JPH0697993B2 - Arginase gene-deficient strain selection medium, breeding of urea-non-producing yeast using it, and method for producing alcoholic beverages using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Use) The present invention is not a laboratory strain yeast (haploid) but a yeast used for actual brewing of food and drink (practical brewing yeast: diploid). , Or higher polyploidy) arginase gene (CAR1) by mutation treatment, or by transformation using DNA containing no heterologous gene,
Urea-non-producing yeast by any of the,
The present invention relates to a breeding method using the yeast and utilization of the yeast.

Urea non-producing practical brewer's yeast bred by the mutation treatment or transformation method according to the present invention is arginase (EC3.5.) Which is an enzyme that decomposes arginine into ornithine and urea.
Urea is not produced due to deletion of 3.1). Therefore,
It is possible to suppress the production of the carcinogen ethyl carbamate derived from urea.

That is, by using the mutant strain or the gene-disrupted strain of the present invention, alcoholic fermentation rate and quality are the same as those of the parent strain, and moreover, safe alcoholic beverages containing no ethyl albamate can be produced. Therefore, the present invention greatly contributes to the production of alcoholic beverages, alcohol and other brewed foods.

(Background of the Invention and Prior Art and Problems Thereof) Generally, the content of brewed foods and drinks (sake, shochu, fruit seeds, beer, whiskey, brandy, soy sauce, miso, old sake, etc.) is considerably high. Despite differences, urea is a problem worldwide because it reacts with ethanol to produce the carcinogen ethyl carbamate (urethane).

Conventionally, as a method for reducing urea, a method using a mutant strain lacking arginine permease involved in the uptake of arginine into yeast cells, or a urea amide lyase action that decomposes urea into ammonia and carbon dioxide Although a method using a desuppression mutant strain has been adopted, even if these strains are used, the amount of urea can be reduced only to about half of the conventional amount. Moreover, since these mutants generally have a sharper fermentation than the parent strain, there is a problem that they are easily contaminated by other yeasts and it is difficult to obtain a reliable effect.

In Japanese Patent Application No. 1-207874, the inventors bred urea-non-producing practical brewer's yeast by disrupting the arginase gene. However, it is in a situation where it cannot be used in the brewery. Further, the gene-disrupted strain has a heterologous gene other than yeast, that is, it has a DNA sequence of Escherichia coli. In Japanese Patent Application No. 63-268097 (JP-A-2-117385), a urea non-producing yeast was bred by disruption or substitution of the arginase gene. The host yeast was provided with a marker for selecting transformants. Since it uses a laboratory strain (1 culture) that is easy to do and is a haploid, there is a problem in that the fermentation rate is practically slow.

Practical brewer's yeast (Saccharomyces cerevisiae), which is usually used for the production of sake, shochu, fruit wine, beer, whiskey, brandy, old sake, etc., has a diploid (or higher polyploid) Besides, it has properties required for the production of each liquor, for example, alcohol resistance in sake and shochu, sulfite resistance in fruit liquor, low temperature fermentability in beer, etc. There is.

In addition, in imparting a marker for transformant selection, an auxotrophic marker by artificial mutation can be imparted to a haploid laboratory strain.

However, since the practical brewer's yeast is diploid (or higher polyploid), it is not possible to obtain such an auxotrophy which is a recessive mutation without impairing its brewing characteristics. There wasn't.

Furthermore, in the practical brewer's yeast, its ploidy is diploid (or higher polyploid), and therefore, in order to destroy or replace the target gene, 2 (or more) It is necessary to destroy or replace the same gene on the chromosome, and it is necessary to construct a plurality of disrupting or replacing plasmids in which the genes of the above selection markers are linked.

(Means for Solving the Problems) As described above, the present inventors create an excellent practical brewing yeast having urea non-productivity in the practical brewing yeast which is completely different from the laboratory strain in various points as described above. This was done to develop a comprehensive system.

From the above viewpoints, the present study was conducted for the purpose of developing an arginase gene-deficient mutant strain by artificial mutation, which is a urea non-producing brewer's yeast that can be practically used at the brewery at the present time. In addition, as soon as the guidelines for recombinant use of alcoholic beverages were established, we conducted an earnest study for the purpose of developing an arginase gene-disrupted strain that does not contain a heterologous gene other than yeast, and that could be put into practical use in a brewery immediately.

Therefore, in order to search for the desired mutant strain, screening was eagerly performed according to a conventional method. That is, since the arginase gene-deficient strain does not have arginase and cannot grow in a medium containing only arginine as a nitrogen source, a method called a replica method, which is a conventional method for selecting such a deficient strain, was used. However, this replica method can examine only about 100 strains on one plate. In other words, it was impossible in reality that 1 million plates had to be used in order to obtain a mutant strain at a rate of 1 in 10 ⁸ .

In this way, in view of the fact that one has to expect the chance to select the target mutant strain, it is necessary to change the conventional idea in order to obtain the target mutant strain. I got the idea that it is more important to change the method and develop a new method. Then, it was decided to prepare a medium suitable for selecting the target mutant strain.

Therefore, using the arginase gene-disrupted strain FERM P-10903 and the parent strain, Sake Yeast Society No. 9 (Saccharomyces cerevisiae) bred by the present inventor in Japanese Patent Application No. 1-207874, only the gene-disrupted strain can grow, and the parent strain Examined various media that could not grow.

As a result, we succeeded for the first time in creating a medium particularly suitable for positively selecting only this arginase gene-disrupted strain, and a medium in which only the arginase gene-deficient strain can selectively grow (hereinafter also referred to as CAO medium). Moreover, it was also confirmed that if this medium is used, it is possible to investigate 5 million strains with one plate.

Next, it was found that an arginase gene-deficient mutant strain obtained by subjecting a practical brewing yeast to an artificial mutation treatment can be obtained by using the medium.

In addition, a DNA fragment encoding the arginase gene of Saccharomyces cerevisiae was cloned to construct plasmid A or plasmid B in which a part of the coding region was deleted. It was also found that an arginase gene-deficient transformant strain containing no heterologous gene other than yeast can be obtained by using any of these plasmids and further using CAO medium.

Then, when using the obtained mutant strain and transformed strain to produce alcoholic beverages, urea production was not observed at all,
Therefore, it was confirmed that harmful ethyl carbamate was not produced, and that the alcohol fermentation rate and the quality of sake were the same as those of the parent strain, and the present invention was completed.

(Detailed Description) In particular, in the Japanese Patent Application No. 1-207874, the present inventors have developed a urea-non-producing brewer's yeast AL-1 strain (FERM P-109) by disrupting the arginase gene.
As a result of conducting various conditions using the yeast, we succeeded in producing a medium in which only the arginase gene-deficient strain could grow selectively.

The mutant strain selection medium according to the present invention contains at least canavanine, arginine, ornithine, and as a preferred example, yeast nitrogen base (amino acid-free) and sugars (glucose, fructose, Maltose, lactose, oligosaccharides, starch, dextrin, etc.) may be added. Further, if necessary, a commonly used selective medium component may be added.

These compounding amounts are 0.1 to 20 ppm canavanine, 0.05 to 50 mM arginine, and 0.25 to 250 mM ornithine.When glucose is used as the yeast nitrogen base and sugar, the former is 0.05 to 0.7% and the latter is 5.5 to Ten
It may be contained in the medium within the range of%.

The selective medium (CAO medium) prepared in this way can be used to investigate about 5 million strains on a single plate, so it is possible to accurately and in a short time without overlooking the target mutant strain that appears at an extremely low frequency. Can be detected. Therefore,
By using this CAO medium, it is possible to efficiently obtain an arginase gene-deficient mutant strain and an arginase gene-disrupted strain containing no heterologous gene other than yeast by transformation.

Regarding the mutant strain, for example, Oda et al.
83, 614 (1988)). That is, the practically brewed yeast is subjected to a mutation treatment using ethyl methane sulfonate and applied on the CAO medium. Among the mutants that have grown, a strain that cannot grow in an arginine single nitrogen source medium but can grow in an ornithine single nitrogen source medium is obtained as an intended arginase-deficient mutant strain.

For the gene-disrupted strain containing no heterologous gene other than yeast by transformation, the genomic DNA donor used was Saccharomyces cerevisiae, specifically, Association Yeast No. 7 (commercially available).

The method for extracting the chromosomal DNA from the bacterium and the method for preparing the chromosomal gene library are, for example, Agric.Biol.Chem., Vol.53,
It is performed according to the method described in 431-436 (1989).

For the isolation of the gene from the chromosomal gene library obtained above, the DNA sequence of the Saccharomyces cerevisiae arginase gene (J. Bacteriol., Vol.
160, 1078 to 1087 (1984)), prepare a DNA oligomer, and use it as a probe for plaque hybridization to clone the DNA of the Saccharomyces cerevisiae arginase gene.

Plasmid A or plasmid B for gene disruption
Is manufactured as follows.

The plasmid A was prepared from 0.87 derived from the arginase gene.
The plasmid pHP-1 in which the HindIII-PstI fragment of Kbp was inserted into pUC119 was cut with Sal I, treated with Klenow fragment and self-ligated.
A plasmid A (pHP-1SH) is prepared by removing the Hinc II-Hinc II (0.15 Kbp) fragment from S (Fig. 1).

Further, the construction of plasmid B was carried out by using plasmid pCAR112 in which a BamHI-BamHI fragment of about 5.5 Kbp containing the arginase gene was incorporated into pUC119 to BglII-BglII (0.74Kp).
b) A plasmid B (pCAR112-GG) excluding the fragment is prepared (Fig. 2).

In the transformation, the practical brewer's yeast is diploid (or higher polyploid), but since an arginase gene-deficient strain selection medium (CAO medium) is used, 2 strains on two chromosomes are used.
A strain in which one gene of interest is disrupted at the same time can be selected by this CAO medium. Also, an arginase gene-deficient strain in which only one of the genes on the chromosome is destroyed, and the two arginase genes, which are generated by causing the gene conversion with the remaining healthy arginase gene, are also destroyed. Can be obtained. Therefore, at least two types of gene disruption plasmids have been required for gene disruption in conventional brewer's yeasts, whereas one type of gene disruption plasmid is sufficient when the present CAO medium is used. That is, the desired gene-disrupted strain can be efficiently obtained by performing gene disruption using either one of the plasmids A and B produced as described above.

That is, transformation is carried out using the plasmid A cleaved at the Hind III and Pst I sites or the plasmid B cleaved at the BamH I site and applied on a CAO medium to obtain the desired transformant. The Hind III-Pst I fragment (0.72 Kbp) of plasmid A or the BamHI-BamHI of plasmid B that rearranges with the arginase gene of the parent strain.
The fragment (4.8 Kbp) does not include the sequences of heterologous genes such as Escherichia coli, but consists only of yeast-derived sequences.

In addition, to determine whether the arginase gene was deficient by mutation treatment or destroyed by transformation, the arginase activity was measured by the following method, and the presence or absence of the activity was examined. Confirm gene disruption by.

The arginase activity, which is a key factor for deficiency of the arginase gene, is measured as follows.

That is, a mutant strain, a transformant strain and a parent strain were treated with an arginase induction medium (yeast nitrogen base (nitrogen source free) 0.17%, arginine hydrochloride 10 mM, glucose 2%,
The cells are inoculated into ammonium sulfate (5 mM) at 4 × 10 ⁴ cells / ml, shake-cultured at 30 ° C. overnight, and then the cells are collected and washed. Suspend the cells in 10 mM Tris-HCl buffer (pH 7.0) and destroy with glass beads. 15,000 rp of this homogenate
Urea produced by reacting arginine as a substrate with a supernatant obtained by centrifugation for 10 minutes at m for 10 minutes is quantified with a urea kit manufactured by Toyo Brewing Co., Ltd. to obtain arginase activity.

Next, using this mutant strain and transformant strain in the usual manner,
By producing alcoholic beverages such as sake, fruit wine, beer, shochu, whiskey, brandy, and old sake, it is possible to produce alcoholic beverages that do not contain urea at all, and it is safe without carcinogenicity that does not produce ethyl albamate. For the first time, it is possible to produce alcoholic beverages and delicious alcoholic beverages whose flavors are exactly the same as those produced by conventional methods.

Next, examples of the present invention will be described.

Example 1 Search of selective medium for arginase gene-deficient strain; since arginase gene-deficient strain does not have arginase, it cannot grow in a medium containing only arginine as a nitrogen source. When selecting such a defective strain, a method usually called a replica method is used. However, this replica method can examine only about 100 strains on one plate. In other words, it is impossible in reality that one million plates have to be used for the acquisition frequency of mutant strains occurring at a rate of 1 in 10 ⁸ .

Therefore, using the arginase gene-disrupted strain FERM P-10903 and the parent strain, Sake Yeast Society No. 9 (Saccharomyces cerevisiae) bred by the present inventor in Japanese Patent Application No. 1-207874, only the gene-disrupted strain can grow, and the parent strain Examined various media that could not grow.

As a result, it was found that the yeast nitrogen base (amino acid-free) 0.17%, canavanine 10 ppm, arginine 1 mM, ornithine 5 mM, glucose 2% was the best medium for positively selecting only this arginase gene-disrupted strain. , CAO selection medium containing this composition
It was named the medium.

An example of the CAO medium formulation is shown in Table 1.

By using this CAO medium, one plate can be used for about 5
It is possible to examine 0,000,000 strains, and it is possible to efficiently select target strains that are included in low frequencies.

Example 2 Breeding of Saccharomyces cerevisiae that does not produce arginase by mutation; Sake yeast (Association No. 9 and No. 10), wine yeast (Geisenheim 74. Eppanay) in YEPD medium (1% yeast extract, 2% polypeptone, Glucose (2%) was inoculated into 10 ml at 4 × 10 ⁴ cells / ml, shake-cultured at 30 ° C. overnight, and the cells were collected and washed. Suspend the cells in 10 ml of 0.1 mM phosphate buffer (pH 7.0), ethyl methane sulfonate
0.3 ml was added, and the mixture was subjected to mutation treatment by gently shaking at 30 ° C. for 45 minutes. The cells collected by centrifugation were washed once with 10 ml of a 5% sodium thiosulfate solution and twice with 10 ml of sterilized water, suspended in 10 ml of sterilized water, and 40 μ of the suspension was applied on a CAO medium.

After culturing at 30 ℃ for 1 week, the emerged colony was isolated as a single colony on CAO medium, and the arginase gene aimed at a strain that could not grow on arginine single nitrogen source medium but could grow on ornithine single nitrogen source medium Obtained as a defective strain (Table 2). In this way I got from Association No. 9
ALM-9 has been deposited with the Institute of Mechanical Engineering as FERM P-11169.

Example 3 Construction of plasmid for disruption of arginase gene; Method for constructing plasmid A: pCAR112 described in FIG.
-207874) and a 0.87 Kbp DNA fragment derived from the arginase gene excised with the restriction enzymes Hind III and Pst I into pUC119.
Ligation was performed using T4 DNA ligase to prepare plasmid pHP-1. This was cleaved with Sal I, treated with Klenow fragment, and self-ligated using T4 DNA ligase to obtain Hinc II-Hinc I of the obtained plasmid pHP-1S.
After cutting out the I fragment (0.15 Kbp) with a restriction enzyme
Re-ligation was performed using T4 DNA ligase to prepare an arginase gene disruption plasmid (pHP-1SH) (Fig. 1).

Construction method of plasmid B: Bgl II-Bgl II fragment (0.74 Kbp) was cut out from pCAR112 with a restriction enzyme, and then T4DNA
Religation was performed using ligase to prepare a plasmid B (pCAR112-GG) for disrupting the arginase gene (Fig. 2).

Example 4 Preparation of Saccharomyces cerevisiae that does not produce arginase by transformation; using plasmids A or B for disrupting the arginase gene, transformation of Saccharomyces cerevisiae sake yeast association No. 9 (2 culture, commercial product) was performed by Ito et al. Method (J. Bacte
riol., Vol.153,163 (1983)).

That is, Sake yeast (Association No. 9) was added to 4 x 10 ml of YEPD medium.
The cells were inoculated at a rate of 10 ⁴ cells / ml, and after shaking culture at 30 ° C. overnight, cells in the logarithmic growth phase were collected by centrifugation. After washing with TE buffer, the suspension was suspended in 0.5 ml of TE buffer, an equal volume of 0.2 M lithium acetate solution was added, and the mixture was shaken at 30 ° C. for 1 hour. Transfer 0.1 ml from this to a 1.5 ml Eppendorf tube, add 0 μ of DNA solution (0.5 μg / μ),
It was allowed to stand at 30 ° C for 30 minutes.

The DNA solution used here was prepared as follows.

When transformation is performed using plasmid A: Plasmid A (pHP-1SH) is treated with restriction enzymes Hind III and Pst I,
Of the two generated fragments (0.72Kbp, 3.2Kbp), 0.72Kbp
After purifying the fragment of
It was dissolved in TE buffer to prepare a DNA solution. In addition, 0.7
The 2 Kbp Hind III-Pst I fragment consists of yeast-derived genes only and does not contain heterologous genes such as Escherichia coli.

When transformation is carried out using plasmid B: Plasmid B (pCAR112-GG) was treated with the restriction enzyme BamH I to generate 2
Of the two fragments (4.8 Kbp and 3.2 Kbp), the 4.8 Kbp fragment was purified by the Geneclean method and then dissolved in TE buffer to prepare a DNA solution. In addition, 4.8 Kbp BamHI-
The BamHI fragment consists only of yeast-derived genes.

Next, add 150μ of sterilized 70% PEG-4000, mix well, leave at 30 ° C for 1 hour, then leave the Eppendorf tube in a constant temperature bath at 42 ° C for 10 minutes, and immediately cool the cells to room temperature. Then, it was washed with sterilized water and applied on the CAO medium. Incubate at 30 ℃ for 1 week, and use plasmid A (pHP-1SH) for 9 cells (9 cells / 100 μg DNA), plasmid B (pCAR112-G).
In G), 16 strains (16 cells / 100 μg DNA) were obtained, which were transformants containing no target heterologous gene other than yeast.

The ALK-9 strain prepared with plasmid A has been deposited with MIC as FERM p-11168.

Example 5 Confirmation of damage due to mutation of arginase gene or destruction by transformation; Table 3 shows the arginase-deficient strain, parent strain and arginase activity thus obtained, which were also mutated in arginine-induced culture. Since no arginase activity was observed in the strain and the transformant strain, it was confirmed that the arginase genes on the two chromosomes were both defective or destroyed.

Furthermore, regarding the transformed strain, it was confirmed by Southern blotting that the arginase gene was disrupted.

Example 7 Brewing of alcoholic beverages with mutant strains and gene-disrupted strains; parent strain (association No. 9), mutant strains and gene-disrupted strains were statically cultivated in a koji extract medium at 30 ° C. for 3 days, and then collected and washed, 200 g of total rice was charged with sake under the mixing formulation and manufacturing conditions shown in the table.

The progress of alcohol fermentation was measured by the weight loss due to the generation of CO ₂ , and is shown in FIG. In addition, Table 5 shows the urea content and various components of the sake. As a result, by using the mutant strain and the transformant strain, alcohol fermentation rate, general components and sensory evaluation were almost the same as those of the parent strain, and it was possible to produce sake containing no urea and ethyl albamate.

(Effect of the invention) According to the present invention, a selective medium capable of efficiently selecting an arginase gene-deficient strain is provided, and by using this medium, urea non-production that does not produce arginase and therefore does not produce urea. Sex yeast can be bred. By using the yeast thus obtained, it is possible to brew safe alcoholic beverages containing no carcinogenic ethyl carbamate.

[Brief description of drawings]

Fig. 1 is an explanatory diagram of the production of a plasmid A for gene disruption of the arginase gene, Fig. 2 is an explanatory diagram of the production of a similar plasmid B, and Fig. 3 is a progress diagram of alcohol fermentation of a parent strain, a mutant strain and a transformant strain. Is.

Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12N 15/81 // (C12N 1/16 C12R 1: 865) (C12N 1/19 C12R 1: 865) (72 ) Inventor Katsuya Gomi 2-6-30 Takinogawa, Kita-ku, Tokyo Inside the National Brewing Agency's brewing laboratory (72) Inventor Chieko Kumagai 2-6-30 Takinogawa, Kita-ku, Tokyo Inside the National Brewery's brewing laboratory (72) Inventor Hara Masamichi Tokyo 2-6-30 Takinogawa, Kita-ku, National Institute of Taxation Brewing Laboratory (72) Inventor, Gakuzo Tamura 1-54-18, Takinogawa, Kita-ku, Tokyo (56) References 117385 (JP, A)

Claims (8)

[Claims] 1. An arginase gene-deficient strain selection medium containing at least canavanine, arginine and ornithine. 2. Canavanine content is 0.1 to 20 ppm, arginine content is 0.05 to 50 mM, ornithine content is 0.25 to 250 m.
The selection medium according to claim 1, which is M. 3. The arginase gene is deficient by a mutation treatment, which is obtained from a diploid or a practical brewer's yeast having a ploidy of 2 or more by using the selective medium according to claim 1 or 2. Urea non-producing yeast belonging to Satsucaromyces cerevisiae. 4. A plasmid shown below, which is obtained from a diploid or a practical brewer's yeast having a polyploidy of 2 or more using the selective medium according to claim 1 or 2.
A urea non-producing yeast in which the arginase gene is disrupted by gene disruption using pHP-1SH or pCAR112-GG and which does not contain a heterologous gene other than yeast. 5. A strain deficient in all of two or more arginase genes of practical brewer's yeast by mutation is obtained by using the selective medium according to claim 1 or 2. Method for breeding urea-non-producing yeast. 6. A DN containing no heterologous gene for all of two or more arginase genes of practical brewer's yeast.
A method for breeding a urea non-producing yeast, which comprises obtaining a strain in which all of the gene has been disrupted by transformation with A, using the selective medium according to claim 1 or 2. 7. A method for producing alcoholic beverages, alcohols and the like, which comprises using the urea-non-producing yeast according to claim 3. 8. A method for producing alcoholic beverages, alcohols, etc., which comprises using the urea-non-producing yeast according to claim 4.