Glycerol-3-Phosphate Dehydrogenase Gene And Use Thereof
The present invention relates to a gene encoding glycerol-3-phosphate dehydrogenase and use thereof, in particular, a brewer's yeast which produces alcoholic beverages with superior body and mellowness, alcoholic beverages produced with said yeast, and a method for producing said beverages. More particularly, the present invention relates to a yeast whose ability of producing glycerol, which contribute to body and mellowness of products, is enhanced by amplifying expression level of GPD1 or GPD2 gene encoding a Gpd1p or Gpd2p which is a glycerol-3-phosphate dehydrogenase in brewer's yeast, especially non-ScGPD1 gene or non-ScGPD2 gene specific to a lager brewing yeast and to a method for producing alcoholic beverages with said yeast, etc.
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 or SEQ ID NO: 3;
(b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4;
(c) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 in which one or more amino acids thereof are deleted, substituted, inserted and/or added, and having a glycerol-3-phosphate dehydrogenase 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 or SEQ ID NO: 4, and said protein having a glycerol-3-phosphate dehydrogenase 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 or SEQ ID NO: 3 under stringent conditions, and which encodes a protein having a glycerol-3-phosphate dehydrogenase 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 or SEQ ID NO: 4 under stringent conditions, and which encodes a protein having a glycerol-3-phosphate dehydrogenase 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 SEQ ID NO: 4, or encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 in which 1 to 10 amino acids thereof are deleted, substituted, inserted, and/or added, and wherein said protein has a glycerol-3-phosphate dehydrogenase 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 or SEQ ID NO: 4, and having a glycerol-3-phosphate dehydrogenase activity; and
(i) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, under high stringent conditions, which encodes a protein having a glycerol-3-phosphate dehydrogenase activity.
3 . The polynucleotide according to claim 1 comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
4 . The polynucleotide according to claim 1 comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
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 yeast into which the vector according to claim 7 has been introduced.
9 . The yeast according to claim 8 , wherein glycerol producing ability is increased.
10 . The yeast according to claim 8 , wherein low-temperature storage property, frozen storage property or drying-resistant property is increased.
11 . The yeast according to claim 8 , wherein osmotic pressure resistant property is increased.
12 . The yeast according to claim 9 , wherein the glycerol producing ability is increased by increasing an expression level of the protein encoded by the polynucleotide.
13 . The yeast according to claim 10 , wherein the low-temperature storage property, frozen storage property or drying-resistant property is increased by increasing an expression level of the protein encoded by the polynucleotide.
14 . The yeast according to claim 11 , wherein the osmotic pressure resistant property is increased by increasing an expression level of the protein encoded by the polynucleotide.
15 . A method for producing an alcoholic beverage by using the yeast according to claim 8 .
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 glycerol producing ability, comprising using a primer or probe designed based on the nucleotide sequence of a glycerol-3-phosphate dehydrogenase gene having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
20 . A method for assessing a test yeast for its glycerol producing ability, comprising: culturing the test yeast; and measuring the expression level of the glycerol-3-phosphate dehydrogenase gene having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
21 . A method for selecting a yeast, comprising: culturing test yeasts; quantifying the protein of claim 6 or measuring the expression level of the glycerol-3-phosphate dehydrogenase gene having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3; and selecting a test yeast having an amount of the protein or the gene expression level according to desired glycerol producing ability.
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 glycerol-3-phosphate dehydrogenase gene having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3; and selecting a test yeast having the gene expression higher 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 amount of the protein than that in the reference yeast.
24 . A method for producing an alcoholic beverage comprising: conducting fermentation using the yeast according to claim 8 or a yeast selected by the methods according to claim 21 .
25 . A method for enhancing low-temperature storage property of yeast by using a vector comprising a polynucleotide 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: 10, or encoding the amino acid sequence of SEQ ID NO: 10 in which 1 to 10 amino acids thereof are deleted, substituted, inserted, and/or added, and wherein said protein has a glycerol-3-phosphate dehydrogenase activity;
(k) a polynucleotide comprising a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 10, and having a glycerol-3-phosphate dehydrogenase activity; and
(l) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 9 or which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 9, under high stringent conditions, which encodes a protein having a glycerol-3-phosphate dehydrogenase activity.