AMMONIA TRANSPORTER GENE AND USE THEREOF
The present invention relates to ammonia transporter genes and use thereof. The invention relates in particular to a brewer's yeast which shows enhanced ammonia assimilation, alcoholic beverages produced using such yeast, and a method of producing such alcoholic beverages. More specifically, the invention relates to the MEP3 gene which codes for the ammonia transporter Mep3 in brewer's yeast, particularly to a yeast which can control the ammonia assimilation ability by controlling the level of expression of the nonScMEP3 gene or ScMEP3 gene characteristic to beer yeast and to a method of producing alcoholic beverages using such yeast.
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 in which one or more amino acids thereof are deleted, substituted, inserted and/or added, and having ammonia transporter activity;
(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 ammonia transporter activity;
(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 having ammonia transporter 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 encoding the protein having the amino acid sequence of SEQ ID NO:2 under stringent conditions, and which encodes a protein having ammonia transporter activity.
2 . The polynucleotide according to claim 1 selected from the group consisting of:
(g) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, or encoding the amino acid sequence of SEQ ID NO: 2 in which 1 to 10 amino acids thereof are deleted, substituted, inserted, and/or added, and wherein said protein has ammonia transporter activity;
(h) a polynucleotide comprising a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 2, and having ammonia transporter activity; and
(i) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1 or which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, under high stringent conditions, which encodes a protein having ammonia transporter activity.
3 . The polynucleotide according to claim 1 comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1.
4 . The polynucleotide according to claim 1 comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2.
5 . The polynucleotide according to claim 1 , wherein the polynucleotide is DNA.
6 . A protein encoded by the polynucleotide according to claim 1 .
7 . A vector containing the polynucleotide according to claim 1 .
8 . A vector containing any one of the polynucleotides selected from the group consisting of:
(j) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4, or encoding the amino acid sequence of SEQ ID NO: 4 in which 1 to 10 amino acids are deleted, substituted, inserted, and/or added, and having ammonia transporter activity;
(k) a polynucleotide comprising a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 4, and having ammonia transporter activity; and
(l) a polynucleotide comprising a polynucleotide which hybridizes to the polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 3 or which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3, under high stringent conditions, which encodes a protein having ammonia transporter activity.
9 . A polynucleotide selected from the group consisting of:
(m) a polynucleotide encoding RNA having a nucleotide sequence complementary to a transcript of the polynucleotide (DNA) according to claim 5 ;
(n) a polynucleotide encoding RNA that represses the expression of the polynucleotide (DNA) according to claim 5 through an RNAi effect;
(o) a polynucleotide encoding RNA having an activity of specifically cleaving a transcript of the polynucleotide (DNA) according to claim 5 ; and
(p) a polynucleotide encoding RNA that represses the expression of the polynucleotide (DNA) according to claim 5 through a co-suppression effect.
(q) a polynucleotide encoding RNA having a nucleotide sequence complementary to a transcript of, a polynucleotide encoding RNA that represses, through an RNAi effect, the expression of, a polynucleotide encoding RNA having an activity of specifically cleaving a transcript of, or a polynucleotide encoding RNA that represses, through a co-suppression effect, the expression of the polynucleotide (DNA) selected from:
(q1) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4, or encoding the amino acid sequence of SEQ ID NO: 4 in which 1 to 10 amino acids are deleted, substituted, inserted, and/or added, and having ammonia transporter activity;
(q2) a polynucleotide comprising a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 4, and having ammonia transporter activity; or
(q3) a polynucleotide comprising a polynucleotide which hybridizes to the polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 3 or which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3, under high stringent conditions, said which encodes a protein having ammonia transporter activity.
10 . A vector containing the polynucleotide according to claim 9 .
11 . A yeast into which the vector according to claim 7 has been introduced.
12 . The yeast according to claim 11 , wherein the ammonia assimilation ability is increased owing to the introduction of the vector containing the polynucleotide.
13 . A yeast, wherein the expression of the polynucleotide selected from the group consisting of the polynucleotide (DNA) according to claim 5 and the following polynucleotides (q1) to (q3) is repressed by:
(A) introducing the vector containing the polynucleotide;
(B) disrupting the gene according to the polynucleotide (DNA) of claim 5 ; (q1) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4, or encoding the amino acid sequence of SEQ ID NO: 4 in which 1 to 10 amino acids are deleted, substituted, inserted, and/or added, and said protein having ammonia transporter activity; (q2) a polynucleotide comprising a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 4, and having ammonia transporter activity; or (q3) a polynucleotide comprising a polynucleotide which hybridizes to the polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 3 or which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3, under high stringent conditions, which encodes a protein having ammonia transporter activity; or
(C) introducing a mutation into a promoter or genetically altering a promoter.
14 . The yeast according to claim 12 , wherein the ammonia assimilation ability is increased by increasing the expression level of the protein encoded by the polynucleotide.
15 . A method for producing an alcoholic beverage by using the yeast according to claim 11 .
16 . The method according to claim 15 , wherein the brewed alcoholic beverage is a malt beverage.
17 . The 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 ammonia assimilation ability, comprising using a primer or probe designed based on the nucleotide sequence of a gene having the nucleotide sequence of SEQ ID NO: 1 or 3 and encoding a protein having ammonia transporter activity.
20 . A method for assessing a test yeast for its ammonia assimilation ability, comprising: culturing the test yeast; and measuring the expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 or 3 and encoding a protein having ammonia transporter activity.
21 . A method for selecting a yeast, comprising: culturing test yeasts; quantifying the protein of claim 6 or measuring the expression level of the gene having the nucleotide sequence of SEQ ID NO: 1 or 3 and encoding a protein having ammonia transporter activity; and selecting a test yeast having the production amount of the protein or the gene expression level according to the ammonia assimilation ability 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 or 3 and encoding a protein having ammonia transporter activity; 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 the yeast according to claim 11 or a yeast selected by the methods according to claim 21 , and adjusting the contents of ammonia and amino acids.