JPWO2021076917A5 - - Google Patents

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JPWO2021076917A5
JPWO2021076917A5 JP2022523282A JP2022523282A JPWO2021076917A5 JP WO2021076917 A5 JPWO2021076917 A5 JP WO2021076917A5 JP 2022523282 A JP2022523282 A JP 2022523282A JP 2022523282 A JP2022523282 A JP 2022523282A JP WO2021076917 A5 JPWO2021076917 A5 JP WO2021076917A5
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fermentation product
beta
enzyme
lyase activity
4mmp
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JP2022523282A
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JP2022553039A (en
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Priority claimed from PCT/US2020/056022 external-priority patent/WO2021076917A1/en
Publication of JP2022553039A publication Critical patent/JP2022553039A/en
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Claims (26)

ベータ-リアーゼ活性をもつ酵素をコードする異種遺伝子を含む、遺伝子学的に修飾された酵母細胞(修飾された細胞)。 A genetically modified yeast cell (modified cell) containing a heterologous gene encoding an enzyme with beta-lyase activity. (a)ベータ-リアーゼ活性をもつ酵素が、配列番号2で表されるとおりの配列と少なくとも90%の配列同一性をもつ配列を有する;または
(b)ベータ-リアーゼ活性をもつ酵素が、配列番号4もしくは5のいずれか1つで表されるとおりの配列と少なくとも90%の配列同一性をもつ配列を有する、請求項1に記載の修飾された細胞。
(a) the enzyme with beta-lyase activity has a sequence having at least 90% sequence identity with the sequence as represented by SEQ ID NO: 2 ; or
(b) the modification of claim 1, wherein the enzyme with beta-lyase activity has a sequence with at least 90% sequence identity to the sequence as represented by either one of SEQ ID NOs: 4 or 5. cells.
ベータ-リアーゼ活性をもつ酵素が、配列番号1、6、7で表されるとおりの配列をいずれも含まない、請求項1または2に記載の修飾された細胞。 3. The modified cell according to claim 1 or 2, wherein the enzyme with beta-lyase activity does not contain any of the sequences as represented by SEQ ID NO: 1, 6, 7. ベータ-リアーゼ活性をもつ酵素が、配列番号2で表されるとおりの配列を有する、請求項1~3のいずれか一項に記載の修飾された細胞。 Modified cell according to any one of claims 1 to 3, wherein the enzyme with beta-lyase activity has a sequence as represented by SEQ ID NO: 2. ベータ-リアーゼ活性をもつ酵素が、配列番号4~7のいずれか1つで表されるとおりの配列を有する、請求項1または2に記載の修飾された細胞。 The modified cell according to claim 1 or 2 , wherein the enzyme with beta-lyase activity has a sequence as represented by any one of SEQ ID NOs: 4 to 7. ベータ-リアーゼ活性をもつ酵素が、配列番号1の位置H463に対応する位置にて置換変異を含み、任意に
配列番号1の位置H463に対応する位置での置換変異が、フェニルアラニン、アルギニン、グルタミン酸、トレオニン、グリシン、イソロイシン、またはバリンである、請求項1~5のいずれか一項に記載の修飾された細胞。
The enzyme with beta-lyase activity contains a substitution mutation at the position corresponding to position H463 of SEQ ID NO: 1, and optionally
Modified cell according to any one of claims 1 to 5 , wherein the substitution mutation at the position corresponding to position H463 of SEQ ID NO: 1 is phenylalanine, arginine, glutamic acid, threonine, glycine, isoleucine, or valine. .
酵母細胞が、属Saccharomycesから成る、請求項1~6のいずれか一項に記載の修飾された細胞。 Modified cell according to any one of claims 1 to 6 , wherein the yeast cell consists of the genus Saccharomyces. 酵母細胞が、種Saccharomyces cerevisiae(S.cerevisiae)または種Saccharomyces pastorianus(S.pastorianus)から成る、請求項7に記載の修飾された細胞。 8. The modified cell according to claim 7 , wherein the yeast cell consists of the species Saccharomyces cerevisiae (S. cerevisiae) or the species Saccharomyces pastorianus (S. pastorianus) . 酵母細胞が、S.cerevisiae California Ale Yeast株WLP001またはRed Star Cote des Blancsである、請求項8に記載の修飾された細胞。 9. The modified cell of claim 8 , wherein the yeast cell is S. cerevisiae California Ale Yeast strain WLP001 or Red Star Cote des Blancs. 請求項1~9のいずれか一項に記載の修飾された細胞を、少なくとも1つの発酵性糖を含む培地と接触させることを含む、発酵産物を産生する方法またはエタノールを含む組成物を産生する方法であって、ここで接触させることが、少なくとも最初の発酵プロセスの最中に実施されることで、発酵産物またはエタノールを含む組成物が産生される、前記方法。 A method for producing a fermentation product or a composition comprising ethanol , comprising contacting a modified cell according to any one of claims 1 to 9 with a medium comprising at least one fermentable sugar. A method, wherein the contacting is performed during at least an initial fermentation process to produce a fermentation product or a composition comprising ethanol . 少なくとも1つの発酵性糖が、少なくとも1つの糖源において提供される;任意に
発酵性糖が、グルコース、フルクトース、スクロース、マルトース、および/またはマルトトリオースである、請求項10に記載の方法。
at least one fermentable sugar is provided in at least one sugar source ; optionally
11. The method according to claim 10 , wherein the fermentable sugar is glucose, fructose, sucrose, maltose, and/or maltotriose .
少なくとも1つの糖源が、少なくとも1つの前駆体を含み、任意に
少なくとも1つの前駆体が、システインに抱合された3-メルカプトヘキサン-1-オール(Cys 3-MH)、システインに抱合された4-メチル-4-メルカプトペンタン-2-オン(Cys 4MMP)、グルタチオンに抱合された3-メルカプトヘキサン-1-オール(Glut-3-MH)、および/またはグルタチオンに抱合された4-メチル-4-メルカプトペンタン2-オン(Glut 4MMP)を含む、請求項10または11に記載の方法。
at least one sugar source comprises at least one precursor, optionally
The at least one precursor is 3-mercaptohexan-1-ol conjugated to cysteine (Cys 3-MH), 4-methyl-4-mercaptopentan-2-one conjugated to cysteine (Cys 4MMP), glutathione 10. 3-mercaptohexan-1-ol conjugated to (Glut-3-MH) and/or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut 4MMP). or the method described in 11 .
1以上の前駆体を培地へ加えることをさらに含み、ここで前駆体が、3-メルカプトヘキサン-1-オール(Cys 3-MH)、システインに抱合された4-メチル-4-メルカプトペンタン-2-オン(Cys 4MMP)、グルタチオンに抱合された3-メルカプトヘキサン-1-オール(Glut-3-MH)、および/またはグルタチオンに抱合された4-メチル-4-メルカプトペンタン2-オン(Glut 4MMP)を含む、請求項1012のいずれか一項に記載の方法。 further comprising adding one or more precursors to the medium, wherein the precursors include 3-mercaptohexan-1-ol (Cys 3-MH), 4-methyl-4-mercaptopentane-2 conjugated to cysteine; -one (Cys 4MMP), 3-mercaptohexan-1-ol conjugated to glutathione (Glut-3-MH), and/or 4-methyl-4-mercaptopentan-2-one conjugated to glutathione (Glut 4MMP). ) The method according to any one of claims 10 to 12 . 前記発酵産物が、異種遺伝子を発現しない同等の細胞またはベータ-リアーゼ活性を有する野生型酵素を発現する同等の細胞によって産生された発酵産物と比較して、増大したレベルの、少なくとも1つの揮発性チオールを含む;任意に
少なくとも1つの揮発性チオールが、3-メルカプトヘキサン-1-オール(3MH)、3-メルカプト酢酸ヘキシル(3MHA)、4-メチル-4-メルカプトペンタ-2-オン(4MMP)、またはそれらの組み合わせを含む、請求項1013のいずれか一項に記載の方法。
said fermentation product has an increased level of at least one volatility as compared to a fermentation product produced by an equivalent cell that does not express the heterologous gene or an equivalent cell that expresses a wild-type enzyme having beta-lyase activity. Contains thiol ; optionally
The at least one volatile thiol is 3-mercaptohexan-1-ol (3MH), hexyl 3-mercaptoacetate (3MHA), 4-methyl-4-mercaptopent-2-one (4MMP), or a combination thereof. 14. A method according to any one of claims 10 to 13 , comprising :
前記発酵産物が、少なくとも200ng/Lの、3-メルカプトヘキサン-1-オール(3MH)、3-メルカプト酢酸ヘキシル(3MHA)、および/または4-メチル-4-メルカプトペンタ-2-オン(4MMP)を含む、請求項14に記載の方法。 The fermentation product contains at least 200 ng/L of 3-mercaptohexan-1-ol (3MH), hexyl 3-mercaptoacetate (3MHA), and/or 4-methyl-4-mercaptopent-2-one (4MMP). 15. The method of claim 14 , comprising: 前記発酵産物が、異種遺伝子を発現しない同等の細胞またはベータ-リアーゼ活性を有する野生型酵素を発現する同等の細胞によって産生された発酵産物と比較して、低減したレベルの、少なくとも1つの望ましくない産物を含む;任意に
少なくとも1つの望ましくない産物が、インドールである、請求項1015のいずれか一項に記載の方法。
said fermentation product has a reduced level of at least one undesirable product compared to a fermentation product produced by an equivalent cell that does not express the heterologous gene or an equivalent cell that expresses a wild-type enzyme having beta-lyase activity. including products ; optionally
16. A method according to any one of claims 10 to 15 , wherein the at least one undesired product is indole .
前記発酵産物または前記エタノールを含む組成物が、発酵飲料である、請求項10~16のいずれか一項に記載の方法。 The method according to any one of claims 10 to 16 , wherein the fermentation product or the composition comprising ethanol is a fermented beverage. 発酵飲料が、ビール、ワイン、スパークリングワイン(シャンパン)、日本酒、蜂蜜酒、昆布茶、または林檎酒である、請求項17に記載の方法。 18. The method according to claim 17 , wherein the fermented beverage is beer, wine, sparkling wine (champagne), sake, mead, kombucha, or apple wine. 糖源が、麦芽汁、果汁、蜂蜜、米デンプン、またはそれらの組み合わせを含む、請求項1018のいずれか一項に記載の方法。 19. A method according to any one of claims 10 to 18 , wherein the sugar source comprises wort, fruit juice, honey, rice starch, or a combination thereof. 果汁が、ブドウ果汁またはリンゴ果汁である、請求項19に記載の方法。 20. The method according to claim 19 , wherein the fruit juice is grape juice or apple juice. 糖源が、麦芽汁であり、ならびに
方法が、培地を産生することをさらに含み、ここで培地を産生することが、以下:
(a)複数の穀物を水と接触させること;ならびに
(b)水および穀物を煮沸するかまたは浸すことで、麦芽汁を産生すること
を含む、請求項1020のいずれか一項に記載の方法。
the sugar source is wort, and the method further comprises producing a culture medium, wherein producing the culture medium comprises:
(a) contacting the grains with water; and
21. A method according to any one of claims 10 to 20 , comprising: (b) boiling or soaking water and grain to produce wort.
少なくとも1つのホップ品種を麦芽汁へ加えることで、ホップが効いた麦芽汁を産生することをさらに含む、請求項21に記載の方法。 22. The method of claim 21 , further comprising adding at least one hop variety to the wort to produce a hopped wort. 少なくとも1つのホップ品種を培地へ加えることをさらに含む、請求項1021のいずれか一項に記載の方法。 22. A method according to any one of claims 10 to 21 , further comprising adding at least one hop variety to the medium. 少なくとも1つの追加の発酵プロセスおよび/または前記発酵産物に炭酸ガスを入れることをさらに含む、請求項1023のいずれか一項に記載の方法。 24. The method according to any one of claims 10 to 23 , further comprising at least one additional fermentation process and/or carbonation of the fermentation product . 請求項1024のいずれか一項に記載の方法によって産生された、発酵産物。 A fermentation product produced by the method according to any one of claims 10 to 24 . 発酵産物が、少なくとも200ng/Lの、3-メルカプトヘキサン-1-オール(3MH)、3-メルカプト酢酸ヘキシル(3MHA)、および/または4-メチル-4-メルカプトペンタ-2-オン(4MMP)を含む、および/または
発酵産物が、500μg/L未満のインドールを含む、請求項25に記載の発酵産物。
The fermentation product contains at least 200 ng/L of 3-mercaptohexan-1-ol (3MH), hexyl 3-mercaptoacetate (3MHA), and/or 4-methyl-4-mercaptopent-2-one (4MMP). including and/or
26. The fermentation product of claim 25 , wherein the fermentation product contains less than 500 μg/L indole .
JP2022523282A 2019-10-17 2020-10-16 Genetically modified yeast cells and methods of use thereof Pending JP2022553039A (en)

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US201962916529P 2019-10-17 2019-10-17
US62/916,529 2019-10-17
US202063086363P 2020-10-01 2020-10-01
US63/086,363 2020-10-01
PCT/US2020/056022 WO2021076917A1 (en) 2019-10-17 2020-10-16 Genetically engineered yeast cells and methods of use thereof

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CN (1) CN114829618A (en)
AU (1) AU2020366440A1 (en)
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