JP2002191355A - Breeding of strain highly producing isoamyl acetate, and application thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for breeding a strain by which a high-quality alcohol drink containing a large amount of isoamyl acetate, and having excellent fragrance can be produced. SOLUTION: A mutated yeast formed by mutation, having high alcohol acetyl transferase activities, and high ratio of isoamyl acetate/isoamyl alcohol, capable of producing a large amount of the isoamyl acetate is bred and isolated. The novel mutated yeast can be obtained from the strain grown in a selective medium containing a steroid having a pregnane skeleton having a hydroxylated 3-position such as pregnenolone, 17-hydroxypregnenolone and pregnanediol, or a sterol such as cholesterol.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the breeding of yeast, and more particularly, the present invention relates to a method for producing a highly fragrant liquor using yeast which is resistant to pregnenolone, pregnenolone analogs or cholesterol. The present invention also relates to a method for easily selecting a yeast which produces a large amount of isoamyl acetate, and further relates to production of alcoholic beverages using the selected yeast.

[0002]

2. Description of the Related Art Among fragrance components of liquor, liquor containing a large amount of ester is liquor of a gorgeous scent type. Among the esters, isoamyl acetate has a fruity scent and is an important scent component known as one of the ginjo scent components.

[0003] Isoamyl acetate is produced from isoamyl alcohol and acetyl-CoA by the catalytic action of alcohol acetyltransferase (hereinafter referred to as AATFase), and the content ratio of isoamyl acetate / isoamyl alcohol is represented by an E / A ratio. Increasing the E / A ratio, that is, increasing the AATFase activity results in high isoamyl acetate productivity.

[0004] AATFase is composed of ATF1 gene, AT
A transformant encoded by the F2 gene and highly expressed using a genetic engineering technique is a yeast having a high E / A ratio and a high productivity of isoamyl acetate. So far, as a method for breeding yeast to increase the amount of isoamyl acetate produced, a method for obtaining a high-producing strain of isoamyl alcohol (Japanese Patent No. 2128265), a low-decomposing strain of isoamyl acetate (Japanese Patent Application Laid-Open No. 10-276767), and the like have been known. There is. However, there are few yeasts that highly produce the components that make up Ginjo incense while keeping them functionally good with the other components, and high yields of isoamyl acetate with a high E / A ratio, and yeast that does not rely on genetic engineering techniques Breeding was waiting.

[0005]

SUMMARY OF THE INVENTION The present invention provides an AAT
It is an object of the present invention to establish a method for efficiently obtaining a mutant strain having a high se activity and a high productivity of isoamyl acetate, and to obtain an alcoholic beverage enriched in isoamyl acetate.

[0006]

Means for Solving the Problems The present invention has been made to achieve the above-mentioned object, and it is intended to breed and isolate a mutant yeast of interest without using genetic engineering techniques, Not only did we focus on the selection method. As a result of various studies, the present inventors focused on pregnenolone, a kind of steroid.

[0007] First, when the growth inhibition of yeast by pregnenolone was examined, it was found that the growth of yeast was inhibited in the minimal medium containing pregnenolone. Therefore, the minimal medium containing pregnenolone was used as the screening medium used in the present invention. And

When the yeast was mutated and then cultured in a medium containing pregnenolone, unexpectedly, not only colonies were formed but also a large amount of AATFase was generated in the resulting colonies, and isoamyl acetate was produced. A new and useful finding that there is a large amount of yeast produced was obtained. As a result of further studies, it was newly found that, in addition to pregnenolone, analogs and sterols can also be used as a drug to be used in the selective medium.

That is, the present inventors have proposed AATase
In order to efficiently select high activity mutants, pregnenolone, screening in a medium containing pregnenolone analogs or cholesterol, the mutants growing in this medium are AATFase activity high isoamyl acetate high productivity mutants, It has been found that a strain having a high E / A ratio can be efficiently obtained by this method.

[0010] Furthermore, using the yeast bred and separated in this manner, alcoholic beverages such as sake and beer were produced in accordance with a conventional method, and it was also confirmed for the first time that a high-quality product excellent in aroma was obtained. Further research based on these new findings has led to the completion of the present invention. Hereinafter, the present invention will be described in detail.

[0011] In practicing the present invention, strains grown and grown on a selective medium containing a drug such as pregnenolone after mutagenizing the yeast or without the mutagenesis treatment (that is, a strain such as pregnenolone or the like). A strain having high AATFase activity and high productivity of isoamyl acetate may be screened from the resistant strains).

As the drug to be contained in the selection medium, various steroids having a pregnane skeleton in which the 3-position is hydroxylated, including pregnenolone analogs such as 17-hydroxypregnenolone and pregnanediol, in addition to pregnenolone, can be used. Sterols can also be used. As the sterol, various sterols can be used in addition to cholesterol. These agents may be used alone or in combination of two or more when added to the medium.

In the present invention, any method may be used to obtain a mutant yeast. Examples of physical methods of mutation include ultraviolet irradiation and irradiation, and examples of chemical methods include mutagens such as ethyl methanesulfonate (hereinafter referred to as EMS), N-methyl-N-nitroguanidine, and nitrite. There is a mutation method in which the suspension is suspended in a solution such as an acridine dye. Although the frequency of acquisition is low, the mutant yeast of interest can be obtained even in spontaneous mutation.

In the present invention, these mutation methods can be used as appropriate. However, the method has a feature of selecting a mutant yeast, and the above-mentioned steroid and / or sterol-containing medium is used as a selection medium. It is necessary to select the drug-resistant yeast of (1). In addition, these selection processes can be repeated as needed, or the combination of the selection media can be freely changed. The drug is used by adding it to a medium for the purpose of selecting the drug-resistant yeast. Therefore, the amount of the drug used is not particularly limited as long as it can achieve the purpose, and is not particularly limited. As a guide,
It is preferable to add 0.01 to 50 mM, preferably 0.1 to 10 mM, and more preferably 0.3 to 1 mM.

In carrying out the method, after the yeast is subjected to mutation treatment or without mutation treatment, the yeast is transferred to the drug-containing selective medium, cultured in an allowable growth range (for example, at about 30 ° C. for about one week), and grown. (A resistant strain), and a mutant yeast having a high AATFase activity and / or a yeast which produces isoamyl acetate well may be collected from the isolated strain.

The mutant yeast obtained here (the drug-resistant mutant strain) produces isoamyl acetate well in any of sake yeast, shochu yeast, beer yeast, wine yeast, and baker's yeast. Sake using
By producing shochu, beer, wine, other alcoholic beverages, etc., it is possible to produce each product containing a large amount of isoamyl acetate, and it is also possible to produce AATFase at a high level.

In the present invention, a target product having a high fragrance component can be produced by using the drug-resistant mutant thus created, but the drug resistance is further imparted with leucine analog resistance. The double resistant mutant thus obtained has more excellent characteristics, and has a remarkable effect of producing isoamyl acetate at a higher level.

Next, breeding of a yeast producing high isoamyl acetate using a medium containing a leucine analog will be described. The inventors of the present invention have found that the rate of isoamyl acetate production is determined by isoamyl alcohol, and have further obtained the following findings. In other words, isoamyl alcohol is produced in the middle of the leucine biosynthesis pathway by yeast, but when leucine accumulates, this leucine inhibits the leucine biosynthesis pathway, so that the biosynthesis of isoamyl alcohol is also suppressed. . Thus, the present inventors have sought a mutant strain that does not inhibit the leucine biosynthetic pathway even when leucine accumulates,
As a result of intensive studies, by using a leucine analog-containing medium, we succeeded in selecting a mutant yeast that produces a large amount of aroma components such as isoamyl acetate and isoamyl alcohol, and further, by using this mutant yeast, Succeeded in producing alcoholic beverages with rich aroma.

In carrying out this method, a method similar to the above-described method for breeding drug-resistant yeast may be used.
The selection medium must contain 5,5,5-trifluoro-DL-leucine. 5,5,5-trifluoro-DL-leucine is contained in a solid medium, for example, YNB agar medium in an amount of 0.01 to 10 mM, preferably 0.5 to 5 mM,
More preferably, about 1 mM is sufficient.
In addition, the drug to be added to the selective medium of the mutant yeast is 5,
Without being limited to 5,5-trifluoro-DL-leucine, any leucine analog can be used as long as it can increase isoamyl alcohol and isoamyl acetate.

In order to breed a target yeast using this method, various yeasts are subjected to 5,5,5-trifluoro-DL after mutation treatment with EMS or the like or without mutation treatment.
A yeast which is added to a leucine-containing solid medium, cultured at 30 ° C. for about 2 days, isolates the resulting colonies, and employs a yeast which produces isoamyl alcohol and isoamyl acetate well therefrom. Since the yeast obtained here produces isoamyl acetate well, any of the above yeasts can produce aromatic alcohols.

Yeast having resistance to only one of the above-mentioned drugs and leucine analog can be a strain producing high isoamyl acetate, but if a yeast having resistance to both drugs is selected, a strain producing high isoamyl acetate is further obtained. .

For these two types of mutations, for example, the above-mentioned drug-resistant yeast may be further subjected to a mutation treatment, for example, to further impart 5,5,5-trifluoro-DL-leucine resistance, or vice versa. Further, for example, yeasts to which 5,5,5-trifluoro-DL-leucine resistance has been imparted may be further subjected to mutation treatment to impart, for example, the drug resistance described above. Of course, the mutant yeast can be cultured in a medium containing both 5,5,5-trifluoro-DL-leucine and the above-mentioned drug to select a yeast having resistance to both drugs.

As another method, after obtaining a 5,5,5-trifluoro-DL-leucine-resistant mutant and the above-mentioned drug-resistant mutant, tetrad separation is carried out for each of the drug-resistant mutants. There is also a method in which spores having resistance to each drug are obtained, and they are conjugated to obtain a diploid having both resistances. Of course, when obtaining each drug-resistant mutant, a method is used in which mutants having resistance to each drug are obtained using haploids of different mating types, and then mated to form a diploid. Is also possible.

In addition, the method is not limited as long as a mutant strain having both drug resistance such as pregnenolone described above and drug resistance such as 5,5,5-trifluoro-DL-leucine can be finally obtained. In consideration of brewing characteristics and the like, it is considered that the obtained mutant is preferably a diploid, but may be a haploid.

As the treated yeast, Saccharomyces (Sac
All yeasts can be used as long as they belong to the genus charomyces. For example, sake yeasts (Kyoto No. 7 yeast, Kyokai No. 9 yeast, Kyokai No. 10 yeast, etc.), wine yeasts (Grape wine No. 1 yeast (Japan The brewing association belongs to the genus Saccharomyces, including practical yeasts such as sake No. 1 yeast, wine No. 3 yeast, wine No. 4 yeast), beer yeast, baker's yeast, and other yeasts commonly used for alcohol fermentation. Any yeast can be used as long as it is yeast, and any yeast can obtain the desired resistance.

In this way, it was possible to obtain the desired yeast which produces isoamyl acetate at a high level. One of the yeasts was named PGR-13, and was named FERM by the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology. Deposited as P-17983.

By using the mutant having a high odor component produced and isolated according to the present invention, it is possible to produce a high-quality alcoholic beverage having a large amount of isoamyl acetate and other odor components. In the present invention, alcoholic beverages include all beverages containing alcohol, such as sake, shochu, brandy, beer, low-malt beer, wine, and the like.

The novel mutant yeast according to the present invention has a high aroma component-producing ability, and can enhance isoamyl acetate, particularly among the aroma components, and can produce isoamyl acetate of 5 ppm or more, preferably 10 ppm or more. Mutants that produced 17 ppm or more were also obtained. Isoamyl acetate-producing mutants were obtained even in the case of selection treatment without prior mutation treatment, and the parent strain showed a production amount of 0.5 ppm, whereas the mutant strain showed a production amount of 3 ppm or more, and 5 ppm As described above, it has been confirmed that it is possible to obtain a mutant showing a remarkable production amount of preferably 6 to 9 ppm or more, more preferably 11 ppm or more.

Further, in the case of sake moromi, a product containing 30 ppm or more, preferably 40 ppm or more, more preferably 50 ppm or more of isoamyl acetate can be obtained. In the case of beer, 3 ppm or more, preferably 5 pp
m or more, more preferably 10 ppm or more.

Further, the mutant yeast according to the present invention comprises AATF
high productivity, 50 pp / mg protein / hour
m, preferably 100 ppm or more, more preferably 120 ppm or more. By using the present mutant yeast, AATFase can be efficiently produced in high yield. In this case, if the mutant yeast is cultured in the presence of the unsaturated fatty acid, the production amount of the present enzyme further increases. As unsaturated fatty acids, oleic acid, linoleic acid, linolenic acid, arachidonic acid and other unsaturated fatty acids are used alone or in combination of two or more, about 10 to 50%,
Enzyme activity can be increased. 0.1 as used amount
Although it is about 10 to 10 mM, a significant activity increasing effect can be obtained even with the use of 1 mM oleic acid.

As described above, when a fermentation test was performed using the yeast obtained by screening according to the present invention, the culture supernatant contained a large amount of isoamyl acetate, had a remarkably high E / A ratio, and had a high AATFase activity. Was. In addition, when a sake brewing test was performed using the obtained mutant strain,
The sake contained a larger amount of isoamyl acetate than the parent strain and had a higher E / A ratio, and confirmed that the sake was sensually superior to the sake obtained using the parent strain, and completed the present invention. Was.

The representative pregnenolone-resistant strain PGR-1 obtained from the haploid yeast strain F-7 by the method of the present invention.
No. 3 was deposited with the Research Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry and obtained a deposit number (FERM P-17983).

Hereinafter, the present invention will be described with reference to examples.
However, the present invention is not limited by these examples.

[0034]

Example 1 Method for Obtaining PGR Mutant Sake Yeast Association No. 7 yeast (hereinafter referred to as K-7), 5,
The 5,5-trifluoro-DL-leucine (TFL) resistant strain F-7 (haploid) and the TFL resistant strain F-19 (diploid) were transformed into a YPD medium (2% Glucose, 2% Po).
Lypepton, 1% Yeast Extract
At t), the cells were cultured at 30 ° C., and the cells were collected by centrifugation. The cells were placed in a 0.2 M phosphate buffer (pH 8.
Suspend the cells in 0) and wash the cells once. Wash the cells,
9.2 ml of phosphate buffer, 0.5 m of 2% glucose
l, suspended in 0.3 ml of EMS, gently shaken at 30 ° C., washed the yeast suspension once with 5% sodium thiosulfate, and washed twice with sterile water. did. The mutated yeast cells were placed in a minimal medium (Difco 0.67% Yeast nitrogen) containing 0.1 to 10 mM pregnenolone dissolved in an appropriate amount of ethanol.
n base, 2% Glycerol, 2% Aga
r) and cultured at 30 ° C for 7 days. Pregnenolone resistant mutants were isolated, and the isolated mutants were designated as PGR strains.

20 from the F-7 strain, 20 from the K-7 strain
From the strain, F-19 strain, 25 strains and PGR mutant strains were obtained.
No significant difference was found in the frequency of obtaining mutants between the haploid and diploid strains. The PGR strain obtained from the F-7 strain showed pregnenolone resistance. (Table 1)

(Table 1) 最 Minimal medium containing strain Pregnenolone minimal medium─────── ─────────────────── PGR-13 ++ PGR-17 ++ PGR-25 ++ F-7 ＋ ─────────── ＋ +: growing,-: not growing

[0037]

Example 2 Selection of Pregnenolone Analogs and Mutants Which Are Resistant to Cholesterol Mutants which are resistant to 17-hydroxypregnenolone and pregnanediol, which are pregnenolone analogs, and mutants which are resistant to cholesterol were obtained. . The F-7 strain was mutated by the method of Example 1, and the minimum medium was 0.1 to 10 mM 17-hydroxypregnenolone, 0.1 to 10 mM pregnanediol, 0.1 to 10 mM.
A resistant strain was obtained using a selective medium containing 10 mM cholesterol alone. As a result, eight 17-hydroxypregnenolone-resistant strains (HPGR), ten pregnanediol-resistant strains (PNR), and eleven cholesterol-resistant strains (CLR) were obtained.

[0038]

Example 3 Fermentation Test The PGR strain obtained in Example 1 was transformed into a YPD10 medium (10%
Glucose, 2% Polypepton, 1%
Yeast Extract) 15 ml
After 5 days of stationary culture at ℃, the culture supernatant was analyzed by gas chromatography. The E / A ratio of the PGR strain and the amount of isoamyl acetate produced are shown. By selecting the PGR strain, a mutant strain having a high E / A ratio and a high productivity of isoamyl acetate was frequently obtained. (Table 2)

A fermentation test was also performed on the pregnenolone analog-resistant strain HPGR strain, PNR strain, and cholesterol-resistant strain CLR strain obtained in Example 2 by the same method. As a result, the mutant strain which becomes resistant to the pregnenolone analog is PG
Like the R strain, the E / A ratio was high, indicating high isoamyl acetate productivity. (Table 3)

(Table 2) {Strain Isoamyl acetate (ppm) E / A ratio} ──────────────────── PGR-13 17.0 2.9 PGR-17 7.3 2.0 PGR-25 5.6 2.0 F-7 2.5 0.7 ──────────────────────────

(Table 3) {Strain Isoamyl acetate (ppm) E / A ratio} ────────────────────────── HPGR-2 5.3 2.2 HPGR-7 6.8 2.4 PNR-5 5.4 2.2 PNR-8 6.2 2.3 CLR-3 5.9 2.5 CLR-5 6.1 2.3 F-7 2.5 0.7 ──────────────────

[0042]

Example 4 Measurement of AATFase Activity (1) Preparation of Crude Enzyme Solution After culturing the cells twice with distilled water, the cells were disrupted with a buffer for cell disruption (25 mM MImidazole-HCl (pH
7.5) After suspending in 1 mM DTT), the cells were disrupted with glass beads so that the disruption rate was 90% or more. Next, Triton X-100 which is a solubilizing agent in the yeast crushed liquid is used.
Was added to a final concentration of 0.5%, and centrifuged (300
The soluble fraction was separated at 0 rpm for 10 min) to obtain a crude enzyme solution. The amount of protein in the crude enzyme solution was Protein A
Quantification was performed using the ssay kit (manufactured by Bio-Rad).

(2) Measurement of AATFase activity 25 mM imidazole-HCl (pH 7.
5), 46 mM Isoamyl alcohol,
1 mM Acetyl-CoA, 0.1% Trito
nX-100, 1 mM DTT, 0.2 M NaC
After 1 ml of a solution containing 1,20% Glycerol and the present enzyme was sealed in a 10 ml vial, the reaction was carried out at 25 ° C. for 1 hour, and then opened and 0.6 g NaCl was added to stop the reaction. As an internal standard, isobutyl n-butyrate was added to a concentration of 10 ppm, and again capped with a Teflon (registered trademark) stopper. Isoamyl acetate was quantified.

Analytical conditions Column; glass column (2.1 mx 3 mm), packing material;
PEG6000 column temperature; 110 ° C, injection temperature; 130 ° C, carrier gas; nitrogen

As a result of measuring the enzymatic activity when the PGR strain was cultivated in a YPD10 medium for 4 days, the following results were obtained.
The AATase activity of the GR strain was found to be 2.5-5 times higher than the parent strain. (Table 4)

(Table 4) Strain AATFase activity (ppm / mg protein / hr) {PGR-13 114 PGR-17 62 PGR-25 52 F-7 21} ──────────────────────

[0047]

Example 5 Acquisition of PGR strain from F-19 strain A PGR strain was also acquired from a diploid F-19 strain according to Example 1. The aroma components of the culture supernatant when a fermentation test was performed with the YPD10 medium were analyzed by gas chromatography. As a result, a high isoamyl acetate producing strain having an E / A ratio of 4.7 to 7.4 times could be obtained. (Table 5) AA of cultured cells
As a result of measuring the TFase activity, it was found that the TFase activity was twice or more higher than that of the parent strain. (Table 6)

(Table 5) {Strain Isoamyl acetate (ppm) E / A ratio} {FPGR-5 10.0 4.3 FPGR-7 13.7 6.7 F-19 2.4 0.9} ───────────────────────

[0049]

[0050]

Example 6 Acquisition of PGR strain from K-7 A PGR strain was selected from K-7 according to Example 1. These mutants were subjected to a fermentation test in a YPD10 medium, and the generated aroma components were measured. As a result, similar to the TFL-resistant strain, E
A high isoamyl acetate producing strain having a high / A ratio was obtained.
(Table 7)

(Table 7) {Strain Isoamyl acetate (ppm) E / A ratio} Ｋ K7-PGR5 3.0 2.5 K7-PGR15 3.8 2.8 K7-PGR17 5.4 3.0 K-7 0.5 0.4 ─────────────────────────────

[0052]

Example 7 Sake preparation test A sake preparation test of a PGR mutant strain obtained from the F-19 strain was performed. 200 g of total rice, 130% of pumping water, koji rate 2
The test was carried out at 0% at a constant temperature of 15 ° C. (prepared in one step). Table 8 shows the results of the aroma components of the upper tank liquor, and Table 9 shows general analysis values. The production amount of isoamyl acetate is higher than that of the parent strain F-19, the E / A ratio is 1.5 times that of the parent strain, and it is possible to produce sake containing a large amount of aroma components by charging using the PGR strain. Was confirmed. (Table 8, Table 9)

(Table 8) ──────────────────────────────── Strain Isoamyl acetate Isoamyl alcohol E / A ratio ( ppm) (ppm) ────────────────────────────────FPGR-5 31.0 580 5.3 F-19 19.7 532 3.7────────────────────────────────

(Table 9) 菌 Strain Alcohol (%) Total acid ( ml) Amino acid (ml) ＰＧ FPGR-5 18.5 2.4 2 .6 F-19 18.4 2.4 2.5 mm

[0055]

Example 8 Beer preparation test A beer preparation test was performed using 600 g of wort.
The beer was prepared in the usual manner, and the aroma components of the obtained beer were measured.
The R strain produced more isoamyl acetate than the parent strain,
The / A ratio also increased, and it was a new type of beer that was also organoleptically different from the conventional beers. (Table 10)

(Table 10) 菌 Bacterial strain Isoamyl acetate Isoamyl alcohol E / A Ratio (ppm) (ppm) Ｆ FPGR-5 5.0 106 4. 7 F-19 3.5 119 2.9──────────────────────────────────

[0057]

According to the present invention, by obtaining a yeast which is resistant to pregnenolone or an analog thereof or sterol, it is possible to increase the E / A ratio of the culture solution and increase the amount of isoamyl acetate produced. It has become possible.
According to the present invention, it has become possible to produce an alcoholic beverage having a high E / A ratio and a high yield of isoamyl acetate by obtaining a yeast which is resistant to pregnenolone or an analog thereof or a sterol.
These effects could be further enhanced by further conferring leucine analog resistance.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 1/16 (C12N 1/16 C12R 1:85) C12R 1:85) (C12C 11/02) (C12C 11/02 C12R 1:85) C12R 1:85) (C12G 3/02 (C12G 3/02 C12R 1:85) C12R 1:85) (72) Inventor Takehiko Ojima Matsugasaki Idega Kaido, Sakyo-ku, Kyoto-shi 10-1 Maison Corza 102 (72) Inventor Tetsuyoshi Mizuzu 20-18 Otani, Iseda-cho, Uji City (72) Inventor Shoji Kawado 1-32 Kibumakumakoji, Uji City, Kyoto Prefecture (72) Inventor Yasuhisa Abe Uji City, Kyoto Prefecture 29 Uji Otani 41 F term (reference) 4B050 CC01 DD04 LL02 4B065 AA79X AC20 BA18 BA22 BB07 BB12 CA29 CA42

Claims (7)

[Claims] Claims: 1. Sake yeast, shochu yeast, beer yeast, wine yeast, at least one yeast belonging to baker's yeast,
After or without mutation treatment, use a medium containing a steroid having a hydroxylated pregnane skeleton at position 3 or a medium containing sterols to select a mutant yeast that produces a large amount of isoamyl acetate. Breeding a yeast with high productivity of isoamyl acetate. 2. The method according to claim 1, further comprising selecting a mutant yeast producing a large amount of isoamyl acetate using a leucine analog-containing medium. 3. The steroid having a pregnane skeleton hydroxylated at the 3-position is at least one selected from pregnenolone, 17-hydroxypregnenolone, and pregnanediol, or the sterol is cholesterol, or the leucine analog is 5,5. The method according to claim 1 or 2, wherein the method is 5,5-trifluoro-DL-leucine. 4. A high productivity isoamyl acetate yeast bred and isolated by the method according to any one of claims 1 to 3. 5. A yeast PGR-1 having high productivity for isoamyl acetate.
3 (FERM P-17983). 6. A method for producing alcohol acetyltransferase, comprising using the yeast according to claim 4 in the presence or absence of an unsaturated fatty acid. 7. An alcoholic beverage produced using the yeast according to claim 4 or 5.