리세롤로부터 1,3-프로판디올과 이소부탄올의 동시 생성능을 가지는 변이 미생물 및 이를 이용한 1,3-프로판디올과 이소부탄올의 동시 제조방법
JP5845210B2 (ja) * 2013-06-13 2016-01-20 トヨタ自動車株式会社 組換え酵母、及びそれを用いたエタノールの製造方法
CN114836332B (zh) * 2022-05-27 2023-10-20 湖北稻花香酒业股份有限公司 高耐受、低产异戊醇的库德里阿兹威毕赤酵母及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070092957A1 (en) * 2005-10-26 2007-04-26 Donaldson Gail K Fermentive production of four carbon alcohols

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2301949T3 (en) * 2003-05-02 2015-07-20 Cargill Inc Genetically modified yeast species and fermentation methods using genetically modified yeast

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070092957A1 (en) * 2005-10-26 2007-04-26 Donaldson Gail K Fermentive production of four carbon alcohols

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ATSUMI SHOTA ET AL: "Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels", NATURE: INTERNATIONAL WEEKLY JOURNAL OF SCIENCE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 451, no. 7174, 1 January 2008 (2008-01-01), pages 86-90, XP002491157, ISSN: 0028-0836, DOI: 10.1038/NATURE06450 *
BENGTSSON O ET AL: "Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae", BIOTECHNOLOGY FOR BIOFUELS, BIOMED CENTRAL LTD, GB, vol. 2, 5 May 2009 (2009-05-05), pages 1-10, XP002612933, ISSN: 1754-6834, DOI: 10.1186/1754-6834-2-9 *
LEE S K ET AL: "Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels", CURRENT OPINION IN BIOTECHNOLOGY, LONDON, GB, vol. 19, no. 6, 1 December 2008 (2008-12-01), pages 556-563, XP025745480, ISSN: 0958-1669, DOI: 10.1016/J.COPBIO.2008.10.014 [retrieved on 2008-11-10] *
M. R. CONNOR ET AL: "Engineering of an Escherichia coli Strain for the Production of 3-Methyl-1-Butanol", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 74, no. 18, 1 August 2008 (2008-08-01), pages 5769-5775, XP055047091, ISSN: 0099-2240, DOI: 10.1128/AEM.00468-08 *
See also references of WO2010151525A1 *
VAN VLEET J ET AL: "Yeast metabolic engineering for hemicellulosic ethanol production", CURRENT OPINION IN BIOTECHNOLOGY, LONDON, GB, vol. 20, no. 3, 1 June 2009 (2009-06-01), pages 300-306, XP026283532, ISSN: 0958-1669, DOI: 10.1016/J.COPBIO.2009.06.001 [retrieved on 2009-06-21] *
ZHONG-PENG GUO ET AL: "Interruption of glycerol pathway in industrial alcoholic yeasts to improve the ethanol production", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER, BERLIN, DE, vol. 82, no. 2, 19 November 2008 (2008-11-19), pages 287-292, XP019705425, ISSN: 1432-0614 *

Also Published As

Publication number Publication date
EP2446043A4 (fr) 2013-02-13
WO2010151525A1 (fr) 2010-12-29

Similar Documents

Publication Publication Date Title
US20180179557A1 (en) Yeast organism producing isobutanol at a high yield
EP2346998B1 (fr) Micro-organismes manipulés capables de produire des composés cibles en conditions anaérobies
CA2710359C (fr) Organisme de levure produisant de l'isobutanol a un rendement eleve
US9506074B2 (en) Yeast microorganisms with reduced by-product accumulation for improved production of fuels, chemicals, and amino acids
EP2446043A1 (fr) Organismes de levure pour la production d'isobutanol
US20120045809A1 (en) Engineered Microorganisms for the Production of One or More Target Compounds
US20120190089A1 (en) Engineered microogranisms capable of producing target compounds under anaerobic conditions
US20140080188A1 (en) Yeast microorganisms with reduced 2,3-butanediol accumulation for improved production of fuels, chemicals, and amino acids
US20140017748A1 (en) Modified alcohol dehydrogenases for the production of fuels and chemicals
US20140295512A1 (en) Ketol-Acid Reductoisomerases With Improved Performance Properties
WO2013003545A1 (fr) Ajustement fin de sous-produits d'alcool de fusel pendant la production d'isobutanol par des micro-organismes recombinants
US20140295513A1 (en) High-Performance Ketol-Acid Reductoisomerases

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20120120

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20130116

RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 1/16 20060101ALI20130110BHEP

Ipc: C12P 7/16 20060101AFI20130110BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20130815