DESCRIPTION

SULFATE ADENYLTRANSFERASE GENE AND USE THEREOF

TECHNICAL FIELD v The present invention relates to a sulfate adenyltransferase gene and use thereof, in particular, a brewery yeast for producing alcoholic beverages with enhanced flavor stability, alcoholic beverages produced with said yeast, and a method for producing said beverages. More particularly, the present invention relates to a yeast, whose sulfite-producing capability that contribute to a product's flavor, is adjusted by controlling expression level of MET3 gene encoding brewery yeast sulfate adenyltransferase Meβp, for example the non-ScMET3 gene specific to a lager brewing yeast, and to a method for producing alcoholic beverages with said yeast.

BACKGROUND ART Sulfite has been known as a compound having high anti-oxidative activity, and thus has been widely used in the fields of food, beverages, pharmaceutical products or the like (for example,

. Japanese Patent Application Laid-Open Nos. H06-040907 and 2000-093096). In alcoholic beverages, sulfite has been used as an anti-oxidant. For example, in view of an important role in quality maintenance for wine that needs long time aging, addition of up to 350 ppm (parts per million) of residual concentration is permitted by the Ministry of Health, Welfare and Labor in Japan. Further, it is also known that shelf life (quality maintained period) varies depending upon sulfite concentration of a product in beer brewing. Thus, it is quite important to increase the content of this compound from the viewpoint of flavor stability or the like.

The easiest way to increase the sulfite content in a product is to add sulfite. However, sulfite is treated as a food additive, resulting in some problems such as constraint of product development and the food additive related negative image of consumers. hi the meanwhile, yeast produces, by biosynthesis, sulfur containing compounds required for yeast life cycle. Sulfite is produced as an intermediate metabolite. Thus, with use of the capability of yeasts, sulfite content in a product can be increased without addition of sulfite. Methods of increasing sulfite content in a fermentation liquor during brewing process include (1) a method based on process control, and (2) a memod based on breeding of yeast. In the methocTbased on a process control, since the amount of sulfite produced is in inverse proportion to

- the amount of initial oxygen supply, amount of oxygenfto be supplied can be reduced to increase amount of sulfite produce4"and to prevent oxidation. On the other hand, gene manipulation techniques are used in the method based on breeding

of yeast. In sulfur metabolism of yeasts, sulfite is an intermediate produced in biosynthesis, of sulfur-containing amino acids or sulfur-containing vitamins. Sulfite is produced by reduction of three step reactions of sulfate ions taken up from outside of cells.

The MET3 gene is a gene encoding an enzyme that catalyzes a first reaction; and the MET 14 gene is a gene encoding an enzyme that catalyzes a second reaction. Korch et al. attempted to increase a sulfite-producing capability of yeasts by increasing the expression level of the MET3 gene and the MET14-gene, and found that MET14 is more effective (C. Korch et al., Proc. Eur. Brew. Conv. Conger., Lisbon, 201-208, 1991). The MET 3 gene used was isolated from wine yeasts. The obtained results indicated about 0.8 ppm which corresponds to about 10% of sulfite content in an actual beer product. Also, Hansen et al. attempted to increase production amount of . sulfite by disrupting a METlO gene encoding a reductase for sulfite to prevent reduction of sulfite produced (J. Hansen et al., Nature Biotech., 1587-1591, 1996).

Further, Fujimura et al. attempted to increase sulfite content in beer by increasing expression level of a non-ScSSUl gene unique to a lager brewing yeast among SSUl genes " encoding sulfite ion efflux pump of yeast to promote excretion of sulfite to outside the fungal body (Fujimura et al., Abstract of 2003 Annual Conference of the Japan Society for Bioscience, Biotechnology and Agrochem, 159, 2003).

DISCLOSURE OF INVENTION Nevertheless, new methods and materials are needed for increasing the yeast-produced amount of sulfite to improve the shelf-life and flavor stability of the alcoholic beverage produced by the yeast.. As mentioned above, the easiest way to increase sulfite content in a product is addition of extraneous- or non-yeast produced sulfite. However, it is desirable to minimize use of food additives in view of recent consumers' preference, i.e., avoidance of food additives and use of natural materials. Thus, it is desirable to achieve sulfite content effective for flavor stability without adding sulfite from outside. However^ the method based on a process controLas described above may not be practical since shortage of oxygen may cause decrease in growth rate, resulting in delay in fermentation and quality loss.

^■ *" Further, in. breeding of yeast using gene manipulation techniques, there is a report stating _m that ten times or more sulfite content was achieved (J. ^' jfiansen et al., Nature Biotech., 1587-1591, 1996). However, there ate problems such as delay in fermentation and increase of undesirable flavor ingredients such as acetaldehyde and 1-propanol. Thus, the yeast is not good for practical

/ use. Thus, there has been a need for a method for breeding yeast capable of producing abundant amount of sulfite without impairing the fermentation rates and quality of the products.

The materials and methods disclosed herein solve the above problems, and as a result succeeded in identifying and isolating a gene encoding sulfate adenyltransferase from lager brewing yeast which has advantageous effects than the existing proteins. Moreover, a yeast was transformed by introducing and expressing with the obtained gene to confirm that the amount of sulfite produced was increased, thereby completing the present invention.

Thus, the present invention relates to a novel sulfate adenyltransferase gene existing specifically in a lager brewing yeast, to a protein encoded by said gene, to a transformed yeast in which the expression of said gene is controlled, to a method for controlling the amount of sulfite in a product by using a yeast in which the expression of said gene is controlled. More specifically, the present invention provides the following polynucleotides, a vector comprising said polynucleotide, a transformed yeast introduced with said vector, a method for producing alcoholic beverages by using said transformed yeast, and the lik e. (1) A polynucleotide selected from the group consisting of:

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

(b) a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO:2; (c)- a polynucleotide comprising a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO:2 with one or more amino acids thereof being deleted, substituted, inserted and/or added, and having a sulfate adenyltransferase activity;

.(d) a polynucleotide, comprising a polynucleotide encoding a protein having an amino acid sequence having 60% or higher identity with the amino acid sequence of SEQ ID NO:2, and having a sulfate adenyltransferase activity;

(e) a polynucleotide . comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to' the nucleotide sequence of SEQ ID NO:1 under stringent conditions, and which encodes a protein having a sulfate adenyltransferase activity; and (f) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide encoding the protein of the amino acid sequence of SEQ ID NO:2 under stringent conditions, and - which encodes a protein having a sulfate adenyltransferase activity.

(2) The polynucleotide of (1) above selected from the group consisting of: (g) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID

ϊ

NO: 2, or encoding an amino acid sequence of SEQ ID NO: 2 wherein 1 to 10 amino acids thereof is deleted, substituted, inserted, and/or added, and wherein said protein has a sulfate adenyltransferase activity;

(h) a polynucleotide encoding a protein having 90% or higher identity with the amino acid sequence of SEQ ID NO: 2, and having a sulfate adenyltransferase activity; and v_ (i) a polynucleotide which hybridizes to SEQ DD NO: 1 or which hybridizes to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and which encodes a protein having a sulfate adenyltransferase activity. .

(3) The polynucleotide of (1) above comprising a polynucleotide consisting of SEQ DD NO: 1.

(4) The polynucleotide of (1) above comprising a polynucleotide encoding a protein consisting of SEQ ID NO: 2.

(5) The polynucleotide of any one of (1) to (4) above, wherein the polynucleotide is DNA.

(6) A polynucleotide selected from the group consisting of: (j) a polynucleotide encoding RNA of a nucleotide sequence complementary to a transcript of the polynucleotide (DNA) according to (5) above;

(k) a polynucleotide encoding RNA that represses the expression of the polynucleotide (DNA) according to (5) above through RNAi effect;

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

(m) a polynucleotide encoding RNA that represses expression of the polynucleotide (DNA) according to (5) above through co-supression effect.

(7) A protein encoded by the polynucleotide of any one of (1) to (5) above.

(8) A vector comprising the polynucleotide of any one of (1) to (5) above. (8a) The vector of (8) above, .which comprises the expression cassette comprising the following components:

(x) a promoter that can be transcribed in a yeast 'cell;

(y) any of the polynucleotides described in (1) to (5) above linked to the promoter in a sense or antisense direction; and* (z) a signal that can function in a yeast with respect to transcription termination and polyadenylation of a RNA molecule.

(9) A vector comprising the polynucleotide of (φ above.

_, (10) A yeast, wherein the vector of (8) or (9) abqve is introduced.

II 1) The yeast oi (10) above, wherein sulfite producing ability is enhanced by introducing the vector .of (8) above.

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(12) A yeast, wherein an expression of the polynucleotide (DNA) of (S) above is repressed by introducing the vector of (9) above, or by disrupting a gene related to the polynucleotide (DNA) of (5) above.

(13) The yeast of (10) above, wherein a sulfite-producing ability is elevated by increasing - , 5 an expression level of the protein of (7) above. v. (14) A method for producing an alcoholic liquor by using the yeast of any one of (10) through (13) above.

(15) The method for producing an alcoholic liquor of (14) above, wherein the brew is a malt liquor.

10 (16) The method for producing an alcoholic liquor of (14) above, wherein the brew is a wine. . ^■

(17) An alcoholic liquor, which is produced by the method of any one of (14) through (16) above.

(18) A method for assessing a test yeast for its sulfite-producing ability, comprising using a 15 primer or a probe designed based on a nucleotide sequence of a sulfate adenyltransferase gene having the nucleotide sequence of SEQ ID NO: 1,

(18a) A method for selecting a yeast having a high or low sulfite-producing ability by using the method in (18) above.

(18b) A method for producing an alcoholic liquor (for example, beer) by using the yeast 20 selected with the method in ( 18a) above.

(19) A method for assessing a test yeast'for it ^' s sulfite-producing capability, comprising: culturing a test yeast; and measuring an expression level of a sulfate adenyltransferase gene having the nucleotide sequence of SEQ ID NO: 1.

(19a) A method for selecting a yeast having a high sulfite-producing ability, which 25 comprises assessing a test yeast by the method described 'in (19) above and selecting a yeast having a high expression level of sulfate adenyltransferase gene.

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

(20) A method for selecting a yeast, comprising: culturing test yeasts; quantifying the 30 protein of (7) above or measuring an expression level of a sulfate adenyltransferase gene having the nucleotide sequence of SEQ ID NO: 1; and selecting a test yeast having said protein amount or said gene expression level according to a target capability of producing sulfite.

, (21) The method for selecting a yeast of (20) ab^ve, comprising: culturing a reference yeast and test yeasts; measuring an expression level of a sulfate adenyltransferase gene having the

35 nucleotide sequence of SEQ ID NO: 1 in each yeast; and selecting a test yeast having the gene

expressed higher or lower than that in the reference yeast.

(22) The method for selecting a yeast of (20) above comprising: culruring a reference yeast and test yeasts; quantifying the protein of (7) above in each yeast; and selecting a test yeast having said protein for a larger or smaller amount than that in the reference yeast. (23) A method for producing an alcoholic beverage comprising: conducting fermentation for producing an alcoholic beverage using the yeast according to any one of (10). to (13) or a yeast selected by the method according to any one of (20) to (22); and adjusting the production amount of sulfite.

According to the method for producing alcoholic beverages by using a yeast transformed with a sulfate adenyltransferase polynucleotide operably linked to a vector, the content of sulfite having an anti-oxidative activity in a product can be increased so that alcoholic beverages can be produced with enhanced flavor and improved shelf life.

BRIEF DESCRIPTION OF DRAWINGS Figure 1 shows the cell growth with time upon beer brewing testing. The horizontal axis represents fermentation time while the vertical axis represents optical density at 660 nm (OD660).

Figure 2 shows the sugar consumption with time upon beer brewing testing. The horizontal axis represents fermentation time while the vertical axis represents apparent extract concentration (w/w%). Figure 3 shows the expression behavior of non-ScMET3 gene in yeasts upon beer brewing testing. The horizontal axis represents fermentation time while the vertical axis represents the brightness of detected signal.

Figure 4 shows the cell growth with time upon brewing testing using non-ScMET3-highly expressed strains. The horizontal axis represents fermentation time while the vertical axis represents optical density at 660 nm (OD660).

Figure 5 shows the. sugar consumption with time upon beer brewing testing using non-ScMET3-highly expressed strains. The horizontal' axis represents fermentation time while the vertical axis represents apparent extract concentration (w/w%).

Figure 6 shows the sulfite concentration in finished beer /using non-ScMET3-highly expressed strains.

BESTføODES FOR CARRYING OUT THE INVENTION

, Irϊ the known method of increasing expression '{level of a sulfite ion efflux pump, suitable fermentation rate can be ljiaintained since superfluous sulfite is not accumulated in a fungal body. However,. there is a possibility that biosynthetic reaction of sulfurous acid in the fungal body can be a

limiting factor. Thus, disclosed herein are materials and methods that enhance sulfite production by enhancing reduction pathway from sulfate ion which is a staring material to sulfurous acid.

The present inventors have studied based on this conception and as a result, isolated and identified non-ScMET3 gene encoding a sulfate adenyltransferase unique to lager brewing yeast based on the lager brewing yeast genome information mapped according to the method disclosed in Japanese Patent Application Laid-OpenNo. 2004-283169. The nucleotide sequence of the gene. is represented by SEQ ID NO: 1. Further, an amino acid sequence of a protein encoded by the gene is represented by SEQ ID NO: 2.

1. Polynucleotide of the invention

First of all, the.present invention provides (a) a polynucleotide comprising a polynucleotide of the nucleotide sequence of SEQ ID NO:1; and (b) a polynucleotide comprising a polynucleotide encoding a protein of the amino acid sequence of SEQ ID NO:2. The polynucleotide can be DNA orRNA. . The target polynucleotide of the present invention is not limited to the polynucleotide encoding a sulfate adenyltransferase gene derived from lager brewing yeast and may include other

- polynucleotides encoding proteins having equivalent functions to said protein. Proteins with equivalent functions include, for example, (c) a protein of an amino acid .sequence of SEQ ID NO: 2 with one or more amino acids thereof being deleted, substituted, inserted and/or added and having sulfate adenyltransferase activity.

Such proteins include a protein consisting of ah amino acid sequence of SEQ ID NO: 2 with, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 39, 1 to 38, 1 to 37, 1 to 36, l.to 35, 1 to 34, 1 to 33, 1 to 32, 1 to 31, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, ' 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to.6 (1 to several amino acids), 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid residues thereof being deleted, substituted, inserted and/or added and having a sulfate adenyltransferase activity. In general, the number of deletions, substitutions, insertions, and/or additions is preferably smaller. In addition, such proteins include (d) a protein having an amino acid sequence with about 60% tor higher, about 70% or higher, 71% or/higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, ^' 80% or higher, 81% or higher, 82% or higher, 83% br 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 higher, 96% or higher, 97% or higher, 98% or higher,

99% or higher, 99.1% orhigher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher identity with the

amino acid sequence of SEQ ID NO: 2, and having a sulfate adenyltransferase activity. In general, the percentage identity is preferably higher.

Sulfate adenyltransferase activity may be measured, for example, by a method of Klonus et al. as described in Plant J. 6(1): 105-12, 1994 M Furthermore, the present invention also contemplates (e) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and which encodes a protein having sulfate adenyltransferase activity; and (f) a polynucleotide comprising a polynucleotide which hybridizes to a polynucleotide complementary to a nucleotide sequence of encoding a protein of SEQ ID NO: 2 under stringent conditions, and which encodes a protein having sulfate adenyltransferase activity. •

Herein, "a polynucleotide that hybridizes under stringent conditions" refers to nucleotide sequence, such as a DNA, obtained by a colony hybridization technique, a plaque hybridization technique, a southern hybridization technique or the like using all or part of polynucleotide of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or DNA encoding the amino acid sequence of SEQ ID NO: 2 as a probe. The hybridization method may be a method described, for example, in MOLECULAR.CLONING 3rd Ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons 1987-1997.

The term "stringent conditions" as used herein may be any of low stringency conditions, moderate stringency conditions or high stringency conditions. "Low stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 32 ⁰ C. "Moderate stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 42°C. "High . stringency conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide at 50°C. Under these conditions, a polynucleotide, such as a DNA, with higher homology is expected to be obtained efficiently at higher temperature, although multiple factors are involved in hybridization stringency including temperature, probe concentration, probe length, ionic strength, time, salt concentration and others, and one skilled in the art may appropriately select these factors to realize similar stringency.

When a commercially*available kit is used for hybridization, for example, Alkphos Direct Labeling Reagents (Amersham Pharmacia) may be used. In this case, according to the attached protocol, after incubation with a labeled probe overnight, the membrane is washed with a primary wash bluffer containing 0.1% (w/v) SDS at 55 ⁰ C, thereby, detecting hybridized DNA.

, Other polynucleotides that can be hybridized include polynucleotides having about 60% or higher, about 70% or higher, 71% or higher, 72% or higher, 73% or higher, 74% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 79% or higher, 80% or higher, 81% or higher,

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 higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher or 99.9% or higher identity to DNA encoding the amino acid sequence of SEQ HD NO: 2 as calculated by homology search software, such as FASTA and BLAST using default parameters.

Identity between amino acid sequences or nucleotide sequences may be determined using algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990; Proc. Natl. Acad. Sd. USA, 90:- 5873, 1993). Programs called BLASTN and BLASTX based on BLAST algorithm have been developed (Altschul SF et al., J. MoI. Biol. 215: 403, 1990). When a nucleotide sequence is sequenced using BLASTN, the parameters are, for example, score = 100 and word length = 12. When an amino acid sequence is sequenced using BLASTX, the parameters are, for example, score = 50 and word length = 3. When BLAST and Gapped BLAST programs are used, default parameters for each of the programs are employed.

The polynucleotide of the present invention includes (j) a polynucleotide encoding RNA

- having a nucleotide sequence complementary to a transcript of the polynucleotide (DNA) according to (5) above; (k) a polynucleotide encoding RNA that represses the expression of the polynucleotide

(DNA) according to (5) above through RNAi effect; (1) a polynucleotide encoding RNA having an activity of specifically cleaving a transcript of the polynucleotide (DNA) according to (5) above; and (m) a polynucleotide encoding RNA that represses expression of the polynucleotide (DNA) according to (5) above through co-supression effect. These polynucleotides may be incorporated into a vector, which can be introduced into a cell for transformation to repress the expression of the polynucleotides (DNA) of (a) to (i) above. Thus, these polynucleotides may suitably be used when repression of the expression of the above DNA is preferable. ■

The phrase "polynucleotide encoding RNA having a nucleotide sequence complementary to the transcript of DNA" as used herein refers to so-called antisense DNA. Antisense technique is known as a method for repressing expression of a particular endogenous gene, and is described in various publications (see e.g., Hϊrajima and Inoue: New Biochemistry Experiment Course 2 Nucleic Acids IV Gene Replication and Expression (Japanese Biochemical Society Ed., Tokyo Kagaku

Dozin Co., Ltd.) pp.319-347, 1993). The sequence of antisense DNA is preferably complementary to all or part of the endogenous gene, but may not be completely complementary as long as it can

" effectively repress the expression of the gene. The transcribed RNA has preferably 90% or higher, and more preferably 95% or higher complementarity to the transcript 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.

The phrase "polynucleotide encoding RNA that represses DNA expression through RNAi effect" as used herein refers to a polynucleotide for repressing expression of an endogenous gene through RNA interference (RNAi). The term "RNAi" refers to a phenomenon where when double-stranded RNA having a sequence identical or similar to the target gene sequence is introduced into a cell, the expressions of both the introduced foreign gene and the target endogenous gene are repressed. RNA as used herein includes, for example, double-stranded RNA that causes RNA interference of 21 to 25 base length, for example, dsRNA (double strand RNA), siRNA (small interfering RNA) or shRNA (short hairpin RNA). Such RNA may be locally delivered to a desired site with a delivery system such as liposome, or a vector that generates the double-stranded RNA described above may be used fcr local expression thereof Methods for producing or using such double-stranded RNA (dsRNA, siRNA or shRNA) are known from many publications (see, e.g.,

, Japanese National Phase PCT Laid-open Patent Publication No. 2002-516062; US 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). The phrase "polynucleotide encoding RNA having an activity of specifically cleaving tr anscript of DNA" as used herein generally refers 'to a ribozyme. Ribozyme is an RNA molecule with a catalytic activity that cleaves a transcript of a target DNA and inhibits the function of that gene. Design o.f ribozymes can be found in various known publications (see, e.g., FEBS Lett. 228: 228, 1988; FEBS Lett. 239: 285, 1988; Nucl. Acids. Res. 17: 7059, 1989; Nature 323: 349, 1986; Nucl.

1 Acids. Res. 19: 6751, 1991; Protein Eng3: 733, 1990; Nucl. Acids Res. 19: 3875, 1991; Nucl. Acids Res. 19: 5125, 1991; Biochem Biophys Res Commun 186: ϊ27ϊ, ^' 1992). In addition, the phrase "polynucleotide encoding RNA that represses DNA expression through co-supression effect" refers to a nucleotide that inhibits functions of target DNA by "co-supression".

The term "co-supression" as used herein, refers to a phenomenon where when a gene having a sequence identical or similar to a target endogenous gene is transformed into a cell, the expressions of both the introduced foreign gene and the^ target endogenous gene are repressed.

Design of polynucleotides having a co-supression effect can also be' found in various publications

- (see, e.g., Smyth DR: Curr. Biol. 7: R793, 1997, Martierifjsen R: Curr. Biol. 6: 810, 1996).

2. Protein of the present invention

The present invention also provides proteins encoded by any of the polynucleotides (a) to (f) above. A preferred protein of the present invention comprises an amino acid sequence of SEQ ID NO:2 with one or several amino acids thereof being deleted, substituted, inserted and/or added, and has sulfate adenyltransferase activity. Such protein includes those having an amino acid sequence of SEQ ID NO: 2 with amino acid residues thereof of the number mentioned above being deleted, substituted, inserted and/or added and having a sulfate adenyltransferase activity. In addition, such protein includes those having homology as described above with the amino acid sequence of SEQ ID NO: 2 and having sulfate adenyltransferase activity. Such proteins may be obtained by employing site-directed mutation described, for example, in MOLECULAR CLONING 3rd Ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, NUC. Acids. Res., 10: 6487 (1982), Proc. Natl. Acad. Sd USA 19: 6409 (1982), Gene 34: 315 (1985), Nuc. Acids. Res., 13: 4431 (1985), Proc. Natl. Acad. Sd. USA 82: 488 (1985).

Deletion, substitution, insertion and/or addition of one or more amino acid residues in an amino acid sequence of the protein of the invention means that one or more amino acid residues are deleted, substituted, inserted and/or added at any one or more positions in the same amino acid

- sequence. Two or more types of deletion, substitution, insertion and/or addition may occur concurrently.

Hereinafter, examples of mutually substitutable amino . acid residues are enumerated. Amino acid residues in the same group are mutually substitutable. The groups are provided below.

Group A: leucine, isoleucine, norleucinej valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine; Group B: asparatic acid, glutamic acid, isoasparatic 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; and Group G: phenylalanine, tyrosine.

^■ The protein of the present invention may also be produced by chemical synthesis methods such as Fmoc method (fluorenylrnethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method). In addition, peptide synthesizers available from, for example, Advanced ChemTech, PerkinElmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimazu

Corp. can also be used for chemical synthesis.

^• * ^• 3. Vector of the invention and yeast transformed withvthe vector

The present invention then provides a vector comprising the polynucleotide described above. The vector of the present invention is directed to a vector including any of the

polynucleotides described in (a) to (i) above or the polynucleotides described in (j) to (m) above. Generally, the vector of the present invention comprises an expression cassette including as components (x) a promoter that can transcribe in a yeast cell; (y) a polynucleotide described in any of (a) to (i) above that is linked to the promoter in sense or antisense direction; and (z) a signal that functions in the yeast with respect to transcription termination and polyadenylation of RNA molecule. According to the present invention, in order to highly express the protein of the invention described above' upon brewing alcoholic beverages (e.g., beer) described below, these polynucleotides are introduced into the promoter in the sense direction to promote expression of the polynucleotide (DNA) described in any of (a) to (i) above. La order to repress the above protein of the invention, the polynucleotide may be introduced such that the polynucleotide of any of the (j) to

(m) is expressed. According to the present invention, the target gene (DNA) may be disrupted to repress the expression of the DNA or the protein. A gene may be disrupted by adding or deleting

. one or more bases to or from a region involved in expression of the gene product in the target gene, for example, a coding region or. a promoter region, or by deleting these regions entirely. Such disruption of gene may be found in known publications (see, e.gi, Proc. Natl. Acad. Sci. USA, 76, 4951(1979) , Methods in Enzymology, 101, 202(1983), Japanese Patent Application Laid-Open ^■ - No.6-253826).

A vector introduced in the yeast may be any of a multicopy type (YEp type), a single copy type (YCp type), or a chromosome integration type (YTp type). For example, YEp24 (J. R. Broach et al, EXPEIUMENTALMANIPUIAΉON OF GENE EXPRESSION, Academic Press, New York, 83, 1983) is known as a YEp type vector, YCp50 (M. D. Rose et al., Gene 60: 237, 1987) is known as a YCp type vector, and YIρ5 (K. Struhl et al., Proc. Natl. Acad. Sci. USA, 76: 1035; 1979) is known as a YIp type .vector, all of which are readily available.

Promoters/terminators for adjusting gene expression in yeast may be in any combination as long as they function in the brewery yeast and they have'no influence on the concentration of amino acid, sugar, higher alcohol or ester in fermentation broth ' For example, a promoter of glyceraldehydes 3-phosphate dehydrogenase gene (TDH3), or a promoter of 3-phosphoglycerate kinase gene (PGKl) may be used. These genes have previously been cloned, described in detail, for example, in M. F. Tuite et*al., EMBO J., 1, 603 (1982), and are /readily .available by known methods.

Since an auxotrophy marker cannot be used as a selective marker upon transformation for a brewety yeast, for example, a geneticin-resistant gene (G418r), a cOpper-resistant gene (CUPl)

~ (Marin et al, Proc. Natl. Acad. Sci. USA, 81, 337 1984) ^r a cerulenin-resistant gene (fas2m, PDR4)

(Junji Inokoshi et al., Biochemistry, 64, 660, 1992; and Hussain et al., Gene, 101: 149, 1991, respectively) may be used.

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A vector constructed as described above is introduced into a host yeast. Examples of the host yeast include any yeast that can be used for brewing, for example, brewery yeasts for beer, wine and sake. Specifically, yeasts such as genus Saccharomyces may be used. According to the present invention, a lager brewing yeast, for example, Saccharomyces pastorianus W34/70, Saccharomyces carlsbergensis NCYC453 or NCYC456, or Saccharomyces cerevisiae NBRC1951, NBRGl 952, NBRCl 953 or NBRCl 954 may be used. In addition, whisky yeasts such as Saccharomyces cerevisiae NCYC90, wine yeasts such as wine yeasts #1, 3 and 4 from the Brewing Society of Japan, and sake yeasts such as sake yeast #7 and 9 from the Brewing Society of Japan may also be used but not limited thereto. In the present invention, lager brewing yeasts such as Saccharomyces pastorianus may be used preferably.

A yeast transformation method may be a generally used known method. For example, methods that can be used include but not limited to an electroporation method {Meth. Enzym., 194: . 182 (1990)), a spheroplast method (Proa Natl. Acad. Sd. USA, 75: 1929(1978)), a lithium acetate method (J Bacteriology, 153: 163 (1983)), and methods described in Proc. Natl. Acad. ScL USA, 75: , 1929 (1978), METHODS IN YEAST GENETICS, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual.

More specifically, a host yeast is. cultured in a standard yeast nutrition medium (e.g., YEPD medium (Genetic Engineering. Vol. 1, Plenum Press, New York, 117(19,79)), etc.) such that OD600 run will be 1 to 6. This culture yeast is collected by centrifogatiQn, washed and pre-treated with alkali ion mdtal ion, preferably lithium ion at a. concentration of about 1 to 2 M. After the cell is left to stand at about 30°C for about 60 minutes, it is left to stand with DNA to be introduced (about 1 to 20 μg) at about 30°C for about another 60 minutes. Polyethyleneglycol, preferably about 4,000 Dalton of polyethyleneglycol, is added to a final concentration of about 20% to 50%. After leaving at about 30°C for about 30 minutes, the cell is heated at about 42°C for about 5 minutes. Preferably, this cell suspension is washed with a standard yeast nutrition medium, added to a predetermined amount of fresh standard yeast nutrition medium and left to stand at' about 30°C for about 60 minutes. Thereafter, it is seeded to a standard agar medium containing an antibiotic or the like as a selective marker to obtain a transformant.

Other general cloning techniques may be found, for example, in MOLECULAR CLONING 3rd Ed., and METHODS IN YEAST GENETICS, A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

^* " 4. Method of producing alcoholic beverages according to the present invention and alcoholic beverages produced by the method The vector of the present invention described above is introduced into a yeast suitable for

/ brewing a target alcoholic product. This yeast can be used to produce a desired alcoholic beverage with enhanced flavor with an increased content of sulfite, hi addition, yeasts to be selected by the yeast assessment method of the present invention can also be used. The target alcoholic beverages include, for example, but not limited to beer, wine, whisky, sake and the like.

5 hi order to produce these alcoholic beverages, a known technique can be used except that a brewery yeast obtained according to the present invention is used in the place of a parent strain. Since materials, manufacturing equipment, manufacturing control and the like may be exactly the same as the conventional ones, there is no need of increasing the cost for producing alcoholic beverages with an increased content of sulfite. Thus, according to the present invention, alcoholic 10 beverages with enhanced flavor can be produced using the existing facility without increasing the cost. ^• . ■

Further, since in a yeast wherein said gene is highly expressed, a sulphate ion in the culture medium is efficiently incorporated, well growth of yeast and/or alcoholic fermentation may be possible when a raw material containing low sulfur source, e.g., a wort having low malt ratio in the ϊ5 case of beer.

Alternatively, in a yeast wherein the function of synthetic system for sulfur-containing amino acid is too active, sulfur-containing compounds including, hydrogen sulfide as an intermediate-metabolite in the pathway, which cause undesirable off-flavor for alcoholic beverages,

^' are sometimes generated in large amounts and accumulated. By suppressing or disrupting said 0 ' gene function of such yeast, incorporation of sulphate ion as a starting material may be suppressed.

As a result, ah alcoholic beverage wherein the off- flavor is reduced, can be produced.

5. Yeast assessment method of the invention

The present invention relates to a method for assessing a test yeast for its sulfite-producing 5 capability by using a primer or a probe designed based on a nucleotide sequence of a sulfate adenyltransferase gene having the nucleotide sequence of SEQ ID NO:1. General techniques for such assessment method is known and is described in, for example, WOO 1/040514, Japanese Laid-Open Patent Application No. 8-205900 or the like. This assessment method is described in below. 0 First, genome of a test yeast is prepared. For this preparation, any known method such as

Hereford method or potassium acetate method may beVused (e.g., METHODS IN YEAST GENETICS, Cold Spring Harbor Laboratory Press, 130 (1990)). Using a primer or a probe designed based on a

nucleotide sequence (preferably, ORF sequence) of the sulfate adenyltransferase gene, the existence of the gene or a sequence specific to the gene is determined in the test yeast genome obtained. The primer or the probe may be designed according to a known technique.

Detection of the gene or the specific sequence may be carried out by employing a known technique. For example, a polynucleotide including part or all of the specific sequence or a polynucleotide including a nucleotide sequence complementary to said nucleotide sequence is used as one primer, while a polynucleotide including part or all of the sequence upstream or downstream from this sequence or a polynucleotide including a nucleotide sequence complementary to said nucleotide sequence, is used as another primer to amplify a nucleic acid of the yeast by a PCR method, thereby determining the existence of amplified products and molecular weight of the amplified products. The number of bases of polynucleotide used for a primer is generally 10 base pairs (bp) or more, and preferably 15 to 25 bp. In general, the number of bases between the primers . is suitably 300 to 2000 bp. . ^'

The reaction conditions, for PCR are not particularly limited but may be, for example, a , denaturation temperature of 90 to 95°C, an annealing temperature of 40 to 60 ⁰ C, an elongation temperature of 60 to 75 ⁰ C, and the number of cycle of 10 or more. The resulting reaction product

. may be separated, for example, by electrophoresis using agarose gel to determine the molecular weight of the amplified product. This method allows prediction and assessment of the capability of the yeast to produce sulfite as determined by whether the molecular weight of the amplified product is a size that contains the DNA molecule of the specific part, hi addition, by analyzing the nucleotide sequence of the amplified product, the capability may be predicted and/or assessed more precisely.

Moreover, in the present invention, a test yeast is cultured to measure an expression level of the sulfate adenyltransferase gene having the nucleotide sequence of SEQ ID NO: 1 to assess the test yeast for its sulfite-producing capability. , hi this case, the test yeast is cultured and then mRNA or a protein resulting from the sulfate adenyltransferase gene is quantified. The quantification of mRNA or protein may be carried out by employing a kriown technique. For example, mRNA may be quantified, by Northern hybridization or quantitative RT-PCR, while protein may be quantified, for example, by Western blotting (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons 1994-2003).

Furthermore, test yeasts are cultured and expression levels of the sulfate adenyltransferase gene having the nucleotide sequence of SEQ ID NO:. 1 are measured' to select a test yeast with the

- gene expression level according to the target capability of producing sulfite, thereby selecting a yeast favorable for brewing desired alcoholic beverages. In addition, a reference yeast and a test yeast may be cultured so as to measure and compare the expression level of the gene in each of the yeasts,

thereby selecting a favorable test yeast. More specifically, for example, a reference yeast and one or more test yeasts are cultured and an expression level of the sulfate adenyltransferase gene having the nucleotide sequence of SEQ ID NO: 1 is measured in each yeast. By selecting a test yeast with the gene expressed higher than that in the reference yeast, a yeast suitable for brewing alcoholic beverages can be selected.

^. Alternatively, test yeasts are cultured and a yeast with a higher sulfite-producing capability or a higher sulfate adenyltransferase activity is selected, thereby selecting a yeast suitable for brewing desired alcoholic beverages.

In these cases, the test yeasts or the reference yeast may be, for example, a yeast introduced with the vector of the invention, a yeast in which an expression of the polynucleotide (DNA) of the present invention is enhanced or repressed, an artificially mutated, yeast or a naturally mutated yeast.

Sulfate adenyltransferase activity may be measured, for example, by a method of Klonus et al. as

, described in Plant J. 6(1): 105-12, 1994 JuI. The mutation treatment may employ any methods including, for example, physical methods such as ultraviolet irradiation and radiation irradiation, and chemical methods associated with treatments with drugs such as EMS (ethylmethane sulphonate) and N-methyl-N-nitrosoguanidine (see, e.g., Yasuji Oshima Ed., BIOCHEMISTRY EXPERIMENTS vol.

- 39, Yeast Molecular Genetic Experiments, pp. 67-75, JSSP).

In addition, examples of yeasts used as the reference yeast or the test yeasts include any yeasts that can be used for brewing, for example, brewery yeasts for beer, wine, sake and the like. More specifically, yeasts such as genus Saccharomyces may be used (e.g., S. pastorianus, S. cerevisiae, and S. carlsbergensis). According to the present invention, a lager brewing yeast, for example, Saccharomyces pastorianus W34/70; Saccharomyces carlsbergensis NCYC453 or NCYC456; ox Saccharomyces cerevisiae NBRC1951, NBRC1952, NBRC1953 or NBRC1954 may be used. _. Further, wine yeasts such as wine yeasts #1, 3 and 4 from the Brewing Society of Japan; and sake yeasts such as sake yeast #7 and 9 from the Brewing Society of Japan may also be used but not limited thereto. In the . present invention, lager brewing ' yeasts such as Saccharomyces pastorianus may preferably be used. The reference ye'ast and the test yeasts may be selected from the above yeasts in any combination.

^' EXAMPLES

Hereinafter, the present invention will be described in more detail with reference to working examples.- The present invention, however, is not . limited to the examples described ~ below.

Example 1: Clnniiψ of Sulfate Adenyltransferase (non-ScMET3) Gene

/

A. specific novel sulfate adenyltransferase gene (non-ScMET3) gene (SEQ ID NO: I)' from a lager brewing yeast were found, as a result of a search utilizing the comparison database described in Japanese Patent Application Laid-Qpen No. 2004-283169. Based on the acquired nucleotide sequence information, primers non-ScMET3_for (SEQ ID NO: 3) and non-ScMET3_rv (SEQ ID ,5 NO: 4) were designed to amplify the full-length genes, respectively. PCR was carried out using chromosomal DNA of a genome sequencing strain, Saccharomyces pastorianus Weihenstephan 34/70 strain, as a template to obtain DNA fragments including the full-length gene of non-ScMET3.

The thus-obtained non-ScMET3 gene fragment was inserted into pCR2.1-TOPO vector (Inyitrogen) by TA cloning. The nucleotide sequences of non-ScMET3 gene were analyzed 10 according to Sanger's method (F. Sanger, Science, 214: 1215, 1981) to confirm the nucleotide sequence.

Example 2: Analysis of Expression of non-ScMET3 Gene during Beer Brewing Testing

A beer brewing testing was conducted using a lager brewing yeast, Saccharomyces

\$ pastorianus Weihenstephan 34/70 strain and then mRNA extracted fpm a beer yeast fungal body during fermentation was detected by a DNA microarray.

. Wort extract concentration 12.69%

Wort content 70 L

20 Wort dissolved oxygen concentration ' 8.6 ppm

Fermentation temperature 15°C

Yeast input 12. ^' 8xl0 ⁶ cells/mL

Sampling of fermentation liquor was performed with time, and variation with time of yeast

25 growth amount (Fig. l) and apparent extract concentration (Fig. 2) was observed. Simultaneously, sampling of a yeast fungal body was performed, and the prepared mRNA was subjected to be biotin-labeled and was hybridized to a beer yeast DNA microarray as described in Japanese Patent

Application Laid-Open No. 2004-283169. The signal was detected using GCOS; GeneChip

Operating Software 1.0 (manufactured by Affymetrix Co.). Expression pattern of non-ScMET3

30 gene is shown in Figure 3. As a result, it was confirmed that non-ScMET3 gene was expressed in the general beer fermentation.

Example 3: Constitutive Expression of noή-ScMET3 Genes

The non-ScMET3/ρCR2.1-TOPO described in Example 1 was digested with restriction

enzymes Sad and Notl to prepare a DNA fragment including non-ScMET3 gene. This fragment was linked to pUP3GLP2 treated with restriction enzymes Sad and Notl, thereby constructing a non-ScMET3 constitutive expression vector, pUP-nonScMET3. The yeast expression vector, pUP3GLP2, is a YIp type (chromosome integration type) yeast expression vector having oiOtidiiie-5-phosphoric acid decarboxylase gene URA3 at the homologous recombinant site. The introduced gene was constitutively expressed by the promoter and terminator of glycerylaldehyde-3-phosphoric acid dehydrogenase gene, TDH3. Drug-resistant gene YAPl as a selective marker for yeast was introduced under the control of the promoter and terminator of galactokinase GALl, whereby the expression is induced in a culture media comprising galactose. Ampicillin-resistant gene Amp ^r as a selective marker for E. coli was also included.

The constitutive expression vector prepared by the method above was used to transform Saccharomyces pastorianus Weihenstephan 34/70 strain according to the method described in Japanese Patent Application Laid-Open No. 07-303475. Right assessment on the non-ScMET3 gene cannot be conducted if sulfite is accumulated within the fungal body since the yeast itself is damaged by sulfite. Thus, first, a strain in which non-ScSSUl gene encoding a sulfite efflux pump is highly expressed, was prepared according to the method described in Japanese Patent Application

- Laid-Open No. 2004-283169. Then, .the obtained strain was used as a parent strain and transformed by pUP-non-ScMED. Cerulenin-resistant strains were selected in a YPGaI plate medium (1% yeast extract, 2% polypeptone, 2% galactose, 2% agar) containing 1.0 mg/L cerulenin.

Example 4: Analysis of Amount of Sulfite Produced during Beer Brewing Testing

The parent strain, and non-ScMET3-highly expressed strains (two strains) obtained in Example 3, were used to carry out beer brewing testing under the following conditions.

Wort extract concentration 11.96% <

Wort content 1 L

Wort dissolved oxygen concentration about 10 ppm

Fermentation temperature 15 ⁰ C constantly

Yeast input 6 g of wet yeast cells/L of wort

The fermentation brotli was sampled with time to observe the cell growth (OD660) (Fig. 4) and sugar consumption with time (Fig. 5). Quantification of the sulfite content upon completion of fermentation was carried out by collecting sulfite in hydrogen peroxide solution by distillation under acidic condition, and titration with alkali (Revised BCOJ Beer Analysis Method by the Brewing Society -of Japan).

With respect to the amount of sulfite produced upon completion of fermentation, while the

parent strain produced 25.4 ppm, the non-ScMET3-highly expressed strains produced 32.3 ppm and 33.8 ppm, respectively (Fig. 6). Thus, it was found that about 30% of the amount of sulfite produced can be increased by high expression of the non-ScMET3. In these cases, differences in the growth rates and the extract consumption rates were little between the parent strain and the constitutively expressed strains.

As can be appreciated from the above results, by constitutively expressing sulfate adenyltransferase unique to a lager brewing yeast as described herein in the yeast with enhanced sulfite-producing capability, it became possible to specifically increase production amount of sulfite functioning as anti-oxidant for alcoholic beverages such as beer without altering the fermentation procedure or time. Thus, alcoholic beverages with enhanced flavor and long shelf life (with good quality), can be produced.

Example 5: Beer Brewing Testing using Wort Containing Low Sulfiir Source Saccharomyces pastorianus Weihenstephan 34/70 strain is transformed with the high expression vector prepared in Example 3 to obtain Sc and nόn-ScMET3 (sole) highly expressed strains, respectively. Then, a wort containing 24% of malt ratio is prepared as a wort containing low sulfur source. Subsequently, using parent and the highly expressed strains obtained, under the following conditions beer brewing testing is carried out.

Wort extract concentration 13%

Wort content 2L

Wort dissolved oxygen concentration about S ppm

Fermentation temperature 15°C constantly

Yeast input 10.5 g of wet yeast cells/2 L of wort

The fermentation broth is sampled with time tp observe the cell growth (OD660) and the sugar consumption with time.

Example 6: Disruption of MET3 Gene

According to the publication (Goldstein et al., yeast. 15 1541 (1999)), PCR using a plasmid including a -drug-resistant marker (pFA6a (G418) or pAG25 (natl)) as a template is conducted to prepare a fragment for MET3 gene disruption.

_* - With the fragment for gene disruption prepared, W34/70 strain or spore cloning strain (W34/70-2) is transformed. The transformation is performed in accordance with the method described in Japanese Patent Application Laid-Open No. H07-303475. The concentrations of the drugs for selection are 300 mg/L for geneticin and 50 mg/L of nourseothricin, respectively.

Example 7: Analysis of Amounts of Sulfur-Containing Compound Produced upon Beer Brewing Testing

Using parent strain and the gene-disrupted strain obtained in Example 6, under the following conditions, beer brewing testing is carried out.

Wort extract concentration 13%

Wort content 2L

Wort dissolved oxygen concentration about δ ppm Fermentation temperature 15 ⁰ C constantly

Yeast input 10.5 g ofwet yeast cells/2 L of wort

The fermentation broth is sampled with time to observe the cell growth (OD660) and the sugar consumption with time. Analysis of sulfur^containing compounds in broth is performed by employing head-space gas chromatography.

Industrial Applicability

According to the method for producing alcoholic beverages of the present invention,

" because of increase in content of sulfite having anti-oxidative action in a product, alcoholic beverages with enhanced flavor and long shelf life (with good quality), can be produced. Also, since the yeast of the present invention can efficiently reduce a sulphate ion as a sulfur source to

■ synthesize a sulfur-containing compound necessary for growth, desirable alcoholic fermentation can be performed by using raw materials with low contents of sulfur-containing amino acid, e.g., sparkling liquor (happoushu) wort.. Moreover, by suppressing an expression of said gene in yeast wherein sulfur-containing compounds as an off-flavor are highly generated, an alcoholic beverage having desirable flavor can be produced.

This application claims benefit of Japanese Patent Application Nos. 2005-235011 filed August 12 ^" , 2005 and 2006-84289 filed March 23, 2006, which are herein incorporated by reference in their entirety for all purposes. All other references cited above are also incorporated herein in their entirety for all purposes.