WO2025144942A1

WO2025144942A1 - Genetically engineered yeast cells and methods of use thereof

Info

Publication number: WO2025144942A1
Application number: PCT/US2024/061971
Authority: WO
Inventors: Daniel Liu; Enzo LONG; Nick Harris; Jeremy ROOP; Charles DEPEW
Original assignee: Berkeley Fermentation Science Inc
Current assignee: Berkeley Fermentation Science Inc
Priority date: 2023-12-26
Filing date: 2024-12-26
Publication date: 2025-07-03
Anticipated expiration: 2026-06-26

Abstract

Provided herein are genetically modified yeast cells that recombinantly express a gene encoding an acyl-activating enzyme (AAE) enzyme or overexpress an AAE enzyme. Also provided are methods of producing fermented beverages and compositions comprising ethanol using the genetically modified yeast cells described herein.

Description

GENETICALLY ENGINEERED YEAST CELLS AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. provisional application No. 63/614,871, filed December 26, 2023, the content of which is hereby incorporated in its entirety.

SEQUENCE LISTING

The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on December 23, 2024, is named “129085.00045. xml” and is 58,977 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

BACKGROUND

Fruity and tropical fruit flavors are highly desirable in the fermented beverage market. Wines that impart fruity flavors, like Chardonnays and Sauvignon Blancs, make up the majority of wine sales in the US (Statista (2019)), Wine Consumption by Category, United States), while the popularity of beers made with fruity flavoring hops has skyrocketed in the last decade (Craft Beer Club (2018)), Your Guide to the Most Popular Beer Hops in the USA;10 Watson (2018), Beer Style Trends). The fruity flavors present in both beer and wine result from the presence of volatile flavor-active molecules that, when present in concentrations above the human detection threshold, impart fruity aromas and tastes.

Some of the most important of these fruity flavor molecules are ethyl esters. Examples of ethyl ester molecules that impart fruit flavors in fermented beverages are ethyl isovalerate (berry flavor), ethyl butanoate (mango flavor) and ethyl propionate (pineapple flavor). Due to the desirable flavors that they impart, fermented beverage producers have a strong interest in developing novel technologies that would allow them to increase the concentrations of these ethyl esters in their products. SUMMARY

In a first aspect, provided herein is 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. In some embodiments, 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 (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. In some embodiments, the cell produces the increased and/or decreased amounts under standard brewing conditions. In some embodiments, the standard brewing conditions comprise fermentation of 100% Rahr-2-Row wort that has not been hopped and has an original gravity of between 12 and 13 degrees Plato and a prefermentation pH between 5.10 and 5.40.

In some embodiments, 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.

In some embodiments, the one or more ethyl esters is ethyl propionate and the one or more fatty acids is propionic acid. In some embodiments of the foregoing aspects, the one or more ethyl esters is ethyl butanoate and the one or more fatty acids is butyric acid. In some embodiments of the foregoing aspects, the one or more ethyl esters is ethyl isovalerate and the one or more fatty acids is isovaleric acid.

In some embodiments, the enzyme having AAE activity is derived from Humulus lupulus or Hypericum caly cinum.

In some embodiments, the enzyme having AAE activity is derived from Humulus lupulus. In some embodiments of the foregoing aspects, the enzyme having AAE activity is H1CCL2. In some embodiments of the foregoing aspects, the enzyme having AAE activity is H1CCL3. In some embodiments, the enzyme having AAE activity is derived from Hypericum calycinum. In some embodiments, the enzyme having AAE activity is HcAAEl. In some embodiments, the enzyme having AAE activity is HcAAEl (R51K).

In some embodiments, a genetically modified yeast cell is provided that comprises a heterologous nucleic acid encoding an enzyme having an acyl activating enzyme (EC 6.2.1.3) activity (AAE), wherein the enzyme having AAE activity comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

In some embodiments, the genetically modified yeast cell comprises a cassette comprising the nucleic acid encoding the enzyme with AAE activity operably linked to a promoter.

In some embodiments, 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.

In some embodiments, the enzyme having AAE activity comprises a substitution mutation at a position corresponding to position R51 of SEQ ID NO: 2. In some embodiments of the foregoing aspects, the substitution mutation at the position corresponding to position R51 of SEQ ID NO: 2 is a lysine.

In some embodiments, 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.

In some embodiments, 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). In some embodiments, the genetically modified yeast cell comprises a cassette comprising the nucleic acid encoding the enzyme with AAT activity operably linked to a promoter.

In some embodiments, 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. In some embodiments, 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. In some embodiments, the enzyme having AAT activity comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 6-14.

In some embodiments, the enzyme having AAT activity has specificity for an acyl- CoA produced by the enzyme having AAE activity. In some embodiments, 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.

In some embodiments, the genetically modified yeast cell further comprises one or more additional heterologous genes operably linked to one or more additional promoters, wherein the one or more additional heterologous genes encode one or more additional enzymes selected from the group consisting of: PDC1, ALD6, ACS1, and ACC1.

In some embodiments, the genetically modified yeast cell further comprises one or more bacterial genes operably linked to one or more additional promoters, wherein the one or more bacterial genes encode one or more bacterial enzymes selected from the group consisting of: BktB, Hbd, Crt, and Ter.

In some embodiments, the promoter operably linked to the nucleic acid encoding the enzyme having AAE activity or the enzyme having AAT activity is selected from the group consisting of pGPMl, pHSP26, pTHD3, pHEM13, pSPGl, pPRBl, pQCRIO, pPGKl, pOLEl, pERG25, and pHHF2. In some embodiments, the one or more additional promoter is selected from the group consisting of pGPMl, pHSP26, pTHD3, pHEM13, pSPGl, pPRBl, pQCRIO, pPGKl, pOLEl, pERG25, and pHHF2.

In some embodiments, 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. cerevisiae California Ale Yeast strain WLP001, EC-1118, Elegance, Red Star Cote des Blancs, Epernay II, WY1056, A07, GY001, London Ale III, A38, S04, Conan, WLP4000, Verdant, WLP002, WLP006, WLP007, OYLOl l, S04, WLP029, WY2565, WY2007, WLP830, OYL071, PYL091, OYL057, OYL061, or OYL090.

In another aspect, a liquid fermentation composition is provided that comprises: (a) a population of genetically modified yeast cells according to the present disclosure and a sugar source. In some embodiments, the liquid fermentation composition further comprises alcohol, e.g. ethanol. In some embodiments, 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. In some embodiments, at least one fermentable sugar is provided in at least one sugar source. In some embodiments, the fermentable sugar is glucose, fructose, sucrose, maltose, and/or maltotriose.

In some embodiments, 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. In some embodiments, the counterpart cell is characteristic of a cell from which the genetically modified yeast cell is derived. In some embodiments, 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. In some embodiments, the desired product is an ethyl ester selected from the group consisting of ethyl propionate, ethyl butanoate, and ethyl isovalerate. In some embodiments, 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. In some embodiments, 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. In some embodiments, the undesired product is selected from the group consisting of propionic acid, butanoic acid, and isolvaleric acid.

In some embodiments, the fermented product is a fermented beverage. In some embodiments, the fermented beverage is beer, wine, sparkling wine (champagne), wine cooler, wine spritzer, hard seltzer, sake, mead, kombucha, or cider. In some embodiments, the sugar source comprises wort, must, fruit juice, honey, rice starch, or a combination thereof. In some embodiments, 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.

In some embodiments, the sugar source is wort, and 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. In some embodiments, the method further comprises adding at least one hop variety to the wort to produce a hopped wort. In some embodiments, the method further comprises adding at least one hop variety to the medium.

In some embodiments, 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.

In some embodiments, the method further comprises at least one additional fermentation process. In some embodiments, the method further comprises carbonating the fermented product.

Aspects of the present disclosure provide a fermented product produced, obtained, or obtainable by any of the methods described herein. In some embodiments, 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. In some embodiments, the fermented product comprises at least 150 pg/L of ethyl propionate, ethyl butanoate, and ethyl isovalerate. In some embodiments, 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. In some embodiments, at least one fermentable sugar is provided in at least one sugar source. In some embodiments, the fermentable sugar is glucose, fructose, sucrose, maltose, and/or maltotriose.

In some embodiments, the composition 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. In some embodiments, 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. In some embodiments, the desired product is an ethyl ester selected from the group consisting of ethyl propionate, ethyl butanoate, and ethyl isovalerate. In some embodiments, the composition 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. In some embodiments, 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. In some embodiments, the at least one undesired product is an acid selected from the group consisting of propionic acid, butanoic acid, and isovaleric acid.

In some embodiments, the composition is a fermented beverage. In some embodiments, the fermented beverage is beer, wine, sparkling wine (champagne), wine cooler, wine spritzer, hard seltzer, sake, mead, kombucha, or cider. In some embodiments, the sugar source comprises wort, must, fruit juice, honey, rice starch, or a combination thereof. In some embodiments, 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.

In some embodiments, the method further comprises at least one additional fermentation process. In some embodiments, further comprises carbonating the composition.

Aspects of the present disclosure provide a composition produced, obtained, or obtainable by any of the methods described herein. In some embodiments, the composition comprises at least 150 pg/L of ethyl propionate, ethyl butanoate, ethyl isovalerate, ethyl octanoate, ethyl decanoate, 2-methylbutyrate, or ethyl crotonate. In some embodiments, the composition 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 composition comprises at least 150 pg/L of ethyl propionate, ethyl butanoate, and ethyl isovalerate. In some embodiments, the composition comprises less than 15 mg/L of propionic acid, butanoic acid, and isovaleric acid. In some embodiments, the amount of ethyl esters and/or amount of one or more fatty acids can be measured by liquid-liquid extraction with ethyl acetate followed by gas chromatography-mass spectrometry (GC-MS).

In some embodiments, the composition comprises between 150 pg/L and 5 mg/L ethyl propionate, while containing less than 10 mg/L propionic acid. In some embodiments, the composition comprises between 150 pg/L and 50 mg/L ethyl butanoate, while containing less than 15 mg/L butanoic acid. In some embodiments, the composition comprises between 150 pg/L and 50 mg/L ethyl butanoate, while containing less than 15 mg/L butanoic acid.

The engineered strains described herein produce reduced concentrations of one or more ethyl esters (e.g., ethyl propionate, ethyl butanoate, ethyl isovalerate, ethyl octanoate, ethyl decanoate, and/or ethyl crotonate) while simultaneously producing reduced concentrations of one or more MCFAs (e.g., propionic acid, butanoic acid, isovaleric acid, octanoic acid, decanoic acid, and/or crotonic acid), as compared to control cells that were not engineered for heterologous AAE expression.

Also provided herein are methods of producing a fermented beverage involving contacting the genetically modified yeast cells with a medium comprising a sugar source comprising at least one fermentable sugar during a fermentation process. Also provided herein are methods of producing ethanol, including composition comprising ethanol, involving contacting the genetically modified yeast cells with a medium comprising a sugar source comprising at least one fermentable sugar during a fermentation process.

The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the disclosure will be readily appreciated upon review of the Detailed Description of its various aspects and embodiments, described below, when taken in conjunction with the accompanying Drawings. FIG. 1 shows the chemical structures of six exemplary ethyl esters frequently found in fermented beverages. These ethyl esters are attributed with imparting fruity flavors and aromas.

FIG. 2 provides a schematic of an endogenous yeast enzymatic pathway that biosynthesizes C4-C10 ethyl esters from fatty acids during fermentation. Yeast enzymes that catalyze the indicated reactions include PDC1, ALD6, ACS1, ACC1, FAS1, FAS2 and EEB1/EHT1.

FIG. 3 provides a schematic of an engineering strategy used for increasing ethyl ester biosynthesis involving increasing expression of yeast alcohol-O-acyltransferase (AAT) enzymes. Endogenous yeast enzymes and native enzymatic pathways include PDC1, ALD6, ACS1, ACC1, FAS1 and FAS2. Modifications generated by genetic engineering include EEB1, EHT1, ATF1 and ATF2.

FIG. 4 provides a schematic of an engineering strategy for increasing ethyl ester biosynthesis involving increasing expression of the yeast AAT enzyme, EHT1, and deletion of the yeast acyl activating enzyme (AAE), FAA1. Native yeast enzymes and enzymatic pathways include PDC1, ALD6, ACS1, ACC1, FAS1 and FAS2. Modifications generated by genetic engineering include EHT1.

FIG. 5 provides a schematic of an engineering strategy for increasing ethyl ester biosynthesis involving overexpression of several enzymes involved in fatty acid synthesis and expression of the heterologous AAT, SAAT1. Native yeast enzymes and enzymatic pathways include PDC1. Modifications generated by genetic engineering include ALD6, ACS1, ACC1, FAS1, FAS2 and SAAT1.

FIG. 6 provides a schematic of an engineering strategy for increasing biosynthesis of various ethyl esters via expression of bacterial enzymes. The heterologous pathway shows biosynthesis of C4 - C10 straight chain esters via expression of bacterial keto-acyl-thiolase (BktB) and associated enzymes, as well as a heterologous AAT. Native yeast enzymes and enzymatic pathways include PDC1, ALD6, ACS1, ACC1, FAS1 and FAS2. Modifications generated by genetic engineering include BktB, Hbd, Crt, Ter and VAAT/VLAAT.

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). 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. Each engineered strain is indicated along the vertical axis, and individual ethyl esters and MCFAs are indicated along the horizontal axis. Heatmap cells colored red indicate molecules produced at higher levels by engineered strains compared to parental strains, and blue cells indicate molecules produced at lower levels by engineered strains relative to parental strains. Gray cells indicate molecules that were not measured in a given experiment. The values in each cell report the log2 fold change of the abundance of each molecule relative to its abundance in the parental strain. Peak areas were only compared between engineered strains and parental strains tested within the same experiment and not across experiments performed at different times.

FIG. 9 shows a scatter plot presenting the data of FIG. 8 as the correlation between ethyl ester and MCFA concentrations in fermentations with engineered strains. The log- transformed fold-change value for each MCFA is plotted as a function of the fold-change value of its corresponding ethyl ester in the same fermentation. Each point shown in the scatter plot indicates fold-changes for one ethyl ester / MCFA pair as measured in a fermentation with one engineered strain, relative to a parental non-engineered strain. The acyl moiety of each ethyl ester / MCFA pair is indicated by the point shape, as shown in the legend. C4; ethyl butanoate and butanoic acid, C5; ethyl isovalerate and isovaleric acid, C6, ethyl hexanoate and hexanoic acid, C8, ethyl octanoate and octanoic acid, CIO; ethyl decanoate and decanoic acid.

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. 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. 11 shows the concentrations of ethyl esters and medium-chain fatty acids (MCFAs) following fermentation with yeast strains that were engineered to express either an AAT enzyme (“+AAT” on the horizontal axis), or to express both an AAT and the AAE enzyme H1CCL2 (+AAT + H1CC2” on the horizontal axis). LA3 is a wild-type strain that was the parent for the engineered strains. Each subplot shows the concentrations of one pair of ester and MCFA molecules, and yeast strains are described on the horizontal axis. FIG. 12 shows the concentrations of ethyl esters and medium-chain fatty acids (MCFAs) following fermentation with S04 yeast strains that were engineered to express either an AAT enzyme (“+AAT” on the horizontal axis), or to express both an AAT and the AAE enzyme H1CCL3 (+AAT + H1CC3” on the horizontal axis). Each subplot shows the concentrations of one pair of ester and MCFA molecules, and yeast strains are described on the horizontal axis.

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.

DETAILED DESCRIPTION

Fruity flavors in beers and wines are very popular among consumers. At a molecular level, many of the most desirable fruity flavors, like pineapple and mango, are imparted by a family of molecules called ethyl esters (Holt et al. FEMS Microbiol. Rev. (2018). 43, 193— 222). 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).

The majority of ethyl esters in beer and wine are produced by yeast during the fermentation process. During fermentation, yeast primarily convert sugars into ethanol and cell biomass, but they also produce a wide variety of additional flavor-active molecules, including ethyl esters.

Several different yeast enzymatic pathways are responsible for ethyl ester biosynthesis, and these have been well studied and characterized (Saerens et al. AppL Environ. Microbiol. (2008). 74, 454-461; Saerens et al. J. Biol. Chem. (2006). 281, 4446- 4456; Saerens et al. Microb. BiotechnoL (2010). 3, 165-177). Among these pathways, one enzymatic pathway is primarily responsible for the biosynthesis of fruit-flavored ethyl esters. This pathway is connected to the native yeast fatty acid biosynthesis pathway (FIG. 2). As shown in FIG. 2, 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. 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. Yeast express several different AAT enzymes, but among these, EEB1 and EHT1 are thought to play a central role in biosynthesis of fruit flavored C4-C10 ethyl esters (Saerens et al. J. Biol. Chem. (2006). 281, 4446-4456).

Through this enzymatic pathway, most yeast strains will produce low levels of fruity ethyl esters during fermentation. The reason that only low levels of ethyl esters are biosynthesized is likely due to a limited release of medium chain acyl-CoAs from the FAS complex, as well as inefficient catalytic activity of the yeast EEB1 and EHT1 enzymes. Because only low levels of ethyl esters are typically produced by fermenting yeast strains, ethyl esters generally make only a low to moderate contribution to the overall flavor and aroma of beers and wines.

Given that ethyl esters impart such desirable fruity flavors, significant research efforts have been made to discover strategies that increase the concentrations of specific ethyl esters produced by yeast during fermentation. The development and use of strains that are genetically engineered to biosynthesize increased concentrations of ethyl esters could allow brewers and winemakers to create strongly fruity-forward beverages in a cost-effective and consistent manner.

Genetic engineering efforts seeking to engineer yeast for increased ethyl ester biosynthesis have been ongoing for several decades. These efforts have succeeded in discovering multiple strategies allowing for the creation of engineered yeast that biosynthesize increased concentrations of fruity ethyl esters.

One of the first strategies attempted for increasing ethyl ester biosynthesis was to overexpress endogenous yeast AAT enzymes (FIG. 3). In these studies, AATs were overexpressed individually, or in some cases multiple AATs were simultaneously overexpressed. In some instances, these modifications resulted in modest 1-3 -fold increases in the concentrations of several ethyl esters, but in other instances the modifications did not affect the final concentration of ethyl esters in the media. See, e.g., Saerens et al. J. Biol. Chem. (2006). 281, 4446-4456; Lilly et al. Appl. Environ. Microbiol. (2000). 66, 744-753; Yin et al. J. Agric. Food Chem. (2019). 67, 5607-5613. The overall conclusion from these studies was that overexpression of endogenous yeast AATs may result in modestly increased concentrations of some ethyl esters, but that these effects are contingent on specific strain backgrounds and fermentation conditions.

Another strategy previously used involves overexpression of the yeast AAT, EHT1, and deletion of the yeast acyl -activating enzyme, FAA1 (FIG. 4). As FAA1 is a known negative regulator of ACC1, FAA1 deletion increased ACC1 activity resulting in increased metabolic flux through the FAS 1/2 complex and an increase in the biosynthesis of acyl-CoA precursors. Together with overexpression of EHT1, these combined modifications resulted in a 3 -fold increase in ethyl hexanoate concentration following fermentation as compared to the parental non-engineered strain. See, e.g., Chen et al. J. Ind. Microbiol. BiotechnoL (2014). 41, 563-572.

Another strategy implemented to increase ethyl ester production involved expression of a heterologous AAT and overexpression of endogenous fatty acid biosynthesis enzymes (FIG. 5). In this study, a Baijiu yeast was engineered to 1) express a heterologous AAT (SAAT1) derived from strawberry and 2) overexpress five endogenous yeast genes involved in key steps in fatty acid biosynthesis. The five overexpressed genes were ALD6, ACS1, ACC 16, FAS1, and FAS2. The rationale for the overexpression of endogenous genes was to increase the metabolic flux towards the biosynthesis of fatty acids, which would also increase formation of medium chain acyl-CoAs. In addition, SAAT1 was expressed as it was thought to more efficiently catalyze the esterification reaction between medium chain acyl-CoAs and ethanol. When combined, these modifications resulted in large increases in the concentrations of ethyl hexanoate, ethyl octanoate, and ethyl decanoate (26-, 7-, and 9-fold increased concentrations relative to the parental strain, respectively). See, Shi et al. LWT. (2021) 145: 111496.

Another strategy included expression of a bacterial keto-acyl-thiolase pathway and expression of a heterologous AAT (FIG. 6), which resulted in a greater than 80-fold increase in concentrations of ethyl butanoate, ethyl hexanoate, and ethyl crotonate. In several studies, Baijiu yeast were engineered to 1) express four bacterial genes that catalyze biosynthesis of C4-C10 acyl CoAs, and 2) express one of several heterologous AATs derived from plants (See, e.g., Shi et al. LWT. (2021) 145: 111496; Zhang et al. LWT. (2022) 168: 113908; Zhang et al. LWT. (2022) 170: 114061; Ma et al. J. Agric. FoodChem. (2020) 68: 4252-4260). Expression of the bacterial genes in the yeast strains allowed for medium chain acyl-CoA biosynthesis via a biochemical pathway that is independent of the yeast enzymes ACC1, FAS1, and FAS2. Acyl-CoAs biosynthesized by this pathway included saturated C4-C10 acyl CoAs, as well as the unsaturated C4 acyl-CoA, crotonyl-CoA. This research also tested several heterologous AATs enzymes to determine which had optimal catalytic activity for the biosynthesis of specific ethyl esters. Among the AATs tested, it was found that an AAT derived from grape (VLAAT) was optimal for biosynthesis of ethyl crotonate, while an AAT derived from wild strawberry (VAAT) was optimal for biosynthesis of ethyl hexanoate. The final strains constructed from these studies were capable of producing multiple ethyl esters at >80-fold higher concentrations than non-engineered parental strains.

As shown in FIG. 7, a further strategy has been implemented in which 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. This strategy was implemented in several different yeast strain backgrounds and resulted in 10-20-fold increased concentrations of ethyl hexanoate.

The strategies described above and in Figures 2 through 7 demonstrate multiple genetic engineering strategies that can allow for the creation of yeast strains that produce elevated concentrations of desirable ethyl esters molecules. However, when used for the production of fermented beverages, all of these strategies suffer from a substantial limitation. This limitation is that in addition to having increased ethyl esters biosynthesis, these engineered strains simultaneously produce elevated concentrations of medium chain fatty acid (MCFA) molecules. As MCFAs impart strong off-flavors in fermented beverages associated with cheese, bile, goat, and bitter flavors, the increase in MCFA concentrations displayed by these strains is a substantial problem.

It has been discovered that among strains engineered for increased ethyl ester production, the degree of MCFA production scales with the degree of ethyl ester production such that there is a correlation between the levels of MCFAs and ethyl esters. Additionally, the specific MCFAs produced at elevated concentrations are associated with their corresponding ethyl esters (i.e., they contained the same acyl moiety). For example, strains biosynthesizing increased levels of ethyl butanoate also produce increased levels of butanoic acid, while strains producing elevated concentrations of ethyl isovalerate produced elevated concentrations of isovaleric acid. The strength of the correlation between ethyl esters and corresponding MCFAs varied across different combinations of strain background, engineering strategy and heterologous gene expression level, but across all variables a robust correlated trend has been observed (Spearman correlation = .60, FIG 9). Notably, all of the MCFAs observed at elevated concentrations (butanoic acid, isovaleric acid, hexanoic acid, octanoic acid, decanoic acid) are strong off-flavor molecules in beer and wine.

Accordingly, there is a need for an improved engineering strategy for increasing the biosynthesis of desirable fruit-flavored ethyl esters that does not simultaneously increase the production of MCFAs.

The present disclosure describes the development of genetically engineered yeast strains that have been genetically engineered to express an acyl-activating enzyme (AAE). In some embodiments, the genetically modified yeast cells have been engineered to express a heterologous AAE in addition to expression of a heterologous AAT. In some embodiments, the engineered cells have been engineered for increased expression of an AAE in addition to other genetic modifications that result in increased ethyl ester biosynthesis. For example, increased expression a nucleic acid encoding the enzyme having AAE activity can be performed by promoter swapping, inhibition of repressors, or incorporation of a heterologous cassette for transcription of the nucleic acid and expression of the enzyme having AAE activity. In some embodiments, 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. For example, 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. It should be understood that 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.

As used herein, a “brewing yeast cell” or a “brewing yeast strain” refers to a yeast strain that has been selected by brewers to have certain properties that make it suitable for brewing alcoholic and non-alcoholic beverages, and that is genetically distinct from nonbrewing yeast strains. Brewing yeast strains include strains of Saccharomyces cerevisiae whose genome sequence places them within the phylogenetic clades of “Beer 1”, “Beer 2”, or “Mixed” as defined in Galione et al. 2016 and Priess et al. 2018. (PMIDs 27610566, 30258422). Alternatively, 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). In addition, 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. In any of the embodiments of the present disclosure, a genetically modified yeast cell can be derived from a brewing yeast strain. Exemplary brewing yeast strains are disclosed further herein.

As described herein, when a percent identity is stated, or a range thereof (e.g., at least, more than, etc.), unless otherwise specified, 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

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% identity) and all increments thereof (e.g., tenths of a percent (i.e., 0.1%), hundredths of a percent (i.e., 0.01%), etc.).

The terms “percent identity,” “sequence identity,” “% identity,” “% sequence identity,” and % identical,” as they may be interchangeably used herein, refer 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. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

The term “heterologous nucleic acid” as used herein 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 (for example, the nucleic acid is recombinant).

As used herein, the term “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.

Acyl-activating Enzymes (AAEs)

As described herein, 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. In some embodiments, the modified cells overexpress an enzyme having AAE activity. In some embodiments, 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. In some embodiments, 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. In some embodiments, the heterologous gene encoding an enzyme with AAE activity is a wild-type (naturally occurring) AAE (e.g., a gene isolated from an organism).

In some embodiments, provided herein is 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. In some embodiments, 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 (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. In some embodiments, the cell produces the increased and/or decreased amounts under standard brewing conditions. In some embodiments, the standard brewing conditions comprise fermentation of unhopped wort.

In some embodiments, the genetic modification comprises the genetically modified yeast cell being modified to comprise a heterologous gene encoding the enzyme having AAE activity.

In some embodiments, the genetically modified yeast cell does not comprise any additional genetic modifications. In some embodiment, the genetically modified yeast cell further comprises additional genetic modification, such as those described herein.

As used herein, 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. For example, 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.

In some embodiments, 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.

Acyl-activating enzymes (AAEs) catalyze the formation of a medium-chain fatty acyl-CoA from a medium-chain fatty acid and a free coenzyme A (CoA). For example, an AAE catalyzes the reaction that forms hexanoyl-CoA (i.e., a medium-chain fatty acyl-CoA) from hexanoic acid (i.e., a medium-chain fatty acid) and a free CoA. Without wishing to be bound to any particular theory, expression of an AAE during fermentation may reduce the final yield of medium-chain fatty acids in a fermented product or beverage. Genetically modified cells expressing an AAE may produce fermented products or beverages with lower concentrations of undesired medium-chain fatty acids, compared to cells that do not overexpress the AAE and/or express a heterologous AAE. In some embodiments, the one or more ethyl esters comprise ethyl propionate and the one or more fatty acids comprise propionic acid. In some embodiments, the ethyl esters comprise ethyl butanoate and the one or more fatty acid comprise butyric acid. In some embodiments, the ethyl esters comprise ethyl isovalerate and the one or more fatty acids comprise isovaleric acid. In some embodiments, the one or more ethyl esters is ethyl propionate and the one or more fatty acids is propionic acid. In some embodiments, the ethyl esters is ethyl butanoate and the one or more fatty acid is butyric acid. In some embodiments, the ethyl esters is ethyl isovalerate and the one or more fatty acids is isovaleric acid.

In some embodiments, the AAE gene is from a plant. In some embodiments, the AAE gene is from a yeast cell. In some embodiments, the AAE gene is from a Saccharomyces species. In some embodiments, the AAE gene is from Saccharomyces cerevisiae. In some embodiments, the AAE gene is from a Humulus species. In some embodiments, the AAE gene is from Humulus lupulus. In some embodiments, the AAE gene is from Hypericum species. In some embodiments, the AAE gene is from Hypericum calycinum.

An exemplary AAE enzyme is H1CCL2 from Humulus lupulus, which is provided by Accession No. M4IRL4-1 and the amino acid sequence set forth as SEQ ID NO: 1.

An exemplary AAE enzyme is H1CCL3 from Humulus lupulus, which is provided by Accession No. M4IS88-1 and the amino acid sequence set forth as SEQ ID NO: 2.

(SEQ ID NO: 2)

An exemplary AAE enzyme is HcAAE from Hypericum calycinum. which is provided by the Accession No. QII68911-1 and the amino acid sequence set forth as SEQ ID NO: 3.

In some embodiments, the enzyme having AAE activity can comprise SEQ ID NO: 3 with a substitution mutation of R51K (HcAAEl (R51K), SEQ ID NO: 4).

In some embodiments, the enzyme having AAE activity can comprise SEQ ID NO: 30 (ScFAAl from Saccharomyces cerevisiae (Accession No. AJT92227-1)).

In some embodiments, the enzyme having AAE activity can comprise SEQ ID NO: 31 (ScFAA4 from Saccharomyces cerevisiae (Accession No. CAI5316849-1)).

Amino acids of the enzyme having AAE activity may be modified (e.g. substituted) to produce an AAE variant. For example, as described herein, the amino acid at position 51, referred to as arginine 51 (R51) of SEQ ID NO: 3 can be mutated to produce an AAE enzyme having a desired activity, such as increased production of an ethyl ester during fermentation, reduced production of a MCFA during fermentation, and/or an increased ratio of ethyl ester to MCFA production. In some embodiments, the amino acid corresponding to R51 of SEQ ID NO: 3 is substitution with a lysine residue (R51K).

In some embodiments, the genetically modified yeast cell expressing the enzyme having AAE activity increases the amount of ethyl esters in a fermented product or beverage without overexpression of or expression of the enzyme having AAE activity. In some embodiments, the genetically modified yeast cell expressing the enzyme having AAE activity reduces the amount of MCFAs in a fermented product or beverage without overexpression of or expression of the enzyme having AAE activity. In some embodiments, the genetically modified yeast cell expressing the enzyme having AAE activity increases the amount of ethyl esters in a fermented product or beverage and reduces the amount of MCFAs in a fermented product or beverage compared to the fermented product or beverage produced using a counterpart cell without overexpression of or expression of the enzyme having AAE activity.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 and 30-31.

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. 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.

In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the enzyme for use in the modified cells and methods described herein has 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 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 in the region of the catalytic domain of the enzyme relative to a reference AAE (e.g., any one of SEQ ID NOs: 1-4). In some embodiments, the enzyme for use in the modified cells and methods described herein has 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 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 in the region of the catalytic domain of the enzyme relative to a reference AAE (e.g., any one of SEQ ID NOs: 1-4).

In some embodiments, the enzyme for use in the modified cells and methods described herein has a relatively high level of sequence identity in the region of the catalytic domain of the enzyme relative to a reference AAE (e.g., any one of SEQ ID NOs: 1-4) and 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. In some embodiments, 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). In some embodiments, 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).

In some embodiments, 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

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. The acyl esters present in a fermented beverage influence its flavor.

As described herein, 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.

In some embodiments, 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). In some embodiments, the genetically modified yeast cell comprises a heterologous nucleic acid encoding the enzyme having AAT activity operably linked to a promoter.

In some embodiments, the modified cells overexpress an enzyme having AAT activity. In some embodiments, 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. In some embodiments, 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. In some embodiments, the heterologous gene encoding an enzyme with AAT activity is a wild-type (naturally occurring) AAT (e.g., a gene isolated from an organism).

In some embodiments, 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.

In some embodiments, the gene encoding an enzyme with alcohol-O-acyltransferase activity is a wild-type AAT gene (e.g., a gene isolated from an organism). In some embodiments, 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. As will be understood by one of ordinary skill in the art, 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.

In some embodiments, 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.

In some embodiments, the AAT is obtained from a bacterium or a fungus, including a yeast. In some embodiments, 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. In some embodiments, 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 MhWESl from Marinobacter hydrocarbonoclasticus, which is provided by Accession No. WP_011783747-1 and the amino acid sequence set forth in SEQ ID NO: 7.

(SEQ ID NO: 7)

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)

An exemplary AAT enzyme is SpAAT from Solanum pennellii. which is provided by Accession No. NP 001310384.1 and the amino acid sequence set forth in SEQ ID NO: 9.

An exemplary AAT enzyme is CmAAT from Cucumis melo. which is provided by Accession No. KAL0536511.1 and the amino acid sequence set forth in SEQ ID NO: 10.

An exemplary AAT enzyme is FcAATl from Fragaria chiloensis. which is provided by Accession No. ACT82247.1 and the amino acid sequence set forth in SEQ ID NO: 11.

An exemplary AAT enzyme is SAAT from Fragaria x ananassa, which is provided by Accession No. AAG13130.1 and the amino acid sequence set forth in SEQ ID NO: 12.

(SEQ ID NO: 12)

An exemplary AAT enzyme is EEB1 from Saccharomyces cerevisiae. which is provided by Accession No. ADD16960.1 and the amino acid sequence set forth in SEQ ID NO: 13.

An exemplary AAT enzyme is EHT1 from Saccharomyces cerevisiae, which is provided by Accession No. CAI6500086.1 and the amino acid sequence set forth in SEQ ID NO: 14.

In some embodiments, the gene encodes an enzyme with AAT activity such that a cell that expresses the enzyme is capable of increased production of one or more ethyl esters as compared to a cell that does not express the heterologous gene. In some embodiments, the gene encodes an enzyme with AAT activity such that a cell that expresses the enzyme is capable of producing increased levels of one or more ethyl esters as compared to a cell that expresses an enzyme with wild-type AAT activity. In some embodiments, the gene encodes an enzyme with AAT activity such that a cell that expresses the enzyme is capable of producing reduced levels of one or more medium-chain fatty acids as compared to a cell that does not express the gene. In some embodiments, the gene encodes an enzyme with AAT activity such that a cell that expresses the enzyme is capable of producing reduced levels of one or more medium-chain fatty acids as compared to a cell that expresses an enzyme with wild-type AAT activity. In some embodiments, the enzyme with AAT activity that is capable of producing increased levels of one or more ethyl esters has the sequence provided by any one of SEQ ID NOs: 6-14.

In some embodiments, the enzyme with AAT 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 the sequence as set forth in any one of SEQ ID NOs: 6- 14.

In some embodiments, the enzyme with AAT activity comprises the amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence as set forth in SEQ ID NO: 7. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence as set forth in SEQ ID NO: 8. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, the enzyme with AAT activity comprises the amino acid sequence as set forth in SEQ ID NO: 14. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence as set forth in SEQ ID NO: 7. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence as set forth in SEQ ID NO: 8. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, the enzyme with AAT activity consists of the amino acid sequence as set forth in SEQ ID NO: 14.

In some embodiments, the enzyme having AAT activity has specificity for an acyl- CoA produced by the enzyme having AAE activity.

Identification of additional enzymes having AAT activity or predicted to have AAT activity may be performed, for example based on similarity or homology with one or more domains of an AAT, such as the AAT provided by any one of SEQ ID NOs: 8-10. In some embodiments, 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, such as a catalytic domain associated with AAT activity. In some embodiments, an enzyme for use in the modified cells and methods described herein may have a relatively high level of sequence identity with a reference AAT, e.g., a wild-type AAT, such as any of SEQ ID NOs: 8-10, in the region of the catalytic domain but a relatively low level of sequence identity to the reference AAT based on analysis of a larger portion of the enzyme or across the full length of the enzyme. In some embodiments, the enzyme for use in the modified cells and methods described herein has 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 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 in the region of the catalytic domain of the enzyme relative to a reference AAT (e.g., any one of SEQ ID NOs: 6-14).

In some embodiments, the enzymes for use in the modified cells and methods described herein have a relatively high level of sequence identity in the region of the catalytic domain of the enzyme relative to a reference AAT (e.g., any of SEQ ID NOs: 6-14) and a relatively low level of sequence identity to the reference AAT based on analysis of a larger portion of the enzyme or across the full length of the enzyme. In some embodiments, the enzymes for use in the modified cells and methods described herein have at least 10%, at least 15%, at least 20%, at least 25%, 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 AAT (e.g, SEQ ID NOs: 6-14).

In some embodiments, the amino acid substitution(s) may be in the active site. As used herein, the term “active site” refers to a region of the enzyme with which a substrate interacts. The amino acids that comprise the active site and amino acids surrounding the active site, including the functional groups of each of the amino acids, may contribute to the size, shape, and/or substrate accessibility of the active site. In some embodiments, the AAT variant contains one or more modifications that are substitutions of a selected amino acid with an amino acid having a different functional group.

General methods of genetic engineering and enzyme modification

As will also be evident to one or ordinary skill in the art, the amino acid position number of a selected residue in an AAE and/or AAT may have a different amino acid position number in another AAE and/or AAT enzyme (e.g, a reference enzyme). Generally, one may identify corresponding positions in other AAE and/or AAT enzymes using methods known in the art, for example by aligning the amino acid sequences of two or more enzymes. Software programs and algorithms for aligning amino acid (or nucleotide) sequences are known in the art and readily available, e.g., Clustal Omega (Sievers et al. 2011).

The AAE and/or AAT variants described herein may further contain one or more additional modifications, for example to specifically alter a feature of the polypeptide unrelated to its desired physiological activity. Alternatively or in addition, the AAE and/or AAT enzymes described herein may contain one or more mutations to modulate expression and/or activity of the enzyme in the cell.

Mutations of a nucleic acid which encodes an AAE and/or AAT preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such a hairpins or loops, which can be deleterious to expression of the enzyme.

Mutations can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. As described herein, variant polypeptides can be expressed and tested for one or more activities to determine which mutation provides a variant polypeptide with the desired properties. Further mutations can be made to variants (or to non-variant polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host (referred to as codon optimization). The preferred codons for translation of a nucleic acid in, e.g., S. cerevisiae, are well known to those of ordinary skill in the art. Still other mutations can be made to the noncoding sequences of a gene or cDNA clone to enhance expression of the polypeptide. The activity of an AAE and/or AAT (enzyme) variant can be tested by cloning the gene encoding the enzyme variant into an expression vector, introducing the vector into an appropriate host cell, expressing the enzyme variant, and testing for a functional capability of the enzyme, as disclosed herein.

The AAE and/or AAT variants described herein may contain an amino acid substitution of one or more positions corresponding to a reference AAE and/or AAT. In some embodiments, the AAE and/or AAT variant contains an amino acid substitution at 1, 2, 3, 4, 5, or more positions corresponding to a reference AAE and/or AAT. In some embodiments, the AAE and/or AAT is not a naturally occurring AAE and/or AAT, e.g., is genetically modified.

In some embodiments, 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. The skilled artisan will also realize that 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. As used herein, 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. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Exemplary functionally equivalent variants of polypeptides include conservative amino acid substitutions in the amino acid sequences of proteins disclosed herein. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H;

(d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

As one of ordinary skill in the art would be aware, 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). By aligning the amino acid sequence of an enzyme with one or more reference enzymes and/or by comparing the secondary or tertiary structure of a similar or homologous enzyme with one or more reference eta lyase, one can determine corresponding amino acid residues in similar or homologous enzymes and can determine amino acid residues for mutation in the similar or homologous enzyme.

Genes associated with the disclosure can be obtained (e.g., by PCR amplification) from DNA from any source of DNA which contains the given gene. In some embodiments, genes associated with the disclosure are synthetic, e.g., produced by chemical synthesis in vitro. Any means of obtaining a gene encoding the enzymes described herein are compatible with the modified cells and methods described herein.

The disclosure provided herein involves recombinant expression of a gene encoding an enzyme, including but not limited to an enzyme having AAE or AAT activity, functional modifications and variants of the foregoing, as well as uses relating thereto. Homologs and alleles of the nucleic acids associated with the disclosure can be identified by conventional techniques. Also encompassed by the disclosure are nucleic acids that hybridize under stringent conditions to the nucleic acids described herein. The term “stringent conditions” as used herein refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.

There are other conditions, reagents, and so forth which can be used, which result in a similar degree of stringency. The skilled artisan will be familiar with such conditions, and thus they are not given here. It will be understood, however, that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of homologs and alleles of nucleic acids of the disclosure (e.g., by using lower stringency conditions). The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid molecule and sequencing.

The disclosure also includes degenerate nucleic acids which include alternative codons to those present in the native materials. For example, 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. Thus, it will be apparent to one of ordinary skill in the art that 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. Similarly, 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. Thus, 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 disclosure also provides modified nucleic acid molecules which include additions, substitutions and deletions of one or more nucleotides. In preferred embodiments, these modified nucleic acid molecules and/or the polypeptides they encode retain at least one activity or function of the unmodified nucleic acid molecule and/or the polypeptides, such as enzymatic activity. In certain embodiments, the modified nucleic acid molecules encode modified polypeptides, preferably polypeptides having conservative amino acid substitutions as are described elsewhere herein. 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.

For example, 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. In the foregoing example, 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.

In some embodiments, one or more of the genes associated with the disclosure is expressed in a recombinant expression vector. As used herein, a “vector” may be any of a number of nucleic acids into which a desired sequence or sequences may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to: plasmids, fosmids, phagemids, virus genomes and artificial chromosomes.

A cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell. In the case of plasmids, 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. In the case of phage, 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.

In some embodiments, the genetically modified cells described herein comprise one or more heterologous “cassette” comprising a gene sequence operably linked to a promoter sequence. As used herein, a “cassette” refers to a nucleotide sequence that may be transferred into a cell and is not naturally present in the cell. For example, in some embodiments, 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.

As used herein, 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.

When the nucleic acid molecule that encodes any of the enzymes of the present disclosure is expressed in a cell, a variety of transcription control sequences (e.g., promoter/enhancer 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. In some embodiments 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.

The precise nature of the 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. In particular, such 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.

Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA). That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell. As one of ordinary skill in the art would appreciate, 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. For example, 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.

The incorporation of 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. In eukaryotic cells, 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). For example, in some embodiments, the gene may include a promoter. In some embodiments, the promoter may be operably joined to the gene. In some embodiments, the cell is an inducible promoter. In some embodiments, the promoter is active during a particular stage of a fermentation process. For example, in some embodiments, 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. In some embodiments, 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.

Conditions in the medium change during the course of the fermentation process, for example the availability of nutrients and oxygen tend to decrease over time during fermentation as sugar source and oxygen become depleted. Additionally, the presence of other factors, such as products produced by metabolism of the cells, increase. In some embodiments, the promoter is regulated by one or more conditions in the fermentation process, such as presence or absence of one or more factors. In some embodiments, 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.

In some embodiments, the promoter is a constitutive promoter. Examples of constitutive promoters for use in yeast cells are known in the art and evident to one of ordinary skill in the art. In some embodiments, the promoter is a yeast promoter, e.g., a native promoter from the yeast cell in which the gene is expressed.

In some embodiments, the promoter is the GPM1 promoter (pGMPl), the HSP26 promoter (pHSP26), the TDH3 promoter, the HEM13 promoter (pHEM13), SPG1 promoter (pSPGl), PRB1 promoter (pPRBl), QCR10 (pQCRIO), PGK1 promoter (pPGKl), OLE1 promoter (pOLEl), ERG25 promoter (pERG25), or the HHF2 promoter (pHHF2).

An exemplary GPM1 promoter is pGPMl from S. cerevisiae. which is provided by the nucleotide sequence set forth as SEQ ID NO: 15.

An exemplary HSP26 promoter is pHSP26 from S. cerevisiae, which is provided by the nucleotide sequence set forth as SEQ ID NO: 16.

An exemplary TDH3 promoter is pTDH3 from S. cerevisiae, which is provided by the nucleotide sequence set forth as SEQ ID NO: 17.

An exemplary HEM13 promoter is pHEM13 from S. cerevisiae, which is provided by the nucleotide sequence set forth as SEQ ID NO: 18.

An exemplary SPG1 promoter is pSPGl from S. cerevisiae, which is provided by the nucleotide sequence set forth as SEQ ID NO: 19.

An exemplary PRB1 promoter is pPRBl from S. cerevisiae, which is provided by the nucleotide sequence set forth as SEQ ID NO: 20.

An exemplary QCR10 promoter is pQCRIO from S. cerevisiae. which is provided by the nucleotide sequence set forth as SEQ ID NO: 21.

In some embodiments, the heterologous nucleic acid comprising a gene encoding the enzyme having AAE activity can be operably linked to a promoter selected from the group consisting of SEQ ID NOs: 15-21. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 21.

Genetically modified yeast cells

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. In some embodiments, 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. In some embodiments, the genetically modified yeast cells described herein are further genetically modified with and a heterologous gene encoding an enzyme with AAT activity.

The terms “genetically modified cell,” “genetically modified yeast cell,” and “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. In some embodiments, 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. In some embodiments, 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. The terms (e.g., 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. In some embodiments, the yeast cell is of the genus Saccharomyces. The Saccharomyces genus includes nearly 500 distinct species, many of which are used in food production. One example species is Saccharomyces cerevisiae (S. cerevisiae), which 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. Other members of the 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. In some embodiments, the yeast is Saccharomyces cerevisiae (S. cerevisiae).

Saccharomyces species may be haploid (i.e., having a single set of chromosomes), diploid (i.e., having a paired set of chromosomes), or polyploid (i.e., carrying or containing more than two homologous sets of chromosomes). Saccharomyces species used, for example for beer brewing, are typically classified into two groups: ale strains (e.g., S. cerevisiae), which are top fermenting, and lager strains (e.g., S. pastorianus, S. carlsbergensis, S. uvarum), which are bottom fermenting. These characterizations reflect their separation characteristics in open square fermentors, as well as often other characteristics such as preferred fermentation temperatures and alcohol concentrations achieved.

Although beer brewing and wine producing has traditionally focused on use of S. cerevisiae strains, other yeast genera have been appreciated in production of fermented beverages. In some embodiments, 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. In some embodiments, 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. Examples of 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.

In some embodiments, the methods described herein involve use of more than one genetically modified yeast. For example, in some embodiments, the methods may involve use of more than one genetically modified yeast belonging to the genus Saccharomyces . In some embodiments, the methods may involve use of more than one genetically modified yeast belonging to a non-Saccharomyces genus. In some embodiments, 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. Alternatively or in addition, 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.

In some embodiments, the yeast is a hybrid strain. As will be evident to one of ordinary skill in the art, the term “hybrid strain” of yeast refers to a yeast strain that has resulted from the crossing of two different yeast strains, for example, to achieve one or more desired characteristics. For example, a hybrid strain may result from the crossing of two different yeast strains belonging to the same genus or the same species. In some embodiments, a hybrid strain results from the crossing of a Saccharomyces cerevisiae strain and a Saccharomyces eubayanus strain. See, e.g., Krogerus et al. Microbial Cell Factories (2017) 16: 66.

In some embodiments, the yeast strain is a wild yeast strain, such as a yeast strain that is isolated from a natural source and subsequently propagated. Alternatively, in some embodiments, the yeast strain is a domesticated yeast strain. Domesticated yeast strains have been subjected to human selection and breeding to have desired characteristics.

In some embodiments, the genetically modified yeast cells may be used in symbiotic matrices with bacterial strains and used for the production of fermented beverages, such as kombucha, kefir, and ginger beers. Saccharomyces fragilis, for example, is part of kefir culture and is grown on the lactose contained in whey. Other bacterial strains that may be used in symbiotic matrices with the genetically modified yeast cells include Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, bacteria in the genus Lactobacillus, and bacteria in the genus Pediococcus.

Methods of genetically modifying yeast cells are known in the art. In some embodiments, the yeast cell is diploid and one copy of a heterologous gene encoding an enzyme with AAE activity as described herein is introduced into the yeast genome.

In some embodiments, the yeast cell is diploid and one copy of a heterologous gene encoding an enzyme with AAE activity as described herein is introduced into both copies of the yeast genome. In some embodiments, the copies of the heterologous gene are identical. In some embodiments, the copies of the heterologous gene are not identical, but the genes encode an identical enzyme having AAE activity. In some embodiments, the copies of the heterologous gene are not identical, and the genes encode enzymes having AAE activity that are different (e.g., mutants, variants, fragments thereof). In some embodiments, the yeast cell is diploid and one copy of a gene encoding an enzyme with AAT activity as described herein is introduced into both copies of the yeast genome. In some embodiments, the copies of the gene encoding an enzyme with AAT activity are identical. In some embodiments, the copies of the gene encoding an enzyme with AAT activity are not identical, but the genes encode an identical enzyme having AAT activity. In some embodiments, the copies of the gene encoding an enzyme with AAT activity are not identical, and the genes encode enzymes having AAT activity that are different (e.g., mutants, variants, fragments thereof). In some embodiments, the cell contains a gene encoding an enzyme with AAT activity, referred to as an endogenous gene, and also contains a second gene encoding an enzyme with AAT activity, which may be the same or different enzyme with AAT activity as that encoded by the endogenous gene.

In some embodiments, the yeast cell is tetrapioid. Tetrapioid yeast cells are cells which maintain four complete sets of chromosomes (i.e., a complete set of chromosomes in four copies). In some embodiments, the yeast cell is tetrapioid and a copy of a heterologous gene encoding an enzyme with AAE activity as described herein is introduced into at least one copy of the genome. In some embodiments, the yeast cell is tetrapioid and a copy of a heterologous gene encoding an enzyme with AAE activity as described herein is introduced into more than one copy of the genome. In some embodiments, the yeast cell is tetrapioid and a copy of a heterologous gene encoding an enzyme with AAE activity as described herein is introduced in all four copies of the genome. In some embodiments, the copies of the heterologous gene are identical. In some embodiments, the copies of the heterologous gene are not identical, but the genes encode an identical enzyme having AAE activity. In some embodiments, the copies of the heterologous gene are not identical, and the genes encode enzymes having AAE activity that are different (e.g., mutants, variants, fragments thereof).

In some embodiments, the yeast cell is tetrapioid and a copy of a gene encoding an enzyme with AAT activity as described herein is introduced into at least one copy of the genome. In some embodiments, the yeast cell is tetrapioid and a copy of a gene encoding an enzyme with AAT activity as described herein is introduced into more than one copy of the genome. In some embodiments, the yeast cell is tetrapioid and a copy of a gene encoding an enzyme with AAT activity as described herein is introduced in all four copies of the genome. In some embodiments, the copies of the gene encoding an enzyme with AAT activity are identical. In some embodiments, the copies of the gene encoding an enzyme with AAT activity are not identical, but the genes encode an identical enzyme having AAT activity. In some embodiments, the copies of the gene encoding an enzyme with AAT activity are not identical, and the genes encode enzymes having AAT activity that are different (e.g., mutants, variants, fragments thereof). In some embodiments, the cell contains a gene encoding an enzyme with AAT activity, referred to as an endogenous gene, and also contains one or more additional copies of a gene encoding an enzyme with AAT activity, which may be the same or different enzyme with AAT activity as that encoded by the endogenous gene.

In some embodiments, the growth rate of the modified cell is not substantially impaired relative to a wild-type yeast cell that does not comprise the genetic modification(s). Methods of measuring and comparing the growth rates of two cells will be known to be an ordinary skill in the art. Non-limiting examples of growth rates that can be measured and compared between two types of cells are replication rate, budding rate, colony-forming units (CFUs) produced per unit of time, and amount of fermentable sugar reduced in a medium per unit of time. The growth rate of a modified cell is “not substantially impaired” relative to a wild-type cell if the growth rate, as measured, is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% of the growth rate of the wild-type cell.

Strains of yeast cells that may be used with the methods described herein will be known to one of ordinary skill in the art and include yeast strains used for brewing desired fermented beverages as well as commercially available yeast strains. Examples of common beer strains include, without limitation, American ale strains, Belgian ale strains, British ale strains, Belgian lambic/sour ale strains, Barleywine/Imperial Stout strains, India Pale Ale strains, Brown Ale strains, Kolsch and Altbier strains, Stout and Porter strains, and Wheat beer strains.

Non-limiting examples of yeast strains for use with the genetically modified cells and methods described herein include Wyeast American Ale 1056, Wyeast American Ale II 1272, Wyeast Denny’s Favorite 50 1450, Wyeast Northwest Ale 1332, Wyeast Ringwood Ale 1187, Siebel Inst. American Ale BRY 96, White Labs American Ale Yeast Blend WLP060, White Labs California Ale V WLP051, White Labs California Ale WLP001, White Labs Old Sonoma Ale WLP076, White Labs Pacific Ale WLP041, White Labs East Coast Ale WLP008, White Labs East Midlands Ale WLP039, White Labs San Diego Super Yeast WLP090, White Labs San Francisco Lager WLP810, White Labs Neutral Grain WLP078, Lallemand American West Coast Ale BRY-97, Lallemand CBC-1 (Cask and Bottle Conditioning), Brewferm Top, Coopers Pure Brewers’ Yeast, Fermentis US-05, Real Brewers Yeast Lucky #7, Muntons Premium Gold, Muntons Standard Yeast, East Coast Yeast Northeast Ale ECY29, East Coast Yeast Old Newark Ale ECY10, East Coast Yeast Old Newark Beer ECY12, Fermentis Safale US-05, Fermentis Safbrew T-58, Real Brewers Yeast The One, Mangrove Jack US West Coast Yeast, Mangrove Jack Workhorse Beer Yeast, Lallemand Abbaye Belgian Ale, White Labs Abbey IV WLP540, White Labs American Farmhouse Blend WLP670, White Labs Antwerp Ale WLP515, East Coast Yeast Belgian Abbaye ECY09, White Labs Belgian Ale WLP550, Mangrove Jack Belgian Ale Yeast, Wyeast Belgian Dark Ale 3822-PC, Wyeast Belgian Saison 3724, White Labs Belgian Saison I WLP565, White Labs Belgian Saison II WLP566, White Labs Belgian Saison III WLP585, Wyeast Belgian Schelde Ale 3655-PC, Wyeast Belgian Stout 1581-PC, White Labs Belgian Style Ale Yeast Blend WLP575, White Labs Belgian Style Saison Ale Blend WLP568, East Coast Yeast Belgian White ECY11, Lallemand Belle Saison, Wyeast Biere de Garde 3725-PC, White Labs Brettanomyces Bruxellensis Trois Vrai WLP648, Brewferm Top, Wyeast Canadian/Belgian Ale 3864-PC, Lallemand CBC-1 (Cask and Bottle Conditioning), Wyeast Farmhouse Ale 3726-PC, East Coast Yeast Farmhouse Brett ECY03, Wyeast Flanders Golden Ale 3739-PC, White Labs Flemish Ale Blend WLP665, White Labs French Ale WLP072, Wyeast French Saison 3711, Wyeast Leuven Pale Ale 3538-PC, Fermentis Safbrew T-58, East Coast Yeast Saison Brasserie Blend ECY08, East Coast Yeast Saison Single-Strain ECY14, Real Brewers Yeast The Monk, Siebel Inst. Trappist Ale BRY 204, East Coast Yeast Trappist Ale ECY13, White Labs Trappist Ale WLP500, Wyeast Trappist Blend 3789-PC, Wyeast British Ale 1098, Wyeast British Ale II 1335, Wyeast British Cask Ale 1026-PC, Wyeast English Special Bitter 1768-PC, Wyeast Irish Ale 1084, Wyeast London Ale 1028, Wyeast London Ale III 1318, Wyeast London ESB Ale 1968, Wyeast Ringwood Ale 1187, Wyeast Thames Valley Ale 1275, Wyeast Thames Valley Ale II 1882-PC, Wyeast West Yorkshire Ale 1469, Wyeast Whitbread Ale 1099, Mangrove Jack British Ale Yeast, Mangrove Jack Burton Union Yeast, Mangrove Jack Workhorse Beer Yeast, East Coast Yeast British Mild Ale ECY18, East Coast Yeast Northeast Ale ECY29, East Coast Yeast Burton Union ECY17, East Coast Yeast Old Newark Ale ECY10, White Labs Bedford British Ale WLP006, White Labs British Ale WLP005, White Labs Burton Ale WLP023, White Labs East Midlands Ale WLP039, White Labs English Ale Blend WLP085, White Labs English Ale WLP002, White Labs Essex Ale Yeast WLP022, White Labs Irish Ale WLP004, White Labs London Ale WLP013, White Labs Manchester Ale WLP038, White Labs Old Sonoma Ale WLP076, White Labs San Diego Super Yeast WLP090, White Labs Whitbread Ale WLP017, White Labs North Yorkshire Ale WLP037, Coopers Pure Brewers’ Yeast, Siebel Inst. English Ale BRY 264, Muntons Premium Gold, Muntons Standard Yeast, Lallemand Nottingham, Fermentis Safale S-04, Fermentis Safbrew T-58, Lallemand Windsor (British Ale), Real Brewers Yeast Ye Olde English, Brewferm Top, White Labs American Whiskey WLP065, White Labs Dry English Ale WLP007, White Labs Edinburgh Ale WLP028, Fermentis Safbrew S-33, Wyeast Scottish Ale 1728, East Coast Yeast Scottish Heavy ECY07, White Labs Super High Gravity WLP099, White Labs Whitbread Ale WLP017, Wyeast Belgian Lambic Blend 3278, Wyeast Belgian Schelde Ale 3655-PC, Wyeast Berliner-Weisse Blend 3191-PC, Wyeast Brettanomyces Bruxellensis 5112, Wyeast Brettanomyces Lambicus 5526, Wyeast Lactobacillus 5335, Wyeast Pediococcus Cerevisiae 5733, Wyeast Roeselare Ale Blend 3763, Wyeast Trappist Blend 3789-Pc, White Labs Belgian Sour Mix Wlp655, White Labs Berliner Weisse Blend Wlp630, White Labs Saccharomyces “Bruxellensis” Trois Wlp644, White Labs Brettanomyces Bruxellensis Wlp650, White Labs Brettanomyces Claussenii Wlp645, White Labs Brettanomyces Lambicus Wlp653, White Labs Flemish Ale Blend Wlp665, East Coast Yeast Berliner Blend Ecy06, East Coast Yeast Brett Anomala Ecy04, East Coast Yeast Brett Bruxelensis Ecy05, East Coast Yeast Brett Custersianus Ecyl9, East Coast Yeast Brett Nanus Ecyl6, Strain #2, East Coast Yeast BugCounty ECY20, East Coast Yeast BugFarm ECY01, East Coast Yeast Farmhouse Brett ECY03, East Coast Yeast Flemish Ale ECY02, East Coast Yeast Oud Brune ECY23, Wyeast American Ale 1056, Siebel Inst. American Ale BRY 96, White Labs American Ale Yeast Blend WLP060, White Labs Bourbon Yeast WLP070, White Labs California Ale V WLP051, White Labs California Ale WLP001, White Labs Dry English ale WLP007, White Labs East Coast Ale WLP008, White Labs Neutral Grain WLP078, White Labs Super High Gravity WLP099, White Labs Tennessee WLP050, Fermentis US-05, Real Brewers Yeast Lucky #7, Fermentis Safbrew S-33, East Coast Yeast Scottish Heavy ECY07, Lallemand Windsor (British Ale), Wyeast American Ale 1056, Wyeast American Ale II 1272, Wyeast British Ale 1098, Wyeast British Ale II 1335, Wyeast Denny’s Favorite 50 1450, Wyeast London Ale 1028, Wyeast London Ale III 1318, Wyeast London ESB Ale 1968, Wyeast Northwest Ale 1332, Wyeast Ringwood Ale 1187, Siebel Inst. American Ale BRY 96, White Labs American Ale Yeast Blend WLP060, White Labs Bedford British Ale WLP006, White Labs British Ale WLP005, White Labs Burton Ale WLP023, White Labs California Ale V WLP051, White Labs California Ale WLP001, White Labs East Coast Ale WLP008, White Labs English Ale WLP002, White Labs London Ale WLP013, White Labs Essex Ale Yeast WLP022, White Labs Pacific Ale WLP041, White Labs San Diego Super Yeast WLP090, White Labs Whitbread Ale WLP017, Brewferm Top, Mangrove Jack Burton Union Yeast, Mangrove Jack US West Coast Yeast, Mangrove Jack Workhorse Beer Yeast, Coopers Pure Brewers’ Yeast, Fermentis US-05, Fermentis Safale S- 04S04, Fermentis Safbrew T-58, Real Brewers Yeast Lucky #7, Real Brewers Yeast The One, Muntons Premium Gold, Muntons Standard Yeast, East Coast Yeast Northeast Ale ECY29, Lallemand Nottingham, Lallemand Windsor (British Ale), Wyeast American Ale 1056, Wyeast American Ale II 1272, Wyeast British Ale 1098, Wyeast British Ale II 1335, Wyeast Thames Valley Ale 1275, Wyeast Thames Valley Ale II 1882-PC, Wyeast West Yorkshire Ale 1469, Wyeast Whitbread Ale 1099, Wyeast British Cask Ale 1026-PC, Wyeast English Special Bitter 1768-PC, Wyeast London Ale 1028, Wyeast London Ale III 1318, Wyeast London ESB Ale 1968, Wyeast Northwest Ale 1332, Wyeast Ringwood Ale 1187, White Labs American Ale Yeast Blend WLP060, White Labs British Ale WLP005, White Labs Bedford British Ale WLP006, White Labs British Ale WLP005, White Labs Burton Ale WLP023, White Labs California Ale V WLP051, White Labs California Ale WLP001, White Labs East Coast Ale WLP008, White Labs English Ale WLP002, White Labs Essex Ale Yeast WLP022, White Labs French Ale WLP072, White Labs London Ale WLP013, White Labs Pacific Ale WLP041, White Labs Whitbread Ale WLP017, Brewferm Top, East Coast Yeast British Mild Ale ECY18, Coopers Pure Brewers’ Yeast, Muntons Premium Gold, Muntons Standard Yeast, Mangrove Jack Newcastle Dark Ale Yeast, Lallemand CBC-1 (Cask and Bottle Conditioning), Lallemand Nottingham, Lallemand Windsor (British Ale), Fermentis Safale S-04, Fermentis US-05, Siebel Inst. American Ale BRY 96, Wyeast American Wheat 1010, Wyeast German Ale 1007, Wyeast Kolsch 2565, Wyeast Kolsch II 2575-PC, White Labs Belgian Lager WLP815, White Labs Dusseldorf Alt WLP036, White Labs European Ale WLP011, White Labs German Ale/Kblsch WLP029, East Coast Yeast Kolschbier ECY21, Mangrove Jack Workhorse Beer Yeast, Siebel Inst. Alt Ale BRY 144, Wyeast American Ale 1056, Wyeast American Ale II 1272, Wyeast British Ale 1098, Wyeast British Ale II 1335, Wyeast Denny’s Favorite 50 1450, Wyeast English Special Bitter 1768- PC, Wyeast Irish Ale 1084, Wyeast London Ale 1028, Wyeast London Ale III 1318, Wyeast London ESB Ale 1968, Wyeast Northwest Ale 1332, Wyeast Ringwood Ale 1187, Wyeast Thames Valley Ale 1275, Wyeast Thames Valley Ale II 1882-PC, Wyeast West Yorkshire Ale 1469, Wyeast Whitbread Ale 1099, White Labs American Ale Yeast Blend WLP060, White Labs Bedford British Ale WLP006, White Labs British Ale WLP005, White Labs Burton Ale WLP023, White Labs California Ale V WLP051, White Labs California Ale WLP001, White Labs East Coast Ale WLP008, White Labs East Midlands Ale WLP039, White Labs English Ale WLP002, White Labs Essex Ale Yeast WLP022, White Labs Irish Ale WLP004, White Labs London Ale WLP013, White Labs Old Sonoma Ale WLP076, White Labs Pacific Ale WLP041, White Labs Whitbread Ale WLP017, Coopers Pure Brewers’ Yeast, Fermentis US-05, Muntons Premium Gold, Muntons Standard Yeast, Fermentis Safale S-04, Lallemand Nottingham, Lallemand Windsor (British Ale), Siebel Inst. American Ale BRY 96, White Labs American Hefeweizen Ale 320, White Labs Bavarian Weizen Ale 351, White Labs Belgian Wit Ale 400, White Labs Belgian Wit Ale II 410, White Labs Hefeweizen Ale 300, White Labs Hefeweizen IV Ale 380, Wyeast American Wheat 1010, Wyeast Bavarian Wheat 3638, Wyeast Bavarian Wheat Blend 3056, Wyeast Belgian Ardennes 3522, Wyeast Belgian Wheat 3942, Wyeast Belgian Witbier 3944, Wyeast Canadian/Belgian Ale 3864-PC, Wyeast Forbidden Fruit Yeast 3463, Wyeast German Wheat 3333, Wyeast Weihenstephan Weizen 3068, Siebel Institute Bavarian Weizen BRY 235, Fermentis Safbrew WB-06, Mangrove Jack Bavarian Wheat, Lallemand Munich (German Wheat Beer), Brewferm Blanche, Brewferm Lager, East Coast Yeast Belgian White ECY11. In some embodiments, the yeast is S. cerevisiae strain WLP001 California Ale (which may be referred to as “CA01”).

In some embodiments, the yeast strain for use with the genetically modified cells and methods described herein is a wine yeast strain. Examples of yeast strains for use with the genetically modified cells and methods described herein include, without limitation, Red Star Montrachet, EC-1118, Elegance, Red Star Cote des Blancs, Epernay II, Red Star Premier Cuvee, Red Star Pasteur Red, Red Star Pasteur Champagne, Fermentis BCS-103, Fermentis VR44, WY1056, A07, GY001, London Ale III, A38, Conan, WLP4000, Verdant, WLP002, WLP006, WLP007, OYL011, S04, WLP029, WY2565, WY2007, WLP830, OYL071, PYL091, OYL057, OYL061, and OYL090. In some embodiments, the yeast is S. cerevisiae strain Elegance. In some embodiments, the yeast is S. cerevisiae strain EC-1118 (also referred to as ECI 118 or Lalvin EC 1118® (Lallemand Brewing).

In some embodiments, the modified cell is an S. cerevisiae cell that expresses ScFAAl, ScFAA4, HcAAEl, HcAAEl(R51K), H1CCL2, or H1CCL3 under control of a GMP1, HSP26, or TDH3 promoter. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl, HcAAEl(R51K), H1CCL2, or H1CCL3 under control of a GMP1, HSP26, or TDH3 promoter.

In some embodiments, the modified cell is an S. cerevisiae cell that expresses MhWES2, MhWESl, MpAATl, SpAAT, CmAAT, FcAATl, SAAT, EEB1, or EHTl under control of a GMP1, HSP26, or TDH3 promoter. In some embodiments, the modified cell is an S. cerevisiae cell that expresses MhWES2, MhWESl, or MpAATl under control of a GMP1, HSP26, or TDH3 promoter. In some embodiments, the modified cell is an S. cerevisiae cell that expresses a gene encoding an enzyme with AAE activity (e.g HcAAEl, HcAAEl(R51K), H1CCL2, or H1CCL3) under control of a GMP1, HSP26, or TDH3 promoter and , SpAAT, CmAAT, FcAATl, SAAT, EEB1, or EHT1 under control of a GMP1, HSP26, or TDH3 promoter. In some embodiments, the modified cell is an S. cerevisiae cell that expresses a gene encoding an enzyme with AAE activity (e.g HcAAEl, HcAAEl(R51K), H1CCL2, or H1CCL3) under control of a GMP1, HSP26, or TDH3 promoter and MhWES2, MhWESl, or MpAATl under control of a GMP1, HSP26, or TDH3 promoter.

In some embodiments, the modified cell is an S. cerevisiae cell that expresses a gene encoding an enzyme with AAE activity (e.g HcAAEl, HcAAEl(R51K), H1CCL2, or H1CCL3) under control of a GMP1, HSP26, or TDH3 promoter and , SpAAT, CmAAT, FcAATl, SAAT, EEB1, or EHT1 under control of a GMP1, HSP26, or TDH3 promoter. In some embodiments, the modified cell is an S. cerevisiae cell that expresses a gene encoding an enzyme with AAE activity (e.g HcAAEl, HcAAEl(R51K), H1CCL2, or H1CCL3) under control of a GMP1, HSP26, or TDH3 promoter and MhWES2, MhWESl, or MpAATl under control of a GMP1, HSP26, or TDH3 promoter.

In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL3 and MhWES2. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL3 and MhWESl. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL3 and MpAATl. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL3 and SpAAT. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL3 and CmAAT. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL3 and FcAATl. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL3 and SAAT. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL3 and EEB1. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL3 and EHTl.

In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL2 and MhWES2. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL2 and MhWESl. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL2 and MpAATl. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL2 and SpAAT. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL2 and CmAAT. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL2 and FcAATl. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL2 and SAAT. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL2 and EEB1. In some embodiments, the modified cell is an S. cerevisiae cell that expresses H1CCL2 and EHT1.

In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl and MhWES2. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl and MhWESl. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl and MpAATl. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl and SpAAT. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl and CmAAT. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl and FcAATl. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl and SAAT. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl and EEB1. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl and EHTl.

In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl(R51K) and MhWES2. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl(R51K) and MhWESl. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl (R5 IK) and MpAATl. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl (R51K) and SpAAT. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl (R51K) and CmAAT. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl (R51K) and FcAATl. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl (R51K) and SAAT. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl (R51K) and EEB1. In some embodiments, the modified cell is an S. cerevisiae cell that expresses HcAAEl (R51K) and EHT1.

In some embodiments, the modified cell further comprises one or more additional heterologous genes that encodes one or more additional enzymes selected from the group consisting of PDC1, ALD6, ACS1, and ACC1. In some embodiments, the genetically modified yeast cell further comprises a heterologous nucleic acid encoding one or more additional enzymes selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and combination thereof.

An exemplary PDC1 is provided by the amino acid sequence set forth as SEQ ID NO: 22. 23.

24.

25.

In some embodiments, the modified cell further comprises one or more bacterial genes that encodes one or more bacterial enzymes selected from the group consisting of BktB, Hbd, Crt, and Ter. In some embodiments, the modified cell further comprises one or more bacterial genes selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and combinations thereof.

An exemplary BktB is provided by the amino acid sequence set forth as SEQ ID NO:

26.

27.

28. 29.

In some embodiments, the modified cell does not comprise a genetic modification that increases fatty acid biosynthesis. In some embodiments, the modified cell does not comprise a genetic modification to increase fatty acid synthetase (FAS) activity. In some embodiments, the modified cell does not comprise a heterologous nucleic acid encoding an enzyme having A AT activity.

Methods and Liquid Fermentation Compositions (and Fermented Products)

Aspects of the present disclosure relate to methods of producing a fermented product using any of the genetically modified yeast cells described herein. Also provided are methods of producing compositions comprising ethanol using any of the genetically modified yeast cells described herein.

The process of fermentation exploits a natural process of using microorganisms to convert carbohydrates into alcohol and carbon dioxide. It is a metabolic process that produces chemical changes in organic substrates through enzymatic action. In the context of food production, fermentation broadly refers to any process in which the activity of microorganisms brings about a desirable change to a food product or beverage. The conditions for fermentation and the carrying out of a fermentation is referred to herein as a “fermentation process.”

In some aspects, the disclosure relates to a method of producing a fermented product, such as a fermented beverage, involving contacting any of the modified cells described herein with a medium comprising at least one fermentable sugar during a first fermentation process, to produce a fermented product. A “medium” as used herein, refers to liquid conducive to fermentation, meaning a liquid which does not inhibit or prevent the fermentation process. In some embodiments, the medium is water. In some embodiments, the methods of producing a fermented product involve contacting purified enzymes (e.g., any of the AAE or AAT enzymes described herein) with a medium comprising at least one fermentable sugar during a first fermentation process, to produce a fermented product.

In some embodiments, a method is provided for producing a fermented product, comprising contacting a genetically modified yeast cell of the present disclosure 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. It should be understood that the same methods can be used to produce a composition comprising alcohol, e.g. ethanol.

As also used herein, the term “fermentable sugar” refers to a carbohydrate that may be converted into an alcohol and carbon dioxide by a microorganism, such as any of the cells described herein. In some embodiments, the fermentable sugar is converted into alcohol and carbon dioxide by an enzyme, such as a recombinant enzyme or a cell that expresses the enzyme. In some embodiments, the fermentable sugar is glucose, fructose, lactose, sucrose, maltose, and maltotriose.

In some embodiments, the fermentable sugar is provided in a sugar source. The sugar source for use in the claimed methods may depend, for example, on the type of fermented product and the fermentable sugar. Examples of sugar sources include, without limitation, wort, grains/cereals, fruit juice (e.g., grape juice and apple juice/cider), honey, cane sugar, rice, and koji. Examples of fruits from which fruit juice can be obtained include, without limitation, grapes, apples, blueberries, blackberries, raspberries, currants, strawberries, cherries, pears, peaches, nectarines, oranges, pineapples, mangoes, and passionfruit.

As will be evident to one of ordinary skill in the art, in some instances, it may be necessary to process the sugar source in order to make available the fermentable sugar for fermentation. Using beer production as an example fermented beverage, grains (cereal, barley) are boiled or steeped in water, which hydrates the grain and activates the malt enzymes converting the starches to fermentable sugars, referred to as “mashing.” As used herein, the term “wort” refers to the liquid produced in the mashing process, which contains the fermentable sugars. The wort then is exposed to a fermenting organism (e.g., any of the cells described herein), which allows enzymes of the fermenting organism to convert the sugars in the wort to alcohol and carbon dioxide. In some embodiments, 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.

In some embodiments, the grains are malted, unmalted, or comprise a combination of malted and unmalted grains. Examples of grains for use in the methods described herein include, without limitation, barley, oats, maize, rice, rye, sorghum, wheat, karasumugi, and hatomugi.

In the example of producing sake, the sugar source is rice, which is incubated with koji mold (Aspergillus oryzae) converting the rice starch to fermentable sugar, producing koji. The koji then is exposed to a fermenting organism (e.g., any of the cells described herein), which allows enzymes of the fermenting organism to convert the sugars in the koji to alcohol and carbon dioxide. In some embodiments, the koji 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 koji to alcohol and carbon dioxide.

In the example of producing wine, grapes are harvested, mashed (e.g., crushed) into a composition containing the skins, solids, juice, and seeds. The resulting composition is referred to as the “must.” The grape juice may be separated from the must and fermented, or the entirety of the must (i.e., with skins, seeds, solids) may be fermented. The grape juice or must then is exposed to a fermenting organism (e.g., any of the cells described herein), which allows enzymes of the fermenting organism to convert the sugars in the grape juice or must to alcohol and carbon dioxide. In some embodiments, the grape juice or must 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 grape juice or must to alcohol and carbon dioxide.

In some embodiments, the methods described herein involve producing the medium, which may involve heating or steeping a sugar source, for example in water. In some embodiments, the water has a temperature of at least 50 degrees Celsius (50°C) and incubated with a sugar source for a period of time. In some embodiments, the water has a temperature of at least 75°C and incubated with a sugar source for a period of time. In some embodiments, the water has a temperature of at least 100°C and incubated with a sugar source for a period of time. Preferably, the medium is cooled prior to addition of any of the cells described herein.

In some embodiments, the methods described herein further comprise adding at least one (e.g., 1, 2, 3, 4, 5, or more) hop variety, for example, to the medium or to a wort during a fermentation process. Hops are the flowers of the hops plant (Humulus lupulus) and are often used in fermentation to impart various flavors and aromas to the fermented product. Hops are considered to impart bitter flavoring in addition to floral, fruity, and/or citrus flavors and aromas and may be characterized based on the intended purpose. For example, bittering hops impart a level of bitterness to the fermented product due to the presence of alpha acids in the hop flowers, whereas aroma hops have lower levels of alpha acids and contribute desirable aromas and flavor to the fermented product.

Whether one or more varieties of hops are added to the medium and/or the wort and at what stage during which the hops are added may be based on various factors, such as the intended purpose of the hops. For example, hops that are intended to impart a bitterness to the fermented product are typically added to during preparation of the wort, for example, during boiling of the wort. In some embodiments, hops that are intended to impart a bitterness to the fermented product are added to the wort and boiled with the wort for a period of time, for example, for about 15-60 minutes. In contrast, hops that are intended to impart desired aromas to the fermented product are typically added later than hops used for bitterness. In some embodiments, hops that are intended to impart desired aromas to the fermented product are added to at the end of the boil or after the wort is boiled (i.e., “dry hopping”). In some embodiments, one or more varieties of hops may be added at multiple times e.g., at least twice, at least three times, or more) during the methods.

In some embodiments, the hops are added in the form of either wet or dried hops and may optionally be boiled with the wort. In some embodiments, the hops are in the form of dried hop pellets. In some embodiments, at least one variety of hops is added to the medium. In some embodiments, the hops are wet (i.e., undried). In some embodiments, the hops are dried, and optionally may be further processed prior to use. In some embodiments, the hops are added to the wort prior to the fermentation process. In some embodiments, the hops are boiled in the wort. In some embodiments, the hops are boiled with the wort and then cooled with the wort.

Many varieties of hops are known in the art and may be used in the methods described herein. Examples of hop varieties include, without limitation, Ahtanum, Amarillo, Apollo, Cascade, Centennial, Chinook, Citra, Cluster, Columbus, Crystal/Chrystal, Eroica, Galena, Glacier, Greenburg, Horizon, Liberty, Millennium, Mosaic, Mount Hood, Mount Rainier, Newport, Nugget, Palisade, Santiam, Simcoe, Sterling, Summit, Tomahawk, Ultra, Vanguard, Warrior, Willamette, Zeus, Admiral, Brewer’s Gold, Bullion, Challenger, First Gold, Fuggles, Goldings, Herald, Northdown, Northern Brewer, Phoenix, Pilot, Pioneer, Progress, Target, Whitbread Golding Variety (WGV), Hallertau, Hersbrucker, Saaz, Tettnang, Spalt, Feux-Coeur Francais, Galaxy, Green Bullet, Motueka, Nelson Sauvin, Pacific Gem, Pacific Jade, Pacifica, Pride of Ringwood, Riwaka, Southern Cross, Lublin, Magnum, Perle, Polnischer Lublin, Saphir, Satus, Select, Strissel spalt, Styrian Goldings, Tardif de Bourgogne, Tradition, Bravo, Calypso, Chelan, Comet, El Dorado, San Juan Ruby Red, Satus, Sonnet Golding, Super Galena, Tillicum, Bramling Cross, Pilgrim, Hallertauer Herkules, Hallertauer Magnum, Hallertauer Taurus, Merkur, Opal, Smaragd, Halleratau Aroma, Kohatu, Rakau, Stella, Sticklebract, Summer Saaz, Super Alpha, Super Pride, Topaz, Wai-iti, Bor, Junga, Marynka, Premiant, Sladek, Styrian Atlas, Styrian Aurora, Styrian Bobek, Styrian Celeia, Sybilla Sorachi Ace, Hallertauer Mittelfrueh, Hallertauer Tradition, Tettnang, Tahoma, Triple Pearl, Yahima Gold, and Michigan Copper.

In some embodiments, the fermentation process of at least one sugar source comprising at least one fermentable sugar may be carried out for about 1 day to about 31 days. In some embodiments, the fermentation process is performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days or longer. In some embodiments, the 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 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. In some embodiments, the fermentation process of one or more fermentable sugars may be performed at a temperature of about 20°C to about 24°C. In some embodiments, 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.

In some embodiments, fermentation results in the reduction of the amount of fermentable sugar present in a medium. In some embodiments, the reduction in the amount of fermentable sugar occurs within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, or longer, from the start of fermentation. In some embodiments, the amount of fermentable sugar is reduced by at least 5%, 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 70%, at least 75%, at least 80%, at least 85%, at least

90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100%. In some embodiments, the modified cell or cells ferment a comparable or greater amount of fermentable sugar, relative to the amount of fermentable sugar fermented by wild-type yeast cells in the same amount of time.

The methods described herein may involve at least one additional fermentation process. Such additional fermentation methods may be referred to as secondary fermentation processes (also referred to as “aging” or “maturing”). As will be understood by one of ordinary skill in the art, secondary fermentation typically involves transferring a fermented beverage to a second receptacle (e.g., glass carboy, barrel) where the fermented beverage is incubated for a period of time. In some embodiments, the secondary fermentation is performed for a period of time between 10 minutes and 12 months. In some embodiments, the secondary fermentation is performed for 10 minutes, 20 minutes, 40 minutes, 50 minutes, 60 minutes (1 hour), 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months,

10 months, 11 months, 12 months, or longer. In some embodiments, 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. In some embodiments, 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. As will be evident to one of ordinary skill in the art, 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.

In some embodiments, one or more additional flavor component may be added to the medium prior to or after the fermentation process. Examples include, hop oil, hop aromatics, hop extracts, hop bitters, and isomerized hops extract.

Products from the fermentation process may volatilize and dissipate during the fermentation process or from the fermented product. For example, ethyl -butanoate produced during fermentation using the cells described herein may volatilize resulting in reduced levels of ethyl -butanoate in the fermented product. In some embodiments, 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. In some embodiments, 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).

In some embodiments, a fermented product or composition comprising ethanol according to the present disclosure can comprise an increased amount 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. In some embodiments, the desired product is an ethyl ester selected from the group consisting of ethyl butanoate, ethyl isovalerate, ethyl octanoate, ethyl decanoate, ethyl 2-methlybutyrate, and ethyl crotonate. In some embodiments, the desired product is an ethyl ester selected from the group consisting of ethyl propionate, ethyl butanoate, and ethyl isovalerate.

In some embodiments, 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. In some embodiments, 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. In some embodiments, the at least one undesired product is an acid selected from the group consisting of propanoic acid, butanoic acid, and isovaleric acid.

Fermented Products

Aspects of the present disclosure relate to fermented products produced by any of the methods disclosed herein. In some embodiments, the fermented product is a fermented beverage. In some embodiments, the fermented beverage is beer, wine, sake, mead, cider, cava, sparkling wine (champagne), kombucha, ginger beer, water kefir. In some embodiments, the beverage is beer. In some embodiments, the beverage is wine. In some embodiments, the beverage is sparkling wine. In some embodiments, the beverage is Champagne. In some embodiments, the beverage is sake. In some embodiments, the beverage is mead. In some embodiments, the beverage is cider. In some embodiments, the beverage is hard seltzer. In some embodiments, the beverage is a wine cooler. In some embodiments, the fermented beverage is beer, wine, sparkling wine (champagne), wine cooler, wine spritzer, hard seltzer, sake, mead, kombucha, or cider.

In some embodiments, the fermented product is a fermented food product. Examples of fermented food products include, without limitation, cultured yogurt, tempeh, miso, kimchi, sauerkraut, fermented sausage, bread, and soy sauce.

In some embodiments, a liquid fermentation composition is provided that comprises: (a) a population of genetically modified yeast cells according to the present disclosure and a sugar source. In some embodiments, the liquid fermentation composition further comprises alcohol, e.g. ethanol. In some embodiments, 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.

According to aspects of the disclosure, 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. As used 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. In some embodiments, 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,

270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,

450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,

630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800,

810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980,

990, 1000, 1050, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 pg L'¹. In some embodiments, 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. In some embodiments, 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, about 150 pg/L to about 40 mg/L, about 150 pg/L to about 30 mg/L, about 150 pg/L to about 20 mg/L, about 150 pg/L to about 10 mg/L, about 150 pg/L to about 5 mg/L, about 150 pg/L to about 4 mg/L, about 150 pg/L to about 3 mg/L, about 150 pg/L to about 2 mg/L, about 150 pg/L to about 1 mg/L, about 150 pg/L to about 900 pg/L, about 150 pg/L to about 900 pg/L, about 150 pg/L to about 900 pg/L, about 150 pg/L to about 900 pg/L, about 150 pg/L to about 900 pg/L, about 150 pg/L to about 900 pg/L, about 150 pg/L to about 800 pg/L, about 150 pg/L to about 700 pg/L, about 150 pg/L to about 600 pg/L, about 150 pg/L to about 500 pg/L, about 150 pg/L to about 400 pg/L, about 150 pg/L to about 300 pg/L, about 150 pg/L to about 200 pg/L, or about 150 pg/L, 200 pg/L, 300 pg/L, 400 pg/L, 500 pg/L, 600 pg/L, 700 pg/L, 800 pg/L, 900 pg/L, 1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L 5 mg/L, 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L, 50 mg/L or any range or value therebetween. In some embodiments, the one or more ethyl esters comprise ethyl butanoate, ethyl isovalerate, ethyl octanoate, ethyl decanoate, 2-methylbutyrate, and/or ethyl crotonate In some embodiments, the one or more ethyl esters comprise ethyl propionate, ethyl butanoate, and/or ethyl isovalerate. In some embodiments, the one or more ethyl esters comprise ethyl propionate. In some embodiments, the one or more ethyl esters comprise ethyl butanoate. In some embodiments, the one or more ethyl esters comprise ethyl isovalerate. In some embodiments, the one or more ethyl esters are ethyl propionate, ethyl butanoate, and/or ethyl isovalerate. In some embodiments, the one or more ethyl esters is ethyl propionate. In some embodiments, the one or more ethyl esters is ethyl butanoate. In some embodiments, the one or more ethyl esters is ethyl isovalerate. In any of the embodiments of the present disclosure, an increase in the production of desired products (e.g., one or more ethyl esters) can be an increase of about 5% to about 200%. In some embodiments, the increase can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, or 200%, or any range or value therebetween relative to a product produced by a counterpart cell that lacks the genetic modification(s), e.g. the genetic modification to increase the expression of a nucleic acid encoding an enzyme having AAE activity. In some embodiments, the desired product is an ethyl ester. In some embodiments, the ethyl ester is ethyl propionate. In some embodiments, the ethyl ester is ethyl butanoate. In some embodiments, the ethyl ester is ethyl isovalerate.

In any of the embodiments of the present disclosure, a decrease in the production undesired products (e.g., byproducts, off-flavors), such as butanoic acid, during fermentation of a product, can be a decrease of about 5% to about 95% or more. In some embodiments, the in a reduction in the production of an undesired product by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to a product produced by a counterpart cell that lacks the genetic modification(s), e.g. the genetic modification to increase the expression of a nucleic acid encoding an enzyme having AAE activity. In some embodiments, the undesired product is a fatty acid. In some embodiments, the fatty acid is propanoic acid. In some embodiments, the fatty acid is butyric acid. In some embodiments, the fatty acid is isovaleric acid.

In some embodiments, the amount of one or more fatty acids, e.g MCFAs, is less than 1000 mg L^-1, for example less than 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 mg L^-1 or less. In some embodiments, the one or more MCFAs comprise propanoic acid, butyric acid, and/or isovaleric acid. In some embodiments, the one or more MCFAs comprise propanoic acid. In some embodiments, the one or more MCFAs comprise butyric acid. In some embodiments, the one or more MCFAs comprise isovaleric acid. In some embodiments, the one or more MCFAs are propanoic acid, butyric acid, and/or isovaleric acid. In some embodiments, the one or more MCFAs is propanoic acid. In some embodiments, the one or more MCFAs is butyric acid. In some embodiments, the one or more MCFAs is isovaleric acid.

Methods of measuring titers/levels of ethyl esters and/or MCFAs will be evident to one of ordinary skill in the art. In some embodiments, the titers/levels of the ethyl ester and/or MCFAs are measured using gas-chromatograph mass-spectrometry (GC/MS). In some embodiments, the titers/levels of the ethyl ester and/or MCFAs are assessed using sensory panels, including for example human taste-testers. In some embodiments, the fermented beverage contains an alcohol by volume (also referred to as “ABV,” “abv,” or “alc/vol”) between 0.1% and 30%. In some embodiments, 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. In some embodiments, the fermented beverage is nonalcoholic (e.g., has an alcohol by volume less than 0.5%).

In some embodiments, the levels of ethyl esters and/or MCFAs are measured using gas-chromatography mass-spectrometry (GC/MS). In some embodiments, the levels of ethyl esters and/or MCFAs are measured using liquid-chromatography mass-spectrometry (LC/MS).

It should be understood that the fermented products, e.g. fermented beverages of the present disclosure can have the properties recited for the fermentation products throughout this disclosure, e.g. levels of acetate esters and/or ethyl esters and excluded compounds or compounds present below defined levels.

Kits

Aspects of the present disclosure also provide kits for use of the genetically modified yeast cells, for example to produce a fermented beverage, fermented product, or ethanol. In some embodiments, the kit contains a modified cell comprising a genetic modification that results in increased expression of a gene encoding an enzyme with AAE activity.

In some embodiments, the kit is for the production of a fermented beverage. In some embodiments, the kit is for the production of beer. In some embodiments, the kit is for the production of wine. In some embodiments, the kit is for the production of sake. In some embodiments, the kit is for the production of mead. In some embodiments, the kit is for the production of cider. The kits may also comprise other components for use in any of the methods described herein, or for use of any of the cells as described herein. For example, in some embodiments, the kits may contain grains, water, wort, must, yeast, hops, juice, or other sugar source(s). In some embodiments, the kit may contain one or more fermentable sugars. In some embodiments, the kit may contain one or more additional agents, ingredients, or components. Instructions for performing the methods described herein may also be included in the kits described herein. The kits may be organized to indicate a single-use composition containing any of the modified cells described herein. For example, the single use compositions (e.g., amount to be used) can be packaged compositions (e.g., modified cells) such as packeted (i.e., contained in a packet) powders, vials, ampoules, culture tube, tablets, caplets, capsules, or sachets containing liquids. The compositions (e.g., modified cells) may be provided in dried, lyophilized, frozen, or liquid forms. In some embodiments, the modified cells are provided as colonies on an agar medium. In some embodiments, the modified cells are provided in the form of a starter culture that may be pitched directly into a medium. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a solvent, such as a medium. The solvent may be provided in another packaging means and may be selected by one skilled in the art.

A number of packages or kits are known to those skilled in the art for dispensing a composition (e.g., modified cells). In certain embodiments, the package is a labeled blister package, dial dispenser package, tube, packet, drum, or bottle. Any of the kits described herein may further comprise one or more vessels for performing the methods described herein, such as a carboy or barrel.

General Techniques

The practice of the subject matter of the disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, but without limiting, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999). Equivalents and Scope

It is to be understood that this disclosure is not limited to any or all of the particular embodiments described expressly herein, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this disclosure are cited to disclose and describe the methods and/or materials in connection with which the publications are cited. All such publications and patents are herein incorporated by references as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and/or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents (i.e., any lexicographical definition in the publications and patents cited that is not also expressly repeated in the disclosure should not be treated as such and should not be read as defining any terms appearing in the accompanying claims). If there is a conflict between any of the incorporated references and this disclosure, this disclosure shall control. In addition, any particular embodiment of this disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Wherever used herein, a pronoun in a gender (e.g., masculine, feminine, neuter, other, etc) the pronoun shall be construed as gender neutral (i.e., construed to refer to all genders equally) regardless of the implied gender unless the context clearly indicates or requires otherwise. Wherever used herein, words used in the singular include the plural, and words used in the plural include the singular, unless the context clearly indicates or requires otherwise. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists (e.g., in Markush group format), each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the disclosure, or aspects of the disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.

It is also noted that the terms “comprising” and “containing” are intended to be open and permit the inclusion of additional elements or steps. Where ranges are given, endpoints are included in such ranges unless otherwise specified. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the disclosure, as defined in the following claims.

EXAMPLES

Example 1:

This example is related to the production of engineered yeast strains to produce elevated concentrations of ethyl esters during fermentation. Specifically, the goal was to generate yeast strains that biosynthesized elevated concentrations and distinct combinations of multiple ethyl ester molecules. The ethyl esters of interest included ethyl butanoate, ethyl isovalerate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate, which are attributed with imparting desirable sensory attributes to fermented beverages.

To achieve this goal, over one hundred new strains were created, each of which was engineered in line with one of the previously described engineering strategies (FIG. 3 or FIG. 5-7). For each of these strategies, multiple strains were created that varied in the promoters used to drive transgene expression and/or the yeast strain background in which the engineering was done, or both. The goal in creating these varied combinations of strain background, gene expression level, and engineering strategy was to discover synergistic interactions between these components that would maximize the biosynthesis of specific combinations of ethyl esters while avoiding off-target effects negatively impacting fermentation performance or sensory characteristics.

In total, over sixty distinct brewing strains were created across six different brewing strain backgrounds, and over forty wine strains were created across two different wine yeast backgrounds. Fermentation characteristics of these strains were initially tested in small scale 80 mL fermentations, and promising brewing strains were tested at a 20 L scale in a pilot brewery. Following each fermentation, sugar attenuation, aroma, and the concentrations or relative abundance of specific ethyl ester and medium-chain fatty acid (MCFA) molecules were tested. A representative subset of the ethyl ester and MCFA abundance data gathered from these experiments is shown in FIG. 8. As shown in FIG. 8, engineered strains and corresponding parental non-engineered strains were tested in 80 mL fermentations in either malt extract media (for brewing strains) or grape juice media (for wine strains). Following fermentation, gas chromatography mass spectrometry (GC-MS) was used to quantify the peak area corresponding to select ethyl esters and MCFAs in these fermentations. Values shown in the heat map in FIG. 8 report the log2-transformed fold-change of a molecule’s peak area in a fermentation with an engineered strain relative to its peak area in a fermentation with the appropriate non-engineered parental strain.

Data gathered from these fermentation experiments supported two broad conclusions: (1) Multiple different engineering approaches allow for the creation of strains that biosynthesize greatly elevated concentrations of one or more ethyl ester molecules. Across all of the strains created, many combinations of strain background, transgene expression level and engineering strategies resulted in strains that biosynthesize > 10-fold increased concentrations of one or more target ethyl esters (e.g., ethyl butanoate, ethyl isovalerate, ethyl hexanoate, ethyl octanoate, ethyl decanoate). In some cases, engineered strains biosynthesized greater than 20-fold increased concentrations of specific ethyl esters (e.g., ethyl decanoate in strain yl272 and yl273 fermentations as shown in FIG. 8).

(2) Across all strains created, there was a correlation between increased ethyl ester biosynthesis and increased production of MCFA off-flavor molecules. In other words, strains that produced greatly elevated concentrations of ethyl esters also produced greatly elevated concentrations of MCFAs, and strains that biosynthesized modestly increased concentrations of ethyl esters also produced moderately increased levels of MCFAs. The specific MCFAs produced at elevated concentrations were associated with their corresponding ethyl esters (i.e., they contained the same acyl moiety). For example, strains biosynthesizing increased levels of ethyl butanoate also produced increased levels of butanoic acid, while strains producing elevated concentrations of ethyl isovalerate produced elevated concentrations of isovaleric acid. The strength of this correlation between ethyl esters and corresponding MCFAs varied across different combinations of strain background, engineering strategy, and transgene expression level, but across all strains a strong correlated trend was observed (Spearman correlation = .60, FIG. 9). The ethyl ester and MCFA data shown in FIG. 9 are the same data as shown in FIG. 8. In FIG. 9, however, for each strain, the log2 -transformed foldchange value for each MCFA is plotted as a function of the fold-change value of its corresponding ethyl ester in the same fermentation. Notably, all of the MCFAs observed at elevated concentrations (e.g., butanoic acid, isovaleric acid, hexanoic acid, octanoic acid, decanoic acid) are strong off-flavor molecules in beer and wine. Prior work demonstrated a correlation between ethyl hexanoate and hexanoic acid production in fermentations using strains engineered with the strategy shown in FIG. 7 (e.g., strains expressing a variant FAS2 and a heterologous AAT). See, PCT Publication No. WO 2022/104106 Al. The data shown in FIGs. 8 and 9 suggested that increased production of multiple MCFAs may be a general feature of yeast engineered for increased ethyl ester biosynthesis, irrespective of the engineering strategy employed or ethyl ester molecule targeted. This observation has not previously been reported.

As the multiple MCFAs produced in elevated concentrations by these engineered strains represented a major roadblock to the commercial use of these strains, the goal was to discover improved methods for reducing MCFA production during fermentation. This work led to the discovery of an engineering approach that allows for yeast biosynthesis of increased levels of multiple ethyl esters while maintaining low concentrations of relevant MCFAs.

Example 2:

Given the desirable flavors imparted by ethyl esters and the undesirable off-flavors imparted by MCFAs, there was a need to create yeast strains that produced elevated levels of ethyl esters without simultaneously producing elevated levels of MCFAs. In an attempt to accomplish this, seven new strains were created that each expressed one AAE in an otherwise wild-type brewing strain parental background. These strains were tested in 80 mL malt extract fermentations alongside the parental strain. After the fermentations were complete, GC-MS was used to measure the concentrations of the target ethyl esters and their corresponding MCFAs in the fermentation media of both engineered and parental strains. Data from these fermentations is shown in FIG. 10 and reveals that multiple strains that expressed distinct AAE enzymes produced increased levels of one or more desirable ethyl esters. Remarkably, unlike strains created by other engineering strategies, these strains also produced similar, or lower concentrations of one or more MCFA molecules as compared to the parental strain (FIG. 10).

Example 3:

To test whether AAE expression would function to increase ethyl esters and decrease MCFA concentrations when combined with other ethyl ester engineering strategies, several new groups of strains were created. The first group of strains consisted of one strain that was engineered for expression of an enzyme with AAT activity (strategy described in FIG. 3), as well as a second strain that was similarly engineered for increased AAT expression but that was also engineered for expression of the AAE enzyme H1CCL2. These strains were both created in the LA3 brewing yeast background. Both engineered strains as well as the LA3 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. 11. As shown in FIG. 11, strains expressing both the AAT and H1CCL2 produced higher concentrations of three of the measured ethyl esters and reduced concentrations of three of the measured MCFAs as compared to the engineered strains expressing AAT alone. The ethyl esters that increased were ethyl propionate, ethyl butanoate, and ethyl isovalerate, and the MCFAs that were reduced were propionic acid, butanoic acid, and isovaleric acid. These data reveal that when combined with prior strategies for increasing ethyl ester biosynthesis, AAE expression can result in reduced production of MCFAs and increased production of desirable ethyl esters.

Example 4

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. 12, 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. These data reveal that the beneficial effects of expressing an AAE in strains that were previously engineered for increased AAT expression is seen across multiple combinations of AAE, AAT, and strain background. Example 5

To determine whether increased AAE expression would similarly be beneficial in the context of strains previously engineered to produce very high levels of ethyl esters, a final group of strains was constructed. 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. These data are shown in FIG. 13. As shown in FIG. 13, 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. For example, expression of H1CCL2, H1CCL3, and HcAAE(R51K) all resulted in increased production of ethyl butyrate and decreased production of butanoic acid relative to y 1810. while expression of H1CCL2, ScFAA4, and HcAAE(R51K), increased production of ethyl isovalerate and decreased production of isovaleric acid relative to y 1810. These data reveal that the beneficial effects of engineering strains for increased AAE activity also extend to strains that were previously engineered for greatly enhanced production of ethyl esters.

SEQUENCE LISTING

Claims

CLAIMS What is claimed is:

1. A genetically modified yeast cell comprising a genetic modification that results in increased expression of a nucleic acid encoding an enzyme having an 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 counterpart cell that does not comprise the genetic modification.

2. The genetically modified yeast cell of claim 1, wherein the genetic modification comprises a heterologous gene encoding the enzyme having AAE activity.

3. The genetically modified yeast cell of claim 1, wherein the one or more ethyl esters comprise ethyl propionate and the one or more fatty acids comprise propionic acid.

4. The genetically modified yeast cell of claim 1, wherein the one or more ethyl esters comprise ethyl butanoate and the one or more fatty acids comprise butyric acid.

5. The genetically modified yeast cell of claim 1, wherein the one or more ethyl esters comprise ethyl isovalerate and the one or more fatty acids comprise isovaleric acid.

6. The genetically modified yeast cell of claim 1, wherein the enzyme having AAE activity is derived from Humulus lupulus or Hypericum caly cinum.

7. The genetically modified yeast cell of claim 1, wherein the enzyme having AAE activity comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

8. The genetically modified yeast cell of claim 1, wherein the enzyme having AAE activity comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

9. The genetically modified yeast cell of claim 6, wherein the enzyme having AAE activity is derived from Humulus lupulus.

10. The genetically modified yeast cell of claim 9, wherein the enzyme having AAE activity is H1CCL2.

11. The genetically modified yeast cell of claim 9, wherein the enzyme having AAE activity is H1CCL3.

12. The genetically modified yeast cell of claim 6, wherein the enzyme having AAE activity is derived from Hypericum caly cinum.

13. The genetically modified yeast cell of claim 12, wherein the enzyme having AAE activity is HcAAEl.

13. The genetically modified yeast cell of claim 12, wherein the enzyme having AAE activity is HcAAEl (R51K).

14. A genetically modified yeast cell comprising a heterologous nucleic acid encoding an enzyme having an acyl activating enzyme (EC 6.2.1.3) activity (AAE), wherein the enzyme having AAE activity comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

15. The genetically modified yeast cell of claim 14, wherein the enzyme having AAE activity comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

16. The genetically modified yeast cell of claim 1, wherein the nucleic acid encoding the enzyme having AAE activity is operably linked to a promoter.

17. The genetically modified yeast cell of claim 1, wherein the genetically modified yeast cell further comprises 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).

18. The genetically modified yeast cell of claim 17, wherein the genetically modified yeast cell comprises a heterologous nucleic acid encoding the enzyme having AAT activity operably linked to a promoter.

19. The genetically modified yeast cell of claim 17, wherein the enzyme having AAT activity is derived from Marinobacter hydrocar bonoclaslicus. 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.

20. The genetically modified yeast cell of claim 17, wherein 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.

21. The genetically modified yeast cell of claim 17, wherein the enzyme having AAT activity comprises the amino acid sequences set forth in any one of SEQ ID NOs: 6-14.

22. The genetically modified yeast cell of claim 17, wherein the enzyme having AAT activity has specificity for an acyl-CoA produced by the enzyme having AAE activity.

23. The genetically modified yeast cell of claim 1, wherein the genetically modified yeast cell does not comprise a genetic modification that increases fatty acid biosynthesis.

24. The genetically modified yeast cell of claim 23, wherein the genetically modified yeast cell does not comprise a genetic modification to increase fatty acid synthetase (FAS) activity.

25. The genetically modified yeast cell of claim 1, further comprising one or more additional heterologous genes operably linked to one or more additional promoters, wherein the one or more additional heterologous genes encode one or more additional enzymes selected from the group consisting of PDC1, ALD6, ACS1, and ACC1.

26. The genetically modified yeast cell of claim 25, wherein the one or more additional heterologous genes are selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and combinations thereof.

27. The genetically modified yeast cell of claim 1, further comprising one or more bacterial genes operably linked to one or more additional promoters, wherein the one or more bacterial genes encode one or more bacterial enzymes selected from the group consisting of: BktB, Hbd, Crt, and Ter.

28. The genetically modified yeast cell of claim 27, wherein the one or more bacterial genes are selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and combinations thereof.

29. The genetically modified yeast cell of any one of claims 25-28, wherein the promoter(s) operably linked to the one or more heterologous genes and/or one or more bacterial genes are selected from the group consisting of pGPMl, pHSP26, pTHD3, pHEM13, pSPGl, pPRBl, pQCRIO, pPGKl, pOLEl, pERG25, and pHHF2.

30. The genetically modified yeast cell of claim 29, wherein the promoter(s) operably linked to the one or more heterologous genes and/or one or more bacterial genes are each independently selected from the group consisting of SEQ ID NOs: 15-21.

31. The genetically modified yeast cell of claim 1 or claim 17, wherein the promoter operably linked to the nucleic acid encoding the enzyme with AAE activity, and/or, if present, the promoter operably linked to the nucleic acid encoding the enzyme with AAT activity, are each independently selected from the group consisting of pGPMl, pHSP26, pTHD3, pHEM13, pSPGl, pPRBl, pQCRIO, pPGKl, pOLEl, pERG25, and pHHF2.

32. The genetically modified yeast cell of claim 31, wherein the promoter operably linked to the nucleic acid encoding the enzyme with AAE activity, and/or, if present, the promoter operably linked to the nucleic acid encoding the enzyme with AAT activity, are each independently selected from the group consisting of SEQ ID NOs: 15-21.

33. The genetically modified yeast cell of claim 1, wherein the genetically modified yeast cell is of the genus Saccharomyces .

34. The genetically modified yeast cell of claim 33, wherein the genetically modified yeast cell is of the species Saccharomyces cerevisiae.

35. The genetically modified yeast cell of claim 34, wherein the genetically modified yeast cell is S. cerevisiae California Ale Yeast strain WLP001, EC-1118, Elegance, Red Star Cote des Blancs, Epemay II, WY1056, A07, GY001, London Ale III, A38, Conan, WLP4000, Verdant, WLP002, WLP006, WLP007, OYLOl l, S04, WLP029, WY2565, WY2007, WLP830, OYL071, PYL091, OYL057, OYL061, or OYL090.

36. The genetically modified yeast cell of claim 33, wherein the genetically modified yeast cell is of the species Saccharomyces pastorianus.

37. The genetically modified yeast cell of any one of claims 1-2, wherein the one or more ethyl esters comprise ethyl propionate and the one or more fatty acids comprise propionic acid.

38. The genetically modified yeast cell of any one of claims 1-2, wherein the one or more ethyl esters comprise ethyl butanoate and the one or more fatty acids comprise butyric acid.

39. The genetically modified yeast cell of any one of claims 1-2, wherein the one or more ethyl esters comprise ethyl isovalerate and the one or more fatty acids comprise isovaleric acid.

40. The genetically modified yeast cell of any one of claims 1-2 and 37-39, wherein the enzyme having AAE activity is derived from Humulus lupulus o Hypericum caly cinum.

41. The genetically modified yeast cell of any one of claims 1-2 and 37-40, wherein the enzyme having AAE activity comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

42. The genetically modified yeast cell of any one of claims 1-2 and 37-41, wherein the enzyme having AAE activity comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

43. The genetically modified yeast cell of claim 40, wherein the enzyme having AAE activity is derived from Humulus lupulus.

44. The genetically modified yeast cell of claim 43, wherein the enzyme having AAE activity is H1CCL2.

45. The genetically modified yeast cell of claim 43, wherein the enzyme having AAE activity is H1CCL3.

46. The genetically modified yeast cell of claim 40, wherein the enzyme having AAE activity is derived from Hypericum caly cinum.

47. The genetically modified yeast cell of claim 46, wherein the enzyme having AAE activity is HcAAEl.

48. The genetically modified yeast cell of claim 46, wherein the enzyme having AAE activity is HcAAEl (R51K).

49. A genetically modified yeast cell comprising a heterologous nucleic acid encoding an enzyme having an acyl activating enzyme (EC 6.2.1.3) activity (AAE), wherein the enzyme having AAE activity comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

50. The genetically modified yeast cell of claim 14, wherein the enzyme having AAE activity comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-4.

51. The genetically modified yeast cell of any one of claims 1-2, 14-15 and 37-50, wherein the nucleic acid encoding the enzyme having AAE activity is operably linked to a promoter.

52. The genetically modified yeast cell of any one of claims 1-2, 14-15 and 37-51, wherein the genetically modified yeast cell further comprises 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).

53. The genetically modified yeast cell of claim 52, wherein the genetically modified yeast cell comprises a heterologous nucleic acid encoding the enzyme having AAT activity operably linked to a promoter.

54. The genetically modified yeast cell of claim 52 or 53, wherein the enzyme having AAT activity is derived from Marinobacter hydrocar bonoclaslicus. 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.

55. The genetically modified yeast cell of any one of claims 52-54, wherein 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.

56. The genetically modified yeast cell of any one of claims 52-55, wherein the enzyme having AAT activity comprises the amino acid sequences set forth in any one of SEQ ID NOs: 6-14.

57. The genetically modified yeast cell of any one of claims 52-56, wherein the enzyme having AAT activity has specificity for an acyl-CoA produced by the enzyme having AAE activity.

58. The genetically modified yeast cell of any one of claims 1-2, 14-15 and 37-57, wherein the genetically modified yeast cell does not comprise a genetic modification that increases fatty acid biosynthesis.

59. The genetically modified yeast cell of claim 58, wherein the genetically modified yeast cell does not comprise a genetic modification to increase fatty acid synthetase (FAS) activity.

60. The genetically modified yeast cell of any one of claims 1-2, 14-15 and 37-59, further comprising one or more additional heterologous genes operably linked to one or more additional promoters, wherein the one or more additional heterologous genes encode one or more additional enzymes selected from the group consisting of PDC1, ALD6, ACS1, and ACC1.

61. The genetically modified yeast cell of claim 60, wherein the one or more additional heterologous genes are selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and combinations thereof.

62. The genetically modified yeast cell of any one of claims 1-2, 14-15 and 37-91, further comprising one or more bacterial genes operably linked to one or more additional promoters, wherein the one or more bacterial genes encode one or more bacterial enzymes selected from the group consisting of: BktB, Hbd, Crt, and Ter.

63. The genetically modified yeast cell of claim 62, wherein the one or more bacterial genes are selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and combinations thereof.

64. The genetically modified yeast cell of any one of claims 60-63, wherein the promoter(s) operably linked to the one or more heterologous genes and/or one or more bacterial genes are selected from the group consisting of pGPMl, pHSP26, pTHD3, pHEM13, pSPGl, pPRBl, pQCRIO, pPGKl, pOLEl, pERG25, and pHHF2.

65. The genetically modified yeast cell of claim 64, wherein the promoter(s) operably linked to the one or more heterologous genes and/or one or more bacterial genes are each independently selected from the group consisting of SEQ ID NOs: 15-21.

66. The genetically modified yeast cell of any one of claims 12, 14-15 and 37-65, wherein the promoter operably linked to the nucleic acid encoding the enzyme with AAE activity, and/or, if present, the promoter operably linked to the nucleic acid encoding the enzyme with AAT activity, are each independently selected from the group consisting of pGPMl, pHSP26, pTHD3, pHEM13, pSPGl, pPRBl, pQCRIO, pPGKl, pOLEl, pERG25, and pHHF2.

67. The genetically modified yeast cell of claim 66, wherein the promoter operably linked to the nucleic acid encoding the enzyme with AAE activity, and/or, if present, the promoter operably linked to the nucleic acid encoding the enzyme with AAT activity, are each independently selected from the group consisting of SEQ ID NOs: 15-21.

68. The genetically modified yeast cell of any one of claims 1-2, 14-15 and 37-67, wherein the genetically modified yeast cell is of the genus Saccharomyces .

69. The genetically modified yeast cell of claim 68, wherein the genetically modified yeast cell is of the species Saccharomyces cerevisiae.

70. The genetically modified yeast cell of claim 69, wherein the genetically modified yeast cell is S. cerevisiae California Ale Yeast strain WLP001, EC-1118, Elegance, Red Star Cote des Blancs, Epemay II, WY1056, A07, GY001, London Ale III, A38, Conan, WLP4000, Verdant, WLP002, WLP006, WLP007, OYLOl l, S04, WLP029, WY2565, WY2007, WLP830, OYL071, PYL091, OYL057, OYL061, or OYL090.

71. The genetically modified yeast cell of claim 68, wherein the genetically modified yeast cell is of the species Saccharomyces pastorianus.

72. A method of producing a fermented product comprising, contacting the genetically modified yeast cell of any one of claims 1-71 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.

73. The method of claim 72, wherein the at least one fermentable sugar is provided in at least one sugar source.

74. The method of claim 72 or 73, wherein the fermentable sugar is glucose, fructose, sucrose, maltose, and/or maltotriose.

75. The method of any one of claims 72-74, wherein the fermented product comprises an increased amount 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.

76. The method of claim 75, wherein the desired product is an ethyl ester selected from the group consisting of ethyl butanoate, ethyl isovalerate, ethyl octanoate, ethyl decanoate, ethyl 2-methlybutyrate, and ethyl crotonate.

77. The method of claim 75, wherein the desired product is an ethyl ester selected from the group consisting of ethyl propionate, ethyl butanoate, and ethyl isovalerate.

78. The method of any one of claims 72-77, wherein the fermented product comprises 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.

79. The method of claim 78, wherein 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.

80. The method of claim 78, wherein the at least one undesired product is an acid selected from the group consisting of propanoic acid, butanoic acid, and isovaleric acid.

81. The method of any one of claims 72-80, wherein the fermented product is a fermented beverage.

82. The method of claim 81, wherein the fermented beverage is beer, wine, sparkling wine (champagne), wine cooler, wine spritzer, hard seltzer, sake, mead, kombucha, or cider.

83. The method of any one of claims 72-82, wherein the sugar source comprises wort, must, fruit juice, honey, rice starch, or a combination thereof.

84. The method of claim 83, wherein 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.

85. The method of claim 83, wherein the sugar source is wort and 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.

86. The method of claim 85m further comprises adding at least one hop variety to the wort to produce a hopped wort.

87. The method of any one of claims 72-86, further comprising adding at least one hop variety to the medium.

88. The method of claim 83, wherein 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.

89. The method of claim 88, further comprising removing solid fruit material from the must to produce a fruit juice.

90. The method of any one of claims 72-89, further comprising at least one additional fermentation process.

91. The method of any one of claims 72-90, further comprising carbonating the fermented product.

92. A fermented product produced, obtained, or obtainable by the method of any one of claims 72-91.

93. The fermented product of claim 92, wherein the fermented product comprises at least 150 pg/L of ethyl butanoate, ethyl isovalerate, ethyl octanoate, ethyl decanoate, 2- methylbutyrate, or ethyl crotonate.

94. The fermented product of claim 92, wherein the fermented product comprises at least 150 pg/L of ethyl propanoate, ethyl butanoate, or ethyl isovalerate.

95. The fermented product of any one of claims 92-94 wherein the fermented product comprises less than 15 mg/L of butanoic acid, isovaleric acid, octanoic acid, decanoic acid, 2-methyl-butyric acid, or crotonic acid.

96. The fermented product of any one of claims 92-94 wherein the fermented product comprises less than 15 mg/L of propanoic acid, butanoic acid, or isovaleric acid.

97. A method of producing a composition comprising ethanol comprising, contacting the genetically modified yeast cell of any one of claims 1-71 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.

98. The method of claim 97, wherein the at least one fermentable sugar is provided in at least one sugar source.

99. The method of claim 97 or 98, wherein the fermentable sugar is glucose, fructose, sucrose, maltose, and/or maltotriose.

100. The method of any one of claims 97-99, wherein the composition comprising ethanol comprises an increased amount of at least one desired product as compared to a composition comprising ethanol produced by a counterpart cell that does not express the enzyme having AAE activity.

101. The method of claim 100, wherein the desired product is an ethyl ester selected from the group consisting of ethyl butanoate, ethyl isovalerate, ethyl octanoate, ethyl decanoate, ethyl 2-methlybutyrate, and ethyl crotonate.

102. The method of claim 100, wherein the desired product is an ethyl ester selected from the group consisting of ethyl propionate, ethyl butanoate, and ethyl isovalerate.

103. The method of any one of claims 97-102, wherein the composition comprising ethanol comprises a reduced amount of at least one undesired product as compared to a composition comprising ethanol produced by a counterpart cell that does not express the enzyme having AAE activity.

104. The method of claim 103, wherein 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.

105. The method of claim 103, wherein the at least one undesired product is an acid selected from the group consisting of propanoic acid, butanoic acid, and isovaleric acid.

106. The method of any one of claims 97-105, wherein the composition comprising ethanol is a fermented beverage.

107. The method of claim 106, wherein the fermented beverage is beer, wine, sparkling wine (champagne), wine cooler, wine spritzer, hard seltzer, sake, mead, kombucha, or cider.

108. The method of any one of claims 97-107, wherein the sugar source comprises wort, must, fruit juice, honey, rice starch, or a combination thereof.

109. The method of claim 108, wherein 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.

110. The method of claim 108, wherein the sugar source is wort and 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.

111. The method of claim 110 further comprises adding at least one hop variety to the wort to produce a hopped wort.

112. The method of any one of claims 97-111, further comprising adding at least one hop variety to the medium.

113. The method of claim 108, wherein 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.

114. The method of claim 113, further comprising removing solid fruit material from the must to produce a fruit juice.

115. The method of any one of claims 97-114, further comprising at least one additional fermentation process.

116. The method of any one of claims 97-115, further comprising carbonating the composition comprising ethanol.

117. A composition comprising ethanol produced, obtained, or obtainable by the method of any one of claims 97-116.

118. The composition comprising ethanol of claim 117, wherein the composition comprising ethanol comprises at least 150 pg/L of ethyl butanoate, ethyl isovalerate, ethyl octanoate, ethyl decanoate, 2-methylbutyrate, or ethyl crotonate.

119. The composition comprising ethanol of claim 117, wherein the composition comprising ethanol comprises at least 150 pg/L of ethyl propanoate, ethyl butanoate, or ethyl isoval erate.

120. The composition comprising ethanol of any one of claims 117-119 wherein the composition comprising ethanol comprises less than 15 mg/L of butanoic acid, isovaleric acid, octanoic acid, decanoic acid, 2-methyl-butyric acid, or crotonic acid.

121. The composition comprising ethanol of any one of claims 117-119 wherein the composition comprising ethanol comprises less than 15 mg/L of propanoic acid, butanoic acid, or isovaleric acid.

122. A liquid fermentation composition comprising a population of genetically modified yeast cells according to any one of claims 1-71 and a sugar source.

123. The liquid fermentation composition of claim 122, further comprising alcohol.

124. The liquid fermentation composition of any one of claims 122-123, further comprising (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.