Genetically modified yeast cells and methods for using them

Abstract

Provided herein are genetically modified yeast cells that recombinantly express a gene encoding a mutant beta-lyase. Also provided are methods for producing fermentation products and methods for producing ethanol.

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 on October 17, 2019, and U.S. Provisional Application No. 63 / 086,363, filed on October 1, 2020, and the entire contents of each of these applications are 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 Art

[0003] Background Within the last decade, tropical fruit flavors have become increasingly popular in the beverage market, not only in the United States but also 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): washingingtonpost.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, which are prized for the aromas of these tropical fruits. Within the wine industry, tropical flavor notes have fueled the popularity of Sauvignon Blanc and Chardonnay styles, and years of effort have been underway 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 revealed that the vast majority of fruit flavors are produced by combining various flavor molecules in diverse ways.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, countless studies also suggest that much of the tropical flavor and aroma of Sauvignon Blanc wine and certain varieties of flavor hops is due to the presence of three specific volatile thiol molecules. The thiols 3-mercaptohexane-1-ol (3MH), 3-mercaptoacetate hexyl (3MHA), and 4-methyl-4-mercaptopentan-2-one (4MMP) are all detectable by human olfactory receptors at extremely low concentrations and impart grapefruit / passionfruit, guava / currant, and passionfruit / blackcurrant flavors, respectively. See Vanzo et al. Sci.Rep. (2017):7; Roland et al. Chem.Rev. (2012) 111, 7355-7376. [Overview of the project]

[0004] overview Aspects of this disclosure provide genetically modified yeast cells (modified cells) containing heterologous genes encoding enzymes having beta-lyase activity. In some embodiments, the enzyme having beta-lyase activity has a sequence having at least 90% sequence identity with the sequence represented by SEQ ID NO: 2. In some embodiments, the enzyme having beta-lyase activity does not contain any of the sequences represented by SEQ ID NOs: 1, 6, and 7. In some embodiments, 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: 3 to 7. In some embodiments, 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: 3 to 5.

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

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

[0007] Aspects of this disclosure provide a method for producing a fermentation product, comprising contacting any of the modified cells described herein with a culture medium containing at least one fermentable sugar, wherein the contact is carried out at least during the initial fermentation process, thereby producing 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, at least one precursor comprises cysteine-conjugated 3-mercaptohexane-1-ol (Cys 3-MH), cysteine-conjugated 4-methyl-4-mercaptopentan-2-one (Cys 4MMP), glutathione-conjugated 3-mercaptohexane-1-ol (Glut-3-MH), and / or glutathione-conjugated 4-methyl-4-mercaptopentan-2-one (Glut 4MMP). In some embodiments, the method further comprises adding one or more precursors to a culture medium, wherein the precursors include 3-mercaptohexane-1-ol (Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexane-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 contains at least one volatile thiol in an increased level compared to a fermentation product produced by equivalent cells that do not express heterologous genes or by equivalent cells that express a wild-type enzyme having beta-lyase activity. In some embodiments, the at least one volatile thiol includes 3-mercaptohexane-1-ol (3MH), 3-mercaptoacetate hexyl (3MHA), 4-methyl-4-mercaptopenta-2-one (4MMP), or a combination thereof. In some embodiments, the fermentation product contains at least 200 ng / L of 3-mercaptohexane-1-ol (3MH), 3-mercaptoacetate hexyl (3MHA), and / or 4-methyl-4-mercaptopenta-2-one (4MMP).

[0009] In some embodiments, the fermentation product includes at least one undesirable product at a reduced level compared to a fermentation product produced by equivalent cells that do not express heterologous genes or by equivalent cells that express a wild-type enzyme having beta-lyase activity.

[0010] In some embodiments, at least one undesirable 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 includes 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 malt, and the method further comprises producing a culture medium, wherein producing a culture medium comprises bringing several grains into contact with water; and producing malt by boiling or soaking the water and grains. In some embodiments, the method further comprises producing hopped malt by adding at least one hop variety to the malt. In some embodiments, the method further comprises adding at least one hop variety to the culture 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 this disclosure provide fermentation products produced by any of the methods described herein. In some embodiments, the fermentation product comprises at least 200 ng / L of 3-mercaptohexane-1-ol (3MH), 3-mercaptoacetate hexyl (3MHA), and / or 4-methyl-4-mercaptopenta-2-one (4MMP). In some embodiments, the fermentation product comprises less than 500 μg / L of indole.

[0013] Aspects of this disclosure provide a method for producing an ethanol-containing composition, comprising contacting one of the modified cells described herein with a culture medium containing at least one fermentable sugar, wherein such contact is carried out at least during the initial fermentation process, thereby producing the ethanol-containing composition. 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, at least one precursor comprises cysteine-conjugated 3-mercaptohexane-1-ol (Cys 3-MH), cysteine-conjugated 4-methyl-4-mercaptopentan-2-one (Cys 4MMP), glutathione-conjugated 3-mercaptohexane-1-ol (Glut-3-MH), and / or glutathione-conjugated 4-methyl-4-mercaptopentan-2-one (Glut 4MMP). In some embodiments, the method further comprises adding one or more precursors to a culture medium, wherein the precursors include 3-mercaptohexane-1-ol (Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexane-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 at least one volatile thiol in an increased level compared to a composition comprising ethanol produced by equivalent cells that do not express heterologous genes or by equivalent cells that express a wild-type enzyme having beta-lyase activity. In some embodiments, the at least one volatile thiol comprises 3-mercaptohexane-1-ol (3MH), 3-mercaptoacetate hexyl (3MHA), 4-methyl-4-mercaptopenta-2-one (4MMP), or a combination thereof. In some embodiments, the ethanol-containing composition further comprises at least 200 ng / L of 3-mercaptohexane-1-ol (3MH), 3-mercaptoacetate hexyl (3MHA), and / or 4-methyl-4-mercaptopenta-2-one (4MMP).

[0015] In some embodiments, the ethanol-containing composition further comprises at least one undesirable product at a reduced level compared to a composition containing ethanol produced by equivalent cells that do not express heterologous genes or by equivalent cells that express a wild-type enzyme having beta-lyase activity. In some embodiments, the at least one undesirable product is indole.

[0016] In some embodiments, the ethanol-containing composition is a fermented beverage. In some embodiments, the fermented beverage is beer, wine, sparkling wine (champagne), sake, mead, kelp tea, or apple cider. In some embodiments, the sugar source includes 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, the sugar source is malt, and the method further comprises producing a culture medium, wherein producing a culture medium comprises bringing several grains into contact with water; and producing malt by boiling or soaking the water and grains. In some embodiments, the method further comprises producing hopped malt by adding at least one hop variety to the malt. In some embodiments, the method further comprises adding at least one hop variety to the culture medium. In some embodiments, the method further comprises at least one additional fermentation process. In some embodiments, the method further comprises introducing carbon dioxide into the fermentation product.

[0018] Aspects of this disclosure provide compositions comprising ethanol, produced by any of the methods described herein. In some embodiments, the composition further comprises at least 200 ng / L of 3-mercaptohexane-1-ol (3MH), 3-mercaptoacetate hexyl (3MHA), and / or 4-methyl-4-mercaptopenta-2-one (4MMP). In some embodiments, the fermentation product contains less than 500 μg / L of indole. [Brief explanation of the drawing]

[0019] Simple description of the drawing Further aspects of this disclosure will be readily apparent upon reviewing the detailed descriptions of its various aspects and embodiments below, in conjunction with the attached drawings.

[0020] [Figure 1A] Figures 1A–1H illustrate illustrative process diagrams of methods for producing fermentation products or ethanol as described herein. Figure 1A shows a method for producing fermentation products by contacting the modified cells of this disclosure with a culture medium during at least the initial fermentation process. [Figure 1B] Figure 1B shows an embodiment of the method in Figure 1A, in which the culture medium is produced by bringing multiple grains into contact with water, boiling the water to produce malt juice, and then cooling it to create the culture medium. [Figure 1C] Figure 1C shows an embodiment of the method of Figure 1A, where at least one variety of hops is added to the medium. [Figure 1D] Figure 1D shows an embodiment of the method of Figure 1A, where at least one additional fermentation process occurs. [Figure 1E] Figure 1E shows an embodiment of the method of Figure 1A, where carbon dioxide is introduced into the fermentation product. [Figure 1F] Figure 1F shows a method for producing a composition containing ethanol, which involves contacting the modified cells of the present disclosure with the medium during at least the first fermentation process. [Figure 1G] Figure 1G shows an embodiment of the method of Figure 1A, where at least one volatile thiol precursor (e.g., Cys-MH, Glu-3MH) is added to the medium. [Figure 1H] Figure 1H shows a method for producing a fermentation product by contacting the purified enzyme of the present disclosure with the medium during at least the first fermentation process.

[0021] [Figure 2] Figure 2 shows the concentrations of 3-mercaptohexanol (3MH) and indole in beer brewed by wild-type yeast strains and modified yeast strains. The left axis indicates the 3MH concentration, and the right axis reports the indole concentration. Strains shown from left to right: 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). Strains shown from left to right: wild-type California Ale Yeast (WLP001); WLP001 (Y319;Trpase WT) overexpressing wild-type TnaA; and WLP001 (Y502;Trpase H463F) overexpressing the TnaA H463F mutant. Figure 3B shows the concentrations of 3-mercaptohexane-1-ol (3MH) and indole in wines fermented 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). Strains shown from left to right: wild-type Red Star Cote 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 indoles in fermentation products produced using the indicated yeast strains, in or without the addition of glutathione-conjugated 3-mercaptohexane-1-ol (Glut-3MH). Figure 4A shows the concentration of 3-mercaptohexane-1-ol (3MH (ng / L)) in beer brewed with wild-type yeast strains and modified yeast strains expressing TnaA or the TnaA H463F mutant. Figure 4B shows the concentration of indole (μg / L) in beer brewed with wild-type yeast strains and modified yeast strains expressing TnaA or the TnaA H463F mutant. The strains shown from left to right are: wild-type California Ale Yeast (WLP001); WLP001 (Y319;Trpase WT) overexpressing wild-type TnaA; and WLP001 (Y502;Trpase H463F) overexpressing the TnaA H463F mutant. For each strain, the right column shows the indole produced in the fermentate containing Glut-3MH added at the start 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-mercaptohexane-1-ol (3MH) and indole in beer brewed using a yeast strain expressing TnaA containing the indicated amino acid mutation. The left axis shows the 3MH concentration (ng / L), and the right axis reports the indole concentration (μg / L). Strains shown from left to right: wild-type California Ale Yeast (WLP001); WLP001 (Trpase WT) overexpressing wild-type TnaA; WLP001 (Trpase H463F) overexpressing TnaA H463F mutant; WLP001 (Trpase H463R) overexpressing wild-type TnaA H463R mutant; WLP001 (Trpase H463E) overexpressing TnaA H463E mutant; WLP001 (Trpase H463T) overexpressing wild-type TnaA H463T mutant; WLP001 (Trpase H463G) overexpressing wild-type TnaA H463G mutant; WLP001 (Trpase H463I) overexpressing wild-type TnaA H463I mutant; and wild-type TnaA WLP001 (Trpase H463V) overexpresses the H463V mutant.

[0025] [Figure 6]Figure 6 shows the concentrations of 3-mercaptohexane-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 the 3MH concentration (ng / L), and the right axis reports the indole concentration (μg / L). Strains shown from left to right: wild-type California Ale Yeast (WLP001); WLP001 (Y319; Trpase WT) overexpressing wild-type (WT) TnaA from C. amalonaticus; WLP001 (Y502; Trpase H463F) overexpressing the TnaA Trpase H463F mutant from C. amalonaticus; WLP001 (Y644; T. asp homolog) overexpressing the TnaA / Trpase homolog from Trichoderma asperellum; WLP001 (Y645; A. sac homolog) overexpressing the TnaA / Trpase homolog from Aspergillus saccharolyticus; and WLP001 (Y646; Z. gang homolog) overexpressing the TnaA / Trpase homolog from Zooshikella ganghwensis. [Modes for carrying out the invention]

[0026] Detailed description Within the last decade, tropical fruit flavors have become increasingly popular in the beverage market. For example, the beer and wine industries have a demand for beverages with flavor notes such as mango, papaya, and pineapple, and this demand has also increased dramatically in recent years. Three flavor molecules that impart tropical notes to fermented beverages are the volatile thiols 3-mercaptohexane-1-ol (3MH), 3-mercaptoacetate hexyl (3MHA), and 4-methyl-4-mercaptopentan-2-one (4MMP). These thiols are produced by enzymes expressed in yeast 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 modify yeast strains for increased thiol production; however, these attempts have been only moderately successful, as the increase in volatile substance production is either moderate (context-dependent) or is marred by the co-release of undesirable products such as indole (e.g., off-flavors). This disclosure provides genetically modified yeast cells that increase such thiols and reduce the production of undesirable products.

[0027] Provided herein are genetically modified yeast cells that have been modified to express an enzyme having beta-lyase activity. In some embodiments, the enzyme having beta-lyase activity is modified to increase the production of desired volatile thiols and decrease the production of undesirable indoles. Also provided herein is a method for producing a fermented beverage, comprising contacting the genetically modified yeast cells with a culture medium containing a sugar source having at least one fermentable sugar during the fermentation process. Also provided herein is a method for producing ethanol, comprising contacting the genetically modified yeast cells with a culture medium containing a sugar source having at least one fermentable sugar during the fermentation process.

[0028] Beta-lyase (β-lyase) The genetically modified cells described herein contain heterogenes that encode enzymes having beta-lyase activity. The term “heterogene,” as used herein, refers to a hereditary unit corresponding to a sequence of nucleic acid (e.g., DNA) containing a genetic instruction, which is introduced into a host organism (e.g., a genetically modified cell) that does not naturally encode such a gene and is expressed by that organism.

[0029] Beta-lyase enzymes are involved in the production of thiols, which are alcohols and phenyls, but contain either a thiol group or a sulfanyl group ("-SH"). Thiols can have a variety of aromas or odors and are generally classified into negative-odor thiols and positive-odor thiols. Some sulfur-containing compounds (such as those that give off a rotten egg smell) are the result of H2S formation by yeast fermentation. Other secondary reductive odors, such as those found in cooked vegetables, onions, and cabbage, are also produced from sulfur-containing compounds such as thioacetic acid esters and mercaptans, which are thought to be due to the low redox potential in the fermentation products (Brajkovich et al., 2005).

[0030] Sulfur-containing compounds that positively contribute to the product are called "volatile thiols" and tend to have a distinctive aroma profile. For example, well-known volatile thiols that contribute to aromas include: 3-mercaptohexane-1-ol (3MH)(C6H) which conveys the aromas of grapefruit, passion fruit, currant, and guava. 14OS, also known as 3-mercapto-1-hexanol, 3-mercaptohexanol, 3-sulfanylhexanol-1-ol, 3-thiohexanol, 1-hexanol, and 3-mercapto-; 3-mercaptohexyl acetate (3MHA) (C8H) which conveys the aroma of passion fruit, grapefruit, boxwood, currant, and guava. 16 O2S, also known as 3-sulfanylhexyl acetate; and 4-methyl-4-mercaptopentan-2-one (4MMP) (C6H), which conveys the aroma of boxwood, passion fruit, broom, and blackcurrant. 12 It includes OS (also known as 4-mercapto-4-methyl-2-pentanone).

[0031] While we do not wish to be bound by any specific theory, the formation of aromatic precursors appears to involve the enzymatic oxidation of unsaturated fatty acids, metabolic processing, cysteinylated or glutathione conjugation to aldehydes, and beta-lyase cleavage during alcoholic fermentation, which releases aromatic compounds. This process, in which fermentative organisms convert odorless precursor molecules (e.g., plant-derived precursors) of sugar sources in culture media (e.g., malt juice, fruit mash, etc.) into active thiols, is called "biotransformation." For example, see 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 the biological conversion of cysteine ​​conjugation precursor molecules Cys-3MH and Cys-4MMP, respectively. For example, see Roland et al. Flavour and Fragrance Journal (2016) 69:1323. The biological conversion is catalyzed by organisms expressing enzymes with beta-lyase activity, which releases volatile thiols by cleaving the cysteine ​​conjugate. 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 then be acetylated by yeast expressing acyl-transferase enzymes to produce 3MHA. See, for example, Roland et al. Chem. Rev. (2011) 111:7355-7376.

[0032] During fermentation, Cys3-MH and Cys-4MMP can be transported from the culture medium into yeast cells and cleaved by enzymes with beta-lyase activity. Alternatively, brewing malt with grape juice or grape must can also contain the glutathione conjugates Glut-3MH and Glut-4MMP. See, for example, 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-3MH and Cys-4MMP, which then become substrates for enzymes with beta-lyase activity. For example, see Howell et al. Appl. Environ. Microbiol (2005) 71:5420-5426; Santiago et al. FEMS Yeast Res. (2015) 15. 3MH and 4MMP are then produced by the cleavage of the cysteine ​​conjugate by an enzyme with beta-lyase activity. Several studies have indicated that this reaction is highly inefficient, and that commonly used yeast strains by winemakers convert only 0.2%–2.0% of the available cysteine ​​conjugation 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 volatile thiol production, beta-lyase expression can also lead to increased production of undesirable molecules such as indole. Indole is formed from an aromatic heterocyclic organic compound with the formula C8H7N and has a bicyclic structure consisting of a six-membered benzene ring condensed to a five-membered pyrrole ring. Indole is widely distributed in the environment, naturally occurring in human feces, and has a strong excrement odor. Consequently, indole production during the production of fermentable 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 the 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 the enzyme with beta-lyase activity is a mutant beta-lyase gene containing 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 compared to the wild-type enzyme or a reference enzyme (e.g., substitution mutations), or they may not alter the amino acid sequence of the translated polypeptide (e.g., silent mutations).

[0035] In some embodiments, the heterologous gene encoding the enzyme having beta-lyase activity is truncated, which preferably has one or more amino acids missing at the N-terminus or C-terminus compared to the wild-type enzyme or reference enzyme.

[0036] In some embodiments, beta-lyase may also be referred to as cystathionine beta-lyase (EC4.4.1.13). 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 endogenous yeast beta-lyase. Examples of endogenous yeast beta-lyase include, but are not limited to, 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 the S. cerevisiae yeast strain VL3.

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

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

[0039] Amino acid sequence of wild-type TnaA from C. amalonaticus [ka] (Sequence ID 1)

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

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

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

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

[0044] MLPDCHLPETWRAKMVERIPSSTKDQRQEWICKADYNLFKLRSNEVRFDLGTDGGSGGMSDNQWSALMRGDSAATRSPSSYRLQEKVKELFGFTYTIPVHRGRAAKHALVQALLNEE SIVPGNAFDTTRANIESQKAIAIDCAIEGAFDIYYQHPFKGNVNLPELEKILQGSGSNVPMIMVSITCDKTGGQPVSMHNLREVKRLAKMFNVPVILDSARFAENAWFIQKNESEY SSQSIPDIVQEMYHHADGMVMSGKTDGLVNAGGFFATNNKDLFDRVGKYANLFCGLAGRDMEALTVGLGEVTQQEYLDDRIRQIHRFGMRLMAANVPIQQPIGGHAIVIDASLFLPLVPREEYVAKTLAVELYVEAGIRGAGMETVIGGGNPITGINRNRSNAKDFLYLAIPRQAYTNDQLSFVANALIQIFERRFTITRGLYVVHEDAILRYLTIQLKKADGKSIA(Sequence ID 3)

[0045] The amino acids of beta-lyases may be modified (e.g., substituted) to produce beta-lyase variants. For example, as described herein, a beta-lyase enzyme with desired activity was produced by mutating the amino acid at position 463, referred to as histidine 463 (H463) in SEQ ID NO: 1. In some embodiments, the amino acid corresponding to histidine at position 463 (H463) in 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 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 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). In some embodiments, the amino acid corresponding to 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 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 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 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 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 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 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 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 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 H463F substitution mutation (Y502) in TnaA from C. amalonaticus MDNFKHLPEPFRIRVIEPVKRTTREHRNNAIIKSGMNPFLLDSEDVFIDLLTDSGTGAVTQNMQAAMLRGDEAYSGSRSYYALSEAVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKR EQEKGLDRSKMAVFSNYFFDTTQGHSQINGCAVRNVYIKEAFDTGVRYDFKGNFDLDGLERGIQEVGPNNNVPYIVATITSNSAGGQPVSLANLKAMYNIAKKYDIPVVMDSARFAENA YFIQKREAEYRDWSIEEITRETYKYADMLAMSAKKDAMVPMGGLLCIKDDTYFDVYTECRTLCVVQEGFPTYGGLEGGAMERLAVGLVDGMNQDWLAYRIAQVQYLVDGLEAIGVTCQ QAGGHAAFVDAGKLLPHIPAEQFPAQALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRLTIPRATYTQSHMDFIIEAFEHVKENSMNIKGLTFTYEPKVLRFFTAKLKEV (Sequence 2)

[0049] The amino acid sequence of the TnaA homolog from Z. ganghwensis is provided by accession number WP_094789495.1 -H463F substitution mutation MNNFKHLPEPFRIRVVEPVKRTTLAYREKAILNAGMNPFLLDSKDVFIDLLTDSGTGAITQEMQAAMFIGDEAYSGSRSYYALADAVKDIFGYEYTIPTHQGRGAEQIYIPVLIKKREKE KGLDRTKMVALSNYFFDTTQGHTQLNACVAKNVFTKEAFDTSISADFKGNFDLELLEHAILEAGPQNVPYIVSTITCNSAGGQPVSIANLKAVYEIAQRYEIPVIMDSARFAENAYFIQQ REPEYQDWSIEAITFESYKYADALAMSAKKDAMVQMGGLLCFKDKSMLDVYNECRTLCVVQEGFPTYGGLEGGAMERLAVGLYDGMRQDWLAYRINQVQYLVNGLESIGIVCQQAGGHAAFVDAGKLLPHIPADQFPAHALACELYKVAGIRAVEIGSLLLGRDPTTGKQHPCPAELLRLTIPRATYTQTHMDFIIEAFEKVKENASHVKGLTFTYEPEVLRFFTARLKEVEN (Sequence ID 4)

[0050] The amino acid sequence of the TnaA homolog from A. saccharolyticus is provided by accession number XP_025427068.1 -H463F substitution mutation MPNTATPETWRVKTVEHIRPSTRDQRQQWIEEAGFNLFTLPSDRVFIDLLTDSGTGAMSDRQWAAIMSGDESYAGSTSFHALHEVVQDLFGLEYLLPVHQGRAAENALFSVLVHED QLVPANSHFDTTRAHIEFRKAAAVDCLSSGAYDVTDTNPFKGNMNLDMLRDILQESHARVPFILLTITCNTTGGQPVSLANIAAVKALADRYHKPLVVDAARFAENAWFIQQREPGY RDTSLRDITRQMLGMADAMVMSAKKDGLVNIGGFLATRHREWFDQATEYVILFEGFRTYGGLAGRDLAALAVGLEEVISADYLASRIGQVQRFGQRLIDAGVPIQQPVGGHAVLVDASRFLPEVPREEYVAQTLAVELYLEAGVRGVEIGTLLNGRDPESGEERFAETEWLRLAIPRRVYSNDHLEYVAQALIDLYHRRSEIRAGVRIVEEKPVLRFFTVRLERKTE(Sequence No. 5)

[0051] The amino acid sequence of the TnaA homolog from Z. ganghwensis is provided by accession number WP_094789495.1 - wild-type sequence MNNFKHLPEPFRIRVVEPVKRTTLAYREKAILNAGMNPFLLDSKDVFIDLLTDSGTGAITQEMQAAMFIGDEAYSGSRSYYALADAVKDIFGYEYTIPTHQGRGAEQIYIPVLIKKREKE KGLDRTKMVALSNYFFDTTQGHTQLNACVAKNVFTKEAFDTSISADFKGNFDLELLEHAILEAGPQNVPYIVSTITCNSAGGQPVSIANLKAVYEIAQRYEIPVIMDSARFAENAYFIQQ REPEYQDWSIEAITFESYKYADALAMSAKKDAMVQMGGLLCFKDKSMLDVYNECRTLCVVQEGFPTYGGLEGGAMERLAVGLYDGMRQDWLAYRINQVQYLVNGLESIGIVCQQAGGHAAFVDAGKLLPHIPADQFPAHALACELYKVAGIRAVEIGSLLLGRDPTTGKQHPCPAELLRLTIPRATYTQTHMDFIIEAFEKVKENASHVKGLTFTYEPEVLRHFTARLKEVEN (Sequence ID 6)

[0052] The amino acid sequence of the TnaA homolog from A. saccharolyticus is provided by accession number XP_025427068.1 - wild-type sequence MPNTATPETWRVKTVEHIRPSTRDQRQQWIEEAGFNLFTLPSDRVFIDLLTDSGTGAMSDRQWAAIMSGDESYAGSTSFHALHEVVQDLFGLEYLLPVHQGRAAENALFSVLVHED QLVPANSHFDTTRAHIEFRKAAAVDCLSSGAYDVTDTNPFKGNMNLDMLRDILQESHARVPFILLTITCNTTGGQPVSLANIAAVKALADRYHKPLVVDAARFAENAWFIQQREPGY RDTSLRDITRQMLGMADAMVMSAKKDGLVNIGGFLATRHREWFDQATEYVILFEGFRTYGGLAGRDLAALAVGLEEVISADYLASRIGQVQRFGQRLIDAGVPIQQPVGGHAVLVDASRFLPEVPREEYVAQTLAVELYLEAGVRGVEIGTLLNGRDPESGEERFAETEWLRLAIPRRVYSNDHLEYVAQALIDLYHRRSEIRAGVRIVEEKPVLRHFTVRLERKTE(Sequence No. 7)

[0053] In some embodiments, the enzyme comprises one amino acid sequence from sequence numbers 1 to 7, wherein the amino acid corresponding to histidine at position 463 (H463) of sequence number 1 is substituted with an amino acid that is not a histidine residue (for example, any other amino acid). In some embodiments, the enzyme comprises one amino acid sequence from sequence numbers 1 to 7, wherein the amino acid corresponding to histidine at position 463 (H463) of sequence number 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 contains one of the amino acid sequences of SEQ ID NOs: 1-7, wherein the amino acid corresponding to 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 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 a beta-lyase-active enzyme such that cells expressing the beta-lyase-active enzyme are able to produce increased levels of volatile thiols compared to cells not expressing the heterologous gene. In some embodiments, the heterologous gene encodes a beta-lyase-active enzyme such that cells expressing the beta-lyase-active enzyme are able to produce increased levels of volatile thiols compared to cells expressing a wild-type beta-lyase-active enzyme. In some embodiments, the beta-lyase-active enzyme 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 beta-lyase-active enzyme 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 volatile thiols with increased titer / levels. In some embodiments, the mutant beta-lyase produces 3MH with increased titer / levels. In some embodiments, the mutant beta-lyase produces 3MHA with increased titer / levels. In some embodiments, the mutant beta-lyase produces 4MMP with increased titer / levels. In some embodiments, the mutant beta-lyase produces one or more volatile thiols with increased titer / levels, e.g., 3MH, 3MHA, and / or 4MMP.

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

[0058] In some embodiments, an enzyme possessing beta-lyase activity has an amino acid sequence having 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 with the sequence represented by any one of sequence numbers 1 to 7. In some embodiments, the enzyme possessing 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 with the sequence represented by any one of sequence numbers 1-7, wherein the amino acid corresponding to histidine at position 463 (H463) of sequence number 1 is substituted with an amino acid that is not a histidine residue (for example, any other amino acid). In some embodiments, an enzyme possessing 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, and sequence number 1 The amino acid corresponding to histidine at position 463 (H463) 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” may be used interchangeably herein, but 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, as modified as found in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST protein search can be performed using the XBLAST program with score=50 and word length=3 to obtain amino acid sequences homologous to the protein molecule of interest. If a gap exists between two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST and Gapped BLAST programs, the initial settings parameters of each program (e.g., XBLAST and NBLAST) may be used.

[0060] When a percentage of identity or its range (e.g., at least, more, etc.) is defined, unless otherwise specified, endpoints are also included, and the range (e.g., at least 70% identity) encompasses all ranges within the cited range (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%). This includes percentages of identity (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%) and all their increments (for example, 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 having beta-lyase activity comprises the sequence shown in amino acid sequence number 2. In some embodiments, the enzyme having beta-lyase activity consists of the sequence shown in amino acid sequence number 2. In some embodiments, the enzyme having beta-lyase activity comprises the sequence shown in amino acid sequence number 3. In some embodiments, the enzyme having beta-lyase activity consists of sequence 3 as shown in amino acid sequence number 4. In some embodiments, the enzyme having beta-lyase activity comprises the sequence shown in amino acid sequence number 4. In some embodiments, the enzyme having beta-lyase activity comprises the sequence shown in amino acid sequence number 5. In some embodiments, the enzyme having beta-lyase activity consists of the sequence shown in amino acid sequence number 5.

[0062] In some embodiments, a gene encoding an enzyme having beta-lyase activity includes a nucleic acid sequence encoding an enzyme having at least 80% (for example, 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 some embodiments, a gene encoding an enzyme having beta-lyase activity includes a nucleic acid sequence encoding an enzyme having the sequence represented by amino acid sequence number 2. In some embodiments, a gene encoding an enzyme having beta-lyase activity includes a nucleic acid sequence encoding an enzyme consisting of an amino acid sequence as represented by any one of sequence numbers 2-5.

[0063] The identification of additional enzymes that have or are expected to have beta-lyase activity may be carried out based on similarity or homology to one or more domains of a beta-lyase, such as the beta-lyase 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 active domains, such as catalytic domains, such as catalytic domains associated with beta-lyase activity. In some embodiments, enzymes for use in the modified cells and methods described herein may have a relatively high level of sequence identity with a reference beta-lyase (e.g., a wild-type beta-lyase such as SEQ ID NO: 1) based on analysis of the majority of the enzyme or the full length of the enzyme, but have a relatively low level of sequence identity with the reference beta-lyase in the region of the catalytic domain. In some embodiments, the enzymes for use in the modified cells and methods described herein have 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, based on analysis of the majority of the enzyme or the full length of the enzyme, are compared to a reference beta-lyase (e.g., SEQ ID NOs: 1, 3, 6, or 7), It exhibits a relatively high level of sequence identity in the catalytic domain region of the enzyme, and a relatively low level of sequence identity with respect to the reference beta-lyase. In some embodiments, the enzymes for use in the modified cells and methods described herein have sequence identity of 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%, 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% compared to a reference beta-lyase (e.g., SEQ ID NOs. 1, 3, 6, or 7), based on analysis of the majority of the enzyme or the full length of the enzyme.

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

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

[0067] As will also be apparent to those skilled in the art, the amino acid position number of a selected residue in a beta-lyase may have the position number of a different amino acid in another beta-lyase enzyme (e.g., a reference enzyme). Generally, the corresponding position in other beta-lyase enzymes may be identified by using methods known in the art, for example, by aligning the amino acid sequences of two or more enzymes. Software programs and algorithms for aligning amino acid (or nucleotide) sequences are known in the art and readily available (e.g., Clustal Omega (Sievers et al. 2011)).

[0068] The beta-lyase variants described herein may further contain one or more additional modifications that specifically alter the characteristics of the polypeptide, for example, which are not related 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 cells.

[0069] Mutations in nucleic acids encoding beta-lyases 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 and form secondary structures such as hairpins or loops (which may be detrimental to enzyme expression).

[0070] Mutations can be made in the nucleic acid encoding the polypeptide by selecting amino acid substitutions or by random mutagenesis at selected sites. As described herein, the variant polypeptide may be expressed and tested for one or more activities to determine whether the mutation provides a variant polypeptide with desired properties. Further mutations may be made in the variant (or in the non-variant polypeptide), which are silent with respect to the amino acid sequence of the polypeptide but provide codons preferred for translation in a particular host (referred to as codon optimization). Codons preferred for translation of nucleic acids (e.g., nucleic acids in S. cerevisiae) are well known to those skilled in the art. Further mutations may also be made in the non-coding sequences of gene clones or cDNA clones to enhance polypeptide expression. The activity of a beta-lyase variant may 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 the 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 corresponding to a reference beta-lyase. In some embodiments, the beta-lyase variant contains amino acid substitutions at 1, 2, 3, 4, 5 or more positions corresponding to a reference beta-lyase. In some embodiments, the beta-lyase is a beta-lyase that does not exist in nature and is, for example, 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, the beta-lyase variant 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 conserved amino acid substitutions, when made to a beta-lyase variant, may provide a functionally equivalent variant of the polypeptide, i.e., the variant retains the functional capabilities of the polypeptide. As used herein, “conserved amino acid substitution” means an amino acid substitution that does not alter any characteristics of the protein to which the amino acid substitution is made, such as the relative charge or size. Variants may be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, such methods can be found, for example, in references that compile such methods, such as 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, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Functionally equivalent variants of the polypeptide examples include conserved amino acid substitutions in the amino acid sequences of the proteins disclosed herein. Conserved amino acid substitutions include substitutions made from amino acids in 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 those skilled in the art will know, homologous genes encoding enzymes containing beta-lyases may be derived from other species and can be identified by homology searches, for example, through the protein BLAST search available on the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov). By aligning the amino acid sequence of an enzyme with one or more reference enzymes, and / or by comparing the secondary or tertiary structure of a similar or homologous enzyme with one or more reference beta-lyases, corresponding amino acid residues can be determined in a similar or homologous enzyme, and amino acid residues for mutation can be determined in a similar or homologous enzyme.

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

[0075] The disclosures provided herein involve enzymes having beta-lyase activity, the recombinant expression of genes encoding functional modifications and variants of the above enzymes, and related uses. Homologs and alleles of nucleic acids related to the present invention can be identified by conventional techniques. Also covered by the present invention are nucleic acids that hybridize with the nucleic acids described herein under stringent conditions. The term “stringent conditions,” as used herein, refers to parameters well known in the art. Parameters for nucleic acid hybridization may also be found in references that compile such methods, for example, 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, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York.

[0076] Other conditions, reagents, etc., may be used, but these will result in similar stringency. Those skilled in the art are familiar with such conditions, and therefore they are not given here. However, it is understood that those skilled in the art may manipulate these conditions in a manner that allows for clear identification of homologs and alleles of the nucleic acids of the present invention (for example, by using lower stringency conditions). Those skilled in the art are also familiar with methodologies for screening cells and libraries for the expression of such molecules, then for their systematic isolation, and subsequently for the isolation and sequencing of the relevant nucleic acid molecules.

[0077] The present invention also encompasses degenerate nucleic acids, which include codons that replace codons present in native materials. For example, serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT, and AGC. Each of the six codons is equivalent for the purpose of encoding a serine residue. It will be apparent to those skilled in the art that any of the nucleotide triplets encoding serine may be used to direct protein synthesis machinery to incorporate serine residues into elongated polypeptides in vitro or in vivo. Similarly, nucleotide sequence triplets encoding other amino acid residues include, but are not limited to, CCA, CCC, CCG, and CCT (proline codons); CGA, CGC, CGG, CGT, AGA, and AGG (arginine codons); ACA, ACC, ACG, and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC, and ATT (isoleucine codons). Other amino acid residues may also be encoded by multiple nucleotide sequences. Therefore, the present invention encompasses degenerate nucleic acids that differ from biologically isolated nucleic acids in terms of codon sequences resulting from the degeneracy of the genetic code. The present invention also encompasses codon optimization that is suited to the optimal codon utilization of the host cell.

[0078] The present invention also provides modified nucleic acid molecules that include the addition, substitution, and deletion of one or more nucleotides. In a preferred embodiment, these modified nucleic acid molecules, and / or polypeptides encoded therein, retain at least one activity or function (such as enzymatic activity) of an unmodified nucleic acid molecule and / or polypeptide. In a particular embodiment, a modified nucleic acid molecule encodes a modified polypeptide, preferably a polypeptide having a conserved amino acid substitution as described elsewhere herein. The modified nucleic acid molecule is structurally related to the unmodified nucleic acid molecule, and in a preferred embodiment, sufficiently structurally related to the unmodified nucleic acid molecule, so that the modified nucleic acid molecule and the unmodified nucleic acid molecule can hybridize under stringent conditions known to those skilled in the art.

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

[0080] In some embodiments, one or more genes related to the present invention are expressed in a recombinant expression vector. As used herein, “vector” may be any of a number of nucleic acids into which a desired sequence(s) may be inserted by restriction and ligation for transport between different generative environments or for expression in a host cell. Vectors typically consist of DNA, but RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, viral genomes, and artificial chromosomes.

[0081] Cloning vectors are vectors that can replicate autonomously or are integrated into the genome of a host cell. In the case of plasmids, 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 on the host before the host regenerates through mitosis. In the case of phages, replication may occur actively during the lytic phase or passively during the lysogenic phase.

[0082] An expression vector is a vector in which a desired DNA sequence may be inserted so as to be operably linked to a regulatory sequence by restriction and ligation, and which may be expressed as an RNA transcript. The vector may further contain one or more marker sequences suitable for use in identifying cells that are transformed or transfected with the vector or not. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, genes encoding enzymes whose activity is detectable 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). A preferred vector is one that allows for the autonomous replication and expression of structural gene products present in a DNA segment (these are operably linked to the DNA segment).

[0083] As used herein, coding sequences and regulatory sequences are said to be "operably linked" if they are covalently linked in such a way that the expression or transcription of the coding sequence is under the influence or control of the regulatory sequence. If it is desired that the coding sequence be translated into a functional protein, the two DNA sequences are said to be operationally linked if the induction of a promoter in the 5' regulatory sequence results in the transcription of the coding sequence, and the nature of the link between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the promoter region's ability to direct the coding sequence to transcription, or (3) interfere with the corresponding RNA transcript's ability to translate into a protein. Thus, a promoter region will be operationally linked to a coding sequence such that, if the promoter region is capable of acting on the transcription of its DNA sequence, the resulting transcript can be translated into a desired protein or polypeptide.

[0084] When nucleic acid molecules encoding any of the claimed enzymes are expressed in a cell, various transcriptional regulatory sequences (e.g., promoter / enhancer sequences) may be used to direct that expression. A promoter can be a native promoter, i.e., a promoter of a gene in the context in which it is inherent, providing normal regulation of gene expression. In some embodiments, a promoter can be constitutive, i.e., not regulated to allow continuous transcription of its associated gene (e.g., an enzyme having beta-lyase activity). Various conditional promoters, such as those regulated by the presence or absence of a molecule, may also be used.

[0085] The exact nature of the regulatory sequences required for gene expression may vary between species or cell types, but generally include, as needed, 5' untranscribed and 5' untranslated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, etc. In particular, such 5' untranscribed regulatory sequences will include promoter regions containing promoter sequences for transcriptional control of the gene to which they are functionally linked. The regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the present invention may optionally include 5' leader sequences or signal sequences. The selection and design of appropriate vectors are within the scope of the skill and discretion of those 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 expression of heterologous DNA (RNA) in the host cell is made possible by placing the heterologous DNA (RNA) under the operational control of transcription elements. For example, heterologous expression of a gene encoding an enzyme with beta-lyase activity in genetically modified yeast cells in a method for producing fermented beverages (such as beer) has been demonstrated using the S. cerevisiae strain WLP001. As those skilled in the art will understand, any of the enzymes described herein can also be expressed in other yeast cells, including yeast strains used to produce wine, mead, sake, cider, etc.

[0087] The nucleic acid molecule encoding the claimed enzyme can be introduced into one or more cells using methods and techniques standard in the art. For example, the nucleic acid molecule can be introduced by standard protocols such as chemical transformation and electroporation, transduction, particulate guns, etc. Expression of the nucleic acid molecule encoding the claimed enzyme can also be achieved by integrating the nucleic acid molecule into the genome.

[0088] The integration of heterologous genes can be achieved either by the integration of new nucleic acids into the yeast cell genome, or by the transient or stable maintenance of new nucleic acids as episomal elements. In eukaryotic cells, permanent, heritable genetic changes are generally achieved by introducing DNA into the cell's genome.

[0089] Heterogenes may also encompass various transcription elements required for the expression of their encoded gene product (e.g., an enzyme having beta-lyase activity). For example, in some embodiments, heterogenes may encompass promoters. In some embodiments, promoters may be operably linked to the gene of the heterogene. In some embodiments, the cell is an inductive 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 obvious to those skilled in the art. In some embodiments, the promoter is a yeast promoter, e.g., a native promoter from the yeast cell in which the heterogene is expressed. In some examples, the promoter is a PKG1 promoter (pPGK1) or an HHF2 promoter (pHHF2).

[0090] Genetically modified yeast cells Aspects of this 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 heterologous genes encoding enzymes having beta-lyase activity.

[0091] The terms “genetically modified cell,” “genetically modified yeast cell,” and “modified cell” may be used interchangeably herein, but refer to eukaryotic cells (e.g., yeast cells that are modified by the introduction of heterologous genes, or yeast cells that can soon be modified by such introduction). These terms (e.g., modified cell) encompass the offspring of the original cell that has been genetically modified by the introduction of heterologous genes. It will be understood by those skilled in the art that the offspring of a single cell may not necessarily be completely identical to the original parent in terms of morphology or genomic or total nucleic acid complement due to mutation (i.e., a natural, accidental, or intentional alteration 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 sugar) to produce ethanol (ethyl alcohol) and carbon dioxide. In some embodiments, the yeast cells consist of the genus Saccharomyces. The genus Saccharomyces encompasses approximately 500 distinct species, many of which are used in food production. One example species is Saccharomyces cerevisiae (S. cerevisiae), commonly referred to as "brewer's yeast" or "baker's yeast," which is used in the production of wine, bread, and beer, among other products. Other members of the genus Saccharomyces include, without limitation, wild yeasts closely related to S. cerevisiae, such as Saccharomyces paradoxus; 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 pair of chromosomes), or polyploid (i.e., having or containing more than two homologous sets of 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 rectangular fermenters, as well as other characteristics such as preferred fermentation temperature and the alcohol concentration achieved.

[0094] While beer brewing and wine production have traditionally focused on the use of S. cerevisiae strains, other yeast genera are also highly valued in the production of fermented beverages. In some aspects, yeast cells belong to non-Saccharomyces genera. See, for example, Crauwels et al. Brewing Science (2015) 68:110-121; Esteves et al. Microorganisms (2019) 7(11):478. In some aspects, yeast cells consist of genera Kloeckera, Candida, Starmerella, Hanseniaspora, Kluyveromyces / Lachance, Metschnikowia, Saccharomycodes, Zygosaccharomyce, Dekkera (also known as Brettanomyces), Wickerhamomyces, or Torulaspora. Examples of non-Saccharomyces yeasts include, without limitation, Hanseniaspora uvarum, Hanseniaspora guillermondii, Hanseniaspora vinae, Metschnikowia pulcherrima, Kluyveromyces / Lachancea thermotolerans, Starmerella bacillaris (formerly known 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 one or more genetically modified yeasts. For example, in some embodiments, the method may involve the use of one or more genetically modified yeasts belonging to the genus Saccharomyces. In some embodiments, the method may involve the use of one or more genetically modified yeasts belonging to non-Saccharomyces genus. In some embodiments, the method may involve the use of one or more genetically modified yeasts belonging to the genus Saccharomyces and one genetically modified yeast belonging to non-Saccharomyces genus. Alternatively or in addition, any of the methods described herein may involve the use of one or more genetically modified yeasts and one or more genetically unmodified (wild-type) yeasts.

[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 crossing of two different yeast strains to acquire, for example, one or more desired characteristics. For example, a hybrid strain may result from the crossing of two different yeast strains belonging to the same genus or the same species. In some embodiments, a hybrid strain results from the crossing of Saccharomyces cerevisiae and Saccharomyces eubayanus. See, for example, 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. Domesticated yeast strains are subject to human selection and breeding because they possess desired characteristics.

[0098] In some embodiments, genetically modified yeast cells may be used in a symbiotic matrix with a strain, and may be used in the production of fermented beverages such as kelp tea, kefir, and ginger beer. For example, Saccharomyces fragilis is an essential component 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 having the beta-lyase activity 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 having the beta-lyase activity 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 gene encodes an enzyme having the same beta-lyase activity. In some embodiments, the copies of the heterologous gene are not identical, and the gene encodes a different enzyme having beta-lyase activity (e.g., a mutant, a variant, or a fragment thereof).

[0100] In some embodiments, yeast cells are tetraploid. Tetraploid yeast cells are cells that maintain four complete sets of chromosomes (i.e., four copies of a complete set of chromosomes). In some embodiments, yeast cells are tetraploid, and a copy of a heterologous gene encoding an enzyme having beta-lyase activity as described herein is introduced into at least one copy of the genome. In some embodiments, yeast cells are tetraploid, and a copy of a heterologous gene encoding an enzyme having beta-lyase activity as described herein is introduced into one or more copies of the genome. In some embodiments, yeast cells are tetraploid, and a copy of a heterologous gene encoding an enzyme having 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 having identical beta-lyase activity. In some embodiments, the copies of the heterologous gene are not identical, and the gene encodes different enzymes having beta-lyase activity (e.g., mutants, variants, or fragments thereof).

[0101] Strains of yeast cells that may be used in the methods described herein will be known to those skilled in the art. These strains include yeast strains used to brew desired fermented beverages, 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, and 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 It includes ian White ECY11. In some embodiments, the yeast is S. cerevisiae strain WLP001.

[0103] In some embodiments, the yeast strains for use in the genetically modified cells and methods described herein are wine yeast strains. 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 Cote des Blancs, Red Star Premier Cuvee, Red Star Pasteur Red, Red Star Pasteur Champagne, Fermentis BCS-103, and Fermentis VR44.

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

[0105] The fermentation process utilizes a natural process that uses microorganisms to convert carbohydrates into alcohol and carbon dioxide. This is a metabolic process that produces chemical changes in an organic substrate through enzymatic action. In the context of food production, fermentation broadly refers to any process in which microbial activity brings about desired changes in food or beverage. The conditions for fermentation and the execution of fermentation are referred to herein as the “fermentation process.”

[0106] In some aspects, this disclosure relates to a method for producing fermented products, such as fermented beverages, which involves producing a fermented product by contacting one of the modified cells described herein with a culture medium containing at least one fermentable sugar during the initial fermentation process (Figures 1A-1G). When used herein, “culture medium” refers to a liquid that contributes to fermentation and does not inhibit or prevent the fermentation process. In some embodiments, the culture medium is water. In some embodiments, the method for producing a fermented product involves producing a fermented product by contacting a purified enzyme (e.g., one of the beta-lyase enzymes described herein) with a culture medium containing at least one fermentable sugar during the initial fermentation process (Figure 1H).

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

[0108] In some embodiments, fermentable sugars are provided in sugar sources. The sugar source for use in the claimed method may also depend, for example, on the type of fermentation product and fermentable sugar. Examples of sugar sources include, but are not limited to, malt juice, 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 the sugars available for fermentation. Using the production of beer as an example of a fermented beverage, grains (grasses, barley) are boiled or soaked in water, thereby hydrating the grains and activating malt enzymes, which convert the starch into fermentable sugars; this is called "mashing." As used herein, the term "malt juice" refers to the liquid produced in the mashing process that contains fermentable sugars. The malt juice is then exposed to a fermentable organism (e.g., any of the cells described herein) so that the enzymes of the fermentable organism can convert the sugars in the malt juice into alcohol and carbon dioxide. In some embodiments, the malt juice is brought into contact with a recombinant enzyme (e.g., any of the enzymes described herein), which may optionally be purified or isolated from an enzyme-producing organism so that the enzyme can convert the sugars in the malt juice into alcohol and carbon dioxide.

[0110] In some embodiments, the cereals include malted, unmalted, or combinations of malted and unmalted cereals. Examples of cereals for use in the methods herein include, but are not limited to, barley, oats, maize, rice, rye, sorghum, wheat, wild oats, and Job's tears.

[0111] In the example of sake production, the sugar source is rice, which is incubated with Aspergillus oryzae to convert rice starch into fermentable sugars and produce koji. The koji is then exposed to a fermentable organism (for example, any of the cells described herein) so that the enzymes of the fermentable organism can convert the sugars in the koji into alcohol and carbon dioxide. In some embodiments, the koji is brought into contact with a recombinant enzyme (for example, any of the enzymes described herein), which may optionally be purified or isolated from an enzyme-producing organism so that the enzyme can convert the sugars in the koji into alcohol and carbon dioxide.

[0112] In the example of wine production, grapes are harvested and mashed (e.g., crushed) to obtain a composition containing skins, solids, juice, and seeds. The resulting composition is called "whirlpool." The grape juice may be separated from the whirlpool and fermented, or the entire whirlpool (i.e., skins, seeds, and pulp) may be fermented. The grape juice or whirlpool is then exposed to a fermenting organism (e.g., any of the cells described herein) so that the enzymes of the fermenting organism can convert the sugars in the grape juice or whirlpool into alcohol and carbon dioxide. In some embodiments, the grape juice or whirlpool is brought into contact with a recombinant enzyme (e.g., any of the enzymes described herein), which may optionally be purified or isolated from an enzyme-producing organism so that the enzyme can convert the sugars in the grape juice or whirlpool into alcohol and carbon dioxide.

[0113] In some embodiments, the methods described herein involve producing a culture medium, which may involve heating or immersing a sugar source, for example, in water. In some embodiments, the water is incubated with the sugar source for a period of time at a temperature of at least 50 degrees Celsius (50°C). In some embodiments, the water is incubated with the sugar source for a period of time at a temperature of at least 75°C. In some embodiments, the water is incubated with the sugar source for a period of time at a temperature of at least 100°C. Preferably, the culture 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 (Figure 1G). Examples of precursors include, but are not limited to, 3-mercaptohexane-1-ol (Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexane-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 (e.g., 1, 2, 3, 4, 5, or more) hop varieties to the malt, for example, to the culture medium, during the fermentation process. Hops are the flowers of the hop plant (Humulus lupulus) and are often used in fermentation to impart a variety of flavors and aromas to the fermentation product. Hops are considered to impart floral, fruity, and / or citrus flavors and aromas, as well as bitter flavorings, and may be characterized based on their original purpose. For example, bitter hops impart a certain level of bitterness to the fermentation product due to the presence of alpha acids in the hop flower, while aromatic hops have lower levels of alpha acids and contribute to the desired aroma and flavor of the fermentation product.

[0116] Whether one or more hop varieties are added to the culture medium and / or malt, and at what stage the hops are added, may be based 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 the preparation of the malt, for example, while the malt is boiling. In some embodiments, hops intended to impart bitterness to the fermentation product are added to the malt and boiled with it for a period of time, for example, 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 bitterness. In some embodiments, hops intended to impart a desired aroma to the fermentation product are added at the end of boiling or after the malt has been boiled (i.e., "dry hopping"). In some embodiments, one or more hop varieties may be added multiple times during the process (for example, at least twice, at least three times, or more).

[0117] In some aspects, hops are added in either wet or dry form and optionally boiled with malt. In some aspects, hops are in the form of dry hop pellets. In some aspects, at least one variety of hops is added to the culture medium. In some aspects, hops are wet (i.e., not dry). In some aspects, hops are dry and optionally further processed prior to use. In some aspects, hops are added to malt prior to the fermentation process. In some aspects, hops are boiled in malt. In some aspects, hops are boiled with malt and then cooled with malt.

[0118] Many hop varieties are known in the art and may be used in the methods described herein. Examples of hop varieties, without limitation, include 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 containing at least one fermentable sugar may be carried out for about 1 to about 30 days. In some embodiments, the fermentation process is carried out for about 1, 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 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 approximately 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 1D. Such 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, barrel) in which the fermented beverage is incubated for a period of time. In some embodiments, secondary fermentation is carried out for a period between 10 minutes and 12 months. In some embodiments, secondary fermentation can last 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 The process may be carried out for several days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, thirteen days, fourteen days, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, thirteen weeks, fourteen weeks, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, or longer. In some embodiments, the additional or secondary fermentation process of one or more fermentable sugars may be carried out at a temperature of approximately 4°C to approximately 30°C. In some embodiments, the additional or secondary fermentation process of one or more fermentable sugars may be carried out at a temperature of approximately 8°C to approximately 14°C or approximately 18°C ​​to approximately 24°C. In some embodiments, the additional or secondary fermentation process of one or more fermentable sugars may be carried out at a temperature of approximately 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 obvious to those skilled in the art, the choice of duration and temperature for additional or secondary fermentation processes will depend on factors such as the type of beer, the desired characteristics of the beer, and the yeast strain used in the method.

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

[0123] Following fermentation, various refinement, filtration, and maturation processes may be present, after which the liquid is bottled (e.g., sealed in containers for distribution, storage, or consumption). Any of the methods described herein may further involve distilling, pasteurizing, and / or carbonating the fermentation product. In some embodiments, the method involves carbonating the fermentation product (e.g., as shown in Figure 1E). Methods for carbonating fermented beverages are known in the art and include, for example, forced carbonation with gas (e.g., carbon dioxide, nitrogen), spontaneous carbonation by adding a further sugar source to the fermented beverage to promote further fermentation and carbon dioxide production (e.g., bottle conditioning).

[0124] Fermentation products Aspects of this disclosure relate to fermentation products produced by any of the methods disclosed herein. In some embodiments, the fermentation product is a fermentation beverage. Examples of fermentation beverages include, but are not limited to, beer, wine, sake, mead, cider, cava, sparkling wine (champagne), kelp tea, 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 cider.

[0125] In some aspects, fermented products are fermented foods. Examples of fermented foods include, but are not limited to, cultured yogurt, tempeh, miso, kimchi, sauerkraut, fermented sausages, bread, and soy sauce.

[0126] In accordance with aspects of the present invention, increased titer volatile thiols are produced through recombinant expression of genes relevant to the present invention in yeast cells and in the use of said cells in the methods described herein. As used herein, “increased titer” or “high titer” refers to titer on a nanogram per liter (ng L-1) scale. The titer produced for a given product will be influenced by several factors, including the selection of the culture 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 titers are 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, 6 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 larger than L-1.

[0128] In some embodiments, the titer of the volatile thiol is at least 1 μg L-1, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 , 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 , 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880 The values ​​are 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 the volatile thiol is at least 1 mg L-1, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 , 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 , 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1050, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 mg L-1 or higher.

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

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

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

[0133] In some embodiments, fermented beverages contain alcohol up to between 0.1% and 30% by volume (also referred to as "ABV", "abv", or "alc / vol"). In some embodiments, fermented beverages contain alcohol in volumes of approximately 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. In some embodiments, fermented beverages are non-alcoholic (for example, containing less than 0.5% alcohol by volume).

[0134] kit Aspects of this disclosure also provide kits for the use of genetically modified yeast cells, for example, to produce fermentation products or ethanol. In some embodiments, the kits contain modified cells containing heterologous genes encoding enzymes having beta-lyase activity.

[0135] In some embodiments, the kit is for the production of fermented beverages. In some embodiments, the kit is for the production of beer. In some embodiments, the kit is for the production of wine. In some embodiments, the kit is for the production of sake. In some embodiments, the kit is for the production of mead. In some embodiments, the kit is for the production of cider.

[0136] The kit may also include other components for use in any of the methods described herein or for use in any of the cells as described herein. For example, in some embodiments, the kit may contain grain, water, malt, mash, yeast, hops, fruit juice, or other sugar sources (one or more). In some embodiments, the kit may contain one or more fermentable sugars. In some embodiments, the kit may contain one or more additional agents, ingredients, or components.

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

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

[0139] The composition (e.g., modified cells) may be provided in a dry form, a lyophilized form, a frozen form, or a liquid form. In some embodiments, the modified cells are provided as colonies on an agar plate. In some embodiments, the modified cells are provided in the form of a seed culture, which may be directly added to a culture medium. When the reagent or component is provided in a dry form, it is generally reconstituted by adding a solvent such as a culture medium. The solvent may be provided in a separate packaging means and may be selected by those skilled in the art.

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

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

[0142] general technique To put the subject matter of this disclosure into practice, unless otherwise indicated, conventional techniques within the scope of skills in the relevant arts, such as molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, will be employed.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 This is fully explained in literature such as CC Blackwell, eds.; Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis, et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); and Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0143] Equivalents and range This disclosure is not limited to any or all of the specific embodiments expressly described herein, but it should be understood that these embodiments themselves may, of course, vary. It should also be understood that the terminology used herein is intended only to describe specific embodiments and is not intended to limit them.

[0144] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure pertains. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but 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 relating to the methods and / or materials cited in those publications. All such publications and patents are incorporated herein by reference as if each individual publication or patent were specifically and individually pointed to to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and / or materials of the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents (i.e., any lexicographical definitions of the 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 term appearing in the appended claims). In the event of any conflict between any incorporated reference and this disclosure, this disclosure shall prevail. In addition, any specific aspect of this disclosure that falls within the scope of prior art may be expressly excluded from any one or more of the claims. Such aspects, which are considered to be known to those skilled in the art, may be excluded even if the exclusion is not expressly indicated in the specification. Any specific aspect of this disclosure may be excluded from any claim for any reason, whether or not it relates to the existence of prior art.

[0146] Any citation of a publication is intended to disclose it prior to the filing date and should not be interpreted as an admission that this disclosure does not qualify as prior to such publication due to prior disclosures. Furthermore, the date of the provided publication may differ from the actual publication date, which may need to be independently verified.

[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 features of several other embodiments without departing from the spirit or scope of this disclosure. Any of the enumerated methods may be performed in the order of the enumerated events, or in any other logically feasible order.

[0148] Articles in claims such as “a,” “an,” and “the” may mean one or more unless otherwise obvious from the context, unless otherwise indicated otherwise. Wherever pronouns are used in this specification in terms of gender (e.g., masculine, feminine, neuter, etc.), unless the context explicitly indicates or otherwise requires, the pronoun shall be interpreted as neuter, regardless of the gender it implies (i.e., interpreted as referring equally to all genders). Wherever used herein, unless the context explicitly indicates or otherwise requires, words used in the singular form encompass the plural form, and words used in the plural form encompass the singular form. Claims or statements containing “or” between one or more members of a group shall be deemed satisfied if one, more than one, or all of the group members are present in, adopted into, or otherwise related to the given product or process, unless otherwise obvious from the context, unless otherwise indicated otherwise. This disclosure includes embodiments in which exactly one member of the group is present in, incorporated into, or otherwise related to a given product or process. This disclosure also includes embodiments in which more than one or all of the group members are present in, incorporated into, or otherwise related to a given product or process.

[0149] Furthermore, this 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 dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same underlying claim. Where elements are presented enumerated (for example, in the form of Markush groups), each subgroup of the element is also disclosed, and any element(s) may be removed from the group. In general, where this disclosure or an aspect of this disclosure is referred to as including specific elements and / or features, it should be understood that a certain aspect of this disclosure or an aspect of this disclosure consists of, or essentially consists of, such elements and / or features. For the sake of simplification, such aspects are not specifically represented herein. Also note that the terms “comprising” and “containing” are intended to be open, allowing for the inclusion of additional elements or steps. Where a scope is given, an endpoint is included within that scope unless otherwise specified. Furthermore, unless otherwise indicated or is evident from the context and the understanding of those skilled in the art, any value expressed as a range may, unless otherwise clearly indicated by the context, assume that any particular value or subrange within the defined range in various aspects of this disclosure is up to one-tenth of the lower limit unit of the range.

[0150] A person skilled in the art will recognize many equivalents to the particular embodiments described herein, or will be able to determine them using a few standard experimental methods. The scope of the embodiments described herein is not intended to be limited to the foregoing, but rather as expressed in the appended claims. A person skilled in the art will understand that various modifications and alterations to this description may be made as defined in the following claims, without departing from the spirit or scope of this disclosure.

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

[0152] The WLP001 H463F mutation inhibits indole production while increasing 3MH concentration. We investigated whether overexpression of IRC7, STR3, or TnaA in Saccharomyces brewer yeast enhances volatile thiol release during beer fermentation. Each of these genes was sequentially integrated into the ADE2 locus of California Ale Yeast, WLP001. A strong constitutive promoter, PGK1, was used to promote 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 beers fermented with STR3-overexpressing yeast cells or IRC7-overexpressing strains had odor profiles comparable to the wild-type control. In contrast, beers fermented with TnaA-overexpressing yeast cells had a distinctive, strong odor characterized as tropical / guava and excrement / diaper (i.e., off-odor).

[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 volatile thiols 3MH, 3MHA, and 4MMP in beers brewed with the wild-type strain were extremely low, notably below the assay's detection limit of 5 nanograms per liter (ng / L). Overexpression of STR3 or IRC7 had only 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, beer brewed using a yeast strain overexpressing TnaA (Y182) contained 229 ng / L of 3MH, an increase of more than 45 times compared to beer 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 transmit a strong excrement odor (Figure 3). From these data, it was concluded that TnaA expression in brewing yeast increases the concentrations of 3MH and other volatile thiols that transmit tropical fruit flavors in beer, but also increases the production of indole, an undesirable off-flavor product.

[0156] Previous research found that TnaA catalyzes indole production by cleaving tryptophan. 30 While we do not wish to be bound by any specific theory, we hypothesized that the increased indole production in beer fermented by yeast cells overexpressing TnaA was due to the cleavage of tryptophan by TnaA. Next, we modified TnaA by generating the TnaA-H463F variant by mutating the amino acid H463, thereby reducing its activity with tryptophan as a substrate while maintaining relatively high activity with cysteine ​​conjugate substrates, which are precursors to volatile thiols with tropical fruit flavors.

[0157] The activity of several TnaA mutants with the substrate tryptophan and its 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 more than 2 times, compared to only a 2-fold reduction in activity with S-ethyl-L-cysteine.

[0158] The TnaA-H463F mutation was integrated into the WLP001 strain and used to produce beer via malt 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, contained no excrement odor. GC / MS analysis revealed that the indole concentration in the beer fermented with TnaA-H463F-expressing yeast was negligible (Figure 3, Y502). Surprisingly, although H463F substitution had previously been reported to reduce the activity of the TnaA enzyme with its cysteine ​​conjugate, the 3MH concentration was increased by ~25% in the beer produced using TnaA-H463F-expressing yeast cells compared to the beer produced using wild-type TnaA-expressing yeast cells. The 3MH concentration in beer brewed with the TnaA-H463F mutant was 285 ng / L, which was 1.25 times and 56 times higher than that in beer brewed with Y182 and wild-type yeast strains, respectively.

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

[0160] method Construction of brewing yeast strains The TnaA encoding sequence used here was derived from Citrobacter amalonaticus, with codons optimized for expression in Saccharomyces yeast. This encoding sequence was synthesized by TWIST Bioscience (San Francisco, CA) and cloned into a plasmid sandwiched between the PGK1 promoter sequence and ENO1 terminator sequence derived from Saccharomyces cerevisiae. This plasmid also contained the Saccharomyces cerevisiae ADE2 encoding sequence and regulatory region, as well as a sequence homologous to the ADE2 locus, enabling genome insertion into brewing yeast via 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 the integration plasmid.

[0161] Prior to transformation into yeast, the plasmid was digested with restriction enzymes to produce linear DNA fragments containing ADE2 and the gene of interest alongside the ADE2 homology region. Brewing yeast strain WLP001, carrying a deletion of the ADE2 coding sequence, was transformed with the linear DNA, resulting in homologous recombination of the nucleic acid encoding the TnaA-H463F gene.

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

[0163] Beer brewing The strains were streaked onto YPD medium and grown at 25°C for 3 days. Using a single colony, it was inoculated into an initial 5 ml (mL) malt extract (ME, Sigma-Aldrich, St. Louis, MO, USA) culture in a glass culture tube and grown at 25°C for 1 day with shaking at 200 rpm. The resulting culture was then inoculated into 1 L (L) ME culture in a 2 L glass Erlenmeyer flask and grown at 25°C for 2 days with shaking at 200 rpm. The resulting culture was then inoculated into 20 L beer malt in a conical fermenter and grown at 20°C for 10 days.

[0164] For beer fermentation, 28.6 kilograms (kg) of two-row malt was milled and combined with 2.3 kg of oats, then added to 100 L of water treated with 39 g of brewing salt. Saccharification was carried out at 67°C for 60 minutes (min). The malt was recirculated for 10 minutes and separated by lautering. Sparing was performed for 37 minutes, resulting in a final pre-boiling volume of 146 L in the brewing kettle. The malt was boiled until it reached a final volume of 137 L and a specific gravity of 12.6 Platos. 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 malt from the hot tub, it was transferred to six 20L conical fermentation tanks (SS Brewtech, Temecula, CA, USA). The beer was fermented at 20°C until it reached its final specific gravity, then left in this state for another 24 hours to remove vicinaldiketones (VDK), and then cold-conditioned at 0°C to produce the finished beer. This was then analyzed for volatile thiols and indoles by gas chromatography-mass spectrometry.

[0165] GC / MS analysis 3-mercaptohexanol and indole were quantified by gas chromatography / mass spectrometry (GC / MS) analysis using an Agilent 6890 series GC and 5973N mass-selective detector (Agilent Technologies, Santa Clara, CA, USA) equipped with an electron ionization source operating 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 over an HP-5 ms column (Agilent, length 30 m, inner diameter (id) 0.25 mm, film thickness 0.25 μm). The oven temperature was set to 50°C for 3 min, followed by a 10°C / min gradient to 275°C for 1 min, and then a 50°C / min gradient to a final temperature of 325°C 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: 200 mL of finished beer was used for analysis to quantify 3-mercaptohexanol. 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 phase was combined and then washed with 20 mL NaHCO3 (0.3% w / v by volume, pH 6). Thiols were deprotonated and extracted from the organic phase by back-extraction into 6 mL of cold (4°C) 1N NaOH. The aqueous phase was then transferred to a 20 mL headspace vial. Residual pentane was removed by continuously flowing N2 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, the vial was screw-sealed, and derivatized at room temperature for 20 min. Next, 0.5 g of tartaric acid was added to reduce the sample pH to ~4.5. 2 g of NaCl was added before resealing the vial. The derivatized thiol was adsorbed onto a PDMS / DVB solid-phase microextraction fiber at 70°C for 1 hour. Then, the analyte was desorbed onto the column at 250°C for 10 minutes using a splitless injection. The derivatized thiol was 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). The absolute sample concentration was calculated using a linear model generated from a standard curve composed of true standards, with 181 as the quantitative (quant) ion.

[0167] To quantify indole, a 1 mL sample of beer was taken and extracted by vortexing with 0.5 mL of ethyl acetate for 10 seconds, followed by centrifugation at 15,000 × gravity for 10 minutes. The organic phase was transferred to a 1.5 mL tube, and residual water was removed by adding an excess of Na2SO4. The sample was then briefly vortexed and centrifuged at 15,000 × gravity for 5 minutes. The ethyl acetate was then transferred to a GC vial, and the resulting 1 μL extract was injected onto the column using a splitless injection. Indole was detected by selectively monitoring the m / z ions 63, 90, and 117. The peak area of ​​indole was quantified using Agilent MassHunter qualitative software. The absolute sample concentration was calculated using a linear model generated from a standard curve composed of true standards, with 117 as the quantitative 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, while beers brewed using WLP001(Y502) overexpressing the TnaA-H463F mutant contained low levels of indole.

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

[0170] Production of fermentation products The genetically modified yeast strains described herein were evaluated in the fermentation of beer and wine. 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 cultured and used to inoculate initial cultures. The resulting cultures were then used to inoculate larger cultures, which were grown for several days. These cultures were then used to inoculate fermenters, either in 20 L of malt in the case of beer fermentation, or in grape must or grape juice in the case of wine fermentation, and grown for several days until the desired final specific gravity was reached. As described in Example 1, the beer and wine were subsequently analyzed for volatile thiols and indoles by gas chromatography-mass spectrometry.

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

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

[0173] Yeast strains were cultured as described in Example 1. Early in the fermentation process, 3-mercaptohexane-1-ol (Glut-3-MH) conjugated to glutathione was added to the malt. Yeast strains were inoculated into the malt in the presence or absence of Glut-3-MH. The beer was fermented at 20°C until the final specific gravity was reached, and this state was maintained for another 24 hours to remove vicinaldiketones (VDK). The finished beer was then produced by cooling to 0°C and 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 additional Glut-3-MH in the fermentation process. Indole concentrations were also increased in the fermentation product of beer fermentation using strains expressing wild-type TnaA; however, indole concentrations in beer fermentation using strains expressing the TnaA H463F mutant were comparable to those of the wild-type parent strain (WLP001), in the presence or absence of additional Glut-3-MH in the fermentation process. The addition of precursor Glut-3MH to the fermentation process resulted in increased 3MH production compared to control fermentation without the addition of 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 lower 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 to arginine (H463R), glutamic acid (H463E), threonine (H463T), glycine (H463G), isoleucine (H463I), or valine (H463V) were generated by PCR mutagenesis, cloned into expression plasmids 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 strain was inoculated into malt juice and fermented at 20°C until the final specific gravity was reached. This state was then maintained for another 24 hours to remove vicinaldiketones (VDK), and then the mixture was placed under cooling conditions at 0°C to produce the finished beer. This beer was then 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 fermentation using strains expressing wild-type TnaA or the TnaA H463 mutant. Indole concentrations were also increased in the fermentation products of beer fermentation using strains expressing wild-type TnaA; however, indole concentrations in beer fermentation using strains expressing the TnaA H463 mutant were comparable to those of the wild-type parent strain (WLP001).

[0179] Evaluation of TnaA homologs A homolog of TnaA from C. amalonaticus was identified, cloned into an integration plasmid under the control of the PGK1 promoter, and transformed into yeast strains. See Table 1.

[0180] The yeast strain was inoculated into malt juice and fermented at 20°C until the final specific gravity was reached. This state was then maintained for another 24 hours to remove vicinaldiketones (VDK), and then the mixture was placed under cooling conditions at 0°C to produce the finished beer. This beer was then analyzed for volatile thiols and indoles by gas chromatography-mass spectrometry.

[0181] As shown in Figure 6, the 3MH concentration 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. ganghwensis (each exhibiting 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 fermentation using strains expressing the TnaA H463F mutant, or strains expressing TnaA homologs from A. saccharolyticus and Z. ganghwensis, 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 containing a heterologous gene encoding a beta-lyase variant having beta-lyase activity, wherein the beta-lyase variant contains an amino acid sequence having at least 90% sequence identity, and optionally at least 95% sequence identity, with one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 7, and Here, the amino acid sequence lacks histidine at residue 463 or its corresponding residue with respect to SEQ ID NO:

1. Here, the corresponding residue is determined using sequence alignment. The aforementioned genetically modified yeast cells.

2. The genetically modified yeast cell according to claim 1, wherein the beta-lyase variant having beta-lyase activity does not contain any of the sequences represented by SEQ ID NOs: 1, 6, and 7.

3. The genetically modified yeast cell according to claim 1, wherein the beta-lyase variant having beta-lyase activity has the sequence shown in Sequence ID No.

2.

4. The genetically modified yeast cell according to claim 1, wherein the beta-lyase variant having beta-lyase activity has the sequence represented by SEQ ID NO: 4 or 5.

5. A genetically modified yeast cell according to any one of claims 1 to 3, wherein the beta-lyase variant enzyme having beta-lyase activity comprises a substitution mutation at residue H463 or its corresponding residue with respect to SEQ ID NO: 1, wherein optionally the substitution mutation at residue H463 is phenylalanine, arginine, glutamic acid, threonine, glycine, isoleucine, or valine.

6. A genetically modified yeast cell according to any one of claims 1 to 5, wherein the yeast cell comprises the genus Saccharomyces.

7. The genetically modified yeast cells according to claim 6, wherein the yeast cells consist of the species Saccharomyces cerevisiae (S. cerevisiae) or Saccharomyces pastorianus (S. pastorianus).

8. The genetically modified yeast cell according to claim 7, wherein the yeast cell is S. cerevisiae California Ale Yeast strain WLP001 or Red Star Cote des Blancs.

9. A method for producing a fermentation product or a composition containing ethanol, comprising contacting genetically modified yeast cells according to any one of claims 1 to 8 with a culture medium containing at least one fermentable sugar, wherein the contact is carried out at least during the first fermentation process, thereby producing the fermentation product or the composition containing ethanol.

10. At least one fermentable sugar is provided in at least one sugar source; The method according to claim 9, wherein the fermentable sugar is glucose, fructose, sucrose, maltose, and / or maltotriose.

11. At least one sugar source contains at least one precursor, The method according to claim 10, wherein at least one precursor comprises 3-mercaptohexane-1-ol conjugated to cysteine ​​(Cys3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys4MMP), 3-mercaptohexane-1-ol conjugated to glutathione (Glut3-MH), and / or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut4MMP).

12. The method according to any one of claims 9 to 11, further comprising adding one or more precursors to a culture medium, wherein the precursors include 3-mercaptohexane-1-ol (Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine ​​(Cys 4MMP), 3-mercaptohexane-1-ol conjugated to glutathione (Glut 3-MH), and / or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut 4MMP).

13. The fermentation product contains at least one volatile thiol at an increased level compared to a fermentation product produced by equivalent cells that do not express heterologous genes or by equivalent cells that express a wild-type enzyme having beta-lyase activity; The method according to any one of claims 9 to 11, wherein at least one volatile thiol comprises 3-mercaptohexane-1-ol (3MH), 3-mercaptoacetate hexyl (3MHA), 4-methyl-4-mercaptopenta-2-one (4MMP), or a combination thereof.

14. The method according to claim 13, wherein the fermentation product contains at least 200 ng / L of 3-mercaptohexane-1-ol (3MH).

15. The fermentation product contains at least one undesirable product at a reduced level compared to a fermentation product produced by equivalent cells that do not express heterologous genes or by equivalent cells that express wild-type enzymes having beta-lyase activity; The method according to any one of claims 9 to 14, wherein at least one undesirable product is indole.

16. The method according to any one of claims 9 to 15, wherein the fermentation product or the composition containing ethanol is a fermented beverage.

17. The method according to claim 16, wherein the fermented beverage is beer, wine, sparkling wine (champagne), sake, mead, kelp tea, or apple cider.

18. The method according to any one of claims 9 to 17, wherein the sugar source comprises malt juice, fruit juice, honey, rice starch, or a combination thereof.

19. The method according to claim 18, wherein the fruit juice is grape juice or apple juice.

20. The sugar source is malt juice, and The method further includes producing a culture medium, wherein the production of the culture medium is as follows: (a) bringing multiple grains into contact with water; and (b) producing malt juice by boiling or soaking water and grain. The method according to any one of claims 9 to 19, including the method described in any one of claims 9 to 19.

21. The method according to claim 20, further comprising adding at least one hop variety to the malt to produce a hopped malt.

22. The method according to any one of claims 9 to 20, further comprising adding at least one hop variety to the culture medium.

23. The method according to any one of claims 9 to 22, further comprising at least one additional fermentation process and / or introducing carbon dioxide into the fermentation product.

24. A fermentation product produced by the method described in any one of claims 9 to 23.

25. The fermentation product contains at least 200 ng / L of 3-mercaptohexane-1-ol (3MH), and / or The fermentation product according to claim 24, wherein the fermentation product contains less than 500 μg / L of indole.

Images