Materials and methods for brewing beer

Abstract

Described herein are materials and methods for converting the non-volatile form of 3-sulfanyl-1-hexanol (3SH) to free 3SH during the brewing process.

Description

[Background technology]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 147,964, filed on 10 February 2021, and U.S. Provisional Patent Application No. 63 / 292,226, filed on 21 December 2021, the disclosures of which are incorporated herein by reference in their entirety.

[0002] Integration by referencing electronically submitted information This application includes, as a separate part of the present disclosure, a sequence list in computer-readable format (file name: 56400_Seqlisting.txt; size: 42,808 bytes; creation date: February 9, 2022), which is incorporated in its entirety by reference.

[0003] In the beverage industry, fruity and floral aromas are desired, and continuous efforts are being made to improve the aroma of beer by increasing or diversifying flavor profiles. Thiols, also known as mercaptans, are sulfur-containing organic compounds having a sulfur atom bonded to a hydrogen atom. Winemakers have identified thiols that contribute to the aroma of wine. One is 4-methyl-4-sulfanylpentan-2-one (4MSP, also known as 4-mercapto-4-methylpentan-2-one (4MMP)), which imparts the odor and taste of boxwood, blackcurrant, and ribe. Another is 3-sulfanyl-1-hexanol (3SH; also known as 3-mercaptohexan-1-ol (3MH)), which is often described as exotic and having the odor of passion fruit, rhubarb, and citrus. Thirdly, 3-sulfanylhexyl acetate (3SHA, also known as 3-mercaptohexyl acetate (3MHA)) evokes associations of passion fruit and guava. All of these compounds are prominent in Sauvignon blanc, Riesling, and other wines, but are not abundant as free aromatic thiols in grapes. They are formed during fermentation from precursors present in grape must.

[0004] Several hop varieties contain large amounts of precursor forms of these thiols, but only small amounts of the free aromatic forms that contribute to the aroma or flavor of the product. Therefore, there still exists a need in the art for means to release these volatile aromatic thiols from their precursor forms during fermentation to maximize the aromatic potential of beer. SUMMARY OF THE INVENTION

[0005] The present disclosure also provides a recombinant yeast comprising a polynucleotide encoding yeast β-lyase enzyme Irc7 operably linked to a heterologous promoter, wherein the β-lyase enzyme comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1.

[0006] The present disclosure provides a recombinant Saccharomyces spp. comprising a polynucleotide encoding yeast β-lyase enzyme IRC7 operably linked to a heterologous promoter, wherein the β-lyase enzyme comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1.

[0007] The present disclosure also provides a recombinant yeast comprising a polynucleotide encoding cysteine-thiol lyase operably linked to a heterologous promoter. In some embodiments, the cysteine-thiol lyase is PatB. In some embodiments, PatB is derived from a species of the genus Staphylococcus. In some embodiments, PatB is derived from S. lugdunensis, S. devriesei, S. hominis, S. haemolyticus, S. petrasii, or B. subtilis. In some embodiments, PatB is derived from S. petrasii croceilyticus or S. petrasii petrasii. In some embodiments, PatB comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 8 to 14. In some embodiments, PatB comprises the amino acid sequence set forth in any one of SEQ ID NOs: 8 to 14.

[0008] In some embodiments, the heterologous promoter is the TDH3, TDH2, CCW12, PGK1, ADH1, ADH2, CYC1, HHF1, HHF2, TEF1, TEF2, HTB2, PAB1, ALD6, RNR1, RNR2, POP6, RAD27, PSP2, REV1, MFA1, MFa2, GAL1, CUP1, MET25, ICL1, ICL2, GAL3, HXT1, HXT2, MAL11, MAL31, MAL32, MAL33, MRK1, or SUC2 promoter. In some embodiments, the recombinant Saccharomyces spp is S. cerevisiae or S. pastorianus. In some embodiments, the β-lyase enzyme contains the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the β-lyase enzyme does not contain the amino acid sequence described in SEQ ID NO: 3.

[0009] This disclosure also provides a method for converting a non-volatile form of 3-sulfanyl-1-hexanol (3SH) to free 3SH during the brewing process, the method comprising contacting cooled malt with recombinant Saccharomyces described herein for a period of time and under conditions sufficient to convert the non-volatile form of 3SH to free 3SH. In some embodiments, the non-volatile form of 3SH is glutathione-bound 3SH, cysteine-bound 3SH, or a combination thereof. In some embodiments, this method results in a threefold increase in free 3SH in malt fermented with recombinant Saccharomyces compared to malt fermented with unmodified Saccharomyces. In some embodiments, the malt contains at least 60 ng / L of free 3SH after the contact step.

[0010] This method optionally includes adding hops to cooled malt during the contact step. Examples of hops for use in the method herein include, but are not limited to, Cascade, Calypso, Hallertau Tradition, Hallertau Perle, Triple Pearl, Nugget, Saaz, Columbus / CTZ, Chinook, Nelson Sauvin, Hallertau Blancor, or Simcoe. In some embodiments, the hops contain at least 400 μg / kg of cysteine-linked 3SH.

[0011] The disclosure also provides a method for converting the non-volatile form of 3-sulfanyl-1-hexanol (3SH) to free 3SH during the brewing process, the method comprising: (a) a mash-hopping step, which includes adding a plant containing the non-volatile form of 3SH to milling grain and mashing it to produce malt; (b) boiling the malt produced by (a); (c) cooling the malt; and (d) contacting the cooled malt with the recombinant yeast described herein for a time sufficient to convert the non-volatile form of 3SH to free 3SH. In some embodiments, the recombinant yeast is recombinant Saccharomyces containing a polynucleotide encoding the β-lyase enzyme Irc7 operably linked to a heterologous promoter. In some embodiments, the recombinant yeast is recombinant Saccharomyces spp. containing a polynucleotide encoding a cysteine-thiol lyase operably linked to a heterologous promoter. In some embodiments, the non-volatile form of 3SH is glutathione-linked 3SH, cysteine-linked 3SH, or a combination thereof. In some embodiments, this method results in a six-fold increase in free 3SH in malt fermented with recombinant yeast compared to malt fermented with unmodified yeast. In some embodiments, the malt contains at least 60 ng / L of free 3SH after the contact step.

[0012] In some embodiments, the plant material containing a non-volatile form of 3SH is hops. In some embodiments, hops contain at least 400 μg / kg of cysteine-bound 3SH. In some embodiments, hops contain at least 5000 μg / kg of glutathione 3SH. In some embodiments, hops contain at least 400 μg / kg of cysteine-bound 3SH and at least 5000 μg / kg of glutathione 3SH. In some embodiments, hops are Cascade, Calypso, Hallertau Tradition, Hallertau Perle, Triple Pearl, Nugget, Saaz, Columbus / CTZ, Chinook, Nelson Sauvin, Hallertau Blanc, or Simcoe hops.

[0013] In some embodiments, the plant material containing a non-volatile form of 3SH is a grape-derived product. In some embodiments, the grape-derived product is crushed grapes or grape powder. In some embodiments, the grape-derived product is obtained from white grapes, red grapes, or a combination thereof. Exemplary white grape varieties include, but are not limited to, Sauvignon Blanc, Chardonnay, Chenin Blanc, Colombard, Gewurztraminer, Gros Manseng, Koshu, Maccabeo, Muscat, Petit Manseng, Pinot Blanc, Pinot Gris, Riesling, Scheurebe, Semillon, Sylvaner, and Tokay. Representative red grape varieties include, but are not limited to, Cabernet Franc, Cabernet Sauvignon, Grenache, Merlot, and Pinot Noir.

[0014] In some embodiments, the mash-hopping step includes adding less than 100 grams of plant material per kilogram of milling grain.

[0015] In some embodiments, the mash-hopping step includes adding both hop and grape-derived products to the milling grain.

[0016] In some embodiments, the β-lyase is a bacterial β-lyase or a fungal β-lyase. Exemplary bacterial β-lyases for use in accordance with this disclosure include, but are not limited to, Eschericia sp.; Thermoanaerobacter sp.; Symbiobacterium sp.; Photobacterium sp.; Haemophilus sp.; Vibrio sp.; Proteus sp.; Halobacterium sp.; Desulfitobacterium sp.; and Treponema sp. In some embodiments derived from bacteria, the bacterial β-lyase is E. coli TNaA.

[0017] In some embodiments, the fungal β-lyase is yeast β-lyase. Exemplary fungal β-lyases for use according to this disclosure are derived from Saccharomycotina, Taphrnomycotina, and Schizosaccharomycetes. In some embodiments, the yeast β-lyase is derived from Saccharomyces. In some embodiments, the yeast β-lyase is Irc7. In some embodiments, Irc7 contains an amino acid sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence described in SEQ ID NO: 1. In some embodiments, Irc7 contains the amino acid sequence described in SEQ ID NO: 1.

[0018] In some embodiments, the mash-hopping step includes a protein rest before the boiling step. In some embodiments, the protein rest includes maintaining the mash at a temperature below 140°F for at least 5 minutes before the boiling step. In some embodiments, the protein rest includes maintaining the mash at a temperature between 100°F and 140°F for at least 1 hour before the boiling step. In some embodiments, the mash-hopping step further includes a saccharification rest after the protein rest and before the boiling step.

[0019] In some embodiments, the method further comprises contacting cooled malt with hops to produce a mixture, and then contacting the mixture with recombinant yeast. In some embodiments, the recombinant yeast is S. cerevisiae or S. pastorianus.

[0020] In some embodiments, the contact step (d) occurs in a fermenter at a temperature in the range of 45°F to 100°F. [Brief explanation of the drawing]

[0021] [Figure 1] This is the alignment of the IRC7 allele described in Example 1. [Figure 2] This bar graph shows that free 3SH levels are increased in beer fermented with OYL-088-TDH3-IRC7 compared to the unmodified OYL-088 strain. [Modes for carrying out the invention]

[0022] Many plant-derived products contain volatile thiols bound as cysteine ​​S-conjugated precursors, and the conversion of non-volatile precursors to volatile thiol products contributes to the aroma of food or beverage products. Volatile thiols play a role in imparting the aromas of boxwood, passion fruit, grapefruit, gooseberry, and guava to fermented beverages such as beer.

[0023] This disclosure is partly based on the discovery that yeast (e.g., Saccharomyces spp.) modified to overexpress yeast β-lyase enzyme (or cysteine-thiol lyase) during the fermentation process of a brewing process causes the conversion of available non-volatile thiols (e.g., glutathione-bound thiols or cysteine-bound thiols) in plant material to more desirable aromatic (i.e., volatile or free) forms.

[0024] This disclosure is also partly based on the discovery that modifying conventional extraction methods to include a mashing step, which involves adding a plant material containing the target nonvolatile thiol to milling grain to produce malt juice, and then contacting the malt juice with a modified Saccharomyces spp. that overexpresses a β-lyase enzyme (or cysteine-thiol lyase), results in a high conversion rate of the available nonvolatile thiol to its volatile, aromatic form.

[0025] Recombinant yeast. In one embodiment, the recombinant yeast described herein comprises a polynucleotide encoding a β-lyase (or cysteine-thiol-lyase) enzyme operably linked to a heterologous promoter. The term "heterologous promoter," as used herein, refers to a promoter that is non-natural to the β-lyase (or cysteine-thiol-lyase) enzyme.

[0026] In some embodiments, the yeast does not belong to the genus Saccharomyces. In some embodiments, the yeast belongs to the genera Kloeckera, Candida, Starmerella, Hanseniaspora, Kluyveromyces / Lachance, Metschnikowia, Saccharomycodes, Zygosaccharomyce, Dekkera (also known as Brettanomyces), Wickerhamomyces, or Torulaspora. In some embodiments, the yeast is Hanseniaspora uvarum, Hanseniaspora guillermondii, Hanseniaspora vinae, Metschnikowia pulcherrima, Kluyveromyces / Lachancea thermotolerans, Starmerella bacillaris (formerly known as Candida stellatal or Candida zemplinina), Saccharomycodes ludwigii, Zygosaccharomyces rouxii, Dekkera bruxellensis, Dekkera anomala, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Wickerhamomyces anomalus, or Torulaspora delbrueckii.

[0027] In another embodiment, the recombinant Saccharomyces spp. described herein comprises a polynucleotide encoding a β-lyase (or cysteine-thiol lyase) enzyme operably linked to a heterologous promoter.

[0028] In various embodiments, recombinant Saccharomyces is either S. cerevisiae or S. pastorianus.

[0029] β-lyases are enzymes responsible for the release of polyfunctional thiols, or volatile sulfur compounds called mercaptans, typically associated with tropical aromas. Exemplary β-lyase enzymes for use in accordance with this disclosure include, but are not limited to, those described in International Publication No. 2007 / 095682, the entirety of which is incorporated herein by reference. In some embodiments, the β-lyase is a bacterial β-lyase such as Eschericia sp.; Thermoanaerobacter sp.; Symbiobacterium sp.; Photobacterium sp.; Haemophilus sp.; Vibrio sp.; Proteus sp.; Halobacterium sp.; Desulfitobacterium sp.; or Treponema sp. In some embodiments, the β-lyase is tryptophanase (E. coli) (UniProt accession number P0A853).

[0030] In some embodiments, the β-lyase is a fungal β-lyase, such as yeast β-lyase derived from Saccharomyces cerevisiae (all strains), Saccharomyces bayanus, and species of Brettanomyces and Dekkera; Candida; Cryptococcus; Debaryomyces; Hanseniaspora, Kloeckera; Kluyveromyces; Metschnikowia; Pichia; Rhodotorula; Saccharomyces; Saccharomycodes; Schizosaccharomyces or Zygosaccharomyces. In some embodiments, the β-lyase is IRC7. In some embodiments, the β-lyase contains an amino acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence described in Sequence ID No. 1.

[0031] In some embodiments, cysteine-thiol lyase has a Kcat / Km ratio of 0.7 × 10⁻⁶ to cystathionine. 2 minutes-1 mM -1 below (for example, 0.7×10 2 / -1 mM -1 , 0.6×10 2 / -1 mM -1 , 0.5×10 2 / -1 mM -1 , 0.4×10 2 / -1 mM -1 , 0.3×10 2 / -1 mM -1 , 0.2×10 2 / -1 mM -1 , 0.1×10 2 / -1 mM -1 or less). In some embodiments, the Kcat / Km of cysteine-thiol lyase for Cys-3M3SH is 3×10 2 / -1 mM -1 or more (for example, 3×10 2 / -1 mM -1 , 3.5×10 2 / -1 mM -1 , 4×10 2 / -1 mM -1 , 4.5×10 2 / -1 , 5×10 2 / -1 mM -1 , 5.5×10 2 / -1 mM -1 , 6×10 2 / -1 mM -1 , 6.5×10 2 / -1 mM -1 , 7×10 2 / -1 mM -1 , 7.5×10 2 / -1 mM -1 , 8×10 2 / -1 mM -1, 8.5×10 2 minutes -1 mM -1 , 9×10 2 minutes -1 mM -1 , 9.5×10 2 minutes -1 mM -1 (That's all.)

[0032] In some embodiments, the cysteinethiole lyase is PatB. In some embodiments, PatB is derived from the genus Staphylococcus. In some embodiments, PatB is derived from S. lugdunensis (SEQ ID NO: 9), S. devriesei (SEQ ID NO: 10), S. hominis (SEQ ID NO: 8), S. haemolyticus (SEQ ID NO: 13), S. petrasii (SEQ ID NO: 11 or 12), or B. subtilis (SEQ ID NO: 14).

[0033] In another exemplary embodiment, the Disclosure provides a recombinant yeast comprising a polynucleotide encoding PatB operably linked to a heterologous promoter, wherein PatB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 8 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical). In some embodiments, PatB comprises the amino acid sequence described in SEQ ID NO: 8.

[0034] In another exemplary embodiment, the Disclosure provides a recombinant yeast comprising a polynucleotide encoding PatB operably linked to a heterologous promoter, wherein PatB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 9 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or identical). In some embodiments, PatB comprises the amino acid sequence described in SEQ ID NO: 9.

[0035] In another exemplary embodiment, the Disclosure provides a recombinant yeast comprising a polynucleotide encoding PatB operably linked to a heterologous promoter, wherein PatB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 10 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical). In some embodiments, PatB comprises the amino acid sequence described in SEQ ID NO: 10.

[0036] In another exemplary embodiment, the Disclosure provides a recombinant yeast comprising a polynucleotide encoding PatB operably linked to a heterologous promoter, wherein PatB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 11 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical). In some embodiments, PatB comprises the amino acid sequence described in SEQ ID NO: 11.

[0037] In another exemplary embodiment, the Disclosure provides a recombinant yeast comprising a polynucleotide encoding PatB operably linked to a heterologous promoter, wherein PatB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 12 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical). In some embodiments, PatB comprises the amino acid sequence described in SEQ ID NO: 12.

[0038] In another exemplary embodiment, the Disclosure provides a recombinant yeast comprising a polynucleotide encoding PatB operably linked to a heterologous promoter, wherein PatB comprises an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 13 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical). In some embodiments, PatB comprises the amino acid sequence described in SEQ ID NO: 13.

[0039] In another exemplary embodiment, the Disclosure provides a recombinant yeast comprising a polynucleotide encoding PatB operably bound to a heterologous promoter, wherein PatB comprises an amino acid sequence that is at least 80% identical (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical) to the amino acid sequence described in SEQ ID NO: 14. In some embodiments, PatB comprises the amino acid sequence described in SEQ ID NO: 14.

[0040] In some embodiments, recombinant yeast containing a polynucleotide encoding a cysteinethiol lyase (e.g., PatB) promotes the release of 4-methyl-4-sulfanylpentan-2-one (4MSP) at approximately twice the rate compared to recombinant yeast containing a polynucleotide encoding a TnaA enzyme.

[0041] The terms "operably linked" or "functionally linked" refer to the relationship between nucleic acid sequences on a single nucleic acid fragment such that the function of one influences the function of the other. For example, if two sequences are positioned such that a regulatory DNA sequence influences the expression of a coding DNA sequence (i.e., the coding sequence or functional RNA is under the transcriptional control of a promoter), the regulatory DNA sequence is said to be "operably linked" or "associated" with the DNA sequence encoding the RNA or polypeptide. The coding sequence may be operably linked to a regulatory sequence in either the sense or antisense direction.

[0042] The term "promoter" typically refers to the upstream (5') nucleotide sequence of its coding sequence, which controls the expression of the coding sequence by providing recognition sites for RNA polymerase and other factors necessary for proper transcription. A "promoter" may sometimes include a minimal promoter, which is a short DNA sequence containing a TATA box and other sequences that help identify the transcription start site, to which regulatory elements may be added to enhance expression. A "promoter" can also refer to a nucleotide sequence that includes a minimal promoter + regulatory elements and can control the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter often referred to as an enhancer. Thus, an "enhancer" is a DNA sequence that can stimulate promoter activity and may be an innate or heterologous element of a promoter inserted to increase the promoter's level or tissue specificity. It can function in both orientations (normal or reversed) and can function whether it moves upstream or downstream of the promoter. Both enhancers and other upstream promoter elements bind to sequence-specific DNA-binding proteins that mediate their effects. Promoters may be entirely derived from natural genes, composed of different elements, derived from different promoters found in nature, or composed of synthetic DNA segments.

[0043] The promoter may also contain DNA sequences involved in the binding of protein factors, which regulate the effectiveness of transcription initiation in response to physiological or developmental states. The “initiation site” is the position surrounding the first nucleotide, which is part of the transcription sequence, and is also defined as position + 1. With respect to this site, all other sequences of the gene and its regulatory region are numbered. Downstream sequences (i.e., further protein-coding sequences in the 3′ direction) are considered positive, while upstream sequences (most of the regulatory region in the 5′ direction) are considered negative.

[0044] Examples of promoters include, but are not limited to, the TDH3, TDH2, CCW12, PGK1, ADH1, ADH2, CYC1, HHF1, HHF2, TEF1, TEF2, HTB2, PAB1, ALD6, RNR1, RNR2, POP6, RAD27, PSP2, REV1, MFA1, MFa2, GAL1, CUP1, MET25, ICL1, ICL2, GAL3, HXT1, HXT2, MAL11, MAL31, MAL32, MAL33, MRK1, and SUC2 promoters. In some embodiments, the promoter is the TDH3 promoter.

[0045] In exemplary embodiments, the Disclosure provides recombinant yeast (e.g., Saccharomyces spp.) comprising a polynucleotide encoding the yeast β-lyase enzyme Irc7 operably linked to a heterologous promoter, wherein the β-lyase enzyme comprises an amino acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99% or more identical) to the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the β-lyase enzyme comprises the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the β-lyase enzyme does not comprise the amino acid sequence described in SEQ ID NO: 3.

[0046] Techniques for the recombinant expression of enzymes within cells and for the genetic modification of recombinant yeast cells are well known to those skilled in the art. Typically, such techniques involve the transformation of cells using nucleic acid constructs containing the relevant sequences. Such methods can be found in standard manuals such as Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3rd edition),” Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al., eds., “Current protocols in molecular biology,” Green Publishing and Wiley Interscience, New York (1987). Methods for the transformation and genetic modification of fungal host cells are described, for example, in European Patent Application No. EP-A-0635574, International Patent Publication No. 98 / 46772, International Patent Publication No. 99 / 60102, International Patent Publication No. 00 / 37671, International Patent Publication No. 90 / 14423, European Patent Application No. EP-A-0481008, European Patent Application No. EP-A-0635574, and U.S. Patent No. 6,265,186, the disclosures of which are incorporated herein by reference in their entirety.

[0047] method This disclosure provides a method for converting a non-volatile form of 3-sulfanyl-1-hexanol (3SH) to free 3SH during a brewing process. In one embodiment, the method involves contacting a fermentable sugar source (e.g., chilled malt) with recombinant yeast as described herein (e.g., recombinant Saccharomyces spp. comprising a polynucleotide encoding yeast β-lyase enzyme IRC7 having an amino acid sequence at least 95% identical to the amino acid sequence described in SEQ ID NO: 1, operably linked to a heterologous promoter; (or recombinant Saccharomyces spp. comprising a polynucleotide encoding stain-thiol lyase PatB, operably linked to a heterologous promoter) for a period of time and under conditions sufficient to convert the non-volatile form of 3SH to free 3SH.

[0048] In another embodiment, the disclosure provides a method for converting the non-volatile form of 3-sulfanyl-1-hexanol (3SH) to free 3SH in a brewing process, the method comprising contacting a fermentable sugar source with cysteine-thiol lyase for a period of time and under conditions sufficient to convert the non-volatile form of 3SH to free 3SH. In some embodiments, the cysteine-thiol lyase is purified before the contact step. Purification of the cysteine-thiol lyase may be carried out in accordance with the description in Rudden et al., Scientific Reports, 10:12500, 2020, the disclosure of which is incorporated herein by reference.

[0049] In some embodiments, the fermentable sugar source is malt juice, grains / cereals, fruit juice (e.g., grape juice, apple juice / cider), honey, cane sugar, rice, or koji.

[0050] In some embodiments, the non-volatile form of 3SH is glutathione-linked 3SH, cysteine-linked 3SH, or a combination thereof. In some embodiments, this method results in a threefold increase in free 3SH in a fermentable sugar source (e.g., malt) fermented with recombinant yeast (e.g., Saccharomyces) compared with a fermentable sugar source (e.g., malt) fermented with unmodified yeast (e.g., Saccharomyces).

[0051] In some embodiments, the method results in a threefold increase in free 3SH in a fermentable sugar source (e.g., malt) using cysteine-thiol lyase compared to a fermentable sugar source (e.g., malt) not using cysteine-thiol lyase.

[0052] In some embodiments, the cysteinethiole lyase is PatB. In some embodiments, PatB is derived from a species of the genus Staphylococcus. In some embodiments, PatB is derived from S. lugdunensis (SEQ ID NO: 9), S. devriesei (SEQ ID NO: 10), S. hominis (SEQ ID NO: 8), S. haemolyticus (SEQ ID NO: 13), S. petrasii (SEQ ID NO: 11 or 12), or B. subtilis (SEQ ID NO: 14).

[0053] In some embodiments, PatB includes an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 8 (for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical). In some embodiments, PatB includes the amino acid sequence described in SEQ ID NO: 8.

[0054] In some embodiments, PatB includes an amino acid sequence that is at least 80% identical to the amino acid sequence described in Sequence ID No. 9 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical). In some embodiments, PatB includes the amino acid sequence described in Sequence ID No. 9.

[0055] In some embodiments, PatB includes an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 10 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical). In some embodiments, PatB includes the amino acid sequence described in SEQ ID NO: 10.

[0056] In some embodiments, PatB includes an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 11 (for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical). In some embodiments, PatB includes the amino acid sequence described in SEQ ID NO: 11.

[0057] In some embodiments, PatB includes an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 12 (for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical). In some embodiments, PatB includes the amino acid sequence described in SEQ ID NO: 12.

[0058] In some embodiments, PatB includes an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 13 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical). In some embodiments, PatB includes the amino acid sequence described in SEQ ID NO: 13.

[0059] In some embodiments, PatB includes an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 14 (for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical). In some embodiments, PatB includes the amino acid sequence described in SEQ ID NO: 14.

[0060] The disclosure also provides a method for converting a non-volatile form of 3-sulfanyl-1-hexanol (3SH) to free 3SH during the brewing process, the method comprising: (a) a mash-hopping step, which includes adding a plant containing a non-volatile form of 3SH to milling grains, mashing, and producing malt; (b) boiling the malt produced by (a); (c) cooling the malt; and (d) contacting the cooled malt with recombinant Saccharomyces as described herein, for a period of time and under conditions sufficient to convert the non-volatile form of 3SH to free 3SH. In some embodiments, the non-volatile form of 3SH is glutathione-linked 3SH or cysteine-linked 3SH, or a combination thereof.

[0061] In some embodiments, a method for converting a non-volatile form of 3-sulfanyl-1-hexanol (3SH) to free 3SH during the brewing process may be carried out in the absence of recombinant yeast containing a cysteine-thiol lyase polynucleotide operably linked to a heterologous promoter. In this regard, the present disclosure also provides a method for converting a non-volatile form of 3-sulfanyl-1-hexanol (3SH) to free 3SH during the brewing process, the method comprising: (a) a mash-hopping step, which includes adding a plant containing a non-volatile form of 3SH to milling grain, mashing it, and producing malt; (b) boiling the malt produced by (a); (c) cooling the malt; and (d) contacting the cooled malt with purified cysteine-thiol lyase for a period of time and under conditions sufficient to convert the non-volatile form of 3SH to free 3SH. In some embodiments, the non-volatile form of 3SH is glutathione-linked 3SH or cysteine-linked 3SH, and combinations thereof.

[0062] The phrase “plant material containing a non-volatile form of 3SH” refers to any plant (or plant part) containing glutathione-linked 3SH, cysteine-linked 3SH, or a combination thereof. The term “plant part” encompasses all components of a plant, including seeds, buds, stems, leaves, roots, flowers, and plant tissues. In some embodiments, the plant material is processed before being added to the grain for milling. Exemplary methods of processing the plant material include, but are not limited to, crushing, pressurizing, slicing, mixing, sapling, rolling, grinding, or pulverizing the plant material.

[0063] In some embodiments, the plant material containing the non-volatile form of 3SH is hops. Hops suitable for use in the methods described herein include Amarillo, Apollo, Cascade, Centennial, Chinook, Citra, Cluster, Columbus, Crystal, Eroica, Galena, Glacier, Greenburg, Horizon, Liberty, Millennium, Mount Hood, Mount Rainier, Newport, Nugget, Palisade, Santiam, Simcoe, Sterling, Summit, Tomahawk, Ultra, Vanguard, Warrior, Willamette, Zeus, Admiral, Brewer's Gold, Bullion, Challenger, First Gold, Fuggles, Goldings, Herald, Northdown, Northern Brewer, Phoenix, Pilot, Pioneer, Progress, Target, Whitbread Golding Variety (WGV), Hallertau, Hersbrucker, Saaz, Tettnang, Spalt, and 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, Strissselspalt, 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, HallertauerExamples of hops that may be used include, but are not limited to, Taurus, Merkur, Opal, Smaragd, Hallertau Aroma, Kohatu, Rakau, Stella, Sticklebract, Summer Saaz, Super Alpha, Super Pride, Topaz, Wai-iti, Bor, Junga, Marynka, Premiant, Sladek, Styrian Atlas, Styrian Aurora, Styrian Bobek, Styrian Celeia, Sybilla, and Sorachi Ace hops. In some embodiments, the hops may be Cascade, Calypso, Hallertau Tradition, Hallertau Perle, Triple Pearl, Nugget, Saaz, Columbus / CTZ, Chinook, Nelson Sauvin, Hallertau Blanc, and / or Simcoe hops.

[0064] In some embodiments, the hops contain at least about 400 μg / kg of cysteine-linked 3SH. For example, in some embodiments, the hops contain at least about 400 μg / kg, at least about 450 μg / kg, at least about 500 μg / kg, at least about 550 μg / kg, at least about 600 μg / kg, at least about 650 μg / kg, at least about 700 μg / kg, at least about 750 μg / kg, at least about 800 μg / kg, at least about 850 μg / kg, at least about 900 μg / kg, at least about 950 μg / kg, or at least about 1000 μg / kg of cysteine-linked 3SH. In some embodiments, the hops contain cysteine-linked 3SH in amounts ranging from about 400 μg / kg to about 1000 μg / kg, or about 500 μg / kg to about 900 μg / kg, or about 600 μg / kg to about 800 μg / kg, or about 400 μg / kg to about 600 μg / kg.

[0065] In some embodiments, hops contain at least about 5000 μg / kg of glutathione 3SH. For example, in some embodiments, hops contain at least about 5000 μg / kg, at least about 5500 μg / kg, at least about 6000 μg / kg, at least about 6500 μg / kg, at least about 7000 μg / kg, at least about 7500 μg / kg, at least about 5000 μg / kg, at least about 8500 μg / kg, at least about 9000 μg / kg, at least about 9500 μg / kg, or at least about 10,000 μg / kg, at least about 10,500 μg / kg, at least about 11,000 μg / kg, at least about 11,500 μg / kg, at least about 12,000 μg / kg, at least about 12,500 μg / kg. g contains at least about 13,000 μg / kg, at least about 13,500 μg / kg, at least about 14,000 μg / kg, at least about 14,500 μg / kg, at least about 15,000 μg / kg, at least about 15,500 μg / kg, at least about 16,000 μg / kg, at least about 16,500 μg / kg, at least about 17,000 μg / kg, at least about 17,500 μg / kg, at least about 18,000 μg / kg, at least about 18,500 μg / kg, at least about 19,000 μg / kg, at least about 19,500 μg / kg, or at least about 20,000 μg / kg of glutathione-bound 3SH. In some embodiments, hops contain glutathione-bound 3SH in amounts ranging from approximately 5000 μg / kg to approximately 1000 μg / kg, or approximately 5500 μg / kg to approximately 9000 μg / kg, or approximately 6000 μg / kg to approximately 8000 μg / kg, or approximately 4000 μg / kg to approximately 6000 μg / kg, or approximately 5000 μg / kg to approximately 8000 μg / kg, or approximately 8000 μg / kg to approximately 12,000 μg / kg, or approximately 10,000 μg / kg to approximately 20,000 μg / kg, or approximately 15,000 μg / kg to approximately 20,000 μg / kg.

[0066] In some embodiments, the plant material containing a non-volatile form of 3SH is a grape-derived product. Preferred grape-derived products include, for example, crushed grapes or grape powder. The grape-derived product may be obtained from white grapes, red grapes, or a combination thereof. Exemplary white grape varieties include, but are not limited to, Sauvignon Blanc, Chardonnay, Chenin Blanc, Colombard, Gewurztraminer, Gros Manseng, Koshu, Maccabeo, Muscat, Petit Manseng, Pinot Blanc, Pinot Gris, Riesling, Scheurebe, Semillon, Sylvaner, and Tokay. Representative red grape varieties include, but are not limited to, Cabernet Franc, Cabernet Sauvignon, Grenache, Merlot, and Pinot Noir.

[0067] In some embodiments, the grape-derived product contains at least about 400 μg / kg of cysteine-bonded 3SH. For example, in some embodiments, the grape-derived product contains at least about 400 μg / kg, at least about 450 μg / kg, at least about 500 μg / kg, at least about 550 μg / kg, at least about 600 μg / kg, at least about 650 μg / kg, at least about 700 μg / kg, at least about 750 μg / kg, at least about 800 μg / kg, at least about 850 μg / kg, at least about 900 μg / kg, or at least about 950 μg / kg, or at least about 1000 μg / kg, or less At least approximately 1500 μg / kg, or at least approximately 2000 μg / kg, or at least approximately 2500 μg / kg, or at least approximately 3000 μg / kg, or at least approximately 3500 μg / kg, or at least approximately 4000 μg / kg, or at least approximately 4500 μg / kg, or at least approximately 5000 μg / kg, at least approximately 5500 μg / kg, at least approximately 6000 μg / kg, at least approximately 6500 μg / kg, at least approximately 7000 μg / kg, at least approximately 7500 μg / kg, at least approximately 8000 μg / kg kg, at least about 8500 μg / kg, at least about 9000 μg / kg, at least about 9500 μg / kg, or at least about 10,000 μg / kg, at least about 10,500 μg / kg, at least about 11,000 μg / kg, at least about 11,500 μg / kg, at least about 12,000 μg / kg, at least about 12,500 μg / kg, at least about 13,000 μg / kg, at least about 13,500 μg / kg, at least about 14,000 μg / kg, at least 14,500 μg / kg kg, at least about 15,000 μg / kg, at least about 15,500 μg / kg, at least about 16,000 μg / kg, at least about 16,500 μg / kg, at least about 17,000 μg / kg, at least about 17,500 μg / kg, at least about 18,000 μg / kg, at least about 18,500 μg / kg, at least about 19,000 μg / kg, at least about 19,500 μg / kg, or at least about 20,000 μg / kg, or at least 20,500 μg / kg, or at least 21,000 μg / kg, or at least 21,500 μg / kg, or at least 22,000 μg / kg, or at least 22,500 μg / kg, or at least 23,000 μg / kg, or at least 23,500 μg / kg, or at least 24,000 μg / kg, or at least 24,500 μg / kg, or at least 25,000 μg / kg, or at least 25,500 μg / kg, or at least 26,000 μg / kg, or at least 26,500 μg / kg, or at least 27,000 μg / kg, or at least 27,500 μg / kg, or at least 28,000 μg / kg, or at least 28,500 It contains cysteine-bonded 3SH in μg / kg, or at least 29,000 μg / kg, or at least 29,500 μg / kg, or at least 30,000 μg / kg, or at least 30,500 μg / kg, or at least 31,000 μg / kg, or at least 31,500 μg / kg, or at least 32,000 μg / kg, or at least 33,000 μg / kg, or at least 33,500 μg / kg, or at least 34,000 μg / kg, or at least 34,500 μg / kg, or at least 35,000 μg / kg, or at least 35,500 μg / kg, or at least 36,000 μg / kg. In some embodiments, the grape-derived product contains cysteine-linked 3SH in amounts ranging from approximately 400 μg / kg to approximately 1000 μg / kg, or approximately 500 μg / kg to approximately 900 μg / kg, or approximately 600 μg / kg to approximately 800 μg / kg, or approximately 400 μg / kg to approximately 600 μg / kg, or approximately 400 μg / kg to approximately 36,000 μg / kg, or approximately 20,000 μg / kg to approximately 36,000 μg / kg, or approximately 5,000 μg / kg to approximately 8000 μg / kg, or approximately 8000 μg / kg to approximately 12,000 μg / kg, or approximately 10,000 μg / kg to approximately 20,000 μg / kg, or approximately 15,000 μg / kg to approximately 20,000 μg / kg.

[0068] In some embodiments, the grape-derived product contains at least about 5000 μg / kg of glutathione 3SH. For example, in some embodiments, the grape-derived product contains at least about 5000 μg / kg, at least about 5500 μg / kg, at least about 6000 μg / kg, at least about 6500 μg / kg, at least about 7000 μg / kg, at least about 7500 μg / kg, at least about 8000 μg / kg, at least about 8500 μg / kg, at least about 9000 μg / kg, at least about 9500 μg / kg, at least about 10,000 μg / kg, at least about 10,500 μg / kg, at least about 11,000 μg / kg, at least about 11,500 μg / kg, at least about 12,000 μg / kg, at least about 12,500 μg / kg, at least about 13,000 μg / kg, at least about 13,500 μg / kg, and at least about 14 It contains glutathione-bound 3SH at a concentration of 1,000 μg / kg, at least about 14,500 μg / kg, at least about 15,000 μg / kg, at least about 15,500 μg / kg, at least about 16,000 μg / kg, at least about 16,500 μg / kg, at least about 17,000 μg / kg, at least about 17,500 μg / kg, at least about 18,000 μg / kg, at least about 18,500 μg / kg, at least about 19,000 μg / kg, at least about 19,500 μg / kg, or at least about 20,000 μg / kg, or at least 30,000 μg / kg, or at least 35,000 μg / kg, or at least 40,000 μg / kg, or at least 45,000 μg / kg, or at least 50,000 μg / kg.In some embodiments, the grape-derived product contains glutathione-bound 3SH in amounts ranging from approximately 5000 μg / kg to approximately 1000 μg / kg, or approximately 5500 μg / kg to approximately 9000 μg / kg, or approximately 6000 μg / kg to approximately 8000 μg / kg, or approximately 4000 μg / kg to approximately 6000 μg / kg, or approximately 400 μg / kg to approximately 36,000 μg / kg, or approximately 20,000 μg / kg to approximately 50,000 μg / kg, or approximately 5000 μg / kg to approximately 8000 μg / kg, or approximately 8000 μg / kg to approximately 12,000 μg / kg, or approximately 10,000 μg / kg to approximately 20,000 μg / kg, or approximately 15,000 μg / kg to approximately 20,000 μg / kg.

[0069] In some embodiments, the mash-hopping step includes adding less than 100 grams of plant material per 1 kg of grist (for example, less than 75 grams of plant material per 1 kg of milling grain or less than 50 grams of plant material per 1 kg of milling grain). In some embodiments, the mash-hopping step includes adding 75 to 100 grams of plant material per 1 kg of milling grain. In some embodiments, the mash-hopping step includes adding 50 to 75 grams of plant material per 1 kg of milling grain. In some embodiments, the mash-hopping step includes adding 25 to 50 grams of plant material per 1 kg of milling grain. In some embodiments, the mash-hopping step includes adding 1 to 25 grams of plant material per 1 kg of milling grain. In some embodiments, the mash-hopping step includes adding both hop and grape-derived products to the milling grain.

[0070] Optionally, the mash-hopping step includes a protein rest before the boiling step. For example, in some embodiments, protein rest includes maintaining the mash at a temperature below 120°F (e.g., 100°F to 120°F) for at least 5 minutes (e.g., at least about 5 minutes, or at least about 10 minutes, or at least about 15 minutes, or at least about 20 minutes, or at least about 25 minutes, or at least about 30 minutes, or at least about 5 minutes, or at least about 40 minutes, or at least about 45 minutes, or at least about 50 minutes, or about 1 hour) before the boiling step. In some embodiments, protein rest includes maintaining the mash at a temperature below 120°F (e.g., between 100°F and 120°F) for no more than 1 hour before the boiling step. In some embodiments, protein rest includes maintaining the mash at a temperature below 120°F for a period of time ranging from 5 minutes to 1 hour (or about 5 minutes to about 20 minutes, or about 5 minutes to about 10 minutes, or about 10 minutes to about 30 minutes, or about 10 minutes to about 1 hour) before the boiling step.

[0071] In some embodiments, the mash hopping step further includes a saccharification pause after the protein pause and before the boiling step. For example, in some embodiments, the saccharification pause includes maintaining the mash at a temperature below 160°F (e.g., 140°F to 160°F) for at least 15 minutes of the boiling step. In some embodiments, the saccharification pause includes maintaining the mash at a temperature below 160°F (e.g., 140°F to 160°F, or 148°F to 158°F) for at least about 15 minutes, or at least about 20 minutes, or at least about 25 minutes, or at least about 30 minutes, or at least about 5 minutes, or at least about 40 minutes, or at least about 45 minutes, or at least about 50 minutes, or at least about 60 minutes, or about 65 minutes, or about 70 minutes, or about 75 minutes, or about 80 minutes, or about 85 minutes, or about 90 minutes. In some embodiments, the saccharification pause includes maintaining the mash at a temperature below 160°F (e.g., 140°F to 160°F, or 148°F to 158°F) for a period of time ranging from 15 to 90 minutes (or about 15 to 30 minutes, or about 20 to 60 minutes, or about 20 to 40 minutes, or about 60 to 90 minutes) prior to the boiling step.

[0072] In various embodiments, the method further comprises contacting cooled malt with hops to produce a mixture, and contacting the mixture with recombinant Saccharomyces. In some embodiments, recombinant Saccharomyces is S. cerevisiae or S. pastorianus.

[0073] In some embodiments, the contact process takes place in a fermenter at a temperature in the range of 45°F to 100°F. In some embodiments, the contact process occurs over a period of 3 to 14 days (for example, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days).

[0074] Methods for converting other non-volatile thiols (e.g., glutathione-bound or cysteine-bound, non-3SH thiols) during the extraction process are also intended. For example, the methods described herein are intended to be useful for converting glutathione-bound or cysteine-bound -4MSP, glutathione-bound or cysteine-bound -3SH, or glutathione-bound or cysteine-bound -3SHA) to their aromatic, free forms.

[0075] In any of the methods described herein, the malt juice optionally contains at least 60 ng / L of free 3SH after the contact step. In some embodiments, the malt juice contains about 60 ng / L (or about 65 ng / L, or about 70 ng / L, or about 75 ng / L, or about 80 ng / L, or about 85 ng / L, or about 90 ng / L, or about 95 ng / L, or about 100 ng / L, or about 110 ng / L, or about 120 ng / L, or about 130 ng / L, or about 140 ng / L, or about 150 ng / L, or about 160 ng / L, or about 170 ng / L, or about 180 ng / L) after the contact step. It contains free 3SH of approximately 190 ng / L, or approximately 200 ng / L, or approximately 250 ng / L, or approximately 300 ng / L, or approximately 350 ng / L, or approximately 400 ng / L, or approximately 450 ng / L, or approximately 500 ng / L, or approximately 550 ng / L, or approximately 600 ng / L, or approximately 650 ng / L, or approximately 700 ng / L, or approximately 750 ng / L, or approximately 800 ng / L, or approximately 850 ng / L, or approximately 900 ng / L, or approximately 950 ng / L, or approximately 1000 ng / L. Free thiols in a sample can be analyzed, for example, by stable isotope dilution assay and nano liquid chromatography-tandem mass spectrometry (Nano LC-MS / MS) (Roland et al., J. Chromatography A, 1468:154-163, 2016, the disclosure of which is incorporated herein by reference). Methods for quantifying the amount of free thiols in a sample include, for example, derivatization and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) (Capone et al., Anal. Chem., 87:1226-1231, 2015, the disclosure of which is incorporated herein by reference).

[0076] In some embodiments, this method results in a six-fold increase in free 3SH in malt fermented with recombinant Saccharomyces compared to malt fermented with unmodified Saccharomyces. [Examples]

[0077] Example 1 - Recombinant Saccharomyces An embedded cassette containing the KANMX4 gene along with the TDH3 promoter element was amplified with primers containing SEQ ID NO: 5'-AAAAGGCTCCTGATGAAACTGGAGAGTCTCTTTGTTCTGAAATTTTTAAAGTTTAGCACACCATATAG-3' (forward primer) and SEQ ID NO: 6'-TCCAATAACAGACAGTTGCGTAGTAATACCAAACTTCGATAACTCGGTACGATCAATCATTTTGTTTGTTTATGTGTGTTTATTCGAAAC-3' (reverse primer), which have 80 bp homology to the IRC7 promoter. These cassettes were transformed into the respective S. cerevisiae parental strains OYL-088 (WGS of yeast strain A.US-05 (SRR8173067)) and OYL-011 (WGS of yeast strain YMD1872 (SRR8172941)), and the KANMX-TDH3pr sequence was targeted and embedded upstream of IRC7. PCR confirmation of the obtained G418-resistant colonies verified successful incorporation, resulting in TDH3 promoter-driven overexpression of IRC7. The expressed IRC7 allele was further sequenced and aligned to the wild-type IRC7 sequence (Figure 1). SNPs and resulting amino acid substitutions are listed in Table 1 below.

[0078] [Table 1]

[0079] The results showed that the modified OYL-088 strain expresses the active allele of IRC7 (amino acid sequence shown in SEQ ID NO: 1, polynucleotide sequence shown in SEQ ID NO: 2), while the SNP present in the polynucleotide encoding IRC7 in the modified OYL-011 strain expresses the inactive allele of IRC7 (amino acid sequence shown in SEQ ID NO: 3, polynucleotide sequence shown in SEQ ID NO: 4).

[0080] Example 2 - Extraction Method Using a small pilot brewing system, malt was prepared using either the "bag brewing" or BIAB method. Brewer's Malt (Briess Malting) and Cascade hops (Hopsteiner) were added to the mashed and hopped sample, immersed at 120°F for 15 minutes for protein rest, and then heated at 148°F for 15 minutes for saccharification rest. The grains and hops were removed from the malt, and the volume was adjusted to the target 10°P at the beginning of boiling. The malt was boiled for 30 minutes, then transferred to a vortex stand for another 15 minutes, and finally transferred to a flask for cooling. The resulting malt (12°P) was fermented with either OYL-088, OYL-011, OYL-088 TDH3-IRC7, or OYL-011 TDH3-IRC7. The same process was performed on the dried hopped samples, but the hops were omitted from the mash and then added on the second day of fermentation at the same hopping rate of 8 g / L (approximately 2 lb / bbl). The malt juice and beer samples were collected in 50 ml conical tubes, 1.5 mg / L of sodium metabisulfite was added to the samples, and the samples were immediately frozen. The samples were evaluated by derivatization and HPLC-MS / MS as described in Capone al. (Anal. Chem., 87:1226-1231, 2015), or by stable isotope dilution assay and nanoLC-MS / MS as described in Roland al. (J. Chromatography A, 1468:154-163, 2016).

[0081] As shown in Figure 2, the free 3SH level in beer samples fermented with OYL-088 TDH3-IRC7 was nearly three times higher compared to the unmodified strain (OYL-088). The 3SH levels of the same beer made with OYL-011 and OYL-011 TDH3-IRC7 did not change comparatively (due to the inactive allele of IRC7). Surprisingly, the free 3SH level in mash-hop beer samples fermented with OYL-088 TDH3-IRC7 was nearly six times higher compared to the unmodified strain (OYL-088). The 3SH levels of the same beer made with OYL-011 and OYL-011 TDH3-IRC7 did not change comparatively (due to the inactive allele of IRC7).

[0082] The results described above were, at least in part, surprising, given reports that β-lyase overexpression (specifically, IRC7 overexpression) in Saccharomyces does not produce free 3-SH (Denby et al., WBC Connect 2020, Poster 159). In the course of the experiments described herein, it was determined that previous studies had utilized an inactive form of IRC7. Another study (i.e., not an overexpression study) examining cell extracts for endogenous β-lyase activity determined that Irc7 with the V348L substitution (same as SEQ ID NO: 1) is inactive (Curtin et al., “Mutations in carbon-sulfur β-lyase encoding gene IRC7 affect the polyfunctional thiol-releasing capability of brewers yeast,” World Brewing Congress Connect 2020, Poster 157). Unexpectedly, and as shown herein, Irc7, including the amino acid sequence described in SEQ ID NO: 1, efficiently converts non-volatile thiols available in plant material into their free, aromatic forms. In addition, the use of active β-lyase in the mash-hopping process results in a significant increase in the conversion of non-volatile thiols to their free forms.

[0083] Example 3 - Recombinant Yeast G blocks were synthesized by IDT and cloned into yeast shuttle vectors containing a HYGB selection marker. These G blocks contained polynucleotides encoding PatB from S. hominis (SEQ ID NO: 8), IRC7 from S. cerevisiae (SEQ ID NO: 1), or IRC7 from S. pastorianus (SEQ ID NO: 7), under the control of the PGK1 promoter and CYC1 terminator. Next, the vectors were transformed into a lager brewing strain (OYL-106), and the isolates were grown on YPD-HYGB agar plates. These isolates were inoculated into YPD+HYGB, and sulfur production was assayed using lead acetate paper. Lead acetate paper showed significant blackening with the S. pastorianus IRC7 allele, less with the S. cerevisiae IRC7 allele, but little to no blackening with the S. hominis PatB allele and an empty vector control. The isolates were grown in 250 ml of dry malt extract medium + HYGB for small flask fermentation. 48 hours after growth, each culture was centrifuged and inoculated with 300 ml of 15°P malt juice prepared from two-row barley malt, using 10 million cells / ml. Fermentation was carried out for one week, at which point sensory evaluation was performed. The sensory effects resulting from the S. hominis PatB allele were significantly more pronounced than those from the cerevisiae IRC7 allele and the blank vector control. The main aromatic descriptors of S. hominis PatB fermentation were intense passion fruit, guava, and grapefruit, all of which are characteristic of 3SH thiols.

[0084] An investigation into additional PatB alleles was conducted using the same method as described above. Plasmids containing PatB alleles derived from S. anginosus, S. cohnii, and B. subtilis (SEQ ID NO: 14) were used to transform lager brewing strain (OYL-106) and compared with the S. hominis PatB allele. The resulting malt fermentation was sensory-evaluated. Fermentation with overexpression of the B. subtilis PatB allele yielded levels of passion fruit and guava aromas comparable to those of the S. hominis PatB allele, but fermentation with overexpression of the S. anginosus and S. cohnii PatB alleles did not significantly enhance the aroma compared to control fermentation without overexpression.

[0085] Example 4 - Comparison of the effects of PatB cysteine-thiol lyase, Irc7, and TnaA β-lyase enzymes on thiol release. Test fermentation was performed to evaluate the activity and specificity of Irc7, PatB, and TnaA β-lyase enzymes. For saccharification, malt was prepared in a commercial brewery by mashing two-row barley malt at 148°F for 30 minutes, followed by inactivation of enzyme activity at 180°F for 5 minutes. The mash effluent was collected and boiled for 30 minutes. The malt extract was diluted to 15°P, cooled to 70°C, and then inoculated with one of the following: OYL-011, OYL-011+Irc7, OYL-011+PatB, or OYL-011+TnaA. Fermentation flasks were constructed using hydrogen sulfide detector tubes (4H Gastec) to quantify the cumulative H2S released throughout fermentation. When fermentation was complete (after 11 days), H2S levels were recorded, and the resulting beer was analyzed for free thiols 3SH and 4MSP. The results are shown in Table 2 below.

[0086] [Table 2]

[0087] As shown above, strains expressing S. hominis PatB cysteine-thiol lyase exhibited enhanced 3SH output and decreased H2S output compared to strains expressing S. cerevisiae Irc7 β-lyase. The S. hominis PatB allele also showed enhanced 4MSP compared to another enzyme with known β-lyase activity, TnaA tryptophanase from C. amalonaticus.

Claims

1. Recombinant Saccharomyces spp comprising a polynucleotide encoding the yeast β-lyase enzyme Irc7 operably linked to a heterologous promoter, wherein the β-lyase enzyme comprises the amino acid sequence described in Sequence ID No.

1.

2. The recombinant Saccharomyces spp according to claim 1, wherein the heterogeneous promoter is the TDH3, TDH2, CCW12, PGK1, ADH1, ADH2, CYC1, HHF1, HHF2, TEF1, TEF2, HTB2, PAB1, ALD6, RNR1, RNR2, POP6, RAD27, PSP2, REV1, MFA1, MFa2, GAL1, CUP1, MET25, ICL1, ICL2, GAL3, HXT1, HXT2, MAL11, MAL31, MAL32, MAL33, MRK1, or SUC2 promoter.

3. Recombinant Saccharomyces spp according to claim 1 or 2, wherein S. cerevisiae.

4. Recombinant Saccharomyces spp according to claim 1 or 2, wherein S. pastorianus.

5. A method for converting a non-volatile form of 3-sulfanyl-1-hexanol (3SH) to free 3SH during a brewing process, the method comprising contacting cooled malt with recombinant Saccharomyces according to any one of claims 1 to 4 for a period of time and under conditions sufficient to convert the non-volatile form of 3SH to free 3SH.

6. The method according to claim 5, wherein the non-volatile form of 3SH is glutathione-linked 3SH, cysteine-linked 3SH, or a combination thereof.

7. The method according to claim 5 or 6, wherein the method results in a threefold increase in free 3SH in malt fermented with recombinant saccharomyces compared to malt fermented with unmodified saccharomyces.

8. The method according to any one of claims 5 to 7, wherein the malt juice contains at least 60 ng / L of free 3SH after the contact step.

9. The method according to any one of claims 5 to 8, wherein the method comprises adding hops to the cooled malt juice during the contact step.

10. The method according to claim 9, wherein the hops contain at least 400 μg / kg of cysteine-bonded 3SH.

11. The method according to claim 9 or 10, wherein the hops are Cascade, Calypso, Hallertau Tradition, Hallertau Perle, Triple Pearl, Nugget, Saaz, Columbia / CTZ, Chinook, Nelson Sauvin, Hallertau Blanc, or Simcoe hops.

12. A method for converting the non-volatile form of 3-sulfanyl-1-hexanol (3SH) to free 3SH during the brewing process, wherein the method is (a) A mash-hopping step comprising adding a plant containing a non-volatile form of 3SH, mashing it, and producing malt juice, (b) Boiling the malt juice produced by (a), (c) Cooling the malt juice, (d) Contacting cooled malt juice with recombinant saccharomyces for a time sufficient to convert the non-volatile form of 3SH into free 3SH, wherein the recombinant saccharomyces A method comprising a polynucleotide encoding the β-lyase enzyme Irc7 operably linked to a heterologous promoter, wherein Irc7 comprises the amino acid sequence represented by Sequence ID No.

1.

13. The method according to claim 12, wherein the non-volatile form of 3SH is glutathione-linked 3SH, cysteine-linked 3SH, and combinations thereof.

14. The method according to claim 12 or 13, wherein the plant material containing a non-volatile form of 3SH is hops.

15. The method according to claim 14, wherein the hops contain at least 400 μg / kg of cysteine-bonded 3SH.

16. The method according to claim 14 or 15, wherein the hops contain at least 5000 μg / kg of glutathione 3SH.

17. The method according to any one of claims 14 to 16, wherein the hops are Cascade, Calypso, Hallertau Tradition, Hallertau Perle, Triple Pearl, Nugget, Saaz, Columbia / CTZ, Chinook, Nelson Sauvin, Hallertau Blanc, or Simcoe hops.

18. The method according to claim 12 or 13, wherein the plant material containing a non-volatile form of 3SH is a product derived from grapes.

19. The method according to claim 18, wherein the grape-derived product is crushed grapes or grape powder.

20. The method according to claim 19, wherein the grape-derived product is obtained from white grapes, red grapes, or a combination thereof.

21. The method according to claim 20, wherein the white grape is Sauvignon Blanc, Chardonnay, Chenin Blanc, Colombard, Gewurztraminer, Gros Manseng, Koshu, Maccabeo, Muscat, Petit Manseng, Pinot Blanc, Pinot Gris, Riesling, Scheurebe, Semillon, Sylvener, or Tokay.

22. The method according to claim 20, wherein the red grape is Cabernet Franc, Cabernet Sauvignon, Grenache, Merlot, or Pinot Noir.

23. The method according to any one of claims 12 to 22, wherein the method comprises adding less than 100 grams of hops per 1 kg of milling grain during the mash-hopping process.

24. The method according to any one of claims 12 to 23, wherein the mash hopping step includes protein resting.

25. The method according to claim 24, wherein the protein resting comprises maintaining the mash at a temperature below 130°F for at least 5 minutes prior to the boiling step.

26. The method according to claim 24 or 25, wherein the protein resting comprises maintaining the mash at a temperature of 100°F to 120°F for at least 5 minutes prior to the boiling step.

27. The method according to claim 24 or 25, further comprising saccharification pause after the protein pause and before the boiling step.

28. The method according to claim 27, wherein the saccharification pause includes maintaining the malt juice at a temperature of 120°F to 160°F for at least 15 minutes prior to the boiling step.

29. The method according to any one of claims 12 to 28, further comprising contacting the cooled malt juice with hops to produce a mixture, and contacting the mixture with the recombinant Saccharomyces.

30. The method according to any one of claims 12 to 29, wherein the recombinant Saccharomyces is S. cerevisiae or S. pastorianus.

31. The method according to any one of claims 12 to 30, wherein the malt juice contains at least 60 ng / L of free 3SH after the contact step.

32. The method according to any one of claims 12 to 31, wherein the contact step (d) occurs in a fermentation tank at a temperature in the range of 45°F to 100°F.

33. The method according to any one of claims 12 to 32, wherein the method results in a six-fold increase in free 3SH in malt fermented with recombinant S. cerevisiae compared to malt fermented with unmodified S. cerevisiae.

34. Recombinant yeast comprising a polynucleotide encoding the yeast β-lyase enzyme Irc7 operably linked to a heterologous promoter, wherein the β-lyase enzyme comprises the amino acid sequence described in Sequence ID No.

1.

35. The recombinant yeast according to claim 34, wherein the yeast does not belong to the genus Saccharomyces.

36. The recombinant yeast according to claim 34 or 35, wherein the yeast is of the genera Kloeckera, Candida, Starmerella, Hanseniaspora, Kluyveromyces / Lachance, Metschnikowia, Saccharomycodes, Zygosaccharomyce, Dekkara (also called Brettanomyces), Wickerhamomyces, or Torulaspora.

37. The aforementioned yeasts are Hanseniaspora uvarum, Hanseniaspora guillermondii, Hanseniaspora vinae, Metschnikowia pulcherrima, Kluyveromyces / Lachancea thermotolerans, Starmerella bacillaris (formerly known as Candida stellatal or Candida zemplinina), Saccharomycodes ludwigii, Zygosaccharomyces rouxii, Dekkaera bruxellensis, Dekkaera The recombinant yeast according to claim 34 or 35, which is anomala, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Wickerhamomyces anomalus, or Torulaspora delbrüeckii.

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