Method for breeding yeast having high production of l-cysteine

Abstract

To provide, inter alia, a method for breeding a yeast having high productivity of L-cysteine.SOLUTION: A method for breeding yeast having high productivity of L-cysteine, the method comprising the following steps (A)-(C): (A) a mutation step of causing a mutation in a parent strain of a yeast; (B) a selective culture step of culturing the yeast subjected to the mutation step by using a culture medium containing selenomethionine; and (C) an evaluation step of 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.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[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"). [Background technology]

[0002] Cysteine ​​is a type of amino acid that makes up proteins. It exhibits antioxidant properties and is widely used as a raw material for food additives, pharmaceuticals, and cosmetics. Cysteine ​​is mainly produced by extraction and separation from acid hydrolysates of hair, feathers, etc. In recent years, fermentation using microorganisms has become popular as a method for producing cysteine. It is known that an increase in intracellular cysteine ​​concentration inhibits microbial growth and causes feedback inhibition of biosynthetic enzymes, making it 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. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2010-22215 [Patent Document 2] Japanese Patent Application Laid-Open No. 2009-232844 [Patent Document 3] Japanese Patent Publication No. 2020-71 [Non-patent literature]

[0005] [Non-Patent Document 1] PierrePlateau, Cosmin Saveanu, Roxane Lestini, Marc Dauplais, Laurence Decourty, Alain Jacquier, Sylvain Blanquet, Myriam Lazard: Scientific Reports, 7, 44761,2017, DOI: 10.1038 / srep44761 Summary of the Invention [Problem to be solved by the invention]

[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. [Means for solving the problem]

[0007] The present inventors have exploited the fact that selenomethionine (hereinafter referred to as "SeMet") is converted to selenocysteine ​​in yeast cells, which is toxic, to generate SeMet-resistant strains, and 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 mutation is induced in a parent strain of yeast; (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 in the selective culture step is cultured and the cysteine ​​content in the yeast is evaluated to determine whether it is greater than that in the parent strain.

[0008] In the above-mentioned mutagenesis step, it is preferable to use at least one method selected from the group consisting of ethyl methanesulfonate (EMS) treatment, N-methyl-N-nitrosoguanidine (NTG) treatment, nitrous acid 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 culture step is experimentally determined by culturing the target yeast strain in media containing various concentrations of selenomethionine and observing the growth, to determine the selenomethionine concentration required to inhibit the growth of the target yeast strain. Examples of selenomethionine concentrations include 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 required for the characteristics of the yeast used are added to the medium. Another aspect of the present invention provides a yeast obtained by the above-described breeding method for an L-cysteine-rich yeast, which is characterized by exhibiting resistance to SeMet and containing 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 drink containing the 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 and other applications. In the present invention, yeasts include baker's yeast, sake yeast, wine yeast, brewer's yeast, soy sauce yeast, miso yeast, and bioethanol yeast. Either haploid or diploid yeast can be used as yeast.

[0010] Cysteine ​​exhibits antioxidant properties and is 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 broths containing large amounts of cysteine, which can be used in self-healthcare products. Food and drink items include beverages (dairy drinks, coffee, tea, juice, processed milk, sports drinks, etc.), bakery products (bread, pizza, pie, etc.), Western sweets (cookies, crackers, biscuits, cake, 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 sweets (jelly, pudding, mousse, yogurt, buttercream, candy, etc.), and other items. This includes processed foods such as starched cream, Japanese sweets (gyuhi, uriwara, mochi, ohagi, dorayaki, etc.), processed fruits and vegetables (jam, marmalade, preserved fruits in syrup, candied fruits, etc.), pastes (flower paste, fruit paste, peanut paste, etc.), seasonings (soy sauce, sauce, noodle soup, dashi stock, soup stock, etc.), frozen and refrigerated foods (ham, sausage, bacon, hamburger steak, meatballs, croquettes, gyoza, pilaf, rice balls, etc.), and processed seafood (chikuwa, kamaboko, etc.). Food and beverages are not limited to those for humans, but also include those for pets (dogs, cats, etc.). Food and beverages also include health care products such as supplements. [Effects of the Invention]

[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. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 2 is a schematic diagram showing the procedure of the present embodiment. [Figure 2] FIG. 1 is a photograph showing the results of seeding and culturing a resistant strain isolated using SeMet resistance as an indicator and a parent strain (wild-type strain: WT) on a plate containing SeMet. [Figure 3] 1 is a bar graph showing the results of examining the intracellular cysteine ​​content of 6 strains out of 24 strains. DETAILED DESCRIPTION OF THE INVENTION

[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 embodiments can be implemented without departing from the spirit 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 mutations in a parent yeast strain, followed by a selective culture step in which the yeast is cultured in a medium containing SeMet, and the resulting strain is obtained as a SeMet-resistant strain. Next, an evaluation step is performed in which the cysteine ​​content in the SeMet-resistant strain is measured to determine whether it is higher than the cysteine ​​content in the parent strain. By performing these steps, a yeast with high cysteine ​​production 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 additional components (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) was used for culturing the yeast. To isolate SeMet-resistant mutants, strain X2180 was grown 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 The X2180 strain was treated with 6% ethyl methanesulfonate (EMS) in pH 7.0 phosphate buffer at 30°C for 60 minutes to introduce mutations. EMS-treated cells were washed twice with 10% w / v sodium thiosulfate and plated onto SD+Alt medium containing 0.01 mg / mL SeMet. After 7 days of incubation at 30°C, a selective culture step was performed to obtain 24 SeMet-resistant strains. The survival rate of yeast cells after EMS treatment was approximately 46%. 3. Quantification of Intracellular Amino Acid Content Yeast cells were cultured in SD+Am medium at 30°C for 2 days, then collected by centrifugation and washed twice. The resulting yeast pellet was resuspended in sterilized water and treated at 100°C for 20 minutes to extract the amino acids in the yeast cells. After removing the insoluble fraction by centrifugation, 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 yeast mutants resistant to SeMet Selenocysteine ​​(SeCys), in which the sulfur atom of cysteine ​​is replaced by selenium, is incorporated into proteins in place of cysteine ​​residues, producing structurally abnormal proteins that inhibit yeast growth. Because the toxicity of SeCys can be avoided by increasing intracellular cysteine, 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 is toxic as a cysteine ​​analog. Therefore, we performed a mutation treatment on the diploid laboratory yeast strain X-2180, and isolated 24 mutants (SeMet-resistant strains) that exhibited resistance to SeMet. Figure 2 shows the results of culturing the parent strain and the SeMet-resistant strain on an agar plate containing SeMet. No yeast growth was observed in the parent strain (WT), which is in the upper 1 / 4 of the image. In contrast, growth was observed in the SeMet-resistant strains, which are in the other 3 / 4 of the image.

[0017] 2. Isolation of yeast mutants that accumulate intracellular cysteine Next, the amino acid productivity of the obtained SeMet-resistant strains was evaluated. Six SeMet-resistant strains were isolated, with intracellular cysteine ​​levels at least 1.4 times higher than those of 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) had cysteine ​​levels at least twice that of the parent strain. After culturing the resistant strain in large quantities, cysteine ​​can be efficiently produced by isolating and purifying it. The resistant strain can be consumed as is or as a raw material in food and beverages, allowing for efficient cysteine ​​intake. According to this embodiment, a method for breeding a yeast that produces a large amount of cysteine ​​can be provided.

Claims

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

2. 2. The method for breeding an L-cysteine ​​hyper-producing 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. 3. A yeast obtained by the breeding method of claim 1 or 2, which is resistant to selenomethionine and has an intracellular cysteine ​​content that is 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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