WO2013033097A1 - Altération du rapport nadh/nad+ pour augmenter le flux à travers les voies nadh-dépendantes - Google Patents

Altération du rapport nadh/nad+ pour augmenter le flux à travers les voies nadh-dépendantes Download PDF

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WO2013033097A1
WO2013033097A1 PCT/US2012/052669 US2012052669W WO2013033097A1 WO 2013033097 A1 WO2013033097 A1 WO 2013033097A1 US 2012052669 W US2012052669 W US 2012052669W WO 2013033097 A1 WO2013033097 A1 WO 2013033097A1
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microorganism
recombinant
isobutanol
recombinant microorganism
nadh
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PCT/US2012/052669
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English (en)
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Chistopher SMITH
Stephanie Porter-Scheinman
Peter Meinhold
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Gevo, Inc.
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Publication of WO2013033097A1 publication Critical patent/WO2013033097A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/16—Butanols
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • Recombinant microorganisms and methods of producing such microorganisms are provided. Also provided are methods of producing beneficial metabolites including fuels, and chemicals by contacting a suitable substrate with the recombinant microorganisms and enzymatic preparations there from.
  • microorganisms expressing a heterologous, five-step metabolic pathway See, e.g., WO/2007/050671 to Donaldson ef a/., WO/2008/098227 to Liao et a/., and WO/2009/103533 to Festel ef a/.).
  • the microorganisms produced to date have fallen short of commercial relevance due to their low performance characteristics, including, for example low productivities, low titers, and low yields,
  • NADH necessary cofactors
  • the present inventors have discovered that the overexpression of one or more formate dehydrogenase (FDH) genes in yeast strains engineered for the production of isobutanol increases isobutanol production.
  • the invention relates to recombinant yeast ceils engineered to provide increased heterologous or native expression of FDH1 or homologs thereof.
  • ceils that overexpress FDH1 or homoiogs thereof exhibit an enhanced ability to produce beneficial metabolites, such as isobutanol.
  • One aspect of the invention is directed to a recombinant microorganism comprising an isobutanol producing metabolic pathway which comprises at least one exogenous gene, wherein the microorganism is engineered to overexpress one or more polynucleotides encoding one or more FDH proteins or homologs thereof, in one embodiment, the polynucleotides are native polynucleotides. In another embodiment, the polynucleotides are heterologous polynucleotides.
  • the FDH comprises a cytosoiically-localized FDH. In yet another embodiment, the FDH comprises a mitochondrialiy-locaiized FDH.
  • polynucleotides encoding one or more FDH proteins or homologs thereof are native to the recombinant microorganism. In another specific embodiment, the polynucleotides encoding one or more FDH proteins or homologs thereof are heterologous to the recombinant microorganism.
  • polynucleotide encoding one or more FDH proteins or homologs thereof is selected from SEQ ID NO: 1 to SEQ ID NO: 55.
  • the isobutanol producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanol.
  • the isobutanol producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol.
  • the isobutano! producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol.
  • the isobutanol producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol.
  • the isobutano! producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol.
  • all of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes.
  • one or more of the isobutanol pathway genes encodes an enzyme that is localized to the cytosol.
  • the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol.
  • the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least two isobutanol pathway enzymes localized in the cytosol.
  • the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least three isobutanol pathway enzymes localized in the cytosol.
  • the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least four isobutanol pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with five isobutanol pathway enzymes localized in the cytosol. In yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with all isobutanol pathway enzymes localized in the cytosol.
  • the isobutanol pathway genes may encode enzyme(s) selected from the group consisting of acetolactate synthase (ALS), ketoi-acid reductoisomerase (KARl), dihydroxyacid dehydratase (DHAD), 2- keto-acid decarboxylase, e.g., keto-isovalerate decarboxylase (KIVD), and alcohol dehydrogenase (ADH).
  • the KARl is an NADH-dependent KARl (NKR).
  • the ADH is an NADH-dependent ADH.
  • the KARI is an NADH-dependent KARi (NKR) and the ADH is an NADH-dependent ADH.
  • the recombinant microorganisms of the invention that comprise an isobutanol producing metabolic pathway may be further engineered to reduce or eliminate the expression or activity of one or more enzymes selected from a pyruvate decarboxylase (PDC), a glycerol- 3-phosphate dehydrogenase (GPD), a 3-keto acid reductase (3-KAR), or an aldehyde dehydrogenase (ALDH).
  • PDC pyruvate decarboxylase
  • GPD glycerol- 3-phosphate dehydrogenase
  • 3-KAR 3-keto acid reductase
  • ALDH aldehyde dehydrogenase
  • the recombinant microorganisms may be recombinant yeast microorganisms.
  • the yeast microorganisms may be members of the Saccharomyces clade, Saccharomyces sensu stricto microorganisms, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre- WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.
  • the recombinant microorganisms may be yeast recombinant microorganisms of the Saccharomyces clade.
  • the recombinant microorganisms may be Saccharomyces sensu stricto microorganisms.
  • Saccharomyces sensu stricto is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S, mikatae, S, bayanus, S, uvarum, S. carocanis, and hybrids thereof.
  • the recombinant microorganisms may be Crabtree- negative recombinant yeast microorganisms.
  • the Crabtree- negative yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia s issatchenkia, Hansenula, or Candida.
  • the Crabtree-negative yeast microorganism is selected from Saccharomyces kluyveri, Kluyveromyces iactis, Kluyveromyces marxianus, Pichia anomaia, Pichia siipiiis, 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 Saccha myces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Candida, Pichia and Schizosaccharomyces.
  • the Crabtree-positive yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Sacchammyces uvarum, Sacchammyces bayanus, Sacchammyces paradoxus, Saccharomyces castelli, Kluyveromyces thermotolerans, Candida glabrata, Zygosaccharomyces bailli, Zygosaccharomyces rouxii, Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces uvarum.
  • the recombinant microorganisms may be post- WGD (whole genome duplication) yeast recombinant microorganisms.
  • the post-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces or Candida.
  • the post-WGD yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and Candida glabrata,
  • the recombinant microorganisms may be pre-WGD (whole genome duplication) yeast recombinant microorganisms.
  • the pre-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenuia, 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, issatchenkia orientalis, Issatchenkia occidentalis, Debaryomyces hansenii, Hansenuia anomala, Pachysolen tannophiiis, Yarrowia iipolytica, 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, Myxozy a, or Candida.
  • the non-fermenting yeast is C. xestobii.
  • the present application provides methods of producing isobutanol using a recombinant microorganism as described herein.
  • the method includes cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source until isobutanol is produced and optionally, recovering the metabolite.
  • the microorganism produces the metabolite, such as isobutanol, from a carbon source at a yield of at least about 5 percent theoretical.
  • the microorganism produces the metabolite, such as 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, at least about 95 percent, or at least about 97.5 percent theoretical.
  • the metabolite such as isobutanol
  • the recombinant microorganism converts the carbon source to isobutanol under aerobic conditions. In another embodiment, the recombinant microorganism converts the carbon source to isobutanol under microaerobic conditions. In yet another embodiment, the recombinant microorganism converts the carbon source to isobutanol under anaerobic conditions.
  • the recombinant microorganism described herein further comprises an intracellular NADH/NAD* ratio that is increased compared to a recombinant microorganism not engineered to overexpress one or more FDH genes leading to increased isobutanol production and/or yield.
  • the recombinant microorganism described herein produces isobutanol at a faster rate upon contacting the microorganism with a culture medium comprising formate, as compared to a recombinant microorganism not engineered to overexpress one or more FDH genes.
  • the recombinant microorganism described herein further comprises an intracellular NADH/NAD + ratio that is increased compared to a recombinant microorganism not engineered to overexpress one or more FDH genes when contacting the microorganism with a culture medium comprising formate, leading to increased isobutanol production and/or yield.
  • Figure 1 illustrates an isobutanol pathway.
  • Figure 2 illustrates an NADH-dependent isobutanoi pathway.
  • Figure 3 illustrates a plasrnid map of pGV2196.
  • Figure 4 illustrates a plasmid map of pGV3158.
  • Figure 5 illustrates a plasmid map of pGV3159.
  • Figure 6 illustrates isobutanoi specific productivity and yields in inventive yeast microorganisms overexpressing different forms of FDH.
  • Figure 7 illustrates metabolite generation and consumption profiles of inventive yeast microorganisms overexpressing different forms of FDH.
  • microorganism includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista.
  • microbial cells and “microbes” are used interchangeably with the term microorganism.
  • prokaryotes is art recognized and refers to ceils which contain no nucleus or other cell organelles.
  • the prokaryotes are generally classified in one of two domains, the Bacteria and the Archaea.
  • the definitive difference between organisms of the Archaea and Bacteria domains is based on fundamental differences in the nucleotide base sequence in the 168 ribosomai RNA.
  • the term "Archaea” refers to a categorization of organisms of the division Mendosicutes, typically found in unusual environments and distinguished from the rest of the prokaryotes by several criteria, including the number of ribosoma! proteins and the lack of muramic acid in cell walls.
  • the Archaea consist of two phylogeneticaliy-distinct groups: Crenarchaeota and Euryarchaeota.
  • the Archaea can be organized into three types: methanogens (prokaryotes that produce methane); extreme haiophiies (prokaryotes that live at very high concentrations of salt (NaCi); and extreme (hyper) thermophiles (prokaryotes that live at very high temperatures).
  • methanogens prokaryotes that produce methane
  • extreme haiophiies prokaryotes that live at very high concentrations of salt (NaCi)
  • extreme (hyper) thermophiles prokaryotes that live at very high temperatures.
  • these prokaryotes exhibit unique structural or biochemical attributes which adapt them to their particular habitats.
  • the Crenarchaeota consist mainly of hyperthermophiiic sulfur-dependent prokaryotes and the Euryarchaeota contain the methanogens and extreme haiophiies.
  • Bacteria refers to a domain of prokaryotic organisms. Bacteria include at least eleven distinct groups as follows: (1 ) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1 ) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas)] (2) Proteobacteria, e.g., Purple photosynthetic +non-photosynthetic Gram-negative bacteria (includes most "common" Gram-negative bacteria); (3) Cyanobacteria, e.g.
  • Gram-negative bacteria include cocci, nonenteric rods, and enteric rods.
  • the genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurelia, Brucella, Yersinia, Franciseiia, Haemophilus, Bordeteiia, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.
  • Gram positive bacteria include cocci, nonsporuiating rods, and sporuiating rods.
  • the genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothnx, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus,
  • 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 Tindail, B.J. (2007) The Taxonomic Outline of Bacteria and Archaea. TOBA Release 7.7, March 2007. Michigan State University Board of Trustees.
  • genomic hybridization is defined as a collection of closely related organisms with greater than 97% 16S ribosomai RNA sequence homology and greater than 70% genomic hybridization and sufficiently different from all other organisms so as to be recognized as a distinct unit.
  • recombinant microorganism refers to microorganisms that have been genetically modified to express or to overexpress endogenous polynucleotides, to express heterologous polynucleotides, such as those included in a vector, in an integration construct, or which have an alteration in expression of an endogenous gene.
  • alteration it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the alteration.
  • alter can mean “inhibit,” but the use of the word “alter” is not limited to this definition.
  • the terms “recombinant microorganism” and “recombinant host ceil” refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • expression refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein.
  • expression of a protein results from transcription and translation of the open reading frame sequence.
  • the level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the ceil, or the amount of the desired product encoded by the selected sequence.
  • mRNA transcribed from a selected sequence can be quantitated by qRT-PCR or by Northern hybridization (see Sambrook et a/., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
  • Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that recognize and bind the protein. See Sambrook et al., 1989, supra, [0050]
  • the term "overexpression” refers to an elevated level (e.g., aberrant level) of rnRNAs encoding for a protein(s) (e.g. an FDH gene or homolog thereof), and/or to elevated levels of protein(s) (e.g.
  • FDH or "Fdh1 ") in ceils as compared to similar corresponding unmodified cells expressing basal levels of mRNAs (e.g., those encoding FDH proteins) or having basal levels of proteins.
  • FDH mRNA or FDH proteins or homoiogs thereof may be overexpressed by at least 2-foid, 3-foid, 4-foid, 5-foid, 6-foid, 8-fold, 10-fold, 12-fold, 15 ⁇ foid or more in microorganisms engineered to exhibit increased formate dehydrogenase mRNA, protein, and/or activity.
  • reduced activity and/or expression of a protein such as an enzyme can mean either a reduced specific catalytic activity of the protein (e.g. reduced activity) and/or decreased concentrations of the protein in the cell (e.g. reduced expression).
  • reduced activity of a protein in a cell may result from decreased concentrations of the protein in the ceil.
  • wild-type microorganism describes a ceil that occurs in nature, i.e., a cell that has not been genetically modified.
  • a wild-type microorganism can be genetically modified to express or overexpress a first target enzyme.
  • This microorganism can act as a parental microorganism in the generation of a microorganism modified to express or overexpress a second target enzyme.
  • the microorganism modified to express or overexpress a first and a second target enzyme can be modified to express or overexpress a third target enzyme.
  • a "parental microorganism” functions as a reference cell for successive genetic modification events. Each modification event can be accomplished by introducing a nucleic acid molecule in to the reference cell. The introduction facilitates the expression or overexpression of a target enzyme.
  • the term “facilitates” encompasses the activation of endogenous polynucleotides encoding a target enzyme through genetic modification of e.g., a promoter sequence in a parental microorganism. It is further understood that the term “facilitates” encompasses the introduction of heterologous polynucleotides encoding a target enzyme in to a parental microorganism,
  • engine refers to any manipulation of a microorganism that results in a detectable change in the microorganism, wherein the manipulation includes but is not limited to inserting a polynucleotide and/or polypeptide heterologous to the microorganism and mutating a polynucleotide and/or polypeptide native to the microorganism.
  • mutation indicates any modification of a nucleic acid and/or polypeptide which results in an altered nucleic acid or polypeptide. Mutations include, for example, point mutations, deletions, or insertions of single or multiple residues in a polynucleotide, which includes alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences.
  • a genetic alteration may be a mutation of any type. For instance, the mutation may constitute a point mutation, a frame-shift mutation, a nonsense mutation, an insertion, or a deletion of part or all of a gene.
  • the modified microorganism a portion of the microorganism genome has been replaced with a heterologous polynucleotide.
  • the mutations are naturally-occurring.
  • the mutations are identified and/or enriched through artificial selection pressure.
  • the mutations in the microorganism genome are the result of genetic engineering.
  • biosynthetic pathway also referred to as “metabolic pathway” refers to a set of anabolic or cataboiic biochemical reactions for converting one chemical species into another.
  • Gene products belong to the same “metabolic pathway” if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product.
  • isobutanol producing metabolic pathway refers to an enzyme pathway which produces isobutanol from pyruvate.
  • NADH-dependent refers to an enzyme that catalyzes the reduction of a substrate coupled to the oxidation of NADH with a catalytic efficiency that is greater than the reduction of the same substrate coupled to the oxidation of NADPH at equal substrate and cofactor concentrations.
  • exogenous refers to molecules that are not normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
  • endogenous or “native” as used herein with reference to various molecules refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
  • heterologous refers to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., wherein at least one of the following is true: (a) the molecuie(s) is/are foreign ("exogenous") to (i.e., not naturally found in) the host cell; (b) the molecu!e(s) is/are naturally found in (e.g., is "endogenous to") a given host microorganism or host ceil but is either produced in an unnatural location or in an unnatural amount in the cell; and/or (c) the molecule(s) differ(s) in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence(s) such that the molecule differing in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid as found endogenously is produced in an unnatural (e.
  • feedstock is defined as a raw material or mixture of raw materials supplied to a microorganism or fermentation process from which other products can be made.
  • a carbon source such as biomass or the carbon compounds derived from biomass are a feedstock for a microorganism that produces a biofuel in a fermentation process.
  • a feedstock may contain nutrients other than a carbon source.
  • substrate refers to any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme.
  • the term includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate, or derivatives thereof.
  • substrate encompasses not only compounds that provide a carbon source suitable for use as a starting material, such as any biomass derived sugar, but also intermediate and end product metabolites used in a pathway associated with a recombinant microorganism as described herein.
  • the term "fermentation” or “fermentation process” is defined as a process in which a microorganism is cultivated in a culture medium containing raw materials, such as feedstock and nutrients, wherein the microorganism converts raw materials, such as a feedstock, into products.
  • volumetric productivity or “production rate” is defined as the amount of product formed per volume of medium per unit of time. Volumetric productivity is reported in gram per liter per hour (g/L/h).
  • specific productivity or “specific production rate” is defined as the amount of product formed per volume of medium per unit of time per amount of ceils. Specific productivity is reported in gram (or milligram) per gram ceil dry weight per hour (g/g h).
  • yield is defined as the amount of product obtained per unit weight of raw material and may be expressed as g product per g substrate (g/g). Yield may be expressed as a percentage of the theoretical yield. "Theoretical yield” is defined as the maximum amount of product that can be generated per a given amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to isobutanoi is 0.41 g/g. As such, a yield of isobutanoi from glucose of 0.39 g/g would be expressed as 95% of theoretical or 95% theoretical yield.
  • titer is defined as the strength of a solution or the concentration of a substance in solution.
  • concentration of a substance in solution For example, the titer of a biofuel in a fermentation broth is described as g of biofuei in solution per liter of fermentation broth (g/L).
  • “Aerobic conditions” are defined as conditions under which the oxygen concentration in the fermentation medium is sufficiently high for an aerobic or facultative anaerobic microorganism to use as a terminal electron acceptor.
  • anaerobic conditions are defined as conditions under which the oxygen concentration in the fermentation medium is too low for the microorganism to use as a terminal electron acceptor. Anaerobic conditions may be achieved by sparging a fermentation medium with an inert gas such as nitrogen until oxygen is no longer available to the microorganism as a terminal electron acceptor. Alternatively, anaerobic conditions may be achieved by the microorganism consuming the available oxygen of the fermentation until oxygen is unavailable to the microorganism as a terminal electron acceptor. Methods for the production of isobutanoi under anaerobic conditions are described in commonly owned and copending publication, US 2010/0143997, the disclosures of which are herein incorporated by reference in their entireties for all purposes.
  • Aerobic metabolism refers to a biochemical process in which oxygen is used as a terminal electron acceptor to make energy, typically in the form of ATP, from carbohydrates. Aerobic metabolism occurs e.g. via glycolysis and the TCA cycle, wherein a single glucose molecule is metabolized completely into carbon dioxide in the presence of oxygen.
  • anaerobic metabolism refers to a biochemical process in which oxygen is not the final acceptor of electrons contained in NADH. Anaerobic metabolism can be divided into anaerobic respiration, in which compounds other than oxygen serve as the terminal electron acceptor, and substrate level phosphorylation, in which the electrons from NADH are utilized to generate a reduced product via a "fermentative pathway.”
  • NAD(P)H donates its electrons to a molecule produced by the same metabolic pathway that produced the electrons carried in NAD ⁇ P)H.
  • NAD(P)H generated through glycolysis transfers its electrons to pyruvate, yielding ethanol.
  • Fermentative pathways are usually active under anaerobic conditions but may also occur under aerobic conditions, under conditions where NADH is not fully oxidized via the respiratory chain. For example, above certain glucose concentrations, Crabtree-positive yeasts produce large amounts of ethanol under aerobic conditions.
  • byproduct or "by-product” means an undesired product related to the production of an amino acid, amino acid precursor, chemical, chemical precursor, biofuel, or biofuel precursor.
  • the term "substantially free” when used in reference to the presence or absence of a protein activity means the level of the protein is substantially less than that of the same protein in the wild-type host, wherein less than about 50% of the wild-type level is preferred and less than about 30% is more preferred.
  • the activity may be less than about 20%, less than about 10%, less than about 5%, or less than about 1 % of wild-type activity.
  • Microorganisms which are "substantially free" of a particular protein activity e.g. 3-KAR enzymatic activity, ALDH enzymatic activity, PDC enzymatic activity, GPD enzymatic activity, etc.
  • non-fermenting yeast is a yeast species that fails to demonstrate an anaerobic metabolism in which the electrons from NADH are utilized to generate a reduced product via a fermentative pathway such as the production of ethanol and C0 2 from glucose.
  • Non-fermentative yeast can be identified by the "Durham Tube Test” (J.A. Barnett, R.W. Payne, and D. Yarrow. 2000. Yeasts Characteristics and Identification. 3 rd edition, p. 28-29. Cambridge University Press, Cambridge, UK) or by monitoring the production of fermentation productions such as ethanol and C0 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 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 “p!asmids.”
  • 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, piasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes), and the like, that are "episomes,” that is, that replicate autonomously or can integrate into a chromosome of a host cell.
  • a vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poiy-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-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 ceil. Transformation (or transduction, or transfection), can be achieved by any one of a number of means including chemical transformation (e.g. lithium acetate transformation), electroporation, microinjection, bioiistics (or particle bombardment- mediated delivery), or agrobacterium mediated transformation.
  • chemical transformation e.g. lithium acetate transformation
  • electroporation e.g. lithium acetate transformation
  • microinjection e.g. lithium acetate transformation
  • bioiistics or particle bombardment- mediated delivery
  • agrobacterium mediated transformation e.g., 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 polynucleotides,
  • polypeptide indicates an organic polymer composed of two or more amino acidic monomers and/or analogs thereof.
  • amino acid or “amino acidic monomer” refers to any natural and/or synthetic amino acids including glycine and both D or L optical isomers.
  • amino acid analog refers to an amino acid in which one or more individual have been replaced, either with a different atom, or with a different functional group.
  • polypeptide includes an 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
  • homoiog used with respect to an original polynucleotide or polypeptide of a first family or species, refers to distinct polynucleotides or polypeptides of a second family or species which are determined by functional, structural or genomic analyses to be a polynucleotide or polypeptide of the second family or species which corresponds to the original polynucleotide or polypeptide of the first family or species. Most often, homoiogs will have functional, structural or genomic similarities. Techniques are known by which homoiogs of a polynucleotide or polypeptide can readily be cloned using genetic probes and PGR. Identity of cloned sequences as homoiog can be confirmed using functional assays and/or by genomic mapping of the genes.
  • a polypeptide has "homology” or is “homologous” to a second polypeptide if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene.
  • a polypeptide has homology to a second polypeptide if the two polypeptides have "similar” amino acid sequences.
  • homology to a second polypeptide if the two polypeptides have "similar” amino acid sequences.
  • analogs refers to polynucleotide or polypeptide sequences that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure.
  • microorganisms convert sugars to produce pyruvate, which is then utilized in a number of pathways of cellular metabolism.
  • microorganisms including yeast, have been engineered to produce a number of desirable products via pyruvate-d riven biosynthetic pathways, including isobutanoi, an important commodity chemical and biofuel candidate (See, e.g., commonly owned and co-pending patent publications, US 2009/0226991 , US 2010/0143997, US 201 1/0020889, US 201 1/0078733, and WO 2010/075504).
  • the present application relates to recombinant microorganisms for producing isobutanoi, wherein said recombinant microorganisms comprise an isobutanoi producing metabolic pathway.
  • the isobutanoi producing metabolic pathway to convert pyruvate to isobutanoi can be comprised of the following reactions:
  • these reactions are carried out by the enzymes 1 ) Acetoiactate synthase (ALS), 2) Ketol-acid reductoisomerase (KARI), 3) Dihydroxy- acid dehydratase (DHAD), 4) 2-keto-acid decarboxylase, e.g., Keto-isovalerate decarboxylase (KIVD), and 5) an Alcohol dehydrogenase (ADH) ( Figure 1 ).
  • the recombinant microorganism may be engineered to overexpress one or more of these enzymes.
  • the recombinant microorganism is engineered to overexpress ail of these enzymes.
  • isobutanoi producing metabolic pathway comprises five substrate to product reactions.
  • the isobutanoi producing metabolic pathway comprises six substrate to product reactions.
  • the isobutanol producing metabolic pathway comprises seven substrate to product reactions.
  • the recombinant microorganism comprises an isobutanol producing metabolic pathway.
  • the isobutanol producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanol.
  • the isobutanol producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol.
  • the isobutanol producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, all of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes.
  • one or more of the isobutanol pathway genes encodes an enzyme that is localized to the cytosol.
  • the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol.
  • the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least two isobutanol pathway enzymes localized in the cytosol.
  • the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least three isobutanol pathway enzymes localized in the cytosol.
  • the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least four isobutanol pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with five isobutanol pathway enzymes localized in the cytosol. In yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with all isobutanol pathway enzymes localized in the cytosol. Isobutanoi producing metabolic pathways in which one or more genes are localized to the cytosol are described in commonly owned U.S. Patent No. 8,232,089, which is herein incorporated by reference in its entirety for all purposes.
  • isobutanoi pathway enzymes including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyve myces spp., including K, thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H, poiymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including Y. spp.
  • Sources of genes from anaerobic fungi include, but not limited to, Piromyces spp., Orpinomyces spp., or Neocaiiimastix spp.
  • Sources of prokaryotic enzymes that are useful include, but not limited to, Escherichia spp., Zymomonas spp., Staphylococcus spp., Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Ente bacter spp., Streptococcus spp., Salmonella spp., Siackia spp., Cryptobacterium spp., and Eggerthella spp.
  • one or more of these enzymes can be encoded by native genes.
  • one or more of these enzymes can be encoded by heterologous genes.
  • acetoiactate synthases capable of converting pyruvate to acetolactate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including B, subiilis (GenBank Accession No. Q04789.3), L lactis (GenBank Accession No. NP 267340.1 ), S. mutans (GenBank Accession No. NP__721805.1 ), K. pneumoniae (GenBank Accession No. ZPJ36Q14957.1 ), C. glutamicum (GenBank Accession No. P42483.1 ), E. cloacae (GenBank Accession No. YPJH3381361 1 .1 ), M.
  • sources e.g., bacterial, yeast, Archaea, etc.
  • sources e.g., bacterial, yeast, Archaea, etc.
  • sources e.g., bacterial, yeast, Archaea, etc.
  • sources e.g.,
  • Chipman et a/ A review article characterizing the biosynthesis of acetoiactate from pyruvate via the activity of acetoiactate synthases is provided by Chipman et a/., 1998, Biochimica et Biophysica Acta 1385: 401 -19, which is herein incorporated by reference in its entirety. Chipman et a/, provide an alignment and consensus for the sequences of a representative number of acetolactate synthases. Motifs shared in common between the majority of acetolactate synthases include:
  • a protein harboring one or more of these amino acid motifs can generally be expected to exhibit acetolactate synthase activity.
  • Ketol-acid reductoisomerases capable of converting acetolactate to 2,3- dihydroxyisovalerate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including E. coli (GenBank Accession No. EGB30597.1 ), L. lactis (GenBank Accession No. YPJ3033S371 Q.1 ), S. exigua (GenBank Accession No. ZP_ 06160130.1 ), C. curiam (GenBank Accession No. YP_ 003151266.1 ), Shewanella sp. (GenBank Accession No. YP__732498.1 ), V. fischeri (GenBank Accession No.
  • ketol-acid reductoisomerases An alignment and consensus for the sequences of a representative number of ketol-acid reductoisomerases is provided in commonly owned and co-pending US Publication No. 2010/0143997, which is herein incorporated by reference in its entirety. Motifs shared in common between the majority of ketol-acid reductoisomerases include:
  • V(V/I/F)(M/L/A)(A/C)PK (SEQ ID NO: 82),
  • S(D/N/T)TA(E/Q/R)XG (SEQ ID NO: 64) motifs at amino acid positions corresponding to the 89-94, 175-179, 194-200, 282- 272, and 459-485 residues, respectively, of the £, co!i ketol-acid reductoisomerase encoded by HvC.
  • a protein harboring one or more of these amino acid motifs can generally be expected to exhibit ketol-acid reductoisomerase activity.
  • ketol-acid reductoisomerases are known to use NADPH as a cofactor.
  • a ketol-acid reductoisomerase which has been engineered to used NADH as a cofactor may be utilized to mediate the conversion of acetolactate to 2,3-dihydroxyisovaierate.
  • Engineered NADH-dependent KAR! enzymes (“NKRs") and methods of generating such NKRs are disclosed in commonly owned and co-pending US Publication No. 2010/0143997.
  • any number of mutations can be made to a KAR! enzyme, and in a preferred aspect, multiple mutations can be made to a KARl enzyme to result in an increased ability to utilize NADH for the conversion of acetolactate to 2,3-dihydroxyisovaierate.
  • Such mutations include point mutations, frame shift mutations, deletions, and insertions, with one or more (e.g., one, two, three, four, five or more, etc.) point mutations preferred.
  • Mutations may be introduced into naturally existing KARl enzymes to create NKRs using any methodology known to those skilled in the art. Mutations may be introduced randomly by, for example, conducting a PGR reaction in the presence of manganese as a divalent metal ion cofactor.
  • oligonucleotide directed mutagenesis may be used to create the NKRs which allows for 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 KARl enzyme of interest.
  • the mismatched oligonucleotide is then extended by DNA polymerase, generating a double-stranded DNA molecule which contains the desired change in sequence in one strand.
  • the changes in sequence can, for example, result in the deletion, substitution, or insertion of an amino acid.
  • the double-stranded polynucleotide can then be inserted into an appropriate expression vector, and a mutant or modified polypeptide can thus be produced.
  • the above-described oligonucleotide directed mutagenesis can, for example, be carried out via PGR.
  • Dihydroxy acid dehydratases capable of converting 2,3- dihydroxyisovaierate to a-ketoisovaierate may be derived from a variety of sources ⁇ e.g., bacterial, yeast, Archaea, etc.), including E. coli (GenBank Accession No. YP_026248.1 ), L. lactis (GenBank Accession No. NP__287379.1 ), S. mutans (GenBank Accession No. NP__722414.1 ), M. stadtmanae (GenBank Accession No. YP_ 448586.1 ), M. tractuosa (GenBank Accession No. YP ...
  • CDKXXPG (SEQ ID NO: 66)
  • a protein harboring one or more of these amino acid motifs can generally be expected to exhibit dihydroxy acid dehydratase activity.
  • 2-keto-acid decarboxylases capable of converting a-ketoisovalerate to isobutyraldehyde may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including L. lactis kivD (GenBank Accession No. YP 003353820.1 ), E. cloacae (GenBank Accession No. P23234.1 ), M. smegmatis (GenBank Accession No. A0R480.1 ), M. tuberculosis (GenBank Accession No. 053865.1 ), M. avium (GenBank Accession No. Q742Q2.1 , A.
  • brasiiense (GenBank Accession No. P51852.1 ), L. lactis kdcA (GenBank Accession No. AAS49166.1 ), S. epidermidis (GenBank Accession No. NP__765765.1 ), M. caseotyticus (GenBank Accession No. YPJ302560734.1 ), B. megaterium (GenBank Accession No. YP_003561644.1 ), S. cerevisiae ARO10 (GenBank Accession No. NP 010868.1 ), or S. cerevisiae THI3 (GenBank Accession No. CAA98646.1 ).
  • 2-keto-acid decarboxylases capable of converting a-ketoisovalerate to isobutyraldehyde are described in commonly owned and co-pending US Publication No. 201 1/0078733. Motifs shared in common between the majority of 2-keto-acid decarboxylases include:
  • GDG(S/A)(L/F/A)Q(L/M)T (SEQ ID NO: 78) motifs at amino acid positions corresponding to the 21 -27, 70-78, 81 -89, 93-98, and 428-435 residues, respectively, of the L !actis 2-keto-acid decarboxylase encoded by kivO.
  • An additional "HH"-motif found at amino acids 1 12-1 13 in the L lactis 2- keto-acid decarboxylase encoded by kivD is characteristic of thiamin diphosphate- dependent decarboxylases, a class of enzymes of which 2-keto acid decarboxylases belong.
  • a protein harboring one or more of these amino acid motifs can generally be expected to exhibit 2-keto-acid decarboxylase activity.
  • Alcohol dehydrogenases capable of converting isobutyraldehyde to isobutanoi may be derived from a variety of sources ⁇ e.g. , bacterial, yeast, Archaea, etc.), including L lactis (GenBank Accession No. YP__003354381 ), B. cereus (GenBank Accession No. YP_001374103.1 ), N, meningitidis (GenBank Accession No. CBA03965.1 ), S. sanguinis (GenBank Accession No. YPJ301035842.1 ), L brevis (GenBank Accession No. YP 794451 .1 ), B, thuringiensis (GenBank Accession No.
  • G(L/A )G(G/P)(L/W)G (SEQ ID NO: 82) motifs at amino acid positions corresponding to the 39-44, 59-68, 76-82, 91 -97, 147- 152, and 171 -176 residues, respectively, of the L, lactis alcohol dehydrogenase encoded by adhA.
  • a protein harboring one or more of these amino acid motifs can generally be expected to exhibit alcohol dehydrogenase activity.
  • the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutanoi. In one embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutyraidehyde. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to keto-isovalerate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to 2,3-dihydroxyisovalerate, In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to acetoiactate,
  • any of the genes encoding the foregoing enzymes may be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast.
  • pathway steps 2 and 5 of the isobutanoi pathway may be carried out by KARI and ADH enzymes that utilize NADH (rather than NADPH) as a cofactor.
  • KARI NADH-dependent KARI
  • ADH enzymes to catalyze pathway steps 2 and 5, respectively, enables production of isobutanoi at theoretical yield and/or under anaerobic conditions.
  • An example of an NADH-dependent isobutanoi pathway is illustrated in Figure 2.
  • the recombinant microorganisms of the present invention may use an NKR to catalyze the conversion of acetoiactate to produce 2,3-dihydroxyisovalerate.
  • the recombinant microorganisms of the present invention may use an NADH-dependent ADH to catalyze the conversion of isobutyraidehyde to produce isobutanoi.
  • the recombinant microorganisms of the present invention may use both an NKR to catalyze the conversion of acetoiactate to produce 2,3-dihydroxyisovalerate, and an NADH-dependent ADH to catalyze the conversion of isobutyraidehyde to produce isobutanoi.
  • formate dehydrogenase overexpression is expected to be more pronounced in NADH-dependent isobutanoi pathways, because of the requirement of pathway steps 2 and 5 for NADH, which is generated by the activity of the overexpressed formate dehydrogenase. Accordingly, formate dehydrogenase overexpression in the context of an NADH-dependent pathway is a preferred embodiment of the invention.
  • NADH-dependent pathways and NKR and NADH-dependent ADHs are described in commonly owned and co-pending publication, US 2010/0143997, the disclosures of which are herein incorporated by reference in their entireties for all purposes.
  • the recombinant microorganism comprising an isobutanoi producing metabolic pathway comprising at least one exogenous gene is engineered to overexpress one or more polynucleotides encoding one or more formate dehydrogenase (FDH) proteins or homologs thereof.
  • the one or more polynucleotides encoding one or more formate dehydrogenase (FDH) proteins is/are native to the recombinant microorganism.
  • the one or more polynucleotides encoding one or more FDH proteins or homologs thereof is/are heterologous to the recombinant microorganism.
  • Formate dehydrogenase is an enzyme that catalyzes the oxidation of formate to bicarbonate, and, in doing so, donates electrons to NAD + . Consequently, FDH may generate NADH according to the following reaction: Formate + NAD + ⁇ CO2 + NADH. Accordingly, as described below, overexpression of FDH may be used to increase NADH levels for use in the isobutanoi pathway in the recombinant microorganisms described herein.
  • FDH activity may also alleviate the toxic effects of formate produced by the yeast or of formate present in pretreated lignoceliulosic biomass.
  • formate concentrations have been measured to be in the range of 10-20 m in some pretreated biomass (measured at 1 .4 to 3.1 g/L in spruce, wheat or sugar cane hydroiysates in Almeida et a/. J Chem Techno! Biotechnol 82:340-349 (2007)), which may hamper xylose fermentation in metaboiically engineered yeast (Hasunuma et a!. Appl Microbiol Biotechnol (201 1 ) 90:997-1004).
  • FDH can be found compartmentalized in a ceil, for instance, in the cytosoi or in the mitochondria. Accordingly, in various embodiments, the recombinant microorganisms of the present application may comprise a cytosolicaiiy-iocaiized FDH and/or a mitochondrially-localized FDH.
  • the one or more polynucleotides encoding one or more formate dehydrogenases is/are selected from various polynucleotides encoding FDH or homoiogs thereof.
  • Suitable formate dehydrogenases or homoiogs thereof are available from a variety of organisms and can generally be cataloged by EC Number, including, but not limited to EC Number 1 .2.1 .2.
  • Table 1 provides a representative, non-limiting, list of exemplary formate dehydrogenases.
  • Table 1 Representative Formate Dehydrogenases.
  • nicotinamide adenine dinucieotide is an ubiquitous coenzyme comprised of two nucleotides (i.e., one with adenine base and the other a nicotinamide) that is present in ceils in two forms: NAD + (oxidized) and NADH (reduced).
  • NAD + oxidized
  • NADH NADH
  • the molecule is an oxidizing agent that accepts electrons from other molecules and becomes reduced.
  • NADH the molecule is a reducing agent that may donate electrons to allow for various cellular reactions.
  • the isobutanol pathway utilizes the two pyruvate molecules and the two reduced cofactors (NADH) produced by the glycolytic pathway to produce isobutanol through five enzymatic steps, as described above.
  • NADH reduced cofactors
  • the present inventors have found, among others, an increase in isobutanol production upon overexpression of FDH.
  • the intracellular NADH/NAD * ratio may be increased to stimulate flux through the two NADH-utilizing steps of the isobutanol pathway. Therefore, the NADH/NAD* ratio may be increased, a result which may lead to an increase in isobutanol productivity.
  • a recombinant microorganism comprising an isobutanol producing metabolic pathway comprising at least one exogenous gene, wherein the microorganism is engineered to overexpress one or more polynucleotides encoding one or more formate dehydrogenase (FDH) proteins or homologs which further comprises an intracellular NADH/NAD* ratio that is increased compared to a recombinant microorganism not engineered to overexpress one or more FDH genes, leading to increased isobutanol productivity and/or yield.
  • FDH formate dehydrogenase
  • Formate the substrate for FDH
  • a recombinant microorganism comprising an isobutanol producing metabolic pathway comprising at least one exogenous gene, wherein the microorganism is engineered to overexpress one or more polynucleotides encoding one or more formate dehydrogenase (FDH) proteins or homologs thereof.
  • FDH formate dehydrogenase
  • the recombinant microorganism further comprises an intracellular NADH/NAD* ratio that is increased compared to a recombinant microorganism not engineered to overexpress one or more FDH genes, leading to increased isobutanol productivity and/or yield.
  • the recombinant microorganisms of the present application can express a plurality of heterologous and/or native enzymes involved in pathways for the production of a beneficial metabolite such as isobutanol.
  • engineered or “modified” microorganisms are produced via the introduction of genetic materia! 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 introduction of genetic material into and/or the modification of the expression of native genes in a parental microorganism results in a new or modified ability to produce beneficial metabolites, such as isobutanol, from a suitable carbon source.
  • the genetic materia! 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 one or more metabolites, including isobutanol, and may also include additional elements for the expression and/or regulation of expression of these genes, e.g. promoter sequences.
  • an engineered or modified microorganism can also include the alteration, disruption, deletion or knocking-out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism.
  • the microorganism acquires new or improved properties (e.g., the ability to produce a new metabolite or greater quantities of an intracellular metabolite, to improve the flux of a metabolite down a desired pathway, and/or to reduce the production of by-products).
  • Recombinant microorganisms provided herein may also produce metabolites in quantities not available in the parental microorganism.
  • a "metabolite” refers to any substance produced by metabolism or a substance necessary for or taking part in a particular metabolic process.
  • a metabolite can be an organic compound that is a starting material (e.g., glucose or pyruvate), an intermediate (e.g., 2-ketoisovalerate), or an end product (e.g., isobutanol) of metabolism.
  • Metabolites can be used to construct more complex molecules, or they can be broken down into simpler ones.
  • Intermediate metabolites may be synthesized from other metabolites, perhaps used to make more complex substances, or broken down into simpler compounds, often with the release of chemical energy.
  • the disclosure identifies specific genes useful in the methods, compositions and organisms of the disclosure; however it will be recognized that absolute identity to such genes is not necessary.
  • changes in a particular gene or polynucleotide comprising a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically such changes comprise conservative mutations and silent mutations.
  • modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art.
  • Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence.
  • Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E.
  • DNA compounds differing in their nucleotide sequences can be used to encode a given enzyme of the disclosure.
  • the native DNA sequence encoding the biosynthetic enzymes described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure.
  • a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity.
  • the disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide.
  • the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.
  • homoiogs of enzymes useful for generating metabolites are encompassed by the microorganisms and methods provided herein.
  • two proteins are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and nonhomologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • a "conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (See, e.g., Pearson W.R., 1994, Methods in Mol ⁇ 25: 385-89.
  • 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), Giutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucirie (I), Leucine (L), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  • yeast microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of beneficial metabolites such as isobutanol.
  • microorganisms may be selected from yeast microorganisms.
  • yeast microorganisms for the production of a metabolite such as isobutanol are described in commonly-owned U.S. Patent Nos. 8,017,375, 8,017,376, 8,133,715, 8,153,415, 8,158,404, and 8,232,089.
  • the yeast microorganism has reduced or no pyruvate decarboxylase (PDC) activity.
  • PDC catalyzes the decarboxylation of pyruvate to acetaidehyde, which is then reduced to ethanol by ADH via an oxidation of NADH to NAD+.
  • Ethanol production is the main pathway to oxidize the NADH from glycolysis. Deletion, disruption, or mutation of this pathway increases the pyruvate and the reducing equivalents (NADH) available for a biosynthetic pathway which uses pyruvate as the starting material and/or as an intermediate.
  • NADH reducing equivalents
  • deletion, disruption, or mutation of one or more genes encoding for pyruvate decarboxylase and/or a positive transcriptional regulator thereof can further increase the yield of the desired pyruvate-derived metabolite (e.g., isobutanol).
  • said pyruvate decarboxylase gene targeted for disruption, deletion, or mutation is selected from the group consisting of PDC1, PDC5, and PDC6, or homologs or variants thereof.
  • all three of PDC1, PDCS, and PDC6 are targeted for disruption, deletion, or mutation.
  • a positive transcriptional regulator of the PDC1, PDCS, and/or PDC6 is targeted for disruption, deletion or mutation.
  • said positive transcriptional regulator is PDC2, or homologs or variants thereof.
  • the microorganism has reduced giycerol-3- phosphate dehydrogenase (GPD) activity.
  • GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glyceroi-3-phosphate (G3P) via the oxidation of NADH to NAD+.
  • Glycerol is then produced from G3P by Glycerol-3- phosphatase (GPP).
  • Glycerol production is a secondary pathway to oxidize excess NADH from glycolysis. Reduction or elimination of this pathway would increase the pyruvate and reducing equivalents (NADH) available for the production of a pyruvate-derived metabolite (e.g., isobutanol).
  • NADH pyruvate and reducing equivalents
  • disruption, deletion, or mutation of the genes encoding for giycero!-3-phosphate dehydrogenases can further increase the yield of the desired metabolite (e.g., isobutanol).
  • desired metabolite e.g., isobutanol.
  • Yeast strains with reduced GPD activity are described in commonly owned and co-pending US Patent Publication Nos. 201 1/0020889 and 201 1/0183392.
  • the microorganism has reduced 3-keto acid reductase (3-KAR) activity.
  • 3-KARs catalyze the conversion of 3-keto acids (e.g., acetolactate) to 3-hydroxyacids (e.g., DH2MB).
  • Yeast strains with reduced 3-KAR activity are described in commonly owned U.S. Pat. Nos. 8,133,715, 8,153,415, and 8,158,404, which are herein incorporated by reference in their entireties.
  • the microorganism has reduced aldehyde dehydrogenase (ALDH) activity.
  • Aldehyde dehydrogenases catalyze the conversion of aldehydes (e.g., isobutyraldehyde) to acid by-products (e.g., isobutyrate).
  • Yeast strains with reduced ALDH activity are described in commonly owned U.S. Pat. Nos. 8,133,715, 8,153,415, and 8,158,404, which are herein incorporated by reference in their entireties.
  • any method can be used to identify genes that encode for proteins with FDH activity.
  • genes that are homologous or similar to a known FDH gene e.g., S. cerevisiae FDH1 (SEQ ID NO: 1 ) can be identified by functional, structural, and/or genetic analysis.
  • homologous or similar FDH genes and/or homologous or similar FDH proteins will have functional, structural, or genetic similarities.
  • Techniques known to those skilled in the art may be suitable to identify additional homologous genes and homologous enzymes.
  • analogous genes and/or analogous enzymes can be identified by functional analysis and will have functional similarities. Techniques known to those skilled in the art may be suitable to identify analogous genes and analogous enzymes.
  • techniques may include, but not limited to, cloning a FDH gene by PGR using primers based on a published sequence of a gene/enzyme or by degenerate PGR using degenerate primers designed to amplify a conserved region among FDH genes.
  • PGR primers based on a published sequence of a gene/enzyme
  • degenerate PGR using degenerate primers designed to amplify a conserved region among FDH genes.
  • one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity.
  • the computer program BLAST may be used for such a purpose.
  • 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.
  • Techniques also include examining a ceil or ceil culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity (e.g. as described herein or in Kiritani, K.
  • Overexpression of the FDH1 gene or homoiogs thereof may be accomplished by any number of methods.
  • overexpression of the FDH1 or homoiogs thereof gene may be accomplished with the use of piasmid vectors that function in yeast.
  • the expression of FDH1 and/or homologous genes may be increased by overexpressing the genes on a CEN piasmid or alternative piasmids with a similar copy number.
  • FDH1 or a homolog thereof is overexpressed on a CEN piasmid or alternative piasmids with a similar copy number.
  • expression of genes from single or multiple copy integrations into the chromosome of the ceil may be useful.
  • Use of a number of promoters such as TDH3, TEF1, CCW12, PGK1, and EN02, may be utilized.
  • the expression level may be fine-tuned by using a promoter that achieves the optimal expression (e.g. optimal overexpression) level in a given yeast.
  • Different levels of expression of the genes may be achieved by using promoters with different levels of activity, either in single or multiple copy integrations or on piasmids.
  • An example of such a group of promoters is a series of truncated PDC1 promoters designed to provide different strength promoters.
  • promoters that are active under desired conditions such as growth on glucose
  • a promoter from one of the glycolytic genes, the PDC1 promoter, and a promoter from one of the ADH genes in S. cerevisiae may ail be useful.
  • embodiments are exemplified using the yeast S. cerevisiae.
  • other yeasts such as those from the genera listed herein may also be used.
  • the present invention provides a method of producing isobutanoi.
  • the method includes cultivating a recombinant microorganism comprising a isobutanoi producing biosynthetic pathway in a culture medium containing a feedstock providing a carbon source until the isobutanoi is produced and optionally, recovering the isobutanoi.
  • said recombinant microorganism has been engineered to overexpress one or more polynucleotides encoding one or more formate dehydrogenases.
  • a beneficial metabolite e.g., isobutanoi
  • the recombinant microorganism is cultured in an appropriate culture medium containing a carbon source.
  • the method further includes isolating the beneficial metabolite ⁇ e.g., isobutanoi) from the culture medium.
  • a beneficial metabolite e.g., isobutanoi
  • the recombinant microorganism may produce the beneficial metabolite from a carbon source at a yield of at least 5 percent theoretical.
  • the microorganism may produce the beneficial metabolite from a carbon source at a yield of at least about 10 percent, at least about 15 percent, at 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 85 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5% theoretical.
  • the beneficial metabolite is isobutanoi.
  • the microorganism is selected to produce isobutanoi from a carbon source at a specific productivity of at least about 0.05 g/(g *h). In another embodiment, the microorganism is selected to produce isobutanoi from a carbon source at a specific productivity of at least about 0.01 g/(g*h), at least about 0.03 g/(g*h), at least about 0.08 g/(g*h), at least about 0.1 g/(g*h), at least about 0.2 g/(g*h), at least about 0.5 g/(g*h), or at least about 1 .0 g/(g*h).
  • “specific productivity” is determined relative to cell dry weight.
  • CDW or "ceil dry weight” refers to the weight of the microorganism after water contained in the microorganism has been removed using methods known to one skilled in the art.
  • DDG generally refers to the solids remaining after a fermentation, usually consisting of unconsumed feedstock solids, remaining nutrients, protein, fiber, and oil, as well as spent yeast biocatalysts or cell debris therefrom that are recovered by further processing from the fermentation, usually by a solids separation step such as centrifugation.
  • Distillers dried grains may also include soluble residual material from the fermentation, or syrup, and are then referred to as "distillers dried grains and solubles" (DDGS).
  • DDGS soluble residual material from the fermentation, or syrup
  • Use of DDG or DDGS as animal feed is an economical use of the spent biocatalyst following an industrial scale fermentation process.
  • the present invention provides an animal feed product comprised of DDG derived from a fermentation process for the production of a beneficial metabolite (e.g., isobutanol), wherein said DDG comprise a spent yeast biocatalyst of the present invention, !n an exemplary embodiment, the recombinant microorganism comprises an isobutanol producing metabolic pathway comprising at least one exogenous gene, wherein said microorganism is engineered to overexpress one or more polynucleotides encoding one or more formate dehydrogenase (FDH) proteins or homologs thereof.
  • a beneficial metabolite e.g., isobutanol
  • the recombinant microorganism comprises an isobutanol producing metabolic pathway comprising at least one exogenous gene, wherein said microorganism is engineered to overexpress one or more polynucleotides encoding one or more formate dehydrogenase (FDH) proteins or homologs
  • the DDG comprising a spent yeast biocatalyst of the present invention comprise at least one additional product selected from the group consisting of unconsumed feedstock solids, nutrients, proteins, fibers, and oils.
  • the present invention provides a method for producing DDG derived from a fermentation process using a yeast biocatalyst (e.g., a recombinant yeast microorganism of the present invention), said method comprising: (a) cultivating said yeast biocatalyst in a fermentation medium comprising at least one carbon source; (b) harvesting insoluble material derived from the fermentation process, said insoluble material comprising said yeast biocatalyst; and (c) drying said insoluble material comprising said yeast biocatalyst to produce the DDG.
  • a yeast biocatalyst e.g., a recombinant yeast microorganism of the present invention
  • the method further comprises step (d) of adding soluble residual material from the fermentation process to said DDG to produce DDGS.
  • said DDGS comprise at least one additional product selected from the group consisting of unconsumed feedstock solids, nutrients, proteins, fibers, and oils.
  • YP medium contains 1 % (w/v) yeast extract, 2% (w/v) peptone.
  • YPD is YP containing 2% (w/v) glucose.
  • Plasmids Table 3 details the plasmids disclosed herein:
  • GEVO6014 was transformed with pGV2196, pGV3158, and pGV3159 in one set of transformations, yielding strains GEVO6607- GEV06615. Briefly, GEVO6014 ceils were harvested from an YPD and G418 plate that had incubated at 30°C for 2 days and at room temperature for 5 days with a sterile toothpick. The cells were suspended in 100 pL of 100 mM lithium acetate.
  • a mixture of 15 pL of each piasmid DNA (about 2-5 pg), 72 pL 50 % PEG (MW 3350), 10 pL 1 M lithium acetate, 3 pL denatured salmon sperm DNA, and 15 pL of the cell suspension was prepared, and these "transformation mixes" were incubated at 30°C for 30 minutes.
  • the transformation mixes were vortexed briefly prior to their incubation at 42°C for 22 minutes.
  • the cells were collected from the transformation mixes by centrifugation at 18,000xg for 20 seconds, washed once with 1 mL sterile water, and collected again by centrifugation at 18,000xg for 20 seconds.
  • the cells were resuspended in 400 pL YPD with 5% glucose and 0.2 g/L G418 and incubated at 3Q°C, 250 RPM overnight. The entire cultures were spread onto YPD with 0.2 g/L G418 and 0.1 g/L hygromycin and incubated at 30°C until colonies were isolated.
  • GEVO6014 and GEV06399 were transformed with pGV3201 and pGV3202 in one set of transformations, yielding strains GEV0671 1 -6718 and GEVO6720-GEVO6725.
  • GEVO8014 and GEV06399 cells were incubated at 250 RPM, 30°C overnight in 50 mL YP with 8% dextrose and 200 mM IVIES pH 6.5 and 1 % ethanoi (and 0.2 g/L for GEVO8014), reaching optical densities (ODeoo). The ceils were collected by centrifugation at 1800xg for 2 minutes in two sterile 50 mL tubes.
  • the supernatants were discarded, the ceil pellets were resuspended in 50 mL sterile water, and the cells were collected by centrifugation at 1600xg for 2 minutes.
  • the cell pellets were resuspended in 25 mL sterile wafer, and the cells were collected by centrifugation at 1800xg for 2 minutes.
  • the ceil pellets were resuspended in 1 ml 100mM LiOAc and transferred into an eppendorf tube, followed by centrifugation in a microcentrifuge for 10 seconds at 14,000 rpm. The supernatant was removed and the ceil pellet was resuspended with 4 times pellet volume of 100 mM lithium acetate (ceil suspension).
  • a DNA mix for each transformation was prepared with 5 pL piasmid DNA (about 1 pg), 10 pL sterile water, 72 pL 50% PEG (MW 3350), 10 pL 1 M lithium acetate, and 3 pL denatured salmon sperm DNA. To each DNA mix, 15 pL of the cell suspension was added in 1 .5 mL tubes. The transformations were incubated at 30°C for 30 minutes, and then heat-shocked at 42°C for 22 minutes. The ceils were diluted with 1 mL sterile water, and collected by centrifugation for 20 seconds at 18,000xg.
  • the ceils were resuspended in 2 mL YP with 8% dextrose and 200 mM IVIES pH 8.5 and 1 % ethanoi (and 0.2 g/L for GEVO6014) and incubated at 30°C, 250 RPM overnight.
  • the GEV06399 transformed cells were spread onto YPD with hygromycin (0.5 g/L) plates, and the GEVO8014 transformed ceils were spread onto YPD with hygromycin (0.5 g/L) and G418 (0.2 g/L) plates. The plates were incubated at 30 C C until colonies were isolated.
  • GEV06399 was transformed with pGV2196, yielding strains GEV06717- GEV06719. Briefly, cells were grown to mid-log phase, washed twice with water and resuspended with 4 pellet-volumes of 100 mM LiAc. 15 pL of cells were mixed with 100 pL of the DNA mix containing piasmids, 36% PEG, 100 mM LiAc, and 30 pg of boiled and sonicated salmon sperm DNA. The mixture was incubated at 30°C for 30 minutes and heat shocked for 22 minutes at 42°C.
  • Transformations were recovered in 500 pL YPD overnight at 3Q°C and 250 rpm before plating onto YPD and 0.2 g/L hygromycin. Transformants were single colony isolated on YPD with 0.2 g/L hygromycin plates. Isolates were then patched onto YPD with 0.2 g/L hygromycin plates.
  • FDH Over-expression and Formate Feed Shake Flask Fermentation A shake flask fermentation with GEVO8014 strains transformed with CEN piasmids was performed. Briefly, the strains inoculated at an initial optical density (OD 6 oo) of 0.1 in 50 mL YPD (8%) with 200 mM IVIES pH 8.5 and 1 % ethanol and 0.2 g/L G418 and 0.4 g/L hygromycin in round-bottom flasks.
  • the cultures were incubated at 250 RPM, 30 C C for 28 hours prior to measurement of the optical densities and addition of 0 pL or 103 pL (56 mM) or 205 pL (1 12 mM) formic acid (Fiuka Analytical, Sigma- Aidrich, FW 46.03 g/moi, Density 1 .22 g/mL).
  • the samples were incubated at 75 RPM, 30°C for 22 hours, with samples (1 mL) being removed at 0, 2, and 22 hours incubation.
  • Optical densities of the samples were measured prior to centrifugation of the samples at 18,000xg for 10 minutes.
  • the supernatants were stored at 4°C prior to their submission for GC and LC analysis.
  • optical density The optical density of the yeast cultures was determined at 600 nm using a DU 800 spectrophotometer (Beckman-Couiter, Fulierton, CA, USA). Samples were diluted as necessary to yield optical densities of between 0.1 and 0.8.
  • GC1 Gas Chromatography: Analysis of volatile organic compounds, including ethanol and isobutanol was performed on an Agilent 6890 gas chromatograph (GC) fitted with a 7683B liquid autosampler, a split/spiitiess injector port, a ZB-FFAP column (Phenomenex 30 m length, 0.32 mm ID, 0.25 pM film thickness) connected to a flame ionization detector (FID).
  • the temperature program is as follows: 230 C C for the injector, 300°C for the detector, 100°C oven for 1 minute, 3S°C/minute gradient to 230°C, and then hold for 2.5 min.
  • High Performance Liquid Chromatography Analysis of organic acid was performed on an Agilent 1200 or equivalent High Performance Liquid Chromatography system equipped with a Bio-Rad Micro-guard Cation H Cartridge and two Phenomenex Rezex RFQ-Fast Fruit H+ (8%), 100 x 7.8-mm columns in series, or equivalent.
  • Organic acid metabolites were detected using an Agilent 1 100 or equivalent UV detector (210 nm) and a refractive index detector.
  • the column temperature was 60°C. This method was isocratic with 0.0180 N H 2 SO 4 in Mil!i-Q water as mobile phase. Flow was set to 1 .1 mL/min. Injection volume was 20 pL and run time was 18 min.
  • Cell iysate preparation Ceil pellets containing 20 ODs were thawed on ice and resuspended in 750 pL of universal lysis buffer (100 mM NaPO 4 pH 7.0, 5 mM MgCi2, 1 mM DTT). One mL of glass beads (0.5 mm diameter) were added to a 1 .5 mL microcentrifuge tube and 800 pL of cell suspensions were added. Yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown, PA), mixing 8 X 1 min at 30 cycles/sec, with a 1 min incubation on ice between beatings. The tubes were centrifuged for 10 min at 21 ,S0Qxg at 4 ° C and the supernatant was transferred to a fresh tube. Extracts were held on ice until assayed on the same day.
  • universal lysis buffer 100 mM NaPO 4 pH 7.0, 5 mM MgCi2, 1 m
  • Yeast Iysate protein concentration was determined using the BioRad Bradford Protein Assay Reagent Kit (Cat# 500-0006, BioRad Laboratories, Hercules, CA). Briefly, a standard curve for the assay was made using a dilution series of a standard protein stock (500 pg/mL BSA). Appropriate dilutions of each ceil iysate (1 :10) was made in water to obtain OD 5 9 5 measurements of each Iysate that fell within the linear range of the BioRad protein standard curve.
  • FDH Formate Dehydrogenase Assay: The procedure used for measuring formate dehydrogenase (FDH) enzyme activity was described in Overkamp, et ai. Yeast 19(6):509-2 (April 2002). Briefly, crude eel! lysates from each shake flask fermentation sample pellet was diluted 1 :10 in universal lysis buffer (100 mM NaP0 4 pH 7.0, 5 mM MgC , 1 mM DTT), and 10 L was added to 3 wells in 2 adjacent rows (8 wells total) of a microtiter plate stored on ice. Purified formate dehydrogenase (from Candida boidinii, Product #F8849, Sigma-Aldrich St.
  • reaction buffer with formate 50 m!Vl Potassium Phosphate Buffer pH 7, 20 mM NAD 4' , 50 mM sodium formate
  • 90 L of reaction buffer without formate 50 mM Potassium Phosphate Buffer pH 7, 20 m!Vl NAD ⁇
  • the plate was immediately transferred to a SpectraMax 340PC plate reader (Molecular Devices, Sunnyvale, CA) where the absorbances at 340 nm were measured every 10 seconds for 5 minutes. Reactions were performed at 30°C. The V max for each sample was determined by subtracting the background reading of the no substrate control.
  • EXAMPLE 1 FDH Overexpression increases Specific isobutanoi Activity in Recombinant Yeast.
  • GEVO6014 carrying a CEN p!asmid lacking FDH (no FDH), encoding a cytosolicaliy-locaiized formate dehydrogenase (cyto FDH), or encoding a mitochondrialiy-!ocaiized formate dehydrogenase (mito FDH) were assayed in a 22 hour shake flask fermentation. For each strain, biological duplicates were assayed with the addition of the amount of formate to the flasks indicated in Figure 6 and Fsgure 7.

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Abstract

La présente demande concerne des micro-organismes recombinés comprenant des voies de biosynthèse et des procédés d'utilisation desdits micro-organismes recombinés pour produire divers métabolites bénéfiques, comme l'isobutanol. Dans divers aspects de l'invention, les micro-organismes recombinés peuvent comprendre une ou plusieurs modifications conduisant à des augmentations de l'expression de et/ou de l'activité de formiate déshydrogénase.
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WO2009143495A2 (fr) * 2008-05-22 2009-11-26 President And Fellows Of Harvard College Levure à croissance rapide
US20110201089A1 (en) * 2010-02-23 2011-08-18 Burgard Anthony P Methods for increasing product yields

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WO2009143495A2 (fr) * 2008-05-22 2009-11-26 President And Fellows Of Harvard College Levure à croissance rapide
US20110201089A1 (en) * 2010-02-23 2011-08-18 Burgard Anthony P Methods for increasing product yields

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