JP2009504133A - Adenylyl sulfate transferase gene and use thereof - Google Patents

Abstract

  The present invention relates to an adenylyl sulfate transferase gene and use thereof, and in particular, to a brewing yeast that produces alcoholic beverages excellent in flavor stability, alcoholic beverages produced using the yeast, a method for producing the same. More specifically, the present invention relates to the MET3 encoding gene Met3p, a sulfated adenylyl transferase of brewing yeast, in particular by controlling the expression level of the nonScMET3 gene characteristic of brewer's yeast, thereby stabilizing the flavor of the product. The present invention relates to a yeast in which the ability to produce sulfite that contributes to crystallization is controlled, a method for producing alcoholic beverages using the yeast, and the like.

Description

  The present invention relates to an adenylyl sulfate transferase gene and use thereof, and in particular, to a brewing yeast that produces alcoholic beverages excellent in flavor stability, alcoholic beverages produced using the yeast, a method for producing the same. More specifically, the present invention relates to the MET3 encoding gene Met3p, a sulfated adenylyl transferase of brewing yeast, in particular by controlling the expression level of the nonScMET3 gene characteristic of brewer's yeast, thereby stabilizing the flavor of the product. The present invention relates to a yeast in which the ability to produce sulfite that contributes to crystallization is controlled, a method for producing alcoholic beverages using the yeast, and the like.

  Sulfurous acid is known as a compound having a high antioxidant effect, and is widely used as an antioxidant in the fields of foods, beverages, pharmaceuticals and the like (for example, JP-A 06-040907 (Patent Document 1), JP-A 2000-093096). Gazette (patent document 2) etc.). Sulfurous acid is also used as an antioxidant in alcoholic beverages. For example, wine that requires long-term aging plays an important role in maintaining its quality. Therefore, in Japan, it is added by the Ministry of Health, Labor and Welfare to a residual concentration of 350 ppm or less. Is allowed. Also, it is known that the quality retention period changes depending on the concentration of sulfurous acid contained in the product in beer brewing, and it is very important in terms of flavor stability and the like to strengthen this substance.

The simplest way to increase the sulfurous acid content in a product is to add sulfurous acid. In that case, it will be treated as a food additive, and there will be a degree of freedom in product development and a negative consumer image of the food additive. It becomes a problem.
By the way, yeast biosynthesizes sulfur-containing compounds necessary for its own life activities, and sulfurous acid is produced as its intermediate metabolite. That is, by utilizing this ability of yeast, it is possible to increase the amount of sulfurous acid in the product without adding from the outside.
Methods for increasing the concentration of sulfite in the fermentation liquor during the brewing process include 1) a process control method and 2) a yeast breeding method. The process control method is a method in which the production of sulfite during brewing is inversely proportional to the initial oxygen supply, so the oxygen supply to the fermentation broth is reduced, the production of sulfite is increased and the oxidation is suppressed. It is.

  On the other hand, gene manipulation techniques are used in methods based on yeast breeding. In the sulfur metabolism of yeast, sulfurous acid is an intermediate for biosynthesis of sulfur-containing amino acids and sulfur-containing vitamins, and sulfate ions taken from outside the cells are reduced and produced in a three-step reaction. Here, the MET3 gene is a gene encoding an enzyme that catalyzes the first reaction, and the MET14 gene is a catalyst that catalyzes the second reaction. Korhi et al. Attempted to improve the ability of yeast to produce sulfite by increasing the expression level of these two genes, and revealed that MET14 was more effective (C. Korch et al., Proc. Eur. Brew. Conv. Conger., Lisbon, 201-208, 1991: Non-Patent Document 1). The MET3 gene used at this time was isolated from wine yeast, but the obtained result was about 10% (about 0.8 ppm) of the sulfite concentration contained in the actual beer product. Hansen et al. Also attempted to increase the amount of sulfite produced by disrupting the MET10 gene, which encodes a sulfite ion reductase, and preventing reduction of the produced sulfite ion (J. Hansen et al., Nature Biotech. , 1587-1591, 1996: Non-patent document 2).

Fujimura et al. Also promoted the discharge of sulfite produced inside the cells by increasing the expression level of the nonScSSU1 gene, which is unique to brewer's yeast, among the SSU1 genes encoding the sulfite ion excretion pump of yeast. Then, we tried to increase the concentration of sulfite in beer (Fujimura et al., Abstracts of Annual Meeting of Japanese Society of Agricultural Chemistry 2003, 159, 2003: Non-Patent Document 3).
C. Korch et al., Proc. Eur. Brew. Conv. Conger., Lisbon, 201-208, 1991 J. Hansen et al., Nature Biotech., 1587-1591, 1996 Fujimura et al., Abstracts of Annual Meeting of Japanese Society for Agricultural Chemistry 2003, 159, 2003 Japanese Unexamined Patent Publication No. 06-040907 JP 2000-093096 A

  As mentioned above, the simplest way to increase the sulfurous acid content in the product is to add sulfurous acid. However, in recent years, consumers are strongly interested in additive-free and natural ingredients, and the use of food additives is minimal. It is desirable to do. Therefore, it is effective to achieve a sulfite concentration effective for flavor stability without adding sulfite from the outside by utilizing the life activity of yeast itself. However, the process control method described above is not necessarily practical because the growth rate of yeast decreases due to lack of oxygen, which may cause a delay in fermentation or a decrease in quality.

  In addition, it has been reported that yeast breeding using genetic engineering technology has achieved a sulfite concentration 10 times or more that of the parent strain (J. Hansen et al., Nature Biotech., 1587-1591, 1996) On the other hand, fermentation delay and an increase in unfavorable flavor components, acetaldehyde and 1-propanol, were observed, and there was a problem in making it a practical yeast. For these reasons, a yeast breeding method that can produce a sufficient amount of sulfurous acid without impairing the fermentation rate or the quality of the product has been desired.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have succeeded in identifying and isolating a gene encoding adenylyl sulfate transferase 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 amount of sulfurous acid produced increased, thereby completing the present invention.

  That is, the present invention relates to a novel adenylyl sulfate transferase gene that is 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. The present invention relates to a method for controlling the amount of sulfite produced in a product by using sucrose. 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 following (a) to (f):
(a) a polynucleotide comprising a polynucleotide consisting of 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 polynucleotide encoding a protein 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 and having adenylyl sulfate transferase activity 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 adenylyl sulfate transferase activity;
(e) a polynucleotide comprising a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 and encodes a protein having adenylyl sulfate transferase activity Nucleotides; and
(f) Hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 2, and exhibits adenylyl sulfate transferase activity. A polynucleotide comprising a polynucleotide encoding a protein having the same.

(2) The polynucleotide according to (1) above selected from the following (g) to (i):
(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 adenylyl sulfate transferase activity A polynucleotide comprising a polynucleotide encoding a protein having
(h) a polynucleotide comprising a polynucleotide having a amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 and encoding a protein having adenylyl sulfate transferase 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 highly stringent conditions and adenylyl sulfate A polynucleotide comprising a polynucleotide encoding a protein having transferase activity.
(3) The polynucleotide according to (1) above, comprising 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 polynucleotide selected from the following (j) to (m):
(j) a polynucleotide encoding an RNA having a base sequence complementary to the transcription product of the polynucleotide (DNA) described in (5) above;
(k) a polynucleotide encoding an RNA that suppresses the expression of the polynucleotide (DNA) according to (5) above by an RNAi effect;
(l) a polynucleotide encoding an RNA having an activity of specifically cleaving the transcription product of the polynucleotide (DNA) described in (5) above; and
(m) A polynucleotide encoding an RNA that suppresses the expression of the polynucleotide (DNA) according to (5) above by a co-suppression effect.
(7) A protein encoded by the polynucleotide according to any one of (1) to (5) above.
(8) A vector containing the polynucleotide according to any one of (1) to (5) above.
(8a) The vector according to (8) above, comprising an expression cassette comprising the following components (x) to (z):
(x) 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 a sense direction or an antisense direction; and
(z) Signals that function in yeast for transcription termination and polyadenylation of RNA molecules.
(9) A vector containing the polynucleotide according to (6) above.
(10) Yeast into which the vector according to (8) or (9) is introduced.
(11) The yeast according to (10), wherein the ability to produce sulfite is enhanced by introducing the vector according to (8) above.

(12) The polynucleotide according to (5) above by introducing the vector according to (9) above or by destroying the gene related to the polynucleotide (DNA) according to (5) above ( Yeast with suppressed expression of DNA.
(13) The yeast according to (10), wherein the sulfite production ability is improved by increasing the expression level of the protein according to (7).
(14) A method for producing an alcoholic beverage using the yeast according to any one of (10) to (13) 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.
(17) Alcoholic beverages produced by the method according to any one of (14) to (16) above.
(18) A method for evaluating the ability of a test yeast to produce sulfite using a primer or probe designed based on the base sequence of the adenylyl sulfate transferase gene having the base sequence of SEQ ID NO: 1.
(18a) A method for selecting yeast having high or low sulfite generation ability 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.
(19) A method for evaluating the ability of a test yeast to produce sulfite by culturing a test yeast and measuring the expression level of an adenylyl sulfate transferase gene having the base sequence of SEQ ID NO: 1.
(19a) A method for selecting a yeast having a high ability to produce sulfite, wherein the test yeast is evaluated by the method described in (19) above, and a yeast having a high expression level of the adenylyl sulfate transferase gene is selected.
(19b) A method for producing an alcoholic beverage (for example, beer) using the yeast selected by the method described in (19a) above.

(20) The test yeast is cultured, and the protein described in (7) above is quantified or the expression level of the adenylyl sulfate transferase gene having the nucleotide sequence of SEQ ID NO: 1 is measured, and the target ability to produce sulfite A method for selecting yeast, which comprises selecting a test yeast having the amount of protein or the amount of gene expression according to the method.
(21) Reference yeast and test yeast are cultured, and the expression level of each adenylyl sulfate transferase gene having the nucleotide sequence of SEQ ID NO: 1 in each yeast is measured. The method for selecting yeast according to (20) above, wherein the test yeast that is expressed is selected.
(22) The reference yeast and the test yeast are cultured, the protein according to (7) above in each yeast is quantified, and the test yeast having a larger or smaller amount of the protein than the reference yeast is selected (20 ) Selection method of yeast.
(23) Fermentation for liquor production is performed using any one of the yeast described in (10) to (13) above and the yeast selected by the method described in (20) to (22) above, A method for producing an alcoholic beverage, comprising adjusting a sulfurous acid content.
According to the method for producing alcoholic beverages using the transformed yeast of the present invention, it is possible to increase the content of sulfurous acid having an antioxidative effect in the product, so that alcoholic beverages with excellent flavor stability and a longer quality retention period are produced. It becomes possible.

  In the method of increasing the expression level of a conventionally known sulfite ion excretion pump, a surplus sulfite does not accumulate in the microbial cells, so that a good fermentation rate can be maintained. However, the biosynthesis reaction of sulfite in the microbial cells becomes rate-limiting. The possibility was considered. From this, it was considered that sulfite can be generated more efficiently by strengthening the reduction pathway from the sulfate ion, which is the starting material, to sulfite. The present inventors have conducted research based on such an idea, and based on the beer yeast genome information decoded by the method disclosed in JP-A-2004-283169, nonScMET3 encoding a beer yeast-specific adenylyl sulfate transferase. The gene was isolated and 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 provides (a) a polynucleotide containing a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 (specifically, DNA, hereinafter, these are also simply referred to as “DNA”); And (b) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2.
The DNA of interest in the present invention is not limited to the DNA encoding a protein having adenylyl sulfate transferase activity derived from the above-mentioned brewer's yeast, and other DNA encoding a protein functionally equivalent to this protein. Contains DNA. Functionally equivalent proteins include, for example, (c) an 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 adenyl sulfate. Examples include proteins having ryltransferase activity.

  As such a protein, in the amino acid sequence of SEQ ID NO: 2, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1-39, 1-38, 1-37, 1-36, 1-35, 1-34, 1-33, 1-32, 1-31, 1-3 30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-21 20, 1-19, 1-18, 1-17, 1-16, 1-15, 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, 1-2, 1 Examples thereof include proteins having an amino acid sequence in which one amino acid residue is deleted, substituted, inserted and / or added, and having adenylyl sulfate transferase activity. In general, the smaller the number of amino acid residue deletions, substitutions, insertions and / or additions, the better. In addition, such a protein includes (d) the amino acid sequence of SEQ ID NO: 2 and 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% More than 89%, 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, 99.9% or more of amino acid sequence, and adenylyl sulfate transferase activity The protein which has. In general, the larger the homology value, the better.

The adenylyl sulfate transferase activity can be measured, for example, by the method of Klonus et al. (Plant J. 1994 Jul; 6 (1): 105-12.).
The present invention also relates to (e) a protein that hybridizes with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and has adenylyl sulfate transferase activity. A polynucleotide containing a polynucleotide; and (f) hybridizing under stringent conditions with 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 And a polynucleotide containing a polynucleotide encoding a protein having adenylyl sulfate transferase 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 or DNA encoding the amino acid sequence of 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 a part of the above 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.
Other than this, as DNA that can be hybridized, when calculated by homology search software such as FASTA and BLAST using the default parameters, DNA that encodes the amino acid sequence of SEQ ID NO: 2 and 60% or more, 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%, 99.5%, 99.6%, 99.7%, 99.8% or more And DNA having 99.9% or more 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.

  Furthermore, the polynucleotide of the present invention is (j) a polynucleotide encoding an RNA having a base sequence complementary to the transcription product of the polynucleotide (DNA) described in (5) above; (k) the above (5) (1) a polynucleotide encoding an RNA that suppresses the expression of the polynucleotide (DNA) according to (1) by an RNAi effect; (l) an activity that specifically cleaves the transcription product of the polynucleotide (DNA) according to (5) above; And (m) a polynucleotide encoding RNA that suppresses the expression of the polynucleotide (DNA) described in (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 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 a specific endogenous gene, and is described in various literatures (for example, Hirashima and Inoue: Shinsei Kagaku Kougaku Kenkyu 2 Nucleic acid IV Gene replication and expression (Japan) 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 expression of the gene can be effectively suppressed. The transcribed RNA preferably has a complementarity of 90% or more, more preferably 95% or more, with respect to the transcription product of the target gene. The length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, and more preferably 500 bases or more.

  In the present specification, “polynucleotide encoding RNA that suppresses DNA expression by RNAi effect” refers to a polynucleotide for suppressing the expression of an endogenous gene by RNA interference (RNAi). “RNAi” refers to a phenomenon in which, when a double-stranded RNA having the same or similar sequence as a target gene sequence is introduced into a cell, expression of the introduced foreign gene and target endogenous gene are both suppressed. . 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 a liposome, and can also be locally expressed using a vector capable of producing the 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 Genetics). , 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; Science, 296 (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 RNA having an activity of specifically cleaving a transcript of DNA” generally refers to a ribozyme. A ribozyme is an RNA molecule having catalytic activity, which inhibits the function of the gene by cleaving the target DNA transcript. Various known literatures can also be referred to for ribozyme design (eg, FEBS Lett. 228: 228, 1988; FEBS Lett. 239: 285, 1988; Nucl. Acids. Res. 17: 7059, 1989; Nature 323). : 349, 1986; Nucl. Acids. Res. 19: 6751, 1991; Protein Eng 3: 733, 1990; Nucl. Acids Res. 19: 3875, 1991; Nucl. Acids Res. 19: 5125, 1991; Biochem Biophys Res Commun 186: 1271, 1992 etc.). In addition, “polynucleotide encoding RNA that suppresses DNA expression by co-suppression” refers to a nucleotide that inhibits the function of the target DNA by “co-suppression”.
In the present specification, “co-suppression” refers to the expression of a foreign gene and a target endogenous gene introduced by introducing a gene having a sequence identical or similar to the target endogenous gene into a cell by transformation. Both are phenomena that 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, Martienssen R: Curr. Biol. 6: 810, 1996, etc.) reference).

2. Protein of the present invention The present invention also provides a protein encoded by any of the polynucleotides (a) to (f). A preferred protein of the present invention is a protein having 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 and having adenylyl sulfate transferase activity. is there.

  Such a protein comprises an amino acid sequence in which the number of amino acid residues as described above is deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, and adenylyl sulfate transferase activity. The protein which has is mentioned. Examples of such a protein include a protein having the amino acid sequence having the homology as described above with the amino acid sequence of SEQ ID NO: 2 and having adenylyl sulfate transferase 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). Chemical synthesis using peptide synthesizers such as Advanced Chemtech, PerkinElmer, Pharmacia, Protein Technology Instrument, Syntheselvega, Perceptive, Shimadzu, etc. You can also.

3. Transformed yeast vector and were introduced it the present invention Next, the present invention provides a vector comprising the polynucleotide described above. The vector of the present invention contains the polynucleotide (DNA) described in any of (a) to (i) above or the polynucleotide described in any of (j) to (m) above. In addition, the vector of the present invention usually contains (x) a promoter that can be transcribed in yeast cells; (y) 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 (z) 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. In the present invention, when the protein of the present invention is highly expressed in beer brewing described later, 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. Moreover, when suppressing the expression of the protein of the present invention, 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 DNA or the protein can be suppressed by destroying the target gene (DNA). Gene disruption involves adding or deleting 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 deleting these entire regions. Can be performed. Such gene disruption methods can be referred to known literature (for example, Proc. Natl. Acad. Sci. USA, 76, 4951 (1979), Methods in Enzymology, 101, 202 (1983), (See Kaihei 6-35826).

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 brewing yeast and is not affected by components in 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, so the geneticin resistance gene (G418r) and the copper resistance gene (CUP1) (Marin et al., Proc. Natl. Acad Sci. USA, 81, 337 1984), cerulenin resistance gene (fas2m, PDR4) (Hyogo, et al., Biochemistry, 64, 660, 1992; Hussain et al., Gene, 101, 149, 1991) 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, 4 etc., sake yeasts such as association yeast sake nos. 7, 9 etc. can also 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). Process for producing liquor of the present invention and liquor obtained by the process The above-described vector of the present invention is introduced into a yeast suitable for brewing the liquor to be produced, and the yeast is used to increase the desired liquor and sulfite content. Thus, alcoholic beverages with increased flavor can be produced. Moreover, the yeast selected by the yeast evaluation method of the present invention described below can also be used. 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. Accordingly, the raw materials, production facilities, 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 an increased sulfurous acid content. That is, according to the present invention, alcoholic beverages excellent in flavor stability and the like can be produced using existing facilities without increasing costs.
Furthermore, in yeast in which the gene is highly expressed, sulfate ions in the medium can be efficiently reduced, and sulfur-containing compounds necessary for growth can be biosynthesized. Even when wort with a low malt ratio is used, good yeast growth and alcohol fermentation are possible.

  In addition, in yeast where the function of the sulfur-containing amino acid synthesis system is too strong, hydrogen sulfide, which is an intermediate metabolite of the pathway, and other sulfur-containing compounds that are unfavorable to alcoholic beverages may be produced and accumulated in large quantities. is there. For such yeast, alcohol with reduced off-flavor can be produced by suppressing or destroying the function of the gene and suppressing the metabolic pathway.

5). Method for Evaluating Yeast of the Present Invention The present invention evaluates the ability of a test yeast to produce sulfite using a primer or probe designed based on the base sequence of the adenylyl sulfate transferase gene having the base sequence of SEQ ID NO: 1. On how to do. 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)). Targeting the obtained genome, using the primer or probe designed based on the base sequence (preferably the ORF sequence) of the adenylyl sulfate gene, the gene or the gene is specific to the genome of the test yeast. To see if a specific sequence 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. By this method, the ability of the yeast to produce sulfite is predicted and evaluated 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 ability of the test yeast to produce sulfite can also be evaluated by culturing the test yeast and measuring the expression level of the adenylyl sulfate transferase gene having the base sequence of SEQ ID NO: 1. it can. The expression level of the sulfate adenylyltransferase gene can be measured by culturing the test yeast and quantifying the mRNA or protein that is the product of the sulfate adenylyltransferase gene. Quantification of mRNA or protein can be performed using a known method. For example, 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).

  Furthermore, the test yeast is cultured, the expression level of the adenylyl sulfate transferase gene having the base sequence of SEQ ID NO: 1 is measured, and the yeast having the gene expression level corresponding to the target sulfite production ability is selected. Thus, a yeast suitable for brewing a 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 yeast are cultured, and the expression level in each yeast of the adenylyl sulfate transferase gene having the nucleotide sequence of SEQ ID NO: 1 is measured, and the gene is higher than the reference yeast. A yeast suitable for brewing a desired liquor can be selected by selecting a test yeast that is expressed.

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 sulfite producing ability or a high adenylyl sulfate transferase activity.
In these cases, the test yeast or the reference yeast includes, for example, a yeast into which the above-described vector of the present invention has been introduced, a yeast in which expression of the above-described polynucleotide (DNA) of the present invention is promoted or suppressed, and mutation treatment. Applied yeast, naturally mutated yeast, and the like can be used. The adenylyl sulfate transferase activity can be measured, for example, by the method of Klonus et al. (Plant J. 1994 Jul; 6 (1): 105-12.). For the mutation treatment, any method such as a physical method such as ultraviolet irradiation or radiation irradiation, 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, etc.).

  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, brewing 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, wine yeasts such as association wines No. 1, 3 and 4 can be used, and sake yeasts such as association yeast sakes No. 7 and 9 can be used, but are not limited thereto. In the present invention, beer yeast such as Saccharomyces pastorianus is preferably used. The reference yeast and the test yeast may be selected from the above yeasts in any combination.

EXAMPLES Details of the present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

Cloning of a novel sulfate adenylyltransferase gene (nonScMET3) As a result of searching using a comparative database described in JP-A-2004-283169, a novel sulfate adenylyltransferase gene, nonScMET3, which is unique to brewer's yeast was found (SEQ ID NO: 1). Based on the obtained base sequence information, primers nonScMET3_F (SEQ ID NO: 3) / nonScMET3_R (SEQ ID NO: 4) for amplifying the full-length gene were designed, and the genome decoding strain Saccharomyces pastorianus byhen stephan 34/70 strain A DNA fragment containing the full length gene of nonScMET3 was obtained by PCR using the chromosomal DNA of (W34 / 70 strain) as a template.
The nonScMET3 gene fragment obtained as described above was inserted into the pCR2.1-TOPO vector (Invitrogen) by TA cloning. The base sequence of the nonScMET3 gene was analyzed by Sanger's method (F. Sanger, Science, 214, 1215, 1981) to confirm the base sequence.

Non-ScMET3 gene expression analysis during brewing of beer Beer brewing was performed using brewer's yeast Saccharomyces pastorianus strain W34 / 70, and mRNA extracted from brewer's brewer's yeast cells 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 amount (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 pattern of the nonScMET3 gene is shown in (Fig. 3). From this result, it was confirmed that the nonScMET3 gene was expressed in normal beer fermentation.

Constitutive expression of nonScMET3 gene nonScMET3 / pCR2.1-TOPO described in Example 1 was digested with restriction enzymes SacI and NotI to prepare a DNA fragment containing the nonScMET3 gene. This was ligated to pUP3GLP2 treated with restriction enzymes SacI and NotI to construct a nonScMET3 constitutive expression vector pUP-nonScMET3. pUP3GLP2 is a YIp type (chromosomal integration type) yeast expression vector containing the orotidine 5-phosphate decarboxylase gene URA3 as a homologous recombination site. The introduced gene is the promoter / terminator of the glyceraldehyde 3-phosphate dehydrogenase gene TDH3. Is constitutively expressed by As a selection marker in yeast, the drug resistance gene YAP1 is incorporated under the control of the promoter / terminator of the galactokinase gene GAL1, and expression is induced in a medium containing galactose. Also it contains ^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. First, when the produced sulfite accumulates in the microbial cells, the yeast itself is damaged by sulfite and the effect of the gene cannot be evaluated correctly. Therefore, the sulfite discharge pump is encoded by the method described in JP-A-2004-283169. A nonScSSU1 gene high expression strain was obtained and used as a parent strain. The obtained strain was transformed with pUP-nonScMET3, and a cerulenin resistant strain was selected with a YPGal plate medium (1% yeast extract, 2% polypeptone, 2% galactose, 2% agar) containing cerulenin 1.0 mg / L.

Analysis of sulfite production in beer test brewing A fermentation test using the parent strain and the two nonScMET3 high-expressing strains obtained in Example 3 was performed under the following conditions.
Wort extract concentration 11.96%
Wort capacity 1L
Wort dissolved oxygen concentration about 10ppm
Fermentation temperature 15 ℃ constant Yeast input 6g Wet yeast cells / L wort

The fermented koji was sampled over time, and the yeast growth amount (OD660) (FIG. 4) and the extract consumption over time (FIG. 5) were examined. Sulfurous acid concentration at the end of the fermentation was determined by collecting sulfurous acid in aqueous hydrogen peroxide by distillation under acidic conditions and then titrating with alkali (Japan Brewing Association revised BCOJ beer analysis method).
From FIG. 6, the amount of sulfite produced at the end of fermentation was 32.3 and 33.8 ppm for the non-ScMET3 high-expressing strain relative to 25.4 ppm for the parent strain. From this result, it became clear that the amount of sulfite produced increased by about 30% by high expression of nonScMET3. At this time, there was no difference in growth rate and extract consumption rate between the parent strain and the high expression strain.
From the above results, in yeast in which the ability to produce sulfite was enhanced by constitutively expressing the adenylyl sulfate transferase gene unique to brewer's yeast disclosed in the present invention, without changing the fermentation process and fermentation period, It has become possible to specifically increase the amount of sulfurous acid that acts as an antioxidant for beer. This makes it possible to produce alcoholic beverages with excellent flavor stability and a longer quality retention period.

The W34 / 70 strain is transformed with the high expression vector prepared in Example 3 of beer test brewing using low sulfur source wort , and Sc and non-ScMET3 (single) high expression strains are obtained, respectively. Next, wort with a malt ratio of 24% is prepared as a low sulfur source wort, and beer test brewing is performed under the following conditions using the parent strain and the resulting high expression strain.
Wort extract concentration 13%
Wort capacity 2L
Wort dissolved oxygen concentration about 8ppm
Fermentation temperature 15 ° C constant yeast input 10.5g wet yeast cells / 2L wort Fermented moromi is sampled over time, and yeast growth (OD660) and extract consumption are examined over time.

According to the method of MET3 gene disruption literature (Goldstein et al., Yeast . 15 1541 (1999)), PCR using a plasmid containing a drug resistance marker (pFA6a (G418 ^r ) or pAG25 (nat1)) as a template was performed by PCR. Create a fracture fragment.
The prepared gene disruption fragment is used to transform W34 / 70 strain or spore clone W34 / 70-2 strain. Transformation is performed by the method described in Japanese Patent Application Laid-Open No. 07-303475, and the concentrations of the selected drug are geneticin 300 mg / L and nourseothricin 50 mg / L, respectively.

Analysis of sulfur-containing compound production amount in beer test brewing Using the parent strain and the disrupted strain obtained in Example 6, beer test brewing is performed under the following conditions.
Wort extract concentration 13%
Wort capacity 2L
Wort dissolved oxygen concentration about 8ppm
Fermentation temperature 15 ° C Constant yeast input 10.5g Wet yeast cells / 2L wort fermentation moromi is sampled over time to examine changes in yeast growth (OD660) and extract consumption over time. Analysis of sulfur-containing compounds in moromi is performed by headspace gas chromatography.

According to the method for producing alcoholic beverages of the present invention, since the content of sulfurous acid having an antioxidative action in the product is increased, it is possible to produce alcoholic beverages having excellent flavor stability and a longer quality retention period. In addition, since the yeast according to the present invention can efficiently reduce sulfate ions taken as a sulfur source and biosynthesize sulfur-containing compounds necessary for growth, even a raw material having a low sulfur-containing amino acid content, for example, sparkling wort Good alcohol fermentation can be performed. Furthermore, it is possible to produce a liquor with a preferred flavor by suppressing the expression of the gene, even for yeasts with a high production amount of sulfur-containing compounds that become off-flavors.
This application claims the benefit of the priority of Japanese Patent Application No. 2005-235011 (filed on August 12, 2005) and Japanese Patent Application No. 2006-84289 (filed on March 24, 2006) The entire description of the application is incorporated into the present application. All other cited references are also incorporated into the present application.

FIG. 1 is a diagram showing the change over time in the amount of yeast grown in beer test brewing. The horizontal axis represents the fermentation time, and the vertical axis represents the value of OD660. FIG. 2 is a diagram showing the change over time in the amount of extract consumed in beer test brewing. The horizontal axis represents the fermentation time, and the vertical axis represents the appearance extract concentration (w / w%). FIG. 3 is a diagram showing the expression behavior of the nonScMET3 gene in yeast during beer test brewing. The horizontal axis indicates the fermentation time, and the vertical axis indicates the detected signal luminance. FIG. 4 is a diagram showing the change over time in the amount of yeast grown in test brewing using a non-ScMET3 high expression strain. The horizontal axis represents the fermentation time, and the vertical axis represents the value of OD660. FIG. 5 is a diagram showing a change with time in extract consumption in beer test brewing using a non-ScMET3 high expression strain. The horizontal axis represents the fermentation time, and the vertical axis represents the appearance extract concentration (w / w%). FIG. 6 is a diagram showing the sulfite concentration in the mash at the end of the beer test brewing using the nonScMET3 high expression strain.

[SEQ ID NO: 3] Primer
[SEQ ID NO: 4] Primer

Claims (23)

A polynucleotide selected from the following (a) to (f):
(a) a polynucleotide comprising a polynucleotide consisting of 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 polynucleotide encoding a protein 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 and having adenylyl sulfate transferase activity 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 adenylyl sulfate transferase activity;
(e) a polynucleotide comprising a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 and encodes a protein having adenylyl sulfate transferase activity Nucleotides; and
(f) hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 and has adenylyl sulfate transferase activity; A polynucleotide comprising a polynucleotide encoding a protein having the same. The polynucleotide according to claim 1 selected from the following (g) to (i):
(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 adenylyl sulfate transferase activity A polynucleotide comprising a polynucleotide encoding a protein having
(h) a polynucleotide comprising a polynucleotide having a amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 and encoding a protein having adenylyl sulfate transferase 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 highly stringent conditions and adenylyl sulfate A polynucleotide comprising a polynucleotide encoding a protein having transferase activity.   The polynucleotide according to claim 1, comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1.   2. The polynucleotide of claim 1, comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2.   The polynucleotide according to any one of claims 1 to 4, which is DNA. A polynucleotide selected from the following (j) to (m):
(j) a polynucleotide encoding an RNA having a base sequence complementary to the transcription product of the polynucleotide (DNA) according to claim 5;
(k) a polynucleotide encoding an RNA that suppresses the expression of the polynucleotide (DNA) according to claim 5 by an RNAi effect;
(l) a polynucleotide encoding an RNA having an activity of specifically cleaving the transcript of the polynucleotide (DNA) of 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.   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 vector containing the polynucleotide according to claim 6.   A yeast into which the vector according to claim 8 or 9 has been introduced.   The yeast according to claim 10, wherein the ability to produce sulfite is enhanced by introducing the vector according to claim 8.   The expression of the polynucleotide (DNA) according to claim 5 is suppressed by introducing the vector according to claim 9 or by destroying a gene related to the polynucleotide (DNA) according to claim 5. Yeast.   The yeast of Claim 10 which improved the sulfite production | generation ability by increasing the expression level of the protein of Claim 7.   A method for producing an alcoholic beverage using the yeast according to any one of claims 10 to 13.   The method for producing an alcoholic beverage according to claim 14, wherein the alcoholic beverage to be brewed is a malt beverage.   The method for producing an alcoholic beverage according to claim 14, wherein the alcoholic beverage to be brewed is wine.   The liquor manufactured by the method in any one of Claims 14-16.   A method for evaluating the ability of a test yeast to produce sulfite, using a primer or probe designed based on the base sequence of the adenylyl sulfate transferase gene having the base sequence of SEQ ID NO: 1.   A method for evaluating the ability of a test yeast to produce sulfite by culturing a test yeast and measuring the expression level of an adenylyl sulfate transferase gene having the base sequence of SEQ ID NO: 1.   The test yeast is cultured, and the protein according to claim 7 is quantified or the expression level of the adenylyl sulfate transferase gene having the base sequence of SEQ ID NO: 1 is measured, and according to the target sulfite production ability A method for selecting yeast, comprising selecting a test yeast having a protein amount or a gene expression level.   Reference yeast and test yeast are cultured, and the expression level in each yeast of the adenylyl sulfate transferase gene having the nucleotide sequence of SEQ ID NO: 1 is measured, and the gene is expressed at a higher or lower level than the reference yeast. The method for selecting yeast according to claim 20, wherein the test yeast is selected.   The yeast according to claim 20, wherein the reference yeast and the test yeast are cultured, the protein according to claim 7 in each yeast is quantified, and the test yeast having a larger or smaller amount of the protein than the reference yeast is selected. How to choose.   Fermentation for liquor production is carried out using any one of the yeast according to claims 10 to 13 and the yeast selected by the method according to claims 20 to 22, and the sulfite content is adjusted. And a method for producing alcoholic beverages.

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