WO2007097095A1

WO2007097095A1 - Aromatic amino acid transporter gene and use thereof

Info

Publication number: WO2007097095A1
Application number: PCT/JP2006/324609
Authority: WO
Inventors: Yoshihiro Nakao; Yukiko Kodama; Tomoko Shimonaga
Original assignee: Suntory Limited
Priority date: 2006-02-27
Filing date: 2006-12-04
Publication date: 2007-08-30
Also published as: JPWO2007097095A1

Abstract

The invention relates to an aromatic amino acid transporter gene and use thereof, and particularly relates to a brewing yeast strain whose ability to assimilate an aromatic amino acid is controlled, an alcoholic beverage produced by using the yeast strain and a process for producing the same. More specifically, the invention relates to a yeast strain whose ability to assimilate an aromatic amino acid is controlled by controlling the expression level of TAT1 gene encoding Tat1p which is an aromatic amino acid transporter in a brewing yeast strain, particularly nonScTAT1 gene which is unique to beer yeast, a process for producing an alcoholic beverage by using the yeast strain, etc.

Description

Specification

Aromatic amino acid transfer gene and its uses

The present invention relates to an aromatic amino acid transporter gene and use thereof, and in particular, to a brewing yeast in which aromatic amino acid assimilation is controlled, alcoholic beverages produced using the yeast, a production method thereof, and the like. More specifically, the present invention provides an aromatic amino acid by controlling the expression level of the gene TAT1, which encodes Tatlp, an aromatic amino acid lyssporter of brewer's yeast, particularly the nonScTATl gene characteristic of brewer's yeast. The present invention relates to a yeast whose acid-assimilating ability is controlled and a method for producing alcoholic beverages using the yeast. Background Art-In general, amino acids are important as taste components 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 of liquors.

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 acid as a nitrogen source, it is essential to incorporate the amino acid into the cells. It has been clarified that amino acid transporters existing in the cell membrane of yeast are involved in such amino acid uptake.

As amino acid transporters in yeast, many amino acid 卜 transporters with different substrate specificities such as Gapl and substrate specificities (Aromatic amino acid transporter Tatl, Anoleginin transporter Canl, Proline transporter Put4 etc.) Cell Biol. 14: 6597-6606, 1994, Curr Genet 36: 317-328, 1999).

Examples of using yeast having mutations of genes involved in amino acid uptake (gapl, shr3, canl, put4, uga4) to control the amino acid content in alcoholic beverages (JP 2001-321159) (Patent Publication No. 2000-316559) and examples of highly expressing the branched amino acid transporter BAP2 have been reported. Disclosure of the invention.

Under the circumstances as described above, a yeast with controlled amino acid utilization has been desired in order to produce a liquor having a characteristic quality by modifying the amino acid composition in the liquor. As a result of repeated studies to solve the above problems, the present inventors have succeeded in identifying and isolating a gene encoding an aromatic amino acid transporter from brewer's yeast. In addition, a transformed yeast in which the obtained gene was introduced and expressed in yeast was produced, and it was confirmed that the utilization of aromatic amino acids was promoted, thereby completing the present invention. That is, the present invention uses an aromatic amino acid transporter gene 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. It relates 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) A polynucleotide selected from the group consisting of the following (a) to (f):

(a) a polynucleotide comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;

(b) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;

(c) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino ^ sequence of SEQ ID NO: 2 and having an aromatic amino acid transporter activity A polynucleotide comprising: a polynucleotide comprising:

(d) a polynucleotide comprising a polynucleotide encoding a protein having an amino acid sequence having 60% or more identity to the amino acid sequence of SEQ ID NO: 2 and having aromatic amino acid transporter activity;

(e) a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and encodes a protein having aromatic amino acid transporter activity A polynucleotide; and (f) a protein that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of a polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO: 2 and has an aromatic amino acid transporter activity A polynucleotide containing a polynucleotide encoding.

(2) Polynucleotide of ¾ ^ described in (1) above selected from the group consisting of (g) to (i) below:

(g) The amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2, consisting of an amino acid sequence in which 1 to 10 'amino acids are deleted, substituted, inserted and Z or added, and an aromatic amino acid trans A polynucleotide comprising a polynucleotide encoding a protein having porter activity;

(h) a polynucleotide comprising a polynucleotide encoding a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 and having aromatic amino acid transporter activity; and ■

(i) A polynucleotide comprising a nucleotide sequence of SEQ ID NO: 1 or a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under a high stringent condition, and aromatic A polynucleotide comprising a polynucleotide encoding a protein having amino acid transporter activity. '

(3) The polynucleotide according to (1) above, which comprises a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1.

(4) The polynucleotide according to (1) above, which comprises a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2. .

(5) The polynucleotide according to any one of (1) to (4) above, 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

(7 a) The vector according to (7) above, comprising an expression cassette comprising the following components (x) to (z):

(X) One promoter that can be transcribed in yeast cells (y) the polynucleotide according to any one of (1) to (5), which is bound to the promoter in the sense direction or the antisense direction; and

(z) Signals that function in yeast for transcription termination and polyadenylation of RNA molecules.

(8) A polynucleotide selected from the group consisting of the following (j) to (! II):

(j) a polynucleotide encoding RNA having a base sequence complementary to the transcription product of the polynucleotide (DNA) described in (5) above;

(k) a polynucleotide encoding RNA that suppresses the expression of the polynucleotide (DNA) according to (5) above by an RNAi effect;

(1) a polynucleotide encoding an RNA having an activity of specifically cleaving a transcript of the polynucleotide (DNA) described in (5) above; and

(m) A polynucleotide encoding RNA that suppresses the expression of the polynucleotide (DNA) according to (5) above by a co-suppression effect.

(9) A vector comprising the polynucleotide according to (8) above.

(10) A yeast into which the vector according to (7) is introduced. .

(11) The yeast according to (10), wherein the ability to assimilate aromatic amino acids is enhanced by introducing the vector according to (7) above.

(12) The yeast according to (.1 1) above, wherein the ability to assimilate an aromatic amino acid is improved by increasing the expression level of the protein according to (6) above. -

(1 3) (A) By introducing the vector described in (9) above, (B) by destroying the gene related to the polynucleotide (DNA) described in (5) above,

(C) A yeast that suppresses the expression of the polynucleotide (DNA) described in (5) above by adding mutation to the promoter or recombining the promoter.

(14) A method for producing an alcoholic beverage using the yeast according to any one of (1 0) to (1 2) above. (15) The method for producing an alcoholic beverage according to the above (14), wherein the alcoholic beverage to be brewed is a malt beverage.

(16) The method for producing an alcoholic beverage according to the above (14), wherein the alcoholic beverage to be brewed is wine.

(1 7) Alcoholic beverages produced by the method according to any one of (14) to (16) above.

(1 8) Using a primer or probe designed based on the base sequence of the aromatic amino acid transporter gene having the base sequence of SEQ ID NO: 1, A method for evaluating the ability to assimilate aromatic amino acids.

(18a) A method for selecting yeast having a high ability to assimilate an aromatic amino acid by the method described in (18) above.

(18b) A method for producing an alcoholic beverage (for example, beer) using the yeast selected by the method described in (18a) above.

(1 9) Culturing the test yeast and measuring the expression level of the aromatic amino acid transporter gene having the nucleotide sequence of SEQ ID NO: 1 to evaluate the aromatic amino acid-assimilating ability of the test yeast how to.

(19a) Evaluate the test yeast using the method described in (.19) above, and select yeasts with high or low expression of aromatic amino acid transporter gene. High ability to assimilate aromatic amino acids Or a method of selecting low yeast.

(19b) A method for producing an alcoholic beverage (for example, beer) using the yeast selected by the method described in (19a) above. .

(2.0) The test yeast is cultured, and the protein described in (β) above is quantified or the expression level of the aromatic amino acid transporter gene having the base sequence of SEQ ID NO: 1 is measured, and the target aromatic amino acid A method for selecting a yeast, comprising selecting a test yeast having a protein production amount or a gene expression amount according to assimilation ability. '

(2 1) Reference yeast and test yeast are cultured and the expression level in each yeast of the aromatic amino acid transporter gene having the nucleotide sequence of SEQ ID NO: 1 is measured. The method for selecting yeast according to (17) above, wherein a test yeast having low expression is selected.

(22) 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 higher or lower amount of the protein than the reference yeast is selected. The method for selecting yeast according to 1 7).

(2 3) For the production of alcoholic beverages 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 alcoholic beverages, characterized in that fermentation is performed and the aromatic amino acid content is adjusted.

According to the method for producing alcoholic beverages using the transformed yeast of the present invention, since the assimilation of aromatic amino acids can be controlled, the amino acid composition in the alcoholic beverages can be modified. Brief Description of Drawings

FIG. 1 is a diagram showing the change over time in the amount of yeast grown in beer test brewing. The horizontal axis shows the fermentation time, and the vertical axis shows the value of 0D660.

Fig. 2 is a diagram showing the change over time in the amount of extract consumed in beer test brewing. The horizontal axis shows fermentation time, and the vertical axis shows appearance extract concentration (w / w%).

Fig. 3 shows the expression behavior of the nonScTATl gene in yeast during brewing of beer. The horizontal axis shows the fermentation time, and the vertical axis shows the detected signal brightness.

FIG. 4 is a graph showing the change over time in the amount of yeast grown in beer test brewing. The horizontal axis shows the fermentation time, and the vertical axis shows the value of 0D660. '

FIG. 5 is a graph showing changes in extract consumption over time in beer test brewing. The horizontal axis shows fermentation time, and the vertical axis shows appearance extract concentration (w / V /.). BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors thought that it was possible to assimilate aromatic amino acids more efficiently by increasing the activity of the aromatic amino acid transporter of yeast. Based on such an idea, based on the brewer's yeast genome information decoded by the method disclosed in JP-A-2004-283169, a single non-ScTATl gene encoding an aromatic amino acid transporter unique to brewer's yeast was identified. Identified. This base sequence is shown in SEQ ID NO: 1. The amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO: 2.

1. Polynucleotide of the present invention

First, the present invention relates to (a) a polynucleotide containing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, and (b) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2. I will provide a. The polynucleotide may be DNA or RNA.

The polynucleotide targeted by the present invention is the aromatic amino acid derived from the above brewer's yeast It is not limited to DNA encoding an acid transporter, but includes other polynucleotides that encode proteins functionally equivalent to this protein. For example, (c) the amino acid sequence of SEQ ID NO: 2, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and aromatic Examples include proteins having amino acid transporter activity.

As such a protein, in the amino acid sequence of SEQ ID NO: 2, for example:! ~ 90 pieces; ~ 80 pieces ,! ~ 70 ,! ~ 60 pcs! ~ 50 pieces! ~ 40,:! ~ 39,:! ~ 38 pcs! ~ 37 pcs! ~ 36 pcs! ~ 35 pcs! ~ 34 pcs! ~ 33, 1-32! ~ 31 pcs! ~ 30 pcs! ~ 29 pcs! ~ 28 pcs! ~ 27 pcs! ~ 26 pcs! ~ 25 pcs! ~ 24 pcs! ~ 23, 1 ~ 22 1 ~ 21 {@, G 20 ... ~ 19,:! ~ 18,:! ~ 17 'pieces! ~ 16 pcs! ~ 15 pcs! ~ 14 pcs! ~ 13 pcs! ~ 12,:! ~ 11,:! ~ 10 pieces, :! ~ 9,, 1-8,:! ~ 7, 1-6 (1 ~ several), ';! ~ 5, 1-4, 1-3, 1-2, consisting of amino acid sequence with one amino acid residue deleted, substituted, inserted and / or added, and aromatic amino acid transporter A protein having one activity. In general, the smaller the number of amino acid residue deletions, substitutions, insertions and / or additions, the better. Such proteins include (d) the amino acid sequence of SEQ ID NO: 2, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% More than 76%, 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% or more, 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. A protein having an amino acid sequence having 9% or more identity and having an aromatic amino acid transporter activity. In general, the higher the homology value, the better.

The aromatic amino acid transporter activity can be measured, for example, by the method described in (Mol Cell Biol. 14: 6597-6606, 1994).

The present invention also provides: (e) a poly nucleotide comprising a base sequence complementary to the base sequence of SEQ ID NO: 1. A polynucleotide comprising a polynucleotide r that hybridizes with a nucleotide under stringent conditions and that codes for a protein having aromatic amino acid transporter activity; and (f) from the amino acid sequence of SEQ ID NO: 2. Contains a polynucleotide encoding a protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence of the polynucleotide encoding the protein and has an aromatic amino acid transporter activity. It also includes polynucleotides that Here, “a polynucleotide that hybridizes under stringent conditions” means a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1. or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2. Polynucleotides (for example, DNA) obtained by using the colony hybridization method, plaque hybridization method, Southern hybridization method, etc. with all or part of the nucleotide as a probe Say. As a method for hybridization, methods described in, for example, Molecular Cloning 3rd Ed., Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997 can be used. As used herein, “stringent conditions” may be any of low stringency conditions, medium stringency conditions, and high stringency conditions. “Low-slingen conditions” are, for example, 5 X SSC, 5 X Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C. The “medium stringent conditions” are, for example, the conditions of 5 × SSC, 5 × denhanoleto solution, 0.5% SDS, 50% formamide, and 42 ° C. “High stringency conditions” are, for example, 5 X SS (:, 5 X Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. In these conditions, the temperature is It is expected that polynucleotides with higher homology (for example, DNA) can be obtained more efficiently as the value increases, but factors that affect the stringency of the hybridization include temperature, probe concentration, and probe concentration. Multiple factors such as length, ionic strength, time, and salt concentration can be considered, and those skilled in the art can achieve the same stringency by appropriately selecting these factors.

When using a commercially available kit for hybridization, for example, Alkphos Direct Labeling Reagents (manufactured by Amersham Almacia) can be used. The In this case, follow the protocol supplied with the kit, incubate with the labeled probe, and then wash the membrane with 0.1% (w / v) SDS at 55 ° C. After washing, the hybridized polynucleotide (eg, DNA) can be detected.

Other polynucleotides that can be hybridized include polynucleotides that encode the amino acid sequence of SEQ ID NO: 2, when calculated by default parameters using homology search software such as FASTA and BLAST. And about 60% or more, about '70 ° /. 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% or more, 94% or more, 95 %, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4 ° / ₀ , 99.5%, 99.6%, 99.7%, Polynucleotides having 99.8% or more and 99.9% or more identity can be raised.

The identity of amino acid sequences and nucleotide sequences is determined using the algorithm B LAST (proc: Natl. Acad. Sci. USA 872264-2268, 1990; proc Natl Acad Sci USA 90: 5873, 1993) by Carlin and Arthur. it can. Programs called B LAS TN and B LAS TX based on the B AST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When the base sequence is analyzed using B LASTN, the parameter 1 is set to, for example, s corre = 100 and wor d lengt h = 12. In addition, when the amino acid sequence is analyzed using B L A S TX, the parameter 1 is set to, for example, s corre = 50 and wo rd lengh t = 3. When using BLAST and G a p p e d B L A S T programs, use the default parameters of each program.

Further, the polynucleotide of the present invention comprises: (j) a polynucleotide encoding an RNA having a base sequence complementary to the polynucleotide (DNA) transcription product described in (5) above; (k) the above ( 5) a polynucleotide encoding an RNA that suppresses the expression of the polynucleotide (DNA) according to the RNAi effect; (1) specifically cleaves the transcription product of the polynucleotide (DNA) according to (5) above; Coat active RNA Or (m) a polynucleotide that encodes an RNA that suppresses the expression of the polynucleotide (DNA) according to (5) above by a co-suppression effect. These polynucleotides are incorporated into a vector, and can further suppress the expression of the polynucleotides (DNA) (a;) to (i) above in a transformed cell into which the vector has been introduced. Therefore, it can be suitably used when it is preferable to suppress the expression of the polynucleotide (DNA).

In the present specification, “polynucleotide encoding RNA having a base sequence complementary to a transcription product of DNA” refers to so-called antisense DNA. Antisense technology is known as a method for suppressing the expression of specific endogenous genes, and is described in various documents (eg, Hirashima and Inoue: Laboratory for Neonatal Chemistry 2 Nucleic Acid IV Gene Replication and Expression) (See Japan Biochemical Society, Tokyo Kagaku Dojin) 'pp. 319-347, 1993, etc.). The sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene or a part thereof, but may not be completely complementary as long as the gene expression can be effectively suppressed. The transcribed RNA preferably has a complementarity of 90% or more, more preferably 95% or more, to the target gene transcript. The length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more ', more preferably 500 bases or more. '

In the present specification, the “polynucleotide encoding RNA that suppresses DNA expression by the RNAi effect” refers to a polynucleotide for suppressing the expression of an endogenous gene by RNA interference (RNAi). “RNAi” is a phenomenon in which when a double-stranded RNA having the same or similar sequence as the target gene sequence is introduced into a cell, both the introduced foreign gene and the expression of the target endogenous gene are suppressed. Refers to that. Examples of RNA used herein include double-stranded RNA that causes RNA interference of 21 to 25 bases in length, such as dsRNA (double strand RNA), siRNA (small interfering RNA), or shRNA (short hairpin RNA). . Such RNA can be locally delivered to a desired site by a delivery system such as ribosome, and can be locally expressed using a vector that produces the above double-stranded RNA. . Methods for preparing and using such double-stranded RNA (dsRNA, siRNA or shRNA) are known from many literatures (Japanese Patent Publication No. 2002-516062; US Publication No. 2002 / 086356A). Nature Genet ics, 24 (2), 180-183, 2000 Feb .; Genesis, 26 (4), 240-244, 2000 April; Nature, 407: 6802, 319-20, 2002 Sep. 21; Genes & Dev., Vol. 16, (8), 948-958, 2002 Apr. 15; Proc. Natl. Acad. Sci. USA., 99 (8), 5515-5520, 2002 Apr. 16; (5567), 550-553, 2002 Apr. 19; Proc Natl. Acad. Sci. USA, 99: 9, 6047-6052, 2002 Apr. 30; Nature Biotechnology, Vol. 20 (5), 497-500, 2002 May; Nature Biotechnology, Vol. 20 (5), 500-505, 2002 May; Nucleic Acids Res., 30: 10, e46, 2002 May 15 etc.).

In the present specification, “polynucleotide encoding RA having an activity of specifically cleaving a DNA transcript” generally refers to a ribozyme. A ribozyme is an RNA molecule that has catalytic activity and inhibits the function of the gene by cleaving the target DNA transcript. Various known literatures can also be referred to for the design of lipozymes (for example, FEBS Lett. 228: 228, 1988; FEBS Lett. 239: 285, 1988; Nucl. Acids. Res. 17: 7059, 1989; Nature 323). Nucl. Acids Res. 19: 6751, 1991; Protein Eng 3: 733, 1990; Nucl. Acids Res. 19: 3875, 1991; Nucl. Acids Res. 19: 5125, 1991; Biochera Biophys Res Commun 186: 1271, 1992 etc.). In addition, “polynucleotide encoding RNA that suppresses DNA expression by co-suppression effect” refers to a nucleotide that inhibits the function of the target DNA by “co-suppression”. ''

In this specification, “co-suppression” means expression of introduced foreign gene and target endogenous gene by introducing a gene having a sequence identical or similar to the target endogenous gene into cells by transformation. Refers to a phenomenon in which both are suppressed. Various known literatures can also be referred to for designing a polynucleotide having a co-suppression effect (for example, Smyth DR: Curr. Biol. 7: R793, 1997, art ienssen R: Curr. Biol. 6: 810, (See 1996).

2. Protein of the present invention

The present invention also provides a protein encoded by any of the polynucleotides (a) to (i). A preferred protein of the present invention is an amino acid in which one or more amino acids are deleted, substituted, inserted and added or deleted in the amino acid sequence of SEQ ID NO: 2. It is a protein having a nonacid sequence and having an aromatic amino acid transporter activity.

Such a protein comprises an amino acid sequence in which the number of amino acid residues as described above is deleted, substituted, inserted and Z or added in the amino acid sequence of SEQ ID NO: 2, and an aromatic amino acid transporter. Active. Proteins are listed. Examples of such a protein include a protein having an amino acid sequence having the homology as described above with the amino acid sequence of SEQ ID NO: 2 and having an aromatic amino acid transporter activity.

Such proteins are described in "Molecular Cloning 3rd Edition", "Current Protocol" in Molecular "Nuology", "Nu Acids. Res., 10, 6487 (1982)", "Proc. Natl Acad. Sc i. USA, 79, 6409 (1982) ".," Gene, 34: 315 (1985) "," Nuc. Acids. Res., .4, 4431 (1985) "," Proc. Natl. Acad. Sci. USA, 82, 488 (1985) "etc., can be obtained using the site-directed mutagenesis method.

In the amino acid sequence of the protein of the present invention, one or more amino acid residues are deleted, substituted. Insertion and / or addition means that 1 and / or a position in one or more amino acid sequences in the same sequence is 1 Or it means that there are deletion, substitution, insertion and / or addition of a plurality of amino acid residues, and two or more of deletion, substitution, insertion and addition may occur at the same time. '

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, Norin, Norpaline, Alanine, 2-Aminobutanoic acid, Methionine, 0-Methylserine, 1-Butylglycine, t-Butylalanin, Cyclohexylalanin; Group B: Aspartic acid, Glutamic acid, Isoaspartic acid, isoglutamic acid, 2-aminoaminodipic acid, 2-aminosuberic acid; Group C: asparagine, glutamine; Group D: lysine, arginine, ornithine, 2, 4-diaminobutanoic acid, 2, 3-diaminobutanoic acid Aminopropionic acid; Group E: proline, 3-hydroxyproline, 4-hydroxyproline; Group F: serine, threonine, homoserine; Group G: phenylenolanine, tyrosine.

In addition, the protein of the present invention is obtained by Fmoc method (fluorenylmethyloxycarbonyl). Method) and tBoc method (t-butyloxycarbonyl method). Chemical synthesis using peptide synthesizers such as Advanced Dogemtechne, Perkin Elma, Pharmacia, Protein Technology Instrument, Synthecel Vega, Perceptive, Shimadzu, etc. You can also.

3. Vector of the present invention and transformed yeast introduced with the vector

Next, the present invention provides a vector containing the polynucleotide described above. The vector of the present invention contains the polynucleotide (DNA) according to any one of the above (a) to (i). The vector of the present invention is usually (X) a promoter that can be transcribed in yeast cells; (y) the promoters (a) to (i) bound to the promoter in a sense direction or an antisense direction. A polynucleotide (DNA) according to any of the above; and (z) an transcription cassette for RNA molecules, and an expression cassette comprising a signal that functions in yeast as a component for termination and boriadenylation.

In the present invention, in the brewing of beer described later, when the protein of the present invention is highly expressed, the expression of the polynucleotide (DNA) according to any one of the above (a) to (i) is promoted. These polynucleotides are introduced in the sense direction with respect to the promoter. In addition, in beer brewing, when the expression of the protein of the present invention is to be suppressed, the expression of the polynucleotide (DNA) should be suppressed as described in any of the above (a) to (i). These polynucleotides are introduced in the antisense direction with respect to the promoter. In addition, when the expression of the protein of the present invention is suppressed, it can be introduced into a vector so that the polynucleotide described in any of (j) to (m) above can be expressed. In the present invention, the expression of the polynucleotide (DNA) or the expression of the protein can be suppressed by destroying the gene (DNA) as a target. Gene disruption involves the addition or deletion of single or multiple bases within a region involved in the expression of a gene product in a target gene, such as the coding region or promoter region, or lack of these entire regions. It can be done by losing. For such gene disruption methods, known literature can be referred to (for example, Proc. Natl. Acad. Sci. USA, 76, 4951 (1979), Methods in Enzymology, 101, 202 (1983), JP-A-6-253826, etc.). In the present invention, the expression level of the target gene can also be controlled by adding mutations to the promoter or by recombining the promoter by homologous recombination. Nucleic Acids Res. 29, 4238-4250 (2001) for introducing such mutations, and methods for controlling gene expression by converting promoters are described in, for example, Appl Environ Mi crobiol., 72, 5266-5273 (2006). )It is described in.

As a vector used for introduction into yeast, any of a multicopy type (ΥΕρ type), a single copy type (YCp type), and a chromosome integration type (Yip type) can be used. For example, as a YEp type base-click data one YEp24 (JR Broach et al., Experimental Manipulat ion of Gene Expression, Academi c Press, New York, 83, 1983), as is YCp type vector _YCp50 _(M. _{D Rose et} al., gene, 60, 237, 1987), YIp5 (K. Struhl et al., Pro Natl. Acad. Sci. USP, '76, 1035, 1979) is known as one of the Yip type vectors. And can be easily obtained.

As a promoter terminator for regulating gene expression in yeast, any combination can be used as long as it functions in brewing yeast and is not affected by the components in the mash. For example, the promoter of the 'Dari' cellular aldehyde 3 phosphate dehydrogenase gene (TDH3) and the promoter of the 3-phosphoglycerate kinase gene (PGK1) can be used. These genes are already black one ring. For example MF Tuite et al., EMB0 J. , 1, 603 (1982) to have been described in detail, are readily available by known methods ₉ As the selection marker used for transformation, since the auxotrophic force is not available for brewing yeast, the dieneticin resistance gene (G418r), the copper resistance gene (CUP1) (Marin et al., Proc Natl. Acad. Sci. USA, 81, 337 1984), senorelenin resistance gene (fas2m, PDR4) (Ashigaki, et al., Biochemistry, 64, 660, 1992; Hussain et et al., Gene, 101, 149, 1991) can be used.

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 ^ Saccharomyces. However, in the present invention, beer yeasts such as Saccharomyces pas tori anus W34 / 70, Saccharomyces carols benore gensis NCYC453, NCYC456, Saccharomyces cc Z NBRC1951, NBRC1952, NBRC1953, NBRC1954, etc. can be used. In addition, whiskey yeasts such as Saccharomyces cerevisiae NCYC90, wine yeasts such as association wines 1, 3 and 4, sake yeasts such as association yeasts sake 7 and 9 can be used. However, it 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, the electroporation method “Meth. Enzyra., 194, ρ182 (1990)”, the Spheroplast method “Proc. Natl. Acad. Sci. USA, 75 pl929 (1978),”, the lithium acetate method “J. Bacteriology, 153, pl63 (198,3) ", Proc. Natl. Acad. Sci. USA, 75 pl929 (1978), Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual The method can be implemented, but is not limited to this.

More specifically, the host yeast is a standard yeast nutrient medium (for example, YEPD medium “Genetic Engineering. Vol. 1, Plenum Press, New York, 117 (1979)” etc.), and the value of 0D600nm is 1-6. Incubate to The cultured yeast is collected by centrifugation, washed, and pretreated with an alkali metal ion having a concentration of about 1 to 2M, preferably lithium ion. The cells are allowed to stand at about 30 ° C. for about 60 minutes, and then with the DNA to be introduced (about 1 to 20 _μ§ ) at about 30 ° C. for about 60 minutes. Polyethylene glycol, preferably about 4,000 danoleton polyethylene glycol, is added to a final concentration of about 20% to 50%. After leaving at about 30 ° C for about 30 minutes, heat the cells 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. After that, a transformant is obtained by planting on a standard agar medium containing antibiotics used as a selection marker. For other general cloning techniques, see “Molecular Cloning 3rd Clothing”, lvlethods in Yeast (jenitics, A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) ".

4. The method for producing the alcoholic beverage of the present invention and the alcoholic beverage obtained by the method

By introducing the above-described vector of the present invention into a yeast suitable for brewing alcoholic beverages to be produced, and using the yeast, alcoholic beverages characterized by amino acid composition can be produced. Moreover, the yeast selected by the yeast evaluation method of the present invention described below can be used in the same manner. Examples of alcoholic beverages that can be used include, but are not limited to, beer-taste drinks such as beer and happoshu, wine, whiskey, and sake. In the present invention, alcoholic beverages in which the composition of amino acids is adjusted with desired alcoholic beverages can also be produced by using a brewing yeast in which the expression of a target gene is suppressed as necessary. That is, it was selected by the yeast introduced with the vector of the above-mentioned date of origin, the yeast in which the expression of the polynucleotide (DNA) of the present invention described above was suppressed, or the yeast evaluation method of the present invention described below. Fermentation for the production of alcoholic beverages using yeast and adjusting (increasing or decreasing) the content of amino acids to produce alcoholic beverages with the desired alcoholic acid and with the amino acid content adjusted (increased or decreased). You can. ■ '

In producing these alcoholic beverages, a known method can be used except that the brewing mother obtained in the present invention is used in place of the parent strain. Therefore, the raw materials, 'production facilities, production management, etc. may be exactly the same as the conventional method, and there is no increase in costs for producing alcoholic beverages with a reduced fermentation period. That is, according to the present invention, the existing facility can be used without increasing the cost. .

5. Yeast evaluation method of the present invention

The present invention relates to a method for evaluating the ability to assimilate an aromatic amino acid in a test yeast using a primer or probe designed based on the base sequence of an aromatic amino acid transporter gene having the base sequence of SEQ ID NO: 1. About. A general method of such an evaluation method is known, and is described in, for example, WO 0 1 Z 0 4 0 5 14, Japanese Patent Application Laid-Open No. 8-205 0 00, and the like. This evaluation method is briefly described below. First, prepare the test yeast genome. Methods of preparation, such as Hereford method or acetic Kariumu method, any known method can be used (e.g., Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, pl30 (1990)) directed to the ₀ resulting genome Whether the gene of the test yeast genome or a sequence specific to the gene exists using a primer or probe designed based on the base sequence of the aromatic amino acid transporter gene (preferably the 0RF sequence) Check for no. 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; a polynucleotide containing a nucleotide or a nucleotide sequence complementary to the nucleotide sequence used as one primer, and the other primer upstream from this sequence. Alternatively, a polynucleotide containing a part or all of the downstream sequence or a polynucleotide containing a base sequence complementary to the base sequence is used to amplify a yeast nucleic acid by PCR ¾, Measure the molecular weight of the amplified product, etc. The number of bases of the polynucleotide used for the primer is usually 10 bp or more, and preferably 15 to 25 bp. Usually, 300 to 2000 bp is appropriate.

The reaction conditions of the PCR method are not particularly limited. For example, denaturation temperature: 90 to 95 ° C, annealing temperature: 40 to 60 ° C, extension temperature: 60 to 75 ° C, number of cycles: 10 times or more, etc. Conditions can be used. 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. By this method, the ability to assimilate the aromatic amino acid of the yeast is predicted and evaluated depending on whether the molecular weight of the amplification product is large enough to include the DNA molecule of the specific part. In addition, by analyzing the base sequence of the amplified product, the above performance can be predicted and evaluated more accurately.

In the present invention, the test yeast is cultured, and the expression level of the aromatic amino acid transporter gene having the base sequence of SEQ ID NO: 1 is measured to thereby improve the aromatic amino acid assimilation ability of the test fermentation mother. It can also be evaluated. The expression level of the aromatic amino acid transporter gene can be measured by culturing the test yeast and quantifying the mRNA or protein that is the transcription product of the aromatic amino acid transporter gene. Noh. Quantification of mRNA or protein can be performed using a known method. Quantification of mRNA can be performed by, for example, Northern hybridization quantitative RT-PCR, and protein can be quantified by, for example, western blotting. (Current Protocols in Molecular Biology, John Wiley & Sons 1994-2003). It is also possible to predict the expression level of the gene in the test yeast by measuring the aromatic amino acid concentration in the fermentation broth obtained when the test yeast is cultured.

Furthermore, the test yeast is cultured, the expression level of the aromatic amino acid transporter gene having the nucleotide sequence of SEQ ID NO: 1 is measured, and the gene expression according to the target ability to assimilate the aromatic amino acid is obtained. By selecting the amount of yeast, a yeast suitable for brewing the desired liquor can be selected. 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, a standard yeast and a test mother are cultured, and the expression level in each yeast of the aromatic amino acid transporter gene having the base sequence of SEQ ID NO: 1 is measured. By selecting a test yeast having a high or low gene expression, a yeast suitable for producing a desired alcoholic beverage 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 or low aromatic amino acid assimilation ability.

In these cases, as the test yeast or the reference yeast, for example, yeast introduced with the above-mentioned vector of the present invention, yeast subjected to mutation treatment, naturally-mutated yeast, and the like can be used. Mutation treatment uses any method, for example, a physical method such as ultraviolet irradiation or radiation irradiation, a chemical method using chemical treatment such as EMS (ethyl methanesulfonate), N-methyl-N-nitrosoguanidine, etc. which may be (e.g., Taiji Oshima edited, Biological chemistry experiment method 39 yeast molecular genetics experimentation, P67-75, Gakkai Shuppan see, Center) _c the evaluation of aromatic amino acid catabolism, for example, after the end of fermentation The amino acid composition of beer was analyzed using an amino acid analyzer (for example, L-8800 high-speed amino acid analyzer; manufactured by Hitachi), standard amino acid analysis column P / N855-3506; manufactured by Hitachi, Ltd.) composition This can be done by evaluating the aromatic amino acid concentration in the medium.

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 iS _a cch _a romyces ~ (for example, Saccharomyces pastorianus, Saccharomyces cerevisiae and Saccharomyces virulence). In the present invention, beer yeasts such as Saccharomyces pass Trianus Saccharomyces pastorianus W34 / 70, etc., Saccharomyces carlsbergensis) NCYC453, NCYC456, etc. In addition, whiskey yeasts such as Saccharomyces cerevisiae NCYC90, wine yeasts such as Association Wines No. 1, No. 3, No. 4, etc., sake yeasts such as Association Yeast Kiyo '? 酉 No. 7, No. 9 etc. I can, but I'm not limited to this. In the present invention, beer yeast such as Saccharomyces pastorianus is preferably used. The reference yeast and the test yeast may be selected in any combination from the above yeast group. Example ''

Hereinafter, the details of the present invention will be described by way of examples, but the present invention is not limited to the following examples. 'Example 1: Cloning of Aromatic Amino Acid Transporter Gene (nonScTATl) As a result of searching using a comparative database described in JP-A-2004-283169, an aromatic amino acid transporter gene, nonScTATl, unique to brewer's yeast was found. (SEQ ID NO: 1). Based on the obtained nucleotide sequence information, the primers nonScTATl_F (SEQ ID NO: 3) / nonScTATl_R (SEQ ID NO: 4) were designed to amplify the full-length gene, and the genome decoding strain Saccharomyces pas trianus by Henstefan 34 / A DNA fragment containing the full length gene of nonScTATl was obtained by PCR using chromosomal DNA of 70 strains (sometimes abbreviated as Γ 34/70 strain).

The nonScTATl gene fragment obtained as described above was obtained by TA cloning. It was inserted into pCR2. 1-T0P0 vector (Invitrogen). The nucleotide sequence of the nonScTATl gene was analyzed by Sanger (F. Sanger, Science, 214, 1215, 1981) to confirm the nucleotide sequence. Example 2: Analysis of nonScTATl gene expression during beer brewing

Beer brewing 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 8.6 ppm

Fermentation temperature 15 ° C

Yeast input amount 12.8 × 10 ⁶ cells / mL The fermentation broth was sampled over time, and changes in yeast growth (Fig. 1) and appearance extract concentration (Fig. 2) over time were observed. At the same time, yeast cells were sampled, and the prepared mRNA was labeled with piotin and hybridized with a brewer's yeast DNA microarray described in JP-A-2004-283169. Signal detection was performed using a GeneChip operating system (GC0S; GeneChip Operating Software 1.0, manufactured by Affymetritas). The expression pattern of the nonScTATl gene is shown in FIG. From this result, it was confirmed that the nonScTATl gene was expressed in normal beer fermentation. Example 3: Production of nonScTATl high expression strain

Example 1 nonScTATl / _P CR2. The 1-T0P0 restriction enzymes Sacl and Notl digestion described to prepare a DNA fragment containing the protein coding region length. This fragment was ligated with restriction enzymes Sacl and NotY-treated pYCGPYNot to construct nonScTATl high expression vector nonScTATl / pYCGPYNot. pYCGPYNot is a YCp-type yeast expression vector. The introduced gene is highly expressed by the pyruvate kinase gene PYK1 promoter. Appear. It contains the gene for resistance to dieneticin G418 ¹ "as a selectable marker in yeast, and the ampicillin-resistant gene gene Amp ^r as a selectable marker in Escherichia coli. -303475 was transformed Nsutefuan 34/70 strain into Saccharomyces path Torianusu by in a way described in. YPD plate medium containing Jienechishin 300mg / L (1 ° /. yeast Ekisu, 2% polypeptone, ₂ ./ ₀ glucose , 2% agar) 'Example 4: Measurement of amino acid utilization in beer test brewing

A fermentation test using the parent strain and the nonScTATl high expression strain obtained in Example 3 was performed under the following conditions. Wort extract concentration 12%.

Wort capacity 1L

Wort dissolved oxygen concentration about 8ppm

Fermentation temperature 15 ° C—Constant

Yeast input 5g Wet yeast cells ZL wort Fermented rice Sampling was conducted over time, and changes in yeast growth (0D660) (Fig. 4) and kiss consumption (Fig. 5) over time were examined. As shown in Table 1, the amino acid composition of beer produced using nonScTATl high-expressing strains is reduced in aromatic amino acids such as tryptophan, tyrosine, and phenylalanine compared to beer produced using the parent strain. It was. In addition, while glutamic acid, glycine, alanine, etc. increased, ammonia decreased, beer produced using nonScTATl high-expressing strains showed different characteristics from those produced using the parent strain. It was. table 1

Amino acid content (mM) Parent strain NonScTATl high expression strain

Aspartic acid 0. 0056 0. 02375

Threonine 0. 0552 0. 09065

Serine not detected 0. 0197

Gnoretamic acid 0. 0998 0. 2436

Glycine 0. 2385 0. 3345

Alanine 0 6723 1. 39315

Sistine 0. 0133 0. 0224

Valine 0. 3689 0. 40705

Methionine 0. 0056 0. 00575

Isoleucine 0. 0438 0. 03215

Leucine. 0. 0753 0. 05355.

Tyrosine 0. 2918 Not out

Felanalanin 0. 3599 0. 0958

Ornitine 0. 0428 0. 04345

Lysine 0. 1036 0. 11145

Histidine 0. 1225 0. 224

Tribto fan 0 1387 0. 0817

Arginine 0. 0088 0. 03365

Proline 3. 9525 3. 98815

Total amount of amisic acid (mM) 6. 5985 7. 2045 Ammonia (mg / L) 13. 464 4. 488 Example 5 ■: NonScTATl gene disruption.

The plasmids (pFA6a (G418r), pAG25 (natl), pAG32 (hph)) containing drug resistance markers were used as templates according to the method of the literature (Goldstein et al., Yeast. 15 1541 (1999)). Gene disruption fragments are prepared by PCR. -Transform W34 / 70 strain or spore clone W34 / 70-2 strain with the gene disruption fragment. Transformation is carried out by the method described in Japanese Patent Application Laid-Open No. 07-303475, and the concentration of the selected drug is set to 300 mg / L for geneticin and 50 mg / L for noseothricin, respectively. Example 6: Measurement of ammonia and amino acid utilization in beer test brewing

A fermentation test using the parent strain and the nonScTATl disrupted strain obtained in Example 5 is performed under the following conditions. Wort extract concentration 12%

Wort capacity 1L

Wort dissolved oxygen concentration about 8ppm

Fermentation temperature 15 ° C—Constant

Yeast input 5g Wet yeast cells / L wort As in Example 4, sample fermented koji over time, and examine changes over time in yeast growth (0D660), extract consumption, ammonia and various amino acids . Industrial use available

According to the method for producing alcoholic beverages of the present invention, the aromatic amine / acid assimilation ability of yeast can be controlled, so that alcoholic beverages having a characteristic amino acid composition with an adjusted aromatic amino acid content are produced. It becomes possible.

Claims

The scope of the claims

1. A polynucleotide selected from the group consisting of (a) and (f):

'(a) a polynucleotide containing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;

(c) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 2 and having an aromatic amino acid transporter activity A polynucleotide containing a polynucleotide that:

(d) an amino acid having 60% or more identity to the amino acid sequence of SEQ ID NO: 2. a polynucleotide comprising a polynucleotide having a sequence and coding for a protein having a force aromatic amino acid transporter activity ;

(e) Contains a polynucleotide that encodes a protein that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 and has an aromatic amino acid lancer activity and activity. Polynucleotide to be used; and '

(f) It is hybridized under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 and has an aromatic amino acid transporter activity. A polynucleotide comprising a polynucleotide encoding a protein having the same.

2. The polynucleotide of claim 1 selected from the group consisting of (g) to (i) below:

(g) The amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2, consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added, and an aromatic amino A polynucleotide comprising a polynucleotide encoding a protein having acid transporter activity;

(h) Amino acids having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 A polynucleotide comprising a polynucleotide having a sequence and encoding a protein having aromatic amino acid transporter activity; and

(i) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under high stringency conditions, and an aromatic amino acid trans A polynucleotide comprising a polynucleotide encoding a protein having porter activity.

3. The polynucleotide according to claim 1, comprising a polynucleotide comprising 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 any one of claims 1 to 4, which is DNA.

6. A protein encoded by the polynucleotide according to any one of claims 1 to 5.

7. A vector containing the polynucleotide according to any one of claims 1 to 5. .

8. A polynucleotide selected from the group consisting of the following (j) to (m):

(j) a polynucleotide encoding RA having a base sequence complementary to the transcript of the polynucleotide (DNA) of claim 5;

(k) a polynucleotide encoding RNA that suppresses the expression of the polynucleotide (DNA) according to claim 5 by an RNAi effect;

(1) a polynucleotide encoding an RNA having an activity of specifically cleaving a transcript of the polynucleotide (DNA) according to claim 5; and

(m) A polynucleotide encoding RNA that suppresses the expression of the polynucleotide (DNA) according to claim 5 by a co-suppression effect.

9. A vector containing the polynucleotide according to claim 8.

'1 0. Yeast into which the vector according to claim 7 has been introduced.

11. The yeast according to claim 10, wherein the ability to assimilate an aromatic amino acid is improved by introducing the vector according to claim 7.

1 2. The yeast according to claim 11, wherein the ability to assimilate an aromatic amino acid is improved by increasing the expression level of the protein according to claim 6.

1 3. (A) By introducing the vector according to claim 9, (B) by disrupting the gene related to the polynucleotide (DNA) according to claim 5, (C) adding a mutation to the promoter Or by recombining the promoter ^ "

A yeast which suppresses the expression of the polynucleotide (DNA) according to claim 5 by:

14. A method for producing an alcoholic beverage using the yeast according to any one of claims 10 to 12. 15. The method for producing an alcoholic beverage according to claim 14, wherein the alcoholic beverage to be brewed is a malt beverage. .1 6. The method for producing an alcoholic beverage according to claim 14, wherein the brewed alcoholic beverage is wine.

1 7. Alcoholic beverages produced by the method according to any one of claims 14 to 16.

1 8. A method for evaluating the ability to assimilate an aromatic amino acid in a test yeast using a primer or probe designed based on the base sequence of an aromatic amino acid transporter gene having the base sequence of SEQ ID NO: 1.

1 9. Culturing the test yeast, and measuring the expression level of the aromatic amino acid transporter gene having the base sequence of SEQ ID NO: 1, thereby increasing the ability of the test yeast to assimilate the aromatic amino acid. How to evaluate.

20. Culture the test yeast, quantify the protein according to claim 6 or measure the expression level of an aromatic amino acid transporter gene having a base sequence of SEQ ID NO: 1. 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 according to amino acid assimilation ability.

2 1. Cultivate the reference yeast and the test yeast and measure the expression level of the aromatic amino acid transporter gene having the nucleotide sequence of SEQ ID NO: 1 in each yeast. The method for selecting a yeast according to claim 20, wherein a test yeast having high expression or low expression is selected.

2 2. The reference yeast and the test yeast are cultured, and the protein according to claim 6 in each yeast is quantified, and the test yeast having a higher or lower amount of the protein than the reference yeast is selected. 20. The method for selecting yeast according to 20.

2 3. Yeast according to claims 10 to 12 and fermentation for liquor production using any one of yeast selected by the method according to claims 20 to 22. A method for producing an alcoholic beverage, comprising adjusting the aromatic amino acid content.