Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
  • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/16—Butanols
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01044—Phosphogluconate dehydrogenase (decarboxylating) (1.1.1.44)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• Recombinant microorganisms and methods of producing such microorganisms are provided. Also provided are methods of producing beneficial metabolites derived from pyruvate by contacting a suitable substrate with the recombinant microorganisms and enzymatic preparations therefrom.
• Wild-type yeasts such as Saccharomyces cerevisiae produce ethanol from pyruvate via the activity of an endogenous enzymatic conversion mediated by pyruvate decarboxylase (PDC).
• PDC pyruvate decarboxylase
• the present inventors have identified a number of genetic modifications which allow PDC-deficient mutants to exhibit C2- supplement-independence and tolerance to high glucose concentrations.
• the resulting yeast strains exhibit improved growth characteristics, allowing for more economical production of desired pyruvate-derived metabolites ⁇ e.g., isobutanol).
• PDC-deficient yeast strains to (a) improve growth in the absence of C2-supplementation; and/or (b) improve tolerance to high glucose concentrations.
• the present application relates to a recombinant yeast microorganism comprising a metabolic pathway for the production of a pyruvate- derived metabolite, wherein said metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the production of the pyruvate-derived metabolite, wherein said recombinant yeast microorganism is engineered to comprise reduced PDC activity, and wherein said recombinant yeast microorganism is engineered to (a): comprise at least one first modification which results in improved growth in the absence of C2-supplementation as compared to the corresponding recombinant yeast microorganism without said first modification; and/or (b) comprise at least one second modification which results in improved tolerance to glucose as compared to the corresponding recombinant yeast microorganism without said second modification.
• the present application relates to a recombinant yeast microorganism comprising a metabolic pathway for the production of a pyruvate- derived metabolite, wherein said metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the production of the pyruvate-derived metabolite, wherein said recombinant yeast microorganism exhibits reduced PDC activity, and wherein said recombinant yeast microorganism is engineered to (a): comprise at least one first modification which results in improved growth in the absence of C2-supplementation as compared to the corresponding recombinant yeast microorganism without said first modification; and/or (b) comprise at least one second modification which results in improved tolerance to glucose as compared to the corresponding recombinant yeast microorganism without said second modification.
• said first modification is a mutation of GND1 (SEQ ID NO: 1 ) or homologs and variants thereof.
• said first modification is a point mutation of GND1.
• said first modification is a point mutation of GND1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of L340 of Gndlp (SEQ ID NO: 2).
• said first modification is a point mutation of GND1 which results in an amino acid substitution of L340 of Gndl p, wherein said L340 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of GND1 which results in an amino acid substitution of L340 of Gndlp, wherein said L340 residue is replaced with a tryptophan residue.
• said first modification is an increase in the heterologous or native expression of a Gnd1 protein. Accordingly, in one embodiment, said first modification is the overexpression of one or more polynucleotides encoding one or more Gnd1 proteins or homologs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Gnd1 proteins or homologs thereof is an endogenous polynucleotide. In another embodiment, one or more of the polynucleotides encoding said one or more Gnd1 proteins or homologs thereof is an exogenous polynucleotide.
• the Gnd1 protein is selected from SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , and SEQ ID NO: 22.
• the Gnd1 protein is a mutant Gnd1 protein.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L340 residue of Gndlp (SEQ ID NO: 2), wherein said L340 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L340 residue of Gndl p (SEQ ID NO: 2), wherein said L340 residue is replaced with a tryptophan residue.
• the mutant Gnd1 protein comprises the amino acid sequence of SEQ ID NO: 23.
• the present application is directed to an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 23.
• said first modification is a mutation of GND2 (SEQ ID NO: 24) or homologs and variants thereof.
• said first modification is a point mutation of GND2.
• said first modification is a point mutation of GND2 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of L343 of Gnd2p (SEQ ID NO: 25).
• said first modification is a point mutation of GND2 which results in an amino acid substitution of L343 of Gnd2p, wherein said L343 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of GND2 which results in an amino acid substitution of L343 of Gnd2p, wherein said L343 residue is replaced with a tryptophan residue.
• said first modification is an increase in the heterologous or native expression of a Gnd2 protein. Accordingly, in one embodiment, said first modification is the overexpression of one or more polynucleotides encoding one or more Gnd2 proteins or homologs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Gnd2 proteins or homologs thereof is an endogenous polynucleotide. In another embodiment, one or more of the polynucleotides encoding said one or more Gnd2 proteins or homologs thereof is an exogenous polynucleotide.
• the Gnd2 protein is selected from SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
• the Gnd2 protein is a mutant Gnd2 protein.
• the mutant Gnd2 protein comprises an amino acid substitution at an amino acid position corresponding to the L343 residue of Gnd2p (SEQ ID NO: 25), wherein said L343 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the mutant Gnd2 protein comprises an amino acid substitution at an amino acid position corresponding to the L343 residue of Gnd2p (SEQ ID NO: 25), wherein said L343 residue is replaced with a tryptophan residue.
• the mutant Gnd2 protein comprises the amino acid sequence of SEQ ID NO: 28.
• the present application is directed to an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 28.
• said first modification is a mutation, disruption, or deletion of UME6 (SEQ ID NO: 3). In one embodiment, said first modification is a mutation of UME6. In another embodiment, said first modification is a point mutation of UME6. In a specific embodiment, said first modification is a point mutation of UME6 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of R768 of Ume6p (SEQ ID NO: 4).
• said first modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a residue selected from the group consisting of histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a cysteine residue.
• said first modification is a mutation, disruption, or deletion of SEC27 (SEQ ID NO: 5). In one embodiment, said first modification is a mutation of SEC27. In another embodiment, said first modification is a point mutation of SEC27. In a specific embodiment, said first modification is a point mutation of SEC27 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of C145 of Sec27p (SEQ ID NO: 6).
• said first modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a serine residue.
• said first modification is a mutation, disruption, or deletion of ZRT1 (SEQ ID NO: 7). In one embodiment, said first modification is a mutation of ZRT1. In another embodiment, said first modification is a point mutation of ZRT1. In a specific embodiment, said first modification is a point mutation of ZRT1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of D245 of Zrt1 p (SEQ ID NO: 8).
• said first modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrt1p, wherein said D245 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrtlp, wherein said D245 residue is replaced with a glutamic acid residue.
• said first modification is a mutation, disruption, or deletion of YLL056C (SEQ ID NO: 9). In one embodiment, said first modification is a mutation of YLL056C. In another embodiment, said first modification is a point mutation of YLL056C. In a specific embodiment, said first modification is a point mutation of YLL056C which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of I5 of YLL056c (SEQ ID NO: 10).
• said first modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056c, wherein said I5 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056C, wherein said I5 residue is replaced with a valine residue.
• said first modification is a mutation, disruption, or deletion of YOL014W (SEQ ID NO: 11). In one embodiment, said first modification is a mutation of YOL014W. In another embodiment, said first modification is a point mutation of YOL014W. In a specific embodiment, said first modification is a point mutation of YOL014W which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of A39 of YOL014w (SEQ ID NO: 12).
• said first modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with an aspartic acid residue.
• said first modification is a point mutation at a position corresponding to the nucleotides listed in Table 2.
• said second modification is a mutation, disruption, or deletion of MTH1 (SEQ ID NO: 13) or homologs or variants thereof.
• said second modification is a partial deletion of MTH1.
• said second modification is a partial deletion of MTH1, wherein said partial deletion results in at least one amino acid deletion between amino acid positions 25 to 100 of Mthl p (SEQ ID NO: 14).
• said second modification is a partial deletion of ⁇ 1 ⁇ 1, wherein said partial deletion results in at least one amino acid deletion between amino acid positions 40 to 80 of Mth1 p.
• said second modification is a partial deletion of MTH1, wherein said partial deletion results in the deletion of amino acid residues 41 to 78 of Mthl p.
• said second modification is a point mutation of MTH1.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of I85 of Mthlp, wherein said 185 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of I85 of Mthl p, wherein said 185 residue is replaced with an asparagine or serine residue.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of S102 of Mthlp, wherein said S102 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of S102 of Mthlp, wherein said S102 residue is replaced with a glycine residue.
• said second modification is a mutation, disruption, or deletion of UME6 (SEQ ID NO: 3). In one embodiment, said second modification is a mutation of UME6. In another embodiment, said second modification is a point mutation of UME6. In a specific embodiment, said second modification is a point mutation of UME6 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of R768 of Ume6p (SEQ ID NO: 4).
• said second modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a residue selected from the group consisting of histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a cysteine residue.
• said second modification is a mutation, disruption, or deletion of SEC27 (SEQ ID NO: 5). In one embodiment, said second modification is a mutation of SEC27. In another embodiment, said second modification is a point mutation of SEC27. In a specific embodiment, said second modification is a point mutation of SEC27 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of C145 of Sec27p (SEQ ID NO: 6).
• said second modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a serine residue.
• said second modification is a mutation, disruption, or deletion of ZRT1 (SEQ ID NO: 7). In one embodiment, said second modification is a mutation of ZRT1. In another embodiment, said second modification is a point mutation of ZRT1. In a specific embodiment, said second modification is a point mutation of ZRT1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of D245 of Zrt1p (SEQ ID NO: 8).
• said second modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrtlp, wherein said D245 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrt1 p, wherein said D245 residue is replaced with a glutamic acid residue.
• said second modification is a mutation, disruption, or deletion of YLL056C (SEQ ID NO: 9). In one embodiment, said second modification is a mutation of YLL056C. In another embodiment, said second modification is a point mutation of YLL056C. In a specific embodiment, said second modification is a point mutation of YLL056C which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of I5 of YLL056c (SEQ ID NO: 10).
• said second modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056c, wherein said I5 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056c, wherein said I5 residue is replaced with a valine residue.
• said second modification is a mutation, disruption, or deletion of YOL014W (SEQ ID NO: 11 ).
• said second modification is a mutation of YOL014W.
• said second modification is a point mutation of YOL014W.
• said second modification is a point mutation of YOL014W which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of A39 of YOL014w (SEQ ID NO: 12).
• said second modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with an aspartic acid residue.
• said second modification is a point mutation at a position corresponding to the nucleotides listed in Table 2.
• the present application relates to a recombinant yeast microorganism comprising a metabolic pathway for the production of a pyruvate- derived metabolite, wherein said metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the production of the pyruvate-derived metabolite, wherein said yeast microorganism is engineered to comprise reduced pyruvate decarboxylase (PDC) activity, and wherein said yeast microorganism comprises at least one genetic modification which increases the 6-phosphogluconate dehydrogenase (GND) activity in the recombinant yeast microorganism as compared with the 6-phosphogluconate dehydrogenase (GND) activity in a yeast microorganism lacking said genetic modification.
• said metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the
• the genetic modification results in improved growth in the absence of C2- supplementation in the recombinant yeast microorganism as compared to a yeast microorganism lacking said genetic modification.
• said genetic modification is a mutation of GND1 (SEQ ID NO: 1 ) or homologs and variants thereof.
• said genetic modification is a point mutation of GND1.
• said genetic modification is a point mutation of GND1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of L340 of Gndlp (SEQ ID NO: 2).
• said genetic modification is a point mutation of GND1 which results in an amino acid substitution of L340 of Gndlp, wherein said L340 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said genetic modification is a point mutation of GND1 which results in an amino acid substitution of L340 of Gndl p, wherein said L340 residue is replaced with a tryptophan residue.
• said genetic modification is an increase in the heterologous or native expression of a Gnd1 protein.
• said genetic modification is the overexpression of one or more polynucleotides encoding one or more Gnd1 proteins or homologs thereof.
• one or more of the polynucleotides encoding said one or more Gnd1 proteins or homologs thereof is an endogenous polynucleotide.
• one or more of the polynucleotides encoding said one or more Gnd1 proteins or homologs thereof is an exogenous polynucleotide.
• the Gnd1 protein is selected from SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , and SEQ ID NO: 22.
• the Gnd1 protein is a mutant Gnd1 protein.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L340 residue of Gndlp (SEQ ID NO: 2), wherein said L340 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L340 residue of Gndlp (SEQ ID NO: 2), wherein said L340 residue is replaced with a tryptophan residue.
• the mutant Gnd1 protein comprises the amino acid sequence of SEQ ID NO: 23.
• said genetic modification is a mutation of GND2 (SEQ ID NO: 24) or homologs and variants thereof.
• said genetic modification is a point mutation of GND2.
• said genetic modification is a point mutation of GND2 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of L343 of Gnd2p (SEQ ID NO: 25).
• said genetic modification is a point mutation of GND2 which results in an amino acid substitution of L343 of Gnd2p, wherein said L343 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said genetic modification is a point mutation of GND2 which results in an amino acid substitution of L343 of Gnd2p, wherein said L343 residue is replaced with a tryptophan residue.
• said genetic modification is an increase in the heterologous or native expression of a Gnd2 protein.
• said genetic modification is the overexpression of one or more polynucleotides encoding one or more Gnd2 proteins or homologs thereof.
• one or more of the polynucleotides encoding said one or more Gnd2 proteins or homologs thereof is an endogenous polynucleotide.
• one or more of the polynucleotides encoding said one or more Gnd2 proteins or homologs thereof is an exogenous polynucleotide.
• the Gnd2 protein is selected from SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
• the Gnd2 protein is a mutant Gnd2 protein.
• the mutant Gnd2 protein comprises an amino acid substitution at an amino acid position corresponding to the L343 residue of Gnd2p (SEQ ID NO: 25), wherein said L343 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• mutant Gnd2 protein comprises an amino acid substitution at an amino acid position corresponding to the L343 residue of Gnd2p (SEQ ID NO: 25), wherein said L343 residue is replaced with a tryptophan residue.
• mutant Gnd2 protein comprises the amino acid sequence of SEQ ID NO: 28.
• the present application relates to a recombinant yeast microorganism comprising a metabolic pathway for the production of a pyruvate- derived metabolite, wherein said metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the production of the pyruvate-derived metabolite, wherein said yeast microorganism is engineered to comprise reduced pyruvate decarboxylase (PDC) activity, and wherein said yeast microorganism comprises at least one mutation, disruption, or deletion of MTH1.
• PDC reduced pyruvate decarboxylase
• the mutation, disruption, or deletion of MTH1 results in improved tolerance to high glucose concentrations in the recombinant yeast microorganism as compared to a yeast microorganism lacking said mutation, disruption, or deletion of MTH1.
• the yeast microorganism comprises a partial deletion of MTH1.
• the yeast microorganism comprises a partial deletion of MTH1, wherein said partial deletion results in at least one amino acid deletion between amino acid positions 25 to 100 of Mthl p (SEQ ID NO: 14).
• the yeast microorganism comprises a partial deletion of MTH1, wherein said partial deletion results in at least one amino acid deletion between amino acid positions 40 to 80 of Mthlp.
• the yeast microorganism comprises a partial deletion of ⁇ 7 ⁇ 1, wherein said partial deletion results in the deletion of amino acid residues 41 to 78 of Mthlp. In one embodiment, the yeast microorganism comprises a point mutation of MTH1.
• the yeast microorganism comprises a point mutation of MTH1 which results in an amino acid substitution of I85 of Mthlp, wherein said 185 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the yeast microorganism comprises a point mutation of MTH1 which results in an amino acid substitution of I85 of Mthlp, wherein said 185 residue is replaced with an asparagine or serine residue.
• the yeast microorganism comprises a point mutation of MTH1 which results in an amino acid substitution of S102 of Mthlp, wherein said S102 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the yeast microorganism comprises a point mutation of MTH1 which results in an amino acid substitution of S102 of Mthlp, wherein said S102 residue is replaced with a glycine
• the modifications identified herein are relevant to improving growth characteristics in PDC-deficient yeast engineered for the production of variety of pyruvate-derived metabolites.
• the pyruvate-derived metabolite is selected from the group consisting of isobutanol, 2- butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, coenzyme A, lactic acid, and malic acid.
• the pyruvate-derived metabolite is isobutanol.
• the present application relates to a recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, wherein said recombinant yeast microorganism is engineered to comprise reduced PDC activity, and wherein said recombinant yeast microorganism is engineered to (a): comprise at least one first modification which results in improved growth in the absence of C2-supplementation as compared to the corresponding recombinant yeast microorganism without said first modification; and/or (b) comprise at least one second modification which results in improved tolerance to glucose as compared to the corresponding recombinant yeast microorganis
• the present application relates to a recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, wherein said recombinant yeast microorganism exhibits reduced PDC activity, and wherein said recombinant yeast microorganism is engineered to (a): comprise at least one first modification which results in improved growth in the absence of C2-supplementation as compared to the corresponding recombinant yeast microorganism without said first modification; and/or (b) comprise at least one second modification which results in improved tolerance to glucose as compared to the corresponding recombinant yeast microorganism without said second modification.
• said first modification is a mutation of GND1 (SEQ ID NO: 1 ) or homologs and variants thereof.
• said first modification is a point mutation of GND1.
• said first modification is a point mutation of GND1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of L340 of Gndlp (SEQ ID NO: 2).
• said first modification is a point mutation of GND1 which results in an amino acid substitution of L340 of Gndl p, wherein said L340 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of GND1 which results in an amino acid substitution of L340 of Gndlp, wherein said L340 residue is replaced with a tryptophan residue.
• said first modification is an increase in the heterologous or native expression of a Gnd1 protein. Accordingly, in one embodiment, said first modification is the overexpression of one or more polynucleotides encoding one or more Gnd1 proteins or homologs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Gnd1 proteins or homologs thereof is an endogenous polynucleotide. In another embodiment, one or more of the polynucleotides encoding said one or more Gnd1 proteins or homologs thereof is an exogenous polynucleotide.
• the Gnd1 protein is selected from SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , and SEQ ID NO: 22.
• the Gnd1 protein is a mutant Gnd1 protein.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L340 residue of Gndlp (SEQ ID NO: 2), wherein said L340 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L340 residue of Gndl p (SEQ ID NO: 2), wherein said L340 residue is replaced with a tryptophan residue.
• the mutant Gnd1 protein comprises the amino acid sequence of SEQ ID NO: 23.
• the present application is directed to an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 23.
• said first modification is a mutation of GND2 (SEQ ID NO: 24) or homologs and variants thereof.
• said first modification is a point mutation of GND2.
• said first modification is a point mutation of GND2 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of L343 of Gnd2p (SEQ ID NO: 25).
• said first modification is a point mutation of GND2 which results in an amino acid substitution of L343 of Gnd2p, wherein said L343 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of GND2 which results in an amino acid substitution of L343 of Gnd2p, wherein said L343 residue is replaced with a tryptophan residue.
• said first modification is an increase in the heterologous or native expression of a Gnd2 protein. Accordingly, in one embodiment, said first modification is the overexpression of one or more polynucleotides encoding one or more Gnd2 proteins or homologs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Gnd2 proteins or homologs thereof is an endogenous polynucleotide. In another embodiment, one or more of the polynucleotides encoding said one or more Gnd2 proteins or homologs thereof is an exogenous polynucleotide.
• the Gnd2 protein is selected from SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
• the Gnd2 protein is a mutant Gnd2 protein.
• the mutant Gnd2 protein comprises an amino acid substitution at an amino acid position corresponding to the L343 residue of Gnd2p (SEQ ID NO: 25), wherein said L343 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the mutant Gnd2 protein comprises an amino acid substitution at an amino acid position corresponding to the L343 residue of Gnd2p (SEQ ID NO: 25), wherein said L343 residue is replaced with a tryptophan residue.
• the mutant Gnd2 protein comprises the amino acid sequence of SEQ ID NO: 28.
• the present application is directed to an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 28.
• said first modification is a mutation, disruption, or deletion of UME6 (SEQ ID NO: 3). In one embodiment, said first modification is a mutation of UME6. In another embodiment, said first modification is a point mutation of UME6. In a specific embodiment, said first modification is a point mutation of UME6 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of R768 of Ume6p (SEQ ID NO: 4).
• said first modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a residue selected from the group consisting of histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a cysteine residue.
• said first modification is a mutation, disruption, or deletion of SEC27 (SEQ ID NO: 5). In one embodiment, said first modification is a mutation of SEC27. In another embodiment, said first modification is a point mutation of SEC27. In a specific embodiment, said first modification is a point mutation of SEC27 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of C145 of Sec27p (SEQ ID NO: 6).
• said first modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a serine residue.
• said first modification is a mutation, disruption, or deletion of ZRT1 (SEQ ID NO: 7). In one embodiment, said first modification is a mutation of ZRT1. In another embodiment, said first modification is a point mutation of ZRT1. In a specific embodiment, said first modification is a point mutation of ZRT1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of D245 of Zrtl p (SEQ ID NO: 8).
• said first modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrt1 p, wherein said D245 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrtlp, wherein said D245 residue is replaced with a glutamic acid residue.
• said first modification is a mutation, disruption, or deletion of YLL056C (SEQ ID NO: 9). In one embodiment, said first modification is a mutation of YLL056C. In another embodiment, said first modification is a point mutation of YLL056C. In a specific embodiment, said first modification is a point mutation of YLL056C which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of 15 of YLL056c (SEQ ID NO: 10).
• said first modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056c, wherein said I5 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056C, wherein said I5 residue is replaced with a valine residue.
• said first modification is a mutation, disruption, or deletion of YOL014W (SEQ ID NO: 11). In one embodiment, said first modification is a mutation of YOL014W. In another embodiment, said first modification is a point mutation of YOL014W. In a specific embodiment, said first modification is a point mutation of YOL014W which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of A39 of YOL014w (SEQ ID NO: 12).
• said first modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with an aspartic acid residue.
• said first modification is a point mutation at a position corresponding to the nucleotides listed in Table 2.
• said second modification is a mutation, disruption, or deletion of MTH1 (SEQ ID NO: 13) or homologs or variants thereof.
• said second modification is a partial deletion of MTH1.
• said second modification is a partial deletion of MTH1, wherein said partial deletion results in at least one amino acid deletion between amino acid positions 25 to 100 of Mthl p (SEQ ID NO: 14).
• said second modification is a partial deletion of ⁇ 1, wherein said partial deletion results in at least one amino acid deletion between amino acid positions 40 to 80 of Mth1 p.
• said second modification is a partial deletion of MTH1, wherein said partial deletion results in the deletion of amino acid residues 41 to 78 of Mthl p.
• said second modification is a point mutation of MTH1.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of I85 of Mthlp, wherein said 185 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of I85 of Mthl p, wherein said 185 residue is replaced with an asparagine or serine residue.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of S102 of Mthlp, wherein said S 02 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of S102 of Mthlp, wherein said S102 residue is replaced with a glycine residue.
• said second modification is a mutation, disruption, or deletion of UME6 (SEQ ID NO: 3).
• said second modification is a mutation of UME6.
• said second modification is a point mutation of UME6.
• said second modification is a point mutation of UME6 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of R768 of Ume6p (SEQ ID NO: 4).
• said second modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a residue selected from the group consisting of histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a cysteine residue.
• said second modification is a mutation, disruption, or deletion of SEC27 (SEQ ID NO: 5). In one embodiment, said second modification is a mutation of SEC27. In another embodiment, said second modification is a point mutation of SEC27. In a specific embodiment, said second modification is a point mutation of SEC27 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of C145 of Sec27p (SEQ ID NO: 6).
• said second modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a serine residue.
• said second modification is a mutation, disruption, or deletion of ZRT1 (SEQ ID NO: 7). In one embodiment, said second modification is a mutation of ZRT1. In another embodiment, said second modification is a point mutation of ZRT1. In a specific embodiment, said second modification is a point mutation of ZRT1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of D245 of Zrtlp (SEQ ID NO: 8).
• said second modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrtlp, wherein said D245 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrt1 p, wherein said D245 residue is replaced with a glutamic acid residue.
• said second modification is a mutation, disruption, or deletion of YL Q56C (SEQ ID NO: 9). In one embodiment, said second modification is a mutation of YLL056C. In another embodiment, said second modification is a point mutation of YLL056C. In a specific embodiment, said second modification is a point mutation of YLL056C which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of I5 of YLL056c (SEQ ID NO: 10).
• said second modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056c, wherein said I5 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056c, wherein said I5 residue is replaced with a valine residue.
• said second modification is a mutation, disruption, or deletion of YOL014W (SEQ ID NO: 11 ).
• said second modification is a mutation of YOL014W.
• said second modification is a point mutation of YOL014W.
• said second modification is a point mutation of YOL014W which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of A39 of YOL014w (SEQ ID NO: 12).
• said second modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with an aspartic acid residue.
• said second modification is a point mutation at a position corresponding to the nucleotides listed in Table 2.
• the present application relates to a recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, wherein said yeast microorganism is engineered to comprise reduced pyruvate decarboxylase (PDC) activity, and wherein said yeast microorganism comprises at least one genetic modification which increases the 6-phosphogluconate dehydrogenase (GND) activity in the recombinant yeast microorganism as compared with the 6-phosphogluconate dehydrogenase (GND) activity in a yeast microorganism lacking said genetic modification.
• PDC reduced pyruvate decarboxylase
• the genetic modification results in improved growth in the absence of C2-supplementation in the recombinant yeast microorganism as compared to a yeast microorganism lacking said genetic modification.
• said genetic modification is a mutation of GND1 (SEQ ID NO: 1 ) or homologs and variants thereof.
• said genetic modification is a point mutation of GND1.
• said genetic modification is a point mutation of GND1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of L340 of Gndlp (SEQ ID NO: 2).
• said genetic modification is a point mutation of GND1 which results in an amino acid substitution of L340 of Gndlp, wherein said L340 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said genetic modification is a point mutation of GND1 which results in an amino acid substitution of L340 of Gndlp, wherein said L340 residue is replaced with a tryptophan residue.
• said genetic modification is an increase in the heterologous or native expression of a Gnd1 protein.
• said genetic modification is the overexpression of one or more polynucleotides encoding one or more Gnd1 proteins or homologs thereof.
• one or more of the polynucleotides encoding said one or more Gnd1 proteins or homologs thereof is an endogenous polynucleotide.
• one or more of the polynucleotides encoding said one or more Gnd1 proteins or homologs thereof is an exogenous polynucleotide.
• the Gnd1 protein is selected from SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , and SEQ ID NO: 22.
• the Gnd1 protein is a mutant Gnd1 protein.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L340 residue of Gndlp (SEQ ID NO: 2), wherein said L340 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L340 residue of Gndl p (SEQ ID NO: 2), wherein said L340 residue is replaced with a tryptophan residue.
• the mutant Gnd1 protein comprises the amino acid sequence of SEQ ID NO: 23.
• said genetic modification is a mutation of GND2 (SEQ ID NO: 24) or homologs and variants thereof.
• said genetic modification is a point mutation of GND2.
• said genetic modification is a point mutation of GND2 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of L343 of Gnd2p (SEQ ID NO: 25).
• said genetic modification is a point mutation of GND2 which results in an amino acid substitution of L343 of Gnd2p, wherein said L343 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said genetic modification is a point mutation of GND2 which results in an amino acid substitution of L343 of Gnd2p, wherein said L343 residue is replaced with a tryptophan residue.
• said genetic modification is an increase in the heterologous or native expression of a Gnd2 protein.
• said genetic modification is the overexpression of one or more polynucleotides encoding one or more Gnd2 proteins or homologs thereof.
• one or more of the polynucleotides encoding said one or more Gnd2 proteins or homologs thereof is an endogenous polynucleotide.
• one or more of the polynucleotides encoding said one or more Gnd2 proteins or homologs thereof is an exogenous polynucleotide.
• the Gnd2 protein is selected from SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
• the Gnd2 protein is a mutant Gnd2 protein.
• the mutant Gnd2 protein comprises an amino acid substitution at an amino acid position corresponding to the L343 residue of Gnd2p (SEQ ID NO: 25), wherein said L343 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the mutant Gnd2 protein comprises an amino acid substitution at an amino acid position corresponding to the L343 residue of Gnd2p (SEQ ID NO: 25), wherein said L343 residue is replaced with a tryptophan residue.
• the mutant Gnd2 protein comprises the amino acid sequence of SEQ ID NO: 28.
• the present application is directed to an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 28.
• the present application relates to a recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, and wherein said yeast microorganism comprises at least one mutation, disruption, or deletion of ⁇ 1 ⁇ 1.
• the mutation, disruption, or deletion of MTH1 results in improved tolerance to high glucose concentrations in the recombinant yeast microorganism as compared to a yeast microorganism lacking said mutation, disruption, or deletion of MTH1.
• the yeast microorganism comprises a partial deletion of MTH1.
• the yeast microorganism comprises a partial deletion of MTH1, wherein said partial deletion results in at least one amino acid deletion between amino acid positions 25 to 100 of Mthl p (SEQ ID NO: 14).
• the yeast microorganism comprises a partial deletion of MTH1, wherein said partial deletion results in at least one amino acid deletion between amino acid positions 40 to 80 of Mthl p.
• the yeast microorganism comprises a partial deletion of MTH1, wherein said partial deletion results in the deletion of amino acid residues 41 to 78 of Mthlp.
• the yeast microorganism comprises a point mutation of MTH1.
• the yeast microorganism comprises a point mutation of MTH1 which results in an amino acid substitution of I85 of Mthlp, wherein said 185 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the yeast microorganism comprises a point mutation of MTH1 which results in an amino acid substitution of I85 of Mthlp, wherein said 185 residue is replaced with an asparagine or serine residue.
• the yeast microorganism comprises a point mutation of MTH1 which results in an amino acid substitution of S102 of Mthlp, wherein said S102 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the yeast microorganism comprises a point mutation of MTH1 which results in an amino acid substitution of S102 of Mthl p, wherein said S102 residue is replaced with a gly
• the recombinant yeast microorganisms of the application that comprise an isobutanol producing metabolic pathway may be further engineered to reduce or eliminate the expression and/or activity of one or more enzymes selected from a glycerol-3-phosphate dehydrogenase (GPD), a 3-keto acid reductase (3-KAR), or an aldehyde dehydrogenase (ALDH).
• GPD glycerol-3-phosphate dehydrogenase
• 3-KAR 3-keto acid reductase
• ALDH aldehyde dehydrogenase
• the isobutanol producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanol.
• the isobutanol producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol.
• the isobutanol producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol.
• the isobutanol producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, all of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes.
• one or more of the isobutanol pathway genes encodes an enzyme that is localized to the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least two isobutanol pathway enzymes localized in the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least three isobutanol pathway enzymes localized in the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least four isobutanol pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with five isobutanol pathway enzymes localized in the cytosol. In yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with all isobutanol pathway enzymes localized in the cytosol.
• the isobutanol pathway genes may encode enzyme(s) selected from the group consisting of acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2- keto-acid decarboxylase, e.g., keto-isovalerate decarboxylase (KIVD), and alcohol dehydrogenase (ADH).
• the KARI is an NADH-dependent KARI (NKR).
• the ADH is an NADH-dependent ADH.
• the KARI is an NADH-dependent KARI (NKR) and the ADH is an NADH-dependent ADH.
• the recombinant microorganisms of the application are recombinant yeast microorganisms.
• the recombinant yeast microorganisms may be members of the Saccharomyces clade, Sacchammyces sensu stricto microorganisms, Crabtree- negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.
• the recombinant microorganisms may be yeast recombinant microorganisms of the Saccharomyces clade.
• the recombinant microorganisms may be Saccharomyces sensu stricto microorganisms.
• Saccharomyces sensu stricto is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrids thereof.
• the recombinant microorganisms may be Crabtree- negative recombinant yeast microorganisms.
• the Crabtree- negative yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Hansenula, or Candida.
• the Crabtree-negative yeast microorganism is selected from Saccharomyces kluyveri, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Pichia kudriavzevii, Hansenula anomala, Candida utilis and Kluyveromyces waltii.
• the recombinant microorganisms may be Crabtree- positive recombinant yeast microorganisms.
• the Crabtree- positive yeast microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Candida, Pichia and Schizosaccharomyces.
• the Crabtree-positive yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, Kluyveromyces thermotolerans, Candida glabrata, Z. bailli, Z. rouxii, Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces uvarum.
• the recombinant microorganisms may be post- WGD (whole genome duplication) yeast recombinant microorganisms.
• the post-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces or Candida.
• the post-WGD yeast is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and Candida glabrata.
• the recombinant microorganisms may be pre-WGD (whole genome duplication) yeast recombinant microorganisms.
• the pre-WGD yeast recombinant microorganism is classified into a genera selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula, Pachysolen, Yamowia and Schizosaccharomyces.
• the pre-WGD yeast is selected from the group consisting of Saccharomyces kluyveri, Kluyveromyces thermotolerans, Kluyveromyces marxianus, Kluyveromyces waltii, Kluyveromyces lactis, Candida tropicalis, Pichia pastoris, Pichia anomala, Pichia stipitis, Issatchenkia orientalis, Issatchenkia occidentalis, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yarrowia lipolytica, and Schizosaccharomyces pombe.
• the recombinant microorganisms may be microorganisms that are non-fermenting yeast microorganisms, including, but not limited to those, classified into a genera selected from the group consisting of Tricosporon, Rhodotorula, Myxozyma, or Candida.
• the non-fermenting yeast is C. xestobii.
• the present invention provides methods of producing a pyruvate-derived metabolite using a recombinant microorganism as described herein.
• the method includes cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source until the pyruvate-derived metabolite is produced and optionally, recovering the pyruvate-derived metabolite.
• the microorganism produces the pyruvate-derived metabolite from a carbon source at a yield of at least about 5 percent theoretical.
• the microorganism produces the pyruvate-derived 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 60 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5 percent theoretical.
• the pyruvate-derived metabolite is isobutanol.
• the recombinant microorganism converts the carbon source to the pyruvate-derived metabolite under aerobic conditions. In another embodiment, the recombinant microorganism converts the carbon source to the pyruvate-derived metabolite under microaerobic conditions. In yet another embodiment, the recombinant microorganism converts the carbon source to the pyruvate-derived metabolite under anaerobic conditions.
• Figure 1 illustrates an exemplary embodiment of an isobutanol pathway.
• Figure 2 illustrates an exemplary embodiment of an NADH-dependent isobutanol pathway.
• Figure 3 illustrates various biosynthetic pathways which use pyruvate as an intermediate (adapted from Liu et al., 2010, Annu. Rev. Genet.44: 53-69).
• Figure 4 illustrates the evolution of GEVO2302 for C2 supplement- independence. Changes in glucose feed concentration, vessel ethanol concentration, OD600 and acetate feed concentration are plotted over the time frame of evolution until the acetate feed concentration was 0 g/L. The x-axis represents time in hours (0, 24, 48, 72, 96, and 120 hours).
• Figure 5 illustrates the evolution of the C2 supplement-independent evolved GEVO2302 population for glucose tolerance. Changes in glucose feed concentration, vessel residual glucose concentration and OD600 are plotted over the time frame of the evolution for glucose tolerance. The x-axis represents time in increments of 5 days (0, 5, 10, 15, 20, 25, and 30 days).
• Figure 6 illustrates the amount of glucose transported as measured after 15 seconds in a variety of Pdc-deficient strains pre-grown in YPEtOH. Glucose tolerant cells carrying the MTH1 partial deletion transported significantly less glucose than cells harboring a wild-type MTH1 or a full MTH1 deletion.
• Figures 7A and 7B illustrate growth curves of parent strains (GEV09137 and GEV09138), GND1 deletion strains (GEVO9407 and GEVO9408), and GND1 L340w strajns (GEVO9411 and GEV09412) in the presence and absence of 1 % acetate (i.e., C2-supplementation), respectively.
• Figures 8A and 8B illustrate the results of a second experiment designed to measure growth of parent strains (GEV09137 and GEV09138), GND1 deletion strains (GEVO9407 and GEVO9408), and GND1 L340W strains (GEV09411 and GEV09412) in the presence and absence of 1% acetate (i.e., C2-supplementation), respectively.
• 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 and/or activity of an endogenous gene.
• alteration it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the alteration.
• alter can mean “inhibit,” but the use of the word “alter” is not limited to this definition.
• the terms “recombinant microorganism” and “recombinant host cell” refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
• expression refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein.
• expression of a protein results from transcription and translation of the open reading frame sequence.
• the level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the cell, or the amount of the desired product encoded by the selected sequence.
• mRNA transcribed from a selected sequence can be quantitated by qRT-PCR or by Northern hybridization (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
• Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that recognize and bind the protein. See Sambrook et al., 1989, supra.
• over-expression refers to an elevated level (e.g., aberrant level) of mRNAs encoding for a protein(s), and or to elevated levels of protein(s) in cells as compared to similar corresponding unmodified cells expressing basal levels of mRNAs or having basal levels of proteins.
• mRNA(s) or protein(s) may be overexpressed by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8- fold, 10-fold, 12-fold, 15-fold or more in microorganisms engineered to exhibit increased gene mRNA, protein, and/or activity.
• reduced activity and/or expression of a protein such as an enzyme can mean either a reduced specific catalytic activity of the protein (e.g., reduced activity) and/or decreased concentrations of the protein in the cell ⁇ e.g., reduced expression).
• reduced activity of a protein in a cell may result from decreased concentrations of the protein in the cell.
• wild-type microorganism describes a cell that occurs in nature, i.e., a cell that has not been genetically modified.
• a wild-type microorganism can be genetically modified to express or overexpress a first target enzyme.
• This microorganism can act as a parental microorganism in the generation of a microorganism modified to express or overexpress a second target enzyme.
• the microorganism modified to express or overexpress a first and a second target enzyme can be modified to express or overexpress a third target enzyme.
• a "parental microorganism” functions as a reference cell for successive genetic modification events. Each modification event can be accomplished by introducing a nucleic acid molecule in to the reference cell. The introduction facilitates the expression or overexpression of a target enzyme.
• the term “facilitates” encompasses the activation of endogenous polynucleotides encoding a target enzyme through genetic modification of e.g., a promoter sequence in a parental microorganism. It is further understood that the term “facilitates” encompasses the introduction of heterologous polynucleotides encoding a target enzyme in to a parental microorganism
• engine refers to any manipulation of a microorganism that results in a detectable change in the microorganism, wherein the manipulation includes but is not limited to inserting a polynucleotide and/or polypeptide heterologous to the microorganism and mutating a polynucleotide and/or polypeptide native to the microorganism.
• mutation indicates any modification of a nucleic acid and/or polypeptide which results in an altered nucleic acid or polypeptide. Mutations include, for example, point mutations, deletions, or insertions of single or multiple residues in a polynucleotide, which includes alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences.
• a genetic alteration may be a mutation of any type. For instance, the mutation may constitute a point mutation, a frame-shift mutation, a nonsense mutation, an insertion, or a deletion of part or all of a gene.
• the modified microorganism a portion of the microorganism genome has been replaced with a heterologous polynucleotide.
• the mutations are naturally-occurring.
• the mutations are identified and/or enriched through artificial selection pressure.
• the mutations in the microorganism genome are the result of genetic engineering.
• biosynthetic pathway also referred to as “metabolic pathway” refers to a set of anabolic or catabolic biochemical reactions for converting one chemical species into another.
• Gene products belong to the same “metabolic pathway” if they, in parallel or in series, act on the same substrate, produce the same product, or act on or produce a metabolic intermediate (i.e., metabolite) between the same substrate and metabolite end product.
• Isobutanol producing metabolic pathway refers to an enzyme pathway which produces isobutanol from pyruvate.
• NADH-dependent refers to an enzyme that catalyzes the reduction of a substrate coupled to the oxidation of NADH with a catalytic efficiency that is greater than the reduction of the same substrate coupled to the oxidation of NADPH at equal substrate and cofactor concentrations.
• exogenous refers to molecules that are not normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
• endogenous or “native” as used herein with reference to various molecules refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
• heterologous refers to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., wherein at least one of the following is true: (a) the molecule(s) is/are foreign ("exogenous") to (i.e., not naturally found in) the host cell; (b) the molecule(s) is/are naturally found in (e.g., is "endogenous to") a given host microorganism or host cell but is/are either produced in an unnatural location or in an unnatural amount in the cell; and/or (c) the molecule(s) differ(s) in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence(s) such that the molecule differing in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid as found endogenously is produced in an unnatural (e.g., greater than naturally
• feedstock is defined as a raw material or mixture of raw materials supplied to a microorganism or fermentation process from which other products can be made.
• a carbon source such as biomass or the carbon compounds derived from biomass are a feedstock for a microorganism that produces a biofuel in a fermentation process.
• a feedstock may contain nutrients other than a carbon source.
• substrate refers to any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme.
• the term includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate, or derivatives thereof.
• substrate encompasses not only compounds that provide a carbon source suitable for use as a starting material, such as any biomass derived sugar, but also intermediate and end product metabolites used in a pathway associated with a recombinant microorganism as described herein.
• the term "fermentation” or “fermentation process” is defined as a process in which a microorganism is cultivated in a culture medium containing raw materials, such as feedstock and nutrients, wherein the microorganism converts raw materials, such as a feedstock, into products.
• volumetric productivity or “production rate” is defined as the amount of product formed per volume of medium per unit of time. Volumetric productivity is reported in gram per liter per hour (g/L/h).
• specific productivity or “specific production rate” is defined as the amount of product formed per volume of medium per unit of time per amount of cells. Specific productivity is reported in gram (or milligram) per gram cell dry weight per hour (g/g h).
• yield is defined as the amount of product obtained per unit weight of raw material and may be expressed as g product per g substrate (g/g). Yield may be expressed as a percentage of the theoretical yield. "Theoretical yield” is defined as the maximum amount of product that can be generated per a given amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to isobutanol is 0.41 g/g. As such, a yield of isobutanol from glucose of 0.39 g/g would be expressed as 95% of theoretical or 95% theoretical yield.
• titer is defined as the strength of a solution or the concentration of a substance in solution.
• concentration of a substance in solution For example, the titer of a biofuel in a fermentation broth is described as g of biofuel in solution per liter of fermentation broth (g/L).
• “Aerobic conditions” are defined as conditions under which the oxygen concentration in the fermentation medium is sufficiently high for an aerobic or facultative anaerobic microorganism to use as a terminal electron acceptor.
• anaerobic conditions are defined as conditions under which the oxygen concentration in the fermentation medium is too low for the microorganism to use as a terminal electron acceptor. Anaerobic conditions may be achieved by sparging a fermentation medium with an inert gas such as nitrogen until oxygen is no longer available to the microorganism as a terminal electron acceptor. Alternatively, anaerobic conditions may be achieved by the microorganism consuming the available oxygen of the fermentation until oxygen is unavailable to the microorganism as a terminal electron acceptor. Methods for the production of isobutanol under anaerobic conditions are described in commonly owned and copending publication, US 2010/0143997, the disclosures of which are herein incorporated by reference in its entirety for all purposes.
• NAD(P)H donates its electrons to a molecule produced by the same metabolic pathway that produced the electrons carried in NAD(P)H.
• NAD(P)H generated through glycolysis transfers its electrons to pyruvate, yielding ethanol.
• Fermentative pathways are usually active under anaerobic conditions but may also occur under aerobic conditions, under conditions where NADH is not fully oxidized via the respiratory chain. For example, above certain glucose concentrations, Crabtree positive yeasts produce large amounts of ethanol under aerobic conditions.
• byproduct or "by-product” means an undesired product related to the production of an amino acid, amino acid precursor, chemical, chemical precursor, biofuel, biofuel precursor, higher alcohol, or higher alcohol precursor.
• substantially free when used in reference to the presence or absence of a protein activity (3-KAR enzymatic activity, ALDH enzymatic activity, PDC enzymatic activity, GPD enzymatic activity, etc.) means the level of the protein is substantially less than that of the same protein in the wild-type host, wherein less than about 50% of the wild-type level is preferred and less than about 30% is more preferred.
• the activity may be less than about 20%, less than about 10%, less than about 5%, or less than about 1% of wild-type activity.
• Microorganisms which are "substantially free" of a particular protein activity may be created through recombinant means or identified in nature.
• polynucleotide is used herein interchangeably with the term “nucleic acid” and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single stranded or double stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or antisense ribonucleic acid (RNA) of any length, including siRNA.
• DNA single stranded or double stranded
• RNA ribonucleic acid
• nucleotide refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or a pyrimidine base and to a phosphate group, and that are the basic structural units of nucleic acids.
• nucleoside refers to a compound (as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids.
• nucleotide analog or “nucleoside analog” refers, respectively, to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called nucleotidic oligomer or oligonucleotide.
• the polynucleotides described herein include “genes” and that the nucleic acid molecules described herein include “vectors” or “plasmids.”
• the term “gene”, also called a “structural gene” refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non- transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed.
• the transcribed region of the gene may include untranslated regions, including introns, 5'-untranslated region (UTR), and 3'-UTR, as well as the coding sequence.
• enzyme refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide or polypeptides, but can include enzymes composed of a different molecule including polynucleotides.
• polypeptide indicates an organic polymer composed of two or more amino acidic monomers and/or analogs thereof.
• amino acid or “amino acidic monomer” refers to any natural and/or synthetic amino acids including glycine and both D or L optical isomers.
• amino acid analog refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, or with a different functional group.
• polypeptide includes amino acidic polymer of any length including full length proteins, and peptides as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called a protein oligomer or oligopeptide
• homolog used with respect to an original polynucleotide or polypeptide of a first family or species, refers to distinct polynucleotides or polypeptides of a second family or species which are determined by functional, structural or genomic analyses to be a polynucleotide or polypeptide of the second family or species which corresponds to the original polynucleotide or polypeptide of the first family or species. Most often, homologs will have functional, structural or genomic similarities. Techniques are known by which homologs of a polynucleotide or polypeptide can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.
• a polypeptide has "homology” or is “homologous” to a second polypeptide if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene.
• a polypeptide has homology to a second polypeptide if the two polypeptides have "similar” amino acid sequences.
• homology to a second polypeptide if the two polypeptides have "similar” amino acid sequences.
• analogs refers to polynucleotide or polypeptide sequences that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure.
• Crabtree-positive yeast such as S. cerevisiae which have been engineered to disrupt, mutate, or delete one or more endogenous PDC genes cannot grow in medium containing elevated concentrations of glucose.
• PDC-deficient yeast are generally considered “C2-dependent,” meaning they require supplementation with C2 compounds, namely ethanol and acetate. While growth of PDC-deficient yeast in medium comprising ethanol and/or acetate is possible, the addition of such exogenous C2 compounds can be cost-prohibitive. Therefore, the ideal yeast biocatalyst would be capable of growth in the absence of exogenously added ethanol and/or acetate, i.e., "C2-independence.”
• the present inventors have identified a number of genetic modifications which allow PDC-deficient yeast mutants to exhibit (a) improved growth in the absence of C2-supplementation; and (b) improved tolerance to high glucose concentrations.
• the resulting PDC-deficient yeast strains generally exhibit improved growth characteristics, allowing for more economical production of desired pyruvate- derived metabolites (e.g., isobutanol).
• a "pyruvate-derived metabolite” can include any biosynthetic pathway which uses pyruvate as the starting material and/or as an intermediate.
• yeast cells convert sugars to produce pyruvate, which is then utilized in a number of pathways of cellular metabolism.
• yeast cells have been engineered to produce a number of desirable products via pyruvate-driven biosynthetic pathways.
• biosynthetic pathways which derive from pyruvate include pathways for the production of isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene, 3-methyl-l-butanol, 4-methyl-1- pentanol, coenzyme A, lactic acid, and malic acid.
• Engineered biosynthetic pathways for the synthesis of these beneficial pyruvate-derived metabolites are found in Table 1 and Figure 3.
• each of the products listed in Table 1 are derived from pyruvate.
• another compound which is derived from pyruvate is ethanol, the production of which can divert carbon flow away from the desired pyruvate-derived compound, thereby reducing optimal yield of the desired pyruvate- derived compound.
• recombinant yeast strains with reduced PDC activity have been constructed. See, e.g., commonly-owned US Patent No. 8,017,375.
• the commercial production of desired pyruvate-derived metabolites further depends in part, on the ability of the yeast to grow under conditions which are economically favored, i.e., growth on glucose and/or growth without the requirement for exogenous supplementation with C2-compounds, such as ethanol and/or acetate.
• C2-compounds such as ethanol and/or acetate.
• PDC-deficient yeast generally do not tolerate growth on glucose and require supplementation with C2-compounds.
• PDC-deficient yeast strains to (a) improve growth in the absence of C2-supplementation; and/or (b) improve tolerance to high glucose concentrations.
• the present application relates to a recombinant yeast microorganism comprising a metabolic pathway for the production of a pyruvate- derived metabolite, wherein said metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the production of the pyruvate-derived metabolite, wherein said recombinant yeast microorganism is engineered to comprise reduced PDC activity, and wherein said recombinant yeast microorganism is engineered to (a): comprise at least one first modification which results in improved growth in the absence of C2-supplementation as compared to the corresponding recombinant yeast microorganism without said first modification; and/or (b) comprise at least one second modification which results in improved tolerance to glucose as compared to the corresponding recombinant yeast microorganism without said second modification.
• the phrase "improved growth in the absence of C2- supplementation” refers to a yeast which grows to a higher optical density and/or exhibits a higher maximum specific growth rate in medium substantially devoid of exogenously added C2 compounds (i.e., medium containing less than about 0.1% v/v of the combined sum of one or more exogenously added C2 compounds, e.g., ethanol, acetate, etc.) as compared to the corresponding yeast microorganism that has not been engineered or selected to comprise a modification which improves growth in the absence of C2-supplementation.
• medium substantially devoid of exogenously added C2 compounds i.e., medium containing less than about 0.1% v/v of the combined sum of one or more exogenously added C2 compounds, e.g., ethanol, acetate, etc.
• growth may be determined by measuring yeast cell optical density in liquid media, e.g., an optical density of 600 nanometers (ODeoo")-
• a yeast with improved growth in the absence of C2-supplementation may grow to optical density which is 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 100%, 200%, 400%, 1000% or greater in medium substantially devoid of exogenous ethanol and/or acetate than the corresponding yeast microorganism that has not been engineered or selected to comprise a modification which improves growth in the absence of C2-supplementation.
• a yeast with improved growth in the absence of C2-supplementation may exhibit a maximum specific growth rate which is 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 100%, 200%, 400%, 1000% or greater in medium substantially devoid of exogenously added C2 compounds, e.g., ethanol and/or acetate, than the corresponding yeast microorganism that has not been engineered or selected to comprise a genetic modification which improves growth in the absence of C2- supplementation, wherein the maximum specific growth rate is defined as the maximum increase in cell mass per unit time, e.g., grams cells (g) per gram cells (g) per houn g x g "1 x h -1 .
• the phrase "improved tolerance to glucose” refers to a yeast which grows to a higher optical density and/or exhibits a higher maximum specific growth rate in 20 g/L glucose as compared to the corresponding yeast microorganism that has not been engineered or selected to comprise a modification which improves tolerance to glucose.
• a yeast with improved tolerance to glucose may grow to an optical density which is 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 100%, 200%, 400%, 1000% or greater in 20 g/L glucose than the corresponding yeast microorganism that has not been engineered or selected to comprise a modification which improves tolerance to glucose.
• a yeast with improved tolerance to glucose may exhibit a maximum specific growth rate which is 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 100%, 200%, 400%, 1000% or greater in 20 g/L glucose than the corresponding yeast microorganism that has not been engineered or selected to comprise a genetic modification which improves tolerance to glucose, wherein the maximum specific growth rate is defined as the maximum increase in cell mass per unit time, e.g., grams cells (g) per gram cells (g) per hour: g x g "1 x h '1 .
• tine present inventors have found that modification of GND1 (SEQ ID NO: 1) can be made to improve growth in the absence of C2-supplementation.
• the present inventors have identified a L340W mutation in Gndl p which is responsible, in part, for an improved ability to grow in the absence of C2-supplementation.
• the Gnd1 protein mutation, L340W represents a gain-of-activity mutation as compared to the Gnd1 wild-type protein.
• mutant protein GND1 L340W expression of the mutant protein GND1 L340W , overexpression of the wild-type Gndl p, and/or overexpression of mutant protein GND1 L340W can be utilized to improve host cell growth in the absence of C2-supplementation in strains engineered to harbor reduced pyruvate decarboxylase activity.
• said first modification is a mutation of GND1 (SEQ ID NO: 1 ) or homologs and variants thereof.
• said first modification is a point mutation of GND1.
• said first modification is a point mutation of GND1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of L340 of Gndlp (SEQ ID NO: 2).
• said first modification is a point mutation of GND1 which results in an amino acid substitution of L340 of Gndl p, wherein said L340 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of GND1 which results in an amino acid substitution of L340 of Gndlp, wherein said L340 residue is replaced with a tryptophan residue.
• said first modification is an increase in the heterologous or native expression of a Gnd1 protein. Accordingly, in one embodiment, said first modification is the overexpression of one or more polynucleotides encoding one or more Gnd1 proteins or homologs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Gnd1 proteins or homologs thereof is an endogenous polynucleotide. In another embodiment, one or more of the polynucleotides encoding said one or more Gnd1 proteins or homologs thereof is an exogenous polynucleotide.
• the Gnd1 protein is selected from SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , and SEQ ID NO: 22.
• the Gnd1 protein is a mutant Gnd1 protein.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L340 residue of Gndlp (SEQ ID NO: 2), wherein said L340 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L340 residue of Gnd p (SEQ ID NO: 2), wherein said L340 residue is replaced with a tryptophan residue.
• the mutant Gnd1 protein comprises the amino acid sequence of SEQ ID NO: 23.
• the present application is directed to an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 23.
• Gndlp is a 6-phosphogluconate dehydrogenase (decarboxylating) that has been reported to catalyze an NADPH regenerating reaction in the pentose phosphate pathway.
• GND1 encodes the major isoform of 6- phosphogluconate dehydrogenase, accounting for approximately 80% of the cell's 6- phosphogluconate dehydrogenase activity ⁇ i.e., "GND" activity
• GND2 encodes the minor isoform, accounting for the other 20% of the cell's 6-phosphogluconate dehydrogenase activity ⁇ i.e., "GND” activity).
• GND1 Homologs of GND1 are known to occur in yeast other than S. cerevisiae.
• GND2 encodes the minor isoform of 6- phosphogluconate dehydrogenase, Gnd2p (SEQ ID NO: 25).
• Gnd2p accounts for approximately 20% of the cell's 6-phosphogluconate dehydrogenase activity and is relatively homologous to Gndlp - SEQ ID NO: 2 (Gndlp) and SEQ ID NO: 25 (Gnd2p) exhibit 88% amino acid identity and are conserved at the L340 identified by the present inventors as representing a gain of activity mutation (L340 in Gndl p corresponds to L343 in Gnd2p).
• mutant Gnd2 protein Gnd2 L343W
• said first modification is a mutation of GND2 (SEQ ID NO: 24) or homologs and variants thereof.
• said first modification is a point mutation of GND2.
• said first modification is a point mutation of GND2 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of L343 of Gnd2p (SEQ ID NO: 25).
• said first modification is a point mutation of GND2 which results in an amino acid substitution of L343 of Gnd2p, wherein said L343 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of GND2 which results in an amino acid substitution of L343 of Gndlp, wherein said L343 residue is replaced with a tryptophan residue.
• said first modification is an increase in the heterologous or native expression of a Gnd2 protein. Accordingly, in one embodiment, said first modification is the overexpression of one or more polynucleotides encoding one or more Gnd2 proteins or homologs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Gnd2 proteins or homologs thereof is an endogenous polynucleotide. In another embodiment, one or more of the polynucleotides encoding said one or more Gnd2 proteins or homologs thereof is an exogenous polynucleotide.
• the Gnd1 protein is selected from SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
• the Gnd2 protein is a mutant Gnd2 protein.
• the mutant Gnd2 protein comprises an amino acid substitution at an amino acid position corresponding to the L343 residue of Gnd2p (SEQ ID NO: 25), wherein said L343 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the mutant Gnd2 protein comprises an amino acid substitution at an amino acid position corresponding to the L343 residue of Gnd2p (SEQ ID NO: 25), wherein said L343 residue is replaced with a tryptophan residue.
• the mutant Gnd2 protein comprises the amino acid sequence of SEQ ID NO: 28.
• the present application is directed to an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 28.
• GND2 Homologs of GND2 are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, an endogenous Gnd2p protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neumspora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces,
• Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Thchoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces may be (1) mutated as described above, e.g., be mutated to comprise a mutation at a position corresponding to the L343 position of Gnd2p (SEQ ID NO: 25); (2) overexpressed as described above, e.g., overexpressed in a recombinant yeast cell engineered to have reduced pyruvate decarboxylase activity; and/or (3) mutated as described above, e.g., be mutated to comprise a mutation at a position corresponding to the L343 position of Gnd2p (SEQ ID NO: 25) and overexpressed as described above, e.g., overexpressed in a recombinant yeast cell engineered to have reduced pyruvate decarboxylase activity.
• the present inventors have found that a modified UME6 gene is present in mutants with improved growth in the absence of C2-supplementation. Specifically, the present inventors have identified a R768C mutation in Ume6p which is present in mutants with improved growth in the absence of C2-supplementation.
• said first modification is a mutation, disruption, or deletion of UME6 (SEQ ID NO: 3).
• said first modification is a mutation of UME6.
• said first modification is a point mutation of UME6.
• said first modification is a point mutation of UME6 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of R768 of Ume6p (SEQ ID NO: 4).
• said first modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a residue selected from the group consisting of histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a cysteine residue.
• Ume6p (Unscheduled Meiotic gene Expression) is a key transcriptional regulator of early meiotic genes, such as SP011, SP013, and IME2. Ume6p is required for the repression of these genes during mitosis and its destruction during meiosis is required for their meiotic induction.
• This C6 zinc cluster DNA binding protein provides target specificity by binding to the URS1 sequence element that is located upstream of many early meiosis-specific genes. See Strich et a/., 994, Genes Dev 8(7): 796-810. Homologs of UME6 are known to occur in yeast other than S. cerevisiae.
• the present inventors have found that a modified SEC27 gene is present in mutants with improved growth in the absence of C2-supplementation. Specifically, the present inventors have identified a C145S mutation in Sec27p which is present in mutants with improved growth in the absence of C2-supplementation.
• said first modification is a mutation, disruption, or deletion of SEC27 (SEQ ID NO: 5).
• said first modification is a mutation of SEC27.
• said first modification is a point mutation of SEC27.
• said first modification is a point mutation of SEC27 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of C145 of Sec27p (SEQ ID NO: 6).
• said first modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a serine residue.
• Sec27p is an essential beta-coat protein of the COPI coatomer which is involved in ER-to-Golgi and Golgi-to-ER transport. Sec27p is known to contain WD40 domains that mediate cargo selective interactions. See Eugster et a/., 2004, Mol. Biol. Cell 15(3): 1011-23. Homologs of SEC27 are known to occur in yeast other than S. cerevisiae.
• the present inventors have found that a modified ZRT1 gene is present in mutants with improved growth in the absence of C2-supplementation. Specifically, the present inventors have identified a D245E mutation in Zrt1 p which is present in mutants with improved growth in the absence of C2-supplementation .
• said first modification is a mutation, disruption, or deletion of ZRT1 (SEQ ID NO: 7).
• said first modification is a mutation of ZRT1.
• said first modification is a point mutation of ZRT1.
• said first modification is a point mutation of ZRT1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of D245 of Zr p (SEQ ID NO: 8).
• said first modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of ZrMp, wherein said D245 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zr p, wherein said D245 residue is replaced with a glutamic acid residue.
• ZrM p encodes a high-affinity zinc transporter. See Zhao et al., 1996, PNAS 93(6): 2454-8. There are two systems for zinc uptake in yeast, one high- affinity and one lower-affinity. ZrMp and the lower-affinity zinc transporter Zrt2p are similar to each other, and are members of the ZIP family of heavy metal ion transporters. Zap1 p is a transcriptional regulator that controls levels of ZRT1 and ZRT2 mRNAs in response to intracellular zinc levels. Levels of ZrMp are further controlled by regulated degradation. Homologs of ZRT1 are known to occur in yeast other than S. cerevisiae.
• an endogenous ZrM p protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, 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, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium,
• said first modification is a mutation, disruption, or deletion of YLL056C (SEQ ID NO: 9).
• said first modification is a mutation of YLL056C.
• said first modification is a point mutation of YLL056C.
• said first modification is a point mutation of YLL056C which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of I5 of YLL056c (SEQ ID NO: 10).
• said first modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056c, wherein said I5 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056c, wherein said I5 residue is replaced with a residue selected from the group consisting of arginine,
• YLL056c is a putative protein of unknown function. Transcription of YLL056c is activated by paralogous transcription factors Yrm1p and Yr p and genes involved in pleiotropic drug resistance, while expression is induced in cells treated with the mycotoxin patulin. See Iwahashi et al., 2006, J. Agric. Food Chem. 54(5): 1936-42. Homologs of YLL056C are known to occur in yeast other than S. cerevisiae.
• the present inventors have found that a modified YOL014W gene is present in mutants with improved growth in the absence of C2-supplementation. Specifically, the present inventors have identified a A39D mutation in YOL014w which is present in mutants with improved growth in the absence of C2-supplementation.
• said first modification is a mutation, disruption, or deletion of YOL014W (SEQ ID NO: 11).
• said first modification is a mutation of YOL014W.
• said first modification is a point mutation of YOL014W.
• said first modification is a point mutation of YOL014W which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of A39 of YOL014W (SEQ ID NO: 12).
• said first modification is a point mutation of YOL074W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with an aspartic acid residue.
• YOL014w is a putative protein of unknown function. Homologs of YOL014W are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, an endogenous YOL014w protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces,
• said first modification is a point mutation at a position corresponding to the nucleotides listed in Table 2.
• said point mutation occurs in a chromosome selected from chromosomes I, IV, V, VII, VIII, X, XII, XIII, and XV.
• modification of MTH1 SEQ ID NO: 13
• modification of MTH1 SEQ ID NO: 13
• the present inventors have identified a partial deletion ( ⁇ 41-78) in Mthlp which is responsible, in part, for an improved ability to grow on glucose.
• said second modification is a mutation, disruption, or deletion of MTH1 (SEQ ID NO: 13) or homologs or variants thereof.
• said second modification is a partial deletion of MTH1.
• said second modification is a partial deletion of MTH1, wherein said partial deletion results in at least one amino acid deletion between amino acid positions 25 to 100 of Mthlp (SEQ ID NO: 14).
• said second modification is a partial deletion of MTH1, wherein said partial deletion results in at least one amino acid deletion between amino acid positions 40 to 80 of Mthlp.
• said second modification is a partial deletion of MTH1, wherein said partial deletion results in the deletion of amino acid residues 41 to 78 of Mthlp.
• said second modification is a point mutation of MTH1.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of I85 of Mthlp, wherein said 185 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of I85 of Mthlp, wherein said 185 residue is replaced with an asparagine or serine residue.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of S102 of Mthlp, wherein said S102 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of S102 of Mthlp, wherein said S102 residue is replaced with a glycine residue.
• Mthl p is a negative regulator of the glucose-sensing signal transduction pathway and is required for repression of Rgt1 p. It is known to interact with Rgt1 p and the Snf3p and Rgt2p glucose sensors. See Lafuente et al., 2000, Mol. Microbiol. 35(1): 161-72. Homologs of MTH1 are known to occur in yeast other than S. cerevisiae.
• an endogenous Mthl p protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberelia, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotica, Sorda a, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium,
• Mthlp has a syntenic homolog known as Stdlp, encoded by STD1 (also known as MSN3). See Lafuente et al., 2000, Mol. Microbiol. 35: 161-72. Mthl p and Stdlp share approximately 60% sequence identity, and both act as transcription co-repressor proteins. Given the similar roles of Mthl p and Stdlp, and their sequence identity, one skilled in the art, equipped with the present disclosure, would understand that the Mthl p mutations described herein as relevant to glucose tolerance may similarly be made to the corresponding positions of Stdlp (GenBank Accession No. NP_014690.1).
• said second modification is a partial deletion of STD1.
• Said partial deletion may occur between amino acid positions 49 to 89 of Stdlp, which correspond to positions 40 to 80 of Mthl p.
• said partial deletion of Stdlp may result in the deletion of amino acid residues 50 to 87 of Mthlp.
• said second modification is a point mutation of STD1.
• said second modification is a point mutation of STD1 which results in an amino acid substitution of 194 of Stdlp, which corresponds to I85 of Mthlp.
• said second modification is a point mutation of Stdlp which results in an amino acid substitution of 194 of Std1 p, wherein said I94 residue is replaced with an asparagine or serine residue.
• the Mthl p mutation and the associated glucose-tolerant phenotype described in Example 2 modify key aspects of the yeast signaling and transport system for glucose, a preferred carbon source for growth.
• This glucose signaling and transport system represses the genes required for the utilization of alternative carbon sources, gluconeogenesis and respiration when growth is readily available. Kaniak et al., 2004, Euk. Cell 3(1 ): 221 -231.
• the repressor proteins Rgt1 can no longer repress the genes encoding hexose transporters (HXT4, HXT3, HXT2, HXT1, HXT5, and HXT8) and a suite of other genes (YKR075C, YGL157W, STD1, MIG2, YNL234W, YOR062C, AQR1, AHP1, PHM8, YOR338W, YKL036C, CIT2, MIG3, YGL039W, UGP1, CMK2, YHR087W, DIA1, HOR2, VID24, CAD1, YHR097C, YOL046C, TIP1, MTH1, PFK2, YFL054C, NTH1, SKS1, YLR413W, YDL062W, GAT2, YFR016C, UBP11, ECM4, YLR194C, and YAL0
• MTH1 partial deletion described in Example 2 results in a Mth1 protein that is not capable of being targeted for degradation, thereby resulting in a constitutively active allele of Mth1.
• this constitutively active allele of Mth1 may subsequently cause the Rgt1 protein to remain as a repressor of many genes including those that encode hexose transporters.
• hexose transporter expression will remain unchanged. Consequently, there will be no increase in the intracellular glucose concentration and no repression of genes via the SNF1 protein kinase signaling pathway, allowing the genes required for growth on alternative carbon sources, gluconeogenesis and respiration to remain expressed.
• Pdc-deficient cells can now grow on high glucose medium because the expression of respiratory metabolism genes occurs.
• SNF3/RGT2 signaling pathway mutations with similar effects to the Mthlp mutation described herein should result in constitutive repression of Rgt1 targets.
• Such SNF3 RGT2 signaling pathway mutations include the disruption, deletion, or mutation of Snf3p and/or Rgt2p. Mutations in these genes that prevent the cells from sensing high extracellular glucose concentrations include Sn ⁇ 3 null mutants described in Neigebom et al., 1986, Mol. Cell. Biol. 6(11 ): 3569-74 and Rgt2 disruptions described in Ozcan et ai, 1996, PNAS 93(22): 12428-32.
• mutations in Snf3 and/or Rgt2 that prevent interaction of their gene products with Yck1 may reduce and/or eliminate the phosphorylation of Std1 and/or Mth1.
• the phosphorylation targets these two co-repressor proteins for degradation resulting in constitutive repression of the genes downstream of Rgt1 including the hexose transporters.
• mutations in Snf3, Rgt2, Std1 , and/or Mth1 that eliminate or reduce the interaction of the glucose sensors with the co-repressors may also restore growth on glucose by Pdc- cells. This will reduce or eliminate the degradation of Std1 and/or Mth1 resulting in constitutive repression of the genes downstream of Rgt1 including the hexose transporters.
• Mutations in STD1 and/or MTH1 that result in the proteins becoming constitutive repressors may also enable Pdc-deficient cells to grow on glucose. These mutations could include the overexpression of one or both proteins (Hubbard, ef a/., 994, Mol. Cell. Biol. 14(3): 1972-78, mutant alleles that cannot be degraded via the ubiquitin mediated pathway or constitutively active alleles such as HTR1-23 (I85N), HTR1-19 (I85N) or HTR1-5 (I85S). Schulte et al., 2000, J. Bacteriol. 182(2): 540-2.
• Mutations in RTG1 make this transcriptional repressor constitutively active may additionally enable Pdc-deficient cells to grow on glucose.
• These mutations could include alleles that no long require the interaction with Std1 and/or Mth1 , as well as alleles with increased affinity for the c/s-acting promoter elements upstream of Rtg1 genes.
• mutations in glucose uptake signaling pathway should also prevent the repression of respiration - these include mutations in hexose transporter genes that prevent and/or reduce high intracellular glucose concentrations.
• Mutant alleles can also include promoter mutations that prevent high levels of expression, mutations that make the encoded-proteins less stable, and/or the deletion of subset of hexose-transporter-encoding genes.
• mutations that make the kinase Snfl constitutively active regardless of the glucose concentrations may also be used to allow Pdc-deficient cells to grow on high glucose media.
• Such mutations include the overexpression of SNF1, point mutations that alter the protein's activity (See, e.g., Leech et ai, 2003, Euk. Cell 2(2): 265-273), and/or mutations in its associated proteins Snf4 and/or Gal83.
• the present inventors have found that a modified UME6 gene is present in mutants with improved tolerance to growth on glucose. Specifically, the present inventors have identified a R768C mutation in Ume6p which is present in mutants with an improved ability to grow on glucose.
• said second modification is a mutation, disruption, or deletion of UME6 (SEQ ID NO: 3).
• said second modification is a mutation of UME6.
• said second modification is a point mutation of UME6.
• said second modification is a point mutation of UME6 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of R768 of Ume6p (SEQ ID NO: 4).
• said second modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a residue selected from the group consisting of histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a cysteine residue.
• the present inventors have found that a modified SEC27 gene is present in mutants with improved tolerance to growth on glucose. Specifically, the present inventors have identified a C145S mutation in Sec27p which is present in mutants with an improved ability to grow on glucose.
• said second modification is a mutation, disruption, or deletion of SEC27 (SEQ ID NO: 5).
• said second modification is a mutation of SEC27.
• said second modification is a point mutation of SEC27.
• said second modification is a point mutation of SEC27 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of C145 of Sec27p (SEQ ID NO: 6).
• said second modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a serine residue.
• the present inventors have found that a modified ZRT1 gene is present in mutants with improved tolerance to growth on glucose. Specifically, the present inventors have identified a D245E mutation in Zrt1 p which is present in mutants with an improved ability to grow on glucose.
• said second modification is a mutation, disruption, or deletion of ZRT1 (SEQ ID NO: 7).
• said second modification is a mutation of ZRT1.
• said second modification is a point mutation of ZRT1.
• said second modification is a point mutation of ZRT1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of D245 of Zrt1 p (SEQ ID NO: 8).
• said second modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrtlp, wherein said D245 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrt1 p, wherein said D245 residue is replaced with a glutamic acid residue.
• the present inventors have found that a modified YLL056C gene is present in mutants with improved tolerance to growth on glucose. Specifically, the present inventors have identified a I5V mutation in YLL056c which is present in mutants with an improved ability to grow on glucose.
• said second modification is a mutation, disruption, or deletion of YLL056C (SEQ ID NO: 9).
• said second modification is a mutation of YLL056C.
• said second modification is a point mutation of YLL056C.
• said second modification is a point mutation of YLL056C which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of I5 of YLL056c (SEQ ID NO: 10).
• said second modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056c, wherein said I5 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056c, wherein said I5 residue is replaced with a valine residue.
• the present inventors have found that a modified YOL014Wgene is present in mutants with improved tolerance to growth on glucose. Specifically, the present inventors have identified an A39D mutation in YOL014w which is present in mutants with an improved ability to grow on glucose.
• said second modification is a mutation, disruption, or deletion of YOL014W (SEQ ID NO: 11).
• said second modification is a mutation of YOL014W.
• said second modification is a point mutation of YOL014W.
• said second modification is a point mutation of YOL014W which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of A39 of YOL014w (SEQ ID NO: 12).
• said second modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with an aspartic acid residue.
• said second modification is a point mutation at a position corresponding to the nucleotides listed in Table 2.
• said point mutation occurs in a chromosome selected from chromosomes I, IV, V, VII, VIII, X, XII, XIII, and XV.
• the yeast microorganism has reduced or no pyruvate decarboxylase (PDC) activity.
• PDC catalyzes the decarboxylation of pyruvate to acetaldehyde, which is then reduced to ethanol by ADH via an oxidation of NADH to NAD+.
• Ethanol production is the main pathway to oxidize the NADH from glycolysis. Deletion, disruption, or mutation of this pathway increases the pyruvate and the reducing equivalents (NADH) available for a biosynthetic pathway which uses pyruvate as the starting material and/or as an intermediate.
• NADH reducing equivalents
• deletion, disruption, or mutation of one or more genes encoding for pyruvate decarboxylase and/or a positive transcriptional regulator thereof can further increase the yield of the desired pyruvate-derived metabolite ⁇ e.g., isobutanol).
• said pyruvate decarboxylase gene targeted for disruption, deletion, or mutation is selected from the group consisting of PDC1, PDC5, and PDC6, or homologs or variants thereof.
• all three of PDC1, PDC5, and PDC6 are targeted for disruption, deletion, or mutation.
• a positive transcriptional regulator of the PDC1, PDC5, and/or PDC6 is targeted for disruption, deletion or mutation.
• said positive transcriptional regulator is PDC2, or homologs or variants thereof.
• strains that naturally produce low levels of pyruvate decarboxylase can also have applicability for producing increased levels of a pyruvate-derived metabolite.
• strains that naturally produce low levels of pyruvate decarboxylase may inherently exhibit low or undetectable levels of pyruvate decarboxylase activity, a trait which may be favorable for the production of a pyruvate-derived metabolite not requiring such pyruvate decarboxylase activity for metabolite synthesis.
• the present application relates to a recombinant yeast microorganism comprising an engineered isobutanol producing metabolic pathway.
• yeast cells have been engineered to produce increased quantities of isobutanol, an important commodity chemical and biofuel candidate (See, e.g., commonly owned US Patent Nos. 8,017,375, 8,017,376, 8,071 ,358, 8,097,440, 8,133,175, 8,153,415, 8, 58,404, 8,232,089, and 8,273,565).
• the present invention relates to recombinant microorganisms for producing isobutanol, wherein said recombinant microorganisms comprise an isobutanol producing metabolic pathway.
• the isobutanol producing metabolic pathway to convert pyruvate to isobutanol can be comprised of the following reactions:
• these reactions are carried out by the enzymes 1) Acetolactate synthase (ALS), 2) Ketol-acid reductoisomerase (KARI), 3) Dihydroxy- acid dehydratase (DHAD), 4) 2-keto-acid decarboxylase, e.g., Keto-isovalerate decarboxylase (KIVD), and 5) an Alcohol dehydrogenase (ADH) ( Figure 1).
• the recombinant microorganism may be engineered to overexpress one or more of these enzymes.
• the recombinant microorganism is engineered to overexpress all of these enzymes.
• isobutanol producing metabolic pathway comprises five substrate to product reactions.
• the isobutanol producing metabolic pathway comprises six substrate to product reactions.
• the isobutanol producing metabolic pathway comprises seven substrate to product reactions.
• the present inventors have discovered that one or more genetic modifications can be introduced into PDC-deficient yeast strains to (a) improve growth in the absence of C2-supplementation; and/or (b) improve tolerance to high glucose concentrations, thereby resulting in PDC-deficient yeast strains with improved growth characteristics, allowing for more economical production of desired pyruvate-derived metabolites.
• the pyruvate-derived metabolite is isobutanol.
• the present application relates to a recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, wherein said recombinant yeast microorganism is engineered to comprise reduced PDC activity, and wherein said recombinant yeast microorganism is engineered to (a): comprise at least one first modification which results in improved growth in the absence of C2-supplementation as compared to the corresponding recombinant yeast microorganism without said first modification; and/or (b) comprise at least one second modification which results in improved tolerance to glucose as compared to the corresponding recombinant yeast microorganism without said second modification.
• said first modification is a mutation of GND1 (SEQ ID NO: 1 ) or homologs and variants thereof.
• said first modification is a point mutation of GND1.
• said first modification is a point mutation of GND1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of L340 of Gndlp (SEQ ID NO: 2).
• said first modification is a point mutation of GND1 which results in an amino acid substitution of L340 of Gndl p, wherein said L340 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of GND1 which results in an amino acid substitution of L340 of Gndlp, wherein said L340 residue is replaced with a tryptophan residue.
• said first modification is an increase in the heterologous or native expression of a Gnd1 protein. Accordingly, in one embodiment, said first modification is the overexpression of one or more polynucleotides encoding one or more Gnd1 proteins or homologs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Gnd1 proteins or homologs thereof is an endogenous polynucleotide. In another embodiment, one or more of the polynucleotides encoding said one or more Gnd1 proteins or homologs thereof is an exogenous polynucleotide.
• the Gnd1 protein is selected from SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , and SEQ ID NO: 22.
• the Gnd1 protein is a mutant Gnd1 protein.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L340 residue of Gndlp (SEQ ID NO: 2), wherein said L340 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L340 residue of Gndl p (SEQ ID NO: 2), wherein said L340 residue is replaced with a tryptophan residue.
• the mutant Gnd1 protein comprises the amino acid sequence of SEQ ID NO: 23.
• the present application is directed to an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 23.
• GND1 Homologs of GND1 are known to occur in yeast other than 5. cerevisiae. Accordingly, in additional embodiments, an endogenous Gndlp protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, 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, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces may be (1) mutated as described above, e.g., be mutated to comprise a mutation at a position corresponding to the L340 position of Gndlp (SEQ ID NO: 2); (2) overexpressed as described above, e.g., overexpressed in a recombinant yeast cell engineered to have reduced pyruvate decarboxylase activity; and/or (3) mutated as described above, e.g., be mutated to comprise a mutation at a position corresponding to the L340 position of Gndlp (SEQ ID NO: 2) and overexpressed as described above, e.g., overexpressed in a recombinant yeast cell engineered to have reduced pyruvate decarboxylase activity.
• said first modification is a mutation of GND2 (SEQ ID NO: 24) or homologs and variants thereof.
• said first modification is a point mutation of GND2.
• said first modification is a point mutation of GND2 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of L343 of Gnd2p (SEQ ID NO: 24).
• said first modification is a point mutation of GND2 which results in an amino acid substitution of L343 of Gndlp, wherein said L343 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of GND2 which results in an amino acid substitution of L343 of Gnd2p, wherein said L343 residue is replaced with a tryptophan residue.
• said first modification is an increase in the heterologous or native expression of a Gnd2 protein. Accordingly, in one embodiment, said first modification is the overexpression of one or more polynucleotides encoding one or more Gnd2 proteins or homologs thereof. In one embodiment, one or more of the polynucleotides encoding said one or more Gnd2 proteins or homologs thereof is an endogenous polynucleotide. In another embodiment, one or more of the polynucleotides encoding said one or more Gnd2 proteins or homologs thereof is an exogenous polynucleotide.
• the Gnd2 protein is selected from SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
• the Gnd2 protein is a mutant Gnd2 protein.
• the mutant Gnd2 protein comprises an amino acid substitution at an amino acid position corresponding to the L343 residue of Gnd2p (SEQ ID NO: 25), wherein said L343 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine glycine, proline, alanine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• the mutant Gnd1 protein comprises an amino acid substitution at an amino acid position corresponding to the L343 residue of Gnd2p (SEQ ID NO: 25), wherein said L343 residue is replaced with a tryptophan residue.
• the mutant Gnd2 protein comprises the amino acid sequence of SEQ ID NO: 28.
• the present application is directed to an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 28.
• GND2 Homologs of GND2 are known to occur in yeast other than S. cerevisiae. Accordingly, in additional embodiments, an endogenous Gndlp protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Glomerella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neumspora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces,
• Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Thchoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium, Yarrowia or Zygosaccharomyces may be (1) mutated as described above, e.g., be mutated to comprise a mutation at a position corresponding to the L343 position of Gnd2p (SEQ ID NO: 25); (2) overexpressed as described above, e.g., overexpressed in a recombinant yeast cell engineered to have reduced pyruvate decarboxylase activity; and/or (3) mutated as described above, e.g., be mutated to comprise a mutation at a position corresponding to the L343 position of Gnd2p (SEQ ID NO: 25) and overexpressed as described above, e.g., overexpressed in a recombinant yeast cell engineered to have reduced pyruvate decarboxylase activity.
• said first modification is a mutation, disruption, or deletion of UME6 (SEQ ID NO: 3). In one embodiment, said first modification is a mutation of UME6. In another embodiment, said first modification is a point mutation of UME6. In a specific embodiment, said first modification is a point mutation of UME6 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of R768 of Ume6p (SEQ ID NO: 4).
• said first modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a residue selected from the group consisting of histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a cysteine residue.
• Homologs of UME6 are known to occur in yeast other than S. cerevisiae.
• said first modification is a mutation, disruption, or deletion of SEC27 (SEQ ID NO: 5). In one embodiment, said first modification is a mutation of SEC27. In another embodiment, said first modification is a point mutation of SEC27. In a specific embodiment, said first modification is a point mutation of SEC27 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of C145 of Sec27p (SEQ ID NO: 6).
• said first modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a serine residue.
• said first modification is a mutation, disruption, or deletion of ZRT1 (SEQ ID NO: 7). In one embodiment, said first modification is a mutation of ZRT1. In another embodiment, said first modification is a point mutation of ZRT1. In a specific embodiment, said first modification is a point mutation of ZRT1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of D245 of Zrt1 p (SEQ ID NO: 8).
• said first modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrt1p, wherein said D245 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrtlp, wherein said D245 residue is replaced with a glutamic acid residue.
• Homologs of ZRT1 are known to occur in yeast other than S. cerevisiae.
• an endogenous Zrtl p protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, 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, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium
• said first modification is a mutation, disruption, or deletion of YLL056C (SEQ ID NO: 9). In one embodiment, said first modification is a mutation of YLL056C. In another embodiment, said first modification is a point mutation of YLL056C. In a specific embodiment, said first modification is a point mutation of YLL056C which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of 15 of YLL056c (SEQ ID NO: 10).
• said first modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056c, wherein said I5 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056C, wherein said I5 residue is replaced with a valine residue.
• Homologs of YLL056C are known to occur in yeast other than S. cerevisiae.
• an endogenous YLL056c protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Glomemlla, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotica, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticili
• said first modification is a mutation, disruption, or deletion of YOL014W (SEQ ID NO: 11). In one embodiment, said first modification is a mutation of YOL014W. In another embodiment, said first modification is a point mutation of YOL014W. In a specific embodiment, said first modification is a point mutation of YOL014W which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of A39 of YOL0 4w (SEQ ID NO: 12).
• said first modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said first modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with an aspartic acid residue.
• Homologs of YOL014W are known to occur in yeast other than S. cerevisiae.
• said first modification is a point mutation at a position corresponding to the nucleotides listed in Table 2.
• said point mutation occurs in a chromosome selected from chromosomes I, IV, V, VII, VIII, X, XII, XIII, and XV.
• said second modification is a mutation, disruption, or deletion of MTH1 (SEQ ID NO: 13) or homologs or variants thereof.
• said second modification is a partial deletion of MTH1.
• said second modification is a partial deletion of MTH1, wherein said partial deletion results in at least one amino acid deletion between amino acid positions 25 to 100 of Mthl p (SEQ ID NO: 14).
• said second modification is a partial deletion of MTH1, wherein said partial deletion results in at least one amino acid deletion between amino acid positions 40 to 80 of Mth1 p.
• said second modification is a partial deletion of MTH1, wherein said partial deletion results in the deletion of amino acid residues 41 to 78 of Mthl p.
• said second modification is a point mutation of MTH1.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of I85 of Mthlp, wherein said 185 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of I85 of Mthl p, wherein said I85 residue is replaced with an asparagine or serine residue.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of S102 of Mthlp, wherein said S102 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of MTH1 which results in an amino acid substitution of S102 of Mthlp, wherein said S102 residue is replaced with a glycine residue.
• Homologs of MTH1 are known to occur in yeast other than S. cerevisiae.
• an endogenous Mthlp protein derived from a yeast selected from Ajellomyces, Arthroderma, Ashbya, Aspergillus, Botryotinia, Candida, Chaetomium, Clavispora, Coccidioides, Debaryomyces, Gibberella, Glo erella, Grosmannia, Issatchenkia, Kluyveromyces, Leptosphaeria, Lodderomyces, Magnaporthe, Metarhizium, Meyerozyma, Mycosphaerella, Nectria, Neosartorya, Neurospora, Paracoccidioides, Penicillium, Phaeosphaeria, Pichia, Podospora, Pyrenophora, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sclerotinia, Sordaria, Talaromyces, Trichoderma, Trichophyton, Tuber, Uncinocarpus, Verticillium,
• said second modification is a mutation, disruption, or deletion of UME6 (SEQ ID NO: 3). In one embodiment, said second modification is a mutation of UME6. In another embodiment, said second modification is a point mutation of UME6. In a specific embodiment, said second modification is a point mutation of UME6 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of R768 of Ume6p (SEQ ID NO: 4).
• said second modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a residue selected from the group consisting of histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of UME6 which results in an amino acid substitution of R768 of Ume6p, wherein said R768 residue is replaced with a cysteine residue.
• said second modification is a mutation, disruption, or deletion of SEC27 (SEQ ID NO: 5). In one embodiment, said second modification is a mutation of SEC27. In another embodiment, said second modification is a point mutation of SEC27. In a specific embodiment, said second modification is a point mutation of SEC27 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of C145 of Sec27p (SEQ ID NO: 6).
• said second modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of SEC27 which results in an amino acid substitution of C145 of Sec27p, wherein said C145 residue is replaced with a serine residue.
• said second modification is a mutation, disruption, or deletion of ZRT1 (SEQ ID NO: 7). In one embodiment, said second modification is a mutation of ZRT1. In another embodiment, said second modification is a point mutation of ZRT1. In a specific embodiment, said second modification is a point mutation of ZRT1 which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of D245 of Zrtlp (SEQ ID NO: 8).
• said second modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrt1 p, wherein said D245 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of ZRT1 which results in an amino acid substitution of D245 of Zrt1 p, wherein said D245 residue is replaced with a glutamic acid residue.
• said second modification is a mutation, disruption, or deletion of YLLQ56C (SEQ ID NO: 9). In one embodiment, said second modification is a mutation of YLL056C. In another embodiment, said second modification is a point mutation of YLL056C. In a specific embodiment, said second modification is a point mutation of YLL056C which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of 15 of YLL056c (SEQ ID NO: 10).
• said second modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056C, wherein said I5 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of YLL056C which results in an amino acid substitution of I5 of YLL056c, wherein said I5 residue is replaced with a valine residue.
• said second modification is a mutation, disruption, or deletion of YOL014W (SEQ ID NO: 11 ).
• said second modification is a mutation of YOL014W.
• said second modification is a point mutation of YOL014W.
• said second modification is a point mutation of YOL014W which results in an amino acid substitution at an amino acid position which is within 5 Angstroms of A39 of YOL014w (SEQ ID NO: 12).
• said second modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with a residue selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.
• said second modification is a point mutation of YOL014W which results in an amino acid substitution of A39 of YOL014w, wherein said A39 residue is replaced with an aspartic acid residue.
• said second modification is a point mutation at a position corresponding to the nucleotides listed in Table 2.
• said point mutation occurs in a chromosome selected from chromosomes I, IV, V, VII, VIII, X, XII, XIII, and XV
• the yeast microorganism comprising an isobutanol producing metabolic pathway 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 an isobutanol biosynthetic pathway. Accordingly, 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 isobutanol.
• said pyruvate decarboxylase gene targeted for disruption, deletion, or mutation is selected from the group consisting of PDC1, PDC5, and POC6, 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.
• strains that naturally produce low levels of pyruvate decarboxylase can also have applicability for producing increased levels of isobutanol.
• strains that naturally produce low levels of pyruvate decarboxylase may inherently exhibit low or undetectable levels of pyruvate decarboxylase activity, a trait which may be favorable for the production of isobutanol.
• the recombinant microorganism comprises an engineered isobutanol producing metabolic pathway.
• the isobutanol producing metabolic pathway comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanol.
• the isobutanol producing metabolic pathway comprises at least two exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol.
• the isobutanol producing metabolic pathway comprises at least three exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol.
• the isobutanol producing metabolic pathway comprises at least four exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, the isobutanol producing metabolic pathway comprises at least five exogenous genes encoding polypeptides that catalyze steps in the conversion of pyruvate to isobutanol. In yet another embodiment, all of the isobutanol producing metabolic pathway steps in the conversion of pyruvate to isobutanol are converted by exogenously encoded enzymes.
• one or more of the isobutanol pathway genes encodes an enzyme that is localized to the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least two isobutanol pathway enzymes localized in the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least three isobutanol pathway enzymes localized in the cytosol.
• the recombinant microorganisms comprise an isobutanol producing metabolic pathway with at least four isobutanol pathway enzymes localized in the cytosol. In an exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with five isobutanol pathway enzymes localized in the cytosol. In yet another exemplary embodiment, the recombinant microorganisms comprise an isobutanol producing metabolic pathway with all isobutanol pathway enzymes localized in the cytosol. Isobutanol producing metabolic pathways in which one or more genes are localized to the cytosol are described in commonly owned U.S. Patent No. 8,232,089, which is herein incorporated by reference in its entirety for all purposes.
• isobutanol pathway enzymes including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including Y. spp.
• Sources of genes from anaerobic fungi include, but not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp.
• Sources of prokaryotic enzymes that are useful include, but not limited to, Escherichia spp., Zymomonas spp., Staphylococcus spp., Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Slackia spp., Lactococcus spp., Enterobacter spp., Streptococcus spp., Salmonella spp., Bacteroides spp., Methanococcus spp., Erythrobacter spp., Sphingomonas spp., Sphingobium spp., and Novosphingobium 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 acetolactate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including B. subtilis (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. ZP_06014957.1 ), C. glutamicum (GenBank Accession No. P42463.1), E. cloacae (GenBank Accession No. YP_003613611.1), M. maripaludis (GenBank Accession No.
• Chipman et al. provide an alignment and consensus for the sequences of a representative number of acetolactate synthases. Motifs shared in common between the majority of acetolactate synthases include:
• a protein harboring one or more of these amino acid motifs can generally be expected to exhibit acetolactate synthase activity.
• Ketol-acid reductoisomerases capable of converting acetolactate to 2,3- dihydroxyisovalerate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including E. coli (Gen Bank Accession No. EGB30597.1), L. lactis (GenBank Accession No. YP_003353710.1 ), S. exigua (GenBank Accession No. ZP_06160130.1 ), C. curiam (GenBank Accession No. YP_003151266.1 ), Shewanella sp. (GenBank Accession No. YP_732498.1), V. fischeri (GenBank Accession No.
• ketol-acid reductoisomerases An alignment and consensus for the sequences of a representative number of ketol- acid reductoisomerases is provided in commonly owned and co-pending US Publication No. 2010/0143997. Motifs shared in common between the majority of ketol-acid reductoisomerases include:
• V(V/l/F)(M/LyA)(A/C)PK SEQ ID NO: 35
• S(D/N/T)TA(E/Q/R)XG (SEQ ID NO: 37) motifs at amino acid positions corresponding to the 89-94, 175-179, 194-200, 262- 272, and 459-465 residues, respectively, of the E. coli ketol-acid reductoisomerase encoded by HvC.
• a protein harboring one or more of these amino acid motifs can generally be expected to exhibit ketol-acid reductoisomerase activity.
• ketol-acid reductoisomerases are known to use NADPH as a cofactor.
• a ketol-acid reductoisomerase which has been engineered to used NADH as a cofactor may be utilized to mediate the conversion of acetolactate to 2,3-dihydroxyisovalerate.
• Engineered NADH-dependent KARI enzymes (“NKRs") and methods of generating such NKRs are disclosed in commonly owned and co-pending US Publication No. 2010/0143997.
• any number of mutations can be made to a KARI enzyme, and in a preferred aspect, multiple mutations can be made to a KARI enzyme to result in an increased ability to utilize NADH for the conversion of acetolactate to 2,3-dihydroxyisovalerate.
• Such mutations include point mutations, frame shift mutations, deletions, and insertions, with one or more ⁇ e.g., one, two, three, four, five or more, etc.) point mutations preferred.
• Mutations may be introduced into naturally existing KARI enzymes to create NKRs using any methodology known to those skilled in the art. Mutations may be introduced randomly by, for example, conducting a PCR reaction in the presence of manganese as a divalent metal ion cofactor.
• oligonucleotide directed mutagenesis may be used to create the NKRs which allows for all possible classes of base pair changes at any determined site along the encoding DNA molecule. In general, this technique involves annealing an oligonucleotide complementary (except for one or more mismatches) to a single stranded nucleotide sequence coding for the KARI enzyme of interest.
• the mismatched oligonucleotide is then extended by DNA polymerase, generating a double-stranded DNA molecule which contains the desired change in sequence in one strand.
• the changes in sequence can, for example, result in the deletion, substitution, or insertion of an amino acid.
• the double-stranded polynucleotide can then be inserted into an appropriate expression vector, and a mutant or modified polypeptide can thus be produced.
• the above-described oligonucleotide directed mutagenesis can, for example, be carried out via PCR.
• Dihydroxy acid dehydratases capable of converting 2,3- dihydroxyisovalerate to a-ketoisovalerate may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including E. coli (GenBank Accession No. YP_026248.1), L. lactis (GenBank Accession No. NP_267379.1), S. mutans (GenBank Accession No. NP_722414.1), M. stadtmanae (GenBank Accession No. YP_448586.1), M. tractuosa (GenBank Accession No. YP_004053736.1 ), Eubacterium SCB49 (GenBank Accession No.
• CDKXXPG (SEQ ID NO: 39),
• GGSTN SEQ ID NO: 41
• a protein harboring one or more of these amino acid motifs can generally be expected to exhibit dihydroxy acid dehydratase activity.
• 2-keto-acid decarboxylases capable of converting a-ketoisovalerate to isobutyraldehyde may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including L. lactis kivD (GenBank Accession No. YP_003353820.1), E. cloacae (GenBank Accession No. P23234.1), M. smegmatis (GenBank Accession No. A0R480.1), M. tuberculosis (GenBank Accession No. 053865.1), M. avium (GenBank Accession No. Q742Q2.1 , A. brasilense (GenBank Accession No.
• GDG(S/A)(LJF/A)Q(UM)T (SEQ ID NO: 49) motifs at amino acid positions corresponding to the 21-27, 70-78, 81-89, 93-98, and 428-435 residues, respectively, of the L. lactis 2-keto-acid decarboxylase encoded by kivD.
• a protein harboring one or more of these amino acid motifs can generally be expected to exhibit 2-keto-acid decarboxylase activity.
• Alcohol dehydrogenases capable of converting isobutyraldehyde to isobutanol may be derived from a variety of sources (e.g., bacterial, yeast, Archaea, etc.), including L lactis (GenBank Accession No. YP_003354381 ), B. cereus (GenBank Accession No. YP_001374103.1), N. meningitidis (GenBank Accession No. CBA03965.1 ), S. sanguinis (GenBank Accession No. YP_001035842.1), L b vis (GenBank Accession No. YP_794451.1 ), B. thuringiensis (GenBank Accession No. ZP_04101989.1 ), P.
• sources e.g., bacterial, yeast, Archaea, etc.
• L lactis GenBank Accession No. YP_003354381
• B. cereus GenBank Accession No. YP_001374103.1
• G(L A/C)G(G/P)(UI/V)G SEQ ID NO: 55 motifs at amino acid positions corresponding to the 39-44, 59-66, 76-82, 91-97, 147- 152, and 171-176 residues, respectively, of the L. lactis alcohol dehydrogenase encoded by adhA.
• a protein harboring one or more of these amino acid motifs can generally be expected to exhibit alcohol dehydrogenase activity.
• 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. See, e.g., commonly owned and co-pending patent publication US 2010/0143997. An example of an NADH-dependent isobutanol pathway is illustrated in Figure 2.
• the recombinant microorganisms of the present invention may use an NKR to catalyze the conversion of acetolactate to produce 2,3-dihydroxyisovalerate.
• the recombinant microorganisms of the present invention may use an NADH-dependent ADH to catalyze the conversion of isobutyraldehyde to produce isobutanol.
• the recombinant microorganisms of the present invention may use both an NKR to catalyze the conversion of acetolactate to produce 2,3- dihydroxyisovalerate, and an NADH-dependent ADH to catalyze the conversion of isobutyraldehyde to produce isobutanol.
• the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutanol. In one embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to isobutyraldehyde. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to keto-isovalerate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to 2,3-dihydroxyisovalerate. In another embodiment, the yeast microorganism may be engineered to have increased ability to convert pyruvate to acetolactate.
• any of the genes encoding the foregoing enzymes may be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast.
• 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 pyruvate-derived metabolite (e.g., isobutanol).
• a pyruvate-derived metabolite e.g., isobutanol
• engineered or “modified” microorganisms are produced via the introduction of genetic material into a host or parental microorganism of choice and/or by modification of the expression of native genes, thereby modifying or altering the cellular physiology and biochemistry of the microorganism.
• the parental microorganism acquires new properties, e.g., the ability to produce a new, or greater quantities of, an intracellular and/or extracellular metabolite.
• the introduction of genetic material into and/or the modification of the expression of native genes in a parental microorganism results in a new or modified ability to produce a desired pyruvate- derived metabolite (e.g., isobutanol) from a suitable carbon source.
• the genetic material introduced into and/or the genes modified for expression in the parental microorganism contains gene(s), or parts of genes, coding for one or more of the enzymes involved in a biosynthetic pathway for the production of a pyruvate-derived metabolite ⁇ e.g., isobutanol) and may also include additional elements for the expression and/or regulation of expression of these genes, e.g., promoter sequences.
• an engineered or modified microorganism can also include the alteration, disruption, deletion or knocking-out of a gene or polynucleotide to alter the cellular physiology and biochemistry of the microorganism.
• the microorganism acquires new or improved properties (e.g., the ability to produce a new metabolite or greater quantities of an intracellular metabolite, to improve the flux of a metabolite down a desired pathway, and/or to reduce the production of by-products).
• Recombinant microorganisms provided herein may also produce metabolites in quantities not available in the parental microorganism.
• a "metabolite” refers to any substance produced by metabolism or a substance necessary for or taking part in a particular metabolic process.
• a metabolite can be an organic compound that is a starting material (e.g., glucose or pyruvate), an intermediate (e.g., 2-ketoisovalerate), or an end product (e.g., isobutanol) of metabolism.
• Metabolites can be used to construct more complex molecules, or they can be broken down into simpler ones.
• Intermediate metabolites may be synthesized from other metabolites, perhaps used to make more complex substances, or broken down into simpler compounds, often with the release of chemical energy.
• the disclosure identifies specific genes useful in the methods, compositions and organisms of the disclosure; however it will be recognized that absolute identity to such genes is not necessary.
• changes in a particular gene or polynucleotide comprising a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically such changes comprise conservative mutations and silent mutations.
• modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art.
• Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence.
• Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E.
• DNA compounds differing in their nucleotide sequences can be used to encode a given enzyme of the disclosure.
• the native DNA sequence encoding the biosynthetic enzymes described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure.
• a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity.
• the disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide.
• the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.
• homologs of enzymes useful for generating a pyruvate-derived metabolite are encompassed by the microorganisms and methods provided herein.
• two proteins are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
• the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and nonhomologous sequences can be disregarded for comparison purposes).
• the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence.
• the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").
• the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• Sequence homology for polypeptides is typically measured using sequence analysis software. See commonly owned and co-pending application US 2009/0226991.
• a typical algorithm used comparing a molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST. When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms described in commonly owned U.S. Pat. No. 8,017,375.
• microorganisms can be modified to include a recombinant metabolic pathway suitable for the production of a desired pyruvate- derived metabolite (e.g., isobutanol).
• microorganisms may be selected from yeast microorganisms.
• yeast microorganisms for the production of a desired pyruvate-derived metabolite (e.g., isobutanol) may be selected based on certain characteristics:
• One characteristic may include the property that the microorganism is selected to convert various carbon sources into a desired pyruvate-derived metabolite (e.g., isobutanol).
• carbon source generally refers to a substance suitable to be used as a source of carbon for prokaryotic or eukaryotic cell growth. Examples of suitable carbon sources are described in commonly owned U.S. Pat. No. 8,017,375.
• the recombinant microorganism herein disclosed can convert a variety of carbon sources, including but not limited to glucose, galactose, mannose, xylose, arabinose, lactose, sucrose, and mixtures thereof, to one or more pyruvate-derived metabolites (e.g., isobutanol).
• carbon sources including but not limited to glucose, galactose, mannose, xylose, arabinose, lactose, sucrose, and mixtures thereof.
• pyruvate-derived metabolites e.g., isobutanol
• the recombinant microorganism may thus further include a pathway for the production of a desired pyruvate-derived metabolite (e.g., isobutanol) from five- carbon (pentose) sugars including xylose.
• a desired pyruvate-derived metabolite e.g., isobutanol
• pentose five- carbon 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.
• XR xylose reductase
• XDH xylitol dehydrogenase
• the xylulose is then phosphorylated via a xylulokinase (XK) enzyme.
• XK xylulokinase
• This pathway operates inefficiently in yeast species because it introduces a redox imbalance in the cell.
• the xylose-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 anaerobically on xylose or other pentose sugars. Accordingly, a yeast species that can efficiently ferment xylose and other pentose sugars into a desired fermentation product is therefore very desirable.
• the recombinant microorganism is engineered to express a functional exogenous xylose isomerase.
• Exogenous xylose isomerases (XI) functional in yeast are known in the art. See, e.g., Rajgarhia et al., 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 xylulokinase (XK) gene operatively linked to promoter and terminator sequences that are functional in the yeast cell.
• XK xylulokinase
• the microorganism has reduced glycerol-3-phosphate dehydrogenase (GPD) activity.
• GPD catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD+.
• DHAP dihydroxyacetone phosphate
• G3P glycerol-3-phosphate
• Glycerol is then produced from G3P by Glycerol-3-phosphatase (GPP).
• Glycerol production is a secondary pathway to oxidize excess NADH from glycolysis. Reduction or elimination of this pathway would increase the pyruvate and reducing equivalents (NADH) available for the production of a pyruvate-derived metabolite (e.g., isobutanol).
• NADH pyruvate and reducing equivalents
• disruption, deletion, or mutation of the genes encoding for glycerol-3-phosphate dehydrogenases can further increase the yield of the desired metabolite (e.g., isobutanol).
• desired metabolite e.g., isobutanol
• the microorganism has reduced 3-keto acid reductase (3-KAR) activity, e.g., reduced YMR226c 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, e.g., reduced ALD6 activity.
• ALDH aldehyde dehydrogenase
• 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 Clade", as described in commonly owned U.S. Pat. No. 8,017,375.
• Saccharomyces sensu stricto yeast species include but are not limited to S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrids derived from these species (Masneuf et al., 1998, Yeast 7: 61- 72).
• the yeast microorganism may be selected from a post-WGD yeast genus, including but not limited to Saccharomyces and Candida.
• the favored post-WGD yeast species include: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S. castelli, and C. 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 include: S. kluyveri, K. thermotolerans, K. marxianus, K. waltii, K. lactis, C. tropicalis, P. pastoris, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I. scutulata, D. hansenii, H. anomala, Y. lipolytica, and S. pombe.
• a yeast microorganism may be either Crabtree-negative or Crabtree- positive as described in described in commonly owned U.S. Pat. No. 8,017,375.
• the yeast microorganism may be selected from yeast with a Crabtree-negative phenotype including but not limited to the following genera: Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida.
• Crabtree-negative species include but are not limited to: S. kluyveri, K. lactis, K. marxianus, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I. scutulata, H. anomala, and C.
• 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. thermotolemns, C. glabrata, Z. bailli, 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 anaerobically while the yeast is able to metabolize a carbon source in the presence of oxygen.
• Nonfermenting yeast refers to both naturally occurring yeasts as well as genetically modified yeast.
• Ethanol is produced by alcohol dehydrogenase (ADH) via the reduction of acetaldehyde, 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 isobutanol pathway. Deletion of this pathway increases the pyruvate and the reducing equivalents available for the biosynthetic pathway.
• Fermentative pathways contribute to low yield and low productivity of pyruvate-derived metabolites such as isobutanol. Accordingly, deletion of one or more PDC genes may increase yield and productivity of a desired pyruvate-derived 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.
• genes that encode for enzymes that are homologous to the genes described herein may be identified by functional, structural, and/or genetic analysis. In most cases, homologous or similar genes and/or homologous or similar enzymes will have functional, structural, or genetic similarities.
• Techniques known to those skilled in the art may be suitable to identify additional homologous genes and homologous enzymes.
• analogous genes and/or analogous enzymes can be identified by functional analysis and will have functional similarities. Techniques known to those skilled in the art may be suitable to identify analogous genes and analogous enzymes.
• techniques may include, but not limited to, cloning a gene by PCR using primers based on a published sequence of a gene/enzyme or by degenerate PCR using degenerate primers designed to amplify a conserved region among ketol-acid reductoisomerase genes.
• techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity (e.g. as described herein or in Kiritani, K.
• the endogenous nucleic acid or polypeptide identified herein is the S. cerevisiae version of the nucleic acid or polypeptide (e.g., Gndlp, Gnd2p, Ume6p, Sec27p, Zrt1 p,YLL056c, YOL014w, and Mthlp).
• Any method can be used to identify genes that encode for the endogenous polypeptide of interest in a variety of yeast strains.
• 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): 1456-61. Scannell, D. R., K. P. Byrne, J. L. Gordon, S. Wong, and K.
• yeast including but not limited to, Ashbya gossypii, Candida glabrata, Kluyveromyces lactis, Kluyveromyces polyspora, Kluyveromyces thermotolerans, Kluyveromyces waltii, Saccharomyces reteyveri, Saccharomyces castelli, Saccharomyces bayanus, and Zygosaccharomyces rouxii.
• this technique is therefore additionally suitable for the identification homologous Gndlp, Gnd2p, Ume6p, Sec27p, Zrt1p,YLL056c, YOL014w, and Mthlp polypeptides in yeast other than S. cerevisiae.
• 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 al., 1992, Nuc Acids Res. 27: 69-74; Ito et al., 1983, J. Bacteriol. 153: 163-8; and Becker et al., 1991, Methods in Enzymology 194: 182-7.
• the integration of a gene of interest into a DNA fragment or target gene of a yeast microorganism occurs according to the principle of homologous recombination.
• an integration cassette containing a module comprising at least one yeast marker gene and/or the gene to be integrated is flanked on either side by DNA fragments homologous to those of the ends of the targeted integration site (recombinogenic sequences).
• recombinogenic sequences DNA fragments homologous to those of the ends of the targeted integration site
• the integration cassette for integration of a gene of interest into a yeast microorganism includes the heterologous gene under the control of an appropriate promoter and terminator together with the selectable marker flanked by recombinogenic sequences for integration of a heterologous gene into the yeast chromosome.
• the heterologous gene includes an appropriate native gene desired to increase the copy number of a native gene(s).
• the selectable marker gene can be any marker gene used in yeast, including but not limited to, HIS3, TRP1, LEU2, URA3, bar, ble, hph, and kan.
• the recombinogenic sequences can be chosen at will, depending on the desired integration site suitable for the desired application.
• integration of a gene into the chromosome of the yeast microorganism may occur via random integration (Kooistra et al., 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 ef 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 cell in any form.
• exogenous nucleic acid molecules can be integrated into the genome of the cell or maintained in an episomal state that can stably be passed on ("inherited") to daughter cells.
• extra-chromosomal genetic elements such as plasmids, mitochondrial genome, etc.
• the yeast cells can be stably or transiently transformed.
• the yeast cells described herein can contain a single copy, or multiple copies of a particular exogenous nucleic acid molecule as described above.
• Yeast microorganisms within the scope of the invention may have reduced enzymatic activity such as reduced PDC, GPD, ALDH, or 3-KAR activity.
• reduced as used herein with respect to a particular polypeptide activity refers to a lower level of polypeptide activity than that measured in a comparable yeast cell of the same species.
• reduced also refers to the elimination of polypeptide activity as compared to a comparable yeast cell of the same species.
• yeast cells lacking activity for an endogenous PDC, GPD, ALDH, or 3-KAR are considered to have reduced activity for PDC, GPD, ALDH, or 3-KAR since most, if not all, comparable yeast strains have at least some activity for PDC, GPD, ALDH, or 3- KAR.
• Such reduced PDC, GPD, ALDH, or 3-KAR activities can be the result of lower PDC, GPD, ALDH, or 3-KAR concentration (e.g., via reduced expression), lower specific activity of the PDC, GPD, ALDH, or 3-KAR, or a combination thereof.
• Many different methods can be used to make yeast having reduced PDC, GPD, ALDH, or 3-KAR activity.
• a yeast cell can be engineered to have a disrupted PDC-, GPD-, ALDH-, or 3-KAR-encoding locus using common mutagenesis or knock-out technology. See, e.g., Methods in Yeast Genetics (1997 edition), Adams, Gottschling, Kaiser, and Stems, Cold Spring Harbor Press (1998).
• a yeast cell can be engineered to partially or completely remove the coding sequence for a particular PDC, GPD, ALDH, or 3-KAR.
• the promoter sequence and/or associated regulatory elements can be mutated, disrupted, or deleted to reduce the expression of a PDC, GPD, ALDH, or 3-KAR.
• yeast strains which when found in nature, are substantially free of one or more PDC, GPD, ALDH, or 3-KAR activities.
• antisense technology can be used to reduce PDC, GPD, ALDH, or 3-KAR activity.
• yeasts can be engineered to contain a cDNA that encodes an antisense molecule that prevents a PDC, GPD, ALDH, or 3-KAR from being made.
• antisense 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.
• Methods for overexpressing a polypeptide from a native or heterologous nucleic acid molecule are well known. Such methods include, without limitation, constructing a nucleic acid sequence such that a regulatory element promotes the expression of a nucleic acid sequence that encodes the desired polypeptide.
• regulatory elements are DNA sequences that regulate the expression of other DNA sequences at the level of transcription.
• regulatory elements include, without limitation, promoters, enhancers, and the like.
• the exogenous genes can be under the control of an inducible promoter or a constitutive promoter.
• methods for expressing a polypeptide from an exogenous nucleic acid molecule in yeast are well known.
• nucleic acid constructs that are used for the expression of exogenous polypeptides within Kluyveromyces and Saccharomyces are well known (see, e.g., U.S. Pat. Nos. 4,859,596 and 4,943,529, for Kluyveromyces and, e.g., Gellissen et ai, Gene 190(1):87-97 (1997) for Saccharomyces).
• Yeast plasmids have a selectable marker and an origin of replication.
• certain plasmids may also contain a centromeric sequence. These centromeric plasmids are generally a single or low copy plasmid.
• Plasmids without a centromeric sequence and utilizing either a 2 micron (S. cerevisiae) or 1.6 micron (K. lactis) replication origin are high copy plasmids.
• the selectable marker can be either prototrophic, such as HIS3, TRP1, LEU2, URA3 or ADE2, or antibiotic resistance, such as, bar, ble, hph, or kan.
• heterologous control elements can be used to activate or repress expression of endogenous genes. Additionally, when expression is to be repressed or eliminated, the gene for the relevant enzyme, protein or RNA can be eliminated by known deletion techniques.
• any yeast within the scope of the disclosure can be identified by selection techniques specific to the particular polypeptide ⁇ e.g. an isobutanol pathway enzyme) being expressed, over-expressed or repressed.
• Methods of identifying the strains with the desired phenotype are well known to those skilled in the art. Such methods include, without limitation, PCR, RT-PCR, and nucleic acid hybridization techniques such as Northern and Southern analysis, altered growth capabilities on a particular substrate or in the presence of a particular substrate, a chemical compound, a selection agent and the like.
• immunohistochemistry and biochemical techniques can be used to determine if a cell contains a particular nucleic acid by detecting the expression of the encoded polypeptide.
• an antibody having specificity for an encoded enzyme can be used to determine whether or not a particular yeast cell contains that encoded enzyme.
• biochemical techniques can be used to determine if a cell contains a particular nucleic acid molecule encoding an enzymatic polypeptide by detecting a product produced as a result of the expression of the enzymatic polypeptide. For example, transforming a cell with a vector encoding acetolactate synthase and detecting increased acetolactate concentrations compared to a cell without the vector indicates that the vector is both present and that the gene product is active. Methods for detecting specific enzymatic activities or the presence of particular products are well known to those skilled in the art. For example, the presence of acetolactate can be determined as described by Hugenholtz and Starrenburg, 1992, Appl. Micro. Biot. 38:17-22.
• Yeast microorganisms of the invention may be further engineered to have increased activity of enzymes (e.g., increased activity of enzymes involved in an isobutanol producing metabolic pathway).
• increased activity of enzymes e.g., increased activity of enzymes involved in an isobutanol producing metabolic pathway.
• the term "increased” as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured in a comparable yeast cell of the same species. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Increased activities for enzymes involved in glycolysis or the isobutanol pathway would result in increased productivity and yield of isobutanol.
• Methods to increase enzymatic activity are known to those skilled in the art. Such techniques may include increasing the expression of the enzyme by increased copy number and/or use of a strong promoter, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the KM for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press (2003). Methods of Using Recombinant Yeast Microorganisms for Production of Pyruvate- Derived Metabolites
• 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 pyruvate-derived metabolite from a recombinant microorganism described herein.
• the recombinant microorganism comprises a metabolic pathway for the production of a pyruvate-derived metabolite, wherein said metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the production of the pyruvate-derived metabolite, wherein said recombinant yeast microorganism is engineered to comprise reduced PDC activity, and wherein said recombinant yeast microorganism is engineered to (a): comprise at least one first modification which results in improved growth in the absence of C2-supplementation as compared to the corresponding recombinant yeast microorganism without said first modification; and/or (b) comprise at least one second modification which results in improved tolerance to glucose as compared to the corresponding
• the pyruvate-derived metabolite is isobutanol.
• the present invention provides a method of producing isobutanol from a recombinant yeast microorganism described herein.
• the recombinant yeast microorganism comprises an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises at least one exogenous gene and/or at least one overexpressed endogenous gene encoding an enzyme that catalyzes a pathway step in the conversion of pyruvate to isobutanol, wherein said recombinant yeast microorganism is engineered to comprise reduced PDC activity, and wherein said recombinant yeast microorganism is engineered to (a): comprise at least one first modification which results in improved growth in the absence of C2-supplementation as compared to the corresponding recombinant yeast microorganism without said first modification; and/or (b) comprise at least one second modification which results in improved tolerance to glucose as compared to the corresponding recombinant yeast microorganism without said second modification.
• a method to produce a pyruvate-derived metabolite from a carbon source
• the recombinant yeast microorganism is cultured in an appropriate culture medium containing a carbon source.
• the method further includes isolating the pyruvate-derived metabolite (e.g., isobutanol) from the culture medium.
• a pyruvate-derived metabolite e.g., isobutanol
• the recombinant microorganism may produce the pyruvate-derived metabolite (e.g., isobutanol) from a carbon source at a yield of at least 5 percent theoretical.
• Hie microorganism may produce the pyruvate-derived metabolite (e.g., isobutanol) from a carbon source at a yield of at least about 10 percent, at least about 15 percent, about least about 20 percent, at least about 25 percent, at least about 30 percent, at least about 35 percent, at least about 40 percent, at least about 45 percent, at least about 50 percent, at least about 55 percent, at least about 60 percent, at least about 65 percent, at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, or at least about 97.5% theoretical.
• the pyruvate-derived metabolite is isobutanol.
• DDG generally refers to the solids remaining after a fermentation, usually consisting of unconsumed feedstock solids, remaining nutrients, protein, fiber, and oil, as well as spent yeast biocatalysts or cell debris therefrom that are recovered by further processing from the fermentation, usually by a solids separation step such as centrif Ligation.
• 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 pyruvate-derived 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 reduced PDC activity.
• said spent yeast biocatalyst has additionally been engineered to (a): comprise at least one first modification which results in improved growth in the absence of C2-supplementation as compared to the corresponding recombinant yeast microorganism without said first modification; and/or (b) comprise at least one second modification which results in improved tolerance to glucose as compared to the corresponding recombinant yeast microorganism without said second modification.
• 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.
• Example 1 Evolution of C2-lndependent. Glucose-Tolerant Yeast Mutant with Reduced PDC Activity
• YNB + histidine medium 6.7 g L Yeast Nitrogen Base without amino acids and 0.076 g/L histidine. Carbon sources (ethanol, acetate or glucose) were added as indicated.
• YPD medium 10 g/L yeast extract, 20 g/L peptone and 20 g/L dextrose. Agar plates contain the indicated medium plus 20 g agar per liter.
• a DasGip fermentor vessel was sterilized and filled with 1 L of YNB + histidine medium, adjusted to pH 5, containing 2% w/v ethanol as carbon source. Air was sparged into the fermentor at 12 standard liters per hour (slph) at all times. The temperature of the vessel was held constant at 30.0°C and the agitation rate was set at a minimum of 500 rpm, with a cascade control to adjust the agitation to maintain 50% dissolved oxygen in the culture. The off-gas from the vessel was sent to an off-gas analyzer of the DasGip system as well as a mass spectrometer.
• the initial dilution rate was set to 0.06 h -1 to avoid washout. This was achieved by using a two pump system, effectively producing a gradient pumping scheme. Initially pump A was pumping YNB + histidine medium with 10 g L glucose at a rate of 35.5 mL/h and pump B was pumping YNB + histidine medium with only 1 g/L acetate at a rate of 20.3 mL/h. After the culture in the chemostat was stabilized at the 0.06 h -1 dilution rate, the concentration of acetate in the feed was slowly decreased by lowering the pump rate of the feed containing acetate until the acetate concentration in the chemostat was 0 g/L. The same dilution rate was maintained by concomitantly increasing the pump rate of the feed containing glucose.
• Evolution for Glucose Tolerance Evolution of the strain for growth on increased glucose concentrations was performed by slowly increasing the concentration of glucose in the chemostat with the evolved strain that no longer required a C2 supplement. This was achieved by using a two pump system, effectively producing a gradient pumping scheme. Initially pump A was pumping YNB + histidine medium with 10 g/L glucose at a rate of 35.2 mL/h and pump B was pumping YNB + histidine medium with 15 g/L glucose at a rate of 32.9 mlJh. The total glucose going into the vessel was 12.4 g/L.
• the medium reservoirs for pump A and pump B were replaced with reservoirs containing increased concentrations of glucose until the reservoir for pump A contained 80 g/L glucose and the reservoir for pump B contained 100 g/L glucose. During this period, the combined rate of feeding maintained a dilution rate of 0.04 h -1 . At the end of this period, the rate of pump A was finally reduced to 0, resulting in a feed of 100 g/L glucose.
• Evolution of the strain for increased growth rate was performed by slowly increasing the dilution rate in the chemostat with the evolved strain that no longer required a C2 supplement and could grow with a feed of 37.8 g/L glucose with a residual glucose level in the chemostat of 18.8 g/L. Over a period of 13 days, the dilution rate was gradually increased from 0.04 h -1 to 0.14 h- by alternately increasing the rates of pump A, from the 0 g/L glucose reservoir, and pump B, from the 100 g/L reservoir, to maintain a glucose feed concentration of 21-24 g/L glucose while increasing the dilution rate.
• Growth Rate Determination Inocula for growth rate determination experiments were grown in the same medium as for the actual experiment, either YNB medium + 50 g/L glucose + histidine (YNBSODHis) or YPD medium. Overnight cultures for each isolate were prepared by inoculating from isolated colonies from streak plates into 3 ml of medium in 14 ml round-bottom snap-cap tubes. These cultures were incubated overnight shaking at an angle at 250 rpm at 30°C. After overnight growth, samples of the cultures were diluted into a final volume of 200 ⁇ in 96-well flat-bottom polystyrene multiwell plates (Greiner Bio-One) and the OD600 measured on a Spectra max 340PC 384 plate reader.
• YNBSODHis glucose + histidine
• the volume of each culture needed to give an OD600 measurement of 0.01 above the background reading on the plate reader when this volume was added to 15 ml of medium was calculated.
• this volume of culture was added to 15 ml of the growth medium, mixed and then aliquotted into three 5 ml volumes in separate 50 ml Falcon disposable conical screw-cap centrifuge tubes to generate replicate cultures. These cultures were incubated shaking upright at 250 rpm at 30°C with the caps tightly closed. Samples of 100 ⁇ were taken from each tube every 2-3 hours beginning at the time of inoculation to give at least four timepoints and the OD600 was measured.
• GEVO2302 was grown in batch mode in a DasGip fermenter vessel in YNB medium + histidine (YNB+His) with 1.5% w/v ethanol as sole carbon source to an OD600 of 8.
• the vessel was switched to continuous culture fchemostat") mode by pumping in YNB+His + 10 g/L glucose medium at 35.5 ml/h (Pump A) and YNB+His + 1 g/L acetate at 20.3 ml/h (Pump B), resulting in a dilution rate of 0.06 h ⁇ 1 with a 5% carbon equivalent of acetate (C2-supplement) in the feed.
• the glucose feed to the chemostat over this period was increased from 6.4 g/L to 10 g/L and the evolved strain was able to grow on glucose only, with the residual glucose in the chemostat vessel during this period of ⁇ 1 g/L. Also during this period, the OD600 of the culture dropped back to 8.
• the OD600 of the culture dropped from 8 to 2 and the residual glucose concentration rose over the next 24 hours before the culture again began to grow.
• the medium reservoirs for Pump A and Pump B were replaced with reservoirs containing increased concentrations of glucose over the next 6 days until the reservoir for Pump A contained 80 g/L glucose and the reservoir for Pump B contained 100 g/L glucose.
• the combined rate of feeding maintained a dilution rate of 0.04 h -1 .
• the rate of Pump A was finally reduced to 0, resulting in a feed of 100 g/L glucose.
• Evolution of the C2 Supplement-Independent and Glucose Tolerant Evolved GEV2302 Population for Improved Growth Rate Evolution of the strain for increased growth rate was performed by slowly increasing the dilution rate in the chemostat with the evolved strain that no longer required a C2 supplement and could grow with a feed of 37.8 g/L glucose with a residual glucose level in the chemostat of 18.8 g/L.
• the dilution rate was gradually increased from 0.04 h -1 to 0.14 h -1 by alternately increasing the rates of Pump A, from the 0 g/L glucose reservoir, and Pump B, from the 100 g/L reservoir, to maintain a glucose feed concentration of 21-24 g/L glucose while increasing the dilution rate.
• a biomass of OD600 1.6 - 2.0 was maintained at dilution rates of 0.13 h -1 to 0.14 h -1 .
• GEV02712 segregates of a crossing between GEVO2302 and GEV02712 were isolated that are as C2-independent and glucose tolerant as GEV02712.
• the genomic DNA of pooled selected segregants was sequenced, together with genomic DNA of GEV2302 and GEV02712. This information was used to identify mutations linked to the glucose tolerance and C2-independence of GEV02712.
• Genomic DNA from pooled progeny that were both glucose tolerant and C2-supplementation independent was sequence and these data were compared to the genomic DNA sequence from GEVO2302.
• a partial deletion of MTH1 ( ⁇ 41-78 of Mthl p) originally identified by comparative genomic hybridization was detected in the pooled glucose-tolerant segregant's genomic DNA but not in the unevolved parent. These data indicate that this deletion is tightly linked to the glucose tolerant phenotype.
• a partial deletion of MTH1 has previously been attributed to glucose tolerance in the context of malic acid production. Specifically, Winkler et al. observed a 225 base pair deletion of an internal segment of the Mthlp coding region, from nucleotides 169-393, resulting in the in-frame deletion of amino acids 57-131. See US2011/0039327, which is herein incorporated by reference in its entirety.
• a surprising observation described in the instant application is that the deletion shown herein ( ⁇ 41-78 of Mthlp) does not alter the yeast casein kinase I (Yck1) recognition domain - found at residues 118-137 and identified by Moriya et al., 2004, PNAS 101(6): 1572-77. Thus, its effect of activating glucose tolerance cannot be due to inactivation of this known domain. Therefore, the dominant, constitutively activating effect of the allele described herein is a surprising feature of the present invention.
• the partial deletion described herein confers a different phenotype upon yeast cells compared to yeast harboring the I85 mutation. Specifically, the partial deletion mutation described herein is not associated with glucose repression of SUC2 (data not shown). In contrast, the I85 mutations described in the literature caused strong derepression of SUC2 (and other glucose repression targets).
• the chemostat evolution system While the partial deletion of the MTH1 locus is at least responsible, in part, for the glucose tolerant phenotype of GEV02712, the chemostat evolution system also selected for improvements in both the growth rate and for the ability to grow without C2-compound supplementation.
• the pooled segregant sequencing project identified several additional mutations that contribute to one or more phenotypes (glucose-tolerance, C2-independence, and improved growth rate).
• a total of 30 possibly linked single nucleotide polymorphisms (SNPs) were found (Table 2) and six of the 30 SNPs result in an amino acid change in the encoded protein.
• MTH1 Mutation Results in Less Glucose Transport Experiments were performed to measure the impact of the MTH1 partial deletion on glucose transport in multiple Pdc-deficient strains that were pre-grown on YPEtOH. Results are shown in Figure 6. Of note, yeast strains harboring the partial deletion allele (e.g., GEV03672 MTHIpD and GEVO2302 MTHIpD) transported significantly less glucose than comparator strains with a wild-type Mth1 sequence or comprising a full deletion.
• yeast strains harboring the partial deletion allele e.g., GEV03672 MTHIpD and GEVO2302 MTHIpD
• This example describes how recombinant yeast with reduced pyruvate decarboxylase activity exhibit improved growth in the absence of C2- supplementation when transformed with a mutant Gnd1 protein.
• S. cerevisiae strains were generated in which the wild- type Gndlp was replaced with a mutant version of Gndlp comprising an L340W mutation. Replacement of the wild-type Gndlp with the mutant version of Gndlp was confirmed by DNA sequencing.
• the growth of the progeny strains along with parental strains was measured in the minimal medium, YNB (yeast nitrogen base), comprising uracil, tryptophan, casaminoacids, and 1% raffinose in the presence and absence of 1% acetate (i.e., presence or absence of C2-supplementation).
• YNB yeast nitrogen base
• E. coli NKR P2D1-A1
• L. lactis DHAD L lactis KIVD
• L. lactis ADH RE1
• deletions in the endogenous PDC1, PDC5, PDC6 See US Patent No. 8,017,375
• YMR226c See US Patent No. 8,133,715) genes.
• strains comprising a mutant Gnd1 protein showed significantly enhanced growth as compared to the parental strains (approximately 2-fold more growth at intermediate timepoints, e.g., 32h and 38h) and the Gnd1 deletion strains (approximately 2.5- to 3-fold more growth at intermediate timepoints, e.g., 32h and 38h).
• mutant protein GND1 L3 0W expression of the mutant protein GND1 L3 0W , overexpression of the wild-type Gnd1 p, and/or overexpression of mutant protein GND1 L340W can be utilized to improve host cell growth in the absence of C2-supplementation in strains engineered to harbor reduced pyruvate decarboxylase activity.

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