JP3100697B2 - Food and beverage manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a food or drink, such as an alcoholic beverage, characterized by using a yeast belonging to the genus Saccharomyces into which a resistance gene derived from the genus is introduced.
The present invention relates to a method for producing a fermented seasoning (also referred to as a brewed seasoning) and the like, and a food or beverage produced by the method.

[0002]

2. Description of the Related Art Aroma in foods and drinks such as alcoholic beverages and fermented seasonings is an important property for these products. Important components that determine its odor are esters and alcohols. In particular, β-phenethyl alcohol and β-phenethyl acetate are one of the high-boiling aroma components produced by yeast, and exhibit aroma similar to that of rose flower and hyacinth flower, respectively. These odorants are
It is contained in various brewed products and characterizes the product, but it is particularly present in large quantities in ginjo sake and shochu (Journal of the Brewing Association,
74, 173, 1979], and is reported to have an important role in the flavor of alcoholic beverages (Journal of the Japanese Society of Agricultural Chemistry, 35, 829, 1962;
Journal of the Brewing Association, 66, 176, 1971]. Among them, sake is considered to contribute to the mellowness of the taste and the incense. If yeast can produce these aroma substances in large quantities, it is expected that it will be possible to produce sake with a mellow taste and increased incense, as well as brewed beverages and foods with the aroma of rose flowers. You.

[0003] β-phenethyl alcohol is produced by decarboxylation of phenylpyruvic acid, a precursor of phenylalanine, by yeast of the genus Saccharomyces [Journal of the Institute of Brewing (Journal of the Institute of Brewing). Brewing) 71
Volume, 341, 1965; Journal of the Brewing Association, 61, 481, 1966;
76, 629, 1981]. Β-phenethyl acetate is obtained from acetyl-CoA and β-phenethyl alcohol.
It is known to be produced by alcohol acetyltransferase (Journal of the Brewing Association, 82, 369, 1987)
Year〕.

[0004] Conventionally, as a method for increasing the amount of higher alcohols in foods and drinks, a method of adding leucine, valine, or the like in a liquor production process [Journal of the Institute of Brewing,
59, 421-429, 1981].

The present inventors have already reported a method for increasing branched chain alcohols such as isobutyl alcohol or isoamyl alcohol and aromatic alcohols such as β-phenethyl alcohol in food and drink using Saccharomyces cerevisiae. I have. For example, azaleucine-resistant strains (JP-A-1-257423), glyphosate-resistant strains, thienylalanine-resistant strains, thiazolealanine-resistant strains, chloroalanine-resistant strains, propargylglycine-resistant strains (JP-A-3-7579) and amino acids An ethylcysteine-resistant strain (Japanese Patent Application No. 1-192234), a fluorophenylalanine-resistant strain [(JP-A-2-92265, Agricultural and Biological Chemistry (Agricul
tural and Biological Chemistry) 54, 269-271, 19
1990), ibid., 54, 3151-3156, 1990], and azetidine carboxylate resistant strains (Japanese Patent Application No. 2-2061010).

[0006] It is not known to use a transformed yeast belonging to the genus Saccharomyces and obtained by introducing a gene relating to the expression of resistance to fluorophenylalanine for the production of foods and drinks.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a fragrance component such as higher alcohols, which are important as a fragrance component in foods and drinks, and in particular, a trait capable of producing β-phenethyl alcohol and β-phenethyl acetate in larger amounts. It is an object of the present invention to provide a fragrant food or drink by using a converted yeast.

[0008]

Means for Solving the Problems When breeding yeast for brewing, performing mutation treatment may cause mutations in genes other than the target and change the brewing characteristics. Is desired. According to the present invention, a fragrant alcoholic beverage characterized by using a transformed yeast obtained by introducing a gene involved in the expression of fluorophenylalanine resistance belonging to the genus Saccharomyces, and producing a food or drink such as a fermented seasoning. And a food or drink produced by the method.

The transformed yeast used in the present invention belongs to the genus Saccharomyces, and has introduced thereinto a gene involved in the expression of resistance to fluorophenylalanine such as p-fluorophenylalanine (PFP) or o-fluorophenylalanine (OFP). Any yeast can be used. An example of a gene involved in the expression of PFP resistance is the pfp1 gene. OFP
As a gene involved in the development of resistance, the gene represented by SEQ ID NO: 19 can be mentioned. If it contains the gene represented by SEQ ID NO: 19, it may contain a gene necessary for expression before and after the gene. For example, OF
P1 gene (SEQ ID NO: 18). Such a transformed yeast is, for example, a commercially available yeast (for example, brewer's yeast, wine yeast, sake yeast, whiskey yeast, shochu yeast, baker's yeast, etc.) or a yeast isolated from the natural world. , A protoplast method, an electrofusion method or a transformation method in the presence of an alkali metal such as lithium.

[0010] Specific examples of the transformed yeast to be used in the present invention, Saccharomyces Serebije (Saccharomy
ces cerevisiae ) K-9H14-U7 / pKF101 strain (hereinafter K-9H
14-U7 / pKF101 strain) and Saccharomyces cerevisiae FD-8 / pKF201 strain
(Hereinafter referred to as FD-8 / pKF201 strain). The K-9H14-U7 / pKF101 strain and the FD-8 / pKF201 strain were transferred to the Institute of Microbial Industry and Technology on March 22, 1991 based on the Budapest Treaty by FER
MBP-3330) and Deposit No. 3329 (FERM BP-3329).

Using these transformed yeasts, alcoholic beverages and fermented seasonings are produced. The method for producing the alcoholic beverage and the fermented seasoning can be carried out by conventionally known methods. The following alcoholic beverages (distilled spirits such as shochu and whiskey, wine, sake, etc.)
The production of the fermented seasoning will be described. In any of the production methods, alcohol fermentation by yeast is involved.

[Raw Materials] Any carbohydrates and starches can be used, but the raw materials specified by the Liquor Tax Law and the Enforcement Order of the Liquor Tax Law depending on the type of alcoholic beverage to be used are used. For example, fruits such as grapes, molasses, glucose, potatoes, buckwheat, rice, barley, bubble, corn, koryun, cane, fin and rice and barley koji can be used. Fruits such as grapes, molasses, and sugars such as glucose are directly fermented by yeast directly (single fermentation). First, starch from cereals such as potatoes, barley, buckwheat, and corn is decomposed into fermentable sugars by saccharifying enzymes. Then, a method of performing alcohol fermentation with yeast (parallel double fermentation) and the like are known. In general, saccharifying enzymes include those contained in malt, for example, those produced by Aspergillus oryzae such as filamentous fungus Aspergillus oryzae, or enzyme preparations such as α-amylase, glucoamylase, and protease.

[Sake mother] As for the yeast used for fermentation, the number of yeasts is increased from a storage state such as an agar slant medium through an appropriate liquid medium. For example, in the last stage of sake brewing mother,
Yeast is cultivated using rice and rice koji, which are about 7% of moromi.

[Moromi] A raw material as a carbon source, water and inorganic salts are charged into a temperature-controllable tank, and fermentation is started by adding sake brewer. In the production of sake and shochu, in the first stage of fermentation, a part of the carbon raw material is charged, and a stage is prepared in which the remainder is added during fermentation. In sake, the additional stage is called the first stage (with supplement), the second stage (middle), and the third stage (stop). In the fermentation process, the fermentation is performed while stirring at a pH of 3.5 to 5.0 and a temperature of 6 to 20 ° C, and the fermentation is managed with care for microorganisms and the like. Moromi temperature should be between 10 ° C and 18 ° C for sake, preferably 1 ° C.
Controlled at 5 ° C. In the Moromi period, fermentation is completed in about 16 to 25 days. In shochu production, the moromi temperature is slightly higher than sake and the moromi period is shortened.
The moromi is appropriately stirred so that the fermentation proceeds smoothly.

[Upper tank or distillation] The process of converting ethanol in moromi produced by fermentation of yeast into an alcoholic beverage may be carried out in various ways depending on the desired beverage. In the sake, after completion of the moromi fermentation, the sake is separated from the sake basin by squeezing filtration or the like to obtain the original sake. In the case of distilled liquor, a distillate is obtained from moromi using a simple distillation machine or the like. In the case of shochu, moromi is distilled to obtain shochu raw wine in which ethanol is concentrated. In the fermented seasoning, a treatment of adding salt, acetic acid, and the like to the moromi before the end of fermentation (a non-drinking treatment prescribed by the Liquor Tax Law) is performed, followed by filtration.

Examples of the present invention and reference examples are shown below.

[0017]

【Example】

Embodiment 1 FIG. Sake brewing K-9H14-U7 / pKF101, K-9H14-U7, FD-8 / pKF201,
Sake was brewed using FD-8 strain and F44 strain (JP-A-2-92265). Moromi was prepared as shown in Table 1.

[0018]

[Table 1]

Temperature control of Moromi: 16 ° C. for garnishing, dancing, and mashing;
℃, and furthermore, after the preparation, the temperature is 13 ℃.
The temperature of the product was raised at 1 ° C every day, and after the start of falling bubbles, the temperature was lowered by 1 ° C per day to 10 ° C. Table 2 shows the components of the brewed sake.

[0020]

[Table 2]

Transformed yeast K-9H14-U in moromi after fermentation
7 / pKF101 strain (100 strains) uracil requirement and OF
When the P resistance was examined, the plasmid was retained at a high frequency of 90% or more.

Embodiment 2 FIG. Production of barley shochu Shochu was brewed using K-9H14-U7 / pKF101, K-9H14-U7, FD-8 / pKF201 and FD-8. Moromi was prepared as shown in Table 3 and rice koji was used for the primary preparation and barley having a degree of whitening of 75% was used for the secondary preparation.

[0023]

[Table 3]

Moromi management: Moromi temperature is 20 ° C.
And Five days after the first charge, a second charge was performed, and further 10 days later, distillation was performed. 4th component of brewed aged moromi
Table 5 shows the aroma components of shochu obtained by further distillation.

[0025]

[Table 4]

[0026]

[Table 5]

Embodiment 3 FIG. Production of brewed seasonings Fermented seasonings were produced using yeast strains K-9H14-U7 / pKF101 and K-9H14-U7. Moromi was prepared as shown in Table 6.

[0028]

[Table 6]

Fermentation control: The fermentation temperature was 25 ° C. throughout. On the 3rd day after the primary charging, the secondary charging was performed, and 5 days later, the drinking-proof treatment was performed by adding 65 g of salt. Two days later, the insoluble components were removed by press filtration to obtain a fermented seasoning. Table 7 shows the components of the manufactured fermented seasonings.

[0030]

[Table 7]

Embodiment 4 FIG. Acquisition of K-9H14-U7 / pKF101 strain and determination of base sequence of OFP1 gene (1) Cloning of resistance gene OFP resistance gene (OFP1) is 3-deoxy-D-
Arabino-heptulophosphate-7-phosphate synthase (D
This is an allele of the gene ARO4 encoding AHP synthase). The complete nucleotide sequence of ARO4 of Saccharomyces cerevisiae already reported (Swiss Technical University, Paravicini dissertation, 1989) is included in BamH.
Since there are no I and SalI cleavage sites, Sa
The 29-mer oligonucleotides represented by SEQ ID NOS: 1 and 2 having an I1 site and a BamHI site were synthesized using a DNA synthesizer, respectively. The oligonucleotide represented by SEQ ID NO: 1 is an upstream primer having a SalI site, and the oligonucleotide represented by SEQ ID NO: 2 is a downstream primer having a BamHI site. The region between the two primers obtained by synthesis is an approximately 1.9 kb region containing the ARO4 promoter, open reading frame (ORF) and terminator.

Separately, Saccharomyces cerevisiae X2
The 180-1B strain (ATCC26787) was given OFP resistance as follows. YPD medium (1% yeast extract, peptone 2
%, Glucose 2%, pH 6.0) and cultured at 30 ° C for 16 hours with 0.2M phosphate buffer (p
H8.0) to a cell concentration of 10 ⁸ cells / ml.

Then, ethyl methanesulfonate was added to the suspension to a final concentration of 30 μl / ml.
, And the cells were collected by centrifugation. The cells were washed with a 5% aqueous solution of sodium thiosulfate, and further washed with a phosphate buffer (pH 6.0). The cells were cultured at 30 ° C. for 7 days on a plate medium containing 0.7 mg / ml of OFP (Bacto yeast nitrogen base 0.67%, glucose 2%, agar 1.5%, manufactured by Difco). Mutant strain Saccharomyces cerevisiae X2180A having OFP resistance from colonies that appeared on agar plates
The strain was selected.

Based on the Polymerase Chain Reaction (PCR) method (Science, Vol. 239, 487, 1988), using the obtained upstream primer, downstream primer and the whole chromosomal DNA of the mutant having OFP resistance. 1.9 kb of DNA, that is, OFP1 gene was amplified.

The amplified DNA was subjected to agarose electrophoresis to separate a 1.9 kb band, and the DNA was extracted.
Next, the obtained DNA was treated with BamHI and SalI, followed by phenol treatment and ethanol precipitation to obtain DNA.
And pUC19 treated with BamHI and SalI
After ligation with a plasmid, E. coli [ E. coli HB101 strain, Journal of Molecular Biology (Journal
f Molecular Biology) 41, 459, 1969] as a host (Molecular Cloning, Experiment, Second Edition, Cold Spring Harbor
Laboratory Press (Molecular Cloning, A Laboratory
manual, Cold Spring Harbor Laboratory Press, 198
9); Hereinafter, molecular cloning. ].

The obtained plasmid was subjected to electrophoresis, and p
A plasmid which was longer than the UC19 plasmid by a base chain (about 1.9 kb) corresponding to the OFP1 gene was recovered, and the plasmid was named pKF100. When the restriction enzyme cleavage site was examined for the inserted clone of pKF100, it was confirmed that the restriction enzyme cleavage site was present at the same site as the known ARO4 (FIG. 1).

(2) Introduction of OFP1 gene into sake yeast and production of β-phenethyl alcohol: obtained pKF1
Vector Y treated with BamHI and SalI
Cp50 (ura3 CEN4; Amp ^R Tet ^R ) [Gene
60, 237, 1987], and OFP as before
A plasmid which was longer by a base chain (about 1.9 kb) corresponding to one gene was recovered from the electrophoresis gel, and the plasmid was designated as pKF101. Next, 0.5 μg of pKF101 was prepared by the lithium method by the brewing association No. 9 yeast haploid strain Saccharomyces cerevisiae K-9H14-U7 (MATαura3).
After transformation into the strain (molecular cloning), a uracil-free CMM agar medium [2% glucose, 24 μg / ml uracil, tryptophan, histidine, arginine, 0.67% Difco yeast nitrogen base (amino acid free), Methionine, 36μ
g / ml of tyrosine, leucine, isoleucine, lysine,
100 μg / ml asparagine, 150 μg / ml valine, 200 μg / ml threonine, and 60 μg / ml phenylalanine), and cultured at room temperature to obtain a growing transformant. 9H14-U7 / pKF1
Named 01.

(3) Determination of base sequence and amino acid sequence of OFP1 gene Various primers were prepared using a DNA synthesizer, and the dideoxy method [Messing J .: Methods in Enzymology, vol. 20-78, 1983
Year], the nucleotide sequence of the extended DNA fragment was determined.

With reference to the base sequence of ARO4 reported by Paravicini, seven types of primers (SEQ ID NOS: 5 to 11) were translated from the primers of 20 to 21 bases every 300 to 400 bases. It was synthesized using a synthesizer. Furthermore, DN on the non-translated side of this gene
For the A chain (antisense), six primers of 20 to 21 bases were prepared for every 300 to 400 bases (SEQ ID NOS: 12 to 17). The position of the primer in the untranslated strand was selected so as to avoid duplication with the primer on the translation side, and care was taken not to overlook even if there was a mutation in the synthesized primer region.

Sequence kit (Taq DyeDeoxyTM Term
inator Cycle Sequencing Kit, Applied Biosystems)
A DNA fragment of one gene was reacted, and a DNA fragment up to about 400 bases in length was amplified. The nucleotide sequence of these fluorescently labeled DNA fragments was determined using a DNA sequencer (ABI model 373A DNA sequence system, Applied Biosystems). The base sequences of the OFP1 gene were determined by joining the base sequences with sufficient duplication to determine the entire base sequence (SEQ ID NO: 1).
8). Using the same primers and the ARO4 gene as a template, a DNA fragment was amplified by PCR, and the nucleotide sequence was determined by a sequencer. In both cases, there was no contradiction with the result of determining the base sequence by the same experiment using the untranslated DNA strand as a template.

As a result, the difference between the ARO4 gene and the OFP1 gene was only one base. That is, in the OFP1 gene, cytosine (C) at the 496th base (1058th in SEQ ID NO: 18) from the translation start point was replaced with adenine (A). In this substitution, glutamine (Gln) at the 166th amino acid on the protein is
This corresponds to substitution with lysine (Lys).

Reference Example 1 Transformant K-9H14-U7 / pKF101
Strain properties K-9H14-U7, K-9H14-U7 / pKF101 and plasmid YC
After dissolving fluorophenylalanine in water, the K-9H14-U7 / YCp50 strain containing only p50 was dissolved at a concentration of 2 mg / ml.
After inoculation on an agar plate medium prepared by mixing with Bact East Nitrogen Base (manufactured by Difco), the growth was observed at 28 ° C for 4 days. Table 8 shows the results.

Furthermore, K-9H14-U7 strain, K-9H14-U7 / pKF101
The strain and the K-9H14-U7 / YCp50 strain were cultured in YPD with shaking at 30 ° C. for 48 hours, and the results of measurement of β-phenethyl alcohol produced in the culture solution are also shown in Table 8.

[0044]

[Table 8]

Plasmid stability: No dropout of the plasmid was observed in the transformant even after subculture in a nutrient medium.

Reference Example 2 Comparison of ARO4 gene and OFP1 gene A 1.9 kb DNA fragment corresponding to the ARO4 gene was
It was obtained from the 80-1B strain, ligated to pUC19, and further ligated to YCp50 to obtain plasmid pKF401. Saccharomyces cerevisiae K-9H14-U
Saccharomyces cerevisiae PU-2 instead of 7 strains
The (MAT3, aro3, aro4, ura3) strain was treated in the same manner as in Example 4 (2) using pKF401 instead of pKF101 to obtain a transformant strain PU-2 / pKF401.

Further, the PU-2 strain was used in place of the K-9H14-U7 strain, and the same procedure as described in Example 4 (2) was carried out to obtain a transformant strain PU-2 / pKF101. PU-2 strain, PU-2 / pK
Agar prepared by dissolving F101 strain and PU-2 / pKF401 strain in water and dissolving fluorophenylalanine in water and mixing with Bacto yeast nitrogen base (manufactured by Difco) to a concentration of 2 mg / ml. After inoculating the plate medium,
The growth after 4 days at 28 ° C was observed. Table 9 shows the results.

Further, PU-2 strain, PU-2 / YCp50 strain, PU-2 / pKF
Table 9 also shows the results of measuring the β-phenethyl alcohol produced in the culture after shaking culture of strain 101 and strain PU-2 / pKF401 in YPD at 30 ° C. for 48 hours.

[0049]

[Table 9]

Reference Example 3 Use of OFP1 Gene as Dominant Positive Marker For breeding of practical yeast, a dominant resistance marker that expresses a trait in one copy is required. Since the OFP1 gene shows dominant OFP resistance in one copy, its possibility as a dominant positive marker in transformation was examined.
After transforming 0.5 μg of pKF101 and 0.5 μg of YCp50 into the K-9H14-U7 strain and the yeast-derived SK-16-U5 (MATαura3) strain for alcohol fermentation by the lithium method, 2 mg / ml of uracil was supplemented. The cells were spread on an MM2 plate containing OFP, and uracil requirement of the growing strain was examined.
As a result, K-9H14-U transformed with pKF101
All seven strains and the SK-16-U5 strain had complementary uracil auxotrophy (Table 10). Further, as a result of a plasmid drop-out test, the plasmid-dropped bacteria did not grow in the absence of uracil, and the pKF101 plasmid disappeared.
FP resistance and PFP resistance in the presence of tyrosine were also lost, indicating that these traits are linked.

[0051]

[Table 10]

The use of a positive marker derived from Saccharomyces cerevisiae facilitates molecular breeding in practical yeast.

Reference Example 4 Acquisition of FD-8 / pKF201 strain (1) Cloning of resistance gene The PFP resistance gene (pfp1) is an allele of the gene tyr1 encoding prephenate dehydrogenase. The total nucleotide sequence of TYR1 of Saccharomyces cerevisiae already reported [Gene, 85, 303,
1989], a 26-mer oligonucleotide represented by SEQ ID NOs: 3 and 4 having a SalI site and a BamHI site was synthesized using a DNA synthesizer, respectively. The oligonucleotide represented by Formulation No. 3 is an upstream primer having a SalI site, and the oligonucleotide represented by SEQ ID
It is a downstream primer having an amHI site. The region between the two primers obtained by synthesis is the TYR1 promoter and open reading frame (ORF).
And about 2.4 kb including the terminator.

Separately, Saccharomyces cerevisiae X2
The 180-1B strain was given PFP resistance as follows.
X2180- cultured in YPD medium at 30 ° C. for 16 hours
The 1B strain was suspended in a 0.2 M phosphate buffer (pH 8.0) to a cell concentration of 10 ⁸ cells / ml.

Then, ethyl methanesulfonate was added to the suspension to a final concentration of 30 μl / ml.
After standing at 60 ° C. for 60 minutes, the cells were collected by centrifugation. After washing the cells with a 5% aqueous sodium thiosulfate solution,
Further, it was washed with a phosphate buffer (pH 6.0). The cells were plated on a plate medium containing 0.7 mg / ml of PFP (Bacto yeast nitrogen base 0.67, manufactured by Difco).
%, Glucose 2%, agar 1.5%) at 30 ° C. for 7 days. Mutants having PFP resistance were selected from the colonies that appeared on the agar plate.

Using the obtained upstream primers, downstream primers and all chromosomal DNAs of the mutants having PFP resistance, the desired 2.4 kb DNA, that is, the pfp1 gene, was amplified based on the PCR method. This amplified DNA
Was subjected to agarose electrophoresis to separate a 2.4 kb band, and DNA was extracted. Next, the obtained DNA was subjected to Bam
After treatment with HI and SalI, the DNA was recovered by treatment with phenol and ethanol precipitation, ligated to pUC19 plasmid treated with BamHI and SalI, and transformed into E. coli ( E.
coli HB101) as a host (molecular cloning).

The obtained plasmid was subjected to electrophoresis, and p
From the UC19 plasmid, a plasmid which was longer by a base chain (about 2.4 kb) corresponding to the pfp1 gene was recovered, and the plasmid was named pKF200. When the restriction enzyme cleavage site was examined for the inserted clone of pKF200, it was confirmed that the restriction enzyme cleavage site was present at the same site as the known TYR1 (FIG. 2).

(2) Introduction of pfp1 gene into sake yeast and production of β-phenethyl alcohol: obtained pKF2
Vector Y treated with BamHI and SalI
After reconnecting to Cp50, a plasmid which was longer by a base chain (about 2.4 kb) corresponding to the pfp1 gene as described above was recovered from the electrophoresis gel, and the plasmid was designated as pKF201. Next, 0.5 μg of pKF201 was prepared by the lithium method using the Saccharomyces cerevisiae ATCC 44337 strain of Saccharomyces cerevisiae FD-8 (MATauratyty).
r1 his3 lys2) (molecular cloning), spread on a uracil-free CMM agar medium, cultivate at room temperature, and obtain a growing transformant, FD-8 / p
Named KF201.

Reference Example 5 Properties of Transformant FD-8 / pKF201 Strain FD-8 strain, FD-8 / pKF201 strain and FD-8 / YCp50 strain incorporating only plasmid YCp50 were dissolved in water after dissolving fluorophenylalanine in water. After inoculating an agar plate medium prepared by mixing with a uracil-free CMM agar medium so as to be 1 mg / ml,
Table 11 shows the results of observing the growth after 4 days at 28 ° C.

Further, the FD-8 strain, FD-8 / pKF2
01 strain and FD-8 / YCp50 strain in YPD
Table 11 also shows the results of measurement of β-phenethyl alcohol produced in the culture after shaking culture at 48 ° C. for 48 hours.

[0061]

[Table 11]

Plasmid stability: No dropout of the plasmid was observed in the transformant even after subculture in a nutrient medium.

[0063]

EFFECT OF THE INVENTION By using the yeast obtained by the present invention, foods and drinks having a high fragrance and different aroma characteristics from conventional ones can be produced.

[0064]

[Sequence list]

SEQ ID NO: 1 Sequence length: 29 Sequence type: Nucleic acid number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Characteristic symbol: Primer region Upstream location: -556. . -536 Method used to determine features: E

SEQ ID NO: 2 Sequence length: 29 Sequence type: number of nucleic acid strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: Terminator region Downstream location: 1331. . 1350 Method for Determining Features: E

SEQ ID NO: 3 Sequence length: 26 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: Primer region Upstream location: -805. . -789 Method used to determine features: E

SEQ ID NO: 4 Sequence length: 26 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: Terminator region Downstream location: 1542. . 1558 Method for determining features: E

SEQ ID NO: 5 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: Primer region Upstream Location: 210..230 Method used to determine characteristics: E sequence CCACCCGCTG CACCCTGTGC 20

SEQ ID NO: 6 Sequence length: 21 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Characteristic symbol: Primer region Existence Position: 430..451 Method for determining characteristics: E sequence CGTGTTAATT CCTCTTTCGT C21

SEQ ID NO: 7 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA Sequence characteristics Characteristic symbols: Translation region Position: 650..671 Characterized method: E sequence CATTAGCTTC TCCAGCTCTC C 21

SEQ ID NO: 8 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: translation region Position: 896..917 Method for determining characteristics: E sequence AAGCCAAGAA CAACCGTCGG C21

SEQ ID NO: 9 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Characteristic symbol: Translation region Position: 1133..1153 Characteristic determination method: E sequence GCCTCCGGTT TGTCTTTCCC 20

SEQ ID NO: 10 Sequence length: 20 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Characteristic symbol: Translation region Existence Position: 1369..1389 Method for determining characteristics: E sequence GCCTGCCGGT TCCAACGGTC 20

SEQ ID NO: 11 Sequence length: 21 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: Translation region Existence Position: 1623..1642 Method used to determine characteristics: E sequence TGAGGAAATT GGCTGCTGCT G 21

SEQ ID NO: 12 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: Translation region Position: 367..346 Method Determined: E Antisense Yes Sequence TATGAAATTA AACATTGATT GG 22

SEQ ID NO: 13 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: Translation region Existence Position: 663..641 Characterization method: E Antisense Yes sequence GGAGAAGCTA ATGGGTCGTA ACC 23

SEQ ID NO: 14 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Sequence features Characteristic symbols: Translation region Existence Position: 919..898 Method used to determine characteristics: E Antisense Yes sequence CCAGCCGACG GTTGTTCTTG GC 22

SEQ ID NO: 15 Sequence length: 23 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Characteristic symbol: Translation region Existence Position: 1154..1132 Characterization method: E Antisense Yes sequence CTGGGAAAGA CAAACCGGAG GCC 23

SEQ ID NO: 16 Sequence length: 22 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Characteristic symbol: Translation region Position: 1429..1408 Characterization method: E Antisense Yes sequence GAAATCCTTA TTGGAGTTAC CG 22

SEQ ID NO: 17 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA Sequence characteristics Characteristic symbol: translation region Position: 1721..1700 Characteristic determination method: E Antisense Yes sequence GTGGTAGAGG AAAGAATGTA CG 22

SEQ ID NO: 18 Sequence length: 1927 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Genomic DNA Origin Organism: Saccharomyces cerevisiae
S. cerevisiae) Strain name: X2180A Sequence characteristics Characteristic symbol: TATA signal Location: 369..373 Method for determining characteristics: S Symbol for characteristic: TATA signal Location: 413..416 Method for determining characteristics : Symbol representing the S feature: TATA signal Location: 421..427 Method determining the feature: S Symbol representing the feature: peptide Location: 563..1672 Method determining the feature: S Symbol representing the feature: Terminator position: 1673..1675 method to determine the characteristics: S sequence GTCGACTGAA TCACGTGATC AACAGCAAAT TATGTACTCG TATATATGCA AGCGCATTCC 60 TTATATTGAC ACTCTTTCAT TGGGCATGAG GCTGTGTAAA CATAAGCTGT AACGGTCTCA 120 CGGAACACTG TGTAGTTGCA TTACTGTCAG GCAGTTATGT TGCTTAATAT AAAGGCAAAG 180 GCATGGCAGA ATCACTTTAA AACGTGGCCC CACCCGCTGC ACCCTGTGCA TTTTGTACGT 240 TACTGCGAAA TGACTCAACG ATGAAATGAA AAAATTTTGC TTGAAATTTT GAAAAAAAGA 300 TGTGCGGGAC GCATTGTTAG CTCATTGAAT ACATCGTGAT CGAATCCAAT CAATGTTTAA 360 TTTCATATTA ATACAGAAAC TTTTTCTCAT ACTTTCTTCT TCTTTTCATT GGT ATATTAT 420 CTATATATCG TGTTAATTCC TCTTTCGTCA TTTTTAGCAT CGTTATAAGA GTAATTAAGA 480 ATAACTAGAA GAGTCTCTCT TTATATTCGT TTATTTTATA TATTTAACCG CTAAATTTAG 540 TAAACAAAAG AATCTATCAG AA 562 ATG AGT GAA TCT CCA GAG ATC GTC ATC GTC AGC GTC AGC GTC ATC Lys Val Asn 1 5 10 15 CAA GGT GCT GAA GAA GAT GTC AGA ATT TTA GGT TAC GAC CCA TTA GCT 658 Gln Gly Ala Glu Glu Asp Val Arg Ile Leu Gly Tyr Asp Pro Leu Ala 20 25 30 TCT CCA GCT CTC CTT CAA GTG CAA ATC CCA GCC ACA CCA ACT TCT TTG 706 Ser Pro Ala Leu Leu Gln Val Gln Ile Pro Ala Thr Pro Thr Ser Leu 35 40 45 GAA ACT GCC AAG AGA GGT AGA AGA GAA GCT ATA GAT ATT ATT ACC GGT 754 Glu Thr Ala Lys Arg Gly Arg Arg Glu Ala Ile Asp Ile Ile Thr Gly 50 55 60 AAA GAC GAC AGA GTT CTT GTC ATT GTC GGT CCT TGT TCC ATC CAT GAT 802 Lys Asp Asp Arg Val Leu Val Ile Val Gly Pro Cys Ser Ile His Asp 65 70 75 80 CTA GAA GCC GCT CAA GAA TAC GCT TTG AGA TTA AAG AAA TTG TCA GAT 850 Leu Glu Ala Ala Gln Glu Tyr Ala Leu Arg Le u Lys Lys Leu Ser Asp 85 90 95 GAA TTA AAA GGT GAT TTA TCC ATC ATT ATG AGA GCA TAC TTG GAG AAG 898 Glu Leu Lys Gly Asp Leu Ser Ile Ile Met Arg Ala Tyr Leu Glu Lys 100 105 110 CCA AGA ACA ACC GTC GGC TGG AAA GGT CTA ATT AAT GAC CCT GAT GTT 946 Pro Arg Thr Thr Val Gly Trp Lys Gly Leu Ile Asn Asp Pro Asp Val 115 120 125 AAC AAC ACT TTC AAC ATC AAC AAG GGT TTG CAA TCC GCT AGA CAA TTG 994 Asn Asn Thr Phe Asn Ile Asn Lys Gly Leu Gln Ser Ala Arg Gln Leu 130 135 140 TTT GTC AAC TTG ACA AAT ATC GGT TTG CCA ATT GGT TCT GAA ATG CTT 1042 Phe Val Asn Leu Thr Asn Ile Gly Leu Pro Ile Gly Ser Glu Met Leu 145 150 155 160 GAT ACC ATT TCT CCT AAA TAC TTG GCT GAT TTG GTC TCC TTC GGT GCC 1090 Asp Thr Ile Ser Pro Lys Tyr Leu Ala Asp Leu Val Ser Phe Gly Ala 165 170 175 ATT GGT GCC AGA ACC ACC GAA TCT CAA CTG CAC AGA GAA TTG GCC TCC 1138 Ile Gly Ala Arg Thr Thr Glu Ser Gln Leu His Arg Glu Leu Ala Ser 180 185 190 GGT TTG TCT TTC CCA GTT GGT TTC AAG AAC GGT ACC GAT GGT ACC TTA 1186 Gly Leu Ser Phe Pro Val Gly P he Lys Asn Gly Thr Asp Gly Thr Leu 195 200 205 AAT GTT GCT GTG GAT GCT TGT CAA GCC GCT GCT CAT TCT CAC CAT TTC 1234 Asn Val Ala Val Asp Ala Cys Gln Ala Ala Ala His Ser His His Phe 210 215 220 ATG GGT GTT ACT AAG CAT GGT GTT GCT GCT ATC ACC ACT ACT AAG GGT 1282 Met Gly Val Thr Lys His Gly Val Ala Ala Ile Thr Thr Thr Lys Gly 225 230 235 240 AAC GAA CAC TGC TTC GTT ATT CTA AGA GGT GGT AAA AAG GGT ACC AAC 1330 Asn Glu His Cys Phe Val Ile Leu Arg Gly Gly Lys Lys Gly Thr Asn 245 250 255 TAC GAC GCT AAG TCC GTT GCA GAA GCT AAG GCT CAA TTG CCT GCC GGT 1378 Tyr Asp Ala Lys Ser Val Ala Glu Ala Lys Ala Gln Leu Pro Ala Gly 260 265 270 TCC AAC GGT CTA ATG ATT GAC TAC TCT CAC GGT AAC TCC AAT AAG GAT 1426 Ser Asn Gly Leu Met Ile Asp Tyr Ser His Gly Asn Ser Asn Lys Asp 275 280 285 TTC AGA AAC CAA CCA AAG GTC AAT GAC GTT GTT TGT GAG CAA ATC GCT 1474 Phe Arg Asn Gln Pro Lys Val Asn Asp Val Val Cys Glu Gln Ile Ala 290 295 300 305 AAC GGT GAA AAC GCC ATT ACC GGT GTC ATG ATT GAA TCA AAC ATC AAC 1522 Asn Gl y Glu Asn Ala Ile Thr Gly Val Met Ile Glu Ser Asn Ile Asn 310 315 320 GAA GGT AAC CAA GGC ATC CCA GCC GAA GGT AAA GCC GGC TTG AAA TAT 1570 Glu Gly Asn Gln Gly Ile Pro Ala Glu Gly Lys Ala Gly Leu Lys Tyr 325 330 335 GGT GTT TCC ATC ACT GAT GCT TGT ATA GGT TGG GAA ACT ACT GAA GAC 1618 Gly Val Ser Ile Thr Asp Ala Cys Ile Gly Trp Glu Thr Thr Glu Asp 340 345 350 GTC TTG AGG AAA TTG GCT GCT GCT GTC AGA CAA AGA AGA GAA GTT AAC 1666 Val Leu Arg Lys Leu Ala Ala Ala Val Arg Gln Arg Arg Glu Val Asn 355 360 365 AAG AAA 1672 Lys Lys 370 TAGATGTT 1680 TTTTTAATGA TATATGTAAC GTACATTCTT TCCTCTCATTCATTTCATTACGTTGTATTACGATGTATTGATT TGGATGTTAT ACTATCAGTT ACTCTTCTGC AAAAAAAAAT 1860 TGAGTCATAT CGTAGCTTTG GGATTATTTT TCTCTCTCTC CACGGCTAAT TAGGTGATCA 1920 TGGATCC 1927

SEQ ID NO: 19 Sequence length: 1113 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Genomic DNA Origin Organism: Saccharomyces cerevisiae
S. cerevisiae) Strain name: X2180A sequence ATG AGT GAA TCT CCA ATG TTC GCT GCC AAC GGC ATG CCA AAG GTA AAT 48 Met Ser Glu Ser Pro Met Phe Ala Ala Asn Gly Met Pro Lys Val Asn 1 5 10 15 CAA GGT GCT GAA GAA GAT GTC AGA ATT TTA GGT TAC GAC CCA TTA GCT 96 Gln Gly Ala Glu Glu Asp Val Arg Ile Leu Gly Tyr Asp Pro Leu Ala 20 25 30 TCT CCA GCT CTC CTT CAA GTG CAA ATC CCA GCC ACA CCA ACT TCT TTG 144 Ser Pro Ala Leu Leu Gln Val Gln Ile Pro Ala Thr Pro Thr Ser Leu 35 40 45 GAA ACT GCC AAG AGA GGT AGA AGA GAA GCT ATA GAT ATT ATT ACC GGT 192 Glu Thr Ala Lys Arg Gly Arg Arg Glu Ala Ile Asp Ile Ile Thr Gly 50 55 60 AAA GAC GAC AGA GTT CTT GTC ATT GTC GGT CCT TGT TCC ATC CAT GAT 240 Lys Asp Asp Arg Val Leu Val Ile Val Gly Pro Cys Ser Ile His Asp 65 70 75 80 CTA GAA GCC GCT CAA GAA TAC GCT TTG AGA TTA AAG AAA TTG TCA GAT 288 Leu Glu Ala Ala Gln Glu Tyr Ala Leu Arg Leu Lys Lys Leu Ser Asp 85 90 95 GAA TTA AAA GGT GAT TTA TCC ATC ATT ATG AGA GCA TAC TTG GAG AAG 336 Glu Leu Lys Gly Asp Leu Ser Ile Ile Met Arg A la Tyr Leu Glu Lys 100 105 110 CCA AGA ACA ACC GTC GGC TGG AAA GGT CTA ATT AAT GAC CCT GAT GTT 384 Pro Arg Thr Thr Val Gly Trp Lys Gly Leu Ile Asn Asp Pro Asp Val 115 120 125 AAC AAC ACT TTC AAC ATC AAC AAG GGT TTG CAA TCC GCT AGA CAA TTG 432 Asn Asn Thr Phe Asn Ile Asn Lys Gly Leu Gln Ser Ala Arg Gln Leu 130 135 140 TTT GTC AAC TTG ACA AAT ATC GGT TTG CCA ATT GGT TCT GAA ATG CTT 480 Phe Val Asn Leu Thr Asn Ile Gly Leu Pro Ile Gly Ser Glu Met Leu 145 150 155 160 GAT ACC ATT TCT CCT AAA TAC TTG GCT GAT TTG GTC TCC TTC GGT GCC 528 Asp Thr Ile Ser Pro Lys Tyr Leu Ala Asp Leu Val Ser Phe Gly Ala 165 170 175 ATT GGT GCC AGA ACC ACC GAA TCT CAA CTG CAC AGA GAA TTG GCC TCC 576 Ile Gly Ala Arg Thr Thr Glu Ser Gln Leu His Arg Glu Leu Ala Ser 180 185 190 GGT TTG TCT TTC CCA GTT GGT TTC AAG AAC GGT ACC GAT GGT ACC TTA 624 Gly Leu Ser Phe Pro Val Gly Phe Lys Asn Gly Thr Asp Gly Thr Leu 195 200 205 AAT GTT GCT GTG GAT GCT TGT CAA GCC GCT GCT CAT TCT CAC CAT TTC 672 Asn Val Ala Val Asp Ala Cys Gln A la Ala Ala His Ser His His Phe 210 215 220 ATG GGT GTT ACT AAG CAT GGT GTT GCT GCT ATC ACC ACT ACT AAG GGT 720 Met Gly Val Thr Lys His Gly Val Ala Ala Ile Thr Thr Thr Thrs Lys Gly 225 230 235 240 AAC GAA CAC TGC TTC GTT ATT CTA AGA GGT GGT AAA AAG GGT ACC AAC 768 Asn Glu His Cys Phe Val Ile Leu Arg Gly Gly Lys Lys Gly Thr Asn 245 250 255 TAC GAC GCT AAG TCC GTT GCA GAA GCT AAG GCT CAA TTG CCT GCC GGT 816 Tyr Asp Ala Lys Ser Val Ala Glu Ala Lys Ala Gln Leu Pro Ala Gly 260 265 270 TCC AAC GGT CTA ATG ATT GAC TAC TCT CAC GGT AAC TCC AAT AAG GAT 864 Ser Asn Gly Leu Met Ile Asp Tyr Ser His Gly Asn Ser Asn Lys Asp 275 280 285 TTC AGA AAC CAA CCA AAG GTC AAT GAC GTT GTT TGT GAG CAA ATC GCT 912 Phe Arg Asn Gln Pro Lys Val Asn Asp Val Val Cys Glu Gln Ile Ala 290 295 300 305 AAC GGT GAA AAC GCC ATT ACC GGT GTC ATG ATT GAA TCA AAC ATC AAC 960 Asn Gly Glu Asn Ala Ile Thr Gly Val Met Ile Glu Ser Asn Ile Asn 310 315 320 GAA GGT AAC CAA GGC ATC CCA GCC GAA GGT AAA GCC GGC TTG AAA TAT 1008 Glu Gly Asn Gln Gly Ile Pro Ala Glu Gly Lys Ala Gly Leu Lys Tyr 325 330 335 GGT GTT TCC ATC ACT GAT GCT TGT ATA GGT TGG GAA ACT ACT GAA GAC 1056 Gly Val Ser Ile Thr Asp Ala Cys Ile Gly Trp Glu Thr Thr Glu Asp 340 345 350 GTC TTG AGG AAA TTG GCT GCT GCT GTC AGA CAA AGA AGA GAA GTT AAC 1104 Val Leu Arg Lys Leu Ala Ala Ala Val Arg Gln Arg Arg Glu Val Asn 355 360 365 AAG AAA TAG 1113 Lys Lys 370

[Brief description of the drawings]

FIG. 1 is a restriction map of a known ARO4 gene and a restriction enzyme map of an OFP1 resistance gene introduced into a plasmid pKF100. In the figure, S is SalI, Sp is Sph
I and X represent XbaI, K represents KpnI, and B represents BamHI.

FIG. 2 shows a restriction map of a known TRY1 gene and a restriction map of a pfp1 resistance gene introduced into a plasmid pKF200. In the figure, S is SalI, St is Stu
I and H are HindIII, E is EcoRI, B is BamH.
Represents I.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12N 15/09 ZNA C12N 1/19 // C12N 1/19 15/00 ZNAA (C12N 1/19 C12R 1: 865) (58) Fields investigated (Int. Cl. ⁷ , DB name) A23L 1/00 A23L 1/23 C12G 3/02 C12N 15/31 BIOSIS (DIALOG) WPI (DIALOG) CA (STN) REGISTRY (STN) GenBank / EMBL / DDBJ / GeneSeq SwissProt / PIR / GeneSeq JICST file (JOIS)

Claims (3)

(57) [Claims] 1. A method for producing a food or drink using a microorganism, wherein the method belongs to the genus Saccharomyces and comprises SEQ ID NO: 18 or
Is a method for producing a food or drink, comprising using a yeast into which the gene represented by SEQ ID NO: 19 has been introduced. 2. A process of claim 1 food or drink is an alcoholic beverage or a fermented seasoning. 3. The gene represented by SEQ ID NO: 18 or SEQ ID NO: 19.