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WO2025144942A1 - Cellules de levure génétiquement modifiées et procédés d'utilisation associés - Google Patents

Cellules de levure génétiquement modifiées et procédés d'utilisation associés Download PDF

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Publication number
WO2025144942A1
WO2025144942A1 PCT/US2024/061971 US2024061971W WO2025144942A1 WO 2025144942 A1 WO2025144942 A1 WO 2025144942A1 US 2024061971 W US2024061971 W US 2024061971W WO 2025144942 A1 WO2025144942 A1 WO 2025144942A1
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Prior art keywords
enzyme
genetically modified
yeast cell
modified yeast
activity
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Inventor
Daniel Liu
Enzo LONG
Nick Harris
Jeremy ROOP
Charles DEPEW
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Berkeley Fermentation Science Inc
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Berkeley Fermentation Science Inc
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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
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C12/00—Processes specially adapted for making special kinds of beer
    • C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
    • C12C12/004—Genetically modified microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/52—Propionic acid; Butyric acids
    • 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
    • C12P7/62—Carboxylic acid esters
    • 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
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01084—Alcohol O-acetyltransferase (2.3.1.84)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • C12Y602/01003—Long-chain-fatty-acid-CoA ligase (6.2.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C2200/00—Special features
    • C12C2200/05—Use of genetically modified microorganisms in the preparation of beer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G2200/00—Special features
    • C12G2200/11—Use of genetically modified microorganisms in the preparation of wine
    • 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
    • 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

Definitions

  • a genetically modified yeast cell e.g. a brewing yeast cell, comprising a genetic modification that results in increased expression of a nucleic acid encoding an enzyme having acyl activating enzyme (EC 6.2.1.3) activity (AAE), wherein the cell produces (i) an increased amount of one or more ethyl esters selected from the group consisting of ethyl propionate, ethyl butanoate, and ethyl isovalerate, and/or (ii) a decreased amount of one or more fatty acids selected from the group consisting of propionic acid, butyric acid, and isovaleric acid, compared to a cell that does not comprise the genetic modification.
  • EC 6.2.1.3 acyl activating enzyme
  • the one or more ethyl esters comprise ethyl propionate and the one or more fatty acids comprise propionic acid. In some embodiments of the foregoing aspects, the one or more ethyl esters comprise ethyl butanoate and the one or more fatty acids comprise butyric acid. In some embodiments of the foregoing aspects, the one or more ethyl esters comprise ethyl isovalerate and the one or more fatty acids comprise isovaleric acid.
  • the enzyme having AAE activity is derived from Humulus lupulus or Hypericum caly cinum.
  • the enzyme having AAE activity comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequences set forth in any one of SEQ ID NOs: 1-4. In some embodiments, the enzyme having AAE activity comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.
  • the enzyme having AAE activity comprises a substitution mutation at a position corresponding to position R51 of SEQ ID NO: 2.
  • the substitution mutation at the position corresponding to position R51 of SEQ ID NO: 2 is a lysine.
  • the enzyme having AAE activity is not derived from Cannabis sativa. In some embodiments, the enzyme having AAE activity does not comprise the amino acid sequence of SEQ ID NO: 5.
  • the genetically modified yeast cell further comprises a genetic medication that results in increased expression of a nucleic acid encoding an enzyme having alcohol-O-acyltransferase (EC 2.3.1.84) activity (AAT).
  • the genetically modified yeast cell comprises a cassette comprising the nucleic acid encoding the enzyme with AAT activity operably linked to a promoter.
  • the enzyme having AAT activity is derived from Marinobacter hydrocarbonoclasticus, Fragaria x ananassa, Saccharomyces cerevisiae, Neurospora sitophila, Actinidia deliciosa, Actinidia chinensis, Marinobacter aquaeolei, Saccharomycopsis fibuligera, Malus x domestica, Solanum pennellii, or Solanum lycopersicum.
  • the enzyme having AAT activity comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequences set forth in any one of SEQ ID NOs: 6-14.
  • the enzyme having AAT activity comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 6-14.
  • the enzyme having AAT activity has specificity for an acyl- CoA produced by the enzyme having AAE activity.
  • the genetically modified yeast cell does not comprise a genetic modification that increases fatty acid biosynthesis. In some embodiments, the genetically modified yeast cell does not comprise a genetic modification to increase fatty acid synthetase (FAS) activity.
  • the genetically modified yeast cell is of the genus Saccharomyces. In some embodiments, the genetically modified yeast cell is of the species Saccharomyces cerevisiae (S. cerevisiae). In some embodiments, the genetically modified yeast cell is of the species Saccharomyces pastorianus (S. pastorianus). In some embodiments, the genetically modified yeast cell is S.
  • a liquid fermentation composition comprising: (a) a population of genetically modified yeast cells according to the present disclosure and a sugar source.
  • the liquid fermentation composition further comprises alcohol, e.g. ethanol.
  • the liquid fermentation composition further comprises (i) an increased amount of one or more ethyl esters selected from the group consisting of ethyl propionate, ethyl butanoate, and ethyl isovalerate, and/or (ii) a decreased amount of one or more fatty acids selected from the group consisting of propionic acid, butyric acid, and isovaleric acid compared to a liquid fermentation composition produced by the same method using a counterpart cell that does not overexpress or comprise the enzyme having AAE activity.
  • Additional aspects of the present disclosure provide methods of producing a fermented product comprising, contacting any of the genetically modified yeast cells described herein with a medium comprising at least one fermentable sugar, wherein the contacting is performed during at least a first fermentation process, to produce a fermented product.
  • at least one fermentable sugar is provided in at least one sugar source.
  • the fermentable sugar is glucose, fructose, sucrose, maltose, and/or maltotriose.
  • the fermented product comprises an increased level of at least one desired product as compared to a fermented product produced by a counterpart cell that does not express the enzyme having AAE activity.
  • the counterpart cell is characteristic of a cell from which the genetically modified yeast cell is derived.
  • the desired product is an ethyl ester selected from the group consisting of ethyl propionate, ethyl butanoate, ethyl isovalerate, ethyl octanoate, ethyl decanoate, ethyl 2- methlybutyrate, and ethyl crotonate.
  • the desired product is an ethyl ester selected from the group consisting of ethyl propionate, ethyl butanoate, and ethyl isovalerate.
  • the fermented product comprises a reduced level of at least one undesired product as compared to a fermented product produced by a counterpart cell that does not express the enzyme having AAE activity.
  • the at least one undesired product is an acid selected from the group consisting of propionic acid, butanoic acid, isovaleric acid, octanoic acid, decanoic acid, 2-methyl-butyric acid, and crotonic acid.
  • the undesired product is selected from the group consisting of propionic acid, butanoic acid, and isolvaleric acid.
  • the fermented product is a fermented beverage.
  • the fermented beverage is beer, wine, sparkling wine (champagne), wine cooler, wine spritzer, hard seltzer, sake, mead, kombucha, or cider.
  • the sugar source comprises wort, must, fruit juice, honey, rice starch, or a combination thereof.
  • the fruit juice is a juice obtained from at least one fruit selected from the group consisting of grapes, apples, blueberries, blackberries, raspberries, currants, strawberries, cherries, pears, peaches, nectarines, oranges, pineapples, mangoes, and passionfruit.
  • the sugar source is wort
  • the method further comprises producing the medium, wherein producing the medium comprises: (a) contacting a plurality of grains with water; and (b) boiling or steeping the water and grains to produce wort.
  • the method further comprises adding at least one hop variety to the wort to produce a hopped wort.
  • the method further comprises adding at least one hop variety to the medium.
  • the sugar source is must, and the method further comprises producing the medium, wherein producing the medium comprises crushing a plurality of fruits to produce the must. In some embodiments, the method further comprises removing solid fruit material from the must to produce a fruit juice.
  • the method further comprises at least one additional fermentation process. In some embodiments, the method further comprises carbonating the fermented product.
  • the fermented product comprises at least 150 pg/L of ethyl propionate, ethyl butanoate, ethyl isovalerate, ethyl octanoate, ethyl decanoate, 2-methylbutyrate, or ethyl crotonate.
  • the fermented product comprises at least 150 pg/L of ethyl propionate, ethyl butanoate, and ethyl isovalerate.
  • the fermented product comprises less than 15 mg/L of propionic acid, butanoic acid, isovaleric acid, octanoic acid, decanoic acid, 2-methyl- butyric acid, or crotonic acid. In some embodiments, the fermented product comprises less than 15 mg/L of propionic acid, butanoic acid, and isovaleric acid.
  • aspects of the present disclosure provide methods of producing a composition comprising ethanol comprising, contacting any of the genetically modified yeast cells described herein with a medium comprising at least one fermentable sugar, wherein the contacting is performed during at least a first fermentation process, to produce a composition comprising ethanol.
  • at least one fermentable sugar is provided in at least one sugar source.
  • the fermentable sugar is glucose, fructose, sucrose, maltose, and/or maltotriose.
  • FIG. 7 provides a schematic of an engineering strategy for increasing ethyl ester biosynthesis involving expression of a variant fatty acid synthetase, FAS2 (e.g., FAS2(G1250S)), and a heterologous alcohol-O-acyltransferase (AAT).
  • FAS2 e.g., FAS2(G1250S)
  • AAT heterologous alcohol-O-acyltransferase
  • Native yeast enzymes and enzymatic pathways include PDC1, ALD6, ACS1, ACC1, FAS1 and FAS2.
  • Modifications generated by genetic engineering include FAS2(G1250S) and AAT.
  • FIG. 8 shows the relative abundance of ethyl esters and medium-chain fatty acids (MCFAs) following fermentation with a subset of engineered yeast strains.
  • MCFAs medium-chain fatty acids
  • FIG. 10 shows the concentrations of ethyl esters and medium-chain fatty acids (MCFAs) following fermentation with yeast strains that were engineered to express a single acyl-activating enzyme (AAE) and carry no additional genetic modifications.
  • AAE acyl-activating enzyme
  • Each subplot shows the concentrations of one pair of ester and MCFA molecules, and yeast strains are described on the horizontal axis.
  • LA3 is a wild-type parental strain and each additional strain is an LA3 strain that was engineered to express the indicated AAE gene.
  • FIG. 13 shows the concentrations of ethyl esters and medium-chain fatty acids (MCFAs) following fermentation with yeast strains that were engineered to express a heterologous keto-acyl -thiolase pathway in addition to an AAE enzyme.
  • LA3 is the wild-type strain used as a parent for all engineered strains.
  • Y1810 is an engineered strain that expresses a keto-acyl-thiolase pathway (as described in Figure 6; “BktB” pathway). Expression of the keto-acyl-thiolase pathway results in increased biosynthesis of etyl esters and MCFAs.
  • Each additional strain is the same as y 1810 except that each expresses the indicated AAE enzyme.
  • Each subplot shows the concentrations of one pair of ester and MCFA molecules, and yeast strains are described on the horizontal axis.
  • Ethyl esters are composed of an ethanol moiety that is bound to a fatty acid (acyl) moiety via an ester bond. The length and structure of the acyl moiety determines many of the chemical and sensorial properties of the ester (FIG. 1).
  • yeast primarily convert sugars into ethanol and cell biomass, but they also produce a wide variety of additional flavor-active molecules, including ethyl esters.
  • the direct metabolic precursors for ethyl ester biosynthesis are medium chain acyl-CoAs.
  • “Medium chain” refers to an acyl moiety containing 4-10 carbon atoms.
  • Most of these medium chain acyl-CoAs are produced by the fatty acid synthase (FAS) complex composed of yeast FAS1 and FAS2 enzymes, and as such are byproducts of long- chain fatty acid biosynthesis.
  • FES fatty acid synthase
  • the final step in ethyl ester biosynthesis the condensation of an acyl-CoA precursor with ethanol — is catalyzed by one of several yeast alcohol acyltransferase (AAT) enzymes.
  • AAT yeast alcohol acyltransferase
  • yeast are engineered to 1) express a variant of the endogenous FAS2 enzyme (FAS2(G1250S)) that releases higher levels of hexanoy 1 -Co A, and 2) express a heterologous AAT enzyme that efficiently esterifies hexanoyl-CoA and ethanol to produce ethyl-hexanoate.
  • FAS2(G1250S) endogenous FAS2 enzyme
  • heterologous AAT enzyme that efficiently esterifies hexanoyl-CoA and ethanol to produce ethyl-hexanoate.
  • strains engineered for increased ethyl ester production scales with the degree of ethyl ester production such that there is a correlation between the levels of MCFAs and ethyl esters.
  • the specific MCFAs produced at elevated concentrations are associated with their corresponding ethyl esters (i.e., they contained the same acyl moiety).
  • strains biosynthesizing increased levels of ethyl butanoate also produce increased levels of butanoic acid
  • strains producing elevated concentrations of ethyl isovalerate produced elevated concentrations of isovaleric acid.
  • the genetic modification to increase the expression of the nucleic acid encoding the enzyme having AAE activity can result in expression and/or increased expression of the enzyme having AAE activity.
  • expression of an endogenous AAE can be increased or a heterologous AAE not naturally encoded by the genome of the cell can be expressed.
  • the genetically modified cells that express an enzyme with AAE activity and methods of use thereof described herein are capable of producing alcoholic and non-alcoholic fermented products having an increased amount of ethyl esters and a reduced amount of MCFAs compared to a counterpart cell that does not express the enzyme with AAE activity.
  • the counterpart cell can be a cell that includes the same genotype as the genetically modified yeast cell with the exception of the genetic modification to increase expression of the nucleic acid encoding the enzyme having AAE activity.
  • a brewing yeast strain can be a strain of the Saccharomyces pastoriamis, a hybrid species that originated from a hybridization between strains of S. cerevisiae and S. eubayanus during brewing (PMID 26269586).
  • brewing yeast strains have or are likely to have one or more of the following characteristics: 1) they are able to metabolize maltotriose, 2) their genomes contain alleles of PAD 1 and FDC1 that have no/reduced function due to loss-of-function mutations, 3) their genomes encode the AGT1 allele of MALI 1, and/or they display efficient growth on maltose and maltotriose media.
  • a genetically modified yeast cell can be derived from a brewing yeast strain. Exemplary brewing yeast strains are disclosed further herein.
  • the endpoints shall be inclusive and the range (e.g., at least 70% identity) shall include all ranges within the cited range (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least
  • Percent identity refers to a quantitative measurement of the similarity between two sequences (e.g., nucleic acid or amino acid). Percent identity can be determined using the algorithms of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990.
  • heterologous nucleic acid refers to a nucleic acid wherein at least one of the following is true: (a) the nucleic acid is foreign (“exogenous”) to (that is, not naturally found in) a given host cell; (b) the nucleic acid comprises a nucleotide sequence that is naturally found in (that is, is “endogenous to”) a given host cell, but the nucleotide sequence is present in an unnatural amount in the cell (for example, greater than expected or greater than naturally found); (c) the nucleic acid comprises a nucleotide sequence that differs in sequence from an endogenous nucleotide sequence, but the nucleotide sequence encodes the same protein (having the same or substantially the same amino acid sequence) and is present in an unnatural amount in the cell (for example, greater than expected or greater than naturally found); or (d) the nucleic acid comprises two or more nucleotide sequences that are not found in the same relationship to each other in nature
  • endogenous gene refers to a hereditary unit corresponding to a sequence of nucleic acid (e.g., DNA) that contains the genetic instruction, which originates within a host organism (e.g., a genetically modified cell) and is expressed by the host organism.
  • a host organism e.g., a genetically modified cell
  • the genetically modified cells comprise a genetic modification that results in increased expression of a nucleic acid encoding an enzyme with acyl-activating enzyme (AAE) activity.
  • the modified cells overexpress an enzyme having AAE activity.
  • the modified cells express a heterologous gene encoding an enzyme having AAE activity.
  • the heterologous gene can encode an enzyme that is not typically expressed by the cell, a variant of an enzyme that the cell does not typically express (e.g., a mutated enzyme), an additional copy of a gene encoding an enzyme that is typically expressed in the cell, or a gene encoding an enzyme that is typically expressed by the cell but under different regulation.
  • a genetically modified yeast cell e.g. a brewing yeast cell, comprising a genetic modification that results in increased expression of a nucleic acid encoding an enzyme having acyl activating enzyme (EC 6.2.1.3) activity (AAE), wherein the cell produces (i) an increased amount of one or more ethyl esters selected from the group consisting of ethyl propionate, ethyl butanoate, and ethyl isovalerate, and/or (ii) a decreased amount of one or more fatty acids selected from the group consisting of propionic acid, butyric acid, and isovaleric acid, compared to a cell that does not comprise the genetic modification.
  • EC 6.2.1.3 acyl activating enzyme
  • a “counterpart” cell that does not express an enzyme refers to a cell characteristic of the cell from which a genetically modified yeast cell is derived or which is identical to the genetically modified yeast cell except for the genetic modification(s) recited.
  • a counterpart cell that that does not include an enzyme having AAE activity can refer to a cell that does not include a genetic modification that either overexpresses an enzyme having AAE activity or lacks a heterologous nucleic acid encoding the enzyme having AAE activity.
  • the modified cell comprises a heterologous cassette comprising a promoter operably linked to a gene encoding an enzyme with AAE activity, such as a heterologous gene encoding the AAE or an endogenous gene encoding the AAE.
  • the enzyme having AAE activity can comprise SEQ ID NO: 3 with a substitution mutation of R51K (HcAAEl (R51K), SEQ ID NO: 4).
  • the enzyme having AAE activity can comprise SEQ ID NO: 30 (ScFAAl from Saccharomyces cerevisiae (Accession No. AJT92227-1)).
  • the enzyme with AAE activity has an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-6.
  • the enzyme with AAE activity has an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-4.
  • the enzyme with AAE activity comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-4. In some embodiments, the enzyme with AAE activity consists of the amino acid sequence as set forth in any one of SEQ ID NOs: 1-4. In some embodiments, the enzyme with AAE activity comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-4 and 30-31. In some embodiments, the enzyme with AAE activity consists of the amino acid sequence as set forth in any one of SEQ ID NOs: 1-4 and 30-31.
  • the enzyme with AAE activity comprises the amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the enzyme with AAE activity comprises the amino acid sequence as set forth in SEQ ID NO: 2. In some embodiments, the enzyme with AAE activity comprises the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, the enzyme with AAE activity comprises the amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the enzyme with AAE activity comprises the amino acid sequence as set forth in SEQ ID NO: 30. In some embodiments, the enzyme with AAE activity comprises the amino acid sequence as set forth in SEQ ID NO: 31.
  • the enzyme with AAE activity consists of the amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the enzyme with AAE activity consists of the amino acid sequence as set forth in SEQ ID NO: 2. In some embodiments, the enzyme with AAE activity consists of the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, the enzyme with AAE activity consists of the amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the enzyme with AAE activity consists of the amino acid sequence as set forth in SEQ ID NO: 30. In some embodiments, the enzyme with AAE activity consists of the amino acid sequence as set forth in SEQ ID NO: 31.
  • Identification of additional enzymes having AAE activity or predicted to have AAE activity may be performed, for example based on similarity or homology with one or more domains of an AAE, such as an AAE provided by any one of SEQ ID NOs: 1-4 and 30-31.
  • an enzyme for use in the modified cells and methods described herein may be identified based on similarity or homology with an active domain, such as a catalytic domain associated with AAE activity.
  • an enzyme for use in the modified cells and methods described herein may have a relatively high level of sequence identity with a reference AAE, e.g., a wild-type AAE, such as any one of SEQ ID NOs: 1-4 and 30-31, in the region of the catalytic domain but a relatively low level of sequence identity to the reference AAE based on analysis of a larger portion of the enzyme or across the full length of the enzyme.
  • a reference AAE e.g., a wild-type AAE, such as any one of SEQ ID NOs: 1-4 and 30-31
  • the enzyme for use in the modified cells and methods described herein has at least 10%, at least 15%, at least 20%, at least 25%, at least 30% at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity based on a portion of the enzyme or across the full length of the enzyme relative to a reference AAE (e.g., any one of SEQ ID NOs: 1-4).
  • a reference AAE e.g., any one of SEQ ID NOs: 1-4.
  • the enzyme with AAE activity does not comprises CsAAEl from Cannabis sativa, which is provided by the Accession No. H0A1 V3-1 and the amino acid sequence set forth as SEQ ID NO: 5.
  • Alcohol-O-acyltransferase (AAT) enzymes are Alcohol-O-acyltransferase (AAT) enzymes
  • AATs which may also be referred to as acetyl-CoA: acetyltransferases or alcohol acetyltransferases, are bi-substrate enzymes that catalyze the transfer of acyl chains from an acyl-coenzyme A (CoA) donor to an acceptor alcohol, resulting in the production of an acyl ester.
  • CoA acyl-coenzyme A
  • the genetically modified cells comprise a genetic modification that results in increased expression of a gene encoding an enzyme with acyl-activating enzyme (AAE) activity.
  • the genetically modified cells described herein may also comprise a second genetic modification that results in increased expression of a second gene encoding an enzyme with alcohol-O-acyltransferase (AAT) activity.
  • a genetically modified yeast cell of the present disclosure can further comprise a genetic modification that results in increased expression of a nucleic acid encoding an enzyme having alcohol-O-acyltransferase (EC 2.3.1.84) activity (AAT).
  • the genetically modified yeast cell comprises a heterologous nucleic acid encoding the enzyme having AAT activity operably linked to a promoter.
  • the genetic modification comprises the genetically modified yeast cell being modified to comprise a heterologous gene encoding the enzyme having AAE activity.
  • the modified cells overexpress an enzyme having AAT activity.
  • the modified cells express a heterologous gene encoding an enzyme having AAT activity.
  • the heterologous gene can encode an enzyme that is not typically expressed by the cell, a variant of an enzyme that the cell does not typically express (e.g., a mutated enzyme), an additional copy of a gene encoding an enzyme that is typically expressed in the cell, or a gene encoding an enzyme that is typically expressed by the cell but under different regulation.
  • the modified cells express an endogenous gene at a level that is increased as compared to expression in a counterpart cell that is not genetically modified.
  • the heterologous gene encoding an enzyme with AAT activity is a wild-type (naturally occurring) AAT (e.g., a gene isolated from an organism).
  • the gene encoding the enzyme having AAT activity is a heterologous gene. In some embodiments, the gene encoding the enzyme having AAT activity is an endogenous gene. In some embodiments, the modified cell comprises a heterologous cassette comprising a promoter operably linked to a gene encoding an enzyme with alcohol-O-acyltransferase enzyme (AAT) activity, such as a heterologous gene encoding the AAT or an endogenous gene encoding the AAT.
  • AAT alcohol-O-acyltransferase enzyme
  • the gene encoding an enzyme with alcohol-O-acyltransferase activity is a wild-type AAT gene (e.g., a gene isolated from an organism).
  • the gene encoding an enzyme with AAT activity is a mutant AAT gene and contains one or more mutations (e.g., substitutions, deletions, insertions) in the nucleic acid sequence of the AAT gene and/or in amino acid sequence of the enzyme having AAT activity.
  • mutations in a nucleic acid sequence may change the amino acid sequence of the translated polypeptide (e.g., substitution mutation) or may not change the amino acid sequence of the translated polypeptide (e.g., silent mutations) relative to a wild-type enzyme or a reference enzyme.
  • the gene encoding an enzyme with AAT activity is a truncation, which is deficient in one or more amino acids, preferably at the N-terminus or the C-terminus of the enzyme, relative to a wild-type enzyme or a reference enzyme.
  • the AAT is obtained from a bacterium or a fungus, including a yeast.
  • the enzyme having AAT activity is derived from Marinobacter hydrocarbonoclasticus, Fragaria x ananassa, Saccharomyces cerevisiae, Neurospora sitophila, Actinidia deliciosa, Actinidia chinensis, Marinobacter aquaeolei, Saccharomycopsis fibuligera, Malus x domestica, Solanum pennellii, Solanum lycopersicum, Cucumis melo, or Fragaria chiloensis.
  • the AAT is obtained from Marinobacter hydrocarbonoclasticus or Malus x domestica.
  • An exemplary AAT enzyme is MhWES2 from Marinobacter hydrocarbonoclasticus, which is provided by Accession No. ABO21021-1 and the amino acid sequence set forth in SEQ ID NO: 6.
  • An exemplary AAT enzyme is MpAATl from Malus x domestica, which is provided by Accession No. NP_001315675-1 and the amino acid sequence set forth in SEQ ID NO: 8. (SEQ ID NO: 8)
  • the AAE and/or AAT variant may also contain one or more amino acid substitutions that do not substantially affect the activity and/or structure of the AAE and/or AAT enzyme.
  • conservative amino acid substitutions may be made in the enzyme to provide functionally equivalent variants of the foregoing polypeptides, i.e., the variants retain the functional capabilities of the polypeptides.
  • a “conservative amino acid substitution” refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • homologous genes encoding an enzyme having AAE and/or AAT could be obtained from other species and could be identified by homology searches, for example through a protein BLAST search, available at the National Center for Biotechnology Information (NCBI) internet site (ncbi.nlm.nih.gov).
  • NCBI National Center for Biotechnology Information
  • the disclosure also includes degenerate nucleic acids which include alternative codons to those present in the native materials.
  • serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC.
  • Each of the six codons is equivalent for the purposes of encoding a serine residue.
  • any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue into an elongating polypeptide.
  • nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); AC A, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons).
  • Other amino acid residues may be encoded similarly by multiple nucleotide sequences.
  • the disclosure embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.
  • the disclosure also embraces codon optimization to suit optimal codon usage of a host cell.
  • the modified nucleic acid molecules are structurally related to the unmodified nucleic acid molecules and in preferred embodiments are sufficiently structurally related to the unmodified nucleic acid molecules so that the modified and unmodified nucleic acid molecules hybridize under stringent conditions known to one of skill in the art.
  • modified nucleic acid molecules which encode polypeptides having single amino acid changes can be prepared. Each of these nucleic acid molecules can have one, two or three nucleotide substitutions exclusive of nucleotide changes corresponding to the degeneracy of the genetic code as described herein. Likewise, modified nucleic acid molecules which encode polypeptides having two amino acid changes can be prepared which have, e.g., 2-6 nucleotide changes. Numerous modified nucleic acid molecules like these will be readily envisioned by one of skill in the art, including for example, substitutions of nucleotides in codons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2 and 6, and so on.
  • each combination of two amino acids is included in the set of modified nucleic acid molecules, as well as all nucleotide substitutions which code for the amino acid substitutions.
  • Additional nucleic acid molecules that encode polypeptides having additional substitutions (i.e., 3 or more), additions or deletions (e.g., by introduction of a stop codon or a splice site(s)) also can be prepared and are embraced by the disclosure as readily envisioned by one of ordinary skill in the art. Any of the foregoing nucleic acids or polypeptides can be tested by routine experimentation for retention of structural relation or activity to the nucleic acids and/or polypeptides disclosed herein.
  • a cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell.
  • replication of the desired sequence may occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis.
  • replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript.
  • Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., P-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein).
  • Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • the genetically modified cells described herein comprise one or more heterologous “cassette” comprising a gene sequence operably linked to a promoter sequence.
  • a “cassette” refers to a nucleotide sequence that may be transferred into a cell and is not naturally present in the cell.
  • the genetically modified cell comprises a heterologous cassette comprising a heterologous promoter operably linked to a gene, e.g., a heterologous gene or an endogenous gene.
  • a coding sequence and regulatory sequences are said to be “operably” joined or operably linked when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined or operably linked if induction of a promoter in the 5’ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.
  • a variety of transcription control sequences can be used to direct its expression.
  • the promoter can be a native promoter, i.e., the promoter of the gene in its endogenous context, which provides normal regulation of expression of the gene.
  • the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of its associated gene (e.g., an enzyme having AAE or AAT activity).
  • a variety of conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule.
  • regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcrib ed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the disclosure may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • RNA heterologous DNA
  • any of the enzymes described herein can also be expressed in other yeast cells, including yeast strains used for producing wine, mead, sake, cider, etc.
  • a nucleic acid molecule that encodes the enzyme of the present disclosure can be introduced into a cell or cells using methods and techniques that are standard in the art.
  • nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc.
  • Expressing the nucleic acid molecule encoding the enzymes of the disclosure also may be accomplished by integrating the nucleic acid molecule into the genome.
  • genes can be accomplished either by incorporation of the new nucleic acid into the genome of the yeast cell, or by transient or stable maintenance of the new nucleic acid as an episomal element.
  • a permanent, inheritable genetic change is generally achieved by introduction of the DNA into the genome of the cell.
  • the gene may also include various transcriptional elements required for expression of the encoded gene product (e.g., enzyme having AAE and/or AAT activity).
  • the gene may include a promoter.
  • the promoter may be operably joined to the gene.
  • the cell is an inducible promoter.
  • the promoter is active during a particular stage of a fermentation process.
  • peak expression from the promoter is during an early stage of the fermentation process, e.g., before >50% of the fermentable sugars have been consumed.
  • peak expression from the promoter is during a late stage of the fermentation process e.g., after 50% of the fermentable sugars have been consumed.
  • the promoter is regulated by one or more conditions in the fermentation process, such as presence or absence of one or more factors.
  • the promoter is regulated by hypoxic conditions. Examples of promoters of hypoxia activated genes are known in the art. See, e.g., Zitomer et al. Kidney Int. (1997) 51(2): 507-13; Gonzalez Siso et al. Biotechnol. Letters (2012) 34: 2161-2173.
  • the promoter is a constitutive promoter.
  • constitutive promoters for use in yeast cells are known in the art and evident to one of ordinary skill in the art.
  • the promoter is a yeast promoter, e.g., a native promoter from the yeast cell in which the gene is expressed.
  • TDH3 promoter is pTDH3 from S. cerevisiae, which is provided by the nucleotide sequence set forth as SEQ ID NO: 17.
  • aspects of the present disclosure relate to genetically modified yeast cells (modified cells) and use of such modified cells in methods of producing a fermented product (e.g., a fermented beverage) and methods of producing ethanol.
  • the genetically modified yeast cells described herein are genetically modified with a gene encoding an enzyme with AAE activity or to overexpress an enzyme with AAE activity.
  • the genetically modified yeast cells described herein are further genetically modified with and a heterologous gene encoding an enzyme with AAT activity.
  • genetically modified cell As may be used interchangeably herein, to refer to a eukaryotic cell (e.g., a yeast cell) which has been, or may be presently, modified by the introduction of a genetic modification that results in increased expression of a gene encoding an enzyme having AAE activity.
  • the genetically modified yeast cells comprise one or more additional genetic modifications, for example, a genetic modification that results in increased expression of a gene encoding an enzyme having AAT activity.
  • the genetically modified cell comprises a single modification that results in increased expression of a gene encoding an enzyme having AAE activity and increased expression of a gene encoding an enzyme having AAT activity.
  • modified cell include the progeny of the original cell which has been genetically modified by the introduction of a heterologous gene. It shall be understood by the skilled artisan that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total nucleic acid complement as the original parent, due to mutation (i.e., natural, accidental, or deliberate alteration of the nucleic acids of the modified cell).
  • Yeast cells for use in the methods described herein are preferably capable of fermenting a sugar source (e.g., a fermentable sugar) and producing ethanol (ethyl alcohol) and carbon dioxide.
  • the yeast cell is of the genus Saccharomyces.
  • Saccharomyces genus includes nearly 500 distinct species, many of which are used in food production.
  • Saccharomyces cerevisiae S. cerevisiae
  • “brewer’s yeast” or “baker’s yeast” is commonly referred to as “brewer’s yeast” or “baker’s yeast,” and is used in the production of wine, bread, beer, among other products.
  • Saccharomyces genus include, without limitation, the wild yeast Saccharomyces paradoxus, which is a close relative to S. cerevisiae Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces uvarum, Saccharomyces cerevisiae var boulardii, Saccharomyces eubayanus.
  • the yeast is Saccharomyces cerevisiae (S. cerevisiae).
  • the yeast cell belongs to a non-Saccharomyces genus. See, e.g., Crauwels et al. Brewing Science (2015) 68: 110-121; Esteves et al. Microorganisms (2019) 7(11): 478.
  • the yeast cell is of the genus Kloeckera, Candida, Starmerella, Hanseniaspora, Kluyveromyces/Lachance, Metschnikowia, Saccharomycodes, Zygosaccharomyce, Dekker a (also referred to as Brettanomyces), Wickerhamomyces, or Torulaspora.
  • non-Saccharomyces yeast include, without limitation, Hanseniaspora uvarum, Hanseniaspora guillermondii, Hanseniaspora vinae, Metschnikowia pulcherrima, Kluyveromyces/Lachancea thermotolerans, Starmerella bacillaris (previously referred to as Candida stellata!
  • Candida zemplinina Saccharomycodes ludwigii, Zygosaccharomyces rouxii, Dekkera bruxellensis, Dekkera anomala, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Wickerhamomyces anomalus, and Torulaspora delbrueckii.
  • the methods described herein involve use of more than one genetically modified yeast.
  • the methods may involve use of more than one genetically modified yeast belonging to the genus Saccharomyces .
  • the methods may involve use of more than one genetically modified yeast belonging to a non-Saccharomyces genus.
  • the methods may involve use of more than one genetically modified yeast belonging to the genus Saccharomyces and one genetically modified yeast belonging to a non-Saccharomyces genus.
  • the any of the methods described herein may involve use of one or more genetically modified yeast and one or more non-genetically modified (wildtype) yeast.
  • the wort is contacted with a recombinant enzyme (e.g., any of the enzymes described herein), which may optionally be purified or isolated from an organism that produces the enzyme, allowing the enzyme to convert the sugars in the wort to alcohol and carbon dioxide.
  • a recombinant enzyme e.g., any of the enzymes described herein
  • the fermentation process of one or more fermentable sugars may be performed at a temperature of about 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C.
  • the additional or secondary fermentation process of the one or more fermentable sugars may be performed at a temperature of about 4°C to about 30°C. In some embodiments, the additional or secondary fermentation process of one or more fermentable sugars may be carried out at a temperature of about 8°C to about 14°C or about 18°C to about 24°C.
  • the additional or secondary fermentation process of one or more fermentable sugars may be performed at a temperature of about 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C.
  • selection of a time period and temperature for an additional or secondary fermentation process will depend on factors such as the type of beer, the characteristics of the beer desired, and the yeast strain used in the methods.
  • Products from the fermentation process may volatilize and dissipate during the fermentation process or from the fermented product.
  • ethyl -butanoate produced during fermentation using the cells described herein may volatilize resulting in reduced levels of ethyl -butanoate in the fermented product.
  • volatilized ethyl- butanoate is captured and re-introduced after the fermentation process.
  • Various refinement, filtration, and aging processes may occur subsequent fermentation, after which the liquid is bottled (e.g., captured and sealed in a container for distribution, storage, or consumption).
  • Any of the methods described herein may further involve distilling, pasteurizing and/or carbonating the fermented product.
  • the methods involve carbonating the fermented product.
  • Methods of carbonating fermented beverages are known in the art and include, for example, force carbonating with a gas (e.g., carbon dioxide, nitrogen), naturally carbonating by adding a further sugar source to the fermented beverage to promote further fermentation and production of carbon dioxide (e.g., bottle conditioning).
  • a fermented product or composition comprising ethanol according to the present disclosure can comprise a reduced amount of at least one undesired product as compared to a fermented product produced by a counterpart cell that does not express the enzyme having AAE activity.
  • the at least one undesired product is an acid selected from the group consisting of butanoic acid, isovaleric acid, octanoic acid, decanoic acid, 2-methyl-butyric acid, and crotonic acid.
  • the at least one undesired product is an acid selected from the group consisting of propanoic acid, butanoic acid, and isovaleric acid.
  • the fermented product is a fermented beverage.
  • the fermented beverage is beer, wine, sake, mead, cider, cava, sparkling wine (champagne), kombucha, ginger beer, water kefir.
  • the beverage is beer.
  • the beverage is wine.
  • the beverage is sparkling wine.
  • the beverage is Champagne.
  • the beverage is sake.
  • the beverage is mead.
  • the beverage is cider.
  • the beverage is hard seltzer.
  • the beverage is a wine cooler.
  • the fermented beverage is beer, wine, sparkling wine (champagne), wine cooler, wine spritzer, hard seltzer, sake, mead, kombucha, or cider.
  • the fermented product is a fermented food product.
  • fermented food products include, without limitation, cultured yogurt, tempeh, miso, kimchi, sauerkraut, fermented sausage, bread, and soy sauce.
  • a liquid fermentation composition comprises: (a) a population of genetically modified yeast cells according to the present disclosure and a sugar source.
  • the liquid fermentation composition further comprises alcohol, e.g. ethanol.
  • the liquid fermentation composition further comprises (i) an increased amount of one or more ethyl esters selected from the group consisting of ethyl propionate, ethyl butanoate, and ethyl isovalerate, and/or (ii) a decreased amount of one or more fatty acids selected from the group consisting of propionic acid, butyric acid, and isovaleric acid compared to a liquid fermentation composition produced by the same method using a counterpart cell that does not overexpress or comprise the enzyme having AAE activity.
  • increased titers of ethyl esters are produced through the recombinant expression of genes associated with the disclosure, in yeast cells and use of the cells in the methods described herein.
  • an “increased titer” or “high titer” refers to a titer in the nanograms per liter (ng L' 1 ) scale. The titer produced for a given product will be influenced by multiple factors including the choice of medium and conditions for fermentation.
  • the titer of one or more ethyl esters is at least 1 pg L’ 1 , for example at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260,
  • the amount of one or more ethyl esters in the fermented product or composition comprising ethanol is from about 150 pg/L to about 50 mg/L.
  • the fermented beverage contains an alcohol by volume of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.07%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2 %, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or higher.
  • the fermented beverage is nonalcoholic (e.g., has an alcohol by volume less than 0.5%).
  • Example 3 To determine whether the desirable effects of AAE expression described in Example 3 were specific to one combination of AAE, AAT, and parental strain, or whether they would be true across multiple AAE, AAT, and parental strain backgrounds, an additional group of stains were constructed.
  • This strain group was composed of one strain that was engineered for expression of an enzyme with AAT activity that was distinct from that of Example 3 as well as a second strain that was engineered for increased AAT expression as well as expression of the AAE enzyme H1CCL3. These strains were both created in the S04 brewing yeast background. Both engineered strains as well as the S04 parental strains were tested in 80ml malt extract fermentations, after which the concentrations of target ethyl esters and MCFA were measured. These data are shown in FIG. 12. As shown in FIG.
  • strains expressing both the AAT and H1CCL3 produced higher concentrations of two of the measured ethyl esters and reduced concentrations of two of the measured MCFAs as compared to the engineered strains expressing AAT alone.
  • the ethyl esters that increased were ethyl propionate and ethyl butanoate, and the MCFAs that were reduced were propionic acid and butanoic acid.
  • the first strain in this group named yl810, expressed the bacterial BktB pathway and a heterologous AAT, in line with the engineering strategy described in FIG. 6. Seven other strains were also created that carried the same engineered modifications as y 1810, and that additionally were engineered to express a distinct AAE. All of these strains were created in the LA3 brewing yeast background. All eight engineered strains as well as the LA3 parental strain were tested in 80 ml malt extract fermentations, after which the concentrations of target ethyl esters and MCFA were measured in the fermentation media.
  • FIG. 13 shows that y 1810 produced greatly elevated concentrations of ethyl butanoate and ethyl hexanoate, as well as greatly elevated concentrations of the MCFAs butanoic acid and hexanoic acid, compared to the parental LA3 strain.
  • FIG. 13 further shows that expression of many AAEs resulted in reduced concentrations of multiple MCFAs and increased concentrations of multiple ethyl esters.
  • the specific ethyl esters that increased and MCFAs that decreased in each AAE expression strain varied with the identity of the AAE.
  • H1CCL2, H1CCL3, and HcAAE(R51K) all resulted in increased production of ethyl butyrate and decreased production of butanoic acid relative to y 1810.
  • expression of H1CCL2, ScFAA4, and HcAAE(R51K) increased production of ethyl isovalerate and decreased production of isovaleric acid relative to y 1810.

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Abstract

L'invention concerne des cellules de levure génétiquement modifiées qui expriment de manière recombinante un gène codant une enzyme d'enzyme activant l'acyle (AAE) ou surexprimant une enzyme AAE. L'invention concerne également des procédés de production de boissons fermentées et des compositions comprenant de l'éthanol à l'aide des cellules de levure génétiquement modifiées présentement décrites.
PCT/US2024/061971 2023-12-26 2024-12-26 Cellules de levure génétiquement modifiées et procédés d'utilisation associés Pending WO2025144942A1 (fr)

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