Branched-chain amino acid aminotransferase gene and use thereof
The present invention relates to a branched-chain amino acid aminotransferase gene and a use thereof, particularly, to a brewery yeast for producing alcoholic drinks with enhanced flavor, to alcoholic drinks produced with said yeast and to a method for producing said drinks. More particularly, the present invention relates to a yeast whose capacity of producing amyl alcohol and/or isobutanol and/or isoamyl acetate that contribute to the product flavor is controlled by controlling the expression levels of BAT1 and BAT2 genes coding for brewery yeast branched-chain amino acid aminotransferases Bat1p and Bat2p, respectively, particularly nonScBAT1 or nonScBAT2 gene specific to lager brewing yeast, and to a method for producing alcoholic drinks using said yeast.
1 : A polynucleotide selected from the group consisting of the following polynucleotides (a) to (f):
(a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3;
(b) a polynucleotide comprising a polynucleotide coding for a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4;
(c) a polynucleotide comprising a polynucleotide coding for a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 with one or more amino acids thereof being deleted, substituted, inserted and/or added, and having a branched-chain amino acid aminotransferase activity;
(d) a polynucleotide comprising a polynucleotide coding for a protein having an amino acid sequence having 60% or higher identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, and having a branched-chain amino acid aminotransferase activity;
(e) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions, and which codes for a protein having a branched-chain amino acid aminotransferase activity; and
(f) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide coding for the protein of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 under stringent conditions, and which codes for a protein having a branched-chain amino acid aminotransferase activity.
2 : A polynucleotide according to claim 1 , selected from the group consisting of the following polynucleotides (g) to (i):
(a) a polynucleotide comprising a polynucleotide coding for a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 or of an amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 with 1 to 10 amino acids thereof being deleted, substituted, inserted and/or added, and having a branched-chain amino acid aminotransferase activity;
(b) a polynucleotide comprising a polynucleotide coding for a protein of an amino acid sequence having 90% or higher identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, and having a branched-chain amino acid aminotransferase activity; and
(c) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide of the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 or to a polynucleotide of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions, and which codes for a protein having a branched-chain amino acid aminotransferase activity.
3 : A polynucleotide according to claim 1 , comprising a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3.
4 : A polynucleotide according to claim 1 , comprising a polynucleotide coding for a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4.
5 : A polynucleotide according to claim 1 , wherein the polynucleotide is DNA.
6 : A polynucleotide selected from the group consisting of the following polynucleotides (j) to (m):
(a) a polynucleotide coding for RNA having a nucleotide sequence complementary to a transcript of the polynucleotide (DNA) according to claim 5 ;
(b) a polynucleotide coding for RNA that represses the expression of the polynucleotide (DNA) according to claim 5 through RNAi effect;
(c) a polynucleotide coding for RNA having an activity of specifically cleaving a transcript of the polynucleotide (DNA) according to claim 5 ; and
(d) a polynucleotide coding for RNA that represses expression of the polynucleotide (DNA) according to claim 5 through co-repression effect.
7 : A protein coded by the polynucleotide according to claim 1 .
8 : A vector comprising the polynucleotide according to claim 1 .
9 : A vector comprising the polynucleotide according to claim 6 .
10 : A yeast introduced with the vector according to claim 8 .
11 : A yeast according to claim 10 , having an enhanced capacity of producing amyl alcohol and/or isobutanol and/or isoamyl acetate.
12 : A yeast in which the expression of the polynucleotide (DNA) is repressed by introducing the vector according to claim 9 or by disrupting the gene related to the polynucleotide (DNA).
13 : A yeast according to claim 11 , having an enhanced capacity of producing amyl alcohol and/or isobutanol and/or isoamyl acetate by increasing the expression level of the protein encoded by the polynucleotide.
14 : A method for producing an alcoholic drink comprising using the yeast according to claim 10 .
15 : A method for producing an alcoholic drink according to claim 14 , wherein the brewed alcoholic drink is a malt drink.
16 : A method for producing an alcoholic drink according to claim 14 , wherein the brewed alcoholic drink is wine.
17 : An alcoholic drink produced by a method according to claim 14 .
18 : A method for assessing a test yeast for its capacity of producing amyl alcohol and/or isobutanol and/or isoamyl acetate, comprising using a primer or a probe designed based on a nucleotide sequence of a branched-chain amino acid aminotransferase gene having the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3.
19 : A method for assessing a test yeast for its capacity of producing amyl alcohol and/or isobutanol and/or isoamyl acetate, comprising: culturing a test yeast; and determining an expression level of a branched-chain amino acid aminotransferase gene having the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3.
20 : A method for selecting a yeast, comprising: culturing test yeasts; quantifying the protein according to claim 7 or determining an expression level of a branched-chain amino acid aminotransferase gene having the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3; and selecting a test yeast having said protein amount or said gene expression level according to the capacity of producing target amyl alcohol and/or isobutanol and/or isoamyl acetate.
21 : A method for selecting a yeast according to claim 20 , comprising: culturing a reference yeast and test yeasts; determining expression level of a branched-chain amino acid aminotransferase gene having the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 in each yeast; and selecting a test yeast having the gene expressed higher or lower than that in the reference yeast.
22 : A method for selecting a yeast according to claim 20 , comprising: culturing a reference yeast and test yeasts; quantifying the protein encoded by the polynucleotide in each yeast; and selecting a test yeast having said protein for a larger or smaller amount than that in the reference yeast.
23 : A method for producing an alcoholic drink comprising:
(a) conducting fermentation for producing an alcoholic drink using the yeast according to claim 10 , or a yeast selected by the method * for selecting a yeast, comprising: (i) culturing test yeasts; (ii) quantifying the protein encoded by the polynucleotide or determining an expression level of a branched-chain amino acid aminotransferase gene having the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3; and (iii) selecting a test yeast having said protein amount or said gene expression level according to the capacity of producing target amyl alcohol and/or isobutanol and/or isoamyl acetate; and
(b) adjusting the production amount of amyl alcohol and/or isobutanol and/or isoamyl acetate.