WO2024040001A1 - Levure génétiquement modifiée et processus de fermentation pour la production d'éthanol - Google Patents

Levure génétiquement modifiée et processus de fermentation pour la production d'éthanol Download PDF

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WO2024040001A1
WO2024040001A1 PCT/US2023/072055 US2023072055W WO2024040001A1 WO 2024040001 A1 WO2024040001 A1 WO 2024040001A1 US 2023072055 W US2023072055 W US 2023072055W WO 2024040001 A1 WO2024040001 A1 WO 2024040001A1
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yeast cell
engineered yeast
enzyme
seq
gapn
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PCT/US2023/072055
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English (en)
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Christopher Kenneth MILLER
Gregory Michael POYNTER
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Cargill, Incorporated
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Publication of WO2024040001A1 publication Critical patent/WO2024040001A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • 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
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae
    • 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)
    • C12Y101/05—Oxidoreductases acting on the CH-OH group of donors (1.1) with a quinone or similar compound as acceptor (1.1.5)
    • C12Y101/05003—Glycerol-3-phosphate dehydrogenase (1.1.5.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01009—Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (1.2.1.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03021—Glycerol-1-phosphatase (3.1.3.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase

Definitions

  • Typical treatments include acid and/or enzymatic hydrolysis where the polymer chain is hydrolyzed to generate the sugars that can be used by the yeast.
  • Starch degrading enzymes such as alpha amylases and glucoamylases can be added to convert the polymer to simple sugars.
  • alpha amylases and glucoamylases can be added to convert the polymer to simple sugars.
  • enzyme additions can add significant cost and complexity to the fermentation process.
  • Heterologous expression and functionality of enzymes in yeast to aid in starch hydrolysis can be challenging, as it is difficult to know if the nucleic acid will be expressed properly and a functional enzyme will form, and if an active form of the enzyme will be secreted from the cell.
  • yeast it is also challenging to engineer yeast for growth and bioproduct production at non- optimal conditions, such as high temperatures, and in high bioproduct titers.
  • non-optimal conditions e.g., temperature
  • by-product formation can be technically difficult.
  • Increased ethanol concentration and accumulation of undesirable byproducts can also be detrimental to cell health.
  • the present disclosure provides a genetically engineered yeast cell capable of producing ethanol, the engineered yeast cell comprising an exogenous polynucleotide encoding a glyceraldehyde-3-phosphate dehydrogenase (gapN) enzyme at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:28, 32, 48, 52, 64, 68, 80, 92, and 96.
  • gapN glyceraldehyde-3-phosphate dehydrogenase
  • the gapN enzyme may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:28, 32, 48, 52, and 64.
  • the gapN enzyme may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:28, 32, 52, and 64.
  • the gapN enzyme may be at least 85% identical to SEQ ID NO:28; the gapN enzyme may be at least 85% identical to SEQ ID NO:32; the gapN enzyme may be at least 85% identical to SEQ ID NO:52; and/or the gapN enzyme may be at least 85% identical to SEQ ID NO:64.
  • the engineered yeast cell may be capable of producing ethanol at a titer of at least 60, at least 80, at least 100, or at least 120 g/L ethanol after 48 hours and wherein glycerol production by the engineered yeast cell is reduced relative to glycerol production in an equivalent yeast cell lacking the gapN enzyme.
  • the engineered yeast cell may additionally comprise an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:98 and 100.
  • the engineered yeast cell may comprise a deletion or disruption of a native glycerol-3-phosphate phosphatase (GPP) gene.
  • GDP native glycerol-3-phosphate dehydrogenase
  • the engineered yeast cell may additionally comprise an exogenous polynucleotide sequence encoding a glucoamylase (GA) enzyme.
  • the encoded GA enzyme may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:1, 122, 123, and 124.
  • the engineered yeast cell may be capable of producing ethanol at a titer of at least 60, at least 80, at least 100, or at least 120 g/L ethanol after 48 hours and wherein glycerol production by the engineered yeast cell is reduced relative to glycerol production in an equivalent yeast cell lacking the gapN enzyme.
  • a genetically engineered yeast cell capable of producing ethanol, the engineered yeast cell comprising an exogenous polynucleotide encoding a glyceraldehyde-3- phosphate dehydrogenase (gapN) enzyme at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID PT-1298-WO-PCT NOs:28, 32, 48, 52, 64, 68, 80, 92, and 96; an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:98 and 100; an exogenous polynucleotide sequence encoding a glu
  • the encoded GA enzyme may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:1, 122, 123, and 124.
  • One or more of the exogenous polynucleotide sequences may be operably linked to a heterologous or artificial promoter.
  • the promoter may be selected from the group consisting of a pyruvate decarboxylase (PDC) promoter, a glyceraldehyde-3-phosphate dehydrogenase GAPDH (TDH3) promoter, a translation elongation factor 1 (TEF1) promoter, a URA3 promoter, an S- adenosyl methionine transferase 2 (SAM2) promoter; an alcohol dehydrogenase 1 (ADH1) promoter, and a 3-phosphoglycerate kinase (PGK1) promoter.
  • PDC pyruvate decarboxylase
  • TDH3 glyceraldehyde-3-phosphate dehydrogenase GAPDH
  • TEZ1 translation elongation factor 1
  • URA3 promoter
  • SAM2 S- adenosyl methionine transferase 2
  • ADH1 alcohol dehydrogenase 1
  • PGK1 3-phosphog
  • the terminator may be selected from the group consisting of an iso-1-cytophrome c (CYC1) terminator, a URA3 terminator, a PDC terminator, an ADH1 terminator, a TEF1 terminator, or a GAL10 terminator.
  • the yeast cell may be selected from the group consisting of Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., and Yarrowia spp..
  • the yeast cell may be a Saccharomyces cerevisiae cell.
  • the disclosure also provides a method for producing ethanol, the method comprising contacting a substrate with an engineered yeast as described herein, where the engineered yeast cell produces at least 60, at least 80, at least 100, or at least 120 g/L ethanol after 48 hours and wherein glycerol production by the engineered yeast cell is reduced relative to glycerol production in an equivalent yeast cell lacking the gapN enzyme.
  • the substrate may comprise starch, glucose, sucrose, cellulosic biomass, or combinations thereof.
  • the substrate may be obtained from wheat, corn, or a combination thereof.
  • FIG.1 shows show screening of gapN biodiversity as outlined in Example 1.
  • FIG.2 shows glycerol production in the deep well plate assays outlined in Example 3.
  • FIG.3 shows ethanol production in the deep well plate assays outlined in Example 3.
  • FIG.4 shows NADP redox balancing using a combination of an NADP dependent gapN and an NADP dependent ADH enzyme in the production of ethanol from glucose.
  • FIG.5 shows the results of the ADH enzyme assay outlined in Example 4.
  • FIG.1 shows show screening of gapN biodiversity as outlined in Example 1.
  • PT-1298-WO-PCT shows glycerol production in the deep well plate assays outlined in Example 3.
  • FIG.3 shows ethanol production in the deep well plate assays outlined in Example 3.
  • FIG.4 shows NADP redox balancing using a combination of an NADP dependent gapN and an NADP dependent ADH enzyme in the production of ethanol from glucose.
  • FIG.5 shows the results of the ADH enzyme assay
  • FIG. 6 shows the glycerol and ethanol titers from shake flask assays outlined in Example 6.
  • FIG. 7 shows the glycerol and ethanol titers from shake flask assays outlined in Example 6.
  • FIG. 8 shows the glycerol and ethanol titers from shake flask assays outlined in Example 6.
  • FIG. 9 shows the glycerol and ethanol titers from the Ambr15 assays outlined in Example 7.
  • a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the PT-1298-WO-PCT individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range.
  • the statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise.
  • the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.
  • ppm parts per million
  • percentage percentage
  • ratios are on a by weight basis. Percentage on a by weight basis is also referred to as wt% or % (wt) below.
  • wt% Percentage on a by weight basis is also referred to as wt% or % (wt) below.
  • This disclosure relates to various recombinant cells engineered to produce ethanol via glucose, said recombinant cells also having reduced glycerol production.
  • the recombinant cells described herein include a heterologous nucleic acid encoding a gapN enzyme, for example the gapN enzyme of at least one of SEQ ID NOs:28, 32, 48, 52, 64, 68, 80, and 92.
  • the recombinant cell may additionally include a heterologous nucleic acid encoding an ADH enzyme, for example, the ADH enzyme of at least one of SEQ ID NO:99 and 100.
  • the disclosure further provides fermentation methods for the production of ethanol using the genetically engineered cells described herein.
  • recombinant cells described herein are yeast cells.
  • yeast cells include yeast cells obtained from, e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., Yarrowia spp. and industrial polyploid yeast strains.
  • Suitable yeast cells may include, but are not limited to, Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia galeiformis, Pichia sp.
  • yeast cell may be an ethanol tolerant yeast strain, for example, a commercially available ethanol tolerance yeast such as RED STARTM and ETHANOL REDTM yeast (Fermentis/Lesaffre, USA), FALITM (Fleischmann's Yeast, USA), SUPERSTART and THERMOSACCTM yeast (Ethanol Technology, Wis., USA), BIOFERMTM AFT and XR (NABC-North American Bioproducts Corporation, GA, USA), GERT STRAND (Gert Strand AB, Sweden), SUPERSTARTTM (Alltech), ANGELTM (Angel Yeast Ltd, China) and FERMIOLTM (DSM Specialties).
  • RED STARTM and ETHANOL REDTM yeast Fermentis/Lesaffre, USA
  • FALITM Feleischmann's Yeast, USA
  • SUPERSTART and THERMOSACCTM yeast Ethanol Technology, Wis., USA
  • BIOFERMTM AFT and XR NABC-North American Bioproducts Corporation, GA,
  • recombinant yeast cells of the present disclosure are not limited to those expressly recited herein.
  • suitable host cells and examples of recombinant cells capable of producing ethanol are described in US Patent No. 10,724,023, US Patent No. 10,334,288, US Patent Publication No. 20200270644A1, US Patent No. 11,111,482, US Patent Publication No. 20190345471A1, US PT-1298-WO-PCT Patent No. 11,041,218, US Patent No. 11,306,330, and US Patent Publication No. 20210062230A1, each of which is incorporated herein by reference in its entirety.
  • the recombinant cells described herein include one or more exogenous polynucleotide sequences encoding one or more exogenous polypeptides that, when expressed improve the fermentation of sucrose to lactate by the recombinant cells.
  • the recombinant cell may alternatively or additionally include one or more genetic modifications that increases expression of a native polypeptide, wherein said increase in expression improves the fermentation of sucrose to lactate by the recombinant cell.
  • exogenous refers to genetic material or an expression product thereof that originates from outside of the host organism.
  • the exogenous genetic material or expression product thereof can be a modified form of genetic material native to the host organism, it can be derived from another organism, it can be a modified form of a component derived from another organism, or it can be a synthetically derived component.
  • a K. lactis invertase gene is exogenous when introduced into I. orientalis.
  • “native” refers to genetic material or an expression product thereof that is found, apart from individual-to-individual mutations which do not affect function or expression, within the genome of wild-type cells of the host cell.
  • polypeptide and “peptide” are used interchangeably and refer to the collective primary, secondary, tertiary, and quaternary amino acid sequence and structure necessary to give the recited macromolecule its function and properties.
  • enzyme or “biosynthetic pathway enzyme” refer to a protein that catalyzes a chemical reaction. The recitation of any particular enzyme, either independently or as part of a biosynthetic pathway is understood to include the co-factors, co-enzymes, and metals necessary for the enzyme to properly function.
  • Table 1 A summary of the amino acids and their three and one letter symbols as understood in the art is presented in Table 1.
  • amino acid name, three letter symbol, and one letter symbol are used interchangeably herein.
  • Table 1 Amino Acid three and one letter symbols Amino Acid Three letter symbol One letter symbol PT-1298-WO-PCT Aspartic acid Asp D Cysteine Cys C
  • Varian ts or sequences having substantial identity or homology with the polypeptides described herein can be utilized in the disclosed engineered cells, compositions, and methods. Such sequences can be referred to as variants or modified sequences. That is, a polypeptide sequence can be modified yet still retain the ability to exhibit the desired activity.
  • the variant or modified sequence may include or greater than about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with the wild-type, naturally occurring polypeptide sequence, or with a variant polypeptide as described herein.
  • the phrases “% sequence identity,” “% identity,” and “percent identity,” are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods of amino acid and nucleic acid sequence alignment are well-known. Sequence alignment and generation of sequence identity include global alignments and local alignments which are carried out using computational approaches.
  • BLAST National Center for Biological Information (NCBI) Basic Local Alignment Search Tool
  • Amino acid % sequence identity between amino acid sequences can be determined using standard protein BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 6; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: (Existence: 11, Extension: 1); Compositional adjustments: Conditional compositional score matrix adjustment; Filter: none selected; Mask: none selected.
  • Nucleic acid % sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1, -2; Gap costs: Linear; Filter: Low complexity regions; Mask: Mask for lookup table only.
  • a sequence having an identity score of XX% (for example, 80%) with regard to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX% identical or, equivalently, have XX% sequence identity to the reference sequence.
  • Polypeptide or polynucleotide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • polypeptides disclosed herein may include “variant” polypeptides, “mutants,” and “derivatives thereof.”
  • wild-type is a term of the art understood by skilled persons and means the typical form of a polypeptide as it occurs in nature as distinguished from variant or mutant forms.
  • a “variant,” “mutant,” or “derivative” refers to a polypeptide molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule.
  • a variant or mutant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule.
  • the amino acid sequences of the polypeptide variants, mutants, derivatives, or fragments as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence.
  • a variant, mutant, derivative, or fragment polypeptide may include conservative amino acid substitutions relative to a reference molecule.
  • PT-1298-WO-PCT “Conservative amino acid substitutions” are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide.
  • amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge and/or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • polynucleotide polynucleotide sequence
  • nucleic acid sequence and “nucleic acid,” are used interchangeably and refer to a sequence of nucleotides or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent the sense or the antisense strand.
  • the DNA polynucleotides may be a cDNA or a genomic DNA sequence.
  • a polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof.
  • the anti-sense strand of such a polynucleotide is also said to encode the sequence.
  • Those of skill in the art understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide.
  • the polynucleotides may be codon-optimized for expression in a particular cell including, without limitation, a plant cell, bacterial cell, fungal cell, or animal cell. While polypeptides encoded by polynucleotide sequences found in coral are disclosed herein any polynucleotide sequences may be used which encodes a desired form of the polypeptides described herein. Thus, non-naturally occurring sequences may be used. These may be desirable, for example, to enhance expression in heterologous expression systems of polypeptides or proteins.
  • the recombinant cells described herein may include deletions or disruptions in one or more native genes.
  • the phase “deletion or disruption” refers to the status of a native gene in the recombinant cell that has either a completely eliminated coding region (deletion) or a modification of the gene, its promoter, or its terminator (such as be a deletion, insertion, or mutation) so that the gene no longer produces an active expression product, produces severely reduced quantities PT-1298-WO-PCT of the expression product (e.g., at least a 75% reduction or at least a 90% reduction) or produces an expression product with severely reduced activity (e.g., at least 75% reduced or at least 90% reduced).
  • the deletion or disruption can be achieved by genetic engineering methods, forced evolution, mutagenesis, and/or selection and screening.
  • the native gene to be deleted or disrupted may be replaced with an exogenous nucleic acid of interest for the expression of an exogenous gene product (e.g., polypeptide, enzyme, and the like).
  • an exogenous gene product e.g., polypeptide, enzyme, and the like.
  • the recombinant cell described herein may have a deletion or disruption of one or more native genes encoding an enzyme involved in the synthesis of glycerol. Deletion or disruption of one or more of these glycerol biosynthetic pathway enzymes decreases the ability of the cell to product glycerol, thereby increasing fermentation production of ethanol.
  • the recombinant cells described herein may include a deletion or disruption of a native glycerol-3- phosphate phosphatase (GPP) gene.
  • GPP glycerol-3- phosphate phosphatase
  • the native GPP gene(s) encode an enzyme that catalyzes the hydrolysis of glycerol-3-phosphate into glycerol.
  • the native GPP gene(s) encode an enzyme that catalyzes the hydrolysis of glycerol-3-phosphate into glycerol.
  • the host cell contains multiple GPP genes, it is preferred to delete or disrupt at least one of them and more preferred to disrupt all of them to more completely eliminate the host cell’s ability to product glycerol.
  • Gpp1p SEQ ID NO:120
  • Gpp2p SEQ ID NO:121
  • the cell may include a deletion or disruption of a GPP gene encoding an amino acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs:120 and 121.
  • Methods for the deletion or disruption of the GPP genes of S. cerevisiae are known and described in the art and are exemplified herein.
  • the recombinant cells described herein may include a deletion or disruption of a native glycerol-3-phosphate dehydrogenase (GPD) gene.
  • GPD glycerol-3-phosphate dehydrogenase
  • GPD1p glycerol-3-phosphate dehydrogenases
  • suitable interaction loci may include, but are not PT-1298-WO-PCT limited to, the GPP1 loci (defined as the loci flanked by SEQ ID NO:136 and SEQ ID NO:137), the DLD1 loci (defined as the loci flanked by SEQ ID NO:138 and SEQ ID NO:139), and the GPD1 loci (defined as the loci flanked by SEQ ID NO:140 and SEQ ID NO:141).
  • GPP1 loci defined as the loci flanked by SEQ ID NO:136 and SEQ ID NO:137
  • DLD1 loci defined as the loci flanked by SEQ ID NO:138 and SEQ ID NO:139
  • GPD1 loci defined as the loci flanked by SEQ ID NO:140 and SEQ ID NO:141.
  • Other suitable integration loci may be determined one of skill in the art.
  • the recombinant cells described herein are capable of producing ethanol and include an exogenous polynucleotide sequence encoding a glyceraldehyde-3-phosphate dehydrogenase (gapN) enzyme.
  • the gapN enzyme may be any suitable enzyme with glyceraldehyde-3-phosphate dehydrogenase activity.
  • the exogenous polynucleotide sequence may be an exogenous glyceraldehyde-3-phosphate dehydrogenase (gapN) gene.
  • a “glyceraldehyde-3-phosphate dehydrogenase gene” and “gapN gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with glyceraldehyde-3-phosphate dehydrogenase activity.
  • glyceraldehyde-3- phosphate dehydrogenase activity refers to the ability to catalyze the conversion of D- glyceraldehyde 3-phosphate and NADP + to 3-phospho-D-glycerate and NADPH.
  • the gapN enzyme can be from any suitable source organism or may be synthetic.
  • Suitable gapN enzymes may include, but are not limited to, enzymes categorized under Enzyme Commission (EC) number 1.2.1.9, also known in the art as “NADP-dependent non-phosphorylating glyceraldehyde-3- phosphate dehydrogenase.”
  • Suitable gapN enzymes may be the gapN enzymes from Streptococcus pyogenes, Pseudomonas fluorescens, Brevibacillus laterosporus, Arabidopsis thaliana, Chryseobacterium gleum, Streptococcus mutans, Streptococcus henryi, Lactobacillus delbrueckii, Bacillus cereus, and the like.
  • the gapN gene may encode an amino acid at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:28, 32, 48, 52, 64, 68, 80, 92, and 96.
  • the gapN gene may encode an amino acid at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:28, 32, 48, 52, and 64.
  • the gapN gene may encode an amino acid at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:28, 32, 52, and 64.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Streptococcus pyogenes gene encoding the amino acid sequences of SEQ ID NO:28.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at PT-1298-WO-PCT least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:28.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Pseudomonas fluorescens gene encoding the amino acid sequences of SEQ ID NO:32.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:32.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Brevibacillus laterosporus gene encoding the amino acid sequences of SEQ ID NO:48.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:48.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Arabidopsis thaliana gene encoding the amino acid sequences of SEQ ID NO:52.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:52.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Chryseobacterium gleum gene encoding the amino acid sequences of SEQ ID NO:64.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:64.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Streptococcus mutans gene encoding the amino acid sequences of SEQ ID NO:68.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:68.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Streptococcus henryi gene encoding the amino acid sequences of SEQ ID NO:80.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:80.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Lactobacillus delbrueckii gene encoding the amino acid sequences of SEQ ID NO:92.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:92.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Bacillus cereus gene encoding the amino acid sequences of SEQ ID NO:96.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:96.
  • the recombinant cells described herein are capable of producing ethanol, include an exogenous polynucleotide sequence encoding a gapN enzyme, and may additionally include an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme.
  • the ADH enzyme may be any suitable enzyme with NADP-dependent alcohol dehydrogenase activity.
  • the exogenous polynucleotide sequence may be an exogenous alcohol dehydrogenase (ADH) gene.
  • An “alcohol dehydrogenase gene” and “ADH gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with alcohol dehydrogenase activity.
  • alcohol dehydrogenase activity refers to the ability to catalyze the conversion of acetaldehyde and NADH or NADPH to ethanol and NAD + or NADP + .
  • NADP-dependent alcohol dehydrogenase activity refers to the ability to catalyze the conversion of acetaldehyde and NADPH to ethanol and NADP + .
  • the ADH enzyme may be derived from any suitable source or may be synthetic. Suitable ADH enzymes may be the ADH enzymes from Rhodotorula toruloides, Candida maltosa, and the like.
  • the ADH gene may encode an amino acid at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:98 and 100.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Candida maltosa gene encoding the amino acid sequences of SEQ ID NO:98.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:98.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rhodotorula toruloides gene encoding the amino acid sequences of SEQ ID PT-1298-WO-PCT NO:100.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:100.
  • the recombinant cells described herein are capable of producing ethanol, include an exogenous polynucleotide sequence encoding a gapN enzyme, may optionally include an exogenous polynucleotide sequence encoding an ADH enzyme, and may additionally include an exogenous polynucleotide sequence encoding a glucoamylase (GA) enzyme.
  • the GA enzyme may be any suitable enzyme with glucoamylase activity.
  • the exogenous polynucleotide sequence may be an exogenous glucoamylase (GA) gene.
  • a “glucoamylase gene” and “GA gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with glucoamylase activity.
  • “glucoamylase activity” refers to the ability to catalyze the hydrolysis of the terminal 1,4-linked alpha-D-glucose residue from the non-reducing end of an amylose chain to release free glucose.
  • the GA enzyme can be from any suitable source organism or may be synthetic. Suitable glucoamylase enzymes may include, but are not limited to, enzymes of EC 3.2.1.3.
  • Suitable GA enzymes may be the GA enzymes from Saccharomycopsis fibuligera, Rhizopus delemar, Rhizopus microspores, Rhizopus oryzae, and the like.
  • the GA gene may encode an amino acid at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:1, 122, 123, and 124.
  • the GA gene may encode an amino acid at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs:1, 123, and 124.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rhizopus microspores gene encoding the amino acid sequences of SEQ ID NO:1.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:124.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Saccharomycopsis fibuligera gene encoding the amino acid sequences of SEQ ID PT-1298-WO-PCT NO:122.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:122.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rhizopus delemar gene encoding the amino acid sequences of SEQ ID NO:123.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:123.
  • the recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rhizopus oryzae gene encoding the amino acid sequences of SEQ ID NO:124.
  • the exogenous polynucleotide may encode an amino acid sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:124.
  • the exogenous nucleic acids in the recombinant cells described herein may be under the control of a promoter.
  • the exogenous nucleic acid may be operably linked to a heterologous or artificial promoter.
  • Promoters may include, but are not limited to, pyruvate decarboxylase (PDC1), glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) (TDH3 herein; annotated in EC 1.2.1.12; SEQ ID NO:125), translational elongation factor 1 (TEF1; SEQ ID NO:128), URA3 (SEQ ID NO:126), S-adenosyl methionine transferase 2 (SAM2; SEQ ID NO:129), alcohol dehydrogenase 1 (ADH1; SEQ ID NO:130) and 3-phosphoglycerate kinase (PGK1; SEQ ID NO:127).
  • PDC1 pyruvate decarboxylase
  • GPDH glyceraldehyde- 3-phosphate dehydrogenase
  • TEZH3 translational elongation factor 1
  • SAM2 S-adenosyl methionine transferase
  • the exogenous nucleic acids in the recombinant cells described herein may be under the control of a terminator.
  • the exogenous nucleic acid may be operably linked to a heterologous or artificial terminator.
  • Suitable terminators are known and described in the art. Terminators may include, but are not limited to, iso-1-cytochrome c (CYC1; SEQ ID NO:131), URA3 (SEQ ID NO:132), PDC, ADH1 (SEQ ID NO:134), TEF1 (SEQ ID NO:135), and ScGAL10 (SEQ ID NO:133).
  • a promoter or terminator is “operably linked” to a given polynucleotide (e.g., a gene) if its position in the genome or expression cassette relative to said polynucleotide is such that the promoter or terminator, as the case may be, performs its transcriptional control function.
  • the polypeptides described herein may be provided as part of a construct.
  • the term “construct” refers to recombinant polynucleotides including, without limitation, DNA and RNA, which may be single-stranded or double-stranded and may represent the sense or PT-1298-WO-PCT the antisense strand.
  • Recombinant polynucleotides are polynucleotides formed by laboratory methods that include polynucleotide sequences derived from at least two different natural sources or they may be synthetic. Constructs thus may include new modifications to endogenous genes introduced by, for example, genome editing technologies. Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies.
  • the construct may be a vector including a promoter operably linked to the polynucleotide encoding the thermolabile non-heme iron-binding polypeptide.
  • the term “vector” refers to a polynucleotide capable of transporting another polynucleotide to which it has been linked.
  • the vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments may be integrated.
  • the disclosure also provides fermentation methods for the production of ethanol using the recombinant cells described herein.
  • the fermentation methods include the step of fermenting a substrate using the genetically engineered yeasts described herein to product ethanol.
  • the fermentation method can include additional steps, as would be understood by a person skilled in the art. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, and isolating the ethanol from the fermentation broth.
  • the fermentation substrate can comprise a starch. Starch can be obtained from a natural source, such as a plant source.
  • Starch can also be obtained from a feedstock with high starch or sugar content, including, but not limited to corn, sweet sorghum, fruits, sweet potato, rice, barley, sugar cane, sugar beets, wheat, cassava, potato, tapioca, arrowroot, peas, or sago.
  • the fermentation substrate may be from lignocellulosic biomass such as wood, straw, grasses, or algal biomass, such as microalgae and macroalgae.
  • the fermentation substrate may include cellulosic or lignocellulosic biomass.
  • the fermentation substrate may be from grasses, trees, or agricultural and forestry residues, such as corn cobs and stalks, rice straw, sawdust, and wood chips.
  • the fermentation substrate can also comprise a sugar, such as glucose (dextrose) or sucrose.
  • the fermentation substrate may comprise a dry grind ethanol feedstock, such as corn mash.
  • the fermentation substrate can comprise a liquefied corn mash (LCM).
  • the fermentation substrate may comprise a corn wet mill feedstock, such as Light Steep Water/Liquifact (LSW/LQ).
  • LSW/LQ Light Steep Water/Liquifact
  • Media for fermentation of the engineered yeast described herein can be supplemented with various components.
  • media for fermentation of the engineered yeast described herein can be supplemented with a glucoamylase, e.g., the glucoamylase SpirizymeTM (Novozymes, Bagsvaerd, Denmark).
  • the fermentation process can be run under various conditions.
  • the fermentation temperature i.e., the temperature of the fermentation broth during processing, may be ambient temperature. Alternatively, or additionally, the fermentation temperature may be maintained within a predetermined range. For example, the fermentation temperature can be maintained in the range of 25 °C to 40 °C, 27 °C to 38 °C, or 30 °C to 35 °C.
  • the fermentation temperature may be maintained at a temperature of, e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C, or any value in between.
  • the pH of a culture medium described herein may be controlled for optimal ethanol production.
  • the pH of the culture or a fermentation mixture of an engineered cell described herein may be in the range of between 4.0 and 6.0.
  • the pH may be maintained for at least part of the incubation at 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.
  • the pH may be maintained at a range between 5.0 and 5.5.
  • the engineered yeast may be cultured for approximately 24-72 hours.
  • the engineered yeast may be cultured for approximately 12, 18, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 78, 80, 90, 96 hours, or more than 96 hours.
  • the engineered yeast described herein may be cultured for approximately 48 to 72 hours.
  • a culture (fermentation) time of about 48 hours is a representative time for commercial-scale ethanol fermentation processes. Accordingly, a 48 hour time point can be used to compare the fermentation performance of different yeast strains. [0075] Reaction parameters can be measured or adjusted during the production of ethanol.
  • reaction parameters include biological parameters (e.g., growth rate, cell size, cell number, cell density, cell type, or cell state, etc.), chemical parameters (e.g., pH, redox- potential, concentration of reaction substrate and/or product, concentration of dissolved gases, such as oxygen concentration and CO 2 concentration, nutrient concentrations, metabolite concentrations, ethanol concentration, fermentation substrate concentration, concentration of an oligopeptide, concentration of an amino acid, concentration of a vitamin, concentration of a hormone, concentration of an additive, serum concentration, ionic strength, concentration of an ion, relative humidity, molarity, osmolarity, concentration of other chemicals, for example buffering agents, adjuvants, or reaction by-products), physical/mechanical parameters (e.g., density, conductivity, degree of agitation, pressure, and flow rate, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, conversion rate, as well as thermodynamic PT-1298-WO-PCT
  • biological parameters
  • the fermentation process can be associated with various characteristics, such as, but not limited to, fermentation production rate, pathway fermentation yield, final titer, and peak fermentation rate. These characteristics can be affected by the selection of the yeast and/or genetic modification of the yeast used in the fermentation process. These characteristics can be affected by adjusting the fermentation process conditions. These characteristics can be adjusted via a combination of yeast selection or modification and the selection of fermentation process conditions.
  • the ethanol production rate of the process may be at least 1.0, at least 1.5, or at least 2.0, at least 2.5, at least 3.0, or at least 3.5 g L -1 h -1 .
  • the final ethanol titer of the process may beat least 60 g/L, at least 80, at least 100, or at least 120 g/L.
  • the final glycerol titer of the process may be less than 10 g/L, less than 8 g/L, less than 6 g/L, less than 4 g/L, or less than 3 g/L.
  • Example 1 gapN Biodiversity
  • the biodiversity of gapN was surveyed, revealing fewer than 1,00 annotated gapN genes, primarily in the Streptococcus (230 gapN genes), Pseudomonas (94), Bacillus (95), and Clostridium genera (FIG. 1).
  • Strain 1-2 [0081] Strain 1-18 described by Poynter et al. (US Patent Application Publication No. US 2021/006230 published March 4, 2021, incorporated herein by reference in its entirety) is a Saccharomyces cerevisiae host strain that is ura3 negative (ura3-) and amdS negative (amdS-) and in which both alleles of the cytosine deaminase (FCY1) gene are knocked out and replaced with an expression cassette for the Rhizopus microsporus glucoamylase of SEQ ID NO:1 with a TDH3 promoter and a CYC1 terminator.
  • Strain 1-2 refers to Strain 1-18 of Poynter et al.
  • Strain 1-3 refers to Strain 1-25 of Poynter et al. US Application Publication No. US 2021/006230. Strains 1-4 through 1-27 [0083] Strain 1-2 was transformed with the “Upstream Fragment” and “Downstream Fragment” as indicated in Table 2 to produce strains 1-4 through 1-27.
  • Each “Upstream Fragment” PT-1298-WO-PCT contained i) a 5’ GPP1 flanking sequence; ii) a ScTDH3 promoter; iii) a gene encoding the indicated gapN; iv) an ScCYC1 terminator; v) a loxP site; vi) a ScURA3 promoter; and vii) a 5’ portion of the ScURA open reading frame.
  • Each “Downstream Fragment” contained i) a 3’ GPP1 flanking sequence; ii) a ScTDH3 promoter; iii) a gene encoding the indicated gapN; iv) a ScPGK1 terminator, v) a loxP site; vi) a ScURA3 terminator; and vii) a 3’ portion of the ScURA open reading frame.
  • Transformants were selected on synthetic dropout media lacking uracil (ScD-Ura) plates. Resulting transformants were streaked for single colony isolation on ScD-Ura plates. A single colony was selected.
  • Example 3 Deep Well Assays
  • Strains 1-1 negative control
  • 1-3 positive control
  • 1-4 through 1-27 were run in 96-deep well plates to assay ethanol and glycerol production.
  • Strains were struck to a ScD-ura plate and incubated at 30°C until single colonies were visible (2-3 days). Cells from the ScD-ura plate were scraped into 20 g/L YPD media and grown overnight in 96-deep well plates as 30 oC and 800 rpm.
  • 2 mL shake flask medium containing sterilized canola oil was added to each well of a 96-deep well plate.
  • the shake flask medium consisted of 725g partially hydrolyzed corn starch, 150g filtered sterilized (0.2 ⁇ m) light steep water, 10g water, 25g glucose, and 1g urea. Strains were incubated at 35 °C with shaking in an orbital shake at 250 rpm for about 70 hours. Samples were taken and analyzed for ethanol and glycerol concentrations by HPLC. The average over 6 wells per strain is reported in Table 3 and FIGS.2 and 3. PT-1298-WO-PCT Table 3.
  • Strains 1-10, 1-11, 1- 20, 1-23, and 1-26 showed improved glycerol reduction relative to strain 1-1 but at an equivalent level to strain 1-3. Comparing strains 1-27 and 1-3, both have two copies of the B. cereus gapN expression set, however one has one copy at each of the two GPP1 alleles (1-3) while the other has both copes in tandem on a single allele (1-27). Reduced glycerol production in both 1-3 and 1-27 relative to 1-1 demonstrates that the location of gapN expression cassettes can be variable and the beneficial reduction in glycerol production can be seen even when one GPP1 allele is present.
  • Example 4 ADH Biodiversity [0087] The biodiversity of alcohol dehydrogenase (ADH) enzymes was surveyed and 41 were selected for testing. Genes encoding the selected ADH enzymes were cloned into Saccharomyces cerevisiae, biomass was grown, the resulting cells were lysed, and cell free extracts were assayed using ethanol and NAD/NADP as the substrate. While this reaction is the opposite of the desired in vivo activity of the ADH, it is a suitable characterization of the NAD vs NADP preference of the enzyme.
  • ADH alcohol dehydrogenase
  • Each “5’ gapN and ADH Expression Cassette” contained i) a 5’ GPP1 flanking sequence; ii) a PGK1 promoter; iii) a gene encoding the indicated gapN enzyme; iv) a ADH1 terminator; v)a TDH3 promoter; vi) a gene encoding the indicated ADH enzyme; vii)a CYC terminator; and viii) a 5’ portion of the ScURA open reading frame.
  • the “3’ Selectable Marker Cassette” contained i) a 3’ GPP1 flanking sequence; ii) a loxP site; iii) a ScURA terminator; and vi) a 3’ portion of the ScURA open reading frame. Transformants were selected on ScD-Ura plates. Resulting transformants were streaked for single colony isolation on ScD-Ura plates. Single colonies were selected. Correct integration of the indicated gapN and ADH expression cassettes was verified by PCR and the PCR verified isolates were designated with the strain identifier outlined in Table 2. More than one PCR verified isolate, e.g., “sister” isolates, are indicated by letters following the strain number.
  • strain 2-1 has three sister isolates, strains 2-1a, 2-1b, and 2-1c.
  • Resulting strains 2-1 through 2-8 included a single copy of the gapN and ADH expression cassettes in tandem on a single allele of the GPP1 gene.
  • PT-1298-WO-PCT Strains 2-9 through 2-16 [0091] Parent strains indicated in Table 5 were transformed with the “5’ gapN and ADH Expression Cassette” and “3’ Selectable Marker Cassette” as indicated in Table 5 to produce strains 2-9 through 2-16.
  • Each “5’ gapN and ADH Expression Cassette” contained i) a 5’ GPP1 flanking sequence; ii) a PGK1 promoter; iii) a gene encoding the indicated gapN enzyme; iv) a ADH1 terminator; v) a TDH3 promoter; vi) a gene encoding the indicated ADH enzyme; vii) a CYC terminator; and viii) a 5’ portion of the amdS open reading frame.
  • the “3’ Selectable Marker Cassette” contained i) a 3’ GPP1 flanking sequence; ii) a loxP site; iii) an amdS terminator; and vi) a 3’ portion of the amdS open reading frame. Transformants were selected on YNB + acetamide plates. Resulting transformants were streaked for single colony isolation on YNB + acetamide plates. Single colonies were selected. Correct integration of the indicated gapN and ADH expression cassettes was verified by PCR and the PCR verified isolates were designated with the strain identifier outlined in Table 2. More than one PCR verified isolate, e.g., “sister” isolates, are indicated by letters following the strain number.
  • strain 2-9 has two sister isolates, strains 2-9a and 2-9b.
  • Resulting strains 2-9 through 2-16 included two copies of the gapN and ADH expression cassettes, one on each of the two alleles of the GPP1 gene.
  • the shake flask medium consisted of 725g partially hydrolyzed corn starch, 150g filtered sterilized (0.2 ⁇ m) light steep water, 10g water, 25g glucose, and 1g urea. Strains were incubated at 30°C with shaking in an orbital shake at 100 rpm for 72 hours. Samples were taken and analyzed for metabolite concentrations in the broth at the end of fermentation by HPLC. [0095] Fermentation results are reported in Tables 6-8 and in FIGS. 6-8. Many of the tested strains showed improved ethanol titers even through the glycerol titer was not reduced. This may be use to the increased utilization of the acetaldehyde with the NADPH-dependent ADH enzyme.
  • Optical density is measured at a wavelength of 600 nm with a 1 cm path length using a model Genesys 20 spectrophotometer (Thermo Scientific).
  • An Ambr15 reaction vessel is inoculated with the cell slurry to reach an initial OD600 of 0.2.
  • the fermentation medium consisted of 295g partially hydrolyzed corn starch, 90g filtered sterilized (0.2 ⁇ m) light steep water, 79g sterile water, and 36g 500g/L sterile glucose. With continuous stirring, 12mL of fermentation media was added to each bioreactor. Strains were incubated at 30 °C, 450rpm of agitation.

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Abstract

Sont divulguées ici des cellules de levure génétiquement modifiées capables de produire de l'éthanol. Les cellules de levure génétiquement modifiées comprennent une séquence polynucléotidique exogène codant pour une enzyme glycéraldéhyde-3-phosphate déshydrogénase (gapN) d'au moins 60 %, au moins 70 %, au moins 80 %, au moins 85 %, au moins 90 %, au moins 95 %, au moins 97 %, au moins 99 %, ou 100 % identique à au moins l'une des SEQ ID NOs:28, 32, 48, 52, 64, 68, 80, 92 et 96.
PCT/US2023/072055 2022-08-17 2023-08-11 Levure génétiquement modifiée et processus de fermentation pour la production d'éthanol WO2024040001A1 (fr)

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