WO2013003545A1 - Ajustement fin de sous-produits d'alcool de fusel pendant la production d'isobutanol par des micro-organismes recombinants - Google Patents

Ajustement fin de sous-produits d'alcool de fusel pendant la production d'isobutanol par des micro-organismes recombinants Download PDF

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Publication number
WO2013003545A1
WO2013003545A1 PCT/US2012/044563 US2012044563W WO2013003545A1 WO 2013003545 A1 WO2013003545 A1 WO 2013003545A1 US 2012044563 W US2012044563 W US 2012044563W WO 2013003545 A1 WO2013003545 A1 WO 2013003545A1
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Prior art keywords
recombinant microorganism
conversion
microorganism
catalyzing
yeast
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PCT/US2012/044563
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English (en)
Inventor
Peter Meinhold
Matthew W. Peters
Andrew C. Hawkins
Reid M. FELDMAN
Jun Urano
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Gevo, Inc.
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Publication of WO2013003545A1 publication Critical patent/WO2013003545A1/fr

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    • 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
    • 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
    • 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 organisms are provided. Also provided are methods of producing beneficial metabolites including fuels, chemicals, and amino acids by contacting a suitable substrate with recombinant microorganisms and enzymatic preparations therefrom.
  • fuels such as isobutanoi have been produced recombinantiy in microorganisms expressing a heterologous metabolic pathway (See, e.g., WO/2007/050671 to Donaldson, et a/., and WO/2008/098227 to Liao, et a/.).
  • engineered microorganisms represent potentially useful tools for the renewable production of fuels, chemicals, and amino acids, many of these microorganisms have fallen short of commercial relevance due to their low performance characteristics, including low- productivity, low titers, and low yields.
  • the present invention results from the study of enzymes that form this molecule and shows that the suppression of one or more of these enzymes considerably reduces or eliminates the formation of 3-methy!-1 - butanoi, and concomitantly improves the yields and titers of beneficial metabolites derived from 2-ketoisovalerate.
  • 3-methyl-1 -butanol may be beneficial.
  • 3-methyl-1 -butanol may be produced alongside a beneficial metabolite, for instance, isobutanol, in a quantitated manner such that a mixture of products is isolated. Therefore, one may tune a recombinant microorganism to produce a blended product that is composed partially of 3-methyl-1 -butanol.
  • the present inventors have discovered that the occasionally unwanted byproduct 3-methy!-1 -butanol can accumulate during various fermentation processes, including fermentation of the biofuei candidate, isobutanol.
  • the accumulation of this unwanted by-product may result from the undesirable conversion of 2- ketoisovalerate, an intermediate in a number of biosynthetic pathways.
  • the conversion of 2-ketoisovalerate to this sometimes unwanted by-product can hinder the optimal productivity and yield of a desirable product. Therefore, the present inventors have developed methods for reducing the conversion of 2-ketoisovalerate to 3-methyl-1 -butanol during processes where 2-ketoisovaierate acts as a pathway intermediate.
  • the accumulation of this unwanted by-product may also result from the undesirable conversion of leucine, an amino acid that may be present in a fermentation medium.
  • the present inventors have developed methods for reducing the conversion of leucine to 3-methyl-1 -butanoi during processes where leucine is present in the fermentation medium. Also, however, the present inventors have recognized that, in certain situations, it may be desirable to produce a mixture of products that can comprise 3-methyi ⁇ 1 -butanoL Therefore, the present inventors have developed methods for enhancing the conversion of 2-ketoisovalerate to 3- methyl-1 -butanol during processes where 2-ketoisovalerate acts as a pathway intermediate.
  • the present invention relates to a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovalerate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 3-methyl-1 -butanol.
  • the present invention relates to a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovalerate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 2 ⁇ isopropyimaiate; one or more enzymes catalyzing the conversion of 2-isopropyimaiate to 3-isopropyimalate; one or more enzymes catalyzing the conversion of 3-isopropylmalate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methyibutanal; and one or more enzymes catalyzing the conversion of 3-methy!butanal to 3-methyl- 1 -butanol.
  • the present invention relates to a recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 3- methyl-1 -butanoi.
  • the present invention relates to a recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovaierate to 2-isopropyimaiate; one or more enzymes catalyzing the conversion of 2-isopropylmaiate to 3- isopropy!ma!ate; one or more enzymes catalyzing the conversion of 3- isopropylmaiate to ⁇ -ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocapr lodge to 3-methylbutanal; and one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methyl-1 -butanoi.
  • the present invention relates to a recombinant microorganism comprising a biosynthetic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of leucine to 3-methy!-1 -butanol.
  • the present invention relates to a recombinant microorganism comprising a biosynthetic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression of one or more of the following: one or more enzymes catalyzing the conversion of leucine to a- ketoisocaproate; one or more enzymes catalyzing the conversion of a- ketoisocaproate to 3-methylbutanal; and one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methyi-1 -butanol,
  • the present invention relates to a recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of leucine to 3 ⁇ methy!-1 ⁇ butanoi.
  • the present invention relates to a recombinant microorganism comprising an isobutanol producing metabolic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression of one or more of the following: one or more enzymes catalyzing the conversion of leucine to a-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methylbutanai; and one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methyl-1 -butano!.
  • the recombinant microorganisms are engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 3-methyl-1 - butanoi.
  • the enzyme is a 2-isopropylmalate synthase, catalyzing the conversion of 2-ketoisovaierate to 2-isopropyimalate.
  • the 2-isopropylmalate synthase is the S. cerevisiae Leu4 (SEQ ID NO: SEQ ID NO: 2) or the S. cerevisiae Leu9 (SEQ ID NO: SEQ ID NO: 4) or a homoiog or variant thereof.
  • the enzyme is an isopropyimalate isomerase, catalyzing the conversion of 2-isopropylmalate to 3-isopropy!malate.
  • the isopropyimalate isomerase is the S. cerevisiae Leu1 (SEQ ID NO: 8) or a homo!og or variant thereof.
  • the enzyme is a 3- isopropyimaiate dehydrogenase, catalyzing the conversion of 3-isopropylmalate to a- ketoisocaproate.
  • the 3-isopropylmalate dehydrogenase is the S. cerevisiae Leu2 (SEQ ID NO: 8) or a homolog or variant thereof.
  • the enzyme is a branched-chain amino acid transaminase, catalyzing the conversion of leucine to a-kefoisocaproate.
  • the branched-chain amino acid transaminase is the S. cerevisiae Bat1 (SEQ ID NO: 10) or the S. cerevisiae Bat2 (SEQ ID NO: 12) or a homoiog or variant thereof.
  • the enzyme is a keto-isocaproate decarboxylase, catalyzing the conversion of ⁇ -ketoisocaproate to 3-methylbutanai. !n a specific embodiment, the keto-isocaproate decarboxylase is the S.
  • the enzyme is an alcohol dehydrogenase, catalyzing the conversion of 3-methylbutanal to 3-methyl-1 -butanoi.
  • the alcohol dehydrogenase is the S. cerevisiae Adh6 (SEQ ID NO: 18) or the S. cerevisiae Adh7 (SEQ ID NO: 20) or a homoiog or variant thereof.
  • the recombinant microorganisms are engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the initial steps in the conversion of 2- ketoisovalerate to 3-methyi-1 -butanoi.
  • the enzyme is a 2- isopropylmaiate synthase, catalyzing the conversion of 2-ketoisovalerate to 2 ⁇ isopropylmalate.
  • the 2-isopropylmalate synthase is the S, cerevisiae Leu4 (SEQ ID NO: 2) or the S. cerevisiae Leu9 (SEQ ID NO: 4) or a homoiog or variant thereof.
  • the enzyme is a branched-chain amino acid transaminase, catalyzing the conversion of leucine to a-ketoisocaproate.
  • the branched-chain amino acid transaminase is the S. cerevisiae Bat1 (SEQ ID NO: 10) or the S. cerevisiae Bat2 (SEQ ID NO: 12) or a homoiog or variant thereof.
  • the recombinant microorganism comprises an isobutanoi producing metabolic pathway.
  • the isobutanoi producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanoi.
  • the isobutanoi producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol.
  • the isobutanol 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. 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, ail 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 (KARI), dihydroxyacid dehydratase (DHAD), 2 ⁇ keto-acid decarboxylase, e.g., keto-isovalerate decarboxylase (KIVD), and alcohol dehydrogenase (ADH).
  • the KARI is an NADH-dependent KARI (NKR).
  • the ADH is an NADH-dependent ADH.
  • the KAR! is an NADH-dependent KAR! (NKR) and the ADH is an NADH-dependent ADH.
  • the present invention also provides for a recombinant microorganism comprising a biosynthetic pathway which uses 2- ketoisovalerate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following and/or is substantially free of an enzyme catalyzing the conversion of one or more of the following: one or more enzymes catalyzing the conversion of 2 ⁇ ketoisovalerate to 2-isopropyimaiate; one or more enzymes catalyzing the conversion of 2-isopropylmalate to 3-isopropylmalate; one or more enzymes catalyzing the conversion of 3-isopropylmalate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of leucine to a-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methylbutanal; and one or more enzymes catalyzing the conversion of a
  • the present invention also provides for a recombinant microorganism comprising an isobutanoi producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following and/or is substantially free of an enzyme catalyzing the conversion of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovaierate to 2-isopropyimaiate; one or more enzymes catalyzing the conversion of 2-isopropyimaiate to 3-isopropylmalate; one or more enzymes catalyzing the conversion of 3-isopropyimaiate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of leucine to ⁇ -ketoisocaproate; one or more enzymes catalyzing the conversion of ⁇ -ketoisocaproate to 3-methylbutanal; and one or more enzymes catalyzing the conversion of 3-methylbutanal
  • the present invention also provides for a recombinant microorganism for the production of isobutanoi and 3-methyi-l -butanol, wherein said recombinant microorganism comprises an isobutanoi producing metabolic pathway and overexpresses one or more enzymes capable of converting 2-ketoisovaierate to 3-methyl-1 -butanoi.
  • one or more enzymes catalyze the conversion of 3-methylbutanal to 3-methy!-1 -butanol.
  • the enzyme is an alcohol dehydrogenase.
  • the alcohol dehydrogenase is NADH-dependent.
  • 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 of the invention produce a 2-ketoisovaierate-derived product.
  • the 2-ketoisovalerate-derived product is selected from isobutanol, valine, pantothenate, and coenzyme A.
  • production pathway enzymes may be overexpressed to yield a desired product.
  • the recombinant microorganism is a bacterium.
  • the recombinant microorganism may be selected from a genus of Citrobacter, Corynebacterium, Lactobacillus, Lactococcus, Salmonella, Enterobacter, Enterococcus, Erwinia, Pantoea, Morganella, Pectobacterium, Proteus, Serratia, Shigella, and Klebsiella.
  • the recombinant microorganism is a lactic acid bacterium such as, for example, a microorganism derived from the Lactobacillus or Lactococcus genus.
  • the recombinant microorganisms may be yeast microorganisms.
  • the recombinant microorganisms may be yeast recombinant microorganisms of the Saccharomyces ciade.
  • the recombinant microorganisms may be Saccharomyces sensu stricto microorganisms.
  • Saccharomyces sensu stricto microorganism is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis, and hybrids thereof.
  • the recombinant 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, Hansenula, issatchenkia, or Candida.
  • the Crabtree-negative yeast microorganism is selected from Saccharomyces kluyveri, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Pichia kudriavzevii, issatchenkia orientalis, 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, Pichia, Candida, and Schizosaccharomyces.
  • the Crabtree-positive yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelii, Kluyveromyces thermotolerans, Candida glabrata, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii, Pichia pastorius, and Schizosaccharomyces pombe.
  • 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 castelii, 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, Issatchenkia, Pachysolen, Yarrowia and Schizosaccharomyces.
  • the pre-WGD yeast is selected from the group consisting of Saccharomyces kluyveri, Kluyveromyces thermotolerans, Kiuyveromyces marxianus, Kiuyveromyces waltii, Kiuyveromyces lactis, Candida tropicaiis, Pichia pastoris, Pichia anomala, Pichia stipitis, Pichia kudriavzevii, Issatchenkia orientalis, Issatchenkia occidentaiis, Debaryomyces hansenii, Hansenula 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, Myxozyma, or Candida.
  • the non-fermenting yeast is C. xestobii.
  • the present invention provides methods of producing beneficial metabolites including fuels, chemicals, and amino acids using a recombinant microorganism as described herein.
  • the method includes cultivating the recombinant microorganism in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the metabolite is produced.
  • the microorganism produces the metabolite from a carbon source at a yield of at least about 5 percent theoretical.
  • the microorganism produces the metabolite at a yield of at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 80 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5 percent theoretical.
  • the metabolite may be derived from a biosynthetic pathway which uses 2-ketoisovalerate as an intermediate, including, but not limited to, isobutanoi, valine, pantothenate, and coenzyme A pathways.
  • the metabolite is isobutanoi.
  • the recombinant microorganism converts the carbon source to the beneficial metabolite under aerobic conditions. In another embodiment, the recombinant microorganism converts the carbon source to the beneficial metabolite under microaerobic conditions. In yet another embodiment, the recombinant microorganism converts the carbon source to the beneficial metabolite under anaerobic conditions.
  • Figure 1 illustrates an exemplary embodiment of an isobutanoi pathway.
  • Figure 2 illustrates an exemplary embodiment of an NADH-dependent isobutanol pathway.
  • Figure 3 illustrates exemplary biosynthetic pathways utilizing 2- ketoisovalerate as an intermediate.
  • Figure 4 illustrates the conversion of 2-ketoisovalerate to 3-methyl-1 - butanol.
  • Figure 5A illustrates the production of 3-methyl-1 -butanol by an S. cerevissae parental strain (GEVO8014), and progeny strains in which Leu4 or Leu9 have been disrupted.
  • the S. cerevisiae strains were grown in medium void of leucine.
  • Figure 5B illustrates the production of 3-methyl-1 -butanoi by an S. cerevisiae parental strain (GEVO8014), and progeny strains in which Leu4 or Leu9 have been disrupted.
  • the S. cerevisiae strains were grown in medium supplemented with leucine.
  • the present inventors have discovered that the occasionally unwanted byproduct . , 3-methyi-l -butanol, can accumulate during various fermentation processes, including fermentation of the biofuei candidate, isobutanol.
  • the accumulation of this unwanted by-product may result from the undesirable conversion of 2- ketoisovalerate, an intermediate in a number of biosynthetic pathways.
  • the conversion of 2-ketoisovalerate to this sometimes unwanted by-product can hinder the optimal productivity and yield of a desirable product. Therefore, the present inventors have developed methods for reducing the conversion of 2-ketoisovalerate to 3-methyl-1 -butano! during processes where 2-ketoisovaierate acts as a pathway intermediate.
  • the accumulation of this unwanted by-product may also result from the undesirable conversion of leucine, an amino acid that may be present in a fermentation medium.
  • the present inventors have developed methods for reducing the conversion of leucine to 3-methyl-1 -butanoi during processes where leucine is present in the fermentation medium. Also, however, the present inventors have recognized that, in certain situations, it may be desirable to produce a mixture of products that can comprise 3-methyi-1 -butanoi. Therefore, the present inventors have developed methods for enhancing the conversion of 2-ketoisovalerate to 3- methyl-1 -butanoi during processes where 2-keioisovalerate acts as a pathway intermediate.
  • 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, among others.
  • microbial ceils and “microbes” are used interchangeably with the term microorganism.
  • the term "genus” is defined as a taxonomic group of related species according to the Taxonomic Outline of Bacteria and Archaea (Garrity, G.M., et a/. The Taxonomic Outline of Bacteria and Archaea. TOBA Release 7.7, March 2007. Michigan State University Board of Trustees, [http://www.taxonomicoutiine.org/]).
  • genomic hybridization is defined as a collection of closely related organisms with greater than 97% 16S ribosomai RNA sequence homology and greater than 70% genomic hybridization and sufficiently different from ail other organisms so as to be recognized as a distinct unit.
  • recombinant microorganism refers to microorganisms that have been genetically modified to express or to overexpress endogenous polynucleotides, to express heterologous polynucleotides, such as those included in a vector, in an integration construct, or which have an alteration in expression of an endogenous gene.
  • alteration it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the alteration.
  • alter can mean “inhibit,” but the use of the word “alter” is not limited to this definition.
  • the terms “recombinant microorganism” and “recombinant host cell” refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • expression refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein.
  • expression of a protein results from transcription and translation of the open reading frame sequence.
  • the level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the 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 a/., 1989, supra, [0049]
  • the term "overexpression” refers to an elevated level (e.g., aberrant level) of mRNAs encoding for a protein(s), and/or to elevated levels of protein(s) in ceils as compared to similar corresponding unmodified ceils expressing basal levels of mRNAs or having basal levels of proteins.
  • mRNA(s) or protein(s) may be overexpressed by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8- fold, 10-fold, 12-fold, 15-fold or more in microorganisms engineered to exhibit increased gene mRNA, protein, and/or activity.
  • reduced activity and/or expression of a protein such as an enzyme can mean either a reduced specific catalytic activity of the protein (e.g. reduced activity) and/or decreased concentrations of the protein in the cell (e.g. reduced expression).
  • the reduced activity of a protein in a cell may result from decreased concentrations of the protein in the 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 into 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 ail of a gene.
  • the modified microorganism a portion of the microorganism genome has been replaced with a heterologous polynucleotide.
  • the mutations are naturally-occurring.
  • the mutations are identified and/or enriched through artificial selection pressure.
  • the mutations in the microorganism genome are the result of genetic engineering.
  • biosynthetic pathway also referred to as “metabolic pathway” refers to a set of anabolic or catabolic biochemical reactions for converting one chemical species into another.
  • Gene products belong to the same “metabolic pathway” if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product.
  • isobutanol producing metabolic pathway refers to an enzyme pathway which produces isobutanol from pyruvate.
  • NADH-dependent refers to an enzyme that catalyzes the reduction of a substrate coupled to the oxidation of NADH with a catalytic efficiency that is greater than the reduction of the same substrate coupled to the oxidation of NADPH at equal substrate and cofactor concentrations.
  • exogenous refers to molecules that are not normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
  • endogenous or “native” as used herein with reference to various molecules refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
  • heterologous refers to various molecules, e.g. , polynucleotides, polypeptides, enzymes, etc., wherein at least one of the following is true: (a) the molecule(s) is/are foreign ("exogenous") to (i.e.
  • the molecule(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 ceil; 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.g., greater than naturally found) amount in the ceil.
  • 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 grams 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 grams or milligrams per liter per hour per OD (g/L/h/OD).
  • yield is defined as the amount of product obtained per unit weight of raw material and may be expressed as grams product per grams 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 grams of biofuel in solution per liter of fermentation broth (g/L).
  • “Aerobic conditions” are defined as conditions under which the oxygen concentration in the fermentation medium is sufficiently high for an aerobic or facultative anaerobic microorganism to use as a terminal electron acceptor.
  • anaerobic conditions are defined as conditions under which the oxygen concentration in the fermentation medium is too low for the microorganism to use as a terminal electron acceptor. Anaerobic conditions may be achieved by sparging a fermentation medium with an inert gas such as nitrogen until oxygen is no longer available to the microorganism as a terminal electron acceptor. Alternatively, anaerobic conditions may be achieved by the microorganism consuming the available oxygen of the fermentation until oxygen is unavailable to the microorganism as a terminal electron acceptor. Methods for the production of isobutanol under anaerobic conditions are described in commonly owned and copending publication, US 2010/0143997, the disclosures of which are herein incorporated by reference in its entirety for all purposes.
  • Aerobic metabolism refers to a biochemical process in which oxygen is used as a terminal electron acceptor to make energy, typically in the form of ATP, from carbohydrates. Aerobic metabolism occurs e.g. via glycolysis and the TCA cycle, wherein a single glucose molecule is metabolized completely into carbon dioxide in the presence of oxygen.
  • anaerobic metabolism refers to a biochemical process in which oxygen is not the final acceptor of electrons contained in NADH. Anaerobic metabolism can be divided into anaerobic respiration, in which compounds other than oxygen serve as the terminal electron acceptor, and substrate level phosphorylation, in which the electrons from NADH are utilized to generate a reduced product via a "fermentative pathway.”
  • NAD(P)H donates its electrons to a molecule produced by the same metabolic pathway that produced the electrons carried in NAD(P)H.
  • NAD(P)H generated through glycolysis transfers its electrons to pyruvate, yielding ethanoi.
  • Fermentative pathways are usually active under anaerobic conditions but may also occur under aerobic conditions, under conditions where NADH is not fully oxidized via the respiratory chain. For example, above certain glucose concentrations, Crabtree positive yeasts produce large amounts of ethanoi 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 enzymatic activities include, 2-isopropylmalate synthase, isopropy!ma!ate isomerase, 3-isoproy!ma!ate dehydrogenase, branched- chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, GPD, etc.) in carbon pathways that compete with the desired metabolic pathway (e.g., an isobutanol-producing metabolic pathway) means the level of the enzyme activity is substantially less than that of the same enzyme activity 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 enzymatic activity include, 2-isopropylmalate synthase, isopropy!ma!ate isomerase, 3-isoproylmalate dehydrogenase, branched- chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, GPD, etc.
  • a particular enzymatic activity include, 2-isopropylmalate synthase, isopropy!ma!ate isomerase, 3-isoproylmalate dehydrogenase, branched- chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, GPD, etc.
  • non-fermenting yeast is a yeast species that fails to demonstrate anaerobic metabolism in which the electrons from NADH are utilized to generate a reduced product via a fermentative pathway such as the production of ethanoi and CO2 from glucose.
  • Non-fermentative yeast can be identified by the "Durham Tube Test” (Barnett J.A., et a/. 2000, Yeasts Characteristics and Identification. 3 rd edition, p. 28-29.
  • 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 a 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, 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 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 ceil. Transformation (or transduction, or transfection), can be achieved by any one of a number of means including chemical transformation (e.g. lithium acetate transformation), eiectroporation, microinjection, bioHstics (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 polynucleotides.
  • polypeptide indicates an organic polymer composed of two or more amino acidic monomers and/or analogs thereof.
  • amino acid or “amino acidic monomer” refers to any natural and/or synthetic amino acids including glycine and both D or L optical isomers.
  • amino acid analog refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, or with a different functional group.
  • polypeptide includes amino acidic polymers 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 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 PGR. Identity of cloned sequences as homologs 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 amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene.
  • 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.
  • yeast ceils convert sugars to produce pyruvate, which is then utilized in a number of pathways of cellular metabolism, !n recent years, yeast cells have been engineered to produce a number of desirable products via pyruvate-d riven biosynthetic pathways. In many of these biosynthetic pathways, 2-ketoisovalerate is involved.
  • 2-ketoisovalerate formation is a conversion from 2,3- dihydroxyisova!erate via a dihydroxyacid dehydratase in a pathway for the formation of isobutanol.
  • isobutanoi production pathway a number of other biosynthetic pathways also use 2-ketoisovalerate as an intermediate, including, but not limited to, valine, pantothenate, and coenzyme A pathways.
  • Engineered biosynthetic pathways for the synthesis of these beneficial 2-ketoisovalerate-derived metabolites are found in Table 1 and Figure 2,
  • Each of the biosynthetic pathways shares the common 2-ketoisovalerate intermediate ( Figure 3) and therefore, the product yield from these biosynthetic pathways will in part depend upon the amount of 2-ketoisovalerate that is available to downstream enzymes of said biosynthetic pathways.
  • the present inventors have characterized the enzymes responsible for the accumulation of 3-methyl-1 -butanoi. In some circumstances, these enzymatic activities may hinder the optimal productivity and yield of 2-ketoisovalerate-derived products, including, but not limited to, isobutanoi, valine, pantothenate, and coenzyme A. The present inventors have found that suppressing these newly-characterized enzymatic activities considerably reduces or eliminates the formation of 3-methyi-1 -butanol, and concomitantly improves the yields and titers of beneficial metabolites.
  • the present inventors have found that the deletion of the pathway steps by which 3-methyi-1 -butanoi is produced may remove competition for 2-ketoisovalerate and may further removes competition in engineered biosynthetic pathways for reduced co-factors, which increases the NAD(P)H/NAD(P ratio such that flux through an engineered biosynthetic pathway (e.g., an isobutanoi producing metabolic pathway) may increase.
  • an engineered biosynthetic pathway e.g., an isobutanoi producing metabolic pathway
  • the present inventors describe herein multiple strategies for reducing the conversion of 2-ketoisovaierate to 3-methyl-1 -butano!, a process which is accompanied by an increase in the yield of desirable metabolites.
  • reducing the conversion of 2-ketoisovalerate to 3-methy!-1 -butano! enables the increased production of beneficial metabolites such as isobutanoi, valine, pantothenate, and coenzyme A, which are derived from biosynthetic pathways using 2-ketoisovalerate as an intermediate.
  • the present invention relates to a recombinant microorganism comprising a biosynthetic pathway which uses 2- ketoisovalerate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 3-methyi-1 -butanoi.
  • the present invention relates to a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovaierate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovaierate to 2-isopropyimalafe; one or more enzymes catalyzing the conversion of 2-isopropy!malate to 3-isopropy!ma!ate; one or more enzymes catalyzing the conversion of 3-isopropyimaIate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3- methylbutanal; and one or more enzymes catalyzing the conversion of 3- methylbutanal to 3-methyi ⁇ 1 -butanoL
  • the present invention relates to a recombinant microorganism comprising an isobutanoi producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 2-isopropyimaiate; one or more enzymes catalyzing the conversion of 2-isopropyimaiate to 3-isopropylmalate; one or more enzymes catalyzing the conversion of 3-isopropylmalate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methyibutana!; and one or more enzymes catalyzing the conversion of 3-methyibutana! to 3-methyl- 1 -butanol.
  • the present invention relates to a recombinant microorganism comprising a biosynthetic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of leucine to 3-methyi-1 -butanoI.
  • the present invention relates to a recombinant microorganism comprising a biosynthetic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression of one or more of the following: one or more enzymes catalyzing the conversion of leucine to a- ketoisocaproate; one or more enzymes catalyzing the conversion of a- ketoisocaproate to 3-methylbutanai; and one or more enzymes catalyzing the conversion of 3-methylbutanal to 3-methy!-1 -butanol.
  • the present invention relates to a recombinant microorganism comprising an isobutanoi producing metabolic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of leucine to 3-methy!-1 - butanoi.
  • the present invention relates to a recombinant microorganism comprising an isobutanoi producing metabolic pathway wherein said recombinant microorganism is engineered to reduce or eliminate the expression of one or more of the following: one or more enzymes catalyzing the conversion of leucine to ⁇ -ketoisocaproate; one or more enzymes catalyzing the conversion of ⁇ -ketoisocaproate to 3-methylbutanal; and one or more enzymes catalyzing the conversion of 3-methy!butanai to 3-methyl-1 -butanol.
  • the recombinant microorganisms are engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the initial steps in the conversion of 2-ketoisovalerate to 3-methyi-1 -butanoi.
  • the enzyme is a 2-isopropyimaiate synthase, catalyzing the conversion of 2-ketoisovaierate to 2-isopropyimaiate.
  • the 2- isopropylmaiate synthase is the S. cerevisiae Leu4 (SEQ ID NO: 2) or the S. cerevisiae Leu9 (SEQ ID NO: 4) or a homolog or variant thereof.
  • the enzyme is a branched-chain amino acid transaminase, catalyzing the conversion of leucine to a-ketoisocaproate.
  • the branched-chain amino acid transaminase is 8. cerevisiae Bail (SEQ ID NO: 10) or S. cerevisiae Bat2 (SEQ ID NO: 12) or a homolog or variant thereof. Homologs of Leu4, Leu9, Bat1 , and Bat2 are known to occur in yeast other than S. cerevisiae.
  • Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces may be disrupted, deleted, or mutated.
  • the invention is directed to a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovaierate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 3-methyl-1 -butano!.
  • the enzyme catalyzing the conversion of 2 ⁇ ketoisovalerate to 2-isopropy!malate is a 2-isopropyimaiate synthase.
  • the 2-isopropy!ma!ate synthase is the S. cerevisiae Leu4 (SEQ ID NO: 2) or S. cerevisiae Leu9 (SEQ ID NO: 4) or a homolog or variant thereof. Homologs of Leu4 and Leu9 are known to occur in yeast other than S.
  • a Leu4 and/or Leu9 polypeptide derived from a yeast selected from Ajeilomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibbereila, Giomereila, Grosmannia, issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosaiiorya, Neurospora, Paracoccidioides, Peniciliiurn, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces,
  • Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticiliium, Yarrowia or Zygosaccharomyces may be disrupted, deleted, or mutated.
  • 2-isopropyimaiate synthase refers to a polypeptide having an enzymatic activity that catalyzes the conversion of 2- ketoisovalerate to 2-isopropylmalate.
  • Exemplary 2-isopropy!ma!ate synthases are found in a variety of microorganisms, e.g., S. cerevisiae (see above), £. coti LeuA and L !actis LeuA,
  • the enzyme catalyzing the conversion of 2 ⁇ isopropyimalate to 3-isopropylmaiate is a isopropy!ma!ate isomerase.
  • the isopropyimalate isomerase is the S. cerevisiae Leu1 (SEQ ID NO: 8) or a homolog or variant thereof. Homoiogs of Leu4 and Leu9 are known to occur in yeast other than S. cerevisiae.
  • a Leu4 and/or Leu9 polypeptide derived from a yeast selected from Ajeliomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaeiomium, Ciavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Lepiosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerelia, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Scierotinia, Sordaria, Taiaromyces, Trichoderma, Trichophyton, Tuber, Uncino
  • isopropyimalate isomerase refers to one or more a polypeptides having an enzymatic activity that catalyzes the conversion of 2- isopropyimaiate to 3-isopropy!malate.
  • Exemplary isopropyimalate isomerases are known as EC 4.2.1 .33 and are found in a variety of microorganisms, e.g., S. cerevisiae (see above), £. coti LeuC and LeuD and L, iactis LeuG and LeuD.
  • the enzyme catalyzing the conversion of 3- isopropylmaiate to a-ketoisocaproate is an 3-isoproyimaiate dehydrogenase.
  • the 3-isoproyimalate dehydrogenase is the S. cerevisiae Leu2 (SEQ ID NO: 8) or a homolog or variant thereof. Homoiogs of Leu2 are known to occur in yeast other than S. cerevisiae.
  • a Leu2 polypeptide derived from a yeast selected from Ajeliomyces, Arthroderma, Ashbya, Aspergillus, Botsyotinia, Candida, Chaeiomium, Ciavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Scierotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verti
  • 3-isoproylmaiate dehydrogenase reiers to a polypeptide having an enzymatic activity that catalyzes the conversion of 3 ⁇ isopropylmaiate to a-ketoisocaproate.
  • Exemplary 3-isoproylmalate dehydrogenases are known as EC 1 .1 .1 .85 and are found in a variety of microorganisms, e.g. S. cerevssiae (see above) and E. cols LeuB and L lactis LeuB.
  • the enzyme catalyzing the conversion of leucine to a-ketoisocaproate is a branched-chain amino acid transaminase.
  • the branched-chain amino acid transaminase is S. cerevssiae Bat1 (SEQ ID NO: 10) or S. cerevssiae Bat2 (SEQ ID NO: 12) or a homo!og or variant thereof.
  • Homologs of Bat1 and/or Bat2 are known to occur in yeast other than S. cerevssiae.
  • a Bat1 and/or Bat2 polypeptide derived from a yeast selected from Ajeliomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Ciavispora, Coccidioides, Debaryomyces, Gibbereila, Giomereiia, Grosmannia, issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces,
  • Schizosaccharomyces, Scierotinia, Sordaria, Talarosvyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticiliium, Yarrowsa or Zygosaccharomyces may be disrupted, deleted, or mutated.
  • branched-chain amino acid transaminase refers to a polypeptide having an enzymatic activity that catalyzes the conversion of leucine to a-ketoisocaproate.
  • exemplary branched-chain amino acid transaminases are known as EC 2.6.1 .42 and are found in a variety of microorganisms, e.g. S. cerevssiae (see above) and £. coii llvE and L lactis HvE.
  • the enzyme catalyzing the conversion of a- ketoisocaproate to 3-methy!butanal is a keto-isocaproate decarboxylase.
  • the keto-isocaproate decarboxylase is S. cerevssiae Aro10 (SEQ ID NO: 14), Ths3 (SEQ ID NO: 18) or homologs or variants thereof. Homologs of Aro10 and Thi3 are known to occur in yeast other than S. cerevisiae.
  • an Aro10 and/or Thi3 polypeptide derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberel!a, G!omere!la, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meye zyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Peniciiiium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Taiaromyces, Trichoderma, Trichophyton, Tuber,
  • keto-isocaproate decarboxylase refers to a polypeptide having an enzymatic activity that catalyzes the conversion of a- kefoisocaproate to 3-methy!butanai.
  • exemplary keto-isocaproate decarboxylases are known as EC 4.1 .1 .1 or EC 4.1 .1 .72 and are found in a variety of microorganisms, e.g. S. cerevisiae (see above) and L. lactis KivD and L. lactis KdcA.
  • alteration of keto-isocaproate decarboxylase is done so in a manner that ensures that the isobutanoi pathway genes for the enzymes converting 2-ketoisovaierate to isobutyraldehyde are not reduced or deleted.
  • the keto-isocaproate decarboxylase is altered such that its substrate specificity for a-ketoisocaproate or 2-ketoisovalerate is changed relative to a parental strain.
  • the enzyme is engineered to favor 2-ketoisovalerate over a-ketoisocaproate, for instance, when isobutanoi is being produced and 3-methyl-1 -butanoi is to be reduced.
  • the enzyme catalyzing the conversion of 3- methylbutanal to 3-methyl-1 -butanoi is an alcohol dehydrogenase.
  • the alcohol dehydrogenase is the S. cerevisiae Adh6 (SEQ ID NO: 18), Adh7 (SEQ ID NO: 20) or homologs or variants thereof, as summarized below:
  • alcohol dehydrogenase refers to a polypeptide having an enzymatic activity that catalyzes the conversion of 3-methylbutanal to 3- methyM -butanoi.
  • exemplary alcohol dehydrogenases are known as EC 1 .1 .1 .1 or EC 1 .1 .1 .2 and are found in a variety of microorganisms, e.g. S. cerevisiae Adh6 (see above) and L. iactis AdhA.
  • alteration of alcohol dehydrogenase is done so in a manner that ensures that the isobutanol pathway genes for the enzymes converting isobutyraldehyde to isobutanol are not reduced or deleted.
  • the alcohol dehydrogenase is altered such that its substrate specificity for 3-methylbutanal or isobutyraldehyde is changed relative to a parental strain.
  • the enzyme is engineered to favor isobutyraldehyde over 3-methylbutanal, for instance, when isobutanol is being produced and 3-methyl-1 -butanoi is to be reduced.
  • the invention is directed to a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovaierate as an intermediate, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the initial steps in the conversion of 2-ketoisovalerate to 3-methyl-1 -butanol.
  • the enzyme is a 2-isopropylma!ate synthase, cataiyzing the conversion of 2-ketoisovalerate to 2-isopropylmalate.
  • the 2- isopropylmaiate synthase is the S.
  • the enzyme is a branched-chain amino acid transaminase, catalyzing the conversion of leucine to a-ketoisocaproate.
  • the branched-chain amino acid transaminase is S. cerevisiae Bat1 (SEQ ID NO: 10) or S. cerevisiae Bat2 (SEQ ID NO: 12) or a homoiog or variant thereof.
  • the recombinant microorganism of the invention includes a mutation in at least one gene encoding for at least any of the enzymes mentioned herein resulting in a reduction of enzymatic activity of a polypeptide encoded by said gene.
  • the recombinant microorganism includes a partial deletion of a gene encoding for at least any of the enzymes mentioned herein resulting in a reduction of enzymatic activity of a polypeptide encoded by the gene.
  • the recombinant microorganism comprises a complete deletion of a gene encoding for at least any of the enzymes mentioned herein resulting in a reduction of enzymatic activity of a polypeptide encoded by the gene.
  • the recombinant microorganism includes a modification of the regulatory region associated with the gene encoding for at least any of the enzymes mentioned herein resulting in a reduction of expression of an enzyme polypeptide encoded by said gene.
  • the recombinant microorganism comprises a modification of a transcriptional regulator resulting in a reduction of transcription of a gene encoding for any of the enzymes mentioned herein.
  • the recombinant microorganism comprises mutations in ail genes encoding for at least any of the enzymes mentioned herein resulting in a reduction of activity of a polypeptide encoded by the gene(s).
  • homo!ogs of any of the enzymes mentioned herein in yeast other than S. cerevisiae can similarly be inactivated using the methods of the present invention. Homoiogs of the enzymes mentioned herein and methods of identifying such homoiogs are described herein.
  • the recombinant microorganisms of the present invention are engineered to produce less 3-methyl-1 -butanol than an unmodified parental microorganism.
  • the recombinant microorganism produces 3-methyl-1 -butanol from a carbon source at a carbon yield of less than about 20 percent.
  • the microorganism produces 3-methyi-1 - butanoi from a carbon source at a carbon yield of less than about 10, less than about 5, less than about 2, less than about 1 , less than about 0.5, less than about 0.1 , or less than about 0.01 percent.
  • the 3-methyl-1 -butanol derived from 2-ketoisovalerate is reduced by at least about 50% in a recombinant microorganism as compared to a parental microorganism that does not comprise a reduction or deletion of the activity or expression of one or more of the enzymes described herein.
  • the 3-methyl-1 -butanoi derived from 2-ketoisovaierate is reduced by at least about 60%, by at least about 65%, by at least about 70%, by at least about 75%, by at least about 80%, by at least about 85%, by at least about 90%, by at least about 95%, by at least about 99%, by at least about 99.9%, or by at least about 100% as compared to a parental microorganism that does not comprise a reduction or deletion of the activity or expression of one or more of the enzymes described herein.
  • the yield of a desirable fermentation product is increased in the recombinant microorganisms comprising a reduction or elimination of the activity or expression of one or more of the enzymes described herein. In one embodiment, the yield of a desirable fermentation product is increased by at least about 0.1 % as compared to a parental microorganism that does not comprise a reduction or elimination of the activity or expression of one or more of the enzymes described herein.
  • the yield of a desirable fermentation product is increased by at least about 0.5%, by at least about 1 %, by at least about 5%, or by at least about 10% as compared to a parental microorganism that does not comprise a reduction or elimination of the activity or expression of one or more of the enzymes described herein.
  • the desirable fermentation product is derived from any biosynthetic pathway in which 2- ketoisovalerate acts as an intermediate, including, but not limited to, isobutanol, valine, pantothenate, and coenzyme A pathways.
  • endogenous yeast genes coding for potential proteins with the ability to undertake any of the reactions described herein are deleted from the genome of a yeast strain comprising a biosynthetic pathway in which 2-ketoisovalerate is an intermediate. These deletion strains are compared to the parent strain by fermentation and analysis of the fermentation broth for the presence and concentration of the 3-methyl-1 -butanol by-product. In S. cerevisiae, deletions that reduce the production of the 3-methyl-1 -butanoi by-product are combined by construction of strains carrying multiple deletions. Many of these deletion strains are available commercially (for example Open Biosystems YSC1054).
  • deletion strains are transformed with a piasmid pGV2435 from which the ALS gene (e.g., the B. subtiiis alsS) is expressed under the control of the CUP1 promoter.
  • the transformants are cultivated in YPD medium containing 150 g/L glucose in shake flasks at 30°C, 75 rpm in a shaking incubator for 48 hours. After 48 hours samples from the shake flasks are analyzed by HPLC for the concentration of the 3 ⁇ methyl-1 ⁇ butanol by-product.
  • naturally occurring homoiogs of any of the enzymes described herein in yeast other than S. cerevisiae can similarly be inactivated using the methods of the present invention. These homoiogs and methods of identifying such homoiogs are described herein.
  • Another way to screen the deletion library is to incubate yeast cells with 2- ketoisovalerate or leucine and analyze the broth for the production of the 3-methyi-1 - butanol by-product.
  • An alternative approach for finding additional endogenous activity responsible for the production of the 3-methy!-1 -butano! by-product derived from 2- ketoisovalerate or from leucine is to analyze yeast strains that overexpress the genes suspected of encoding the enzyme responsible for production of the 3-methyl- 1 -butanol by-product.
  • Such strains are commercially available for many of the candidate genes listed above (for example Open Biosystems YSC3870).
  • the ORF overexpressing strains are processed in the same way as the deletion strains. They are transformed with a piasmid for ALS expression and screened for 3-methy!-1 - butanol by-product production levels.
  • strains that naturally produce low levels of 3-methyl- 1 -butanol can also have applicability for producing increased levels of desirable fermentation products that are derived from biosynthetic pathways comprising a 2- ketoisovalerate intermediate.
  • strains that naturally produce low levels of 3- methyl-1 -butanol may inherently exhibit low or undetectable levels of endogenous enzyme activity, resulting in the reduced conversion of 2-ketoisovalerate to 3-methyl- 1 -butanol or leucine to 3-methyl-1 -butanol, a trait favorable for the production of a desirable fermentation product such as isobutanol.
  • Described herein are several approaches for identifying a native host microorganism which is substantially free of activity of any of the enzymes described herein.
  • one approach to finding a host microorganism which exhibits inherently low or undetectable endogenous enzyme activity responsible for the production of 3-methyi-1 -butanol is to analyze yeast strains by incubating the yeast ceils with 2-ketoisovaierate or leucine and analyze the broth for the production of 3-methyl-1 -butanol.
  • the present invention also provides for, in some embodiments, the production of 3-methy!-1 -butanol as a product or a metabolic intermediate.
  • the recombinant microorganisms described herein are used to produce 3-methy!-1 -butanol.
  • a blend of isobutanol and 3- methyl-1 -butanoi is produced.
  • one or more enzymes required for the production of 3- methyl-1 -butanol is overexpressed, as described herein, to yield increased amounts of 3-methy!-1 -butanol.
  • the enzyme to be overexpressed is a 2- isopropy!ma!ate synthase, catalyzing the conversion of 2-ketoisovalerate to 2- isopropyimaiate.
  • the 2-isopropylmalafe synthase is the S. cerevisiae Leu4 (SEQ ID NO: 2) or the S. cerevisiae Leu9 (SEO ID NO: 4) or a homoiog or variant thereof.
  • the enzyme to be overexpressed is a isopropylmalate isomerase, catalyzing the conversion of 2-isopropylmalate to 3- isopropylrnalate.
  • the isopropylmalate isomerase is the S, cerevisiae Leu1 (SEQ ID NO: 8) or a homoiog or variant thereof.
  • the enzyme to be overexpressed is a 3-isopropylmalate dehydrogenase, catalyzing the conversion of 3-isopropyimaiate to a-ketoisocaproate.
  • the 3-isopropylmalate dehydrogenase is the S.
  • the enzyme to be overexpressed is a keto-isocaproate decarboxylase, catalyzing the conversion of a-ketoisocaproate to 3-methyibutanai.
  • the keto-isocaproate decarboxylase is the S. cerevisiae Aro10 (SEQ ID NO: 14) or the S. cerevisiae Thi3 (SEQ ID NO: 16) or homologs or variants thereof.
  • the enzyme is an alcohol dehydrogenase, catalyzing the conversion of 3-methylbutanal to 3-methyl-1 -butano!.
  • the alcohol dehydrogenase is the S. cerevisiae Adh6 (SEQ ID NO: 18) or the S. cerevisiae Adh7 (SEQ ID NO: 20) or homologs or variants thereof.
  • the overexpressed ADH may be engineered to require an NADH, as opposed to NADPH, cofactor.
  • a blend of isobutanoi and 3-methyl-1 -butanol is produced that comprises a ratio of at least about 90:10 isobutanoi to 3 ⁇ methyl-1 ⁇ butanoi.
  • a blend of isobutanoi and 3-methyi-1 -butanoi that comprises a ratio of at least about 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, or 10:90 isobutanoi to 3-methyl ⁇ 1 -butanoi is produced.
  • 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 biofuei candidate (See, e.g., commonly owned and co-pending patent publications, US 2009/0228991 , US 2010/0143997, US 201 1/0020889, US 201 1/0076733, and WO 2010/075504).
  • the present invention relates to recombinant microorganisms for producing 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 ) Acetolactate synthase (ALS), 2) Ketol-acid reductoisomerase (KARI), 3) Dihydroxy- acid dehydratase (DHAD), 4) 2-keto-acid decarboxylase, e.g., Keto-isovalerate decarboxylase (KIVD), and 5) an Alcohol dehydrogenase (ADH) ( Figure 1 ).
  • the recombinant microorganism may be engineered to overexpress one or more of these enzymes.
  • the recombinant microorganism is engineered to overexpress 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 isobutanoi producing metabolic pathway comprises seven substrate to product reactions.
  • the recombinant microorganism comprises an isobutanoi producing metabolic pathway.
  • the isobutanoi producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanoi.
  • the isobutanoi producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi.
  • the isobutanoi producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi.
  • the isobutano! producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi.
  • the isobutanoi producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanoi.
  • all of the isobutanoi producing metabolic pathway steps in the conversion of pyruvate to isobutanoi are converted by exogenously encoded enzymes.
  • one or more of the isobutanoi pathway genes encodes an enzyme that is localized to the cytosol.
  • the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least one isobutanoi pathway enzyme localized in the cytosol.
  • the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least two isobutanoi pathway enzymes localized in the cytosol.
  • the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least three isobutanoi pathway enzymes localized in the cytosol.
  • the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with at least four isobutanoi pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with five isobutanoi pathway enzymes localized in the cytosol. In yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanoi producing metabolic pathway with all isobutanoi pathway enzymes localized in the cytosol. Isobutanoi producing metabolic pathways in which one or more genes are localized to the cytosol are described in commonly owned and copending U.S. Publication No. 201 1/0078733, which is herein incorporated by reference in its entirety for all purposes.
  • isobutanoi pathway enzymes including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. iactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including V. spp.
  • Sources of genes from anaerobic fungi include, but not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp.
  • Sources of prokaryotic enzymes that are useful include, but not limited to, Escherichia spp., Zymomonas spp., Staphylococcus spp., Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., Streptococcus spp., Salmonella spp., 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.
  • acetolactate synthases capable of converting pyruvate to acetoiactate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including B, subiiiis (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. ZPJ36014957.1 ), C. glutamicum (GenBank Accession No. P42483.1 ), E, cloacae (GenBank Accession No. YP_00361361 1 .1 ), M.
  • sources e.g., bacterial, yeast, Archaea, etc.
  • sources e.g., bacterial, yeast, Archaea, etc.
  • sources e.g., bacterial, yeast, Archaea, etc.
  • B subiiiis
  • Chipman et a/ A review article characterizing the biosynthesis of acetoiactate from pyruvate via the activity of acetolactate 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 are disclosed in commonly owned and co-pending PCT Application No. PCT/US12/42824. Thus, a protein harboring one or more of these amino acid motifs can generally be expected to exhibit acetoiactate synthase activity.
  • Ketol-acid reductoisomerases capable of converting acetoiactate 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.
  • sources e.g., bacterial, yeast, Archaea, etc.
  • E. coli GenBank Accession No. EGB30597.1
  • L. lactis GenBank Accession No. YPJ3033S371 Q.1
  • S. exigua GenBank Accession No.
  • Dihydroxy acid dehydratases capabie of converting 2,3- dihydroxyisova!erate 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 iactis (GenBank Accession No. NP_267379.1 ), S. mutans (GenBank Accession No. NP__722414.1 ), M. stadtmanae (GenBank Accession No. YP__448586.1 ), M. iractuosa (GenBank Accession No.
  • YP_004053736.1 Eubacterium SCB49 (GenBank Accession No. ZP_01890126.1 ), G, forsetti (GenBank Accession No. YP__862145.1 ), Y. lipolytica (GenBank Accession No. XP__502180.2), N. crassa (GenBank Accession No. XP_963045.1 ), or S. cerevisiae ILV3 (GenBank Accession No. NP__012550.1 ). Additional dihydroxy acid dehydratases capable of 2,3-dihydroxyisovalerate to a-ketoisovalerate are described in commonly owned and co-pending US Publication No. 201 1/0076733.
  • 2-keio-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 laciis kivD (GenBank Accession No. YPJ3033S382Q.1 ), £. cloacae (GenBank Accession No.
  • Alcohol dehydrogenases capable of converting isobutyraldehyde to isobutanol may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including L lactis (GenBank Accession No. YP_003354381 ), S. cereus (GenBank Accession No. YP 001374103.1 ), N. meningitidis (GenBank Accession No. CBA03985.1 ), S. sanguinis (GenBank Accession No. YP_001035842.1 ), L, brevis (GenBank Accession No. YP__794451 .1 ), B. thuringiensis (GenBank Accession No.
  • pathway steps 2 and 5 of the isobutanol pathway may be carried out by KARI and ADH enzymes that utilize NADH (rather than NADPH) as a cofactor. It has been found previously that utilization of NADH-dependent KARI (NKR) and ADH enzymes to catalyze pathway steps 2 and 5, respectively, surprisingly enables production of isobutanol at theoretical yield and/or under anaerobic conditions.
  • 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 isobutanol.
  • 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 isobutanol.
  • the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutanol. In one embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutyraidehyde. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to keto-isovaierate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to 2,3-dihydroxyisovalerate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to 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.
  • the invention is directed to a recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovalerate to 2-isopropyimaiate; one or more enzymes catalyzing the conversion of 2-isopropyimaiate to 3-isopropylmalate; one or more enzymes catalyzing the conversion of 3-isopropylmalate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of leucine to a-ketoisocaproate; one or more enzymes catalyzing the conversion of ⁇ -ketoisocaproate to 3-methy!butanal; and one or more enzymes catalyzing the conversion of 3-methyibutana! to 3-methyl- -butanoi.
  • the recombinant microorganisms comprising an isobutanol producing metabolic pathway are engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the conversion of 2- ketoisovalerate to 3-methyl-1 -butanoI.
  • the enzyme is a 2- isopropylmalate synthase, catalyzing the conversion of 2-ketoisovalerate to 2- isopropyimaiate.
  • the 2-isopropyimaiate synthase is the S. cerevisiae Leu4 (SEO ID NO: 2) or S. cerevisiae Leu9 (SEQ ID NO: 4) or a homoiog or variant thereof.
  • the enzyme is a isopropyimalate isomerase, catalyzing the conversion of 2-isopropyimaiate to 3-isopropylmalate.
  • the isopropyimalate isomerase is the S. cerevisiae Leu1 (SEQ ID NO: 8) or a homoiog or variant thereof.
  • the enzyme is an 3- isoproyimaiate dehydrogenase, catalyzing the conversion of 3-isopropylmalate to Q- ketoisocaproate.
  • the 3-isoproylmalate dehydrogenase is the S.
  • the enzyme is a branched-chain amino acid transaminase, catalyzing the conversion of leucine to ⁇ -kefoisocaproate.
  • the branched-chain amino acid transaminase is S. cerevisiae Bat1 (SEQ ID NO: 10) or S. cerevisiae Bat2 (SEQ ID NO: 12) or a homoiog or variant thereof.
  • the enzyme is a keto-isocaproate decarboxylase, catalyzing the conversion of a-ketoisocaproate to 3-methy!butanai.
  • the keto-isocaproate decarboxylase is S. cerevisiae Aro10 (SEQ ID NO: 14), Ths3 (SEQ ID NO: 16), or homoiogs or variants thereof.
  • the enzyme is an alcohol dehydrogenase, catalyzing the conversion of 3-methylbutanal to 3-methyl-1 - butanoi.
  • the alcohol dehydrogenase is the S. cerevisiae Adh6 (SEQ ID NO: 18), Adh7 (SEQ ID NO: 20), or homoiogs or variants thereof.
  • the present invention also provides for a recombinant microorganism comprising an isobutanoi producing metabolic pathway, wherein said recombinant microorganism is engineered to reduce or eliminate the expression or activity of one or more of the following and/or substantially free of an enzyme catalyzing the conversion of one or more of the following: one or more enzymes catalyzing the conversion of 2-ketoisovaierate to 2-isopropy!malate; one or more enzymes catalyzing the conversion of 2-isopropyimaiate to 3-isopropylmalate; one or more enzymes catalyzing the conversion of 3-isopropyima!ate to a-ketoisocaproate; one or more enzymes catalyzing the conversion of leucine to a-ketoisocaproate; one or more enzymes catalyzing the conversion of a-ketoisocaproate to 3-methylbutana!; and one or more enzymes catalyzing the conversion of 3-methylbutan
  • the recombinant microorganisms are engineered to reduce or eliminate the expression or activity of one or more enzymes catalyzing the initial steps in the conversion of 2-ketoisovalerate to 3-methy!-1 -butanol.
  • the enzyme is a 2-isopropylmaiate synthase, catalyzing the conversion of 2-ketoisovaierate to 2-isopropylmalate,
  • the 2- isopropylmaiate synthase is the S. cerevisiae Leu4 (SEQ ID NO: 2) or the S. cerevisiae Leu9 (SEQ ID NO: 4) or a homolog or variant thereof.
  • the enzyme is a branched-chain amino acid transaminase, catalyzing the conversion of leucine to ⁇ -ketoisocaproate.
  • the branched-chain amino acid transaminase is S. cerevisiae Bat1 (SEQ ID NO: 10) or S. cerevisiae Bat.2 (SEQ ID NO: 12) or a homolog or variant thereof.
  • the recombinant microorganisms of the present invention can express a plurality of heterologous and/or native enzymes involved in pathways for the production of a beneficial metabolite such as isobutanoi,
  • engineered or “modified” microorganisms are produced via the introduction of genetic material into a host or parental microorganism of choice and/or by modification of the expression of native genes, thereby modifying or altering the cellular physiology and biochemistry of the microorganism, Through the introduction of genetic material and/or the modification of the expression of native genes the parental microorganism acquires new properties, e.g., the ability to produce a new, or greater quantities of, an intracellular and/or extracellular metabolite.
  • 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 isobutanoi, valine, pantothenate, and coenzyme A, from a suitable carbon source.
  • the genetic material introduced into and/or the genes modified for expression in the parental microorganism contains gene ⁇ s), or parts of genes, coding for one or more of the enzymes involved in a biosynthetic pathway for the production of one or more metabolites selected from isobutanoi, valine, pantothenate, and coenzyme A and may also include additional elements for the expression and/or regulation of expression of these genes, e.g., promoter sequences.
  • an engineered or modified microorganism can also include the alteration, disruption, deletion or knocking-out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism.
  • the microorganism acquires new or improved properties (e.g., the ability to produce a new metabolite or greater quantities of an intracellular metabolite, to improve the flux of a metabolite down a desired pathway, and/or to reduce the production of by-products).
  • Recombinant microorganisms provided herein may also produce metabolites in quantities not available in the parental microorganism.
  • a "metabolite” refers to any substance produced by metabolism or a substance necessary for or taking part in a particular metabolic process.
  • a metabolite can be an organic compound that is a starting material (e.g., glucose or pyruvate), an intermediate (e.g., 2-ketoisovaierate), or an end product (e.g., isobutanoi) 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 identify to such genes is not necessary. For example, changes in a particular gene or polynucleotide comprising a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically, such changes comprise conservative mutations and silent mutations. Such modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art.
  • Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence.
  • Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and £.
  • 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.
  • homo!ogs 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 80%, 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.
  • 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) Isoleucine (I), Leucine (L), Alanine (A), Valine (V), and 8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  • Sequence homology for polypeptides is typically measured using sequence analysis software. See commonly owned U.S. Pat. No. 8,017,375.
  • a typical algorithm used comparing a molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST. When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms described in commonly owned U.S. Pat. No. 8,017,375.
  • microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of beneficial metabolites from biosynthetic pathways requiring the use of 2-ketoisovalerate as an intermediate.
  • microorganisms may be selected from yeast microorganisms.
  • yeast microorganisms for the production of a metabolite such as isobutanoi, valine, pantothenate, and coenzyme A may be selected based on certain characteristics.
  • One characteristic may include the property that the microorganism is selected to convert various carbon sources into beneficial metabolites such as isobutanoi, valine, pantothenate, and coenzyme A.
  • carbon source generally refers to a substance suitable to be used as a source of carbon for prokaryotic or eukaryotic ceil growth. Examples of suitable carbon sources are described in commonly owned U.S. Pat. No. 8,017,375. Accordingly, in one embodiment, the recombinant microorganism herein disclosed can convert a variety of carbon sources to products, including but not limited to glucose, galactose, mannose, xylose, arabinose, lactose, sucrose, and mixtures thereof.
  • the recombinant microorganism may thus further include a pathway for the production of isobutanol from five-carbon (pentose) sugars including xylose.
  • Most yeast species metabolize xylose via a complex route, in which xylose is first reduced to xylitol via a xylose reductase (XR) enzyme. The xylitol is then oxidized to xylulose via a xylitol dehydrogenase (XDH) enzyme. The xylulose is then phosphoryiated via a xyiulokinase (XK) enzyme.
  • XR xylose reductase
  • XDH xylitol dehydrogenase
  • XK xyiulokinase
  • This pathway operates inefficiently in yeast species because it introduces a redox imbalance in the ceil.
  • the xyiose-to- xylitol step uses primarily NADPH as a cofactor (generating NADP+), whereas the xylitol-to-xylulose step uses NAD+ as a cofactor (generating NADH).
  • Other processes must operate to restore the redox imbalance within the cell. This often means that the organism cannot grow anaerobicaily on xylose or other pentose sugars. Accordingly, a yeast species that can efficiently ferment xylose and other pentose sugars into a desired fermentation product is therefore very desirable.
  • the recombinant microorganism is engineered to express a functional exogenous xylose isomerase.
  • Exogenous xylose isomerases (XI) functional in yeast are known in the art. See, e.g., Rajgarhia et ai., U.S. Pat. No. 7,943,366, which is herein incorporated by reference in its entirety.
  • the exogenous XI gene is operatively linked to promoter and terminator sequences that are functional in the yeast cell.
  • the recombinant microorganism further has a deletion or disruption of a native gene that encodes for an enzyme (e.g., XR and/or XDH) that catalyzes the conversion of xylose to xylitol.
  • the recombinant microorganism also contains a functional, exogenous xyiulokinase (XK) gene operatively linked to promoter and terminator sequences that are functional in the yeast ceil.
  • XK xyiulokinase
  • the yeast microorganism has reduced or no pyruvate decarboxylase (PDC) activity.
  • PDC catalyzes the decarboxylation of pyruvate to acetaldehyde, which is then reduced to ethanol by ADH via an oxidation of NADH to NAD+.
  • Ethanol production is the main pathway to oxidize the NADH from glycolysis. Deletion, disruption, or mutation of this pathway increases the pyruvate and the reducing equivalents (NADH) available for a biosynthetic pathway which uses pyruvate as the starting material and/or as an intermediate.
  • NADH reducing equivalents
  • deletion, disruption, or mutation of one or more genes encoding for pyruvate decarboxylase and/or a positive transcriptional regulator thereof can further increase the yield of the desired pyruvate-derived metabolite ⁇ e.g., isobutanoi).
  • said pyruvate decarboxylase gene targeted for disruption, deletion, or mutation is selected from the group consisting of PDC1, PDC5, and PDC6, or homologs or variants thereof.
  • all three of PDC1, PDC5, and PDC6 are targeted for disruption, deletion, or mutation.
  • a positive transcriptional regulator of the PDC1, PDC5, and/or PDC6 is targeted for disruption, deletion or mutation.
  • said positive transcriptional regulator is PDC2, or homologs or variants thereof,
  • the microorganism has reduced giycerol-3- phosphate dehydrogenase (GPD) activity.
  • GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD+.
  • DHAP dihydroxyacetone phosphate
  • G3P glycerol-3-phosphate
  • Glycerol is then produced from G3P by Glycerol-3- phosphatase (GPP).
  • Glycerol production is a secondary pathway to oxidize excess NADH from glycolysis. Reduction or elimination of this pathway would increase the pyruvate and reducing equivalents (NADH) available for the production of a pyruvate-derived metabolite (e.g., isobutanoi).
  • NADH pyruvate and reducing equivalents
  • 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.
  • the yeast microorganisms may be selected from the "Saccharomyces Yeast C!ade", as described in commonly owned U.S. Pat. No. 8,017,375.
  • Saccharomyces sensu stricto taxonomy group is a cluster of yeast species that are highly related to S. cerevssiae (Rainier! et ai., 2003, J. Biosci Bioengin 96: 1 -9). Saccharomyces sesisu stricto yeast species include but are not limited to S. cerevssiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarurn, S. carocanis and hybrids derived from these species (Masneuf et ai., 1998, Yeast 7: 61 - 72).
  • the yeast microorganism may be selected from a post-WGD yeast genus, including but not limited to Saccharomyces and Candida.
  • the favored post-WGD yeast species include: S. cerevisiae, S. uvarurn, S. bayanus, S. paradoxus, S. casie!li, 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.
  • pre-WGD yeast species inciude S. kluyveri, K. thermotolerans, K. marxianus, K. waltii, K. iactis, C. tmpicalis, P. pastoris, P, anoma!a, 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 as described in described in commonly owned U.S. Pat. No. 8,017,375.
  • the yeast microorganism may be selected from yeast with a Crabtree-negative phenotype including but not limited to the following genera: Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida.
  • Crabtree-negative species include but are not limited to: S. kluyveri, K. Iactis, K. marxianus, P. anomala, P. stipitis, I. orientaiis, I. occidentalis, i scutulata, H.
  • the yeast microorganism may be selected from yeast with a Crabtree-positive phenotype, including but not limited to Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Pichia and Schizosaccharomyces.
  • Crabtree-positive yeast species include but are not limited to: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S. castelli, K, thermotolerans, C. glabrata, Z. basils ' , Z. rouxii, D. hansenii, P, pastorius, and S. pombe.
  • Another characteristic may include the property that the microorganism is that it is non-fermenting. In other words, it cannot metabolize a carbon source anaerobicaliy while the yeast is able to metabolize a carbon source in the presence of oxygen.
  • Nonfermenting yeast refers to both naturally occurring yeasts as well as genetically modified yeast.
  • Ethanol is produced by alcohol dehydrogenase (ADH) via the reduction of acetaidehyde, which is generated from pyruvate by pyruvate decarboxylase (PDC).
  • 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 isobutanoi pathway. Deletion of this pathway increases the pyruvate and the reducing equivalents available for the biosynthetic pathway.
  • Fermentative pathways contribute to low yield and low productivity of pyruvate-derived metabolites such as isobutanol. Accordingly, deletion of one or more PDC genes may increase yield and productivity of a desired metabolite ⁇ e.g., isobutanol).
  • the recombinant microorganisms may be microorganisms that are non-fermenting yeast microorganisms, including, but not limited to those, classified into a genera selected from the group consisting of Tricosporon, Rhodotorula, Myxozyma, or Candida.
  • the non-fermenting yeast is C. xestobii.
  • the recombinant microorganisms may be derived from bacterial microorganisms.
  • the recombinant microorganism may be selected from a genus of Citrobacter, Corynebacterium, Lactobacillus, Lactococcus, Salmonella, Enterobacter, Enterococcus, Erwinia, Pantoea, Morganella, Pectobacterium, Proteus, Serratia, Shigella, and Klebsiella.
  • the recombinant microorganism is a Iactic acid bacteria such as, for example, a microorganism derived from the Lactobacillus or Lactococcus genus.
  • Any method can be used to identify genes that encode for enzymes that are homologous to the genes described herein ⁇ e.g., 2-isopropyImalate synthase homoiogs, isopropyimaiate isomerase homoiogs, 3-isoproyimaiate dehydrogenase homoiogs, branched-chain amino acid transaminase homoiogs, keto-isocaproate decarboxylase homoiogs, and alcohol dehydrogenase homoiogs, etc.).
  • genes that are homologous or similar to the 2-isopropyimalate synthases, isopropyimaiate isomerases, 3-isoproy!maiate dehydrogenases, branched-chain amino acid transaminases, keto-isocaproate decarboxylases, and alcohol dehydrogenases described herein may be identified by functional, structural, and/or genetic analysis. In most cases, homologous or similar genes and/or homologous or similar enzymes will have functional, structural, or genetic similarities.
  • Techniques 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. Branched- Chain Amino Acids Methods Enzymoiogy, 1970), then isolating the enzyme with said activity through purification, determining the protein sequence of the enzyme through techniques such as Edman degradation, design of PGR primers to the likely nucleic acid sequence, amplification of said DNA sequence through PGR, and cloning of said nucleic acid sequence.
  • analogous genes and/or analogous enzymes or proteins techniques also include comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC,
  • the candidate gene or enzyme may be identified within the above mentioned databases in accordance with the teachings herein.
  • HMM Hidden Markov Model
  • profile HMM is statistical descriptions of the consensus of a multiple sequence alignment. They use position-specific scores for amino acids (or nucleotides) and position specific scores for opening and extending an insertion or deletion. Compared to other profile based methods, HMMs have a formal probabilistic basis. Profile HMMs for a large number of protein families are publicly available in the PFAM database (Janelia Farm Research Campus, Ashburn, Va.).
  • any homologous protein that matches the Profile HMM with an E value of ⁇ 10 "3 using hmmsearch program in the HMMER package is expected to be a functional homoiog.
  • the present invention provides in some embodiments, microorganisms with the reduced expression and/or activity of 2-isopropylmalate synthase homologs, isopropylmalate isomerase homologs, 3-isoproylmalate dehydrogenase homologs, branched-chain amino acid transaminase homologs, keto-isocaproate decarboxylase homologs, and alcohol dehydrogenase homologs, wherein said homologs have an MM search profile E value of ⁇ 10 "3 using the hmmsearch program.
  • the endogenous nucleic acid or polypeptide identified herein is the S. cerevisiae version of the nucleic acid or polypeptide ⁇ e.g., Leu4, Leu9, Leu1 , Leu2, Bat1 , Bat2, Aro10, Thi3, Adh8, Adh7, etc.). Any method can be used to identify genes that encode for the endogenous polypeptide of interest in a variety of yeast strains. Generally, genes that are homologous or similar to the endogenous polypeptide of interest can be identified by functional, structural, and/or genetic analysis. Homologous or similar polypeptides will generally have functional, structural, or genetic similarities.
  • the chromosomal location of the genes encoding endogenous S. cerevisiae polypeptides may be syntenic to chromosomes in many related yeast [Byrne, K.P. and K. H. Wolfe (2005) "The Yeast Gene Order Browser: combining curated homology and syntenic context reveals gene fate in polyploid species.” Genome Res. 15(10):1458-61 . Scannell, D. R., K, P. Byrne, J. L. Gordon, S. Wong, and K. H.
  • yeast including but not limited to, Ashbya gossypii, Candida glabrata, Kiuyveromyces lactis, Kiuyveromyces polyspora, Kiuyveromyces thermotolerans, Kiuyveromyces waitii, Saccharomyces kiuyveri, Saccharomyces casteiii, Saccharomyces bayanus, and Zygosaccharomyces rouxii.
  • this technique is therefore additionally suitable for the identification homologous ⁇ e.g., Leu4, Leu9, Leu1 , Leu2, Bati , Bat2, Aro10, Thi3, Adh8, Adh7, etc.) polypeptides in yeast other than S. cerevisiae. Genetic insertions and Deletions
  • 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 ai., 1992, Nuc Acids Res. 27: 69-74; Ito et ai, 1983, J. Bacterioi. 153: 183-8; and Becker et al., 1991 , Methods in Enzymo!ogy 194: 182-7,
  • the integration of a gene of interest into a DNA fragment or target gene of a yeast microorganism occurs according to the principle of homologous recombination.
  • an integration cassette containing a module comprising at least one yeast marker gene and/or the gene to be integrated is flanked on either side by DNA fragments homologous to those of the ends of the targeted integration site (recombinogenic sequences).
  • recombinogenic sequences recombinogenic sequences
  • 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, hie, hph, and kan.
  • the recombinogenic sequences can be chosen at will, depending on the desired integration site suitable for the desired application.
  • integration of a gene into the chromosome of the yeast microorganism may occur via random integration (Kooistra et ai., 2004, Yeast 21 : 781 -792).
  • URA3 marker loss can be obtained by plating URA3 containing cells in FOA (5 ⁇ fluoro ⁇ orotic acid) containing medium and selecting for FOA resistant colonies (Boeke et al., 1984, MoL Gen. Genet 197: 345-47).
  • exogenous nucleic acid molecule contained within a yeast cell of the disclosure can be maintained within that ceil in any form,
  • exogenous nucleic acid molecules can be integrated into the genome of the cell or maintained in an episoma! state that can stably be passed on ("inherited") to daughter cells.
  • extra-chromosomal genetic elements such as piasmids, mitochondrial genome, etc.
  • the yeast cells can be stably or transiently transformed, !n addition, 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, for example, reduced 2-isopropylmalafe synthase, isopropylmalate isomerase, 3-isoproylmaiate dehydrogenase, branched-chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, or GPD activity.
  • reduced as used herein with respect to a particular enzymatic activity refers to a lower level of enzymatic activity than that measured in a comparable yeast cell of the same species.
  • yeast cells lacking 2-isopropylmalate synthase, isopropylmalate isomerase, 3-isoproylmalate dehydrogenase, branched- chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, or GPD activity are considered to have reduced 2-isopropylmaiate synthase, isopropylmalate isomerase, 3-isoproylmalate dehydrogenase, branched-chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, or GPD activity since most, if not ail, comparable yeast strains have at least some 2-isopropylmalate synthase, isopropyl
  • 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, et a!., Cold Spring Harbor Press (1998).
  • certain poinf- mutation(s) can be introduced which results in an enzyme with reduced activity.
  • yeast strains which when found in nature, are substantially free of one or more activities selected from 2- isopropyimaiate synthase, isopropylmalate isomerase, 3-isoproylmalate dehydrogenase, branched-chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, or GPD activity.
  • anfisense 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.
  • yeasts having a reduced enzymatic activity can be identified using many methods. For example, yeasts having reduced 2-isopropyimalate synthase, isopropyimaiate isomerase, 3-isoproylmaiate dehydrogenase, branched-chain amino acid transaminase, keto-isocaproate decarboxylase, alcohol dehydrogenase, 3-KAR, ALDH, PDC, or GPD activity can be easily identified using common methods, which may include, for example, measuring for the formation of the by-products produced by such enzymes via liquid 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 a/., Gene 190(1 ):87-97 (1997) for Saccharomyces).
  • Yeast piasmids have a selectable marker and an origin of replication.
  • certain piasmids may also contain a centromeric sequence. These centromeric piasmids are generally a single or low copy plasmid.
  • Piasmids without a centromeric sequence and utilizing either a 2 micron (S. cerevisiae) or 1 .8 micron (K. lactls) replication origin are high copy piasmids.
  • the selectable marker can be either prototrophic, such as HIS3, TRP1, Leu2, URA3 or ADE2, or antibiotic resistance, such as, bar, ble, hph, or kan,
  • heterologous control elements can be used to activate or repress expression of endogenous genes. Additionally, when expression is to be repressed or eliminated, the gene for the relevant enzyme, protein or RNA can be eliminated by known deletion techniques,
  • any yeast within the scope of the disclosure can be identified by selection techniques specific to the particular enzyme being expressed, over-expressed or repressed. Methods of identifying the strains with the desired phenotype are well known to those skilled in the art. Such methods include, without limitation, PGR, RT-PCR, and nucleic acid hybridization techniques such as Northern and Southern analysis, altered growth capabilities on a particular substrate or in the presence of a particular substrate, a chemical compound, a selection agent and the like. 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 ceil contains that encoded enzyme.
  • biochemical techniques can be used to determine if a ceil 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 aceto!aciate synthase and detecting increased acetoiactate 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 acetoiactate can be determined as described by Hugenho!tz and Starrenburg, 1992, Appl. Micro. Blot. 38:17-22.
  • Yeast microorganisms of the invention may be further engineered to have increased activity of enzymes (e.g., increased activity of enzymes involved in an isobutanoi producing metabolic pathway).
  • increased activity of enzymes e.g., increased activity of enzymes involved in an isobutanoi producing metabolic pathway.
  • the term "increased” as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured in a comparable yeast ceil of the same species. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Increased activities for enzymes involved in glycolysis or the isobutanoi pathway would result in increased productivity and yield of isobutanoi.
  • Methods to increase enzymatic activity are known to those skilled in the art. Such techniques may include increasing the expression of the enzyme by increased copy number and/or use of a strong promoter, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the 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 only product produced is the desired metabolite, as extra products (i.e. by-products) lead to a reduction in the yield of the desired metabolite and an increase in capital and operating costs, particularly if the extra products have little or no value. These extra products also require additional capital and operating costs to separate these products from the desired metabolite.
  • the present invention provides a method of producing a beneficial metabolite derived from a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovalerate as an intermediate in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the beneficial metabolite is produced.
  • the present invention provides a method of producing a isobutanol derived from a recombinant microorganism comprising a biosynthetic pathway which uses 2-ketoisovaierate as an intermediate in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the isobutanol is produced
  • said recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of 2- ketoisovalerate to 2-isopropylma!ate.
  • the recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of 2-isopropylmalate to 3-isopropylmaiate.
  • the recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of 3- isopropylmaiate to a-ketoisocaproate.
  • the recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of leucine to a-ketoisocaproate.
  • the recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of a- ketoisocaproate to 3-methyibutanal. In yet another embodiment, the recombinant microorganism is engineered to reduce or eliminate the expression or activity of an enzyme catalyzing the conversion of 3-methylbutanal to 3-methy!-1 -butanol.
  • the beneficial metabolite may be derived from any biosynthetic pathway which uses 2-ketoisovalerate as intermediate, including, but not limited to, biosynthetic pathways for the production of isobutanol, valine, pantothenate, and coenzyme A.
  • the beneficial metabolite is isobutanol.
  • 3-methyl-1 -butanol may be desired. Further, it may be desired to produce a blend of 3-methyl-1 -butanol and a beneficial metabolite. Further still, it may be desired to produce a blend of 3-methyl-1 -butanol and isobutanol.
  • a method of producing isobutanol and 3-methyi-1 -butanol comprising cultivating a recombinant microorganism described above and herein in a culture medium containing a feedstock providing the carbon source until a recoverable quantity of the isobutanoi and 3-methyl-1 -butano! is produced, is provided.
  • a blend product of isobutanoi and 3-methyl-1 - butanol is produced.
  • a blend of isobutanoi and 3-methyl-1 - butanoi is produced that comprises a ratio of at least about 90:10 isobutanoi to 3- methyl-1 -butanoi.
  • a blend of isobutanoi and 3-methyl-1 - butanoi that comprises a ratio of at least about 80:20, 70:30, 80:40, 50:50, 40:60, 30:70, 20:80, or 10:90 isobutanoi to 3-methyi is produced.
  • the yeast microorganism is cultured in an appropriate culture medium containing a carbon source.
  • the method further includes isolating the beneficial metabolite 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.
  • the recombinant microorganism may produce the beneficial metabolite from a carbon source at a yield of at least 5 percent theoretical.
  • the microorganism may produce the beneficial metabolite from a carbon source at a yield of at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 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.
  • DDG generally refers to the solids remaining after a fermentation, usually consisting of unconsumed feedstock solids, remaining nutrients, protein, fiber, and oil, as well as spent yeast biocatalysts or cell debris therefrom that are recovered by further processing from the fermentation, usually by a solids separation step such as centrifugation.
  • Distillers dried grains may also include soluble residual material from the fermentation, or syrup, and are then referred to as "distillers dried grains and solubles" (DDGS).
  • DDGS soluble residual material from the fermentation, or syrup
  • Use of DDG or DDGS as animal feed is an economical use of the spent biocatalyst following an industrial scale fermentation process.
  • the present invention provides an animal feed product comprised of DDG derived from a fermentation process for the production of a beneficial metabolite (e.g., isobutanol), wherein said DDG comprise a spent yeast biocatalyst of the present invention.
  • said spent yeast biocatalyst has been engineered to comprise an isobutanol producing metabolic pathway.
  • 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.
  • Media Medium used is standard yeast medium (see, for example Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3rd ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press and Guthrie, C. and Fink, G.R. eds. Methods in Enzymoiogy Part B: Guide to Yeast Genetics and Molecular and Cell Biology 350:3-823 (2002)).
  • YP medium contains 1 % (w/v) yeast extract, 2% (w/v) peptone.
  • YPD is YP containing 2% glucose unless specified otherwise.
  • YPE is YP containing 25 mL/L ethanoi.
  • SC medium is 8.7 g/L DifcoTM Yeast Nitrogen Base, 14g/L SigmaTM Synthetic Dropout Media supplement (includes amino acids and nutrients excluding histidine, tryptophan, uracil, and leucine), 0.076 g/L histidine, 0.078 g/L tryptophan, 0.380 g/L leucine, and 0.078 g/L uracil.
  • SCD is SC containing 2% (w/v) glucose unless otherwise noted. Drop-out versions of SC and SCD media are made by omitting one or more of histidine (-H), tryptophan (-W), leucine (-L), or uracil (-U). Solid versions of the above described media contain 2% (w/v) agar.
  • Cloning techniques Standard molecular biology methods for cloning and plasmid construction are generally used, unless otherwise noted (Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).
  • Cloning techniques included digestion with restriction enzymes, PGR to generate DNA fragments (KOD Hot Start Polymerase, Cat# 71086, Merck, Darmstadt, Germany), ligations of two DNA fragments using the DNA Ligation Kit (Mighty Mix Cat# TAK 6023, Clontech Laboratories, Madison, Wl), and bacterial transformations into competent E.coii cells (Xtreme Efficiency DH5a Competent Cells, Cat# ABP-CE-CC02098P, Allele Biotechnology, San Diego, CA). Plasmid DNA is purified from E. coil ceils using the Qiagen QIAprep Spin Miniprep Kit (Cat# 27106, Qiagen, Valencia, CA). DNA is purified from agarose gels using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, CA; Catalog #04002) according to manufacturer's protocols.
  • Colony PGR Yeast colony PGR uses the FailSafeTM PGR System (EPICENTRE ⁇ Biotechnologies, Madison, Wl; Catalog #FS99250) according to manufacturer's protocols.
  • a PGR cocktail containing 15 pL of Master Mix E buffer, 10.5 pL water, 2 pL of each primer at 10 pM concentration, 0.5 pL polymerase enzyme mix from the kit is added to a 0.2 mL PGR tube for each sample (30 pL each).
  • For each candidate a sma!i amount of cells is added to the reaction tube using a sterile pipette tip. Presence of the positive PGR product is assessed using agarose gel electrophoresis.
  • SOE PGR The PGR reactions are incubated in a thermocycler using the following PCR conditions: 1 cycle of 94°C x 2 min, 35 cycles of 94°C x 30 s, 53°C x 30 s, 72°C x 2 min and 1 cycle of 72°C x 10 min.
  • a master mix is made such that each reaction contained the following: 3 pL MgSG 4 (25 mM), S pL 10X KOD buffer, 5 pL 50% DMSO, 5 pL dNTP mix (2 mM each), 1 pL KOD, 28 pL dH 2 O, 1 .5 pL forward primer (10 ⁇ ), 1 .5 pL reverse primer (10 pM), 0.5 pL template (plasmid or genomic DNA).
  • Genomic DNA Isolation The Zymo Research ZR Fungal/Bacterial DNA Kit (Zymo Research Orange, CA; Catalog #06005) is used for genomic DNA isolation according to manufacturer's protocols with the following modifications. Following resuspension of pellets, 200 pL is transferred to 2 separate ZR BashingBeadTM Lysis Tubes (to maximize yield). Following lysis by bead beating, 400 pL of supernatant from each of the ZR BashingBeadTM Lysis Tubes is transferred to 2 separate Zymo-SpinTM IV Spin Filters and centrifuged at 7,000 rpm for 1 min.
  • S. cerevisiae Transformations S. cerevisiae strains are grown in YPD containing 1 % ethanoi. Transformation-competent cells are prepared by resuspension of S. cerevisiae cells in 100 mM lithium acetate. Once the cells are prepared, a mixture of DNA (final volume of 15 pL with sterile water), 72 pL 50% PEG, 10 pL 1 M lithium acetate, and 3 pL of denatured salmon sperm DNA (10 mg/mL) is prepared for each transformation.
  • GC Gas Chromatography
  • Analysis of volatile organic compounds, including isobutanol and 3-methy!-1 -butanol is performed on a Agilent 5890/6890/7890 gas chromatograph fitted with an Agilent 7673 Autosampier, a ZB- FFAP column (J&W; 30 m length, 0.32 mm ID, 0.25 ⁇ film thickness) or equivalent connected to a flame ionization detector (FID).
  • the temperature program is as follows: 200°C for the injector, 300°C for the detector, 100°C oven for 1 minute, 70 c C/minute gradient to 230°C, and then hold for 2.5 min. Analysis is performed using authentic standards (>99%, obtained from Sigma-Aldrich, and a 5-point calibration curve with 1 -pentanoi as the internal standard.
  • High Performance Liquid Chromatography Analysis of organic acid metabolites including glucose is 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 are detected using an Agilent 1 100 or equivalent UV detector (210 rim) and a refractive index detector. The column temperature is 60°C. This method is isocratic with 0.0180 N H2SO4 in Miili-Q water as mobile phase. Flow is set to 1 .1 mL/min. Injection volume is 20 pL and run time is 16 min. Quantitation of organic acid metabolites is performed using a 5 ⁇ point calibration curve with authentic standards (>99% or highest purity available).
  • Example 1 Deletion of Enzymes Used in the Production of 3-methyl-1 -butanoi
  • strains in which Leu4 has been eliminated are selected on agar plated containing defined medium lacking uracil. Replacement of Leu4 in the genome GEV03991 is confirmed with PGR with sets of primers designed to verify the absence of Leu4 and the presence of the genetic marker. A confirmed strain is spread onto plates containing 5-FOA to select for loss of the URA3-marker gene to recycle the marker, and the resulting strain is designated GEVO###2 (Table 2).
  • Elimination of Leu9 from the S. cerevisiae strain GEV03991 is accomplished by transformation with a PGR product generated with primers designed to target and replace the Leu Aocus with a genetic marker (URA3) flanked with a repeat sequence (TS C _CYCI) that enables the strain to grow in medium lacking uracil.
  • Primers to amplify the genetic marker sequence targeted for the Leu9-locus are designed with > 40 bp sequence homologous to the region immediately upstream and downstream of the open reading frame of Leu9.
  • the PGR product is introduced into GEV03991 following transformation protocols described. Following transformation, strains in which Leu9 has been eliminated are selected on agar plated containing defined medium lacking uracil.
  • Elimination of Leu2 from the S. cerevisiae strain GEV03991 is accomplished by transformation with a PGR product generated with primers designed to target and replace the Leu2- ⁇ ocus with a genetic marker (URA3) flanked with a repeat sequence (TS C _CYCI) that enables the strain to grow in medium lacking uracil.
  • Primers to amplify the genetic marker sequence targeted for the Let/2 ⁇ locus are designed with > 40 bp sequence homologous to the region immediately upstream and downstream of the open reading frame of Leu2.
  • the PGR product is introduced into GEV03991 following transformation protocols described. Following transformation, strains in which Leu2 has been eliminated are selected on agar plated containing defined medium lacking uracil.
  • strains in which Leu1 has been eliminated are selected on agar plated containing defined medium lacking uracil.
  • Replacement of Leu1 in the genome GEVO3991 is confirmed with PGR with sets of primers designed to verify the absence of Leu1 and the presence of the genetic marker.
  • a confirmed strain is spread onto plates containing 5-FOA to select for loss of the URA3-marker gene to recycle the marker, and the resulting strain is designated GEVO#####5 (Table 2).
  • Elimination of Bat2 from the S. cerevisiae strain GEV03991 is accomplished by transformation with a PGR product generated with primers designed to target and replace the Bat2- ⁇ ocus with a genetic marker (URA3) flanked with a repeat sequence (T SC _CYCI ) that enables the strain to grow in medium lacking uracil.
  • Primers to amplify the genetic marker sequence targeted for the Saf2-locus are designed with > 40 bp sequence homologous to the region immediately upstream and downstream of the open reading frame of Bat2.
  • the PGR product is introduced into GEV03991 following transformation protocols described. Following transformation, strains in which Bat2 has been eliminated are selected on agar plated containing defined medium lacking uracil.
  • a fermentation is performed to determine the amount of isobutanol and 3- methyl-1 -butanoi produced by strains in which a gene or set of genes encoding activities required for production of 3-methyl-1 -butanol production are eliminated
  • Fermenters are operated for an appropriate duration of time under conditions conducive to isobutanol production. Periodically, samples (1 .5 mL) from each fermenter are removed. A portion of each sample is used to determine cell density
  • This example describes overexpression of genes encoding activities required for production of 3-methyl-1 -butanol, including Leu4, LeuQ, Leu2, Leu1,
  • One or more of the following genes are cloned into an appropriate vector that also contains a selectable marker (e.g. URA3) under a promoter that drives transcription to the desired levels: Leu4, Leu9, Leu2, Leu1, Bat1, and/or Bat2,
  • the resulting vector(s) are transformed into GEV03991 (Table 2) as described.
  • a fermentation is performed to determine the amount of isobutanol and 3- methyl-1 -butanoi produced by strains in which a gene or set of genes encoding activities required for production of 3-methyl-1 -butanol production are overexpressed in the isobutano!-production strain, GEV03991 (Table 2).
  • GEV03991 Table 2
  • single isolate cell colonies are transferred to 500 rnL baffled flasks containing 80 mL of YPD containing appropriate medium and incubated at 30°C under temperature and agitation conditions conducive to generate sufficient biomass.
  • the flask cultures are transferred to individual fermenter vessels. Fermenters are operated for an appropriate duration of time under conditions conducive to isobutanol production.
  • samples (1 .5 mL) from each fermenter are removed. A portion of each sample is used to determine cell density (OD 6 oo), and the remainder of each sample is transferred into 1 .5 mL tubes and centrifuged in a microcentrifuge for 10 min at 18,000xg. The supernatants are analyzed by gas chromatography (GC) analysis to determine of the amount of isobutanol and 3-methy!-1 -butanol produced.
  • GC gas chromatography
  • the GEVO6014A/et/4, GEVO6014A/ey9, and GEVO6014 strains were inoculated for overnight growth at 30°C/250 rpm. These strains harbor an engineered isobutanol producing pathway comprising the B, subtilis a!sS gene, an engineered variant of E. coii HvC (EcJ!vC__coScP2D1-A1 , described in commonly-owned US Patent No. 8,097,440), the L lactis IlvD gene, the L.
  • lactis kivD gene and an engineered variant of L lactis adhA gene (U__adhA R£1 , described in commonly owned US Patent Publication No. 201 10201072).
  • Cells were harvested approximately 48 hrs later when the cell density was roughly Cells were transferred into 50 ml Falcon tubes .

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Abstract

La présente invention concerne des micro-organismes recombinants comprenant des voies de biosynthèse et des procédés d'utilisation desdits micro-organismes recombinants pour produire différents métabolites bénéfiques. Dans différents aspects de l'invention, les micro-organismes recombinants peuvent comprendre en outre une ou plusieurs modifications conduisant à la réduction ou l'élimination d'un sous-produit dérivé de 2-cétoisovalérate tel que le 3-méthyl-1-butanol. Les micro-organismes recombinants peuvent être des micro-organismes du clade Saccharomyces, des micro-organismes de levure Crabtree négatif, des micro-organismes de levure Crabtree positif, des micro-organismes de levure post-WGD (duplication du génome total), des micro-organismes de levure pré-WGD (duplication du génome total), et des micro-organismes de levure non fermenteurs.
PCT/US2012/044563 2011-06-28 2012-06-28 Ajustement fin de sous-produits d'alcool de fusel pendant la production d'isobutanol par des micro-organismes recombinants WO2013003545A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9840724B2 (en) 2012-09-21 2017-12-12 Butamax Advanced Biofuels Llc Production of renewable hydrocarbon compositions
US10604774B2 (en) 2012-09-21 2020-03-31 Butamax Advanced Biofuels Llc Production of renewable hydrocarbon compositions
JP2016032436A (ja) * 2014-07-31 2016-03-10 ヤマサ醤油株式会社 不快臭を生成しないチゴサッカロミケス属酵母

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