Method for breeding yeast having high production of l-cysteine

Abstract

[Problem] To provide, inter alia, a method for breeding a yeast that produces a high amount of L-cysteine. [Solution] A method for breeding yeast having high production of L-cysteine, the method comprising the following steps (A)-(C): (A) a mutation step for causing a mutation in a parent strain of a yeast; (B) a selective culture step for culturing the yeast subjected to the mutation step by using a culture medium containing selenomethionine; and (C) an evaluation step for culturing the yeast grown through the selective culture step and evaluating whether the cysteine content in the yeast is greater than the cysteine content in the parent strain.

Description

Method for breeding yeast that produces high levels of L-cysteine

[0001] The present invention relates to a method for breeding yeast capable of producing a large amount of L-cysteine ​​(hereinafter referred to as "cysteine").

[0002] Cysteine ​​is a type of amino acid that constitutes proteins. It exhibits antioxidant properties and is widely used as a raw material for food additives, pharmaceuticals, and cosmetics. Cysteine ​​is mainly produced by separation and extraction from acid hydrolysates of hair, feathers, etc. In recent years, fermentation using microorganisms has been used to produce cysteine. It is known that an increase in intracellular cysteine ​​concentration inhibits microbial growth and causes feedback inhibition of biosynthetic enzymes. For this reason, it has been difficult to obtain mutants with high cysteine ​​productivity using microorganisms (Patent Documents 1-3).

[0003] A method for breeding yeast with high amino acid content is known, using toxic analogs, which are structural analogs of amino acids that become toxic when taken up into cells. Specifically, strains containing high concentrations of a particular amino acid are expected to be resistant to the toxic analog when taken up into cells. By examining such analog-resistant strains, strains with high production of a particular amino acid can be efficiently isolated. To apply this method to cysteine, it is necessary to find a suitable cysteine ​​analog. One known cysteine ​​analog is selenocysteine. Selenocysteine ​​is a cysteine ​​analog in which the sulfur atom is replaced by selenium. Selenocysteine ​​is mistakenly incorporated into proteins in place of cysteine ​​residues, producing structurally abnormal proteins and inhibiting yeast growth. However, because yeast do not take up selenocysteine ​​in the culture medium, it has been difficult to use selenocysteine ​​as a toxic analog.

[0004] JP 2010-22215 A JP 2009-232844 A JP 2020-71 A

[0005] PierrePlateau, Cosmin Saveanu, Roxane Lestini, Marc Dauplais, Laurence Decourty, Alain Jacquier, Sylvain Blanquet, Myriam Lazard: Scientific Reports, 7, 44761,2017, DOI: 10.1038 / srep44761

[0006] The present study was conducted in view of the above-mentioned problems, and its purpose is to provide a method for breeding yeast that produces a large amount of cysteine.

[0007] The present inventors have obtained SeMet-resistant strains by utilizing the fact that selenomethionine (hereinafter referred to as "SeMet") is converted to selenocysteine ​​within yeast cells and exhibits toxicity. From these resistant strains, they have bred yeast strains capable of high cysteine ​​production. The method for breeding an L-cysteine-rich yeast according to the present invention comprises the following steps (A) to (C): (A) a mutagenesis step in which a parent yeast strain is mutated; (B) a selective culture step in which the yeast that has undergone the mutagenesis step is cultured in a medium containing selenomethionine (SeMet); and (C) an evaluation step in which the yeast grown through the selective culture step is cultured and the cysteine ​​content in the yeast is evaluated to determine whether it is higher than that in the parent strain.

[0008] The mutagenesis step preferably employs at least one method selected from the group consisting of ethyl methanesulfonate (EMS) treatment, N-methyl-N-nitrosoguanidine (NTG) treatment, nitrite treatment, ultraviolet irradiation, X-ray irradiation, and gamma ray irradiation. In the above invention, the amount of selenomethionine in the "selenomethionine-containing medium" used in the selective culturing step is experimentally determined by culturing the target yeast strain in media containing various concentrations of selenomethionine and observing the growth. The selenomethionine concentration required to inhibit the growth of the target yeast strain is, for example, 0.01 mg / mL or higher. The medium and culture conditions, other than the amount of selenomethionine, can be those typically used for yeast culture. If necessary, nutrients necessary for the characteristics of the yeast strain used are added to the medium. Another invention relates to a yeast obtained by the above-described method for breeding an L-cysteine-rich yeast, which exhibits resistance to SeMet and contains 1.4 times or more (preferably, 2 times or more) the amount of intracellular cysteine ​​of the parent strain. Another invention relates to a food or beverage containing the above yeast.

[0009] Yeast is a general term for fungi that are eukaryotic, unicellular microorganisms that are non-motile, have cell walls, lack photosynthetic ability, and decompose external organic matter and absorb nutrients. Yeast includes the genus Saccharomyces, a type of budding yeast that has been used in foods. In the present invention, this includes baker's yeast, sake yeast, wine yeast, brewer's yeast, soy sauce yeast, miso yeast, bioethanol yeast, and the like. Either haploid or diploid yeast can be used as yeast.

[0010] Cysteine ​​has antioxidant properties and is therefore widely used as a food additive, pharmaceutical, and supplement. According to the present invention, yeast with a high cysteine ​​content can be obtained, which allows the production of fermentation products and fermentation liquids containing a large amount of cysteine, making them suitable for use in self-healthcare products. Food and drink products include beverages (dairy-containing beverages, coffee, tea, juice, processed milk, sports drinks, etc.), bakery products (bread, pizza, pies, etc.), Western confectionery (cookies, crackers, biscuits, cakes, castella, etc.), noodles, pasta, snacks, confectionery (candy, caramel, gum, chocolate, etc.), frozen desserts (ice cream, sorbet, etc.), dairy products (cream, cheese, mousse, powdered milk, condensed milk, dairy drinks, etc.), Western confectionery (jelly, pudding, mousse, yogurt, buttercream, chocolate, etc.), and other products. This includes processed foods such as starched cream, Japanese sweets (gyuhi, uriwara, mochi, ohagi, dorayaki, etc.), processed fruit and vegetable foods (jam, marmalade, preserved fruits in syrup, candied fruits, etc.), pastes (flower paste, fruit paste, peanut paste, etc.), seasonings (soy sauce, sauce, dipping sauce, noodle soup, dashi stock, soup stock, etc.), frozen and refrigerated foods (ham, sausage, bacon, hamburger steak, meatballs, croquettes, gyoza, pilaf, rice balls, etc.), processed seafood foods (chikuwa, kamaboko, etc.). Food and drink is not limited to food for humans, but also includes food for pets (dogs, cats, etc.). Food and drink also includes health care products such as supplements.

[0011] According to the present invention, a method for breeding a yeast with a high cysteine ​​content can be provided. Because this yeast has a high cysteine ​​content, it can be used directly for cysteine ​​production. By consuming the yeast itself, cysteine ​​can be efficiently ingested.

[0012] Fig. 1 is a schematic diagram showing the procedure of this embodiment. Fig. 2 is a photograph showing the results when resistant strains isolated using SeMet resistance as an indicator and the parent strain (wild-type strain: WT) were seeded and cultured on a SeMet-containing plate. Fig. 3 is a bar graph showing the results of examining the intracellular cysteine ​​content of 6 strains out of 24 strains.

[0013] Next, embodiments of the present invention will be described with reference to the drawings. The technical scope of the present invention is not limited to these embodiments, and various forms can be implemented without changing the gist of the invention. <Test Method> An outline of the test method is shown in Figure 1. In this test, a mutation step is performed to induce mutation in a parent yeast strain, and a selective culture step is performed to culture this yeast in a medium containing SeMet, and the grown yeast is obtained as a SeMet-resistant strain. Next, an evaluation step is performed to measure the cysteine ​​content in the SeMet-resistant strain and evaluate whether it is higher than the cysteine ​​content in the parent strain. By performing these steps, a yeast that produces a high amount of cysteine ​​can be obtained.

[0014] 1. Strains and Media. The diploid laboratory yeast strain X2180 (wild-type (WT), MATa / α) of S. cerevisiae was used as the parent strain for mutant breeding. YPD nutrient medium containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose was used for culturing the yeast. Synthetic dextrose minimal medium plus supplements (1.7 g / L amino acids and nitrogen base for yeast culture without ammonium sulfate (Difco Laboratories), 20 g / L glucose, and 5 g / L ammonium sulfate) were also used for culturing the yeast. For isolating SeMet-resistant mutants, strain X2180 was cultured in synthetic dextrose minimal medium SD plus supplements (SD medium supplemented with 5 g / L allantoin instead of ammonium sulfate as the nitrogen source).

[0015] 2. Isolation of SeMet-Resistant Mutants: Mutations were introduced into strain X2180 by treating it with 6% ethyl methanesulfonate (EMS) in pH 7.0 phosphate buffer at 30°C for 60 minutes. The EMS-treated cells were washed twice with 10% w / v sodium thiosulfate and plated on SD+Alt medium containing 0.01 mg / mL SeMet. After 7 days of incubation at 30°C, a selective incubation step was performed to obtain 24 SeMet-resistant strains. The viability of yeast cells after EMS treatment was approximately 46%. 3. Quantification of Intracellular Amino Acid Content: After incubation in SD+Am medium at 30°C for 2 days, the yeast cells were harvested by centrifugation and washed twice. The resulting yeast pellet was resuspended in sterile water and incubated at 100°C for 20 minutes to extract the amino acids in the yeast cells. After removing the insoluble fraction by centrifugation, an evaluation step was carried out in which the amino acid content in the supernatant was quantified using an amino acid analyzer (JLC-500 / V2: manufactured by JEOL).

[0016] <Test Results> 1. Isolation of SeMet-Resistant Yeast Mutants. Selenocysteine ​​(SeCys), in which the sulfur atom of cysteine ​​is replaced by selenium, is incorporated into proteins in place of cysteine ​​residues, generating structurally abnormal proteins that inhibit yeast growth. Because the toxicity of SeCys can be avoided by increasing intracellular cysteine ​​levels, SeCys-resistant strains are expected to have increased cysteine ​​content. However, extracellular SeCys is not taken up by yeast cells. On the other hand, when SeMet, in which the sulfur atom of methionine (Met) is replaced by selenium, is added to the culture medium, it is taken up into yeast cells and converted to SeCys, which becomes toxic as a cysteine ​​analog. Therefore, we performed a mutation treatment on the diploid laboratory yeast strain X-2180, and isolated 24 SeMet-resistant mutants (SeMet-resistant strains). Figure 2 shows the results of culturing the parent and SeMet-resistant strains on agar plates containing SeMet. No yeast growth was observed in the parent strain (WT) in the upper quarter of the figure, whereas growth was observed in the SeMet-resistant strains (other than WT) in the other three-quarters of the figure.

[0017] 2. Isolation of Yeast Mutant Strains That Accumulate Cysteine ​​Intracellularly Next, the amino acid productivity of the obtained SeMet-resistant strains was evaluated. Six SeMet-resistant strains were obtained, with intracellular cysteine ​​levels increased by more than 1.4 times compared to the parent strain. Figure 3 shows the results of examining the intracellular cysteine ​​content of these six strains and the parent strain. Four of the six strains (resistant strains numbered 1, 3, 4, and 6 in the figure) exhibited cysteine ​​levels more than twice that of the parent strain. Cysteine ​​production can be efficiently achieved by mass-cultivating the resistant strains and isolating and purifying cysteine. Cysteine ​​can be efficiently ingested by consuming the resistant strains directly or as a raw material in foods and beverages. This embodiment provides a method for breeding yeast that produces high levels of cysteine.

Claims

1. A method for breeding an L-cysteine-rich yeast, comprising the following steps (A) to (C): (A) a mutagenesis step of inducing mutations in a parent strain of yeast; (B) a selective culture step of culturing the yeast that has undergone the mutagenesis step in a medium containing selenomethionine; and (C) an evaluation step of culturing the yeast grown in the selective culture step and evaluating whether the cysteine ​​content in the yeast is greater than that in the parent strain.

2. The method for breeding an L-cysteine-rich yeast according to claim 1, wherein the mutagenesis step uses at least one method selected from the group consisting of ethyl methanesulfonate (EMS) treatment, N-methyl-N-nitrosoguanidine (NTG) treatment, nitrite treatment, ultraviolet irradiation, X-ray irradiation, and gamma ray irradiation.

3. A yeast obtained by the breeding method of claim 1 or 2, which is resistant to selenomethionine and has an intracellular cysteine ​​content 1.4 times or more that of the parent strain.

4. A food or drink containing the yeast according to claim 3.

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