Met4 mutant protein, gene encoding Met4 mutant protein, yeast having gene encoding Met4 mutant protein, composition containing these, and method for producing the same.

Abstract

To provide a Met4 mutant protein capable of enhancing sulfur metabolism, a gene encoding the Met4 mutant protein, yeast having the gene encoding the Met4 mutant protein, and compositions containing these. [Solution] Met4 mutant protein, wild type Saccharomyces cerevisiae The amino acid corresponding to lysine position 163 of the Met4 protein is deleted or substituted, and the wild type Saccharomyces cerevisiae Examples include Met4 mutant proteins characterized by the deletion or substitution of at least one amino acid among the amino acids corresponding to positions 189 through 312 of the Met4 protein, and exhibiting improved transcriptional activation ability for sulfur metabolism genes compared to the wild-type Met4 protein.

Description

Technical Field

[0001] The present invention relates to a Met4 mutant protein, a gene encoding the Met4 mutant protein, yeast having a gene encoding the Met4 mutant protein, a composition comprising any of the foregoing, and a method for producing the same. Background Art

[0002] Sulfur atom-containing amino acids such as S-adenosylmethionine and methionine (sulfur-containing amino acids) are known to have various functions in vivo and are widely used as supplements. Met4 is known as a transcriptional activator that governs sulfur metabolism. It is also known that in the presence (high concentration) of methionine, Met4 undergoes ubiquitination to be inactivated or degraded, whereby transcription of a gene group of the sulfur metabolism system is suppressed; and in the absence (low concentration) of methionine, ubiquitin is removed, Met4 enters the nucleus, and the gene group of the sulfur metabolism system is transcribed.

[0003] Patent Document 1 describes mutant MET4 obtained by a random mutagenesis method, in which the 215th serine of MET4 is substituted with proline, and mutant MET4 in which the 156th phenylalanine of MET4 is substituted with serine. It is described that in yeast expressing these mutant MET4, the intracellular glutathione content increases, but the effect was not sufficient.

[0004] Accordingly, there is a strong demand for the prompt provision of a Met4 mutant protein capable of enhancing sulfur metabolism, a gene encoding the Met4 mutant protein, yeast having a gene encoding the Met4 mutant protein, and a composition comprising any of the foregoing. Prior Art Literature Patent Literature

[0005] Patent Document 1 Japanese Patent Application Laid-Open No. 10-033161 Summary of the Invention [Problems that the invention aims to solve]

[0006] The present invention aims to solve the aforementioned problems in the conventional era and achieve the following objectives. Specifically, the present invention aims to provide a Met4 mutant protein capable of enhancing sulfur metabolism, a gene encoding the Met4 mutant protein, yeast having the gene encoding the Met4 mutant protein, and compositions containing these. [Means for solving the problem]

[0007] As a result of diligent research conducted by the present inventors to achieve the above objective, they have found that it is possible to provide a Met4 mutant protein capable of enhancing sulfur metabolism, a gene encoding the Met4 mutant protein, yeast having the gene encoding the Met4 mutant protein, and compositions containing these.

[0008] The present invention is based on the aforementioned findings by the inventors, and the means for solving the aforementioned problems are as follows: <1> Met4 mutant protein, Wild type Saccharomyces cerevisiae The amino acid corresponding to lysine position 163 of the Met4 protein is deleted or substituted, Wild type Saccharomyces cerevisiae In the Met4 protein, at least one amino acid corresponding to positions 189 through 312 is deleted or substituted. This is a Met4 mutant protein characterized by improved transcriptional activation ability for sulfur metabolism genes compared to the wild-type Met4 protein. <2> The aforementioned <1> This gene is characterized by encoding the Met4 mutant protein described in [reference]. <3> The aforementioned <2> This yeast is characterized by having the genes described in [reference]. <4> The aforementioned <1> The Met4 mutant protein described above, <2> The genes described above, and the aforementioned <3> This composition is characterized by containing one or more yeasts selected from those described above. <5> A method for producing a composition comprising two or more components selected from valine, isoleucine, leucine, arginine, γ-aminobutyric acid, trehalose, and SAM, the <3> This is a method for producing a composition, characterized by including a culture step of culturing the yeast described above. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a Met4 mutant protein capable of enhancing sulfur metabolism, a gene encoding the Met4 mutant protein, yeast having the gene encoding the Met4 mutant protein, and compositions containing these. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a graph showing the expression analysis results for the SAM1 gene (sake yeast). [Figure 2] Figure 2 is a graph showing the expression analysis results for the MET17 gene (sake yeast). [Figure 3] Figure 3 is a graph showing the expression analysis results for the CYS3 gene (sake yeast). [Figure 4] Figure 4 is a graph showing the expression analysis results for the SAM1 gene (laboratory yeast). [Figure 5] Figure 5 is a graph showing the expression analysis results for the MET17 gene (experimental yeast). [Modes for carrying out the invention]

[0011] (Met4 mutant protein) The aforementioned Met4 mutant protein (mutant Met4) is a mutant protein of the Met4 protein (wild-type Met4 protein).

[0012] Said Met4 protein (wild-type Met4 protein) is a transcriptional activator. Said Met4 protein controls the transcription of sulfur metabolism-related genes. In an environment rich in methionine, said Met4 protein undergoes ubiquitination and is degraded, thereby suppressing sulfur metabolism.

[0013] There are no particular limitations on said sulfur metabolism-related genes, and they can be appropriately selected according to the purpose. Examples include SAM1, which encodes S-adenosylmethionine (SAM) synthase, MET17, which encodes homocysteine synthase, and CYS3, which encodes cysteine synthase.

[0014] There are no particular limitations on said Met4 protein (wild-type Met4 protein), and it can be appropriately selected according to the purpose. It is preferably derived from yeast, more preferably derived from Saccharomyces cerevisiae, Saccharomyces further preferably derived from yeast belonging to the genus , Saccharomyces cerevisiae particularly preferably derived from , and most preferably has the amino acid sequence represented by SEQ ID NO: 1 or a homolog thereof.

[0015] There are no particular limitations on the homolog of the Met4 protein having the amino acid sequence represented by SEQ ID NO: 1, and it can be appropriately selected according to the purpose. Preference is given to those having a sequence with at least 80% homology to the amino acid sequence represented by SEQ ID NO: 1, more preference to those with at least 85% homology, further preference to those with at least 90% homology, particular preference to those with at least 95% homology, and most preference to those with at least 99% homology.

[0016] Said Met4 mutant protein is a wild-type Saccharomyces cerevisiae Met4 protein wherein the amino acid corresponding to position 163 lysine is deleted or substituted, and the wild-type Saccharomyces cerevisiae Met4 protein wherein at least one amino acid among the amino acids corresponding to positions 189 to 312 is deleted or substituted. In other words, the Met4 mutant protein is a double mutant protein having mutations (two types of mutations) in two regions of the Met4 protein: at position 163 and from position 189 to 312.

[0017] The aforementioned wild type Saccharomyces cerevisiae The lysine at position 163 of the Met4 protein is a site for ubiquitination. The aforementioned wild type Saccharomyces cerevisiae The amino acids corresponding to positions 189 through 312 of the Met4 protein are the ubiquitin ligase interaction region (inhibitory region).

[0018] The aforementioned Met4 mutant protein exhibits improved transcriptional activation ability for sulfur metabolism genes compared to the wild-type Met4 protein. In other words, the Met4 mutant protein is a mutant protein capable of enhancing sulfur metabolism.

[0019] "Compared to the wild-type Met4 protein, the transcriptional activation ability for sulfur metabolism genes is improved" means that in yeast expressing the Met4 mutant protein, the expression level of at least one of the following is increased compared to yeast expressing the wild-type Met4 protein: SAM1, which encodes S-adenosylmethionine (SAM) synthase; MET17, which encodes homocysteine ​​synthase; and CYS3, which encodes cysteine ​​synthase. Among these, the expression levels of at least one of the following—SAM1 encoding S-adenosylmethionine (SAM) synthase, MET17 encoding homocysteine ​​synthase, and CYS3 encoding cysteine ​​synthase—are wild-type Saccharomyces cerevisiae Compared to yeast expressing a Met4 mutant protein (single mutant protein) in which the amino acid corresponding to lysine at position 163 of the Met4 protein is substituted with arginine, an increase is more preferable.

[0020] Furthermore, in yeast expressing the Met4 mutant protein, it is preferable that the expression levels of SAM1 encoding S-adenosylmethionine (SAM) synthase, MET17 encoding homocysteine ​​synthase, and CYS3 encoding cysteine ​​synthase are increased compared to yeast expressing the wild-type Met4 protein. Saccharomyces cerevisiae Compared to yeast expressing a Met4 mutant protein (single mutant protein) in which the amino acid corresponding to lysine at position 163 of the Met4 protein is substituted with arginine, an increase is more preferable.

[0021] The aforementioned Met4 mutant protein is preferably derived from yeast, and more preferably from budding yeast. Saccharomyces cerevisiae The origin is even more appealing. Among these, wild type Saccharomyces cerevisiae Preferably, the amino acid sequence homology with the Met4 protein is 50% or more, more preferably 60% or more, even more preferably 70% or more, and particularly preferably 80% or more.

[0022] The aforementioned wild type Saccharomyces cerevisiae There are no particular restrictions on Met4 mutant proteins in which the amino acid corresponding to the 163rd lysine position of the Met4 protein is deleted or substituted, and they can be appropriately selected depending on the purpose, but the wild type Saccharomyces cerevisiae A Met4 mutant protein in which the amino acid corresponding to the 163rd lysine position of the Met4 protein is substituted is preferred, and the wild type Saccharomyces cerevisiae A Met4 mutant protein in which the amino acid corresponding to lysine at position 163 of the Met4 protein is replaced with arginine is more preferable.

[0023] The aforementioned wild type Saccharomyces cerevisiae There are no particular restrictions on Met4 mutant proteins in which at least one amino acid is deleted or substituted from the amino acids corresponding to positions 189 to 312 of the Met4 protein, and they can be appropriately selected depending on the purpose, but the wild type Saccharomyces cerevisiae A Met4 mutant protein is preferred in which the amino acids corresponding to positions 189 to 312 of the Met4 protein are deleted, or the amino acid corresponding to histidine at position 198, serine at position 210, lysine at position 216, or glycine at position 270 is substituted, and the wild type Saccharomyces cerevisiae More preferably, a Met4 mutant protein is obtained in which the amino acids corresponding to positions 189 to 312 of the Met4 protein are deleted, or the amino acid corresponding to histidine at position 198, the amino acid corresponding to lysine at position 216, or the amino acid corresponding to glycine at position 270 is substituted, and the wild type Saccharomyces cerevisiae A Met4 mutant protein is more preferably one in which the amino acids corresponding to positions 189 to 312 of the Met4 protein are deleted, or the amino acid corresponding to histidine at position 198 or lysine at position 216 is substituted.

[0024] Among these, wild type Saccharomyces cerevisiae The amino acid corresponding to lysine at position 163 of the Met4 protein is substituted with arginine, and the wild type Saccharomyces cerevisiae A preferred Met4 mutant protein is one in which the amino acids corresponding to positions 189 through 312 of the Met4 protein are deleted, or the amino acid corresponding to histidine at position 198 is replaced with leucine, the amino acid corresponding to serine at position 210 is replaced with phenylalanine, the amino acid corresponding to lysine at position 216 is replaced with glutamic acid, or the amino acid corresponding to glycine at position 270 is replaced with arginine. Saccharomyces cerevisiaeThe amino acid corresponding to lysine at position 163 of the Met4 protein is substituted with arginine, and the wild type Saccharomyces cerevisiae More preferably, a Met4 mutant protein is obtained in which the amino acids corresponding to positions 189 to 312 of the Met4 protein are deleted, or the amino acid corresponding to histidine at position 198 is replaced with leucine, the amino acid corresponding to lysine at position 216 is replaced with glutamic acid, or the amino acid corresponding to glycine at position 270 is replaced with arginine, and the wild type Saccharomyces cerevisiae The amino acid corresponding to lysine at position 163 of the Met4 protein is substituted with arginine, and the wild type Saccharomyces cerevisiae A more preferable Met4 mutant protein is one in which the amino acids corresponding to positions 189 through 312 of the Met4 protein are deleted, or the amino acid corresponding to histidine at position 198 is replaced with leucine, or the amino acid corresponding to lysine at position 216 is replaced with glutamic acid.

[0025] (gene) The aforementioned gene is the gene that encodes the Met4 mutant protein. The aforementioned Met4 mutant protein is as described above under (Met4 mutant protein).

[0026] The gene encoding the aforementioned Met4 mutant protein is a MET4 mutant gene, in which some of the bases in the MET4 gene sequence are deleted or substituted.

[0027] (yeast) The aforementioned yeast has a gene that encodes a Met4 mutant protein. The gene encoding the aforementioned Met4 mutant protein is as described above under (gene). The yeast possessing the gene encoding the aforementioned Met4 mutant protein is a double mutant.

[0028] Yeast having the gene encoding the Met4 mutant protein can be obtained by transforming a parent yeast strain that does not have the gene encoding the Met4 mutant protein, by inducing spontaneous mutations or spontaneous mutations, or by genome editing.

[0029] There are no particular restrictions on the parent strain (yeast as a parent strain), and it can be appropriately selected depending on the purpose, but budding yeast is preferred. Saccharomyces Yeast belonging to the genus is more preferable. Saccharomyces cerevisiae This is even more preferable. The parent plants may be used individually or in combination of two or more.

[0030] Specific examples of the aforementioned parent strains (yeast used as parent strains) include sake yeast, shochu yeast, and wine yeast, which are commercially available from the Japan Brewing Association. The aforementioned parent strain (yeast as a parent strain) may be laboratory yeast.

[0031] Examples of sake yeasts commercially available from the aforementioned Japan Brewing Association include foaming yeasts such as Kyokai No. 6, Kyokai No. 7, Kyokai No. 9, Kyokai No. 10, Kyokai No. 11, and Kyokai No. 14; and non-foaming yeasts such as Kyokai No. 601, Kyokai No. 701, Kyokai No. 901, Kyokai No. 1001, Kyokai No. 1401, and Kyokai No. 1501.

[0032] Examples of shochu yeasts commercially available from the aforementioned Japan Brewing Association include Shochu Yeast No. 1, Shochu Yeast No. 2, and Shochu Yeast No. 3.

[0033] Examples of wine yeasts commercially available from the aforementioned Japan Brewing Association include Wine Yeast No. 1, Wine Yeast No. 2, Wine Yeast No. 3, and Wine Yeast No. 4.

[0034] In yeast (double mutant) possessing the gene encoding the aforementioned Met4 mutant protein, sulfur metabolism is enhanced even in the presence of methionine, leading to rapid metabolism (consumption) of intracellular methionine and high production of SAM and other amino acids.

[0035] There are no particular restrictions on the lower limit of the methionine content per unit weight of dry cells of yeast having the gene encoding the Met4 mutant protein, and it can be appropriately selected depending on the purpose, but it is preferably 0.1 ppm (0.0001 mg / g) or more, more preferably 1 ppm (0.001 mg / g) or more, even more preferably 10 ppm (0.01 mg / g) or more, particularly preferably 30 ppm (0.03 mg / g) or more, and most preferably 50 ppm (0.05 mg / g) or more. There are no particular restrictions on the upper limit of the methionine content per unit weight of dry cells in yeast having the gene encoding the Met4 mutant protein, and it can be appropriately selected depending on the purpose, but it is preferably 300 ppm (0.3 mg / g) or less, more preferably 200 ppm (0.2 mg / g) or less, and even more preferably 100 ppm (0.1 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0036] There are no particular restrictions on the lower limit of the valine content per dry cell weight of yeast having the gene encoding the Met4 mutant protein, and it can be appropriately selected depending on the purpose, but 350 ppm (0.35 mg / g) or more is preferred, 400 ppm (0.4 mg / g) or more is more preferred, and 500 ppm (0.5 mg / g) or more is even more preferred. There are no particular restrictions on the upper limit of the valine content per dry cell weight of yeast having the gene encoding the aforementioned Met4 mutant protein, and it can be appropriately selected depending on the purpose, but examples include 10,000 ppm (10 mg / g) or less, 5,000 ppm (5 mg / g) or less, 1,000 ppm (1 mg / g) or less, or 800 ppm (0.8 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0037] There are no particular restrictions on the lower limit of isoleucine content per dry cell weight of yeast having the gene encoding the Met4 mutant protein, and it can be appropriately selected depending on the purpose, but 200 ppm (0.2 mg / g) or more is preferred, 250 ppm (0.25 mg / g) or more is more preferred, and 300 ppm (0.3 mg / g) or more is even more preferred. There are no particular restrictions on the upper limit of isoleucine content per unit weight of dry cells in yeast having the gene encoding the aforementioned Met4 mutant protein, and it can be appropriately selected depending on the purpose, but examples include 10,000 ppm (10 mg / g) or less, 5,000 ppm (5 mg / g) or less, 1,000 ppm (1 mg / g) or less, 800 ppm (0.8 mg / g) or less, or 500 ppm (0.5 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0038] There are no particular restrictions on the lower limit of the leucine content per unit weight of dry cells of yeast having the gene encoding the Met4 mutant protein, and it can be appropriately selected depending on the purpose, but 150 ppm (0.15 mg / g) or more is preferred, 200 ppm (0.2 mg / g) or more is more preferred, and 250 ppm (0.25 mg / g) or more is even more preferred. There are no particular restrictions on the upper limit of leucine content per unit weight of dry cells in yeast having the gene encoding the aforementioned Met4 mutant protein, and it can be appropriately selected depending on the purpose, but examples include 10,000 ppm (10 mg / g) or less, 5,000 ppm (5 mg / g) or less, 1,000 ppm (1 mg / g) or less, 800 ppm (0.8 mg / g) or less, or 500 ppm (0.5 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0039] There are no particular restrictions on the lower limit of the arginine content per unit weight of dry cells of yeast having the gene encoding the Met4 mutant protein, and it can be appropriately selected depending on the purpose, but 7000 ppm (7 mg / g) or more is preferred, 8000 ppm (8 mg / g) or more is more preferred, 10000 ppm (10 mg / g) or more is even more preferred, and 11000 ppm (11 mg / g) or more is particularly preferred. There are no particular restrictions on the upper limit of arginine content per unit weight of dry cells in yeast having the gene encoding the aforementioned Met4 mutant protein, and it can be appropriately selected depending on the purpose, but examples include 100,000 ppm (100 mg / g) or less, 50,000 ppm (50 mg / g) or less, 30,000 ppm (30 mg / g) or less, or 20,000 ppm (20 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0040] There are no particular restrictions on the lower limit of the γ-aminobutyric acid content per dry cell weight of yeast having the gene encoding the Met4 mutant protein, and it can be appropriately selected depending on the purpose, but 20 ppm (0.02 mg / g) or more is preferred, 30 ppm (0.03 mg / g) or more is more preferred, 40 ppm (0.04 mg / g) or more is even more preferred, and 50 ppm (0.05 mg / g) or more is particularly preferred. There are no particular restrictions on the upper limit of the γ-aminobutyric acid content per unit weight of dry cells in yeast having the gene encoding the aforementioned Met4 mutant protein, and it can be appropriately selected depending on the purpose, but examples include 10,000 ppm (10 mg / g) or less, 5,000 ppm (5 mg / g) or less, 1,000 ppm (1 mg / g) or less, 800 ppm (0.8 mg / g) or less, or 500 ppm (0.5 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0041] There are no particular restrictions on the lower limit of the trehalose content per unit weight of dry cells of yeast having the gene encoding the Met4 mutant protein, and it can be appropriately selected depending on the purpose, but 50 ppm (0.05 mg / g) or more is preferred, 80 ppm (0.08 mg / g) or more is more preferred, and 100 ppm (0.1 mg / g) or more is even more preferred. There are no particular restrictions on the upper limit of the trehalose content per unit weight of dry cells in yeast having the gene encoding the Met4 mutant protein, and it can be appropriately selected depending on the purpose, but it is preferable to have a value of 10,000 ppm (10 mg / g) or less, 5,000 ppm (5 mg / g) or less, 1,000 ppm (1 mg / g) or less, 800 ppm (0.8 mg / g) or less, or 500 ppm (0.5 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0042] There are no particular restrictions on the lower limit of the SAM content per dry cell weight of yeast having the gene encoding the Met4 mutant protein, and it can be appropriately selected depending on the purpose, but 25,000 ppm (25 mg / g) or more is preferred, 28,000 ppm (28 mg / g) or more is more preferred, 50,000 ppm (50 mg / g) or more is even more preferred, 60,000 ppm (60 mg / g) or more is even more preferred, 80,000 ppm (80 mg / g) or more is particularly preferred, and 90,000 ppm (90 mg / g) or more is most preferred. There are no particular restrictions on the upper limit of the SAM content per unit weight of dry cells in yeast having the gene encoding the aforementioned Met4 mutant protein, and it can be appropriately selected depending on the purpose, but examples include 500,000 ppm (500 mg / g) or less, 300,000 ppm (300 mg / g) or less, or 200,000 ppm (200 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0043] (composition) The composition comprises a Met4 mutant protein, a gene encoding the Met4 mutant protein, or yeast having a gene encoding the Met4 mutant protein. The aforementioned Met4 mutant protein is as described above under (Met4 mutant protein). The gene encoding the aforementioned Met4 mutant protein is as described above under (gene). The yeast having the gene encoding the aforementioned Met4 mutant protein is as described above under (yeast).

[0044] The aforementioned composition may also contain two or more components selected from valine, isoleucine, leucine, arginine, γ-aminobutyric acid, trehalose, and SAM.

[0045] There are no particular restrictions on the lower limit of the methionine content in the above composition, and it can be appropriately selected depending on the purpose, but it is preferably 0.1 ppm (0.0001 mg / g) or more, more preferably 1 ppm (0.001 mg / g) or more, even more preferably 10 ppm (0.01 mg / g) or more, particularly preferably 30 ppm (0.03 mg / g) or more, and most preferably 50 ppm (0.05 mg / g) or more. There are no particular restrictions on the upper limit of the methionine content in the above composition, and it can be appropriately selected depending on the purpose, but it is preferably 300 ppm (0.3 mg / g) or less, more preferably 200 ppm (0.2 mg / g) or less, and even more preferably 100 ppm (0.1 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0046] There are no particular restrictions on the lower limit of the valine content in the above composition, and it can be appropriately selected depending on the purpose, but 350 ppm (0.35 mg / g) or more is preferred, 400 ppm (0.4 mg / g) or more is more preferred, and 500 ppm (0.5 mg / g) or more is even more preferred. There are no particular restrictions on the upper limit of the valine content in the above composition, and it can be appropriately selected depending on the purpose, but examples include 10,000 ppm (10 mg / g) or less, 5,000 ppm (5 mg / g) or less, 1,000 ppm (1 mg / g) or less, or 800 ppm (0.8 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0047] There are no particular restrictions on the lower limit of the isoleucine content in the above composition, and it can be appropriately selected depending on the purpose, but 200 ppm (0.2 mg / g) or more is preferred, 250 ppm (0.25 mg / g) or more is more preferred, and 300 ppm (0.3 mg / g) or more is even more preferred. There are no particular restrictions on the upper limit of the isoleucine content in the above composition, and it can be appropriately selected depending on the purpose, but examples include 10,000 ppm (10 mg / g) or less, 5,000 ppm (5 mg / g) or less, 1,000 ppm (1 mg / g) or less, 800 ppm (0.8 mg / g) or less, or 500 ppm (0.5 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0048] There are no particular restrictions on the lower limit of the leucine content in the above composition, and it can be appropriately selected depending on the purpose, but 150 ppm (0.15 mg / g) or more is preferred, 200 ppm (0.2 mg / g) or more is more preferred, and 250 ppm (0.25 mg / g) or more is even more preferred. There are no particular restrictions on the upper limit of the leucine content in the above composition, and it can be appropriately selected depending on the purpose, but examples include 10,000 ppm (10 mg / g) or less, 5,000 ppm (5 mg / g) or less, 1,000 ppm (1 mg / g) or less, 800 ppm (0.8 mg / g) or less, or 500 ppm (0.5 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0049] There are no particular restrictions on the lower limit of the arginine content in the above composition, and it can be appropriately selected depending on the purpose, but 7000 ppm (7 mg / g) or more is preferred, 8000 ppm (8 mg / g) or more is more preferred, 10000 ppm (10 mg / g) or more is even more preferred, and 11000 ppm (11 mg / g) or more is particularly preferred. There are no particular restrictions on the upper limit of the arginine content in the above composition, and it can be appropriately selected depending on the purpose, but examples include 100,000 ppm (100 mg / g) or less, 50,000 ppm (50 mg / g) or less, 30,000 ppm (30 mg / g) or less, or 20,000 ppm (20 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0050] There are no particular restrictions on the lower limit of the γ-aminobutyric acid content in the above composition, and it can be appropriately selected depending on the purpose, but 20 ppm (0.02 mg / g) or more is preferred, 30 ppm (0.03 mg / g) or more is more preferred, 40 ppm (0.04 mg / g) or more is even more preferred, and 50 ppm (0.05 mg / g) or more is even more preferred. There are no particular restrictions on the upper limit of the γ-aminobutyric acid content in the above composition, and it can be appropriately selected depending on the purpose, but examples include 10,000 ppm (10 mg / g) or less, 5,000 ppm (5 mg / g) or less, 1,000 ppm (1 mg / g) or less, 800 ppm (0.8 mg / g) or less, or 500 ppm (0.5 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0051] There are no particular restrictions on the lower limit of the trehalose content in the above composition, and it can be appropriately selected depending on the purpose, but 50 ppm (0.05 mg / g) or more is preferred, 80 ppm (0.08 mg / g) or more is more preferred, and 100 ppm (0.1 mg / g) or more is even more preferred. There are no particular restrictions on the upper limit of the trehalose content in the aforementioned composition, and it can be appropriately selected depending on the purpose, but examples include 10,000 ppm (10 mg / g) or less, 5,000 ppm (5 mg / g) or less, 1,000 ppm (1 mg / g) or less, 800 ppm (0.8 mg / g) or less, or 500 ppm (0.5 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0052] There are no particular restrictions on the lower limit of the SAM content in the above composition, and it can be appropriately selected depending on the purpose, but 25,000 ppm (25 mg / g) or more is preferred, 28,000 ppm (28 mg / g) or more is more preferred, 50,000 ppm (50 mg / g) or more is even more preferred, 60,000 ppm (60 mg / g) or more is even more preferred, 80,000 ppm (80 mg / g) or more is particularly preferred, and 90,000 ppm (90 mg / g) or more is most preferred. There are no particular restrictions on the upper limit of the SAM content in the aforementioned composition, and it can be appropriately selected depending on the purpose, but examples include 500,000 ppm (500 mg / g) or less, 300,000 ppm (300 mg / g) or less, or 200,000 ppm (200 mg / g) or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0053] There are no particular restrictions on the lower limit of the mass ratio of valine to methionine in the above composition, and it can be appropriately selected depending on the purpose, but it is preferably 1 or more, more preferably 2 or more, even more preferably 3 or more, particularly preferably 5 or more, and most preferably 7 or more. There are no particular restrictions on the upper limit of the mass ratio of valine to methionine in the above composition, but examples include 100 or less, 50 or less, 30 or less, 20 or less, or 15 or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0054] There are no particular restrictions on the lower limit of the mass ratio of isoleucine to methionine in the above composition, and it can be appropriately selected depending on the purpose, but it is preferably 1 or more, more preferably 1.5 or more, even more preferably 2 or more, particularly preferably 3 or more, and most preferably 5 or more. There are no particular restrictions on the upper limit of the mass ratio of isoleucine to methionine in the above composition, but examples include 100 or less, 50 or less, 30 or less, 20 or less, 15 or less, or 10 or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0055] There are no particular restrictions on the lower limit of the mass ratio of leucine to methionine in the above composition, and it can be appropriately selected depending on the purpose, but it is preferably 1 or more, more preferably 1.5 or more, even more preferably 2 or more, particularly preferably 3 or more, and most preferably 4 or more. There are no particular restrictions on the upper limit of the mass ratio of leucine to methionine in the above composition, but examples include 100 or less, 50 or less, 30 or less, 20 or less, 15 or less, or 10 or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0056] There are no particular restrictions on the lower limit of the mass ratio of arginine to methionine in the above composition, and it can be appropriately selected depending on the purpose, but it is preferably 20 or more, more preferably 50 or more, even more preferably 100 or more, particularly preferably 130 or more, and most preferably 150 or more. There are no particular restrictions on the upper limit of the mass ratio of arginine to methionine in the above composition, but examples include 500 or less, 300 or less, 250 or less, or 200 or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0057] There are no particular restrictions on the lower limit of the mass ratio of γ-aminobutyric acid to the mass of methionine in the above composition, and it can be appropriately selected depending on the purpose, but it is preferably 0.05 or higher, more preferably 0.1 or higher, even more preferably 0.3 or higher, particularly preferably 0.5 or higher, and most preferably 0.8 or higher. There are no particular restrictions on the upper limit of the mass ratio of γ-aminobutyric acid to the mass of methionine in the above composition, but examples include 100 or less, 50 or less, 30 or less, 20 or less, 15 or less, 10 or less, 3 or less, 1.5 or less, or 1 or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0058] There are no particular restrictions on the lower limit of the mass ratio of trehalose to methionine in the above composition, and it can be appropriately selected depending on the purpose, but it is preferably 0.2 or higher, more preferably 0.5 or higher, even more preferably 1 or higher, even more preferably 5 or higher, particularly preferably 10 or higher, and most preferably 13 or higher. There are no particular restrictions on the upper limit of the mass ratio of trehalose to the mass of methionine in the above composition, but examples include 500 or less, 300 or less, 100 or less, 50 or less, 30 or less, 25 or less, or 20 or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0059] There are no particular restrictions on the lower limit of the mass ratio of SAM to the mass of methionine in the above composition, and it can be appropriately selected depending on the purpose, but it is preferably 75 or more, more preferably 100 or more, even more preferably 200 or more, even more preferably 250 or more, particularly preferably 300 or more, and most preferably 400 or more. There are no particular restrictions on the upper limit of the mass ratio of SAM to the mass of methionine in the above composition, but examples include 2000 or less, 1000 or less, 700 or less, or 500 or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0060] Among these, a composition containing 10 ppm (0.01 mg / g) or more of methionine and two or more selected from valine in a mass ratio of 5 to 15 to the amount of methionine, isoleucine in 3 to 10, leucine in 3 to 10, arginine in 100 to 250, γ-aminobutyric acid in 0.5 to 1.5, trehalose in 5 to 25, and SAM in 250 to 700 is preferred. A composition containing 10 ppm (0.01 mg / g) or more of methionine, 100-250 mg of arginine, 0.5-1.5 mg of γ-aminobutyric acid, 5-25 mg of trehalose, and 3 or more selected from 250-700 mg of SAM is more preferable, and a composition containing 10 ppm (0.01 mg / g) or more of methionine, with a mass ratio of 5-15 of valine to methionine, 3-10 mg of isoleucine, 3-10 mg of leucine, 100-250 mg of arginine, 0.5-1.5 mg of γ-aminobutyric acid, 5-25 mg of trehalose, and 4 or more selected from 250-700 mg of SAM is even more preferable. Preferably, the composition contains 10 ppm (0.01 mg / g) or more of methionine, and more preferably contains 5 or more selected from valine, isoleucine, leucine, arginine, 0.5 to 1.5 in mass ratio to methionine, 5 to 25 in trehalose, and 250 to 700 in SAM, and more preferably contains 10 ppm (0.01 mg / g) or more of methionine, and more preferably contains valine, isoleucine, and leucine in mass ratios of 5 to 15 in mass ratio to methionine, and more preferably contains 5 or more selected from valine, isoleucine, and leucine in mass ratios of 5 to 10 in mass ratio to methionine. A composition containing 6 or more selected from leucine, 100-250 arginine, 0.5-1.5 gamma-aminobutyric acid, 5-25 trehalose, and 250-700 SAM is particularly preferred, and a composition containing 10 ppm (0.01 mg / g) or more of methionine, with a mass ratio of 5-15 of valine, 3-10 isoleucine, 3-10 leucine, 100-250 arginine, 0.5-1.5 gamma-aminobutyric acid, 5-25 trehalose, and 250-700 SAM is most preferred.

[0061] (Method of manufacturing the composition) The method for producing the above composition includes a culture step and may further include other steps. The aforementioned composition is as described in (Composition) above.

[0062] -Culture process- The aforementioned culture step is a step of culturing yeast. The yeast in question is a yeast having a gene encoding the Met4 mutant protein, as described above under (Yeast).

[0063] The culture method is not particularly limited and can be appropriately selected depending on the purpose, but shaking culture is preferred.

[0064] There are no particular restrictions on the lower limit of the methionine concentration in the culture medium during the culture process described above, and it can be appropriately selected depending on the purpose. However, a concentration of 0.1 mM or higher is preferred, 0.5 mM or higher is more preferred, 1 mM or higher is even more preferred, 10 mM or higher is particularly preferred, and 20 mM or higher is most preferred. There are no particular restrictions on the upper limit of the methionine concentration in the culture medium during the culture process described above, and it can be appropriately selected depending on the purpose, but it is preferably 100 mM or less, more preferably 50 mM or less, and even more preferably 30 mM or less. Furthermore, a range of values ​​where one of the values ​​indicated as the lower limit and one of the values ​​indicated as the upper limit are used as the preferred range.

[0065] -Other processes- The aforementioned other steps are not particularly limited and can be appropriately selected depending on the purpose. Examples include a yeast recovery step, a yeast washing step, and a yeast freeze-drying step after the culture step. [Examples]

[0066] The following describes embodiments of the present invention, but the present invention is not limited in any way to these embodiments.

[0067] (Production of Met4 mutant protein) (1) Preparation of DNA fragments for transformation 1. DNA fragment D (DNA fragment for HL69 met4 K163R synthesis) Forward primer 1 and reverse primer 2 were used as primers, and HL69 bacterial cells (MATα) were used as template DNA. Colony PCR was performed using KOD One® PCR Master Mix-Blue- manufactured by Toyobo Co., Ltd. ((98°C for 10 seconds, 55°C for 5 seconds, 68°C for 10 seconds) × 40 cycles) to produce DNA fragment A. Forward primer 1 (SEQ ID NO: 2) 5'-ACTAGTCAGCTGATGAAGCAGGAGCAGTCC-3' Reverse Primer 2 (SEQ ID NO: 3) 5'- GACGCTGGATCCTTATTCTTTCTGAACCTTCTC-3'

[0068] DNA fragment A and the pFA6-natNT2 plasmid (JANKE, Carsten, et al. Yeast, 2004, 21.11: 947-962) were restricted enzyme-treated with PvuII and BamHI restriction enzymes from Takara Bio Inc., and ligated using the DNA Ligation Kit Ver.1 from Takara Bio Inc. to obtain plasmid a.

[0069] Next, using forward primer 3 and reverse primer 4 as primers and HL69 bacterial cells (MATα) as template DNA, colony PCR was performed using KOD One® PCR Master Mix-Blue manufactured by Toyobo Co., Ltd. ((98°C for 10 seconds, 55°C for 5 seconds, 68°C for 1 second) × 40 cycles) to produce DNA fragment B. Forward primer 3 (SEQ ID NO: 4) 5'-GCAGCGGAGCTCAATAACAGACTATACAGTTGAATTA-3' Reverse primer 4 (SEQ ID NO: 5) 5'-ACTAGTGATATCAGTGTCTTCATCACTTGAAC-3'

[0070] DNA fragment B and plasmid a were treated with restriction enzymes SacI and EcoRV from Takara Bio Inc., and plasmid b was prepared by ligation using the DNA Ligation Kit Ver.1 from Takara Bio Inc.

[0071] Next, forward primer 5 and reverse primer 6 were used as primers, and plasmid b as template DNA. PCR was performed using KOD One® PCR Master Mix-Blue from Toyobo Co., Ltd. ((98°C for 10 seconds, 55°C for 5 seconds, 68°C for 32 seconds) × 40 cycles) to produce DNA fragment C. Forward primer 5 (SEQ ID NO: 6) 5'-GGATAGGCCGAGCAATAACAATAAC-3' Reverse primer 6 (SEQ ID NO: 7) 5'-TCGGCCTATCCTCGTCTGGGAAG-3'

[0072] Subsequently, plasmid c was prepared by ligation using the DNA Ligation Kit Ver.1 manufactured by Takara Bio Inc.

[0073] Next, forward primer 7 and reverse primer 8 were used as primers, and plasmid c as template DNA. PCR was performed using KOD One® PCR Master Mix-Blue from Toyobo Co., Ltd. ((98°C for 10 seconds, 55°C for 5 seconds, 68°C for 20 seconds) × 40 cycles) to produce DNA fragment D. Forward primer 7 (SEQ ID NO: 8) 5'-ATGAAGCAGGAGCAGTCC-3' Reverse primer 8 (SEQ ID NO: 9) 5'-AGTGTCTTCATCACTTGAAC-3'

[0074] 2. DNA fragment G (DNA fragment for HL69 met4 IRΔ production) Forward primer 7 and reverse primer 9 were used as primers, and plasmid b as template DNA. PCR was performed using KOD One® PCR Master Mix-Blue from Toyobo Co., Ltd. ((98°C for 10 seconds, 55°C for 5 seconds, 68°C for 1 second) × 40 cycles) to produce DNA fragment E. Reverse primer 9 (SEQ ID NO: 10) 5'-GAGGAATTTTGAGCAGAAGTAGGGTCCTCATGCAATACGT-3'

[0075] Next, forward primer 10 and reverse primer 8 were used as primers, and plasmid b as template DNA. PCR was performed using KOD One® PCR Master Mix-Blue from Toyobo Co., Ltd. ((98°C for 10 seconds, 55°C for 5 seconds, 68°C for 15 seconds) × 40 cycles) to produce DNA fragment F. Forward primer 10 (SEQ ID NO: 11) 5'-ACTTCTGCTCAAAATTCCTC-3'

[0076] Next, DNA fragment G was prepared by using DNA fragment E as a forward primer, reverse primer 8 as a reverse primer, and DNA fragment F as template DNA, and performing PCR using KOD One® PCR Master Mix-Blue- manufactured by Toyobo Co., Ltd. ((98°C for 10 seconds, 55°C for 5 seconds, 68°C for 15 seconds) × 40 cycles).

[0077] 3. DNA fragment I (DNA fragment for HL69 met4 K163R_IRΔ production) Forward primer 5 and reverse primer 6 were used as primers, and DNA fragment E was used as template DNA. PCR was performed using KOD One® PCR Master Mix-Blue- manufactured by Toyobo Co., Ltd. ((98°C for 10 seconds, 55°C for 5 seconds, 68°C for 1 second) × 40 cycles) to produce DNA fragment H.

[0078] Next, DNA fragment I was prepared by using DNA fragment H as the forward primer, reverse primer 8 as the reverse primer, and DNA fragment F as the template DNA, and performing PCR using KOD One® PCR Master Mix-Blue- manufactured by Toyobo Co., Ltd. ((98°C for 10 seconds, 55°C for 5 seconds, 68°C for 15 seconds) × 40 cycles).

[0079] 4. DNA fragment J'~M' (for creating HL69 met4 K163R_IR point mutation) To introduce point mutations within the IR region, fragments J'(H198L), K'(S210F), L'(K216E), and M'(G270R) were prepared as follows.

[0080] Forward primer 11 and reverse primer 12 were used as primers for fragment J, forward primer 13 and reverse primer 14 for fragment K, forward primer 15 and reverse primer 16 for fragment L, and forward primer 17 and reverse primer 18 for fragment M. Plasmid c was used as the template DNA in all cases, and PCR was performed using KOD One® PCR Master Mix-Blue from Toyobo Co., Ltd. ((98°C for 10 seconds, 55°C for 5 seconds, 68°C for 32 seconds) × 40 cycles) to produce each DNA fragment J to M. Forward primer 11 (SEQ ID NO: 12) 5'-GAAATCCTCTCTTCTTGACT-3' Reverse primer 12 (SEQ ID NO: 13) 5'-AGTCAAGAAGAGAGGATTTC-3' Forward primer 13 (SEQ ID NO: 14) 5'-CCTAACTTTTCAATACGACC-3' Reverse primer 14 (SEQ ID NO: 15) 5'-GGTCGTATTGAAAAGTTAGG-3' Forward primer 15 (SEQ ID NO: 16) 5'-CGACCAATCAAAGTCTCGATT-3' Reverse primer 16 (SEQ ID NO: 17) 5'-AATCGAGACTTTGATTGGTCG-3' Forward primer 17 (SEQ ID NO: 18) 5'-GACCATGGCCGCTTCACAAA-3' Reverse Primer 18 (SEQ ID NO: 19) 5'-TTTGTGAAGCGGCCATGGTC-3'

[0081] Next, using forward primer 7 and reverse primer 8 as primers and DNA fragments J to M respectively as template DNA, PCR was performed using KOD One® PCR Master Mix-Blue manufactured by Toyobo Co., Ltd. ((98°C for 10 seconds, 55°C for 5 seconds, 68°C for 20 seconds) × 40 cycles) to produce DNA fragments J' to M'.

[0082] (2) Transformation of budding yeast 1. Strain We used HL69 (MATα), a haploid budding yeast, and X2180-1A (MATa), a laboratory yeast from YGSC (Yeast Genetic Stock Center) / ATCC (American Type Culture Collection). HL69 (MATα) was isolated from sake yeast K10 (Kyoukai No. 10 (MATa / α)) by random spore isolation (Matsumoto K, Y Adach, A Toh-e and Y Oshima. 1980. Function of positive regulatory gene gal4 in the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae: evidence that the GAL81 region codes for part of the gal4 protein. J Bacteriol 141:508-527).

[0083] 2. Transformation Method DNA fragments D, G, I, and J'~M' were introduced into HL69(MATα) and DNA fragment I was introduced into X2180-1A using the electroporation method (Masafumi Muraji; Wataru Takebe, On Electroporation of Yeast, Journal of the Brewing Society of Japan, 1996, 91.12:867-872). These strains were inoculated onto a medium containing 25 ppm noseoslysin to obtain transformants. The aforementioned 25 ppm norseothricin-containing medium was prepared by dissolving JenaBioScience GmbH's "Nourseothricin-Solution, NTC or clonNAT, sterile ready-to-go stock solution" in YPD medium to a concentration of 25 ppm.

[0084] (3) Expression analysis of sulfur metabolism-related genes Four strains (H198L, S210F, K216E, G270R) with point mutations in the IR region (189-312a.a.) of HL69 (sake yeast haploid), HL69 met4 K163R, HL69 met4 IRΔ, HL69 met4K163R_IRΔ, and HL69 met4 K163R were cultured overnight in 3 mL of YPD medium at 30°C with shaking. After that, each strain was subcultured in 10 mL of SD medium with an OD600 of 0.5 and cultured at 30°C with shaking.

[0085] After 4 hours, methionine (Fujifilm Wako Pure Chemical Corporation) was added to achieve a final concentration of 1 mM. After another hour, the collected cells were washed with cold water, and 10 OD units of bacterial cells were recovered by centrifugation. After washing with cold water, RNA was extracted using MACHEREY-NAGEL's "NucleoSpin® RNA," and the RNA solution was diluted to a concentration of 50 ng / μL.

[0086] Real-time PCR (42°C for 5 minutes, 95°C for 10 seconds, 95°C for 5 seconds, 60°C for 30 seconds) x 40 cycles was performed using the following forward and reverse primers as primers, Takara Bio Inc.'s "One Step TB Green® PrimeScript® PLUS RT-PCR Kit (Perfect Real Time)" as the real-time PCR kit, and BIO-RAD's "CFX Connect® Real-time PCR Analysis System" as the real-time PCR instrument (N=3).

[0087] The genes analyzed were SAM1, MET17, and CYS3, which encode S-adenosylmethionine (SAM) synthase, homocysteine ​​synthase, and cysteine ​​synthase, respectively. These expression levels were standardized by dividing them by the expression level of ACT1, which encodes an endogenous control actin constituent protein. Furthermore, we similarly performed expression analysis of sulfur metabolism-related genes using two strains of laboratory yeast, X2180-1A and X2180-1A met4K163R_IRΔ. The results are shown in Figures 1 to 5.

[0088] Primer sequence a) SAM1 Forward Primer 19 (SEQ ID NO: 20) 5'-GTAACGTCCTTGTCGCCATTG-3' Reverse primer 20 (SEQ ID NO: 21) 5'-AAGGCAAACCCTCTGGAGTTTC-3' b) MET17 Forward Primer 21 (SEQ ID NO: 22) 5'-AATGGATTGGTGGTCATGGTA-3' Reverse Primer 22 (SEQ ID NO: 23) 5'-GATATCCTTCGGCAGGTTGA-3' c)CYS3 Forward primer 23 (SEQ ID NO: 24) 5'-ATACGGGTTGGCTTTCTCCTC-3' Reverse primer 24 (SEQ ID NO: 25) 5'-TCTGTGGGTACCACCGTACAC-3' d) ACT1 Forward primer 25 (SEQ ID NO: 26) 5'-GGTTGCTGCTTTGGTTATTGA-3' Reverse primer 26 (SEQ ID NO: 27) 5'-TTTTGACCCATACCGACCAT-3'

[0089] As shown in Figures 1 to 3, under conditions of 1 mM methionine addition, the expression levels of SAM1 (Figure 1), MET17 (Figure 2), and CYS3 (Figure 3) in HL69 met4K163R_IRΔ increased by 7.4 times, 12.1 times, and 9.6 times, respectively, compared to HL69. Furthermore, four strains (H198L, S210F, K216E, and G270R) possessing point mutations within the IR region of HL69 met4 K163R also showed higher expression levels of SAM1, MET17, and CYS3 than HL69 or HL69 met4K163R. The above findings indicate that strains possessing both the K163R mutation and a deletion or point mutation in the IR region exhibit improved expression of sulfur metabolism-related genes.

[0090] As shown in Figures 4 and 5, the expression levels of SAM1 (Figure 4) and MET17 (Figure 5) increased 4.0 times and 6.8 times, respectively, in X2180-1A met4K163R_IRΔ compared to laboratory yeast X2180-1A. The above findings demonstrate that the enhancement of sulfur metabolism due to the K163R mutation and deletion or point mutations in the IR region is a universal phenomenon observed even in laboratory yeast.

[0091] (4) Measurement of metabolite levels i) Intracellular amino acid content HL69 and HL69 met4K163R_IRΔ were pre-cultured overnight in YPD medium at 30°C. The following day, after washing with sterile water, the cells were subcultured in 1 L of SD+1 mM methionine medium to an OD600 of 0.1 and cultured at 30°C with shaking at 120 rpm. Cells in the logarithmic growth phase (OD600 = 1-3) were collected by centrifugation, washed, and then freeze-dried to obtain freeze-dried cells. Next, 10 mg of freeze-dried bacterial cells were suspended in 250 μL of sterile water and heated at 99°C for 20 minutes to extract amino acids. After centrifugation, the supernatant was collected, diluted fourfold with lithium citrate buffer (pH 2.2), and filtered through a 0.45 μm microfilter (N=3). Subsequently, the amino acid extract was analyzed using Shimadzu Corporation's Prominence amino acid analysis system under the following analytical conditions. The results are shown in Table 1. In Table 1, "DCW" refers to the dry cell weight.

[0092] Analysis conditions Column: Shim-pack Amino-Li, 6.0 × 100 mm, 5 μm Mobile phase: Amino acid mobile phase kit Li-type Flow rate: 0.6mL / min Column temperature: 39℃ Detection: Excitation wavelength 350 nm, fluorescence wavelength 450 nm

[0093] ii) Trehalose content within the bacterial cell Freeze-dried bacterial cells were obtained using the same method as described in "i) Intracellular amino acid content". 8 mg of freeze-dried bacterial cells were placed in a tube for a multi-bead shocker (Yasui Kikai Co., Ltd.) (N=3), an equal amount of glass beads were added to the bacterial cells, and the cells were crushed using a multi-bead shocker (Yasui Kikai Co., Ltd.) (2800 rpm, ON for 30 seconds, OFF for 30 seconds x 5 times). Next, 1 mL of a methanol:chloroform:water = 2.5:1:1 mixture was added, along with 60 μL of 0.2 mg / mL of ribitol (Fujifilm Wako Pure Chemical Industries, Ltd.) as an internal standard reagent. The mixture was then incubated in an Eppendorf thermomix at 37°C and 1400 rpm for 30 minutes. After centrifugation, 700 μL of the supernatant was transferred to a tube. 400 μL of distilled water was added, and after centrifugation, 700 μL of the supernatant was taken and transferred to another tube. The mixture was concentrated by centrifugation in a centrifugal evaporator for 4 hours and freeze-dried overnight. 100 μL of a 20 mg / mL methoxyamine hydrochloride (Sigma-Aldrich) solution dissolved in pyridine (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the concentrated sample and incubated at 30°C and 1400 rpm for 90 minutes using a thermomixer (Eppendorf). 50 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (GL Sciences Inc.) was added and incubated at 37°C and 1400 rpm for 30 minutes using a thermomixer. Furthermore, 1 mL each of aqueous solutions of trehalose (Fujifilm Wako Pure Chemical Industries, Ltd.) at concentrations of 0, 0.01, 0.025, and 0.05 mg / mL were prepared as calibration curve samples. 60 μL of 0.2 mg / mL of ribitol was added as an internal standard reagent, and the mixtures were incubated in a thermomixer at 37°C and 1400 rpm for 30 minutes. The solution, after being kept warm, was analyzed using a Shimadzu GCMS-QP2010 Plus under the following analytical conditions. The results are shown in Table 1.

[0094] Analysis conditions Column: CP-SIL8CB low bleed Injection temperature: 230℃ Injection volume: 1 μL Split ratio: 25:1 GC oven: 2 minutes at 80°C, then 15°C / minute from 80°C to 320°C, and finally 6 minutes at 320°C. Lancefar Line: 250℃ MS ion source: 200℃ Detector: Scan mode, m / z 85-500, 20 scans / second

[0095] iii) SAM content inside bacteria Freeze-dried bacterial cells were obtained using the same method as described in "i) Intracellular amino acid content". 40 mg of freeze-dried bacterial cells were mixed with 1 mL of 10% perchloric acid (Fujifilm Wako Pure Chemical Industries, Ltd.) (N=3). The mixture was incubated at 30°C and 1400 rpm for 1 hour using a thermomixer. Next, the mixture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. This supernatant was then diluted 10-fold with ultrapure water and filtered through a 0.45 μm filter. This solution was analyzed using an Agilent 7100 capillary electrophoresis system manufactured by Agilent Technologies under the following analytical conditions. The results are shown in Table 1. Furthermore, in order to confirm SAM productivity according to the amount of methionine added, cultured cells were collected and the intracellular SAM content was analyzed in the same manner as before, except that 10 mM methionine-containing SD medium, 25 mM methionine-containing SD medium, or 50 mM methionine-containing SD medium were used instead of "1 mM methionine-containing SD medium". The results are shown in Table 2. In Table 2, "DCW" refers to the dry cell weight.

[0096] Analysis conditions Capillary tube: HPCE standard cap 75μm × 72cm Electrophoresis buffer: Buffer solution pH 2.5 for HPCE (sodium phosphate 50mM) Capillary temperature: 25℃ Applied voltage: positive 20kV Injection method: Pressurized injection method, 4.0 seconds at 50 mbar Detection: Signal; 260nm / 4nm, Reference: 360nm / 10nm

[0097] [Table 1]

[0098] [Table 2]

[0099] As shown in Table 1, the methionine content of HL69 met4K163R_IRΔ decreased to 19% of that of HL69. In contrast, the content of branched-chain amino acids (valine, isoleucine, leucine), arginine, GABA, trehalose, and SAM increased in HL69 met4K163R_IRΔ. Furthermore, the ratio of each component to methionine was shown to be significantly higher in HL69 met4K163R_IRΔ.

[0100] As shown in Table 2, the SAM content in HL69 met4K163R_IRΔ increased dramatically in proportion to the amount of methionine added.

[0101] Based on the above findings, in strains possessing both the K163R mutation and deletion or point mutations in the IR region, not only is the expression of sulfur metabolism-related genes improved, but the production of SAM is significantly increased. Furthermore, surprisingly, the production of branched-chain amino acids (valine, isoleucine, leucine), arginine, GABA, and trehalose is also significantly increased.

[0102] Examples of embodiments of the present invention include the following: <1> Met4 mutant protein, Wild type Saccharomyces cerevisiae The amino acid corresponding to lysine position 163 of the Met4 protein is deleted or substituted, Wild type Saccharomyces cerevisiae In the Met4 protein, at least one amino acid corresponding to positions 189 through 312 is deleted or substituted. This is a Met4 mutant protein characterized by improved transcriptional activation ability for sulfur metabolism genes compared to the wild-type Met4 protein. <2> Wild type Saccharomyces cerevisiaeThe amino acid corresponding to lysine at position 163 of the Met4 protein is substituted with arginine, <1> This is the Met4 mutant protein described in [reference]. <3> Wild type Saccharomyces cerevisiae The amino acids corresponding to positions 189 through 312 of the Met4 protein are deleted, or the amino acid corresponding to histidine at position 198, serine at position 210, lysine at position 216, or glycine at position 270 are substituted. <1> This is the Met4 mutant protein described in [reference]. <4> Wild type Saccharomyces cerevisiae The amino acid corresponding to lysine at position 163 of the Met4 protein is substituted with arginine, and Wild type Saccharomyces cerevisiae The following is a case where the amino acids corresponding to positions 189 through 312 of the Met4 protein are deleted, or the amino acid corresponding to histidine at position 198 is replaced with leucine, the amino acid corresponding to serine at position 210 is replaced with phenylalanine, the amino acid corresponding to lysine at position 216 is replaced with glutamic acid, or the amino acid corresponding to glycine at position 270 is replaced with arginine. <1> This is the Met4 mutant protein described in [reference]. <5> The above-mentioned, derived from budding yeast <1> This is the Met4 mutant protein described in [reference]. <6> Wild type Saccharomyces cerevisiae The amino acid sequence homology with the Met4 protein is 70% or more. <1> This is the Met4 mutant protein described in [reference]. <7> The aforementioned <1> from <6> This gene is characterized by encoding a Met4 mutant protein as described in any of the following. <8> The aforementioned <7> This yeast is characterized by having the genes described in [reference]. <9> The aforementioned <1> from <6> Met4 mutant protein as described in any of the above <7> The genes described above, and the aforementioned <8> This composition is characterized by containing one or more yeasts selected from those described above. <10> Contains 10 ppm (0.01 mg / g) or more of methionine, The above is characterized by containing two or more substances selected from valine in a mass ratio of 5 to 15 to methionine mass, isoleucine in 3 to 10, leucine in 3 to 10, arginine in 100 to 250, γ-aminobutyric acid in 0.5 to 1.5, trehalose in 5 to 25, and SAM in 250 to 700. <9> The composition is as described above. <11> A method for producing a composition comprising two or more components selected from valine, isoleucine, leucine, arginine, γ-aminobutyric acid, trehalose, and SAM, the <8> This is a method for producing a composition, characterized by including a culture step of culturing the yeast described above.

Claims

1. Met4 mutant protein, In wild-type Saccharomyces cerevisiae, the amino acid corresponding to lysine position 163 in the Met4 protein is deleted or substituted, In the wild-type Saccharomyces cerevisiae Met4 protein, at least one amino acid corresponding to positions 189 through 312 is deleted or substituted. A Met4 mutant protein characterized by improved transcriptional activation ability for sulfur metabolism genes compared to the wild-type Met4 protein.

2. The Met4 mutant protein according to claim 1, wherein the amino acid corresponding to lysine at position 163 of the Met4 protein of wild-type Saccharomyces cerevisiae is substituted with arginine.

3. The Met4 mutant protein according to claim 1, wherein the amino acids corresponding to positions 189 to 312 of the wild-type Saccharomyces cerevisiae Met4 protein are deleted, or the amino acid corresponding to histidine at position 198, the amino acid corresponding to serine at position 210, the amino acid corresponding to lysine at position 216, or the amino acid corresponding to glycine at position 270 is substituted.

4. In wild-type Saccharomyces cerevisiae, the amino acid corresponding to lysine at position 163 of the Met4 protein is substituted with arginine, and The Met4 mutant protein according to claim 1, wherein the amino acids corresponding to positions 189 to 312 of the wild-type Saccharomyces cerevisiae Met4 protein are deleted, or the amino acid corresponding to histidine at position 198 is replaced with leucine, the amino acid corresponding to serine at position 210 is replaced with phenylalanine, the amino acid corresponding to lysine at position 216 is replaced with glutamic acid, or the amino acid corresponding to glycine at position 270 is replaced with arginine.

5. The Met4 mutant protein according to claim 1, derived from budding yeast.

6. The Met4 mutant protein according to claim 1, wherein the amino acid sequence homology with the Met4 protein of wild-type Saccharomyces cerevisiae is 70% or more.

7. A gene characterized by encoding the Met4 mutant protein described in any one of claims 1 to 6.

8. A yeast characterized by having the gene described in claim 7.

9. A composition characterized by comprising one or more selected from the Met4 mutant protein described in any one of claims 1 to 6, the gene described in claim 7, and the yeast described in claim 8.

10. It contains 10 ppm (0.01 mg / g) or more of methionine. The composition according to claim 9, characterized by comprising two or more elements selected from valine in a mass ratio of 5 to 15 to methionine, isoleucine in 3 to 10, leucine in 3 to 10, arginine in 100 to 250, γ-aminobutyric acid in 0.5 to 1.5, trehalose in 5 to 25, and SAM in 250 to 700.

11. A method for producing a composition comprising two or more components selected from valine, isoleucine, leucine, arginine, γ-aminobutyric acid, trehalose, and SAM, characterized by comprising a culture step of culturing the yeast described in claim 8.

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