Gene Encoding Protein Responsible for Flocculation Property of Yeast and Use Thereof
The present invention relates to a gene encoding a protein responsible for flocculation property of yeast and use thereof, in particular, a brewery yeast with appropriate flocculation property for producing desired alcoholic beverages, alcoholic beverages produced with said yeast, and a method for producing said beverages. More particularly, the present invention relates to a yeast to which suitable flocculation property for brewing desired alcoholic beverages was imparted by controlling expression level of MHP1 gene encoding a protein responsible for flocculation property of brewery yeast, especially non-ScMHP1 gene specific to a lager brewing yeast, to alcoholic beverages produced with said yeast and to a method for producing said beverages.
1 . A polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;
(b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(c) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 with one or more amino acids thereof being deleted, substituted, inserted and/or added, and having an activity of imparting flocculation property to yeast;
(d) a polynucleotide comprising a polynucleotide encoding a protein having an amino acid sequence having 60% or higher identity with the amino acid sequence of SEQ ID NO: 2, and said protein having an activity of imparting flocculation property to yeast;
(e) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and which encodes a protein an activity of imparting flocculation property to yeast; 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 encoding the protein of the amino acid sequence of SEQ ID NO:2 under stringent conditions, and which encodes a protein having an activity of imparting flocculation property to yeast.
2 . The polynucleotide of claim 1 selected from the group consisting of:
(a) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, or encoding an amino acid sequence of SEQ ID NO: 2 wherein 1 to 10 amino acids thereof is deleted, substituted, inserted, and/or added, and wherein said protein has an activity of imparting flocculation property to yeast;
(b) a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 2, and having an activity of imparting flocculation property to yeast; and
(c) a polynucleotide which hybridizes to SEQ ID NO: 1 or which hybridizes to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and which encodes a protein having an activity of imparting flocculation property to yeast.
3 . The polynucleotide of claim 1 comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1.
4 . The polynucleotide of claim 1 comprising a polynucleotide encoding a protein consisting of the amino acid sequence SEQ ID NO: 2.
5 . The polynucleotide according to claim 1 , wherein the polynucleotide is DNA.
6 . A polynucleotide selected from the group consisting of:
(a) a polynucleotide encoding RNA having a nucleotide sequence complementary to a transcript of the polynucleotide (DNA) according to claim 5 ;
(b) a polynucleotide encoding RNA that represses the expression of the polynucleotide (DNA) according to claim 5 through an RNAi effect;
(c) a polynucleotide encoding RNA having an activity of specifically cleaving a transcript of the polynucleotide (DNA) according to claim 5 ; and
(d) a polynucleotide encoding RNA that represses the expression of the polynucleotide (DNA) according to claim 5 through a co-suppression effect.
7 . A protein encoded by the polynucleotide of claim 1 .
8 . A vector containing the polynucleotide of claim 1 .
9 . A vector containing the polynucleotide according to claim 6 .
10 . A yeast into which the vector according to claim 8 has been introduced.
11 . The yeast according to claim 10 , wherein the flocculation property is increased.
12 . The yeast according to claim 11 , wherein the flocculation property is increased by increasing an expression level of protein encoded by the polynucleotide.
13 . A yeast, wherein the expression of the polynucleotide is DNA and is repressed by:
(A) introducing the vector according to claim 8 ;
(B) disrupting the gene according to the DNA polynucleotide; or
(C) introducing a mutation into a promoter or genetically altering a promoter.
14 . The yeast according to claim 13 , wherein the flocculation property is decreased.
15 . A method for producing an alcoholic beverage by using the yeast according to claim 10 .
16 . A method according to claim 15 , wherein the brewed alcoholic beverage is a malt beverage.
17 . A method according to claim 15 , wherein the brewed alcoholic beverage is wine.
18 . An alcoholic beverage produced by the method according to claim 15 .
19 . A method for assessing a test yeast for its flocculation property, comprising suing a primer or probe designed based on the nucleotide sequence of a gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having an activity of imparting flocculation property to yeast.
20 . A method for assessing a test yeast for its flocculation property, comprising: culturing the test yeast; and measuring the expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having an activity of imparting flocculation property to yeast.
21 . A method for selecting a yeast, comprising: culturing test yeasts; quantifying the protein of claim 7 or measuring the expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having an activity of imparting flocculation property to yeast; and selecting a test yeast having an amount of the protein or the gene expression level according to the flocculation property of interest.
22 . The method for selecting a yeast according to claim 21 , comprising: culturing a reference yeast and test yeasts; measuring for each yeast the expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 and encoding a protein having an activity of imparting flocculation property to yeast; and selecting a test yeast having the gene expression higher or lower than that in the reference yeast.
23 . The method for selecting a yeast according to claim 21 , comprising: culturing a reference yeast and test yeasts; quantifying the protein encoded by the polynucleotide in each yeast; and selecting a test yeast having a larger or smaller amount of the protein than that in the reference yeast.
24 . A method for producing an alcoholic beverage comprising: conducting fermentation for producing an alcoholic beverage using:
(a) a yeast comprising a vector containing a polynucleotide selected from the group consisting of:
(i) a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;
(ii) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(iii) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 with one or more amino acids thereof being deleted, substituted, inserted and/or added, and having an activity of imparting flocculation property to yeast;
(iv) a polynucleotide comprising a polynucleotide encoding a protein having an amino acid sequence having 60% or higher identity with the amino acid sequence of SEQ ID NO: 2, and said protein having an activity of imparting flocculation property to yeast;
(v) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and which encodes a protein an activity of imparting flocculation property to yeast; and
(vi) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide encoding the protein of the amino acid sequence of SEQ ID NO: 2 under stringent conditions, and which encodes a protein having an activity of imparting flocculation property to yeast; or
(b) a yeast selected by the method according to claim 21 , and
adjusting flocculation property of yeast.