JP2009165351A - Gene encoding acetolactate synthase and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To breed a brewer's yeast having a low vicinal diketone or diacetyl-producing ability, and to provide a method for producing a liquor having a reduced content of vicinal diketone or diacetyl. <P>SOLUTION: Provided are an acetolactate synthase gene and a use thereof, especially, the brewer's yeast for producing the liquor excellent in flavor, the liquor produced using the yeast, the method for producing the same, etc. More concretely, provided are a yeast strain whose ability to produce VDK, particularly DA, to cause an off-flavor of a product is reduced by reducing the expression level of ILV6 gene encoding Ilv6p which is an acetolactate synthase in a brewer's yeast strain, particularly nonScILV6 gene which is unique to beer yeast, a process for producing an alcoholic beverage by using the yeast strain, etc. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a gene encoding acetolactate synthase and use thereof, and particularly relates to a brewing yeast that produces an alcoholic beverage with excellent flavor, an alcoholic beverage produced using the yeast, a method for producing the same. More specifically, the present invention relates to vicinal diketones that serve as off-flavors of products by suppressing the expression level of the gene ILV6 encoding acetolactate synthase of brewing yeast, particularly the nonScILV6 gene characteristic of brewer's yeast. The present invention relates to yeast with reduced diacetyl production, a method for producing alcoholic beverages using the yeast, and the like.

  Among the aroma components of alcoholic beverages, the diacetyl (DA) odor is one of the typical off-flavors in brewed liquors such as beer, sake and wine. DA odor (expressed as stuffy odor or butter odor in beer and scented in sake) is caused by the presence of vicinal diketones (hereinafter referred to as VDK) mainly consisting of DA in the product. ppm (Journal of the Institute of Brewing, 76, 486 (1970): Non-Patent Document 1).

  VDKs in liquors are broadly divided into DA and 2,3-pentanedione (PD). DA and PD are produced by non-enzymatic reactions not involving yeast, using valine and isoleucine biosynthesis intermediate products α-acetolactic acid and α-acetohydroxybutyric acid as precursors, respectively.

  From the above, VDK (DA and PD) and its precursor α-acetohydroxy acids (α-acetolactic acid and α-acetohydroxybutyric acid) as a whole can be considered as those that may cause DA odor to the product. The breeding of yeast that stably reduces these not only facilitates the production management of alcoholic beverages, but also expands the possibility of developing new products.

  As a method for controlling the DA odor, for example, JP-A-2001-204457 (Patent Document 1) reports a method for suppressing the production of DA by using a liquor having a low pyruvic acid concentration, which is a precursor of α-acetolactic acid. ing. Acetolactate synthase is an enzyme that converts pyruvate or α-oxobutyric acid to α-acetolactic acid or α-acetohydroxybutyric acid, respectively. However, genes encoding yeast acetolactate synthase include ILV2 and ILV6. It is known that both gene products form a complex. In Journal of Basic Microbiology, 28 (3), 175-183 (1988) (Non-patent Document 2), by introducing mutation or gene disruption to ILV2 encoding the active site, the activity of the above enzyme is suppressed. It has been reported that the synthesis of precursors (α-acetohydroxy acids) is reduced, resulting in a decrease in DA concentration. Enzymatic analysis of acetolactate synthase activity is performed on Ilv6p, which is an activity control site, for example in Biochemistry, 38 (16), 5222-31 (1999) (Non-patent Document 3). The impact is not clear.

Furthermore, in the Journal of American Society of Brewing Chemists, Proceeding, 94-99 (1973) (Non-patent Document 4), it has been reported that the amount of VDK produced in valine, leucine and isoleucine-requiring yeast is reduced. Sexual strains have not been put to practical use because they are prone to growth and fermentation delays. Japanese Patent Application Laid-Open No. 2002-291465 (Patent Document 2) discloses a method in which mutant strains that are sensitive to analogs of these branched amino acids are obtained and a DA low accumulation strain is selected from them. In the Journal of American Society of Brewing Chemists, Proceeding, 81-84 (1987) (Non-patent Document 5), genetically engineered yeasts that regulate the expression level of ILV5 gene derived from laboratory yeasts are also known as European Brewery Convention, Proceedings of The 21st EBC congress, Madrid, 553-560 (1987) (Non-Patent Document 6) also reports genetically engineered yeasts that regulate the expression level of the ILV3 gene. At this time, the enzyme activity of acetohydroxy acid reductoisomerase encoded by the ILV5 gene increased 5-7 times, and the amount of VDK produced decreased to about 40%. In addition, the enzyme activity of dihydroxy acid dehydratase encoded by the ILV3 gene was increased 5-6 times, but no significant decrease was observed in the amount of VDK produced. However, the above two reports use a synthetic medium, and the effect on actual beer brewing has not been analyzed. Dulieu et al., In the European Brewery Convention, Proceedings of the 26th EBC congress, Maastricht, 455-460 (1997) (Non-Patent Document 7), α-acetolactic acid decarboxylase is used to produce α- A method for rapidly converting acetolactate to acetoin was proposed, but in Japan, enzymes cannot be added to the koji during fermentation for tax purposes. JP-A-2-265488 (Patent Document 3) and JP-A-7-171 (Patent Document 4) both report a genetically engineered yeast using a DNA strand encoding α-acetolactic acid decarboxylase. Yes.
Japanese Patent Laid-Open No. 2001-204457 JP 2002-291465 JP JP-A-2-265488 Japanese Unexamined Patent Publication No. 7-171 Journal of the Institute of Brewing, 76, 486 (1970) Journal of Basic Microbiology, 28 (3), 175-183 (1988) Biochemistry, 38 (16), 5222-31 (1999) Journal of American Society of Brewing Chemists, Proceeding, 94-99 (1973) Journal of American Society of Brewing Chemists, Proceeding, 81-84 (1987) European Brewery Convention, Proceedings of the 21st EBC congress, Madrid, 553-560 (1987) European Brewery Convention, Proceedings of the 26th EBC congress, Maastricht, 455-460 (1997)

  An object of the present invention is to produce a VDK, particularly a gene encoding a protein capable of reducing the generation of DA odor in yeast, and to produce a liquor excellent in flavor by breeding a VDK low-productivity yeast using the protein. Is to make it possible.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have succeeded in identifying and isolating a gene encoding acetolactate synthase that has an advantageous effect over known proteins from brewer's yeast. Moreover, the transformed yeast which suppressed the expression of the obtained gene was produced, and it confirmed that VDK density | concentration in beer, especially DA density | concentration reduced, and completed this invention.

  That is, the present invention uses a novel acetolactate synthase gene characteristic of beer 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. This relates to a method for controlling the VDK concentration in the product, especially the DA concentration. Specifically, the present invention specifically includes the following polynucleotides, vectors or DNA fragments containing the polynucleotides, transformed yeasts into which the vectors or DNA fragments have been introduced, methods for producing alcoholic beverages using the transformed yeasts, etc. I will provide a.

(1) The polynucleotide according to any one of the following (a) to (f):
(a) a polynucleotide comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;
(b) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(c) comprising a polynucleotide encoding a protein comprising 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 acetolactate synthase activity Polynucleotides to:
(d) a polynucleotide comprising a polynucleotide having an amino acid sequence having 60% or more identity to the amino acid sequence of SEQ ID NO: 2 and encoding a protein having acetolactate synthase activity;
(e) a polynucleotide comprising 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 acetolactate synthase activity; as well as
(f) a protein that 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 acetolactate synthase activity A polynucleotide containing a polynucleotide encoding.

(2) The polynucleotide according to (1) above, which is any of the following (g) to (i):
(g) a protein having an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2 and having acetolactate synthase activity A polynucleotide comprising a polynucleotide encoding
(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 acetolactate synthase activity; and
(i) Hybridizes with 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 has an acetolactate synthase activity A polynucleotide comprising a polynucleotide encoding a protein having

(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) The polynucleotide according to any one of the following (j) to (m):
(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 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 (7) above, comprising an expression cassette comprising the following components (a) to (c):
(a) Promoter capable of transcription in yeast cells
(b) the polynucleotide according to any one of (1) to (5), which is bound to the promoter in the antisense direction; and
(c) Signals that function in yeast for transcription termination and polyadenylation of RNA molecules. (9) A vector containing the polynucleotide according to (6) above.

(10) A transformed yeast introduced with the vector according to any one of (8) to (9) above.
(11) The yeast for brewing according to (10), wherein the total vicinal diketone producing ability or the total diacetyl producing ability is reduced by introducing the vector according to any one of (8) to (9) above.
(12) The brewery yeast according to (11), wherein the total vicinal diketone production ability or the total diacetyl production ability is reduced by reducing the expression level of the protein according to (7).
(13) By introducing the vector according to any one of (8) to (9) above or by destroying the gene related to the polynucleotide (DNA) according to (5) above (5) ) Yeast for brewing in which expression of the polynucleotide (DNA) described in (1) is suppressed.

(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 total vicinal diketone producing ability or the total diacetyl producing ability of a test yeast using a primer or probe designed based on the base sequence of an acetolactate synthase gene having the base sequence of SEQ ID NO: 1.
(18a) A method for selecting a yeast having a low total vicinal diketone production ability or total diacetyl production 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 total vicinal diketone-producing ability or the total diacetyl-producing ability of a test yeast by culturing the test yeast and measuring the expression level of the acetolactate synthase gene having the base sequence of SEQ ID NO: 1. .
(19a) A method for selecting a yeast, wherein the test yeast is evaluated by the method described in (19) above and a yeast having a low expression level of an acetolactate synthase 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 acetolactate synthase gene having the base sequence of SEQ ID NO: 1 is measured, and the target total vicinal diketone producing ability or 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 the total diacetyl-producing ability.
(21) Culturing the reference yeast and the test yeast to measure the expression level of each acetolactate synthase gene having the nucleotide sequence of SEQ ID NO: 1 in each yeast, and the test yeast in which the gene is less expressed than the reference yeast The yeast selection method according to (20) above, wherein
(22) The reference yeast and the test yeast are cultured, the protein according to (7) in each yeast is quantified, and the test yeast having a smaller amount of the protein than the reference yeast is selected. Yeast selection method. That is, the yeast selection method according to (20) above, wherein a plurality of yeasts are cultured, the protein according to (7) in each yeast is quantified, and a test yeast having a small amount of the protein is selected therein. .

(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 the total vicinal diketone production amount or the total diacetyl production amount.

According to the method for producing alcoholic beverages using the transformed yeast of the present invention, vicinal diketones (VDK) (for example, diacetyl (DA), 2,3-pentanedione (PD), etc.) that become off-flavor in the product or the like Production of precursors (for example, α-acetohydroxy acids), especially diacetyl (DA) or precursors thereof (for example, α-acetolactic acid) can be reduced. Can be manufactured.

The present inventors have isolated and identified a nonScILV6 gene encoding an acetolactate synthase unique to brewer's yeast based on the brewer's yeast genome information decoded by the method disclosed in JP-A-2004-283169. 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.
In the present specification, VDK and its precursor α-acetohydroxy acid may be collectively referred to as “all vicinal diketones”. In addition, DA and its precursor α-acetolactic acid may be collectively referred to as “all diacetyl”.

1. Polynucleotide of the Present Invention First, the present invention provides (a) a polynucleotide containing a polynucleotide comprising 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 the acetolactate synthase derived from the brewer's yeast, but includes other DNAs encoding proteins functionally equivalent to this protein. Examples of functionally equivalent proteins include (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 acetolactate synthase Examples include proteins having 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, 1-14, 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 a protein having an amino acid sequence in which one amino acid residue is deleted, substituted, inserted and / or added and having acetolactate synthase activity. In general, the smaller the number of amino acid residue deletions, substitutions, insertions and / or additions, the better. Such a protein includes (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% 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% 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, A protein having an amino acid sequence having 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 and having acetolactate synthase activity Is mentioned. In general, the larger the homology value, the better. The acetolactate synthase activity can be measured, for example, by the method of Pang et al. (Biochemistry, 38, 5222-5231 (1999)).

  The present invention also relates to (e) a polynucleotide encoding 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 acetolactate synthase activity And (f) hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to a base sequence of a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2, and Also encompassed are polynucleotides containing a polynucleotide encoding a protein having acetolactate synthase 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. “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 using homology search software such as FASTA and BLAST using default parameters, the DNA encoding the amino acid sequence of SEQ ID NO: 2 is 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 higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher 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 DNA having the identity of not less than% and not less than 99.9% can be mentioned.
The identity of the amino acid sequence and base sequence is determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA, 87, 2264-2268, 1990; Proc. Natl. Acad. Sci. USA, 90, 90) by Carlin and Arthur. 5873, 1993). 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. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. 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 for suppressing 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 (i). A preferred protein of the present invention is a protein comprising 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 acetolactate synthase activity. Such a protein has 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 has acetolactate synthase activity. Examples include proteins. 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 acetolactate synthase 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. Next, the present invention provides a vector containing the polynucleotide described above. The vector of the present invention contains the polynucleotide (DNA) described in any of (a) to (i) above. In addition, the vector of the present invention usually comprises (a) a promoter that can be transcribed in yeast cells so as to suppress the expression of the polynucleotide (DNA) described in any of (a) to (i) above; b) The polynucleotide (DNA) according to any one of (a) to (i) above bound to the promoter in the antisense direction; and (c) transcription termination and polyadenylation of RNA molecules, which functions in yeast. It is configured to include an expression cassette that includes a signal as a component. In addition, these polynucleotides are introduced into a vector containing the polynucleotide described in any of (j) to (m) above so that the polynucleotide 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. USA, 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 geneticin resistance gene (G418r), 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 and 4, etc., sake yeasts such as association yeast sake nos. 7 and 9 can be used. However, the present invention is not limited to this. In the present invention, beer yeast such as Saccharomyces pastorianus is preferably used.

As a yeast transformation method, a publicly known method can be used. For example, electroporation method “Meth. Enzymol., 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. In addition, regarding general cloning techniques, “Molecular Cloning 3rd Edition”, “Methods in Yeast Genetics, A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)” and the like can be referred to.

4). In the present invention, by using the brewing yeast in which the expression of the polynucleotide (DNA) of the present invention described above is suppressed, the desired alcoholic beverage can be used as a VDK. The amount of DA produced is reduced, and alcoholic beverages with excellent flavor can be produced. Specifically, a yeast introduced with the above-described vector of the present invention, a yeast in which the expression of the above-described polynucleotide (DNA) of the present invention is suppressed, or a yeast selected by the following yeast evaluation method of the present invention is used. Thus, by performing fermentation for liquor production and reducing the VDK production amount, particularly the DA production amount, it is possible to produce an alcoholic beverage with a desired liquor and a reduced VDK content, particularly a DA content. Examples of alcoholic beverages to be targeted include, but are not limited to, beer-taste drinks such as beer and sparkling wine, wine, whiskey, and sake.
In producing these alcoholic beverages, a known method can be used except that the brewer's yeast obtained in the present invention is used instead of the parent strain. Therefore, the raw materials, production equipment, production management, etc. may be exactly the same as the conventional method, and it does not increase the cost for producing VDK, particularly alcoholic beverages with reduced DA production. That is, according to the present invention, alcoholic beverages with excellent flavor can be produced using existing facilities without increasing costs.

5. Yeast evaluation method of the present invention The present invention evaluates the ability of a test yeast to produce VDK or DA using a primer or probe designed based on the base sequence of an acetolactate synthase gene having the base sequence of SEQ ID NO: 1. On how to do. A general method of such an evaluation method is known, and is described in, for example, WO01 / 040514, JP-A-8-205900, and the like. Hereinafter, this evaluation method will be briefly described.

  First, the genome of the test yeast is prepared. As the preparation method, any known method such as Hereford method or potassium acetate method can be used (for example, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, p130 (1990)). Using the primer or probe designed based on the base sequence of the acetolactate synthase gene (preferably the ORF sequence) for the obtained genome, the gene or the gene specific to the genome of the test yeast Check whether an array exists. The primer or probe can be designed using a known method.

  Detection of a gene or a specific sequence can be performed using a known technique. For example, a polynucleotide containing a part or all of a specific sequence or a polynucleotide containing a base sequence complementary to the base sequence is used as one primer, and the upstream or downstream of this sequence is used as the other primer. Using a polynucleotide containing a part or all of the sequence or a polynucleotide containing a base sequence complementary to the base sequence, a yeast nucleic acid is amplified 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 VDK or DA is predicted / 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 test yeast is cultured, and the expression level of the acetolactate synthase gene having the base sequence of SEQ ID NO: 1 is measured to evaluate the VDK or DA production ability of the test yeast. it can. In this case, it is possible by culturing the test yeast and quantifying the acetolactate synthase gene product mRNA or protein. 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 acetolactate synthase gene having the base sequence of SEQ ID NO: 1 is measured, and the yeast having the gene expression level according to the target VDK or DA production ability is selected. Thus, a yeast suitable for brewing a desired liquor can be selected. In addition, a reference yeast (for example, genome decoding strain Saccharomyces pastorianus bihenstephan 34/70 strain) and a test yeast are cultured, and the expression level of the gene having the base sequence of SEQ ID NO: 1 is measured in each yeast. In addition, by selecting a test yeast in which the expression of the gene is suppressed (that is, low expression), a yeast suitable for brewing alcoholic beverages can be selected.

Alternatively, a test yeast suitable for brewing a desired liquor can be selected by culturing the test yeast and selecting a yeast having a low VDK or DA production ability or a low acetolactate synthase activity.
In these cases, the test yeast or the reference yeast is, 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 suppressed, or a mutation treatment. Yeast, naturally mutated yeast, and the like may be used. VDK or DA production ability can be measured by a known method. For example, the amount of total vicinal diketone can be quantified by the method described in Drews et al., Mon. fur Brau., 34, 1966. The total amount of diacetyl can be quantified, for example, by the method described in J Agric Food Chem. 50 (13): 3647-53, 2002. The acetolactate synthase activity can be measured by, for example, the method of Epelbaum et al. (Anal. Biochem, 191, 96-9 (1990)). 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 Hereinafter, although an Example demonstrates the detail of this invention, this invention is not limited to a following example.

Example 1: Analysis of nonScILV6 gene expression during beer brewing Beer brewing was performed using the brewer's yeast Saccharomyces pastorianus strain W34 / 70.
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, yeast cells were sampled, and the prepared mRNA was labeled with biotin and hybridized with a brewer's yeast DNA microarray. Signal detection was performed using GeneChip Operating System (GCOS; GeneChip Operating Software 1.0, manufactured by Affymetrix). The expression pattern of the nonScILV6 gene is shown in FIG. From this result, it was confirmed that the nonScILV6 gene was expressed in normal beer fermentation.

Example 2: Disruption of nonScILV6 gene Plasmids containing drug resistance markers (pFA6a (G418r), pAG25 (nat1), pAG32 (hph)) according to the method of literature (Goldstein et al., Yeast. 15 1541 (1999)) Gene disruption fragments are prepared by PCR using as a template. As primers for PCR, nonScILV6_delta_for (SEQ ID NO: 3) and nonScILV6_delta_rv (SEQ ID NO: 4) are used.
The spore clone strain (W34 / 70-2) isolated from the brewer's yeast Saccharomyces pastorianus strain W34 / 70 is transformed with the gene disruption fragment prepared by the method described above. Transformation was performed by the method described in Japanese Patent Application Laid-Open No. 07-303475, and YPD plate medium containing Geneticin 300 mg / L or Noseothricin 50 mg / L or Hygromycin B 200 mg / L ( 1% yeast extract, 2% polypeptone, 2% glucose, 2% agar).

Example 3: Analysis of VDK production amount in beer test brewing A fermentation test using the parent strain and the nonScILV6 disrupted strain obtained in Example 2 is performed under the following conditions.
Wort extract concentration 11.85%
Wort capacity 2L
Wort dissolved oxygen concentration 8ppm
Fermentation temperature 15 ° C Constant yeast input 5g Wet yeast cells / L wort Fermented koji is sampled over time, and changes in yeast growth (OD660) and extract consumption over time are examined. Quantification of total VDK in soot is carried out by reacting VDK (DA and PD) with hydroxylamine and measuring the absorbance of the complex formed by the reaction of the glyoxime derivative and divalent iron ions (Drews et al. , Mon. fur Brau., 34, 1966). At this time, the precursors α-acetolactic acid and α-acetohydroxybutyric acid are converted into DA and PD by gas washing method (oxidative decarboxylation reaction) in advance, respectively. To do.

  According to the method for producing an alcoholic beverage of the present invention, the production amount of VDK, which is an off-flavor in the product, in particular, DA production is reduced, and an alcoholic beverage with excellent flavor can be easily produced.

It is a figure which shows the time-dependent change of the yeast growth amount in beer test brewing. The horizontal axis represents the fermentation time, and the vertical axis represents the value of OD660. It is a figure which shows the time-dependent change of the extract consumption in beer test brewing. The horizontal axis represents the fermentation time, and the vertical axis represents the appearance extract concentration (w / w%). It is a figure which shows the expression behavior of the nonScILV6 gene in yeast during beer test brewing. The horizontal axis indicates the fermentation time, and the vertical axis indicates the detected signal luminance.

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

Claims (23)

The polynucleotide according to any one of the following (a) to (f):
(a) a polynucleotide comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1;
(b) a polynucleotide containing a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(c) comprising a polynucleotide encoding a protein comprising 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 acetolactate synthase activity Polynucleotides to:
(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 acetolactate synthase activity;
(e) a polynucleotide comprising 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 acetolactate synthase activity; as well as
(f) a protein that 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 acetolactate synthase activity A polynucleotide containing a polynucleotide encoding. The polynucleotide of any one of the following (g) to (i):
(g) a protein having an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2 and having acetolactate synthase activity A polynucleotide comprising a polynucleotide encoding
(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 acetolactate synthase activity; and
(i) Hybridizes with 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 has an acetolactate synthase activity A polynucleotide comprising a polynucleotide encoding a protein having   The polynucleotide according to claim 1, comprising a polynucleotide comprising the base sequence of SEQ ID NO: 1.   The polynucleotide according to 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. The polynucleotide according to any one of 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 an 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 transformed yeast into which the vector according to claim 8 or 9 has been introduced.   The brewery yeast according to claim 10, wherein the vicinal diketone production ability or the total diacetyl production ability is reduced by introducing the vector according to claim 8 or 9.   The brewery yeast according to claim 11, wherein the total vicinal diketone producing ability or the total diacetyl producing ability is reduced by reducing the expression level of the protein according to claim 7.   Expression of the polynucleotide (DNA) according to claim 5 by introducing the vector according to claim 8 or 9, or by disrupting a gene related to the polynucleotide (DNA) according to claim 5. Yeast for brewing with suppressed   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 total vicinal diketone production ability or the total diacetyl production ability of a test yeast using a primer or probe designed based on the base sequence of an acetolactate synthase gene having the base sequence of SEQ ID NO: 1.   A method for evaluating the total vicinal diketone-producing ability or the total diacetyl-producing ability of a test yeast by culturing the test yeast and measuring the expression level of an acetolactate synthase 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 acetolactate synthase gene having the base sequence of SEQ ID NO: 1 is measured, and the target total vicinal diketone production ability or total diacetyl production 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 the ability.   The reference yeast and the test yeast are cultured, and the expression level in each yeast of the acetolactate synthase gene having the nucleotide sequence of SEQ ID NO: 1 is measured, and the test yeast in which the gene is lower expressed than the reference yeast is selected. The method for selecting yeast according to claim 20.   21. The method for selecting a 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 smaller amount of the protein than the reference yeast is selected. . Fermentation for liquor production is performed 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 total vicinal diketone production or total diacetyl production A method for producing alcoholic beverages, characterized in that the amount is adjusted.