JP2009165353A - Amino acid transporter gene and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a brewer's yeast having an ability to assimilate an amino acid, to provide a method for producing an alcoholic drink low in the content of amino acid and characteristics of an amino acid composition, and the like. <P>SOLUTION: Provided are the yeast having an ability to assimilate the amino acid which is improved by improving the expression amount of gene GAP1, AGP2, MUP1 or MUP3, respectively, encoding brewer's yeast amino acid transporters Gap1p, Agp2p, Mup1p or Mup3p, in particular, gene nonScGAP1, nonScAGP2, nonScMUP1 or nonScMUP3 characteristic of beer yeast; and a method for producing an alcoholic drink by using the above gene. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an amino acid transporter gene and use thereof, and particularly relates to a brewing yeast excellent in amino acid assimilation, alcoholic beverages produced using the yeast, a production method thereof, and the like. More specifically, the present invention relates to genes GAP1, AGP2, MUP1 or MUP3 encoding amino acid transporters Gap1p, Agp2p, Mup1p or Mup3p of brewing yeast, particularly nonScGAP1, nonScAGP2, nonScMUP1 or The present invention relates to a yeast whose amino acid assimilation ability is improved by increasing the expression level of the nonScMUP3 gene, a method for producing alcoholic beverages using the yeast, and the like.

In general, amino acids are important as a taste component of alcoholic beverages, and are known to be important factors affecting quality. Therefore, controlling the amount of amino acids according to the desired liquor quality is important in the development of new types.
However, since amino acids are assimilated as a nitrogen source by yeast during fermentation, it is extremely difficult to control the amino acid content at the end of fermentation.
In order for yeast to use extracellular amino acids as a nitrogen source, it is essential to incorporate amino acids into the cells. It has been clarified that such amino acid uptake involves an amino acid transporter present in the cell membrane of yeast.
As the amino acid transporter of yeast, Gap1 having low substrate specificity and many amino acid transporters having different substrate specificities (amino acid transporter TAT2, arginine transporter Can1, proline transporter Put4 and the like (Non-patent Document 1; Mol Cell Biol. 14: 6597-6606, 1994, Non-Patent Document 2; Curr Genet 36: 317-328, 1999).
Examples of using yeast having mutations in genes involved in amino acid incorporation (gap1, shr3, can1, put4, uga4) in order to control the amino acid content in alcoholic beverages (Patent Document 1; No. 321159) and examples of highly expressing the branched amino acid transporter BAP2 (Patent Document 2; JP 2000-316559 A) have been reported.
JP 2001-321159 JP 2000-316559 A Mol Cell Biol. 14: 6597-6606,1994 Curr Genet 36: 317-328, 1999

  In order to modify the amino acid composition of sake and produce alcoholic beverages with characteristic qualities, yeasts with controlled amino acid utilization have been desired.

  As a result of intensive studies to solve the above problems, the present inventors have succeeded in identifying and isolating a gene encoding an amino acid transporter that has an advantageous effect over known proteins from brewer's yeast. In addition, a transformed yeast in which the obtained gene was introduced into yeast and expressed was produced, and it was confirmed that the utilization of amino acids was promoted, thereby completing the present invention.

  That is, the present invention uses a novel amino acid transporter gene characteristically present in brewer's yeast, a protein encoded by the gene, a transformed yeast in which the expression of the gene is regulated, and a yeast in which the expression of the gene is regulated. Related to a method for producing alcoholic beverages. Specifically, the present invention provides the following polynucleotide, a vector containing the polynucleotide, a transformed yeast introduced with the vector, a method for producing alcoholic beverages using the transformed yeast, and the like.

(1) The polynucleotide according to any one of the following (a) to (f):
(a) a polynucleotide comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7;
(b) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8;
(c) consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, And a polynucleotide containing a polynucleotide encoding a protein having amino acid transporter activity;
(d) a protein having an amino acid sequence having 60% or more identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, and having amino acid transporter activity A polynucleotide comprising a polynucleotide encoding
(e) Hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, and an amino acid transporter A polynucleotide comprising a polynucleotide encoding a protein having activity; and
(f) stringent with a polynucleotide comprising a base sequence complementary to a base sequence of a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 A polynucleotide comprising a polynucleotide that hybridizes under conditions and encodes a protein having amino acid transporter activity.

(2) The polynucleotide according to claim 1, which is any of the following (g) to (i):
(g) In the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. A polynucleotide comprising a polynucleotide encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added, and having amino acid transporter activity;
(h) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, and having amino acid transporter activity A polynucleotide containing a polynucleotide that encodes; and
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 A polynucleotide comprising a polynucleotide comprising a polynucleotide that hybridizes with a polynucleotide consisting of a base sequence complementary to the above base sequence under a highly stringent condition and encodes a protein having amino acid transporter activity.
(3) The polynucleotide according to (1) above, comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7.
(4) The polynucleotide according to (1) above, comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.
(5) The polynucleotide according to any one of claims 1 to 4, which is DNA.
(6) A protein encoded by the polynucleotide according to any one of (1) to (5) above.

(7) A vector containing the polynucleotide according to any one of (1) to (5) above.
(7a) The vector according to (7) above, comprising an expression cassette containing the following components (a) to (c):
(a) Promoter capable of transcription in yeast cells
(b) the polynucleotide according to any one of (1) to (5), which is bound to the promoter in a sense direction or an antisense direction; and
(c) Signals that function in yeast for transcription termination and polyadenylation of RNA molecules.
(8) A transformed yeast introduced with the vector according to (7) above.
(9) The yeast for brewing according to (8) above, wherein the ability to assimilate amino acids is enhanced by introducing the vector according to (7) above.
(10) The yeast for brewing according to (9), wherein the ability to assimilate amino acids is improved by increasing the expression level of the protein according to (6).
(11) A method for producing an alcoholic beverage using the yeast according to any one of (8) to (10) above.
(12) The method for producing an alcoholic beverage according to the above (11), wherein the alcoholic beverage to be brewed is a malt beverage.
(13) The method for producing an alcoholic beverage according to the above (11), wherein the alcoholic beverage to be brewed is wine.
(14) Alcoholic beverages produced by the method according to any one of (11) to (13) above.
(15) Using a primer or probe designed based on the nucleotide sequence of an amino acid transporter gene having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, A method for evaluating amino acid assimilation ability.
(15a) A method for selecting yeast having high amino acid assimilation ability by the method described in (15) above.
(15b) A method for producing an alcoholic beverage (for example, beer) using the yeast selected by the method described in (15a) above.
(16) The test yeast is cultured, and the expression level of the amino acid transporter gene having the base sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 is measured, thereby To evaluate amino acid assimilation ability.
(16a) A method for selecting a yeast having a high amino acid assimilation ability, wherein the test yeast is evaluated and a yeast having a high expression level of an amino acid transporter gene is selected by the method described in (16) above.
(16b) A method for producing an alcoholic beverage (for example, beer) using the yeast selected by the method described in (16a) above.
(17) The test yeast is cultured, the protein according to claim 6 is quantified or the expression level of the amino acid transporter gene having the base sequence of SEQ ID NO: 1 is measured, and the target amino acid assimilation ability is determined. A method for selecting a yeast, comprising selecting a test yeast having a production amount of the protein or an expression level of the gene.
(18) Reference yeast and test yeast are cultured, and the expression level in each yeast of the amino acid transporter gene having the base sequence of SEQ ID NO: 1 is measured, and the test is such that the gene is expressed at a higher level than the reference yeast The method for selecting yeast according to (17) above, wherein the yeast is selected.
(19) The reference yeast and the test yeast are cultured, the protein according to claim 6 in each yeast is quantified, and the test yeast having a larger amount of the protein than the reference yeast is selected. Yeast selection method.
(20) Fermentation for liquor production is performed using any one of the yeast described in (8) to (10) above and the yeast selected by the method described in (17) to (19) above, A method for producing an alcoholic beverage, comprising adjusting an amino acid content.

  According to the method for producing alcoholic beverages using the transformed yeast of the present invention, assimilation of amino acids is promoted, the amino acid composition in the alcohol can be modified, and alcoholic beverages having characteristic qualities can be produced. .

  The present inventors considered that it is possible to assimilate amino acids more efficiently by increasing the activity of the amino acid transporter of yeast. Based on such an idea, based on the beer yeast genome information decoded by the method disclosed in JP-A-2004-283169, nonScGAP1, nonScAGP2, nonScMUP1 and nonScMUP3 genes encoding amino acid transporters unique to brewer's yeast Isolated and identified. The base sequences of these genes are arranged and shown in column No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7, respectively. The amino acid sequences of the proteins encoded by these genes are shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, respectively.

1. First, the present invention relates to (a) a polynucleotide comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 (specifically, DNA, hereinafter, these are simply referred to as “ And (b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8. The DNA targeted by the present invention is not limited to the DNA encoding the amino acid transporter derived from the above-mentioned brewer's yeast, and includes other DNA encoding a protein functionally equivalent to this protein. Examples of the functionally equivalent protein include (c) an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8. And a protein having amino acid transporter activity. As such a protein, in the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 -50, 1-40, 1-39, 1-38, 1-37, 1-36, 1-35, 1-34, 1-33, 1-32, 1 -31, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1 -21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1 -11, 1-10, 1-9, 1-8, 1-7, 1-6 (1 to several), 1-5, 1-4, 1-3, Examples thereof include proteins having an amino acid sequence in which 1 to 2 or 1 amino acid residue is deleted, substituted, inserted and / or added, and having amino acid transporter activity. In general, the smaller the number of amino acid residue deletions, substitutions, insertions and / or additions, the better. In addition, the protein includes (d) the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 and about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more of amino acid sequence and amino acid Examples include proteins having transporter activity. In general, the larger the homology value, the better. The amino acid transporter activity can be measured, for example, by the method described in (J. Bacteriology 121: 562-570,1975).

  The present invention also includes (e) a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 under a stringent condition and having amino acid transporter activity. A polynucleotide comprising a polynucleotide encoding a protein; and (f) a polynucleotide comprising a base sequence complementary to the base sequence of a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 And a polynucleotide containing a polynucleotide encoding a protein that hybridizes under stringent conditions with an amino acid transporter activity.

  Here, “polynucleotide (DNA) that hybridizes under stringent conditions” means DNA consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1, 3, 5 or 7, or SEQ ID NO: 2, DNA obtained by using a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like using all or part of the DNA encoding the amino acid sequence of 4, 6, or 8 as a probe. As a hybridization method, for example, a method described in Molecular Cloning 3rd Ed., Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997 can be used.

  As used herein, the “stringent conditions” may be any of low stringency conditions, moderate stringency conditions, and high stringency conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 32 ° C. The “medium stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. Under these conditions, it can be expected that DNA having higher homology can be efficiently obtained as the temperature is increased. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect hybridization stringency. Those skilled in the art will select these factors as appropriate. It is possible to achieve similar stringency.

  In addition, when using a commercially available kit for hybridization, Alkphos Direct Labeling Reagents (made by Amersham Pharmacia) can be used, for example. In this case, follow the protocol supplied with the kit, incubate with the labeled probe overnight, and then wash the membrane with a primary wash buffer containing 0.1% (w / v) SDS at 55 ° C. , Hybridized DNA can be detected.

As other DNA that can be hybridized, DNA that encodes the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 when calculated using homology search software such as FASTA and BLAST using default parameters. About 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6% or more 99.7% or more, 99.8% or more, or 99.9% or more of DNA having identity.
The identity of amino acid sequences and nucleotide sequences is determined using the algorithm BLAST (proc. Natl. Acad. Sci. USA 872264-2268, 1990; proc Natl Acad Sci USA 90: 5873, 1993) by Carlin and Arthur. it can. Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. When analyzing an amino acid sequence using BLASTX, parameters are set to score = 50 and wordlength = 3, for example. When using BLAST and Gapped BLAST programs, the default parameters of each program are used.

2. Protein of the present invention The present invention also provides a protein encoded by any of the polynucleotides (a) to (e). A preferred protein of the present invention comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, and amino acid transporter activity It is protein which has. Such a protein comprises the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, wherein the number of amino acid residues as described above is deleted, substituted, inserted and / or added, and Examples include proteins having amino acid transporter activity. Examples of such a protein include a protein having an amino acid sequence having homology as described above with the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, and having amino acid transporter activity. Such proteins are described in “Molecular Cloning 3rd Edition”, “Current Protocols in Molecular Biology”, “Nuc. Acids. Res., 10, 6487 (1982)”, “Proc. Natl. Acad. Sci. USA, 79, 6409 (1982) ”,“ Gene, 34, 315 (1985) ”,“ Nuc. Acids. Res., 13, 4431 (1985) ”,“ Proc. Natl. Acad. Sci. USA , 82, 488 (1985) ”and the like.

The deletion, substitution, insertion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of the present invention means that one or more amino acid residues in one and a plurality of amino acid sequences in the same sequence It means that there are amino acid residue deletion, substitution, insertion and / or addition, and two or more of deletion, substitution, insertion and addition may occur simultaneously.
Examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other. Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine; Group B: aspartic acid, glutamic acid, isoaspartic acid , Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid; group C: asparagine, glutamine; group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid; group E : Proline, 3-hydroxyproline, 4-hydroxyproline; Group F: serine, threonine, homoserine; Group G: phenylalanine, tyrosine.

  The protein of the present invention can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). It can also be chemically synthesized using peptide synthesizers such as Advanced Chemtech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Syntheselve Vega, Perceptive, Shimadzu, etc. .

3. Next, the present invention provides a vector containing the polynucleotide described above. The vector of the present invention contains the polynucleotide (DNA) described in any of (a) to (i) above. In addition, the vector of the present invention usually contains (a) a promoter capable of being transcribed in yeast cells; (b) any one of the above (a) to (i) bound to the promoter in a sense direction or an antisense direction. The described polynucleotide (DNA); and (c) with respect to transcription termination and polyadenylation of RNA molecules, configured to include an expression cassette comprising as a component a signal that functions in yeast.

As a vector used for introduction into yeast, any of a multicopy type (YEp type), a single copy type (YCp type), and a chromosome integration type (YIp type) can be used. For example, the YEp type vector is YEp24 (JR Broach et al., Experimental Manipulation of Gene Expression, Academic Press, New York, 83, 1983), and the YCp type vector is YCp50 (MD Rose et al., Gene, 60, 237 YIp5 (K. Struhl et al., Proc. Natl. Acad. Sci. USP, 76, 1035, 1979) is known as a YIp type vector and can be easily obtained.
As a promoter / terminator for regulating gene expression in yeast, any combination may be used as long as it functions in the yeast for brewing and is not affected by the concentration of components such as sugar and amino acid in the mash. For example, a promoter of glyceraldehyde 3-phosphate dehydrogenase gene (TDH3), a promoter of 3-phosphoglycerate kinase gene (PGK1), and the like can be used. These genes have already been cloned and are described in detail, for example, in MF Tuite et al., EMBO J., 1, 603 (1982) and can be easily obtained by known methods.

  As a selection marker used for transformation, an auxotrophic marker cannot be used in the case of yeast for brewing. Sci. USA, 81, 337 1984), cerulenin-resistance gene (fas2m, PDR4) (Hyogo, et al., Biochemistry, 64, 660, 1992; Hussain et et al., Gene, 101, 149, 1991), etc. Is available. The vector constructed as described above is introduced into the host yeast. Examples of the host yeast include any yeast that can be used for brewing, for example, brewery yeast for beer, wine, sake and the like. Specific examples include yeasts of the genus Saccharomyces. In the present invention, beer yeasts such as Saccharomyces pastorianus W34 / 70, Saccharomyces carlsbergensis NCYC453, NCYC456, etc. Saccharomyces cerevisiae NBRC1951, NBRC1952, NBRC1953, NBRC1954, etc. can be used. Furthermore, whiskey yeasts such as Saccharomyces cerevisiae NCYC90, wine yeasts such as association wines No. 1, 3 and 4, etc., sake yeasts such as association yeast sake nos. 7 and 9 can be used. However, the present invention is not limited to this. In the present invention, beer yeast such as Saccharomyces pastorianus is preferably used.

As a yeast transformation method, a publicly known method can be used. For example, electroporation method “Meth. Enzym., 194, p182 (1990)”, spheroplast method “Proc. Natl. Acad. Sci. USA, 75 p1929 (1978)”, lithium acetate method “J. Bacteriology, 153, p163 (1983) ”, Proc. Natl. Acad. Sci. USA, 75 p1929 (1978), Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual, etc. However, the present invention is not limited to this.
More specifically, the host yeast is a standard yeast nutrient medium (for example, YEPD medium “Genetic Engineering. Vo1.1, Plenum Press, New York, 117 (1979)” etc.), and the value of OD600nm is 1-6. Incubate to. The cultured yeast is collected by centrifugation, washed, and pretreated with alkali ion metal ions, preferably lithium ions, at a concentration of about 1-2 M. The cells are allowed to stand at about 30 ° C. for about 60 minutes, and then left at about 30 ° C. for about 60 minutes together with the DNA to be introduced (about 1 to 20 μg). Polyethylene glycol, preferably about 4,000 daltons of polyethylene glycol, is added to a final concentration of about 20% to 50%. After standing at about 30 ° C. for about 30 minutes, the cells are heated at about 42 ° C. for about 5 minutes. Preferably, the cell suspension is washed with a standard yeast nutrient medium, placed in a predetermined amount of fresh standard yeast nutrient medium, and allowed to stand at about 30 ° C. for about 60 minutes. Thereafter, the transformant is obtained by planting on a standard agar medium containing an antibiotic or the like used as a selection marker. For other general cloning techniques, “Molecular Cloning 3rd Edition”, “Methods in Yeast Genitics, A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)” and the like can be referred to.

4). The method for producing the liquor of the present invention and the liquor obtained by the production method The above-described vector of the present invention is introduced into a yeast suitable for brewing the liquor to be produced, and by using the yeast, a liquor having a characteristic amino acid composition is obtained. Can be manufactured. Examples of alcoholic beverages that can be used include, but are not limited to, beer, wine, whiskey, and sake.
In producing these alcoholic beverages, a known method can be used except that the brewer's yeast obtained in the present invention is used instead of the parent strain. Therefore, the raw materials, production equipment, production management, etc. may be exactly the same as in the conventional method, and there is no increase in cost for producing alcoholic beverages with a reduced fermentation period. That is, according to the present invention, the existing facility can be used and manufactured without increasing the cost.

5. Method for Evaluating Yeast of the Present Invention The present invention relates to an amino acid of a test yeast using a primer or probe designed based on the base sequence of an amino acid transporter gene having the base sequence of SEQ ID NO: 1, 3, 5 or 7. It relates to a method for evaluating the assimilation ability. General methods of evaluation methods using primers or probes are known, and are described, for example, in WO01 / 040514, JP-A-8-205900, and the like. Hereinafter, this evaluation method will be briefly described.
First, the genome of the test yeast is prepared. As the preparation method, any known method such as Hereford method or potassium acetate method can be used (for example, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, p130 (1990)). Using the obtained genome as a target, using the primer or probe designed based on the nucleotide sequence (preferably the ORF sequence) of the amino acid transporter gene, the gene or the gene specific to the genome of the test yeast Check whether an array exists. The primer or probe can be designed using a known method.
Detection of a gene or a specific sequence can be performed using a known technique. For example, a polynucleotide containing a part or all of a specific sequence or a polynucleotide containing a base sequence complementary to the base sequence is used as one primer, and the upstream or downstream of this sequence is used as the other primer. A polynucleotide containing a part or all of the sequence or a polynucleotide containing a base sequence complementary to the base sequence is used to amplify a yeast nucleic acid by PCR, and the presence or absence of the amplified product and the molecular weight of the amplified product are determined. Measure the size. The number of bases of the polynucleotide used for the primer is usually 10 bp or more, preferably 15 to 25 bp. In addition, the base number of the sandwiched portion is usually suitably 300 to 2000 bp.

  The reaction conditions for the PCR method are not particularly limited. For example, conditions such as denaturation temperature: 90 to 95 ° C., annealing temperature: 40 to 60 ° C., extension temperature: 60 to 75 ° C., cycle number: 10 times or more should be used. Can do. The obtained reaction product is separated by electrophoresis using an agarose gel or the like, and the molecular weight of the amplified product can be measured. This method predicts and evaluates the amino acid assimilation ability of the yeast depending on whether the molecular weight of the amplified product is large enough to include a specific portion of the DNA molecule. Further, by analyzing the base sequence of the amplified product, it is possible to predict and evaluate the above performance more accurately.

  In the present invention, the test yeast is cultured, and the expression level of the amino acid transporter gene having the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 is measured, whereby the amino acid assimilation ability of the test yeast is determined. Can also be evaluated. The expression level of the amino acid transporter gene can be measured by culturing a test yeast and quantifying mRNA or protein that is a transcription product of the amino acid transporter gene. Quantification of mRNA or protein can be performed using a known method. mRNA can be quantified by, for example, Northern hybridization or quantitative RT-PCR, and protein can be quantified by, for example, western blotting (Current Protocols in Molecular Biology, John Wiley & Sons 1994-2003).

  Further, the test yeast is cultured, the expression level of the amino acid transporter gene having the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 is measured, and the gene expression level according to the target amino acid assimilation ability By selecting this yeast, it is possible to select a yeast suitable for brewing the desired liquor. Alternatively, a reference yeast and a test yeast may be cultured, the gene expression level in each yeast may be measured, and the gene expression levels of the reference yeast and the test yeast may be compared to select a desired yeast. Specifically, for example, the reference yeast and the test yeast are cultured, and the expression level in each yeast of the amino acid transporter gene having the base sequence of SEQ ID NO: 1, 3, 5 or 7 is measured. By selecting a strain in which the gene is highly expressed, a yeast suitable for brewing alcoholic beverages can be selected.

Alternatively, a test yeast suitable for brewing a desired liquor can be selected by culturing the test yeast and selecting a yeast having a high amino acid assimilation ability.
In these cases, as the test yeast or the reference yeast, for example, a yeast introduced with the above-described vector of the present invention, a yeast that has been subjected to a mutation treatment, a naturally-mutated yeast, or the like can be used. For the mutation treatment, any method such as a physical method such as ultraviolet irradiation or radiation irradiation, or a chemical method by chemical treatment such as EMS (ethyl methanesulfonate) or N-methyl-N-nitrosoguanidine may be used. (For example, see Taiji Oshima, Biochemical Experimental Method 39 Yeast Molecular Genetics Experimental Method, p67-75, Academic Publishing Center).

  The yeast that can be used as the reference yeast or the test yeast includes any yeast that can be used for brewing, for example, brewery yeast for beer, wine, sake, and the like. Specific examples include yeasts of the genus Saccharomyces. In the present invention, beer yeasts such as Saccharomyces pastorianus W34 / 70, Saccharomyces carlsbergensis NCYC453, NCYC456, etc. Saccharomyces cerevisiae NBRC1951, NBRC1952, NBRC1953, NBRC1954, etc. can be used. Furthermore, whiskey yeasts such as Saccharomyces cerevisiae NCYC90, wine yeasts such as association wines No. 1, 3 and 4, etc., sake yeasts such as association yeast sake nos. 7 and 9 can be used. However, the present invention is not limited to this. In the present invention, beer yeast such as Saccharomyces pastorianus is preferably used. The reference yeast and test yeast may be selected from the above yeast group in any combination.

  EXAMPLES Hereinafter, although an Example demonstrates the detail of this invention, this invention is not limited to a following example.

Example 1: Cloning of novel amino acid transporters (nonScGAP1, nonScAGP2, nonScMUP1, nonScMUP3) As a result of searching using a comparative database described in JP-A-2004-283169, novel amino acid transporter genes nonScGAP1, nonScAGP2, unique to brewer's yeast NonScMUP1 and nonScMUP3 were found (SEQ ID NOs: 1, 3, 5, and 7). Based on the obtained base sequence information, primers nonScGAP1_F (SEQ ID NO: 9) / nonScGAP1_R (SEQ ID NO: 10), nonSc AGP2_F (SEQ ID NO: 11) / nonSc AGP2_R (SEQ ID NO: 12), nonScMUP1_F (SEQ ID NO: 13) / nonScMUP1_R (SEQ ID NO: 14), nonSc MUP3_F (SEQ ID NO: 15) / nonSc MUP3_R (SEQ ID NO: 16) were designed, and the genome decoding strain Saccharomyces pastorianus byhen stephan 34 DNA fragments containing the full-length genes of nonScGAP1, nonScAGP2, nonScMuP1 and nonScMUP3 were obtained by PCR using chromosomal DNA of the / 70 strain as a template.

  The nonScGAP1, nonScAGP2, nonScMUP1 and nonScMUP3 gene fragments obtained as described above were each inserted into a pCR2.1-TOPO vector (manufactured by Invitrogen) by TA cloning. The nucleotide sequences of the nonScGAP1, nonScAGP2, nonScMUP1 and nonScMUP3 genes were analyzed by the Sanger method (F. Sanger, Science, 214, 1215, 1981) to confirm the nucleotide sequences.

Example 2: Analysis of nonScGAP1 gene expression during beer brewing Beer tasting was performed using the brewer's yeast Saccharomyces pastorianus strain W34 / 70, and mRNA extracted from the brewer's yeast cells during fermentation was detected with a brewer's yeast DNA microarray.

Wort extract concentration 12.69%
Wort capacity 70L
Wort dissolved oxygen concentration 8.6ppm
Fermentation temperature 15 ℃
Yeast input 12.8 × 10 ⁶ cells / mL

The fermentation broth was sampled over time, and changes in the yeast growth (Fig. 1) and appearance extract concentration (Fig. 2) over time were observed. At the same time, the yeast cells were sampled, and the prepared mRNA was labeled with biotin and hybridized to the brewer's yeast DNA microarray described in JP-A-2004-283169. Signal detection was performed using GeneChip Operating System (GCOS; GeneChip Operating Software 1.0, manufactured by Affymetrix). The expression patterns of the nonScGAP1, nonScAGP2, nonScMuP1, and nonScMUP3 genes are shown in FIG. 3, FIG. 4, FIG. 5, and FIG. From this result, it was confirmed that the nonScGAP1, nonScAGP2, nonScMUP1 and nonScMUP3 genes were expressed in normal beer fermentation.

Example 3: Constitutive expression of nonScGAP1, nonScAGP2, nonScMUP1, nonScMUP3 genes nonScGAP1 / pCR2.1-TOPO, nonScAGP2 / pCR2.1-TOPO, nonScMUP1 / pCR2.1-TOPO, nonScMUP3 / pCR2. 1-TOPO is digested with restriction enzymes SacI and NotI to prepare a DNA fragment containing the entire protein coding region. This fragment was ligated to restriction enzymes SacI and NotI-treated pYCGPYNot to construct constitutive expression vectors nonScGAP1 / pYCGPYNot, nonScAGP2 / pYCGPYNot, nonScMUP1 / pYCGPYNot, nonScMUP3 / pYCGPYNot. pYCGPYNot is a YCp type yeast expression vector, and the introduced gene is constitutively expressed by the promoter of the pyruvate kinase gene PYK1. The geneticin-resistant gene G418 ^r is as the selection marker in the yeast, and the ampicillin-resistant gene Amp ^r as a selective marker in Escherichia coli.

  Using the constitutive expression vector prepared by the above-mentioned method, Saccharomyces pastorianus bihenstephan 34/70 strain was transformed by the method described in JP-A-07-303475. Transformants are selected on YPD plate medium (1% yeast extract, 2% polypeptone, 2% glucose, 2% agar) containing geneticin 300 mg / L.

Example 4 Measurement of Amino Acid Utilization in Beer Test Brewing A fermentation test using the parent strain and the nonScGAP1, nonScAGP2, nonScMUP1, and nonScMUP3 high expression strains obtained in Example 3 is performed under the following conditions.

Wort extract concentration 12%
Wort capacity 1L
Wort dissolved oxygen concentration about 8ppm
Fermentation temperature constant 12 ° C Yeast input 5g Wet yeast cells / L wort

Sampling fermented koji over time and examining changes in yeast growth (OD660) and extract consumption over time. Furthermore, the amino acid composition of the beer after fermentation is measured using an L-8800 high-speed amino acid analyzer (manufactured by Hitachi) and a standard amino acid analysis column P / N855-3506 (manufactured by Hitachi, Ltd.).

  According to the method for producing alcoholic beverages of the present invention, it is possible to produce alcoholic beverages having a low amino acid content and a characteristic amino acid composition because the amino acid assimilation ability of yeast is increased.

It is a figure which shows the time-dependent change of the yeast growth amount in beer test brewing. The horizontal axis represents the fermentation time, and the vertical axis represents the value of OD660. It is a figure which shows the time-dependent change of the extract consumption in beer test brewing. The horizontal axis represents the fermentation time, and the vertical axis represents the appearance extract concentration (w / w%). It is a figure which shows the expression behavior of the nonScGAP1 gene in yeast during beer test brewing. The horizontal axis indicates the fermentation time, and the vertical axis indicates the detected signal luminance. It is a figure which shows the expression behavior of the nonScAGP2 gene in yeast during beer test brewing. The horizontal axis indicates the fermentation time, and the vertical axis indicates the detected signal luminance. It is a figure which shows the expression behavior of the nonScMUP1 gene in yeast during beer test brewing. The horizontal axis indicates the fermentation time, and the vertical axis indicates the detected signal luminance. It is a figure which shows the expression behavior of the nonScMUP3 gene in yeast during beer test brewing. The horizontal axis indicates the fermentation time, and the vertical axis indicates the detected signal luminance.

[SEQ ID NO: 9] Primer [SEQ ID NO: 10] Primer [SEQ ID NO: 11] Primer [SEQ ID NO: 12] Primer [SEQ ID NO: 13] Primer [SEQ ID NO: 14] Primer [SEQ ID NO: 15] Primer [ SEQ ID NO: 16] Primer

Claims (20)

The polynucleotide according to any one of the following (a) to (f):
(a) a polynucleotide comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7;
(b) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8;
(c) consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, And a polynucleotide containing a polynucleotide encoding a protein having amino acid transporter activity;
(d) a protein having an amino acid sequence having 60% or more identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, and having amino acid transporter activity A polynucleotide comprising a polynucleotide encoding
(e) Hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, and an amino acid transporter A polynucleotide comprising a polynucleotide encoding a protein having activity; and
(f) It is stringent with a polynucleotide comprising a nucleotide sequence complementary to a nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 A polynucleotide comprising a polynucleotide that hybridizes under conditions and encodes a protein having amino acid transporter activity. The polynucleotide of any one of the following (g) to (i):
(g) In the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. A polynucleotide comprising a polynucleotide encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added, and having amino acid transporter activity;
(h) a protein having an amino acid sequence having 90% or more identity to the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 and having amino acid transporter activity A polynucleotide containing a polynucleotide that encodes; and
(i) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 A polynucleotide comprising a polynucleotide that hybridizes with a polynucleotide consisting of a base sequence complementary to the above base sequence under a highly stringent condition and encodes a protein having amino acid transporter activity.   The polynucleotide according to claim 1, comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.   The polynucleotide according to claim 1, comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.   The polynucleotide according to any one of claims 1 to 4, which is DNA.   A protein encoded by the polynucleotide according to any one of claims 1 to 5.   A vector containing the polynucleotide according to any one of claims 1 to 5.   A transformed yeast into which the vector according to claim 7 has been introduced.   The yeast for brewing according to claim 8, wherein the ability to assimilate amino acids is improved by introducing the vector according to claim 7.   The brewing yeast according to claim 9, wherein the amino acid assimilation ability is improved by increasing the expression level of the protein according to claim 6.   A method for producing an alcoholic beverage using the yeast according to any one of claims 8 to 10.   The method for producing an alcoholic beverage according to claim 11, wherein the alcoholic beverage to be brewed is a malt beverage.     The method for producing an alcoholic beverage according to claim 11, wherein the alcoholic beverage to be brewed is wine.   Alcoholic beverages produced by the method according to any one of claims 11 to 13.   Amino acid assimilation of test yeast using a primer or probe designed based on the nucleotide sequence of the amino acid transporter gene having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. How to evaluate performance.   By culturing the test yeast and measuring the expression level of the amino acid transporter gene having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, A method of evaluating potency. A test yeast is cultured, the protein according to claim 6 is quantified or the expression level of an amino acid transporter gene having the nucleotide sequence of SEQ ID NO: 1 is measured, and the protein according to the target amino acid assimilation ability is measured. A method for selecting yeast, comprising selecting a test yeast having a production amount or an expression level of the gene. Reference yeast and test yeast are cultured and the expression level in each yeast of the amino acid transporter gene having the nucleotide sequence of SEQ ID NO: 1 is measured, and the test yeast in which the gene is expressed higher than the reference yeast is selected. The method for selecting yeast according to claim 17. The method for selecting yeast according to claim 17, wherein the reference yeast and the test yeast are cultured, the protein according to claim 6 in each yeast is quantified, and the test yeast having a larger amount of the protein than the reference yeast is selected. . Fermentation for liquor production is performed using any one of the yeast selected from the yeast according to claims 8 to 10 and the method according to claims 17 to 19, and the amino acid content is adjusted. And a method for producing alcoholic beverages.

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