JP3923102B2 - Production method of yeast haploid strain - Google Patents

Description

【００２１】
【実施例２】
β−アラニン培地で３５℃で生育できる清酒酵母協会７号変異株
【００２２】
実施例１と同様にＥＭＳ処理した清酒酵母協会７号をβ−アラニン培地に塗布し、３５℃で培養した。３日後に形成したコロニーについて胞子形成試験を行い、胞子形成良好な３株、７β−１、７β−２、７β−３を得た。
【００２３】
これらの変異株と親株の性質を酵母の性質の測定法に基づき測定したところ、下記表２に示したように、変異株に於て胞子形成率の改善、四胞子形成頻度の上昇、酵母の性質の変化が認められた。また、変異株の胞子は明らかに胞子生存率が上昇していた。
【００２４】
【表２】

【００２５】
【発明の効果】
本発明は、酵母に高い胞子形成能と胞子の生存率を付与する目的で開発された為に、従来１倍体の取得法のように多数の栄養細胞が混在する実験系を用いることなく、容易に１倍体株を単離できる。またその結果、これまで困難であった実用清酒酵母の遺伝解析が容易になり、醸造酵母としての有用形質の解析や、優良酵母の育種の原株として高い利用価値を持つ。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a yeast haploid strain, and more particularly, to a mutation treatment and a mutant strain selection method for increasing the spore formation rate and spore survival rate of yeast.
[0002]
[Prior art]
It is known that among practical yeasts (diploids), there are many strains with low sporulation rate and low survival rate of the formed spore spores. In order to create a haploid strain from a strain having a problem with such spore formation, a technique called a random spore separation method is generally used. In this method, the spore-formed yeast pupa is treated with a cell wall lytic enzyme, only the pupa wall is lysed, and the resulting spore suspension is heat-treated, whereby an undissolved diploid strain is obtained. Or after separating only spores, and then spreading on a medium such as YPD (2% glucose, 2% polypeptone, 1% yeast extract, 2% agar), and haploid strains from the grown colonies Is a technique for separating
[0003]
As a method for distinguishing haploid strains, there are rapid growth, TTC stainability, and stainability in a dye medium (Nagai et al. Methodology p71-p76 in yeast research). In addition to heat treatment, selective killing of diploid strains includes ether treatment, alcohol treatment (Kurose et al., JP-A-5-317035) and high-pressure treatment method (Nakatomi et al., JP-A-5-236948). As a method, there are a mineral oil method and a polyethylene glycol method.
[0004]
[Problems to be solved by the invention]
However, among practical yeasts, sake yeast, for example, has a low spore formation rate and a low spore survival rate as described above. Although Kurose et al. (Japanese Patent Laid-Open No. 5-317035) has improved the spore formation rate several times and the acquisition frequency of haploid strains has been significantly improved by several percent, the normal spore separation is possible. Compared with laboratory yeast, the frequency is extremely low and a great deal of time is required. Accordingly, an object of the present invention is to provide a method for producing a haploid strain of a practical yeast such as sake yeast more easily and as easily as a laboratory yeast.
[0005]
[Means for Solving the Problems]
The present invention has been made to achieve the above object, and the present inventors have conducted research on the properties of practical yeasts such as general sake yeast having a problem in spore formation. It was found that there is a relationship between the characteristic properties of spore and the spore formation rate and spore survival rate.
[0006]
Commonly used practical yeasts such as sake yeast are known to have one or more of the following properties. (A) resistance to methionine analog ethionine, (B) high intracellular content of methionine metabolite S-adenosylmethinion, (C) constitutive acid phosphatase activity not detected, (D ) Among sake yeasts, Association No. 7 or yeast derived from Association No. 7 cannot grow on a β-alanine agar medium at 35 ° C. As for the properties of these sake yeasts, no knowledge related to the sake brewing characteristics of yeast has been obtained, and they have been used exclusively as an indicator for the purity test of sake lees.
[0007]
Since the strains having a high spore formation rate and spore survival rate do not have the properties found in these sake yeasts, the present inventors have first made ethinion resistance by mutation treatment than a strain having a high spore formation rate and spore survival rate. A mutant strain was prepared. As a result, it was clarified that several of the ethionine resistant mutants have the other properties (B) to (D) at the same time. Next, when an ethionine-sensitive mutant strain was selected from sake yeast having the properties (A) to (D), the spore formation rate and spore survival rate of the mutant strains that lost these properties increased significantly. I found out. That is, it was demonstrated that yeast having good spore formation can be obtained by selecting yeast using these properties as indices, and the present invention has been completed. The range of ethionine sensitivity in the present invention means that it cannot grow on an SD medium containing at least 0.1 mM to 10 mM ethionine.
[0008]
The yeast in the present invention is not limited to any kind of practical yeast such as wine yeast, beer yeast, shochu yeast, etc., as long as it has any of the above properties (A) to (D). As yeast having properties, Sake Yeast Association No.7, Sake Yeast Association No.9, Sake Yeast Association No.10 or No Bubble Mutant Association of these strains No.701, No.901, No.1001 Sake Yeast Association No.11 strain is suitable.
[0009]
In the present invention, a conventional method is appropriately used for the mutation treatment, for example, mutation treatment such as X-ray irradiation, ultraviolet irradiation, N-methyl-N′-nitro-N-nitrosoguanidine, 2-aminopurine, EMS, etc. is appropriately used. Is done. Of course, this method can also be applied to natural mutant strains.
Below, the manufacturing method of the practical yeast haploid which concerns on this invention is demonstrated in detail.
[0010]
(1) Method of measuring yeast spore formation rate, tetraspore formation rate and spore viability YPD agar medium (2% glucose, 2% polypeptone, 1% yeast extract, 2% agar) was used for yeast preculture. After culturing at 30 ° C. for 18 to 24 hours, scrape an appropriate amount, apply to a spore formation medium (2% potassium acetate, 0.05% yeast extract, 0.02% glucose, 2% agar) and culture at 30 ° C. for 3 days. To do. The sporulation rate was expressed as a percentage of the number of offspring per yeast number counted using a hemocytometer. The tetraspore formation rate was expressed as a percentage of the number of offspring that formed four spores per the number of offspring counted. The obtained pupa was suspended in sterilized water and treated with a cell wall lytic enzyme such as zymolyase to lyse the pupa wall, followed by monospore separation on a YPD agar medium using a micromanipulator, and 30 ° C. And culturing for 3 days to obtain monospore-derived integrated yeast. Spore viability was expressed as a percentage of the number of haploid yeast grown per single spore isolate. The mating type (a or α) of the obtained haploid strain was determined by crossing with a known standard haploid strain.
[0011]
(2) Method for measuring the properties of yeast [0012]
(2)-(i) Method for measuring ethionine resistance YPD agar medium (2% glucose, 2% polypeptone, 1% yeast extract, 2% agar) was used for preculture of yeast at 30 ° C. for 18-24 hours. After culturing, an appropriate amount is scraped off and inoculated on SD medium (2% glucose, 0.67% Yeast Nitrogen Base “Difco”, 2% agar) containing 0.1-1 mM ethionine, and cultured at 30 ° C. for 3 days. Colony formation was determined later, and a strain that formed a colony was considered resistant and a strain that did not form was considered sensitive.
[0013]
(2)-(ii) Method for measuring yeast S-adenosylmethionine 1 platinum ear inoculation of yeast precultured on YPD agar medium in 5 ml YPD liquid medium (2% glucose, 2% polypeptone, 1% yeast extract) And cultured at 30 ° C. for 18 hours. Next, the yeast cells obtained by centrifuging the culture solution at 3000 rpm for 10 minutes were washed twice with distilled water. 0.5 ml 10% TCA (trichloroacetic acid) was added to the obtained washed yeast cells, and after stirring, the mixture was kept at 30 ° C. for 1 hour to extract S-adenosylmethionine. S-adenosylmethionine was quantified by high performance liquid chromatography using an Asahipak GS-320 column, 0.1M phosphate buffer (pH 10) was used as the mobile phase, and absorption at a wavelength of 254 nm was measured to determine S-adenosyl. Quantification was performed in comparison with a methionine standard solution.
[0014]
(2)-(iii) Constitutive Acid Phosphatase Activity Assay YPD agar medium (2% glucose, 2% polypeptone, 1% yeast extract, 2% agar) is used for preculture of yeast at 30 ° C. for 18 hours to After culturing for 24 hours, scrape an appropriate amount and use Burkholder's minimum medium (2% glucose, 0.2% asparagine, 0.15% potassium phosphate, 0.033% calcium chloride, 0.05% magnesium sulfate, 60 μg / ml). Boric acid, 30 μg / ml manganese sulfate, 300 μg / ml zinc sulfate, 40 μg / ml copper sulfate, 250 μg / ml ferrous chloride, 25 μg / ml sodium molybdate, 200 μg / l thiamine, 200 μg / l pyridoxine, 200 μg / l nicotinic acid 200 μg / l pantothenic acid, 2 μg / l biotin, 10 mg / l inositol, 2% agar, pH 5. Inoculated in), and cultured for 2 days at 30 ℃. Next, 5 ml of 3% agar dissolved by heating and 5 ml of the staining solution (0.1 M acetate buffer solution, pH 4.0 containing 0.05% sodium α-naphthyl phosphate and 0.05% Fast Blue B Salt) were mixed, and colonies were mixed. Overlay on the formed medium. Incubation was carried out at 30 ° C. for 30 minutes to 1 hour, and a strain in which the colony was stained red was positive, and a strain that remained white was negative.
[0015]
(2)-(iv) Determination of growth at 35 ° C. in β-alanine medium YPD agar medium (2% glucose, 2% polypeptone, 1% yeast extract, 2% agar) was used for yeast pre-culture. After culturing for 18 hours to 24 hours at ° C, an appropriate amount is scraped, and β-alanine medium (2% glucose, 0.05% ammonium sulfate, 0.15% potassium phosphate, 0.05% magnesium sulfate, 200 μg / ml thiamine, 200 μg / l pyridoxine, 200 μg / l nicotinic acid, 0.2 μg / l biotin, 1000 μg / l inositol, 200 μg / l p-aminobenzoic acid, 40 μg / l β-alanine, 2% agar, pH 5.0) And cultured at 35 ° C. for 2 days. After the cultivation, the growth was determined, and the growing strain was positive and the non-growing strain was negative.
[0016]
Examples of the present invention will be described below.
[0017]
[Example 1]
Ethionine-sensitive mutant of Sake Yeast Association No. 7
Sake Yeast Association No. 7 is pre-cultured overnight in YPD medium, and after harvesting and washing, 5 ml of 0.2 M phosphate buffer containing 1 mM EMS is subjected to mutagenesis under shaking conditions at 30 ° C. for 1 hour. did. Next, after washing the yeast four times with sterilized water, suspending in SD medium containing 1 mM ethionine, culturing at 30 ° C. for 5 hours, adding nystatin to a final concentration of 10 μg / ml, and further culturing The yeast showing resistance to ethionine was selectively killed. The surviving yeast was appropriately diluted and then cultured on a YPD agar medium to form colonies. A spore formation test was performed on each of the grown strains. Three strains with good spore formation rates, 7ETS1, 7ETS2, and 7ETS3 were obtained. Obtained.
[0019]
The properties of these mutants and parent strains were measured based on the method for measuring yeast properties. As shown in Table 1 below, in the mutant strains, the spore formation rate was improved, the tetraspore formation frequency was increased, Changes in properties were observed. Moreover, the spore survival rate of the mutant spore clearly increased.
[0020]
[Table 1]

[0021]
[Example 2]
Sake Yeast Association No. 7 mutant that can grow at 35 ° C. in β-alanine medium.
Sake yeast association No. 7 treated with EMS in the same manner as in Example 1 was applied to a β-alanine medium and cultured at 35 ° C. A colony formed after 3 days was subjected to a spore formation test, and three strains with good spore formation, 7β-1, 7β-2, and 7β-3 were obtained.
[0023]
The properties of these mutants and the parent strain were measured based on the method for measuring yeast properties. As shown in Table 2 below, in the mutant strains, the spore formation rate was improved, the tetraspore formation frequency was increased, Changes in properties were observed. In addition, the spore survival rate of the mutant spore was clearly increased.
[0024]
[Table 2]

[0025]
【The invention's effect】
Since the present invention was developed for the purpose of imparting high spore-forming ability and spore survival rate to yeast, without using an experimental system in which a large number of vegetative cells coexist like the conventional haploid acquisition method, A haploid strain can be easily isolated. As a result, genetic analysis of practical sake yeast, which has been difficult until now, becomes easy, and it has high utility value as a source strain for analysis of useful traits as brewing yeast and breeding of excellent yeast.

Claims (1)

Practical sake yeast characterized by selecting a mutant strain having a high spore formation rate by obtaining a strain having a practical brewer's yeast as a parent strain and obtaining a strain having ethionine sensitivity and a growth ability at 35 ° C. in a β-alanine medium from the parent strain. Production method of haploid strains.