Genetically modified yeast cells and methods of use thereof

Abstract

To provide a genetically modified yeast cell recombinantly expressing a gene encoding a mutant beta-lyase, a method for producing a fermentation product, and a method for producing ethanol.SOLUTION: Provided herein are genetically modified yeast cells that recombinantly express a gene encoding a mutant beta-lyase. Also provided are methods of producing a fermentation product and methods of producing ethanol.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62 / 916,529, filed October 17, 2019, and U.S. Provisional Application No. 63 / 086,363, filed October 1, 2020, the entire contents of each of these referenced applications being incorporated herein by reference.

[0002] Government support This invention was made with government support under Award No. 1831242 awarded by the National Science Foundation. The government has certain rights in this invention. [Background technology]

[0003] background Within the last decade, tropical fruit flavors have become increasingly popular in beverage markets, both within the United States and internationally. See Cannon et al. J. Food Drug Anal. (2018) 26:445-468; Watson, B. Early 2018 Beer Style Trends; Hahn et al. Washington Post (2016): washingtonpost.com / lifestyle / food / pineapple-and-mango-in-the-pint-glass-so-hot-right-now / 2016 / 05 / 22 / 73f6c52a-1dd2-11e6-b6e0-c53b7ef63b45_story.html. In the beer industry, this trend is exemplified by the significant increase in the use of flavoring hops, prized for their tropical fruit aromas. Within the wine industry, tropical flavor notes have fueled the popularity of Sauvignon Blanc and Chardonnay styles, and long-standing efforts have sought to further enhance the tropical aromas found in these wines. See Tominaga, et al. Flavour and Fragrance Journal (1998) 13, 159-162; Swiegers, et al. Yeast (2007) 24, 561-574; Howell, et al. Appl. Environ. Microbiol. (2005) 71, 5420-5426; Santiago, et al. FEMS Yeast Res. (2015) 15, fov034; Roland, A., et al. Flavour and Fragrance Journal (2012) 27, 266-272; Jeffery, et al. Australian Journal of Chemistry (2016) 69, 1323. Research has shown that the majority of fruit flavors are produced by various combinations of diverse flavor molecules.See Cannon et al. J. Food Drug Anal. (2018) 26:445-468; Bartowsky et al. Biology of Microorganisms on Grapes, in Must and in Wine, pp:209-231; Holt et al. (FEMS Microbiol. Rev. 2019) 43:193-222. However, numerous studies also attribute much of the tropical flavor and aroma of Sauvignon Blanc wine and certain flavor hop varieties to the presence of three specific volatile thiol molecules. These thiols, 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and 4-methyl-4-mercaptopentan-2-one (4MMP), are all detectable to human olfactory receptors at extremely low concentrations and confer grapefruit / passion fruit, guava / gooseberry, and passion fruit / blackcurrant flavors, respectively. See Vanzo et al. Sci. Rep. (2017): 7; Roland et al. Chem. Rev. (2012) 111, 7355-7376. Summary of the Invention

[0004] overview Aspects of the present disclosure provide genetically modified yeast cells (modified cells) comprising a heterologous gene encoding an enzyme with beta-lyase activity. In some embodiments, the enzyme with beta-lyase activity has a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO:2. In some embodiments, the enzyme with beta-lyase activity does not include any of the sequences as set forth in SEQ ID NOs:1, 6, or 7. In some embodiments, the enzyme with beta-lyase activity has a sequence as set forth in SEQ ID NO:2. In some embodiments, the enzyme with beta-lyase activity has a sequence having at least 90% sequence identity to the sequence as set forth in any one of SEQ ID NOs:3-7. In some embodiments, the enzyme with beta-lyase activity has a sequence as set forth in any one of SEQ ID NOs:3-5.

[0005] In some embodiments, the enzyme with beta-lyase activity comprises a substitution mutation at a position corresponding to position H463 of SEQ ID NO: 1. In some embodiments, the substitution mutation at the position corresponding to position H463 of SEQ ID NO: 1 is phenylalanine, arginine, glutamic acid, threonine, glycine, isoleucine, or valine.

[0006] In some embodiments, the yeast cell is of the genus Saccharomyces. In some embodiments, the yeast cell is of the species Saccharomyces cerevisiae (S. cerevisiae). In some embodiments, the yeast cell is S. cerevisiae California Ale Yeast strain WLP001. In some embodiments, the yeast cell is of the species Saccharomyces pastorianus (S. pastorianus).

[0007] Aspects of the present disclosure provide methods for producing a fermentation product, comprising contacting any of the modified cells described herein with a medium containing at least one fermentable sugar, wherein the contacting is performed during at least an initial fermentation process to produce the fermentation product. In some embodiments, the 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 at least one sugar source comprises at least one precursor, such as a plant-derived precursor or a chemically synthesized precursor. In some embodiments, the at least one precursor comprises 3-mercaptohexan-1-ol conjugated to cysteine ​​(Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexan-1-ol conjugated to glutathione (Glut-3-MH), and / or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut 4MMP). In some embodiments, the method further comprises adding one or more precursors to the culture medium, wherein the precursors comprise 3-mercaptohexan-1-ol (Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexan-1-ol conjugated to glutathione (Glut-3-MH), and / or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut 4MMP).

[0008] In some embodiments, the fermentation product comprises an increased level of at least one volatile thiol compared to a fermentation product produced by an equivalent cell not expressing a heterologous gene or an equivalent cell expressing a wild-type enzyme having beta-lyase activity. In some embodiments, the at least one volatile thiol comprises 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), 4-methyl-4-mercaptopent-2-one (4MMP), or a combination thereof. In some embodiments, the fermentation product comprises at least 200 ng / L of 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and / or 4-methyl-4-mercaptopent-2-one (4MMP).

[0009] In some embodiments, the fermentation product comprises a reduced level of at least one undesirable product compared to a fermentation product produced by an equivalent cell that does not express a heterologous gene or an equivalent cell that expresses a wild-type enzyme having beta-lyase activity.

[0010] In some embodiments, the at least one undesired product is indole. In some embodiments, the fermentation product is a fermented beverage. In some embodiments, the fermented beverage is beer, wine, sparkling wine (champagne), sake, mead, kombucha, or cider. In some embodiments, the sugar source comprises malt juice, fruit juice, honey, rice starch, or a combination thereof. In some embodiments, the fruit juice is grape juice or apple juice.

[0011] In some embodiments, the sugar source is wort, and the method further comprises producing a medium, wherein producing the medium comprises contacting a plurality of grains with water; and boiling or steeping the water and grains to produce the 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 method further comprises at least one additional fermentation process. In some embodiments, the method further comprises carbonating the fermentation product.

[0012] Aspects of the present disclosure provide fermentation products produced by any of the methods described herein. In some embodiments, the fermentation product contains at least 200 ng / L of 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and / or 4-methyl-4-mercaptopent-2-one (4MMP). In some embodiments, the fermentation product contains less than 500 μg / L of indole.

[0013] Aspects of the present disclosure provide methods for producing a composition comprising ethanol, comprising contacting any of the modified cells described herein with a medium comprising at least one fermentable sugar, wherein such contacting is carried out during at least an initial fermentation process to produce a composition comprising ethanol. In some embodiments, the 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 at least one sugar source comprises at least one precursor. In some embodiments, the at least one precursor comprises 3-mercaptohexan-1-ol conjugated to cysteine ​​(Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexan-1-ol conjugated to glutathione (Glut-3-MH), and / or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut 4MMP). In some embodiments, the method further comprises adding one or more precursors to the culture medium, wherein the precursors comprise 3-mercaptohexan-1-ol (Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexan-1-ol conjugated to glutathione (Glut-3-MH), and / or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut 4MMP).

[0014] In some embodiments, the ethanol-containing composition further comprises an increased level of at least one volatile thiol compared to a composition comprising ethanol produced by a comparable cell not expressing a heterologous gene or a comparable cell expressing a wild-type enzyme having beta-lyase activity. In some embodiments, the at least one volatile thiol comprises 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), 4-methyl-4-mercaptopent-2-one (4MMP), or a combination thereof. In some embodiments, the ethanol-containing composition further comprises at least 200 ng / L of 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and / or 4-methyl-4-mercaptopent-2-one (4MMP).

[0015] In some embodiments, the ethanol-containing composition further comprises a reduced level of at least one undesired product compared to a composition comprising ethanol produced by an equivalent cell that does not express a heterologous gene or an equivalent cell that expresses a wild-type enzyme having beta-lyase activity, hi some embodiments, the at least one undesired product is indole.

[0016] In some embodiments, the composition comprising ethanol is a fermented beverage. In some embodiments, the fermented beverage is beer, wine, sparkling wine (champagne), sake, mead, kombucha, or apple juice. In some embodiments, the sugar source comprises malt juice, fruit juice, honey, rice starch, or a combination thereof. In some embodiments, the fruit juice is grape juice or apple juice.

[0017] In some embodiments, wherein the sugar source is wort, and the method further comprises producing a medium, wherein producing the medium comprises contacting a plurality of grains with water; and boiling or steeping the water and grains to produce the wort. In some embodiments, the method further comprises adding at least one hop variety to the wort to produce a hoppy wort. In some embodiments, the method further comprises adding at least one hop variety to the medium. In some embodiments, the method further comprises at least one additional fermentation process. In some embodiments, the method further comprises carbonating the fermentation product.

[0018] Aspects of the present disclosure provide compositions comprising ethanol produced by any of the methods described herein. In some embodiments, the compositions further comprise at least 200 ng / L of 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and / or 4-methyl-4-mercaptopent-2-one (4MMP). In some embodiments, the fermentation product comprises less than 500 μg / L of indole. [Brief explanation of the drawings]

[0019] Brief description of the drawings Further aspects of the present disclosure will be readily appreciated upon review of the following detailed description of its various aspects and embodiments when considered in conjunction with the accompanying drawings.

[0020] [Figure 1A] 1A-1H show exemplary process diagrams for methods of making a fermentation product or methods of making ethanol as described herein. Figure 1A shows a method of producing a fermentation product by contacting modified cells of the present disclosure with a medium during at least an initial fermentation process. [Figure 1B] FIG. 1B illustrates an embodiment of the method of FIG. 1A, where the medium is produced by contacting a plurality of grains with water, boiling the water to produce a wort, which is cooled to produce the medium. [Figure 1C] FIG. 1C shows an embodiment of the method of FIG. 1A, but in which at least one variety of hops is added to the medium. [Figure 1D] FIG. 1D shows an embodiment of the method of FIG. 1A, but in which at least one additional fermentation process occurs. [Figure 1E] FIG. 1E shows an embodiment of the method of FIG. 1A, where the fermentation product is carbon dioxide. [Figure 1F] FIG. 1F shows a method of making ethanol that involves contacting modified cells of the present disclosure with a culture medium during at least an initial fermentation process to produce a composition comprising ethanol. [Figure 1G] FIG. 1G shows an embodiment of the method of FIG. 1A, except that at least one volatile thiol precursor (eg, Cys-MH, Glu-3MH) is added to the medium. [Figure 1H] FIG. 1H illustrates a method of producing a fermentation product by contacting a purified enzyme of the present disclosure with a medium during at least the initial fermentation process.

[0021] [Figure 2] Figure 2 shows the concentrations of 3-mercaptohexanol (3MH) and indole in beer brewed with wild-type and engineered yeast strains. The left axis indicates 3MH concentration, and the right axis reports indole concentration. Shown from left to right are the wild-type California Ale Yeast (WLP001); WLP001 overexpressing IRC7 (Y27); WLP001 overexpressing STR3 (Y33); WLP001 overexpressing TnaA (Y182); and WLP001 overexpressing TnaA-H463F (Y502).

[0022] [Figure 3]Figures 3A and 3B show the concentrations of mercaptohexanol (3MH) and indole in fermentation products produced using the indicated yeast strains. Figure 3A shows the concentrations of 3-mercaptohexanol (3MH) and indole in beer brewed with wild-type yeast strains and modified yeast strains expressing TnaA or the TnaA H463F mutant. The left axis shows the 3MH concentration (ng / L), and the right axis reports the indole concentration (μg / L). Shown from left to right are: wild-type California Ale Yeast (WLP001); WLP001 overexpressing wild-type TnaA (Y319; Trpase WT); and WLP001 overexpressing the TnaA H463F mutant (Y502; Trpase H463F). Figure 3B shows the concentrations of 3-mercaptohexan-1-ol (3MH) and indole in wine fermented with wild-type yeast strains and engineered yeast strains expressing TnaA or the TnaA H463F mutant. The left axis shows 3MH concentration (ng / L), and the right axis reports indole concentration (μg / L). Shown from left to right are the wild-type Red Star Côte des Blanc yeast strain; Red Star overexpressing wild-type TnaA (Y919; Trpase WT); and Red Star overexpressing the TnaA H463F mutant (Y484; Trpase H463F).

[0023] [Figure 4]Figures 4A and 4B show the concentrations of volatile thiols and indole in fermentation products produced using the indicated yeast strains in the presence or absence of added glutathione-conjugated 3-mercaptohexan-1-ol (Glut-3MH). Figure 4A shows the concentration of 3-mercaptohexan-1-ol (3MH (ng / L)) in beer brewed with a wild-type yeast strain and an engineered yeast strain expressing TnaA or the TnaA H463F mutant. Figure 4B shows the concentration of indole (μg / L) in beer brewed with a wild-type yeast strain and an engineered yeast strain expressing TnaA or the TnaA H463F mutant. Shown from left to right are strains: wild-type California Ale Yeast (WLP001); WLP001 overexpressing wild-type TnaA (Y319; Trpase WT); and WLP001 overexpressing the TnaA H463F mutant (Y502; Trpase H463F). For each strain, the right column shows the indole produced in fermentations containing Glut-3MH added at the beginning of the fermentation process; the left column shows the 3MH produced in the absence of Glut-3MH added to the fermentation process.

[0024] [Figure 5]Figure 5 shows the concentrations of 3-mercaptohexan-1-ol (3MH) and indole in beer brewed using yeast strains expressing TnaA containing the indicated amino acid mutations. The left axis shows 3MH concentration (ng / L), and the right axis reports indole concentration (μg / L). Strains shown from left to right are: wild-type California Ale Yeast (WLP001); WLP001 overexpressing wild-type TnaA (Trpase WT); WLP001 overexpressing the TnaA H463F mutant (Trpase H463F); WLP001 overexpressing the wild-type TnaA H463R mutant (Trpase H463R); WLP001 overexpressing the TnaA H463E mutant (Trpase H463E); WLP001 overexpressing the wild-type TnaA H463T mutant (Trpase H463T); WLP001 overexpressing the wild-type TnaA H463G mutant (Trpase H463G); WLP001 overexpressing the wild-type TnaA H463I mutant (Trpase H463I); and wild-type TnaA WLP001 (Trpase H463V) overexpressing the H463V mutant.

[0025] [Figure 6]Figure 6 shows the concentrations of 3-mercaptohexan-1-ol (3MH) and indole in beers brewed using yeast strains expressing tryptophanase TnaA from Citrobacter amalonaticus and homologous enzymes from other species. The left axis shows 3MH concentration (ng / L), and the right axis reports indole concentration (μg / L). Shown from left to right are strains: wild-type California Ale Yeast (WLP001); WLP001 overexpressing wild-type (WT) TnaA from C. amalonaticus (Y319; Trpase WT); WLP001 overexpressing the TnaA Trpase H463F mutant from C. amalonaticus (Y502; Trpase H463F); WLP001 overexpressing a TnaA / Trpase homolog from Trichoderma asperellum (Y644; T. asp homolog); WLP001 overexpressing a TnaA / Trpase homolog from Aspergillus saccharolyticus (Y645; A. sac homolog); and WLP001 overexpressing a TnaA / Trpase homolog from Zooshikella ganghwensis (Y646; Z. gang homolog). DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description Within the last decade, tropical fruit flavors have become increasingly popular in the beverage market. For example, the demand for beverages with flavor notes such as mango, papaya, and pineapple in the beer and wine industries has increased dramatically in recent years. Three flavor molecules that impart tropical notes in fermented beverages are the volatile thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and 4-methyl-4-mercaptopentan-2-one (4MMP). These thiols are produced by yeast-expressed enzymes that convert odorless precursors (e.g., plant-derived precursors) into flavor-active volatile thiols during the fermentation process. Several attempts have been made to identify yeast strains that release high levels of these thiols and to engineer yeast strains for increased thiol production; however, these attempts have met with limited success, with increases in volatile production being moderate, context dependent, or compromised by the concomitant release of undesirable products such as indole (e.g., off-flavors). The present disclosure provides genetically modified yeast cells that have been modified to increase such thiols and reduce the production of undesirable products.

[0027] Provided herein are genetically modified yeast cells engineered to express an enzyme having beta-lyase activity. In some embodiments, the enzyme having beta-lyase activity is modified to increase production of desired volatile thiols and reduce production of undesirable indoles. Also provided herein are methods for producing fermented beverages, involving contacting genetically modified yeast cells with a medium containing a sugar source, including at least one fermentable sugar, during a fermentation process. Also provided herein are methods for producing ethanol, involving contacting genetically modified yeast cells with a medium containing a sugar source, including at least one fermentable sugar, during a fermentation process.

[0028] Beta-lyase (β-lyase) The genetically modified cells described herein contain a heterologous gene encoding an enzyme with beta-lyase activity. The term "heterologous gene," as used herein, refers to a hereditary unit corresponding to a nucleic acid (e.g., DNA) sequence containing genetic instructions that is introduced into and expressed by a host organism (e.g., a genetically modified cell) that does not naturally encode said gene.

[0029] Beta-lyase enzymes are responsible for the production of thiols, which are related to alcohols and phenyls but contain a thiol or sulfanyl group ("-SH"). Thiols can have any of a variety of aromas or odors and are generally classified as negative-smelling thiols and positive-smelling thiols. Some sulfur-containing compounds (such as those that provide a rotten egg odor) are the result of the formation of H2S during yeast fermentation. Other secondary reducing odors, such as those in cooked vegetables, onions, and cabbage, are also produced from sulfur-containing compounds such as thioacetates and mercaptans, which are thought to result from too low a redox potential in the fermentation products (Brajkovich et al., 2005).

[0030] Sulfur-containing compounds that contribute positively to a product are called "volatile thiols" that tend to have unique aroma profiles. For example, well-known aroma-responsible volatile thiols include: 3-mercaptohexan-1-ol (3MH) (CH), which conveys the aroma of grapefruit, passion fruit, gooseberry, and guava. 14OS, also known as 3-mercapto-1-hexanol, 3-mercaptohexanol, 3-sulfanylhexan-1-ol, 3-thiohexanol, 1-hexanol, 3-mercapto-; 3-mercaptohexyl acetate (3MHA) (CH), which imparts aromas of passion fruit, grapefruit, boxwood, gooseberry, and guava. 16 0S, also known as hexyl 3-sulfanylacetate); and 4-methyl-4-mercaptopentan-2-one (4MMP) (CH), which conveys aromas of boxwood, passion fruit, broom, and black currant. 12 OS, also known as 4-mercapto-4-methyl-2-pentanone).

[0031] Without wishing to be bound by any particular theory, the formation of aromatic precursors is believed to involve the steps of enzymatic oxidation of unsaturated fatty acids, metabolic processing, cysteinylated or glutathionylated conjugation to aldehydes, and beta-lyase cleavage during alcoholic fermentation to release aromatic compounds. This process, in which fermenting organisms convert odorless precursor molecules (e.g., plant-derived precursors) of sugar sources in the medium (e.g., wort, must, etc.) to active thiols, is called "biotransformation." See, for example, Swiegers et al. Yeast (2007) 24:561-574; Santiago et al. FEMS Yeast Res. (2015) 15; Holt et al. Appl. Environ. Microbiol. (2011) 77:3626-3632; Thibon et al. FEMS Yeast Res. (2008) 8:1076-1086; Kishimoto et al. J. Am. Soc. Brewing Chemists (2008) 66:192-196. 3MH and 4MMP are produced during fermentation by bioconversion of the cysteine-conjugated precursor molecules Cys-3MH and Cys-4MMP, respectively. See, for example, Roland et al. Flavor and Fragrance Journal (2016) 69:1323. The biotransformation is catalyzed by organisms expressing enzymes with beta-lyase activity, which cleaves cysteine ​​conjugates to release volatile thiols. See, for example, Santiago et al. FEMS Yeast Res. (2015) 15; Roncoroni et al. Food Microbiol. (2011) 926-935; Roland et al. Chem. Rev. (2011) 111:7355-7376. 3MH can subsequently be acetylated by yeast expressing an acyltransferase enzyme to produce 3MHA. See, for example, Roland et al. Chem. Rev. (2011) 111:7355-7376.

[0032] During fermentation, Cys-3-MH and Cys-4-MMP can be transported from the medium into yeast cells and cleaved by enzymes with beta-lyase activity. Alternatively, brewing wort with grape juice or grape must can also contain glutathione conjugates Glut-3-MH and Glut-4-MMP. For examples, see Roland et al. Chem. Rev. (2011) 111:7355-7376; Kishimoto et al. J. Am. Soc. Brewing Chemists (2008) 66:192-196. The glutathione conjugates can be transported into yeast cells and cleaved by transpeptidase enzymes to produce Cys-3-MH and Cys-4-MMP, which then become substrates for enzymes with beta-lyase activity. See, for example, Howell et al. Appl. Environ. Microbiol (2005) 71:5420-5426; Santiago et al. FEMS Yeast Res. (2015) 15. 3MH and 4MMP are then generated by cleavage of the cysteine ​​conjugates by enzymes with beta-lyase activity. Several studies have indicated that this reaction is highly inefficient, and that commonly used yeast strains in winemaking convert only 0.2%-2.0% of the available cysteine ​​conjugate precursors into flavor-active thiols during grape must fermentation. 6,7,21 This inefficiency represents a significant biochemical bottleneck that limits the production of beneficial volatile thiols (e.g., 3MH, 3MHA, 4MMP) in fermented beverages.

[0033] In addition to inefficiencies in the production of volatile thiols, beta-lyase expression can also result in increased production of undesirable molecules such as indole. Indole is formed from aromatic heterocyclic organic compounds with the formula CHN and has a bicyclic structure consisting of a six-membered benzene ring fused to a five-membered pyrrole ring. Indole is widely distributed in the environment, occurs naturally in human feces, and has a strong fecal odor. Consequently, producing indole during the production of fermented products intended for consumption is undesirable.

[0034] Various enzymes exhibit beta-lyase activity, such as beta-lyase and tryptophanase (TnaA). In some embodiments, the heterologous gene encoding an enzyme with beta-lyase activity is a wild-type beta-lyase gene (e.g., a gene isolated from an organism). In some embodiments, the heterologous gene encoding an enzyme with beta-lyase activity is a mutant beta-lyase gene that contains one or more mutations (e.g., substitutions, deletions, insertions) in the nucleic acid sequence of the beta-lyase gene and / or in the amino acid sequence of the enzyme with beta-lyase activity. As will be understood by those skilled in the art, mutations in the nucleic acid sequence may alter the amino acid sequence of the translated polypeptide (e.g., substitution mutations) or may not alter the amino acid sequence of the translated polypeptide (e.g., silent mutations) compared to the wild-type or reference enzyme.

[0035] In some embodiments, the heterologous gene encoding an enzyme with beta-lyase activity is truncated, i.e., lacking one or more amino acids, preferably at the N-terminus or C-terminus of the enzyme, compared to the wild-type or reference enzyme.

[0036] In some embodiments, the beta-lyase may also be referred to as cystathionine beta-lyase (EC 4.4.1.13). In some embodiments, the beta-lyase gene is from a fungus. In some embodiments, the beta-lyase gene is from a Saccharomyces species, such as an endogenous yeast beta-lyase. Examples of endogenous yeast beta-lyases include, without limitation, Irc7p (also referred to as YFR055W) encoded by the gene IRC7 and Str3p encoded by the gene STR3. In some embodiments, the beta-lyase is IRC7 or STR3 from S. cerevisiae yeast strain VL3.

[0037] In some embodiments, the beta-lyase gene is from a bacterium or a fungus. In some embodiments, the beta-lyase gene is from Escherichia coli (E. coli). In some embodiments, the beta-lyase gene is from a Citrobacter species. In some embodiments, the beta-lyase gene is from Citrobacter amalonaticus.

[0038] An exemplary beta-lyase is TnaA from Citrobacter amalonaticus, provided by the amino acid sequence set forth as SEQ ID NO: 1. The consensus motif "MSAKKD" (SEQ ID NO: 8) is shown in bold, and the catalytic residue, a lysine at position 270 (designated K270), is shown in bold and underlined. The conserved motif "IDLLTDSGT" (SEQ ID NO: 9) is shown in bold and italic.

[0039] Amino acid sequence of wild-type TnaA from C. amalonaticus [ka] (SEQ ID NO: 1)

[0040] In some embodiments, the beta-lyase is a homolog of TnaA from C. amalonaticus (SEQ ID NO: 1). Homologs or related enzymes may be identified using methods known in the art, such as those described herein. In some embodiments, the beta-lyase gene is from a Zooshikella species. In some embodiments, the beta-lyase gene is from Zooshikella ganghwensis. The amino acid sequence of a wild-type TnaA homolog from Z. ganghwensis is provided by accession number WP_094789495.1 and has 82% sequence identity over its entire length with TnaA from C. amalonaticus (SEQ ID NO: 1).

[0041] In some embodiments, the beta-lyase gene is from an Aspergillus species. In some embodiments, the beta-lyase gene is from Aspergillus saccharolyticus (e.g., A. saccharolyticus strain JOP 1030-1). The amino acid sequence of a wild-type TnaA homolog from A. saccharolyticus is provided by accession number XP_025427068.1 and has 44% full-length sequence identity with TnaA from C. amalonaticus (SEQ ID NO: 1).

[0042] In some embodiments, the beta-lyase gene is from an Aspergillus species. In some embodiments, the beta-lyase gene is from Trichoderma asperellum (e.g., T. asperellum strain CBS 433.97).

[0043] The amino acid sequence of the wild-type TnaA homolog from T. asperellum is provided by accession number XP_024760083.1 and shares 38% full-length sequence identity with TnaA from C. amalonaticus (SEQ ID NO: 1). The amino acid sequence of the beta-lyase from T. asperellum (SEQ ID NO: 3) contains a tyrosine (Y) at the position corresponding to H463 of TnaA (SEQ ID NO: 1).

[0044] MLPDCHLPETWRAKMVERIPSSTKDQRQEWICKADYNLFKLRSNEVRFDLGTDGGSGGMSDNQWSALMRGDSAATRSPSSYRLQEKVKELFGFTYTIPVHRGRAAKHALVQALLNEE SIVPGNAFDTTRANIESQKAIAIDCAIEGAFDIYYQHPFKGNVNLPELEKILQGSGSNVPMIMVSITCDKTGGQPVSMHNLREVKRLAKMFNVPVILDSARFAENAWFIQKNESEY SSQSIPDIVQEMYHHADGMVMSGKTDGLVNAGGFFATNNKDLFDRVGKYANLFCGLAGRDMEALTVGLGEVTQQEYLDDRIRQIHRFGMRLMAANVPIQQPIGGHAIVIDASLFLPLVPREEYVAKTLAVELYVEAGIRGAGMETVIGGGNPITGINRNRSNAKDFLYLAIPRQAYTNDQLSFVANALIQIFERRFTITRGLYVVHEDAILRYLTIQLKKADGKSIA (SEQ ID NO: 3)

[0045] Amino acids of beta-lyase may be modified (e.g., substituted) to produce beta-lyase variants. For example, as described herein, mutating the amino acid at position 463, designated histidine 463 (H463) of SEQ ID NO: 1, produced a beta-lyase enzyme with desired activity. In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO: 1 is substituted with an amino acid that is not a histidine residue (e.g., any other amino acid). In some embodiments, the amino acid corresponding to histidine at position 463 (H463) of SEQ ID NO: 1 is substituted with an amino acid selected from alanine (A), arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (G), cysteine ​​(C), glycine (G), proline (P), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), or tryptophan (W).

[0046] In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO:1 is substituted with a hydrophobic amino acid (e.g., alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), tryptophan (W)). In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO:1 is substituted with a phenylalanine (F), arginine (R), glutamic acid (E), threonine (T), glycine (G), isoleucine (I), or valine (V). In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO:1 is substituted with a phenylalanine (F) residue (H463F), as provided by SEQ ID NO:2. In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO:1 is substituted with an arginine (R) residue (H463R). In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO:1 is substituted with a glutamic acid (E) residue (H463E). In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO:1 is substituted with a threonine (T) residue (H463T). In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO:1 is substituted with a glycine (G) residue (H463G). In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO:1 is substituted with an isoleucine (I) residue (H463I). In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO: 1 is substituted with a valine (V) residue (H463V).

[0047] In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO: 1 is substituted with a phenylalanine (F) residue (H463F), as provided by SEQ ID NO: 4. In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO: 1 is substituted with a phenylalanine (F) residue (H463F), as provided by SEQ ID NO: 5.

[0048] Amino acid sequence of TnaA from C. amalonaticus - H463F substitution mutation (Y502) MDNFKHLPEPFRIRVIEPVKRTTREHRNNAIIKSGMNPFLLDSEDVFIDLLTDSGTGAVTQNMQAAMLRGDEAYSGSRSYYALSEAVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKR EQEKGLDRSKMAVFSNYFFDTTQGHSQINGCAVRNVYIKEAFDTGVRYDFKGNFDLDGLERGIQEVGPNNNVPYIVATITSNSAGGQPVSLANLKAMYNIAKKYDIPVVMDSARFAENA YFIQKREAEYRDWSIEEITRETYKYADMLAMSAKKDAMVPMGGLLCIKDDTYFDVYTECRTLCVVQEGFPTYGGLEGGAMERLAVGLVDGMNQDWLAYRIAQVQYLVDGLEAIGVTCQ QAGGHAAFVDAGKLLPHIPAEQFPAQALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRLTIPRATYTQSHMDFIIEAFEHVKENSMNIKGLTFTYEPKVLRFFTAKLKEV (SEQ ID NO: 2)

[0049] The amino acid sequence of the TnaA homolog from Z. gangwensis is provided by accession number WP_094789495.1 -H463F substitution mutation MNNFKHLPEPFRIRVVEPVKRTTLAYREKAILNAGMNPFLLDSKDVFIDLLTDSGTGAITQEMQAAMFIGDEAYSGSRSYYALADAVKDIFGYEYTIPTHQGRGAEQIYIPVLIKKREKE KGLDRTKMVALSNYFFDTTQGHTQLNACVAKNVFTKEAFDTSISADFKGNFDLELLEHAILEAGPQNVPYIVSTITCNSAGGQPVSIANLKAVYEIAQRYEIPVIMDSARFAENAYFIQQ REPEYQDWSIEAITFESYKYADALAMSAKKDAMVQMGGLLCFKDKSMLDVYNECRTLCVVQEGFPTYGGLEGGAMERLAVGLYDGMRQDWLAYRINQVQYLVNGLESIGIVCQQAGGHAAFVDAGKLLPHIPADQFPAHALACELYKVAGIRAVEIGSLLLGRDPTTGKQHPCPAELLRLTIPRATYTQTHMDFIIEAFEKVKENASHVKGLTFTYEPEVLRFFTARLKEVEN (SEQ ID NO: 4)

[0050] The amino acid sequence of the TnaA homologue from A. saccharolyticus is provided by accession number XP_025427068.1. The -H463F substitution mutation MPNTATPETWRVKTVEHIRPSTRDQRQQWIEEAGFNLFTLPSDRVFIDLLTDSGTGAMSDRQWAAIMSGDESYAGSTSFHALHEVVQDLFGLEYLLPVHQGRAAENALFSVLVHED QLVPANSHFDTTRAHIEFRKAAAVDCLSSGAYDVTDTNPFKGNMNLDMLRDILQESHARVPFILLTITCNTTGGQPVSLANIAAVKALADRYHKPLVVDAARFAENAWFIQQREPGY RDTSLRDITRQMLGMADAMVMSAKKDGLVNIGGFLATRHREWFDQATEYVILFEGFRTYGGLAGRDLAALAVGLEEVISADYLASRIGQVQRFGQRLIDAGVPIQQPVGGHAVLVDASRFLPEVPREEYVAQTLAVELYLEAGVRGVEIGTLLNGRDPESGEERFAETEWLRLAIPRRVYSNDHLEYVAQALIDLYHRRSEIRAGVRIVEEKPVLRFFTVRLERKTE (SEQ ID NO: 5)

[0051] The amino acid sequence of the TnaA homologue from Z. gangwensis is provided by accession number WP_094789495.1 - wild-type sequence MNNFKHLPEPFRIRVVEPVKRTTLAYREKAILNAGMNPFLLDSKDVFIDLLTDSGTGAITQEMQAAMFIGDEAYSGSRSYYALADAVKDIFGYEYTIPTHQGRGAEQIYIPVLIKKREKE KGLDRTKMVALSNYFFDTTQGHTQLNACVAKNVFTKEAFDTSISADFKGNFDLELLEHAILEAGPQNVPYIVSTITCNSAGGQPVSIANLKAVYEIAQRYEIPVIMDSARFAENAYFIQQ REPEYQDWSIEAITFESYKYADALAMSAKKDAMVQMGGLLCFKDKSMLDVYNECRTLCVVQEGFPTYGGLEGGAMERLAVGLYDGMRQDWLAYRINQVQYLVNGLESIGIVCQQAGGHAAFVDAGKLLPHIPADQFPAHALACELYKVAGIRAVEIGSLLLGRDPTTGKQHPCPAELLRLTIPRATYTQTHMDFIIEAFEKVKENASHVKGLTFTYEPEVLRHFTARLKEVEN (SEQ ID NO: 6)

[0052] The amino acid sequence of the TnaA homologue from A. saccharolyticus is provided by accession number XP_025427068.1 - wild-type sequence MPNTATPETWRVKTVEHIRPSTRDQRQQWIEEAGFNLFTLPSDRVFIDLLTDSGTGAMSDRQWAAIMSGDESYAGSTSFHALHEVVQDLFGLEYLLPVHQGRAAENALFSVLVHED QLVPANSHFDTTRAHIEFRKAAAVDCLSSGAYDVTDTNPFKGNMNLDMLRDILQESHARVPFILLTITCNTTGGQPVSLANIAAVKALADRYHKPLVVDAARFAENAWFIQQREPGY RDTSLRDITRQMLGMADAMVMSAKKDGLVNIGGFLATRHREWFDQATEYVILFEGFRTYGGLAGRDLAALAVGLEEVISADYLASRIGQVQRFGQRLIDAGVPIQQPVGGHAVLVDASRFLPEVPREEYVAQTLAVELYLEAGVRGVEIGTLLNGRDPESGEERFAETEWLRLAIPRRVYSNDHLEYVAQALIDLYHRRSEIRAGVRIVEEKPVLRHFTVRLERKTE (SEQ ID NO: 7)

[0053] In some embodiments, the enzyme comprises the amino acid sequence of any one of SEQ ID NOs: 1-7, wherein the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO: 1 is substituted with an amino acid that is not a histidine residue (e.g., any other amino acid). In some embodiments, the enzyme comprises the amino acid sequence of any one of SEQ ID NOs: 1-7, wherein the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO: 1 is substituted with an amino acid selected from alanine (A), arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (G), cysteine ​​(C), glycine (G), proline (P), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), or tryptophan (W).

[0054] In some embodiments, the enzyme comprises the amino acid sequence of any one of SEQ ID NOs: 1-7, wherein the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO: 1 is substituted with a hydrophobic amino acid (e.g., alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), tryptophan (W)). In some embodiments, the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO: 1 is substituted with phenylalanine (F), arginine (R), glutamic acid (E), threonine (T), glycine (G), isoleucine (I), or valine (V).

[0055] In some embodiments, the heterologous gene encodes an enzyme with beta-lyase activity such that cells expressing the enzyme with beta-lyase activity are capable of producing increased levels of volatile thiols compared to cells not expressing the heterologous gene. In some embodiments, the heterologous gene encodes an enzyme with beta-lyase activity such that cells expressing the enzyme with beta-lyase activity are capable of producing increased levels of volatile thiols compared to cells expressing an enzyme with wild-type beta-lyase activity. In some embodiments, the enzyme with beta-lyase activity capable of producing increased levels of volatile thiols contains an amino acid substitution at the position corresponding to histidine at position 463 (H463) of SEQ ID NO:1. In some embodiments, the enzyme with beta-lyase activity capable of producing increased levels of volatile thiols has a sequence provided by any one of SEQ ID NOs:2-5.

[0056] In some embodiments, the mutant beta-lyase produces increased potency / levels of volatile thiols. In some embodiments, the mutant beta-lyase produces increased potency / levels of 3MH. In some embodiments, the mutant beta-lyase produces increased potency / levels of 3MHA. In some embodiments, the mutant beta-lyase produces increased potency / levels of 4MMP. In some embodiments, the mutant beta-lyase produces increased potency / levels of one or more volatile thiols, such as 3MH, 3MHA, and / or 4MMP.

[0057] In some embodiments, the heterologous gene encodes an enzyme with beta-lyase activity and reduced tryptophanase activity, hi some embodiments, the heterologous gene encodes an enzyme with beta-lyase activity, such that the enzyme with beta-lyase activity produces increased concentrations of volatile thiols compared to an enzyme with wild-type beta-lyase activity and reduced tryptophanase activity.

[0058] In some embodiments, the enzyme with beta-lyase 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 a sequence as set forth in any one of SEQ ID NOs:1-7. In some embodiments, the enzyme with beta-lyase 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 a sequence as set forth in any one of SEQ ID NOs:1-7, wherein the amino acid corresponding to the histidine at position 463 (H463) of SEQ ID NO:1 is substituted with an amino acid that is not a histidine residue (e.g., any other amino acid). In some embodiments, the enzyme with beta-lyase 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 a sequence as set forth in any one of SEQ ID NOs: 1-7, and is substituted with an amino acid selected from alanine (A), arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (G), cysteine ​​(C), glycine (G), proline (P), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y), or tryptophan (W).

[0059] The terms "percent identity," "sequence identity," "% identity," "% sequence identity," and "% identical," which may be used interchangeably herein, refer to a quantitative measurement of similarity between two sequences (e.g., nucleic acids or amino acids). Percent identity can be determined using the algorithm 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 an algorithm has been 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 molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0060] When a percent identity or range (e.g., at least, more, etc.) is specified, unless otherwise specified, the endpoints are intended to be encompassed, and a range (e.g., at least 70% identity) includes all ranges within the recited 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%, and the like). "At least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity" is intended to encompass 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., one-tenth of a percent (i.e., 0.1%), one-hundredth of a percent (i.e., 0.01%), etc.).

[0061] In some embodiments, the enzyme with beta-lyase activity comprises the sequence as represented by amino acid SEQ ID NO:2. In some embodiments, the enzyme with beta-lyase activity consists of the sequence as represented by amino acid SEQ ID NO:2. In some embodiments, the enzyme with beta-lyase activity comprises the sequence as represented by amino acid SEQ ID NO:3. In some embodiments, the enzyme with beta-lyase activity consists of the sequence as represented by amino acid SEQ ID NO:3. In some embodiments, the enzyme with beta-lyase activity comprises the sequence as represented by amino acid SEQ ID NO:4. In some embodiments, the enzyme with beta-lyase activity consists of the sequence as represented by amino acid SEQ ID NO:4. In some embodiments, the enzyme with beta-lyase activity comprises the sequence as represented by amino acid SEQ ID NO:5. In some embodiments, the enzyme with beta-lyase activity consists of the sequence as represented by amino acid SEQ ID NO:5.

[0062] In some embodiments, the gene encoding the enzyme with beta-lyase activity comprises a nucleic acid sequence encoding an enzyme comprising an amino acid sequence having at least 80% (e.g., 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 to a sequence as set forth in any one of SEQ ID NOs:2-5. In some embodiments, the gene encoding the enzyme with beta-lyase activity comprises a nucleic acid sequence encoding an enzyme comprising an amino acid sequence as set forth in SEQ ID NO:2. In some embodiments, the gene encoding an enzyme with beta-lyase activity comprises a nucleic acid sequence encoding an enzyme consisting of an amino acid sequence as set forth in any one of SEQ ID NOs: 2-5.

[0063] Identification of additional enzymes having or predicted to have beta-lyase activity may be performed based on similarity or homology to one or more domains of a beta-lyase, such as, for example, the beta-lyases provided by any one of SEQ ID NOS: 1-7. In some embodiments, enzymes for use in the modified cells and methods described herein may be identified based on similarity or homology to an activity domain, such as a catalytic domain, such as a catalytic domain associated with beta-lyase 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 to a reference beta-lyase (e.g., a wild-type beta-lyase, such as SEQ ID NOS: 1), based on analysis over most or the full length of the enzyme, but a relatively low level of sequence identity to the reference beta-lyase in the region of the catalytic domain. In some embodiments, an 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 compared to a reference beta-lyase (e.g., SEQ ID NO: 1).

[0064] In some embodiments, the enzymes for use in the modified cells and methods described herein have a higher or lower nucleotide sequence than a reference beta-lyase (e.g., SEQ ID NO: 1, 3, 6, or 7) based on analysis of a majority of the enzyme or the full length of the enzyme. It has a relatively high level of sequence identity in the region of the catalytic domain of the enzyme and a relatively low level of sequence identity to the reference beta-lyase. In some embodiments, an enzyme for use in the modified cells and methods described herein has 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 to a reference beta-lyase (e.g., SEQ ID NO: 1, 3, 6 or 7) based on analysis over a majority or the full length of the enzyme.

[0065] In some embodiments, the amino acid substitution(s) may be in the active site. As used herein, the term "active site" refers to the region of an enzyme with which a substrate interacts. The amino acids comprising and surrounding the active site, including the functional groups of each amino acid, may contribute to the size, shape, and / or substrate accessibility of the active site. In some embodiments, a beta-lyase variant contains one or more modifications that are substitutions of selected amino acids with amino acids having different functional groups.

[0066] This information can also be used to identify positions, e.g., corresponding positions, in other enzymes that have or are predicted to have beta-lyase activity. As will be apparent to one of skill in the art, an amino acid substitution at a position identified in one beta-lyase enzyme can also be made at the corresponding amino acid position in another beta-lyase enzyme. In such cases, one of the beta-lyase enzymes may be used as a reference enzyme. For example, as described herein, an amino acid substitution at position H463 in TnaA from Citrobacter amalonaticus has been shown to increase the production of volatile thiols and reduce the production of indole. Similar amino acid substitutions can be made at corresponding positions in other enzymes with beta-lyase activity, using TnaA as a reference (e.g., SEQ ID NO: 1). For example, amino acid substitutions can be made at corresponding positions in beta-lyases from Z. gangwensis or A. saccharolyticus, as described herein, using TnaA as a reference (e.g., SEQ ID NO: 1). In some embodiments, the amino acid in another enzyme (e.g., beta-lyase from T. asperellum) at a position corresponding to position H463 of TnaA from C. amalonaticus (SEQ ID NO: 1) is not histidine. See, e.g., SEQ ID NO: 3.

[0067] As will also be apparent to one of skill in the art, the amino acid position number of a selected residue in a beta-lyase may have a different amino acid position number in another beta-lyase enzyme (e.g., a reference enzyme). Generally, methods known in the art may be used to identify corresponding positions in other beta-lyase enzymes, 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)).

[0068] The beta-lyase variants described herein may further contain one or more additional modifications that, for example, specifically alter a feature of the polypeptide unrelated to its desired physiological activity. Alternatively or in addition, the beta-lyase variants described herein may contain one or more additional mutations that modulate the expression of the enzyme in the cell.

[0069] Mutations in the nucleic acid encoding the beta-lyase preferably preserve the amino acid reading frame of the coding sequence and preferably do not create regions in the nucleic acid that are likely to hybridize to form secondary structures such as hairpins or loops that may be detrimental to expression of the enzyme.

[0070] Mutations can be made by selecting amino acid substitutions in the nucleic acid encoding the polypeptide or by random mutagenesis of selected sites. As described herein, the variant polypeptide can be expressed and tested for one or more activities to determine whether the mutations provide a variant polypeptide with desired properties. Additional mutations can be made to the variant (or to the non-variant polypeptide) that are silent with respect to the amino acid sequence of the polypeptide but provide preferred codons for translation in a particular host (referred to as codon optimization). Preferred codons for translation of nucleic acids (e.g., nucleic acids in S. cerevisiae) are well known to those of skill in the art. Still other mutations can be made in non-coding sequences of a gene clone or cDNA clone to enhance polypeptide expression. The activity of a beta-lyase variant can be tested by cloning the gene encoding the beta-lyase variant into an expression vector, introducing the vector into a suitable host cell, expressing the beta-lyase variant, and testing for functional capability of the beta-lyase as disclosed herein.

[0071] The beta-lyase variants described herein contain amino acid substitutions at one or more positions relative to the reference beta-lyase. In some embodiments, the beta-lyase variants contain amino acid substitutions at 1, 2, 3, 4, 5, or more positions relative to the reference beta-lyase. In some embodiments, the beta-lyase is a non-naturally occurring beta-lyase, e.g., genetically modified. In some embodiments, the beta-lyase does not have the amino acid sequence provided by SEQ ID NO: 1. In some embodiments, the beta-lyase does not have the amino acid sequence provided by SEQ ID NO: 3. In some embodiments, the beta-lyase does not have the amino acid sequence provided by SEQ ID NO: 6. In some embodiments, the beta-lyase does not have the amino acid sequence provided by SEQ ID NO: 7.

[0072] In some embodiments, beta-lyase variants may also contain one or more amino acid substitutions that do not substantially affect the activity and / or structure of the beta-lyase enzyme. Those skilled in the art will also recognize that conservative amino acid substitutions may be made in beta-lyase variants to provide functionally equivalent variants of the polypeptide, i.e., variants that retain the functional capabilities of the polypeptide. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not alter characteristics such as the relative charge or size of the protein in which the amino acid substitution is made. Variants may be prepared according to methods for altering polypeptide sequences known to those skilled in the art, such as those found in references compiling 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.A.usubel, 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 the proteins disclosed herein. Conservative amino acid substitutions include substitutions made from among 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.

[0073] As one skilled in the art will appreciate, homologous genes encoding enzymes having beta-lyase activity can be obtained from other species and identified by homology searches, for example, through protein BLAST searches 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 comparing the secondary or tertiary structure of a similar or homologous enzyme with one or more reference beta-lyases, corresponding amino acid residues in the similar or homologous enzymes can be determined, and amino acid residues for mutation in the similar or homologous enzymes can be determined.

[0074] The genes related to the present disclosure can be obtained from DNA (e.g., by PCR amplification) from any source of DNA containing the desired gene. In some embodiments, the genes related to the present invention are synthetic, e.g., produced in vitro by chemical synthesis. Any means of obtaining genes encoding the enzymes described herein is compatible with the modified cells and methods described herein.

[0075] The disclosure provided herein involves the recombinant expression of genes encoding enzymes having beta-lyase activity, functional modifications and variants of the enzymes, and uses related thereto. Homologs and alleles of the nucleic acids related to the present invention can be identified by conventional techniques. Also encompassed by the present invention are nucleic acids that hybridize to the nucleic acids described herein under stringent conditions. The term "stringent conditions," as used herein, refers to parameters well known in the art. Nucleic acid hybridization parameters may be found in references compiling 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.A.usubel, et al., eds., John Wiley & Sons, Inc., New York.

[0076] Other conditions, reagents, etc. can be used, but will result in a similar degree of stringency. Those skilled in the art are familiar with such conditions, and therefore will not be given here. However, it is understood that those skilled in the art will be able to manipulate the conditions (for example, by using lower stringency conditions) in a manner that allows for the unambiguous identification of homologs and alleles of the nucleic acids of the invention. Those skilled in the art are also familiar with methodologies for screening cells and libraries for the expression of such molecules, which are then routinely isolated, followed by the isolation and sequencing of the relevant nucleic acid molecules.

[0077] The present invention also encompasses degenerate nucleic acids, which include codons that substitute for those present in the native material. 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 purpose of encoding a serine residue. Thus, it will be apparent to those skilled in the art that any of the nucleotide triplets encoding serine may be employed to direct the protein synthesis machinery to incorporate the serine residue into a growing polypeptide in vitro or in vivo. Similarly, nucleotide sequence triplets encoding other amino acid residues include, but are not limited to, the following: CCA, CCC, CCG, and CCT (proline codons); CGA, CGC, CGG, CGT, AGA, and AGG (arginine codons); ACA, ACC, ACG, and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC, and ATT (isoleucine codons). Other amino acid residues may similarly be encoded by multiple nucleotide sequences. Thus, the present invention encompasses degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code. The present invention also encompasses codon optimization to match optimal codon usage in the host cell.

[0078] The present invention also provides modified nucleic acid molecules that 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 (such as enzymatic activity) of the unmodified nucleic acid molecule and / or polypeptide. In some embodiments, the modified nucleic acid molecule encodes a modified polypeptide, preferably a polypeptide with conservative amino acid substitutions as described elsewhere herein. The modified nucleic acid molecule is structurally related to the unmodified nucleic acid molecule, and in preferred embodiments, is sufficiently structurally related to the unmodified nucleic acid molecule, such that the modified and unmodified nucleic acid molecules can hybridize under stringent conditions known to those of skill in the art.

[0079] For example, modified nucleic acid molecules encoding polypeptides with a single amino acid change can be prepared. Each of these nucleic acid molecules can have one, two, or three nucleotide substitutions, excluding nucleotide changes corresponding to the degeneracy of the genetic code as described herein. Similarly, modified nucleic acid molecules encoding polypeptides with two amino acid changes (e.g., having two to six nucleotide changes) can also be prepared. Numerous modified nucleic acid molecules such as these (e.g., including nucleotide substitutions in codons encoding two and three, two and four, two and five, two and six, etc. amino acids) will be readily envisioned by those skilled in the art. In the above example, each combination of two amino acids is encompassed in the series of modified nucleic acid molecules, as well as all nucleotide substitutions encoding amino acid substitutions. Additional nucleic acid molecules encoding polypeptides with additional substitutions (i.e., three or more), additions, or deletions (e.g., by introducing a stop codon or splice site(s)) can also be prepared and are encompassed by the present invention, as will be readily envisioned by those skilled in the art. Any of the above nucleic acids or polypeptides can be tested by routine experimentation for retention of structural relationship or activity with the nucleic acids and / or polypeptides disclosed herein.

[0080] In some embodiments, one or more of the genes related to the present invention are expressed in a recombinant expression vector. As used herein, a "vector" refers to any of a number of nucleic acids into which a desired sequence(s) may be inserted by restriction and ligation for transport between different production 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, the following: plasmids, fosmids, phagemids, viral genomes, and artificial chromosomes.

[0081] A cloning vector is a vector that can replicate autonomously or that integrates into the genome of a host cell. In the case of a plasmid, replication of the desired sequence may occur multiple times as the plasmid increases in copy number within the host cell (such as a host bacterium), or only once per host before the host reproduces by mitosis. In the case of a phage, replication may occur actively during the lytic phase or passively during the lysogenic phase.

[0082] An expression vector is a vector into which a desired DNA sequence may be inserted such that it is operably linked to regulatory sequences by restriction and ligation and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in identifying cells that have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes encoding enzymes whose activities can be detected by standard assays known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase), and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). Preferred vectors are capable of autonomous replication and expression of structural gene products present in the DNA segment to which they are operably linked.

[0083] As used herein, a coding sequence and a regulatory sequence are said to be "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. When it is desired that the coding sequence be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequences results in 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 frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into protein. Thus, a promoter region would be operably linked to a coding sequence if it were capable of effecting transcription of that DNA sequence, such that the resulting transcript could be translated into the desired protein or polypeptide.

[0084] When a nucleic acid molecule encoding any of the enzymes of the claimed invention is expressed in a cell, various transcription control sequences (e.g., promoter / enhancer sequences) can be used to direct its expression. The promoter can be the 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 not regulated to allow continuous transcription of its associated gene (e.g., an enzyme having beta-lyase activity). Various conditional promoters can also be used, such as promoters controlled by the presence or absence of a molecule.

[0085] The precise nature of regulatory sequences required for gene expression may vary between species or cell types, but generally include, as necessary, 5' non-transcribed and 5' non-translated sequences involved in initiation of transcription and translation, respectively, such as the TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed regulatory sequences will include a promoter region containing a promoter sequence for transcriptional control of an operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences, as desired. Vectors of the invention may optionally include 5' leader or signal sequences. The selection and design of an appropriate vector is within the ability and discretion of one skilled in the art.

[0086] Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012. Cells are genetically modified by the introduction of heterologous DNA (RNA) into the cell. The heterologous DNA (RNA) is placed under operable control of transcriptional elements, allowing expression of the heterologous DNA in the host cell. For example, heterologous expression of a gene encoding an enzyme with beta-lyase activity in genetically modified yeast cells in a method for producing a fermented beverage (such as beer) has been demonstrated in an example using S. cerevisiae strain WLP001. As will be appreciated by those skilled in the art, any of the enzymes described herein can also be expressed in other yeast cells, including yeast strains used to produce wine, mead, sake, apple cider, etc.

[0087] Nucleic acid molecules encoding the enzymes of the claimed invention can be introduced into a cell or cells using methods and techniques standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as chemical transformation and transformation, including electroporation, transduction, biolistics, etc. Expressing nucleic acid molecules encoding the enzymes of the claimed invention may also be achieved by integrating the nucleic acid molecules into the genome.

[0088] Integration of heterologous genes can be achieved 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, permanent, heritable genetic changes are generally achieved by introduction of DNA into the genome of the cell.

[0089] A heterologous gene may also include various transcription elements required for expression of the encoded gene product (e.g., an enzyme having beta-lyase activity). For example, in some embodiments, a heterologous gene may include a promoter. In some embodiments, the promoter may be operably linked to the heterologous gene. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is active during a specific stage of the fermentation process. In some embodiments, the promoter is a constitutive promoter. Examples of constitutive promoters for use in yeast cells are known in the art and will be apparent to those of 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 heterologous gene is expressed. In some examples, the promoter is a PKG1 promoter (pPGK1) or a HHF2 promoter (pHHF2).

[0090] Genetically modified yeast cells Aspects of the present disclosure relate to genetically modified yeast cells (modified cells) and the use of such modified cells in methods for producing fermentation products (e.g., fermented beverages) and methods for producing ethanol. The genetically modified yeast cells described herein are genetically modified with a heterologous gene encoding an enzyme with beta-lyase activity.

[0091] The terms "genetically modified cell," "genetically modified yeast cell," and "modified cell," which may be used interchangeably herein, refer to eukaryotic cells (e.g., yeast cells that have been or will soon be modified by the introduction of a heterologous gene). These terms (e.g., modified cell) encompass the progeny of an original cell that has been genetically modified by the introduction of a heterologous gene. It will be understood by those skilled in the art that the progeny of a single cell may not necessarily be completely identical in morphology or genomic or total nucleic acid complement to the original parent due to mutations (i.e., natural, accidental, or deliberate alterations of the nucleic acid of the modified cell).

[0092] Yeast cells for use in the methods described herein are preferably capable of fermenting a sugar source (e.g., fermentable sugars) to produce ethanol (ethyl alcohol) and carbon dioxide. In some embodiments, the yeast cells are of the genus Saccharomyces. The genus Saccharomyces encompasses approximately 500 distinct species, many of which are used in food production. One exemplary 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, and beer, among other products. Other members of the Saccharomyces genus include, but are not limited to, the wild yeast Saccharomyces paradoxus, which is closely related to S. cerevisiae; Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces uvarum, Saccharomyces cerevisiae var boulardii, and Saccharomyces eubayanus. In some embodiments, the yeast is Saccharomyces cerevisiae (S. cerevisiae).

[0093] 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., having or containing more than two sets of homologous chromosomes). Saccharomyces species used (e.g., for beer brewing) are typically classified into two groups: top-fermenting ale strains (e.g., S. cerevisiae) and bottom-fermenting lager strains (e.g., S. pastorianus, S. carlsbergensis, S. uvarum). These characterizations reflect their separation characteristics in open-top square fermenters, as well as often other characteristics such as preferred fermentation temperatures and achieved alcohol concentrations.

[0094] While beer brewing and wine production have traditionally focused on the use of S. cerevisiae strains, other yeast genera are also appreciated in the production of fermented beverages. In some embodiments, the yeast cell belongs to a non-Saccharomyces genus. See, for example, 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, Dekkera (also called Brettanomyces), Wickerhamomyces, or Torulaspora. Examples of non-Saccharomyces yeasts include, but are not limited to, 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.

[0095] In some embodiments, the methods described herein involve the use of more than one genetically modified yeast. For example, in some embodiments, the methods may involve the use of more than one genetically modified yeast belonging to the genus Saccharomyces. In some embodiments, the methods may involve the use of more than one genetically modified yeast belonging to a non-Saccharomyces genus. In some embodiments, the methods may involve the 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 additionally, any of the methods described herein may involve the use of one or more genetically modified yeast and one or more genetically unmodified (wild-type) yeast.

[0096] In some embodiments, the yeast is a hybrid strain. As will be apparent to those skilled in the art, the term "hybrid strain" of yeast refers to a yeast strain resulting from the mating of two different yeast strains, for example, to obtain one or more desired characteristics. For example, a hybrid strain may be the result of mating two different yeast strains belonging to the same genus or species. In some embodiments, a hybrid strain is the result of mating a Saccharomyces cerevisiae strain with a Saccharomyces eubayanus strain. For example, see Krogerus et al. Microbial Cell Factories (2017) 16:66.

[0097] In some embodiments, the yeast strain is a wild yeast strain, such as a yeast strain isolated from a natural source and subsequently propagated. Alternatively, in some embodiments, the yeast strain is a domesticated yeast strain. Environmentally adapted yeast strains have been subject to human selection and breeding for desirable characteristics.

[0098] In some embodiments, genetically modified yeast cells may be used in a symbiotic matrix with strains and used to produce fermented beverages such as kombucha, kefir, and ginger beer. For example, Saccharomyces fragilis is an integral part of kefir cultures and is grown on lactose contained in whey.

[0099] Methods for genetically modifying yeast cells are known in the art. In some embodiments, the yeast cells are diploid and one copy of a heterologous gene encoding an enzyme with beta-lyase activity as described herein is introduced into the yeast genome. In some embodiments, the yeast cells are diploid and one copy of a heterologous gene encoding an enzyme with beta-lyase 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 enzymes with the same beta-lyase activity. In some embodiments, the copies of the heterologous gene are not identical, but the genes encode different enzymes (e.g., mutants, variants, or fragments thereof) with beta-lyase activity.

[0100] In some embodiments, the yeast cell is tetraploid. A tetraploid yeast cell is a cell that maintains four complete sets of chromosomes (i.e., four copies of a complete set of chromosomes). In some embodiments, the yeast cell is tetraploid and a copy of a heterologous gene encoding an enzyme with beta-lyase activity as described herein is introduced into at least one copy of the genome. In some embodiments, the yeast cell is tetraploid and a copy of a heterologous gene encoding an enzyme with beta-lyase activity as described herein is introduced into more than one copy of the genome. In some embodiments, the yeast cell is tetraploid and a copy of a heterologous gene encoding an enzyme with beta-lyase activity as described herein is introduced into 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 gene encodes an enzyme with the same beta-lyase activity. In some embodiments, the copies of the heterologous gene are not identical, but the gene encodes a different enzyme (e.g., mutant, variant, fragment thereof) with beta-lyase activity.

[0101] Strains of yeast cells that may be used in the methods described herein will be known to those skilled in the art, and include yeast strains used to brew the desired fermented beverage, as well as commercially available yeast strains. Examples of common beer strains include, but are not limited to, 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.

[0102] Non-limiting examples of yeast strains for use in 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 CoastYeast、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 AleWLP072、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、WhiteLabs 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 Ale3655-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 Ecy19、East Coast Yeast Brett Nanus Ecy16、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 YeastBlend 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 LondonAle 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-04、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 BlendWLP060、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 Koelsch 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 / Koelsch WLP029、East Coast YeastKoelschbier 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 LabsWhitbread 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 Belg In some embodiments, the yeast is S. cerevisiae strain WLP001.

[0103] In some embodiments, the yeast strain for use in the genetically modified cells and methods described herein is a wine yeast strain. Examples of yeast strains for use in the genetically modified cells and methods described herein include, but are not limited to, Red Star Montrachet, Red Star Côte des Blancs, Red Star Premier Cuvée, Red Star Pasteur Red, Red Star Pasteur Champagne, Fermentis BCS-103, and Fermentis VR44.

[0104] method Aspects of the present disclosure relate to methods of producing fermentation products using any of the genetically modified yeast cells described herein. Also provided are methods of producing ethanol using any of the genetically modified yeast cells described herein.

[0105] The process of fermentation utilizes natural processes that use 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 microbial activity brings about a desired change in a food or beverage. The conditions and execution of fermentation are referred to herein as the "fermentation process."

[0106] In some aspects, the present disclosure relates to methods for producing a fermentation product, such as a fermented beverage, that involve contacting any of the modified cells described herein with a medium comprising at least one fermentable sugar during an initial fermentation process to produce the fermentation product (FIGS. 1A-1G). "Medium," as used herein, refers to a liquid that is conducive to fermentation and does not inhibit or prevent the fermentation process. In some embodiments, the medium is water. In some embodiments, the method for producing a fermentation product involves contacting a purified enzyme (e.g., any of the beta-lyase enzymes described herein) with a medium comprising at least one fermentable sugar during an initial fermentation process to produce the fermentation product (FIG. 1H).

[0107] As also used herein, the term "fermentable sugar" refers to a carbohydrate that can be converted by a microorganism, such as any of the cells described herein, to alcohol and carbon dioxide. In some embodiments, the fermentable sugar is converted by an enzyme, such as a recombinant enzyme, or a cell expressing the enzyme, to alcohol and carbon dioxide. Examples of fermentable sugars include, but are not limited to, glucose, fructose, lactose, sucrose, maltose, and maltotriose.

[0108] In some embodiments, the fermentable sugars are provided in a sugar source. The sugar source for use in the claimed methods can depend, for example, on the type of fermentation product and fermentable sugar. Examples of sugar sources include, but are not limited to, malt, grains / cereals, fruit juices (e.g., grape juice, apple juice / cider), honey, cane sugar, rice, and koji.

[0109] As will be apparent to those skilled in the art, in some cases, it may be necessary to process the sugar source to make fermentable sugars available for fermentation. Using the production of beer as an example of a fermented beverage, grains (cereals, barley) are boiled or steeped in water, which hydrates the grain and activates malt enzymes, converting starch to fermentable sugars, a process known as "mashing." As used herein, the term "wort" refers to the liquid produced in the saccharification process and containing fermentable sugars. The wort is then exposed to a fermenting organism (e.g., any of the cells described herein), allowing the 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 the organism that produces the enzyme, allowing the enzyme to convert the sugars in the wort to alcohol and carbon dioxide.

[0110] 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, but are not limited to, barley, oats, corn, rice, rye, sorghum, wheat, oats, and pearl barley.

[0111] In the example of producing sake, the sugar source is rice, and the rice is incubated with Aspergillus oryzae to convert the rice starch into fermentable sugars, producing koji. The koji is then exposed to a fermenting organism (e.g., any of the cells described herein), allowing enzymes in 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), optionally purified or isolated from the organism that produces the enzyme, allowing the enzyme to convert the sugars in the koji to alcohol and carbon dioxide.

[0112] In the example of producing wine, grapes are harvested and mashed (e.g., crushed) into a composition containing skins, pulp, juice, and seeds. The resulting composition is referred to as "must." The grape juice may be separated from the must and fermented, or the entire must (i.e., skins, seeds, and pulp) may be fermented. The grape juice or must is then exposed to a fermenting organism (e.g., any of the cells described herein), allowing enzymes in the fermenting organism to convert sugars in the grape juice or must into 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), optionally purified or isolated from the organism that produces the enzyme, allowing the enzyme to convert sugars in the grape juice or must into alcohol and carbon dioxide.

[0113] In some embodiments, the methods described herein involve producing a medium, which may involve heating or soaking a sugar source, for example, in water. In some embodiments, the water has a temperature of at least 50 degrees Celsius (50°C) and is incubated with the sugar source for a period of time. In some embodiments, the water has a temperature of at least 75°C and is incubated with the sugar source for a period of time. In some embodiments, the water has a temperature of at least 100°C and is incubated with the sugar source for a period of time. Preferably, the medium is cooled prior to the addition of any of the cells described herein.

[0114] In some embodiments, the methods described herein further include adding at least one precursor (e.g., plant-derived or chemically synthesized) to the culture medium or during the initial fermentation process (FIG. 1G). Examples of precursors include, but are not limited to, 3-mercaptohexan-1-ol (Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexan-1-ol conjugated to glutathione (Glut-3-MH), and / or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut 4MMP). In some embodiments, the precursor is a plant-derived precursor. In some embodiments, the precursor is a chemically synthesized precursor. Methods for producing and / or obtaining precursors are known in the art, for example, in Grant-Preece et al. J. Agric. Food Chem. (2010) 58(3): 1383-1389; Fedrizzi et al. J. Agric. Food Chem. (2009) 57(3): 991-995; Pardon et al. J. Agric. Food Chem. (2008) 56(10): 3758-3763; Howell et al. FEMS Microbiol. Lett. (2004) 240(2): 125-9.

[0115] In some embodiments, the methods described herein further include adding at least one hop variety (e.g., one, two, three, four, five, or more) to the wort, e.g., to the medium, during the fermentation process. Hops are the flowers of the hop plant (Humulus lupulus) and are often used in fermentation to impart various flavors and aromas to the fermentation product. Hops are considered to impart bitter flavoring agents in addition to floral, fruity, and / or citrus flavors and aromas, and may be characterized based on their intended purpose. For example, bittering hops impart a certain level of bitterness to the fermentation product due to the presence of alpha acids in the hop flowers, while aromatic hops have lower levels of alpha acids and contribute desirable aromas and flavors to the fermentation product.

[0116] Whether one or more varieties of hops are added to the medium and / or wort, and at what stage the hops are added, may depend on various factors, such as the intended purpose of the hops. For example, hops intended to impart bitterness to the fermentation product are typically added during wort preparation, e.g., during the boiling of the wort. In some embodiments, hops intended to impart bitterness to the fermentation product are added to the wort and boiled with the wort for a period of time, e.g., about 15 to 60 minutes. In contrast, hops intended to impart a desired aroma to the fermentation product are typically added after the hops used for bittering. In some embodiments, hops intended to impart a desired aroma to the fermentation product are added at the end of the boil or after the wort has been boiled (i.e., "dry hopping"). In some embodiments, one or more varieties of hops may be added multiple times during the process (e.g., at least two times, at least three times, or more times).

[0117] In some embodiments, hops are added in the form of either wet hops or dried hops, and may optionally be boiled with the wort. In some embodiments, 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, hops are wet (i.e., not dried). In some embodiments, hops are dry and may optionally be further processed prior to use. In some embodiments, hops are added to the wort prior to the fermentation process. In some embodiments, hops are boiled in the wort. In some embodiments, hops are boiled with the wort and then cooled with the wort.

[0118] Many varieties of hops are known in the art and may be used in the methods described herein. Examples of hop varieties include, but are not limited to, 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, and 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, Strisselspalt, 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, HallertauerTaurus, Merkur, Opal, Smaragd, Halleratau Aroma, Kohatu, Rakau, Stella, Sticklebract, Summer Saaz, Super Alpha, Super Pride, Topaz, Wai-iti, Bor, Junga, Marynka, Premium, Sladek, Styrian Atlas, Styrian Aurora, Styrian Bobek, Styrian Celeia, Sybilla Sorachi Includes Ace, Hallertauer Mittelfrueh, Hallertauer Tradition, Tettnanger, Tahoma, Triple Pearl, Yahima Gold, and Michigan Copper.

[0119] 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 30 days. In some embodiments, the fermentation process is carried out 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, or longer. In some embodiments, the fermentation process of one or more fermentable sugars may be carried out 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 carried out 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.

[0120] The methods described herein may involve at least one additional fermentation process, for example, as shown in Figure ID. Such an additional fermentation process may be referred to as a secondary fermentation process (also referred to as "aging" or "maturing"). As will be understood by those skilled in the art, secondary fermentation typically involves transferring the fermented beverage to a second container (e.g., a glass carboy, a barrel) where the fermented beverage is incubated for a period of time. In some embodiments, the secondary fermentation is carried out for a period of between 10 minutes and 12 months. In some embodiments, the secondary fermentation is carried out for 10 minutes, 20 minutes, 40 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 ... The fermentation process may be carried out for a period of time ranging from about 4° C. to about 30° C., such as from about 8° C. to about 14° C., or from about 18° C. to about 24° C. In some embodiments, the ... In some embodiments, the fermentation process may be carried out for a period of time ranging from about 4° C. to about 30° C. In some embodiments, the fermentation process may be carried out for a period of time In some embodiments, the additional or secondary fermentation process of one or more fermentable sugars may be carried out 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.

[0121] As will be apparent to one skilled in the art, the selection of the duration and temperature for the additional or secondary fermentation process will depend on factors such as the type of beer, the desired beer characteristics, and the yeast strain used in the process.

[0122] In some embodiments, one or more additional flavor components may be added to the medium prior to or after the fermentation process. Examples include hop oil, hop aromatics, hop extract, hop bitterness, and isomerized hop extract.

[0123] Following fermentation, there may be various refinement, filtering, and aging processes before the liquid is bottled (e.g., retained 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 method involves carbonating the fermented product (e.g., as shown in FIG. 1E). Methods of carbonating fermented beverages are known in the art and include, for example, forced carbonation with a gas (e.g., carbon dioxide, nitrogen), natural carbonation by adding an additional sugar source to the fermented beverage to promote further fermentation and carbon dioxide production (e.g., bottle conditioning).

[0124] fermentation products Aspects of the present disclosure relate to a fermentation product produced by any of the methods disclosed herein. In some embodiments, the fermentation product is a fermented beverage. Examples of fermented beverages include, but are not limited to, beer, wine, sake, mead, apple cider, cava, sparkling wine (champagne), kombucha, ginger beer, and water kefir. In some embodiments, the beverage is beer. In some embodiments, the beverage is wine. In some embodiments, the beverage is sake. In some embodiments, the beverage is mead. In some embodiments, the beverage is apple cider.

[0125] In some embodiments, the fermentation product is a fermented food. Examples of fermented foods include, but are not limited to, cultured yogurt, tempeh, miso, kimchi, sauerkraut, fermented sausage, bread, and soy sauce.

[0126] According to aspects of the present invention, increased titers of volatile thiols are produced through recombinant expression of genes associated with the present invention in yeast cells and use of said cells in the methods described herein. As used herein, "increased titer" or "high titer" refers to titer on the nanograms per liter (ng L) scale. The titer produced for a given product will be influenced by multiple factors, including the selection of the medium and conditions for fermentation.

[0127] In some embodiments, the titer of the volatile thiol (e.g., 3MH, 3MHA, and / or 4MMP) is at least 100 ng L −1 . For example, the titer is at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 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, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 108 80, 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, or 3000ng It can be greater than L-1.

[0128] In some embodiments, the titer of volatile thiol is at least 1 μg L, e.g., 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, and 3000 μg L-1.

[0129] In some embodiments, the titer of volatile thiol is at least 1 mg L, e.g., 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 mg L-1 or more.

[0130] In some embodiments, the titer of the volatile thiol is limited by the amount of precursor added to the fermentation process.

[0131] Aspects of the present disclosure relate to reducing the production of undesired products (e.g., by-products, off-flavors), such as indole, during fermentation of a product. In some embodiments, expression of a beta-lyase described herein reduces the production of the undesired product (e.g., indole) by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to the production of the undesired product (e.g., indole) using a wild-type beta-lyase.

[0132] Methods for measuring the potency / level of volatile thiol and / or indole will be apparent to those skilled in the art. In some embodiments, the potency / level of volatile thiol and / or indole is measured using gas chromatography-mass spectrometry (GC / MS). In some embodiments, the potency / level of volatile thiol and / or indole is assessed using a sensory panel, including, for example, human taste testers.

[0133] In some embodiments, the fermented beverage contains between 0.1% and 30% alcohol by volume (also referred to as "ABV," "abv," or "alc / vol"). In some embodiments, the fermented beverage contains 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 more alcohol by volume. In some embodiments, the fermented beverage is non-alcoholic (eg, having less than 0.5% alcohol by volume).

[0134] kit Aspects of the present disclosure also provide kits for use of genetically modified yeast cells, e.g., to produce fermentation products or ethanol. In some embodiments, the kits contain modified cells that contain a heterologous gene encoding an enzyme with beta-lyase activity.

[0135] 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 apple wine.

[0136] The kits may also include 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 grain, water, wort, must, yeast, hops, fruit juice, or other sugar source(s). In some embodiments, the kits may contain one or more fermentable sugars. In some embodiments, the kits may contain one or more additional agents, ingredients, or components.

[0137] Instructions for practicing the methods described herein may also be included in the kits described herein.

[0138] The kits may be arranged to indicate a single-use composition containing any of the modified cells described herein. For example, the single-use composition (e.g., the amount to be used) may be a packaged composition (e.g., modified cells), such as a packeted (i.e., contained in a packet) powder, vial, ampoule, culture tube, tablet, caplet, capsule, or liquid-containing sachets.

[0139] The composition (e.g., the modified cells) may be provided in a dried, lyophilized, frozen, or liquid form. 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 seed culture that may be directly added to a medium. When a reagent or component is provided in a dried form, it is generally reconstituted 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 of skill in the art.

[0140] Numerous packages or kits for dispensing compositions (e.g., modified cells) are known to those of skill in the art. In some embodiments, the package is a labeled blister package, a dial dispenser package, a tube, a packet, a drum, or a bottle.

[0141] Any of the kits described herein may further include one or more vessels for carrying out the methods described herein, such as a carboy or barrel.

[0142] general technique The practice of the subject matter of the present 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 include, but are not limited to, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press; Animal Cell Culture(RIFreshney,ed.,1987);Introduction to Cell and Tissue Culture (JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-8)J.Wiley and Sons;Methods in Enzymology(Academic Press,Inc.);Handbook of Experimental Immunology(DMWeir and Gene Transfer Vectors for Mammalian Cells (JM Miller and MPCalos, eds., 1987); Current Protocols in Molecular Biology (FMA Musubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999), and other references.

[0143] Equivalence and Scope It is to be understood that the present disclosure is not limited to any or all of the particular embodiments expressly described herein, 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.

[0144] Unless otherwise defined, 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. Also, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described herein.

[0145] All publications and patents cited in this disclosure are cited to disclose and describe methods and / or materials related to the methods and / or materials for which the publications are cited. All such publications and patents are incorporated by reference herein as if each individual publication or patent was 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 lexical definitions from the cited publications and patents (i.e., any lexical definitions from cited publications and patents that are not expressly repeated in this disclosure should not be treated as such and should not be read as defining any terms that appear in the appended claims). In the event of a conflict between any of the incorporated references and this disclosure, the present disclosure shall control. Additionally, any specific aspects of the present disclosure that are within the prior art may be expressly excluded from any one or more of the claims. Such aspects may be excluded, even if the exclusion is not expressly expressed in the specification, as being known to those of ordinary skill in the art. Any specific aspect of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0146] 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 may be different from the actual publication dates which may need to be independently confirmed.

[0147] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that may be readily separated from or combined with any of the features of the other several embodiments without departing from the spirit or scope of the disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically feasible.

[0148] Articles such as "a," "an," and "the" in the claims may mean one or more than one, unless indicated to the contrary or otherwise clear from the context. Whenever pronouns are used herein in a gendered manner (e.g., masculine, feminine, neuter, etc.), the pronouns shall be construed as gender-neutral (i.e., interpreted as referring equally to all genders) regardless of any implied gender, unless the context clearly indicates or requires otherwise. Whenever 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. A claim or description including "or" between one or more members of a group is deemed to be satisfied when one, more than one, or all of the group members are present in, employed in, or otherwise related to a given product or process, unless indicated to the contrary or otherwise clear from the context. The present 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 present 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.

[0149] Furthermore, the present disclosure covers all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends on the same base claim. Where elements are presented as lists (e.g., in Markush group format), each subgroup of elements is also disclosed, and any element(s) can be removed from the group. In general, when the present disclosure or aspects of the disclosure are referred to as comprising specific elements and / or features, it should be understood that certain embodiments of the disclosure or aspects of the disclosure consist of, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been so specifically described herein. Note also that the terms "comprising" and "containing" are intended to be open, allowing for 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 apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges may assume that any particular value or subrange within the stated range in various aspects of the present disclosure is to one-tenth of the unit of the lower limit of the range unless the context clearly dictates otherwise.

[0150] 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 embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to this description may be made, as defined in the following claims, without departing from the spirit or scope of the present disclosure.

[0151] example Example 1 introduction Volatile thiol molecules such as 3MH, 3MHA, and 4MMP are primarily responsible for the tropical fruit flavors found in certain foods and beverages. Within the wine industry, considerable research efforts have been devoted to enhancing the biosynthesis of these volatile thiols by Saccharomyces wine yeasts during grape must fermentation. These efforts have primarily focused on increasing the efficiency of the beta-lyase-catalyzed enzymatic reactions that produce 3MH and 4MMP from their cysteine-conjugated precursors. Several groups have shown that overexpression of the endogenous yeast beta-lyases IRC7 and STR3 can enhance the production of volatile thiols during the fermentation of either grape must or synthetic grape mediza. 7,10 It has also been shown that expression of Escherichia coli (E. coli) tryptophanase / beta-lyase, TnaA, in yeast cells greatly enhances volatile thiol production during both grape must and Sauvignon Blanc must fermentations. 5,30 .

[0152] The WLP001 H463F mutation increases 3MH concentrations while inhibiting indole production We investigated whether overexpression of IRC7, STR3, or TnaA in Saccharomyces brewer's yeast would enhance volatile thiol release during beer fermentation. Each of these genes was sequentially integrated into the ADE2 locus of California Ale Yeast, WLP001. The strong constitutive promoter PGK1 was used to drive heterologous gene expression.

[0153] Beer was brewed using yeast strains overexpressing IRC7, STR3, or TnaA, as well as an unmodified WLP001 control (wild-type). Sensory analysis following fermentation indicated that beer fermented with yeast cells overexpressing STR3 or the IRC7 overexpressing strain had an odor profile comparable to the wild-type control strain. In contrast, beer fermented with yeast cells overexpressing TnaA had a distinctive, strong odor characterized as tropical / guava and excrement / diaper (i.e., off-flavors).

[0154] To quantitatively measure the concentrations of volatile thiols and other flavor molecules produced during fermentation, gas chromatography / mass spectrometry (GC / MS) analysis was performed on these beers. The analysis revealed that the concentrations of the volatile thiols 3MH, 3MHA, and 4MMP in beers brewed with the wild-type strain were extremely low, notably below the detection limit of the assay at 5 nanograms per liter (ng / L). Overexpression of STR3 or IRC7 had a negligible effect on volatile thiol production. The levels of these thiols in beers brewed with these strains were also below the detection limit (Figure 3, Y27 and Y33). In contrast, beers brewed using a yeast strain overexpressing TnaA (Y182) contained 229 ng / L of 3MH, a more than 45-fold increase compared to beers brewed with the wild-type strain (Figure 3).

[0155] TnaA expression also led to increased production of other unidentified thiol molecules (not shown), as well as substantial production (302 μg / L) of indole, an off-flavor molecule known to convey a strong fecal odor (Figure 3). From these data, we conclude that TnaA expression in brewer's yeast increases the concentrations of 3MH and other volatile thiols that convey tropical fruit flavors in beer, but also increases the production of indole, an off-flavor, undesirable product.

[0156] Previous studies have shown that TnaA catalyzes the production of indole by cleaving tryptophan. 30 Without wishing to be bound by any particular theory, we hypothesized that the increased production of indole in beer fermented by yeast cells overexpressing TnaA was due to the cleavage of tryptophan by TnaA. TnaA was then modified to reduce its activity with tryptophan as a substrate while maintaining its relatively high activity with cysteine-conjugated substrates, precursors of volatile thiols with tropical fruit flavors, by mutating amino acid H463 to generate the TnaA-H463F variant.

[0157] The activity of several TnaA mutants with the substrate tryptophan and the cysteine ​​conjugate S-ethyl-L-cysteine ​​has been reported. 25 Data from the literature showed that the introduction of the H463F mutation reduced TnaA activity with tryptophan by >2000-fold, whereas activity with S-ethyl-L-cysteine ​​was reduced only by 2-fold.

[0158] The TnaA-H463F mutation was integrated into strain WLP001 and used to produce beer by wort fermentation as described above. The finished beer was found to have a strong guava / papaya aroma and, in contrast to beer brewed using yeast expressing wild-type TnaA, did not contain any fecal odor. GC / MS analysis revealed that the concentration of indole in beer fermented with yeast expressing TnaA-H463F was negligible (Figure 3, Y502). Surprisingly, although the H463F substitution has previously been reported to reduce the activity of the TnaA enzyme with cysteine ​​conjugates, the concentration of 3MH was increased by ∼25% in beer produced using yeast cells expressing TnaA-H463F compared to beer produced using yeast cells expressing wild-type TnaA. The 3MH concentration in beer brewed with the TnaA-H463F mutant was 285 ng / L, a 1.25-fold and 56-fold increase compared to beer brewed with Y182 and the wild-type yeast strain, respectively.

[0159] These data indicate that expression of TnaA-H463F in brewing yeast strains promotes robust production of 3MH in beer and also reduces production of undesirable indole. In addition, the H463F mutation in TnaA was unexpectedly found to also increase production of 3MH in beer compared to the level of 3MH produced using yeast cells expressing wild-type TnaA.

[0160] method Brewer's yeast strain construction The TnaA-encoding sequence used here was derived from Citrobacter amalonaticus and codon-optimized for expression in Saccharomyces yeast. This coding sequence was synthesized by TWIST Bioscience (San Francisco, CA) and cloned into a plasmid flanked by the PGK1 promoter and ENO1 terminator sequences from Saccharomyces cerevisiae. This plasmid also contained the Saccharomyces cerevisiae ADE2 coding sequence and regulatory regions, and encoded sequences homologous to the ADE2 locus, allowing for genome insertion into brewer's yeast by homologous recombination. Using this plasmid as a template, the TnaA-H463F gene was generated by PCR mutagenesis. The STR3- and IRC7-encoding sequences used in this study were PCR-amplified from the wine yeast strain VL3 and similarly cloned into an integration plasmid.

[0161] Prior to transformation into yeast, the plasmid was digested with restriction enzymes to produce a linear DNA fragment containing ADE2 and the gene of interest flanked by ADE2 homology regions. The linear DNA was transformed into the brewer's yeast strain WLP001, which carries a deletion of the ADE2 coding sequence, resulting in homologous recombination of the nucleic acid encoding the TnaA-H463F gene.

[0162] Three days after transformation, white colonies were selected based on rescue of the adenine biosynthetic pathway by the TnaA-ADE2 nucleic acid and screened by diagnostic PCR for insertion of ADE2 / TnaA-H463F DNA at the ADE2 locus.

[0163] beer brewing The strain was streaked onto YPD medium and grown for 3 days at 25°C. A single colony was used to inoculate an initial 5 milliliter (mL) malt extract (ME, Sigma-Aldrich, St. Louis, MO, USA) culture in a glass culture tube, which was grown for 1 day at 25°C with shaking at 200 revolutions per minute (rpm). The resulting culture was used to inoculate a 1 liter (L) ME culture in a 2 L glass Erlenmeyer flask, which was then grown for 2 days at 25°C with shaking at 200 rpm. The resulting culture was then used to inoculate a 20 L beer wort in a conical fermenter and grown for 10 days at 20°C.

[0164] For beer fermentation, 28.6 kilograms (kg) of two-row malt was milled and combined with 2.3 kg of oats and added to 100 L of water treated with 39 grams (g) of brewer's salt. Mashing was carried out at 67°C for 60 minutes (min). The wort was recirculated for 10 minutes and separated by lautering. Sparging was carried out for 37 minutes, bringing the final pre-boil volume in the brew kettle to 146 L. The wort was boiled to a final volume of 137 L and a specific gravity of 12.6 Plato. 58 grams of Warrior hop pellets were added to the kettle and boiled for 1 hour (h). Ingredients were sourced from Brewers Supply Group (Shakopee, MN, USA) unless otherwise stated. After separating the wort from the hot trub, it was transferred to six 20 L conical fermenters (SS Brewtech, Temecula, CA, USA). The beer was fermented at 20°C until final gravity was reached, held for another 24 h for vicinal diketone (VDK) removal, and then cold conditioned at 0°C to produce the finished beer, which was subsequently analyzed by gas chromatography-mass spectrometry for volatile thiols and indoles.

[0165] GC / MS analysis 3-Mercaptohexanol and indole were quantified by gas chromatography / mass spectrometry (GC / MS) analysis using an Agilent 6890 Series GC equipped with an electron ionization source and a 5973N mass-selective detector (Agilent Technologies, Santa Clara, CA, USA) operated in positive mode. For all experiments, helium (He) was used as the carrier gas and flowed at a constant rate of 1.0 mL / min onto an HP-5ms column (Agilent, 30 m long, 0.25 mm internal diameter (id), 0.25 μm film thickness). The oven temperature was maintained at 50°C for 3 min, followed by a 10°C / min ramp to 275°C, held for 1 min, and a 50°C / min ramp to a final temperature of 325°C, held for 5 min. All reagents and standards were obtained from Sigma-Aldrich, St. Louis, MO, USA.

[0166] Sampling and ion monitoring were optimized for each analyte: To quantify 3-mercaptohexanol, 200 mL of finished beer was used for analysis. 1 g EDTA disodium salt and 2 g NaCl were added, and the sample was extracted twice with 23 mL pentane in a separatory funnel. The organic phases were combined and then washed with 20 mL NaHCO (0.3% weight / volume (w / v), pH 6). Thiols were deprotonated and extracted from the organic phase by back-extraction into 6 mL cold (4 °C) 1 N NaOH. The aqueous phase was then transferred to a 20 mL headspace vial. Residual pentane was removed by passing a steady stream of N gas over the sample for 7 min. 100 microliters (μL) of 2,3,4,5,6-pentafluorobenzyl bromide (0.4% v / v in EtOH) was added, and the vial was sealed with a screw cap and derivatized for 20 min at room temperature. 0.5 g tartaric acid was then added to reduce the sample pH to ∼4.5. 2 g NaCl was added before resealing the vial. The derivatized thiols were adsorbed onto a PDMS / DVB solid-phase microextraction fiber at 70 °C for 1 h. The analytes were then desorbed onto the column at 250 °C for 10 min using splitless injection. The derivatized thiols were detected by selectively monitoring m / z ions 133 and 181. The peak area of ​​3-mercaptohexanol was quantified using MassHunter software (Agilent Technologies, Santa Clara, CA, USA). Absolute sample concentrations were calculated using a linear model generated from a standard curve constructed from authentic standards and using 181 as the quantitation ion.

[0167] To quantify indole, 1 mL of beer was sampled and extracted with 0.5 mL of ethyl acetate by vortexing for 10 seconds (sec), followed by centrifugation at 15,000× gravity for 10 min. The organic phase was transferred to a 1.5 mL tube, and residual water was removed by adding excess NaSO. The sample was then vortexed briefly and centrifuged at 15,000× gravity for 5 min. The ethyl acetate was then transferred to a GC vial, and 1 μL of the resulting extract was injected onto the column using splitless injection. Indole was detected by selectively monitoring m / z ions 63, 90, and 117. The indole peak area was quantified using Agilent MassHunter qualitative software. Absolute sample concentrations were calculated using a linear model generated from a standard curve constructed from authentic standards and using 117 as the quantitation ion.

[0168] As shown in Figure 2, beers brewed using WLP001(Y182) overexpressing wild-type TnaA and WLP001(Y502) overexpressing the TnaA-H463F mutant were found to contain increased concentrations of 3MH compared to beers brewed using wild-type California Ale Yeast (WLP001), WLP001(Y27) overexpressing IRC7, and WLP001(Y33) overexpressing STR3. However, beers brewed using WLP001(Y182) overexpressing wild-type TnaA were also found to contain increased concentrations of indole, whereas beers brewed using WLP001(Y502) overexpressing the TnaA-H463F mutant contained low levels of indole.

[0169] Example 2 Generation of wine-fermenting strains The sequence encoding TnaA was derived from Citrobacter amalonaticus, codon-optimized for expression in Saccharomyces yeast, as described in Example 1, and used for transformation into the wine-fermenting yeast strain Red Star Cote des Blancs.

[0170] Fermentation product production The genetically modified yeast strains described herein were evaluated in beer and wine fermentation. Briefly, the beer-brewing strain California Ale Yeast WLP001 described in Example 1 and the wine-fermenting strain Red Star Cote de Blancs, expressing wild-type TnaA (Y919) and TnaA H463F (Y484), were cultivated and used to inoculate initial cultures. The resulting cultures were used to inoculate larger cultures, which were then grown for several days. The resulting cultures were then used to inoculate 20 L of wort in the case of beer fermentation, or grape must or grape juice for wine fermentation, in fermenters and grown for several days until the desired final gravity was reached. The beer and wine were subsequently analyzed for volatile thiols and indoles by gas chromatography-mass spectrometry, as described in Example 1.

[0171] As shown in Figures 3A and 3B, strains expressing the TnaA H463F mutant produced fermentation products with increased 3MH concentrations while retaining indole concentrations comparable to those of the wild-type parent strains (WLP001 and Red Star, respectively).

[0172] Example 3 Addition of precursors to the fermentation process The yeast strains described herein were further analyzed for production of fermentation products using a process involving the addition of precursors to the fermentation process (as shown in Figure 1G, either to the medium or during the initial fermentation process).

[0173] Yeast strains were cultured as described in Example 1. Glutathione-conjugated 3-mercaptohexan-1-ol (Glut-3-MH) was added to the wort early in the fermentation process. Yeast strains were inoculated into the wort in the presence or absence of Glut-3-MH. The beer was fermented at 20°C until final gravity was reached, held for another 24 h for vicinal diketone (VDK) removal, and then placed under refrigerated conditions at 0°C to produce the finished beer, which was subsequently analyzed for volatile thiols and indoles by gas chromatography-mass spectrometry.

[0174] As shown in Figures 4A and 4B, the concentration of 3MH was increased in the fermentation product of beer fermentation using strains expressing wild-type TnaA or the TnaA H463F mutant in the presence or absence of added Glut-3-MH to the fermentation process. The indole concentration was also increased in the fermentation product of beer fermentation using strains expressing wild-type TnaA; however, the indole concentration in beer fermentation using strains expressing the TnaA H463F mutant was comparable to that of the wild-type parent strain (WLP001) in the presence or absence of added Glut-3-MH to the fermentation process. Addition of the precursor Glut-3MH to the fermentation process resulted in increased production of 3MH compared to control fermentation without added Glut-3MH.

[0175] Example 4 Evaluation of TnaA substitution mutants As described in Example 1, beer brewed using a yeast strain expressing the TnaA H463F mutant was found to contain increased levels of 3MH and reduced levels of indole compared to beer brewed using a yeast strain expressing wild-type TnaA. Additional amino acid substitutions of the histidine residue at position 463 of TnaA were also evaluated. Briefly, mutant TnaA genes containing substitutions of the histidine residue at position 463 with arginine (H463R), glutamic acid (H463E), threonine (H463T), glycine (H463G), isoleucine (H463I), or valine (H463V) were generated by PCR mutagenesis, cloned into an expression plasmid under the control of the HHF2 promoter, and transformed into yeast strains. See Table 1.

[0176] Table 1: Yeast strains [Table 1]

[0177] The yeast strains were inoculated into the wort and fermented at 20°C until final gravity was reached, followed by another 24 h for vicinal diketone (VDK) removal and then chilled to 0°C to produce the finished beer, which was subsequently analyzed for volatile thiols and indoles by gas chromatography-mass spectrometry.

[0178] As shown in Figure 5, the concentration of 3MH was increased in the fermentation products of beer fermentations using strains expressing wild-type TnaA or the TnaA H463 mutant. The indole concentration was also increased in the fermentation products of beer fermentations using strains expressing wild-type TnaA; however, the indole concentration in beer fermentations using strains expressing the TnaA H463 mutant was comparable to that of the wild-type parent strain (WLP001).

[0179] Evaluation of TnaA homologs A homologue of TnaA from C. amalonaticus was identified, cloned into an integration plasmid under the control of the PGK1 promoter, and transformed into a yeast strain (see Table 1).

[0180] The yeast strains were inoculated into the wort and fermented at 20°C until final gravity was reached, followed by another 24 h for vicinal diketone (VDK) removal and then chilled to 0°C to produce the finished beer, which was subsequently analyzed for volatile thiols and indoles by gas chromatography-mass spectrometry.

[0181] As shown in Figure 6, the concentration of 3MH was increased in beers brewed using strains expressing wild-type TnaA, strains expressing the TnaA H463F mutant, or strains expressing TnaA homologs from T. asperellum, A. saccharolyticus, and Z. gangwensis (which share 38%, 44%, and 82% sequence identity with TnaA from C. amalonaticus, respectively). Indole concentrations were also increased in beers brewed using strains expressing wild-type TnaA; however, indole concentrations in beer fermented using strains expressing the TnaA H463F mutant or strains expressing TnaA homologs from A. saccharolyticus and Z. gangwensis were comparable to those of the wild-type parent strain (WLP001).

[0182] References [Table 2-1]

[0183] [Table 2-2]

[0184] [Table 2-3]

Claims

1. A genetically modified yeast cell (modified cell) containing a heterologous gene encoding an enzyme with beta-lyase activity.

2. 2. The modified cell of claim 1, wherein the enzyme having beta-lyase activity has a sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO:

2.

3. The modified cell of claim 1 or 2, wherein the enzyme having beta-lyase activity does not comprise any of the sequences set forth in SEQ ID NOs: 1, 6, and 7.

4. 4. The modified cell according to any one of claims 1 to 3, wherein the enzyme with beta-lyase activity has the sequence as set forth in SEQ ID NO:

2.

5. The modified cell of claim 1, wherein the enzyme having beta-lyase activity has a sequence having at least 90% sequence identity with the sequence represented by any one of SEQ ID NOs: 4 or 5.

6. The modified cell of claim 1 or 5, wherein the enzyme with beta-lyase activity has a sequence as set forth in any one of SEQ ID NOs: 4 to 7.

7. 7. The modified cell of any one of claims 1 to 6, wherein the enzyme with beta-lyase activity comprises a substitution mutation at a position corresponding to position H463 of SEQ ID NO:

1.

8. 8. The modified cell of claim 7, wherein the substitution mutation at the position corresponding to position H463 of SEQ ID NO: 1 is phenylalanine, arginine, glutamic acid, threonine, glycine, isoleucine, or valine.

9. The modified cell of any one of claims 1 to 8, wherein the yeast cell is of the genus Saccharomyces.

10. 10. The modified cell of claim 9, wherein the yeast cell is of the species Saccharomyces cerevisiae (S. cerevisiae).

11. 11. The modified cell of claim 10, wherein the yeast cell is S. cerevisiae California Ale Yeast strain WLP001 or Red Star Cote des Blancs.

12. 10. The modified cell of claim 9, wherein the yeast cell is of the species Saccharomyces pastorianus (S. pastorianus).

13. 13. A method for producing a fermentation product, comprising contacting the modified cells of any one of claims 1 to 12 with a medium comprising at least one fermentable sugar, wherein the contacting is carried out during at least a first fermentation process to produce the fermentation product.

14. 14. The method of claim 13, wherein the at least one fermentable sugar is provided in at least one sugar source.

15. 15. The method of claim 13 or 14, wherein the fermentable sugar is glucose, fructose, sucrose, maltose, and / or maltotriose.

16. 16. The method of any one of claims 13 to 15, wherein the at least one sugar source comprises at least one precursor.

17. 17. The method of claim 16, wherein the at least one precursor comprises 3-mercaptohexan-1-ol conjugated to cysteine ​​(Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexan-1-ol conjugated to glutathione (Glut-3-MH), and / or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut 4MMP).

18. 18. The method of any one of claims 13 to 17, further comprising adding one or more precursors to the culture medium, wherein the precursors comprise 3-mercaptohexan-1-ol (Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexan-1-ol conjugated to glutathione (Glut-3-MH), and / or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut 4MMP).

19. 19. The method of any one of claims 9 to 18, wherein the fermentation product comprises an increased level of at least one volatile thiol compared to a fermentation product produced by an equivalent cell not expressing a heterologous gene or an equivalent cell expressing a wild-type enzyme having beta-lyase activity.

20. 20. The method of claim 19, wherein the at least one volatile thiol comprises 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), 4-methyl-4-mercaptopent-2-one (4MMP), or a combination thereof.

21. 21. The method of claim 20, wherein the fermentation products comprise at least 200 ng / L of 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and / or 4-methyl-4-mercaptopent-2-one (4MMP).

22. 22. The method of any one of claims 13 to 21, wherein the fermentation product comprises a reduced level of at least one undesired product compared to a fermentation product produced by an equivalent cell not expressing a heterologous gene or an equivalent cell expressing a wild-type enzyme having beta-lyase activity.

23. 23. The method of claim 22, wherein at least one undesired product is indole.

24. The method according to any one of claims 13 to 23, wherein the fermentation product is a fermented beverage.

25. 25. The method of claim 24, wherein the fermented beverage is beer, wine, sparkling wine (champagne), sake, mead, kombucha, or apple wine.

26. 26. The method of any one of claims 13 to 25, wherein the sugar source comprises malt, fruit juice, honey, rice starch, or a combination thereof.

27. 27. The method of claim 26, wherein the fruit juice is grape juice or apple juice.

28. the sugar source is malt; and The method further comprises producing a medium, wherein producing the medium comprises: (a) contacting a plurality of grains with water; and (b) The boiling or steeping of water and grain to produce wort The method according to any one of claims 13 to 27, comprising:

29. 30. The method of claim 28, further comprising adding at least one hop variety to the wort to produce a hopped wort.

30. 30. The method of any one of claims 13 to 29, further comprising adding at least one hop variety to the medium.

31. 31. The method of any one of claims 13 to 30, further comprising at least one additional fermentation process.

32. 32. The method of any one of claims 13 to 31, further comprising carbonating the fermentation product.

33. A fermentation product produced by the method of any one of claims 13 to 32.

34. 34. The fermentation product of claim 33, wherein the fermentation product comprises at least 200 ng / L of 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and / or 4-methyl-4-mercaptopent-2-one (4MMP).

35. 35. The fermentation product of claim 33 or 34, wherein the fermentation product contains less than 500 μg / L of indole.

36. 13. A method for producing a composition comprising ethanol, comprising contacting the modified cells of any one of claims 1 to 12 with a medium comprising at least one fermentable sugar, wherein said contacting is carried out during at least an initial fermentation process to produce a composition comprising ethanol.

37. 37. The method of claim 36, wherein the at least one fermentable sugar is provided in at least one sugar source.

38. 38. The method of claim 36 or 37, wherein the fermentable sugar is glucose, fructose, sucrose, maltose, and / or maltotriose.

39. 39. The method of any one of claims 36 to 38, wherein the at least one sugar source comprises at least one precursor.

40. 40. The method of claim 39, wherein the at least one precursor comprises 3-mercaptohexan-1-ol conjugated to cysteine ​​(Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexan-1-ol conjugated to glutathione (Glut-3-MH), and / or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut 4MMP).

41. 41. The method of any one of claims 36 to 40, further comprising adding one or more precursors to the culture medium, wherein the precursors comprise 3-mercaptohexan-1-ol (Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexan-1-ol conjugated to glutathione (Glut-3-MH), and / or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut 4MMP).

42. 42. The method of any one of claims 36-41, wherein the composition comprising ethanol further comprises an increased level of at least one volatile thiol compared to a composition comprising ethanol produced by an equivalent cell that does not express a heterologous gene or an equivalent cell that expresses a wild-type enzyme having beta-lyase activity.

43. 43. The method of claim 42, wherein the at least one volatile thiol comprises 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), 4-methyl-4-mercaptopent-2-one (4MMP), or a combination thereof.

44. 44. The method of claim 43, wherein the composition comprising ethanol further comprises at least 200 ng / L of 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and / or 4-methyl-4-mercaptopent-2-one (4MMP).

45. 45. The method of any one of claims 36-44, wherein the composition comprising ethanol further comprises a reduced level of at least one undesired product compared to a composition comprising ethanol produced by an equivalent cell that does not express a heterologous gene or an equivalent cell that expresses a wild-type enzyme having beta-lyase activity.

46. 46. ​​The method of claim 45, wherein at least one undesired product is indole.

47. 47. The method of any one of claims 36 to 46, wherein the composition comprising ethanol is a fermented beverage.

48. 48. The method of claim 47, wherein the fermented beverage is beer, wine, sparkling wine (champagne), sake, mead, kombucha, or apple wine.

49. 49. The method of any one of claims 36 to 48, wherein the sugar source comprises malt, fruit juice, honey, rice starch, or a combination thereof.

50. 50. The method of claim 49, wherein the fruit juice is grape juice or apple juice.

51. the sugar source is malt; and The method further comprises producing a medium, wherein producing the medium comprises: (a) contacting a plurality of grains with water; and (b) The boiling or steeping of water and grain to produce wort 51. The method of any one of claims 36 to 50, comprising:

52. 52. The method of claim 51, further comprising adding at least one hop variety to the wort to produce a hopped wort.

53. 53. The method of any one of claims 36 to 52, further comprising adding at least one hop variety to the medium.

54. 54. The method of any one of claims 36 to 53, further comprising at least one additional fermentation process.

55. 55. The method of any one of claims 36 to 54, further comprising carbonating the fermentation product.

56. 56. A composition comprising ethanol produced by the method of any one of claims 36 to 55.

57. 57. The composition of claim 56, wherein the composition further comprises at least 200 ng / L of 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and / or 4-methyl-4-mercaptopent-2-one (4MMP).

58. 58. The composition of claim 56 or 57, wherein the fermentation product contains less than 500 μg / L indole.

Images