TW201540832A - Method for producing [gamma]-Glu-Abu and method for producing yeast extract containing [Gamma]-Glu-Abu - Google Patents

Abstract

Provided is a method for producing [gamma]-Glu-Abu and method for producing yeast extract containing [gamma]-Glu-Abu. Yeast, which is capable of producing [gamma]-Glu-Abu modified so as to retain a variant GAP1 gene and/or modified so that the activity of protein coded in the YGL114w gene increases, is cultivated in a culture medium to generate and accumulate [gamma]-Glu-Abu in the culture medium, and [gamma]-Glu-Abu is harvested from the culture medium to thereby produce [gamma]-Glu-Abu. A yeast extract containing [gamma]-Glu-Abu is produced by preparing a yeast extract using yeast as a starting material having the ability to accumulate [gamma]-Abu, the starting-material yeast being modified so that the activity of the protein coded in the YGL114w is reduced.

Description

Method for producing γ-Glu-Abu and method for producing yeast extract containing γ-Glu-Abu

The present invention relates to a method for controlling localization of γ-Glu-Abu inside and outside cells in yeast and its utilization. The present invention relates in particular to a method for producing γ-Glu-Abu and a method for producing a yeast extract containing γ-Glu-Abu. γ-Glu-Abu or a yeast extract containing the same is useful in the food field.

The γ-Glu-Abu system has a peptide that imparts an "alcoholic taste". "Aromatic taste" means that it is not possible to express only five basic tastes of sweet taste, salty taste, sour taste, bitter taste, and umami, and also enhances the thickness. (tickness), growth (mouthfulness), continuity, harmony, etc. The basic taste of marginal tastes or marginal flavors.

γ-Glu-Abu is produced by L-glutamic acid (L-Glu) and α-aminobutyric acid (Abu) by the action of γ-glutamine-based cysteine synthase. Further, Abu is produced by α-butyric acid (α-KB) by the action of an aminotransferase. Therefore, by enhancing, for example, α-butyric acid synthase, Enzyme activity related to γ-Glu-Abu biosynthesis, such as aminotransferase and γ-glutamic acid cysteine synthase, increases the accumulation of γ-Glu-Abu in cells (Patent Document 1) ).

The Gap1 gene is a gene encoding a Gap1 protein of a general amino acid permease of an amino acid. The Gap1 protein has the transport capacity of all natural amino acids and many amino acid analogs. The Gap1 protein is known to be localized in response to the amount of amino acid inside and outside the cell (Non-Patent Document 1). That is, the Gap1 protein is localized in the cell membrane when the amino acid is scarcely present, and when a large amount of amino acid is present, it is ubiquitinated and decomposed in the vacuole. On the other hand, the mutated Gap1 protein in which the quaternary acid of the ninth and the 16th position is substituted with arginine does not have a ubiquitination site, and thus is not decomposed by ubiquitination. Therefore, the variant Gap1 protein is normally localized to the cell membrane regardless of the amount of amino acid inside and outside the cell (Non-Patent Document 2).

The YGL114W gene is a gene encoding a protein predicted to be a peptide transport protein.

However, transport proteins associated with the uptake and/or excretion of gamma-Glu-Abu are not known.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] WO2012/046731

[Non-patent literature]

[Non-Patent Document 1] Cain NE, Kaiser CA., Mol. Biol. Cell., 2011, Jun 1; 22(11): 1919-29.

[Non-Patent Document 2] Soetens O, De Craene JO, Andre B., J. Biol. Chem., 2001, Nov 23; 276(47): 43949-57.

The object of the present invention is to develop a novel technique for controlling the localization of γ-Glu-Abu cells in and out of yeast, and to provide an efficient method for producing γ-Glu-Abu and a yeast extract containing γ-Glu-Abu. law.

As a result of intensive studies to solve the above problems, the present inventors have found that the accumulation of extracellular γ-Glu-Abu is enhanced by the normalized localized GAP1 gene expression in yeast synthesized by the enhanced γ-Glu-Abu. the amount. Further, the present inventors have found that the concentration of γ-Glu-Abu in the cells is decreased or increased by the high expression or destruction of the YGL114w gene in the yeast synthesized by the enhanced γ-Glu-Abu. Based on these findings, the inventors completed the present invention.

That is, the present invention can be exemplified as follows.

[1]

A method for producing γ-Glu-Abu, which comprises culturing a yeast having a γ-Glu-Abu production capacity in a medium to make γ- Glu-Abu accumulates in the medium, and adopts γ-Glu-Abu from the medium, and the yeast has the properties of either or both of the following (1) to (2): (1) retaining the variant The manner of the GAP1 gene is altered; (2) is altered in such a way that the activity of the protein encoded by the YGL114w gene is increased.

[2]

The method according to [1], wherein the variant GAP1 gene is a gene having the following variation of (A) and (B): (A) the quaternary acid residue at position 9 of the wild-type Gap1 protein is substituted with Variation of other amino acid residues; (B) The amino acid residue at position 16 of the wild-type Gap1 protein is substituted with a variation of other amino acid residues.

[3]

The method according to [2], wherein the quaternary acid residue at the 9th and 16th positions is substituted with a arginine residue.

[4]

The method of [2] or [3] wherein the wild type Gap1 protein is a protein selected from the group consisting of (a) to (c): (a) comprising the amino acid represented by SEQ ID NO: 20. a protein of the sequence; (b) an amino acid sequence comprising a substitution, deletion, insertion, or addition of one or more amino acid residues, and having an amino group Acid transport protein active protein; (c) comprises 90% of the amino acid sequence shown in SEQ ID NO: An amino acid sequence of the same identity and having a protein of amino acid transport protein activity.

[5]

The method of any one of [1] to [4], wherein the activity of the protein encoded by the YGL114w gene is increased by increasing the expression of the YGL114w gene.

[6]

The method of [5], wherein the expression of the YGL114w gene is increased by increasing the copy number of the gene and/or changing the expression regulatory sequence of the gene.

[7]

A method for producing a yeast extract containing γ-Glu-Abu, which comprises preparing a yeast extract by using yeast having a γ-Glu-Abu accumulation ability as a raw material, and the aforementioned yeast has the following ( 3) Properties: (3) The manner of the activity of the protein encoded by the YGL114w gene is decreased.

[8]

The method of [7], wherein the activity of the protein encoded by the YGL114w gene is decreased by decreasing the expression of the YGL114w gene or by disrupting the gene.

[9]

The method of any one of [1] to [8], wherein the aforementioned YGL114w base The DNA selected from the group consisting of (A) to (E): (A) DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 31; (B) DNA encoding the protein, The protein line is contained in the amino acid sequence shown in SEQ ID NO: 31, and contains a substitution, deletion, insertion, or additional amino acid sequence of one or several amino acid residues, and has an increased activity in yeast. Yeast γ-Glu-Abu production is improved compared to non-modified strains when the γ-Glu-Abu production is improved compared to non-modified strains and/or when activity is reduced in yeast. (C) a protein-encoding DNA comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 31, and having an increase in yeast When active, the γ-Glu-Abu production of yeast is improved compared to non-modified strains, and/or the activity is reduced in yeast, the γ-Glu-Abu accumulation of yeast is compared with non-change The strain has improved properties; (D) DNA comprising the base sequence shown in SEQ ID NO: 30; (E) and SEQ ID NO: 30 The complementary sequence of the base sequence or the probe which can be prepared by the complementary sequence will hybridize under stringent conditions, and encode the DNA of the protein which has the activity of increasing activity in yeast, γ-Glu-Abu of yeast When the production amount is improved compared to the non-modified strain, and/or the activity is decreased in the yeast, the γ-Glu-Abu accumulation amount of the yeast is improved compared to the non-modified strain.

[10]

The method of any one of [1] to [9], wherein the yeast is further The activity of the enzyme selected from the group consisting of γ-glutamic acid cysteine synthase, α-butyric acid synthase, and aminotransferase is increased.

[11]

The method according to [10], wherein the aforementioned α-butyric acid synthase is an enzyme encoded by the CHA1 gene.

[12]

The method according to [10] or [11] wherein the aforementioned aminotransferase is an enzyme encoded by the BAT1 gene.

[13]

The method according to any one of [1] to [12] wherein the yeast is a yeast of the genus Saccharomyces or a yeast of Candida.

[14]

The method according to [13], wherein the yeast is Saccharomyces cerevisiae or Candida utilis.

[15]

A yeast having the properties of any one of the following (1) to (4) and having γ-Glu-Abu productivity and/or γ-Glu-Abu accumulation ability: (1) to retain the variant GAP1 The manner of the gene is changed; (2) is changed in such a manner that the activity of the protein encoded by the YGL114w gene is increased; (3) is changed in such a manner that the activity of the protein encoded by the YGL114w gene is decreased; (4) A combination of the properties of the above (1) and (2).

[16]

a yeast according to [15], which is further selected from the group consisting of γ-glutamine-based cysteine synthetase, α-butyric acid synthase, and aminotransferase or The manner in which the activity of the above enzymes increases increases.

Fig. 1 is a graph showing the effect of the high expression of the wild-type GAP1 gene and the cell membrane normalized localized GAP1 gene on the production of γ-Glu-Abu. The vertical axis indicates the concentration of γ-Glu-Abu in the culture supernatant. A is expressed at an OD600 = 1.8 time point, and B is a γ-Glu-Abu concentration at an OD600 = 2.5 time point.

Fig. 2 is a graph showing the effect of high expression and destruction of the YGL114w gene on the accumulation of γ-Glu-Abu. The vertical axis indicates the concentration of γ-Glu-Abu in the bacterial extract. A indicates the influence of the high expression of the YGL114w gene, and B indicates the influence of the destruction of the YGL114w gene.

The invention is described in detail below.

<1> Yeast of the present invention <1-1> Yeast of the present invention

The yeast of the present invention is localized inside and outside the cell of γ-Glu-Abu Controlled yeast. In the present invention, localization control of γ-Glu-Abu can be carried out using the GAP1 gene and/or the YGL114w gene. Further, in the present invention, Abu and Glu are L bodies. That is, "γ-Glu-Abu" means L-γ-glutamic acid-L-2-aminobutyric acid.

The yeast of the present invention specifically has the properties of any one of the following (1) to (4). This property is also referred to as "the nature of the invention".

(1) is changed in such a manner as to retain the variant GAP1 gene; (2) is changed in such a manner that the activity of the protein encoded by the YGL114w gene is increased; (3) is changed in such a manner that the activity of the protein encoded by the YGL114w gene is decreased; (4) A combination of the properties of the above (1) and (2).

The nature of the present invention can be appropriately selected in accordance with the localized control of γ-Glu-Abu.

For example, γ-Glu-Abu can be produced extracellularly by altering the yeast in a manner that retains the variant GAP1 gene, and/or by increasing the activity of the protein encoded by the YGL114w gene. That is, one aspect of the yeast of the present invention may be changed in such a manner as to retain the variant GAP1 gene, and/or in a manner to increase the activity of the protein encoded by the YGL114w gene, having γ-Glu- Yeast of Abu production capacity (hereinafter also referred to as "γ-Glu-Abu production type yeast"). The "increased activity of the protein encoded by the YGL114w gene" may specifically be an increase in the expression of the YGL114w gene. The γ-Glu-Abu production type yeast can be used, for example, in the production of γ-Glu-Abu.

Further, for example, by changing the activity of the yeast by the protein encoded by the YGL114w gene, the accumulation of γ-Glu-Abu in the cells can be improved. In other words, the yeast of the present invention may be a yeast having a γ-Glu-Abu accumulation ability in a manner in which the activity of the protein encoded by the YGL114w gene is decreased (hereinafter also referred to as "γ-Glu-". Abu cumulative yeast"). "The activity of the protein encoded by the YGL114w gene is decreased", specifically, the expression of the YGL114w gene may be lowered, or the YGL114w gene may be disrupted. The γ-Glu-Abu accumulation type yeast can be used, for example, in the production of a yeast extract containing γ-Glu-Abu.

That is, the yeast of the present invention may have a γ-Glu-Abu productivity and/or a γ-Glu-Abu accumulation ability. The yeast of the present invention may inherently have γ-Glu-Abu productivity and/or γ-Glu-Abu accumulation ability, and may also be changed to have γ-Glu-Abu productivity and/or γ-Glu-Abu accumulation. ability. The yeast having the γ-Glu-Abu productivity and/or the γ-Glu-Abu accumulation ability can be, for example, γ-Glu-Abu-producing ability and/or γ-Glu-Abu accumulation ability, or borrowed from the yeast described later. It is obtained by enhancing the γ-Glu-Abu productivity and/or the γ-Glu-Abu accumulation ability of the yeast described later. The method for imparting or enhancing the γ-Glu-Abu productivity and/or the γ-Glu-Abu accumulation ability will be described later.

The yeast of the present invention can be obtained by changing the yeast having the γ-Glu-Abu productivity and/or the γ-Glu-Abu accumulation ability to have the properties of the present invention. Further, the yeast of the present invention can also be obtained by changing the yeast to have the properties of the present invention and imparting γ-Glu-Abu productivity and/or γ-Glu-Abu accumulation ability. Furthermore, the yeast of the present invention may also In order to obtain γ-Glu-Abu productivity and/or γ-Glu-Abu accumulation ability by changing to have the properties of the present invention. In the present invention, the modification of the yeast for constructing the present invention can be carried out in any order.

The "γ-Glu-Abu production capacity" refers to the ability of the yeast of the present invention to be cultured in a medium to produce and accumulate γ-Glu-Abu in a medium to a detectable extent. The yeast of the present invention may be a yeast which can accumulate more γ-Glu-Abu than the non-modified strain in the medium. The non-altered strain may be a wild strain or a parent strain. Further, the yeast of the present invention may be a yeast capable of accumulating γ-Glu-Abu in a medium in an amount of 1 mg/L or more, 5 mg/L or more, 10 mg/L or more, or 15 mg/L or more.

The "γ-Glu-Abu accumulation ability" refers to the ability of the yeast of the present invention to be cultured in a medium to produce and accumulate γ-Glu-Abu in a cell to a detectable extent. The yeast of the present invention may be a yeast which accumulates a larger amount of γ-Glu-Abu than the non-modified strain in the cells. The non-altered strain may be a wild strain or a parent strain. Further, the yeast of the present invention may be 0.4 μmol/g-DCW or more, 1 μmol/g-DCW or more, 2 μmol/g-DCW or more, 3 μmol/g-DCW or more, 5 μmol/g-DCW or more, or 10 μmol/ A yeast in which γ-Glu-Abu is accumulated in cells in an amount of g-DCW or more.

The yeast of the present invention may be a budding yeast or a split yeast. The budding yeast can be exemplified by a yeast belonging to the genus Saccharomyces cerevisiae or the like, Candida utilis, such as Candida utilis, and Pichia pastoris. Yeast (Pichia), Hansenula polymorpha (Hansenula polymorpha), etc., of the genus Hansenula. The yeast which is a genus of the genus Schizosaccharomyces, such as Schizosaccharomyces pombe, is exemplified. Among them, beer yeast or high protein Candida which is commonly used for the production of yeast extract is preferred. The yeast of the present invention may be in the form of a haplotype or a doubling of the doubling or more.

The S. cerevisiae may, for example, be Saccharomyces cerevisiae Y006 strain (FERM BP-11299). The Y006 strain was deposited on August 18, 2010, and was deposited in the Patent Biological Depository Center of the Industrial Technology Research Institute (now, the patent administrative biological deposit center of the independent administrative legal person product evaluation technology base institution, postal code: 292-0818, address: Japan It is given the registration number FERM BP-11299 in Kazusa, Kami-Ku, Chiba Prefecture, 2-5-8.

Further, the brewer's yeast may, for example, be Saccharomyces cerevisiae BY4743 strain (ATCC 201390) or Saccharomyces cerevisiae S288C strain (ATCC 26108). Further, as the high protein Candida, for example, the Candida albicans ATCC 22023 strain can be mentioned. Such strains are available, for example, from the American Type Culture Collection (Address 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States of America). That is, the distribution can be accepted using the registration number corresponding to each strain (refer to http://www.atcc.org/). The registration number corresponding to each strain is described in the catalogue of the American Type Culture Collection.

<1-2> Genes used in the present invention <1-2-1> GAP1 gene and Gap1 protein

Hereinafter, the GAP1 gene and the Gap1 protein will be described. The GAP1 gene is a gene encoding a membrane of a total amino acid permease. That is, the Gap1 protein encoded by the gene has an activity of transporting an amino acid and/or a peptide from the cell to the outside of the cell through a cell membrane. This activity is also referred to as "amino acid transport protein activity". The Gap1 protein may specifically have, for example, an activity of expelling γ-Glu-Abu from the outside of the cell.

In the present invention, the GAP1 gene having the "specific variation" described later is also referred to as a variant GAP1 gene, and the protein encoded thereby is also referred to as a variant Gap1 protein. Furthermore, the variant Gap1 protein is localized in the cell membrane. Therefore, the variant GAP1 gene is also called the cell membrane normal localized GAP1 gene, and the variant Gap1 protein is also called the cell membrane normal localized Gap1 protein. Further, in the present invention, the GAP1 gene which does not have the "specific variation" described later is also called the wild type GAP1 gene, and the protein encoded thereby is also called the wild type Gap1 protein. Further, in the Gap1 protein, a change in the amino acid sequence caused by "specific variation" in the GAP1 gene is also referred to as "specific variation".

The wild type GAP1 gene includes, for example, the yeast GAP1 gene. Specific examples of the yeast GAP1 gene include the GAP1 gene of Saccharomyces cerevisiae.

The base sequence of the GAP1 gene of S. cerevisiae is disclosed in the Saccharomyces Genome Database (http://www. Yeastgenome.org/). Further, the GAP1 gene of S. cerevisiae S288C strain (ATCC 26108) was registered in the NCBI database as the chromosome XI sequence of GenBank accession NC_001143, which corresponds to the sequence of 515063 to 516871. Further, the Gap1 protein of S. cerevisiae S288C strain (ATCC 26108) was registered as GenBank accession NP_012965. The base sequence of the GAP1 gene of S. cerevisiae S288C strain (ATCC 26108) and the amino acid sequence of the Gap1 protein encoded by the gene are shown in SEQ ID NOs: 19 and 20, respectively.

Further, the wild type Gap1 protein may be a variant of the above Gap1 protein as long as it does not have the "specific variation" described later and maintains the original function. Furthermore, there is a case where such a variant is referred to as a "preservation variant". Examples of the storage variant include a homolog of the above Gap1 protein or an artificial variant. "Maintaining the original function" means that the variant of the protein has an activity corresponding to the activity of the original protein. That is, the "maintaining function" in the case of the wild-type Gap1 protein means a variant of a protein having an amino acid transport protein activity.

Further, the wild type GAP1 gene may be a variant of the GAP1 gene as long as it encodes the Gap1 protein or a stored variant thereof.

A gene encoding a homolog of the above Gap1 protein can be easily obtained from a public database by, for example, using the above-described yeast GAP1 gene (SEQ ID NO: 19) as a BLAST search or FASTA search of a query sequence. Also, encoding the same Gap1 protein The gene of the source can be obtained, for example, by PCR using a yeast chromosome as a template and using an oligonucleotide prepared based on such well-known gene sequences as a primer.

The wild-type Gap1 protein may be substituted with one or several amino acids in one or several positions in the amino acid sequence as long as it does not have the "specific variation" described later and maintains the original function. A protein that is deleted, inserted, or appended with an amino acid sequence. In addition, the above-mentioned "one or several" differs depending on the position in the steric structure of the protein of the amino acid residue or the type of the amino acid residue, but specifically, for example, means 1 to 50 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, and particularly preferably 1 to 3.

Substitution, deletion, insertion, or addition of one or more of the above amino acids is a preservative variation that normally maintains protein function. Representatives of conservation variables are preservative substitutions. The storage-replacement substitution is between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid; In the case of acid, it refers to Gln and Asn; in the case of basic amino acid, it refers to Lys, Arg, and His; in the case of acidic amino acid, it refers to between Asp and Glu; In the case of acid, it means a variation in which each of Ser and Thr is substituted with each other. Substitutions considered as preservative substitutions, specifically, substitution of Ala for Ser or Thr; substitution of Arg for Gln, His or Lys; substitution of Asn for Glu, Gln, Lys, His or Asp; replacement by Asp Is Asn, Glu or Gln; substituted by Cys to Ser or Ala; replaced by Gln to Asn, Glu, Lys, His, Asp or Arg; taken by Glu Substituting Gly, Asn, Gln, Lys or Asp; substituted by Gly to Pro; replaced by His to Asn, Lys, Gln, Arg or Tyr; substituted by Ile to Leu, Met, Val or Phe; replaced by Leu to Ile, Met , Val or Phe; substituted by Ass for Asn, Glu, Gln, His or Arg; substituted by Met for Ile, Leu, Val or Phe; substituted by Phe for Trp, Tyr, Met, Ile or Leu; replaced by Ser for Thr Or Ala; substituted by Thr to Ser or Ala; substituted by Trp to Phe or Tyr; substituted by Tyr to His, Phe or Trp; and substituted by Val to Met, Ile or Leu. Further, in the case of the above-mentioned amino acid substitution, deletion, insertion, addition, or inversion, etc., the biological individual difference due to the gene, and the naturally occurring variation (mutant or variant) based on the difference of the species are also included. Produced by.

Further, the wild-type Gap1 protein may have 80% or more, preferably 90% or more, based on the entire amino acid sequence as long as it does not have the "specific variation" described later and maintains the original function. More preferably, it is 95% or more, more preferably 97% or more, and particularly preferably 99% or more of the same protein. In addition, in this specification, "homology" also refers to the case of "identity".

Further, the wild type GAP1 gene may be a probe which can be prepared from a well-known gene sequence as long as it does not have a "specific variation" described later and encodes a protein which maintains the original function, for example, the entire base sequence or A portion of the complementary sequence hybridizes to DNA under stringent conditions. "Stringent conditions" refers to conditions under which a so-called specific hybrid is formed and non-specific hybrids are not formed. As an example, DNA having high identity may be, for example, 80% or more, preferably 90% or more, and more preferably DNA which is 95% or more, more preferably 97% or more, and particularly preferably 99% or more, is hybridized to each other, and the DNA having a lower homology does not hybridize with each other, or the washing of the usual Southern hybrid method. The condition is equivalent to 60 ° C, 1 × SSC, 0.1% SDS; preferably 60 ° C, 0.1 × SSC, 0.1% SDS; more preferably 68 ° C, 0.1 × SSC, 0.1% SDS salt concentration and temperature, washed 1 time, preferably 2 to 3 times.

As described above, the probe used in the above hybridization method may also be a part of the complementary sequence of the gene. Such a probe can be produced by PCR using a oligonucleotide prepared based on a well-known gene sequence as a primer and a DNA fragment containing the base sequence as a template. For example, when a DNA fragment having a length of about 300 bp is used as a probe, washing conditions of the hybridization method include 50 ° C, 2 × SSC, and 0.1% SDS.

Further, the wild type GAP1 gene may be a wild type Gap1 protein as described above, and may be any one substituted with an equivalent codon. For example, the wild-type GAP1 gene can be changed to have the most appropriate codon depending on the frequency of codon usage of the host used.

In addition, the description of the variant of the above-mentioned gene or protein may be applied to any protein such as an enzyme related to the synthesis of γ-Glu-Abu, and a gene encoding the same.

The variant Gap1 protein has the "specific variation" described later in the amino acid sequence of the wild type Gap1 protein as described above.

In other words, in other words, the variant Gap1 protein may be the Gap1 protein of the above yeast, in addition to the "specific variation" described later. Quality or its preservation variant.

Specifically, for example, the variant Gap1 protein may be a protein having an amino acid sequence represented by SEQ ID NO: 20, in addition to the "specific variation" described later.

Specifically, for example, the variant Gap1 protein may have a substitution of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 20, in addition to the "specific variation" described later. A protein that is deleted, inserted, or otherwise amino acid sequence.

Specifically, for example, the variant Gap1 protein may have 80% or more, preferably 90%, based on the amino acid sequence shown by SEQ ID NO: 20, in addition to the "specific variation" described later. More than or equal to, more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more of the same amino acid sequence protein.

In other words, the variant Gap1 protein may include a "specific variation" described later in the Gap1 protein of the yeast, and a variant containing a preservative mutation may be further included in the site other than the "specific variation".

Specifically, for example, the variant Gap1 protein may have a "specific variation" to be described later in the amino acid sequence represented by SEQ ID NO: 20, and further include 1 or a number in a portion other than the "specific variation". A protein of a substitution, deletion, insertion, or additional amino acid sequence of an amino acid.

The variant GAP1 gene is not particularly limited as long as it encodes the variant Gap1 protein as described above.

Hereinafter, the "specific variation" of the variant GAP1 gene will be described.

"Specific variation" is the variation of the encoded Gap1 protein into the normal localization of the cell membrane. The "specific variation" may be exemplified by the combination of the following (A) and (B): (A) the quaternary acid residue at the 9th position of the wild type Gap1 protein is substituted with the variation of other amino acid residues; (B) The amino acid residue at position 16 of the wild-type Gap1 protein is substituted with a variation of other amino acid residues.

The type of "other amino acid" is not particularly limited as long as it is other than the amine acid. Examples of the amino acid other than the amino acid include glycine (Gly), alanine (Ala), lysine (Val), leucine (Leu), isoleucine (Ile), and serine ( Ser), threonine (Thr), cysteine (Cys), methionine (Met), aspartame (Asn), glutamine (Gln), proline (Pro), aspartate Amine (Asp), glutamic acid (Glu), arginine (Arg), histidine (His), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp). The quaternary acid residue at position 9 is preferably substituted, for example, with a arginine residue. The amino acid residue at position 16 is preferably substituted, for example, with a arginine residue.

In the present invention, the "amino acid residue at the X position of the wild type Gapl protein" means an amino acid residue corresponding to the amino acid residue at the X position in SEQ ID NO: 20 unless otherwise specified. . Further, the "X-th position" in the amino acid sequence means that the N-terminal end of the amino acid sequence is No. X, and the amino acid residue at the N-terminus is the amino acid residue at the first position. That is, the position of the above amino acid residue indicates the relative position, and the amino group In the absence, insertion, or addition of acid, the position may change before and after. For example, "the lysine residue at position 9 of the wild-type Gap1 protein" means an amino acid residue corresponding to the quaternary acid residue at the 9th position in SEQ ID NO: 20, which is more N-terminal than the ninth position. When the amino acid residue on the side is deleted, the amino acid residue No. 8 from the N-terminus is the "amino acid residue at the ninth position of the wild-type Gap1 protein". When the 1 amino acid residue is inserted at the N-terminal side from the ninth position, the amino acid residue No. 10 from the N-terminus is the "amino acid residue at the 9th position of the wild-type Gap1 protein. "."

In the case of any amino acid sequence, which amino acid residue is "an amino acid residue corresponding to the amino acid residue at the X position in SEQ ID NO: 20", the arbitrary amino acid sequence can be carried out by It is determined by alignment with the amino acid sequence of SEQ ID NO: 20. The alignment can be carried out, for example, using well-known gene analysis software. Specific software includes DNASIS manufactured by Hitachi Solutions, GENETYX manufactured by GENETYX, etc. (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24(1), 72-96, 1991; Barton GJ et al., Journal Of molecular biology, 198(2), 327-37.1987).

The variant GAP1 gene can be obtained by changing the wild type GAP1 gene to have the above-mentioned "specific variation". DNA changes can be made by well-known methods. Specifically, for example, a site-specific variation method for introducing a variation of a target at a target site of DNA can be exemplified by a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HAEds., Stockton press). (1989); Carter, P., Meth.in Enzymol., 154, 382 (1987)), or the method of using phage (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol. , 154, 367 (1987)). Further, the variant GAP1 gene can also be obtained by chemical synthesis.

Hereinafter, a method of changing yeast to a variant GAP1 gene will be described.

The change of yeast to the variant GAP1 gene can be achieved by introducing the variant GAP1 gene into yeast. Further, changing the yeast to the variant GAP1 gene can also be achieved by introducing a mutation into the wild-type GAP1 gene of the yeast by natural variation or mutation treatment.

The method of introducing the variant GAP1 gene into yeast is not particularly limited. The introduction of the variant GAP1 gene can be carried out in the same manner as the method of increasing the copy number of the gene described later. In the yeast of the present invention, the variant GAP1 gene may be expressed under the control of a promoter which can function in the yeast. In the yeast of the present invention, the variant GAP1 gene may be present on a vector that grows autonomously, such as a plastid, and may also be ligated to a chromosome. Regarding the promoter or vector which can be used, it is exemplified in the method of increasing the copy number of the gene described later. The yeast of the present invention may have only one copy of the variant GAP1 gene, and may have two or more copies. The yeast of the present invention may have only one variant of the GAP1 gene, or may have two or more variants of the GAP1 gene.

When the yeast of the present invention has a ploidy of two or more and the variant GAP1 gene is ligated to the chromosome, the yeast of the present invention can Different chromosomes have a chromosome in which the variant GAP1 gene is ligated and a chromosome that does not contain the variant GAP1 gene, and can also have a chromosome in which the variant GAP1 gene is inserted in the same chromosome.

The yeast of the present invention may or may not have a wild-type GAP1 gene. A yeast that does not have a wild-type GAP1 gene can be obtained by disrupting the wild-type GAP1 gene on the chromosome. The disruption of the wild-type GAP1 gene can be carried out, for example, by a method of destroying a gene described later. Specifically, for example, the wild-type GAP1 gene can be disrupted by deleting part or all of the coding region of the wild-type GAP1 gene. Further, a yeast having no wild-type GAP1 gene and having a variant GAP1 gene can be obtained by substituting the wild-type GAP1 gene on the chromosome with the variant GAP1 gene.

<1-2-2> YGL114W gene and YGL114W protein

Hereinafter, the YGL114W gene and the YGL114W protein will be described. The YGL114W gene is a gene encoding a protein predicted to be a peptide transport protein. That is, the YGL114W protein encoded by the gene may have a peptide transport protein activity. The YGL114W protein may specifically have, for example, an activity of excreting γ-Glu-Abu out of the cell.

Further, the YGL114W protein has the property of increasing the activity of the γ-Glu-Abu of the yeast compared to the non-altered strain when the activity is increased in the yeast, and/or the activity of the yeast when the activity is decreased in the yeast. The cumulative amount of γ-Glu-Abu is improved compared to non-modified strains. This property is also known as "the nature of YGL114W protein".

The YGL114W gene may, for example, be a yeast YGL114W gene. Specific examples of the yeast YGL114W gene include the YGL114W gene of Saccharomyces cerevisiae.

The base sequence of the YGL114W gene of S. cerevisiae is disclosed in the Saccharomyces Genome Database (http://www.yeastgenome.org/). Further, the YGL114W gene of S. cerevisiae S288C strain (ATCC 26108) is a sequence corresponding to chromosome VII of GenBank accession NC_001139 in the NCBI database, and corresponds to the sequence of 293460 to 295637. Further, the YGL114W protein of S. cerevisiae S288C strain (ATCC 26108) was registered as GenBank accession NP_011401. The base sequence of the YGL114W gene of S. cerevisiae S288C strain (ATCC 26108) and the amino acid sequence of the YGL114W protein encoded by the gene are shown in SEQ ID NOs: 30 and 31, respectively.

The YGL114W gene is not limited to the above-exemplified gene or a gene having a well-known base sequence, and may be a variant thereof as long as it encodes a protein that maintains the original function. Regarding the variant of the YGL114W gene or the YGL114W protein, the description of the storage variant of the wild type GAP1 gene and the wild type Gap1 protein can be used. The "maintaining function" of the YGL114W protein means that the protein variant has the above-described properties of the YGL114W protein. Further, the "maintaining function" of the YGL114W protein may also mean that the protein variant has an activity of discharging γ-Glu-Abu out of the cell.

Genes encoding the variant can be introduced, for example, into yeast By the means of increasing the activity of the variant in yeast, and confirming whether the production of γ-Glu-Abu is increased, thereby confirming whether the protein variant has an activity of increasing the activity in yeast, and whether or not the yeast has γ-Glu. - Abu production has improved properties compared to non-modified strains.

The activity of the variant in yeast can be reduced by, for example, disrupting the gene encoding the variant in yeast, and whether the accumulation of γ-Glu-Abu is increased can be confirmed, thereby confirming the variant of the protein in the yeast. When the activity is lowered, it has the property of increasing the γ-Glu-Abu accumulation amount of yeast compared to the non-modified strain.

<1-3> Other changes

The yeast of the present invention may further have other modifications. For example, the yeast of the present invention can be modified in such a manner as to impart or enhance γ-Glu-Abu productivity and/or γ-Glu-Abu accumulation ability. γ-Glu-Abu productivity and/or γ-Glu-Abu accumulation ability can reduce γ-glutamine thiol-cysteine synthesis by, for example, increasing the activity of γ-Glu-Abu biosynthesis-related enzymes The activity of the enzyme, the activity of the peptide-degrading enzyme, or a combination thereof, imparted or enhanced. In the present invention, the alteration of the yeast for constructing the present invention can be carried out in any order.

That is, the yeast of the present invention can be changed in such a manner as to increase the activity of the enzyme involved in the synthesis of γ-Glu-Abu in the cell. Examples of the enzymes involved in the synthesis of γ-Glu-Abu include α-butyric acid synthase, aminotransferase, and gamma glutamylcysteine synthetase. In the present invention, γ-Glu-Abu can be made The activity of one of the enzymes involved in the synthesis of γ-Glu-Abu can also increase the activity of two or more enzymes related to the synthesis of γ-Glu-Abu.

The "α-butyric acid synthase" referred to herein means a protein having an activity of catalyzing a reaction for producing α-butyric acid from L-threonine. The α-butyric acid synthetase may, for example, be serine/threonine deaminase or threonine deaminase. The gene encoding a serine/threonine deaminase can be exemplified by the CHA1 gene. The gene encoding the threonine deaminase can be exemplified by the ILV1 gene. Among these, for example, it is preferred to increase the activity of the serine/threonine deaminase encoded by the CHA1 gene. In the present invention, the activity of one α-butyric acid synthase can be increased, and the activity of two or more α-butyric acid synthase can be increased.

The base sequence of the CHA1 gene (system name: YCL064C) and the ILV1 gene (system name: YER086W) of S. cerevisiae is disclosed in the Saccharomyces Genome Database (http://www.yeastgenome.org/). The base sequence of the CHA1 gene of S. cerevisiae S288C strain (ATCC 26108) and the amino acid sequence of Cha1 protein encoded by the gene are shown in SEQ ID NOs: 22 and 23, respectively. The base sequence of the ILV1 gene of the S. cerevisiae S288C strain (ATCC 26108) and the amino acid sequence of the Ilv1 protein encoded by the gene are shown in SEQ ID NOs: 24 and 25, respectively.

The term "aminotransferase" as used herein refers to a protein having an activity of catalyzing a reaction for producing Abu from α-butyric acid by an aminotransfer reaction. The aminotransferase may, for example, be an alanine: a glyoxylamine group Alanine (glyoxylate aminotransferase), branched-chain amino acid transaminase, aspartate amino transferase, γ-aminobutyrate transaminase Gamma-aminobutyrate transaminase). The gene encoding alanine: glyoxylate aminotransferase can be exemplified by the AGX1 gene. The gene encoding the branched amino acid transaminase may, for example, be the BAT1 and BAT2 genes. The gene encoding the aspartate aminotransferase includes the AAT1 and AAT2 genes. The gene encoding γ-aminobutyric acid transaminase can be exemplified by the UGA1 gene. Among these, for example, it is preferred to increase the activity of the branched amino acid transaminase encoded by BAT1. In the present invention, the activity of one aminotransferase can be increased, and the activity of two or more aminotransferases can be increased.

AGX1 gene of Saccharomyces cerevisiae (system name: YFL030W), BAT1 gene (system name: YHR208W), BAT2 gene (system name: YJR148W), AAT1 gene (system name: YKL106W), AAT2 gene (system name: YLR027C), and The base sequence of the UGA1 gene (system name: YGR019W) is disclosed in the Saccharomyces Genome Database (http://www.yeastgenome.org/). The base sequence of the BAT1 gene of S. cerevisiae S288C strain (ATCC 26108) and the amino acid sequence of the Bat1 protein encoded by the gene are shown in SEQ ID NOs: 26 and 27, respectively.

The "gamma-glutamic acid cysteine synthetase" referred to herein is a protein which recognizes the activity of the reaction of γ-Glu-Abu by recognizing L-Glu and Abu as a matrix. Gamma-glutamic acid cysteine Synthetic enzymes may or may not have the activity of recognizing the reaction of γ-glutamic acid cysteine (γ-Glu-Cys) with L-Glu and L-Cys as a matrix. The activity of the γ-glutamine-based cysteine synthase is enhanced, for example, in U.S. Patent No. 7,553,638, and Dazhu Kangzhi et al. (Bioscience and Industry, Vol. 50, No. 10, pp. 989-994, 1992) and so on.

Further, the yeast of the present invention can be changed in such a manner that the activity of glutathione synthase in the cells is lowered. The term "glutathione synthetase" as used herein refers to a protein having an activity of recognizing the reaction of γ-Glu-Abu and Gly as a substrate and catalyzing the formation of γ-Glu-Abu-Gly. The glutathione synthetase may or may not have the activity of recognizing the reaction of γ-Glu-Cys-Gly by γ-Glu-Cys and Gly as a matrix.

The gene encoding the γ-glutamic acid cysteine synthase can be exemplified by the GSH1 gene. The gene encoding the glutathione synthetase can be exemplified by the GSH2 gene. The base sequence of the GSH1 gene and the GSH2 gene of S. cerevisiae is disclosed in the Saccharomyces Genome Database (http://www.yeastgenome.org/). Further, the base sequence of the GSH1 gene and the GSH2 gene of Candida albicans is disclosed in U.S. Patent No. 7,553,638. The base sequence of the GSH1 gene of S. cerevisiae S288C strain (ATCC 26108) and the amino acid sequence of the Gsh1 protein encoded by the gene are shown in SEQ ID NOs: 28 and 29, respectively.

Further, the yeast of the present invention can be changed in such a manner that the activity of the peptide-decomposing enzyme in the cell is lowered. Peptide-decomposing enzyme By the term, it refers to a protein having an activity of catalyzing a reaction of hydrolysis of a peptide bond of a peptide. Examples of the peptide-degrading enzyme include a protein encoded by the DUG1 gene, the DUG2 gene, the DUG3 gene, and the ECM38 gene (JP-A-2012-213376). Furthermore, Dug1p, Dug2p, and Dug3p, which are encoded by the DUG1 gene, the DUG2 gene, and the DUG3 gene, respectively, form a DUG complex and function. Decomposition of glutathione by the DUG complex, all of which are known to be required for Dug1p, Dug2p, and Dug3p (Ganguli D. et al., Genetics. 2007 Mar; 175(3): 1137-51), by reduction The protein activity of one or more selected from Dug1p, Dug2p, and Dug3p can reduce the activity of the DUG complex. Among these, it is preferred to at least reduce the activity of Dug2p. In the present invention, the activity of one type of peptide-degrading enzyme can be lowered, and the activity of two or more peptide-degrading enzymes can also be reduced. The base sequences of the DUG1 gene, the DUG2 gene, the DUG3 gene, and the ECM38 gene of S. cerevisiae are disclosed in the Saccharomyces Genome Database (http://www.yeastgenome.org/).

In addition, a gene that uses such "other changes" as long as it encodes a protein that maintains its original function is not limited to the above-exemplified gene or a gene having a well-known base sequence, and may be a variant thereof. Regarding the variant of the gene or protein, the description of the preservation variant of the wild type GAP1 gene and the wild type Gap1 protein can be used. Further, when the protein functions as a complex composed of a plurality of subunits, the "maintaining function" of each subunit can form a complex for each subunit and the remaining subunits, and the complex Have the right Should be active. That is, for example, regarding the "maintaining function" of each subunit of the DUG complex, a complex may be formed for each subunit and the remaining subunits, and the complex has a peptide decomposition activity.

Various changes as described above can be carried out by mutation processing or genetic engineering. The specific methods of genetic engineering of S. cerevisiae are described in many books. Further, regarding the high protein Candida, various methods have been reported in recent years, and these can be used. For example, in Chemical Engineering, June 1999 issue (23p-28p, Misawa Ryohiko), FEMS Microbiology Letters (Luis Rodriguez et al., 165 (1998) 335-340), WO 98/07873, Japanese Patent Laid-Open No. 8-173170, WO95/32289, Journal of Bacteriology (KEIJI KONDO et al., Vol. 177 No. 24 (1995) 7171-7177), WO 98/14600, JP-A-2006-75122, JP-A-2006-75123, JP-A 2007 A specific method is described in the prior documents of JP-A-2008-101867, and the like.

<1-4> Techniques for increasing the activity of proteins

Hereinafter, a method of increasing the activity of a protein will be described.

"Increased activity of protein" means that the activity per cell of the protein is increased relative to a non-altered strain such as a wild strain or a parent strain. Furthermore, "increased activity of proteins" is also known as "enhanced activity of proteins". "Increased activity of a protein" means, in particular, an increase in the number of molecules per cell of the protein as compared with a non-modified strain, and/or an increase in the function per molecule of the protein. That is, mentioning "protein The "activity" when the activity is increased is not limited to the catalytic activity of the protein, and may also mean the amount of transcription (the amount of mRNA) or the amount of translation (the amount of protein) of the gene encoding the protein. Further, "increased activity of protein" means not only increasing the activity of the protein in a strain having the activity of the target protein, but also imparting activity to the strain which is not active in the target protein. Further, as long as the activity of the protein is increased, the activity of the target protein can be imparted after the activity of the protein originally possessed by the yeast is lowered or disappeared.

The activity of the protein is not particularly limited as long as it is increased compared to the non-modified strain, and for example, it can be increased by 1.5 times or more, 2 times or more, or 3 times or more as compared with the non-modified strain. Further, when the non-modified strain does not have the activity of the target protein, the protein may be produced by introducing a gene encoding the protein. For example, the protein can be produced to such an extent that the activity of the enzyme can be measured.

A change that increases the activity of a protein is achieved, for example, by increasing the expression of a gene encoding the protein. "A rise in the performance of a gene" means that the amount of expression per cell of the gene is increased compared to a non-altered strain such as a wild strain or a parent strain. The "increased expression of a gene" may specifically mean an increase in the amount of transcription of a gene (amount of mRNA) and/or an increase in the amount of translation of a gene (amount of protein). Furthermore, "the increase in the performance of genes" is also known as "enhanced performance of genes". The expression of the gene, for example, can be increased by 1.5 times or more, 2 times or more, or 3 times or more as compared with the non-modified strain. In addition, "increased gene expression" refers not only to the increase in the expression of the gene in the strain in which the original target gene is expressed, but also in the original performance. In the strain of the target gene, the gene is expressed. That is, "increased expression of a gene" includes, for example, introducing a gene into a strain that does not retain a target gene, and expressing the gene.

The increased performance of genes can be achieved, for example, by increasing the copy number of the gene.

The increase in the copy number of the gene can be achieved by introducing the gene into the chromosome of the host microorganism. The introduction of a gene into a chromosome can be performed, for example, by homologous recombination (Miller I, J. H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). The gene may be introduced in only 1 copy or 2 copies or more. For example, a majority copy of a gene can be introduced into a chromosome by homologous recombination with a sequence in which a majority copy is present on the chromosome. There are many copies of the sequence in the chromosome, and an autonomously replicating sequence (ARS) composed of a unique short repeat sequence or an rDNA sequence of about 150 copies may be cited. In the pamphlet of International Publication No. 95/32289, an example in which yeast transformation using a plastid containing ARS is described. Alternatively, the gene can be accessed in a transposon and transferred to a chromosome to introduce a majority of the gene.

The confirmation of introduction of the target gene on the chromosome can be confirmed by Southern hybridization using a probe having a sequence complementary to all or a part of the gene, PCR using a primer based on the sequence of the gene, or the like.

Further, the number of copies of the gene is increased, and it can also be achieved by introducing a vector containing the target gene into the host microorganism. For example, by including the standard The DNA fragment of the gene is linked to a vector functional in the host microorganism to construct a expression vector of the gene, and the expression vector is transformed into the host microorganism to increase the copy number of the gene. The DNA fragment containing the target gene can be obtained, for example, by PCR using the genomic DNA of the microorganism having the target gene as a template. The vector allows the vector to be autonomously replicable in the cells of the host microorganism. The vector is preferably a multicopy vector. The vector which is functional in yeast cells may, for example, be a plastid having a copying initiation point of CEN4 or a multicopy plastid having an origin of replication of 2 μm DNA. Specific examples of the carrier in the yeast cell include pAUR123 (Takara Bio Inc.) or pYES2 (Invitrogen).

In the case of introducing a gene, the gene can be expressed as long as it remains in the yeast of the present invention. Specifically, the gene may be expressed by being controlled by a promoter sequence functional in the yeast of the present invention. The promoter may be a promoter from a host or a promoter derived from a heterologous species. The promoter may be an intrinsic promoter of the introduced gene or a promoter of another gene. The promoter may also be a promoter originally loaded on the vector to be used. As the promoter, for example, a more powerful promoter as described later can be used.

Further, when two or more genes are introduced, each gene can be expressed as long as it remains in the yeast of the present invention. For example, each gene may be retained on a single expression vector or may remain on the chromosome. In addition, each gene may be retained on a plurality of expression vectors, or may be separately retained on a single or a plurality of expression vectors and chromosomes. Also It can be introduced by constructing an operon with two or more genes. In the case of introducing a gene encoding two or more enzymes, for example, a gene encoding two or more subunits constituting a single enzyme may be introduced. And combinations of these.

The gene to be introduced is not particularly limited as long as it encodes a protein that is functional in the host. The gene to be introduced may be a gene derived from a host or a gene derived from a heterologous species. The introduced gene can be obtained, for example, by PCR using a primer designed based on the base sequence of the gene, using the gene DNA of the organism having the gene, or the plastid carrying the gene as a template. Further, the introduced gene can be fully synthesized based on, for example, the base sequence of the gene.

Further, when the protein functions as a complex composed of a plurality of subunits, as long as the activity of the resulting protein increases, all or a plurality of the plurality of subunits may be changed. That is, for example, when the activity of the protein is increased by increasing the expression of the gene, the performance of all of the plurality of genes encoding the subunits can be enhanced, and only a part of the expression can be enhanced. It is generally preferred to enhance the performance of all of the plurality of genes encoding the subunits. Further, each unit constituting the complex may be a single organism or a different organism of two or more types as long as the complex has a function as a target protein. That is, for example, a gene derived from the same organism encoding a plurality of subunits can be introduced into a host, and genes derived from different organisms can also be introduced into a host.

Moreover, the increase in gene expression can be achieved by increasing the transcription of genes. Efficiency is achieved. Moreover, the increase in gene expression can be achieved by improving the efficiency of gene translation. An increase in the transcription efficiency or translation efficiency of a gene can be achieved, for example, by changing the expression regulatory sequence. The "expression regulation sequence" refers to a general term for a promoter or the like that affects the expression of a gene. Specifically, an increase in gene transcription efficiency can be achieved, for example, by substituting a promoter of a gene on a chromosome with a more powerful promoter. "More powerful promoter" means a promoter with a higher transcription of the gene than the wild-type promoter originally present. The promoter of a more potent promoter may, for example, be a promoter of a gene of PGK1, PDC1, TDH3, TEF1, HXT7, ADH1 or the like which is well known as a high expression promoter. Further, in the case of a more potent promoter, a highly active type of the original promoter can be obtained by using various reporter genes.

An increase in the efficiency of translation of a gene can be achieved, for example, by a change in a codon. That is, when the heterogeneous expression of the gene is performed, the translation efficiency of the gene can be improved by replacing the rare codon present in the gene with a synonymous codon used at a higher frequency. The substitution of a codon can be carried out, for example, by a site-specific variation method in which a target mutation is introduced into a target site of DNA. The site-specific variation method can be exemplified by the method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HAEds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987). ). Alternatively, the gene fragment of the substitution codon can be fully synthesized. The frequency of use of codons in various organisms is disclosed in the "Codon Usage Database" (http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 ( 2000)).

Moreover, the increase in gene expression can also be achieved by amplifying a regulator that increases the expression of the gene, or by deleting or weakening the modifier that reduces the expression of the gene.

The method of increasing the gene expression as described above may be used singly or in any combination.

Further, the change in the activity of the protein can be achieved, for example, by increasing the specific activity of the protein. The increase in specific activity also includes the reduction and release of feedback suppression. Proteins with enhanced specific activity, for example, can be obtained by exploring various organisms. Further, it is also possible to obtain a highly active type by introducing a mutation into the original protein. The introduced variation, for example, may be substituted, deleted, inserted, or added to one or several amino acids of one or several positions of the protein. Introduction of the mutation can be carried out, for example, by the site-specific variation method as described above. Further, the introduction of the mutation can be carried out, for example, by a mutation treatment. The mutation treatment may be an irradiation of X-rays, irradiation of ultraviolet rays, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methanesulfonate. Treatment with a variant agent such as an ester (MMS). Alternatively, the DNA can be directly treated with hydroxylamine in vitro to induce random variation. The specific activity can be used singly or in combination with the method of enhancing gene expression as described above.

When the yeast of the present invention has a ploidy of two or more and the activity of the protein is increased by a change in chromosome, the yeast of the present invention can be in a different chromosome manner as long as the result increases the activity of the protein. Chromosomes with wild-type chromosomes that change to increase protein activity can also change to increase protein in the same chromosome The active chromosome.

The transformation method of yeast can be carried out by protoplast method, KU method (H. Ito et al., J. Bateriol., 153-163 (1983)), KUR method (fermentation and industrial vol. 43, p. 630-637). (1985)), electroporation (Luis et al., FEMS Micro biology Letters 165 (1998) 335-340), method using vector DNA (Gietz RD and Schiestl RH, Methods Mol. Cell. Biol. 5: 255- 269 (1995)) and the like which are usually used for yeast transformation. In addition, the operation of the yeast cell formation and the separation of the diploid yeast is described in "Chemical and Biological Experiment Line 31 Yeast Experimental Technology", the first edition, and the Hirokawa Bookstore; "Bio-Operation Manual Series 10 Yeast Genetic Experiment Method The first edition, the sheep community, and so on.

The activity of the protein can be confirmed to be increased by measuring the activity of the protein.

It is also possible to confirm an increase in the activity of the protein by confirming an increase in the expression of the gene encoding the protein. The increase in the expression of the gene can be confirmed by confirming an increase in the amount of transcription of the gene or confirming an increase in the amount of protein expressed by the gene.

The confirmation of the increase in the amount of transcription of the gene can be carried out by comparing the amount of mRNA transcribed from the gene with a non-altered strain such as a wild strain or a parent strain. Examples of the method for evaluating the amount of mRNA include Northern hybridization, RT-PCR, and the like (Sambrook, J., et al., Molecular Cloning A Laboratory Manual/Third Edition, Cold Spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001). The amount of mRNA can be increased by, for example, 1.5 times compared to non-modified strains. Above, 2 times or more, or 3 times or more.

The increase in the amount of protein can be confirmed by using Western blotting using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of protein can be increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more as compared with a non-modified strain.

The above method of increasing the activity of the protein can be utilized for enhancing the activity of any protein, for example, the enzyme related to the synthesis of γ-Glu-Abu; or the expression of any gene, such as a gene encoding the arbitrary protein.

<1-5> Techniques for reducing the activity of proteins

The following describes the method of reducing the activity of a protein.

"Reduced protein activity" means that the activity per cell of the protein is reduced compared to a non-altered strain such as a wild strain or a parent strain, and the activity completely disappears. "Reduced protein activity" means, in particular, a decrease in the number of molecules per cell of the protein and/or a decrease in function per molecule of the protein compared to a non-modified strain. In other words, the "activity" when the "protein activity is lowered" is not limited to the catalytic activity of the protein, and may also mean the amount of transcription (the amount of mRNA) or the amount of translation (the amount of protein) of the gene encoding the protein. Furthermore, "the number of molecules per cell is reduced" means that the protein does not exist at all. Further, "the function of each molecule of protein is lowered" means that the function of each molecule of the protein completely disappears. Protein activity, as long as There is no particular limitation as compared with the non-modified strain, and for example, it can be reduced to 50% or less, 20% or less, 10% or less, 5% or less, or 0% as compared with the non-modified strain.

A change that reduces the activity of a protein is achieved, for example, by reducing the expression of the gene encoding the protein. "Reduced expression of a gene" means that the amount of expression per cell of the gene is reduced compared to a non-altered strain such as a wild strain or a parent strain. "Reduced expression of a gene" means, in particular, a decrease in the amount of transcription (mRNA amount) of a gene, and/or a decrease in the amount of translation of a gene (amount of protein). "Reduced expression of genes" is a case in which the gene is not expressed at all. Furthermore, "the reduction in the performance of genes" is also known as "the weakening of gene expression." The expression of the gene can be reduced to 50% or less, 20% or less, 10% or less, 5% or less, or 0%, for example, compared to the non-modified strain.

The decrease in the expression of the gene can be performed, for example, by a decrease in transcription efficiency, by a decrease in translation efficiency, or by a combination of these. The expression of the gene is reduced, for example, by altering the expression regulatory sequence of the promoter of the gene or the like. When the expression regulatory sequence is changed, the expression regulatory sequence is preferably changed by 1 base or more, more preferably 2 bases or more, and particularly preferably 3 bases or more. Alternatively, the promoter of the gene on the chromosome can be replaced with a weaker promoter. "Weaker promoter" means a promoter whose transcription is weaker than the wild-type promoter that is present. For weaker promoters, for example, various inducible promoters can be utilized. That is, an inducible promoter can function as a weak promoter in the absence of an inducing substance. The inducible promoter may, for example, be galactose-induced galactose The promoter of the glycokinase gene (GAL1). Also, some or all of the expression regulatory sequences may be deleted. Moreover, the reduction in gene expression can be achieved, for example, by operating a factor associated with performance control. Examples of factors related to performance control include low molecular weight (inducing substances, inhibitory substances, etc.), proteins (transcription factors, etc.), nucleic acids (siRNA, etc.) related to transcription or translation control. Further, the decrease in gene expression can be achieved, for example, by a decrease in the expression of a gene introduced into a coding region of a gene. For example, by substituting the codons of the coding region of a gene for synonymous codons that are utilized at a lower frequency in the host, the performance of the gene can be reduced. Further, for example, by the destruction of a gene as described later, the expression of the gene itself can be lowered.

Further, the change in the activity of the protein can be achieved, for example, by disrupting the gene encoding the protein. "Destruction of a gene" means to alter the gene so that it does not produce a protein that functions normally. The "protein that does not function normally" includes a case where the protein is not produced by the gene at all, or a protein in which the function (activity or property) per molecule is reduced or disappeared by the gene.

The disruption of a gene can be achieved, for example, by partially or completely damaging one of the coding regions of the gene on the chromosome. Further, the entire gene, including the sequence before and after the gene on the chromosome, may be deleted. The missing region may be any region such as an N-terminal region, an internal region, or a C-terminal region, as long as the activity of the protein is lowered. Usually, the missing region is long and is more able to reliably disable the gene. Further, the sequence before and after the missing region is preferably inconsistent.

Moreover, the destruction of genes, for example, can also be due to The coding region of the gene is introduced by introducing an amino acid substitution (missense variation), introducing a stop codon (meaningless variation), or introducing a frame shift mutation such as addition or deletion of 1 to 2 bases (Journal of Biological Chemistry 272). : 8611-8617 (1997), Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833-20839 (1991)).

Further, the disruption of the gene can be achieved, for example, by inserting another sequence into the coding region of the gene on the chromosome. The insertion site can be any region of the gene, but the inserted sequence is long and is more capable of reliably inactivating the gene. Further, the sequence before and after the insertion site is preferably inconsistent. The other sequence is not particularly limited as long as the activity of the encoded protein is lowered or disappeared, and examples thereof include a marker gene such as an antibiotic resistance gene or a gene for production of a target substance.

The gene on the chromosome is changed as described above, for example, by deleting a partial sequence of the gene, making a deletion gene which is changed to a protein which does not function normally, and transforming the microorganism with the recombinant DNA containing the deletion gene The deletion gene is caused by homologous recombination with the wild type gene on the chromosome to replace the wild type gene on the chromosome with the deletion type gene. In this case, it is easy to operate by including a marker gene in advance in the recombinant DNA in accordance with the nutritional requirements of the host. The protein encoded by the deletion gene, even if it is produced, has a different stereostructure from the wild-type protein, and its function is reduced or disappeared.

Depending on the structure of the recombinant DNA used, the result of homologous recombination, or the presence of a wild-type gene and a deletion-type gene to sandwich the recombinant DNA The state in which other parts (such as the vector part and the marker gene) are inserted into the chromosome. In this state, the wild-type gene functions, and it is necessary to cause homologous recombination between the two genes, and the one-copy gene is detached from the chromosomal DNA together with the vector portion and the marker gene, and the deletion-type gene is selected.

Further, for example, the linear DNA containing an arbitrary sequence and the linear DNA having the upstream and downstream sequences of the substitution target site on the chromosome at both ends of the arbitrary sequence can be transformed into the yeast upstream of the substitution site and The downstream causes homologous recombination, respectively, and the substitution site portion is substituted with the arbitrary sequence in one step. For this arbitrary sequence, for example, a sequence comprising a marker gene can be used. The marker gene can also be removed as needed thereafter. When the marker gene is removed, the sequence for homologous recombination can be added to both ends of the marker gene in advance so that the marker gene can be efficiently removed.

Further, the change in the activity of the protein can be changed, for example, by a mutation treatment. The mutation treatment may be an irradiation of X-rays, irradiation of ultraviolet rays, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methanesulfonate Treatment of variants such as esters (MMS).

Further, when the protein functions as a complex composed of a plurality of subunits, as long as the activity of the resulting protein is lowered, all of the plurality of subunits may be changed, or only a part of them may be changed. That is, for example, all of the plurality of genes encoding the subunits may be destroyed, or only a part of the genes may be destroyed. Moreover, when there are a plurality of isozymes in the protein, as long as the activity of the resulting protein is lowered, the plurality of isotopes can be reduced. The activity of the enzyme can also reduce only a part of the activity. That is, for example, all of the plurality of genes encoding the isozymes may be destroyed, or only a part of them may be destroyed. Further, for example, some of the plurality of genes encoding the isozymes may be destroyed and the remaining performance may be lowered.

When the yeast of the present invention has a ploidy of a doubling or more, the yeast of the present invention can have a chromosome and a wild-type chromosome which are changed to a protein-reducing activity by different chromosomes as long as the activity of the resulting protein is lowered. Chromosomes that change to reduce the activity of the protein in the same chromosome. In general, the yeast of the present invention preferably has a gene which is altered to reduce the activity of the protein in the same chromosome.

The activity of the protein can be confirmed to be lowered by measuring the activity of the protein.

It is also possible to confirm a decrease in the activity of the protein by confirming a decrease in the expression of the gene encoding the protein. The decrease in the expression of the gene can be confirmed by confirming that the amount of transcription of the gene is lowered or confirming that the amount of the protein expressed by the gene is decreased.

The confirmation of the decrease in the transcription amount of the gene can be carried out by comparing the amount of mRNA transcribed from the gene with the non-modified strain. Examples of the method for estimating the amount of mRNA include Northern hybridization, RT-PCR, and the like (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of mRNA can be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% as compared with the non-modified strain.

The confirmation of the decrease in the amount of protein can be carried out by using Western blotting using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of protein can be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% as compared with the non-modified strain.

By deciding the means used, by deciding one of the genes Part or all of the base sequence, restriction enzyme map, or full length, etc., to confirm that the gene is destroyed.

The above method for lowering the activity of the protein can be utilized for any protein, for example, the activity of the peptide-degrading enzyme is lowered, or the use of any gene, for example, a gene encoding the arbitrary protein, is reduced.

<2> Method of the present invention <2-1> γ-Glu-Abu manufacturing method

As described above, the γ-Glu-Abu-producing yeast can be used, for example, in the production of γ-Glu-Abu. That is, the aspect of the method of the present invention is a method for producing γ-Glu-Abu, which comprises culturing the yeast of the present invention (here, γ-Glu-Abu-producing yeast) in a medium to make γ- Glu-Abu accumulates in this medium and takes γ-Glu-Abu from the medium.

The medium to be used is not particularly limited as long as the yeast of the present invention can be proliferated to produce cumulative γ-Glu-Abu. As the medium, for example, a usual medium for the culture of yeast can be used. Specific examples of the medium include, but are not limited to, SD medium, SG medium, and SDTE medium. The medium can be used, for example, by a carbon source or nitrogen as needed. A medium selected from the group consisting of a source, a source of phosphoric acid, a source of sulfur, and various other organic or inorganic components. The type or concentration of the medium component can be appropriately set depending on various conditions such as the type of yeast to be used.

The carbon source is not particularly limited as long as it is metabolically utilized by the yeast of the present invention to produce γ-Glu-Abu. Specific examples of the carbon source include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, molasses, starch hydrolysate, and hydrolysate of raw materials; acetic acid, fumaric acid, and citric acid; Organic acids such as succinic acid and malic acid; alcohols such as glycerin, crude glycerin and ethanol; and fatty acids. As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

Specific examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen sources such as peptone, yeast extract, meat extract, and soybean protein decomposition product; ammonia and urea. As the nitrogen source, one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

Specific examples of the phosphoric acid source include a phosphate such as potassium hydrogen phosphate or potassium hydrogen phosphate; and a phosphoric acid polymer such as pyrophosphoric acid. As the phosphoric acid source, one type of phosphoric acid source or a combination of two or more kinds of phosphoric acid sources can be used.

Specific examples of the sulfur source include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites; and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one type of sulfur source may be used, or two or more types of sulfur sources may be used in combination.

Specific examples of the other various organic components or inorganic components include inorganic salts such as sodium chloride and potassium chloride; iron, manganese, and magnesium. a trace amount of metal such as calcium; vitamin B1, vitamin B2, vitamin B6, niacin, niacin amide, vitamin B12 and other vitamins; amino acids; nucleic acids; containing such peptone, casein amine An organic component such as an acid, a yeast extract, or a soy protein decomposition product. As the other various organic components or inorganic components, one component may be used, or two or more components may be used in combination.

Further, when a nutrient-requiring mutant such as an amino acid is required to be grown, it is preferred to supplement the nutrients required for the medium. Further, for example, Abu may be supplemented in a medium.

The culture conditions are not particularly limited as long as the yeast of the present invention is proliferated to produce cumulative γ-Glu-Abu. The culture can be carried out, for example, under the usual conditions used for yeast culture. The culture conditions can be appropriately set depending on various conditions such as the type of yeast to be used.

The culture can be carried out, for example, by aeration culture or shaking culture using a liquid medium, in an aerobic environment. The culture temperature may be, for example, 25 to 35 ° C, more preferably 27 to 33 ° C, still more preferably 28 to 32 ° C. The culture period may be, for example, 5 hours or longer, 10 hours or longer, or 15 hours or longer, and may be 120 hours or shorter, 72 hours or shorter, or 48 hours or shorter. The cultivation can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof.

By culturing the yeast of the present invention as described above, γ-Glu-Abu is accumulated in the medium.

Well known for use in the detection or identification of compounds The technique was used to confirm the generation of γ-Glu-Abu. Examples of such a method include HPLC, LC/MS, LC-MS/MS, GC/MS, and NMR. These methods can be combined as appropriate.

The resulting γ-Glu-Abu recovery can be carried out by a well-known method used for separation and purification of the compound. Examples of such a method include an ion exchange resin method, a membrane treatment method, a precipitation method, and a crystallization method. These methods can be combined as appropriate. The recovered γ-Glu-Abu can be a free body, a salt thereof, or a mixture thereof. In the present invention, the term "γ-Glu-Abu" means a free form of γ-Glu-Abu, a salt thereof, or a mixture thereof, unless otherwise specified.

The salt is not particularly limited as long as it can be orally ingested. For example, a salt of an acidic group such as a carboxyl group may, for example, be an ammonium salt or a salt of an alkali metal such as sodium or potassium; a salt with an alkaline earth metal such as calcium or magnesium; an aluminum salt or a zinc salt; a salt of an organic amine such as ethylamine, ethanolamine, morpholine, pyrrolidine, piperidine, piperazine or dicyclohexylamine; or a salt of a basic amino acid such as arginine or lysine. Further, for example, a salt of a basic group such as an amine group may, for example, be a salt of an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid or hydrobromic acid; and acetic acid, citric acid, benzoic acid, and Malay. Acid, fumaric acid, tartaric acid, succinic acid, tannic acid, butyric acid, hibenzate, pamoic acid, heptanoic acid, citric acid, teoclate, salicylic acid, lactic acid, oxalic acid a salt of an organic carboxylic acid such as mandelic acid, malic acid or methylmalonic acid; or a salt of an organic sulfonic acid such as methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid. Further, as the salt, one type of salt may be used, or two or more kinds of salts may be used in combination.

Further, the γ-Glu-Abu recovered may contain components such as yeast cells, medium components, water, and metabolic by-products of yeast, in addition to γ-Glu-Abu. The purity of the recovered γ-Glu-Abu may be, for example, 30% (w/w) or more, 50% (w/w) or more, 70% (w/w) or more, or 80% (w/w) or more. 90% (w/w) or more, or 95% (w/w) or more.

<2-2> Method for producing yeast extract containing γ-Glu-Abu

As described above, the γ-Glu-Abu accumulation type yeast can be used, for example, in the production of a yeast extract containing γ-Glu-Abu. That is, another aspect of the method of the present invention is a method for producing a yeast extract containing γ-Glu-Abu, which comprises using the yeast of the present invention (here, γ-Glu-Abu cumulative yeast) as a raw material. To prepare yeast extracts.

The yeast extract produced by using the yeast of the present invention (here, γ-Glu-Abu cumulative yeast) as a raw material is also referred to as "the yeast extract of the present invention". The yeast extract of the present invention can be produced in the same manner as a usual yeast extract, except that the yeast of the present invention is used as a raw material. The method of producing a yeast extract of the present invention is explained below.

First, the yeast of the present invention is cultured in a medium. Regarding the culture medium and the culture conditions, the description of the culture medium and the culture conditions of the γ-Glu-Abu production method can be used. By culturing the yeast of the present invention, γ-Glu-Abu can be accumulated in the cells of the yeast to obtain a yeast containing γ-Glu-Abu.

The yeast extract is prepared from the obtained yeast in the same manner as in the preparation of a usual yeast extract. Yeast extract, which can be used to treat yeast cells by hot water, or digestive yeast The person who handles it. Further, the obtained yeast extract may be concentrated or dried to form a powder form as needed.

As described above, a yeast extract containing γ-Glu-Abu (the yeast extract of the present invention) can be obtained. The yeast extract of the present invention preferably contains 0.1% by weight or more, more preferably 1.0% by weight or more, and still more preferably 2.0% by weight or more, based on the total amount of the solid content in the yeast extract. Preferably, it contains 20% by weight or more of γ-Glu-Abu.

<3> Food and drink of the present invention

The γ-Glu-Abu and/or yeast extract obtained as described above can be used for the manufacture of foods and drinks. That is, the present invention provides a method for producing a food or drink comprising adding a γ-Glu-Abu and/or a yeast extract obtained by the method of the present invention to a food or beverage or a raw material thereof. The food or drink manufactured in this manner is also referred to as "the food and beverage of the present invention". Further, in one aspect, by adding γ-Glu-Abu and/or a yeast extract, an alcoholic taste can be imparted to a food or beverage. Examples of the food and drink include alcoholic beverages, breaded foods, and fermented food seasonings.

The food or drink of the present invention can be produced by the same method as the usual food and beverage products except for the addition of γ-Glu-Abu and/or yeast extract. Examples of the raw material such as alcoholic beverage include rice, barley, and corn starch; and the bread foods include flour, sugar, salt, cream, and yeast for fermentation; and the fermented food seasoning includes soybean, wheat, and the like. Addition of γ-Glu-Abu and/or yeast extract for food and beverage At any stage of the manufacturing process. In other words, the γ-Glu-Abu and/or the yeast extract may be added to a raw material of a food or beverage, a food or drink that can be added during the production, or may be added to the finished food or beverage. Further, the yeast extract or a concentrate thereof, or the dried ones themselves may be used as a fermented food seasoning.

The amount of the γ-Glu-Abu and/or the yeast extract to be added is not particularly limited, and may be appropriately set depending on the type of the food or drink or the state of intake of the food or beverage. The γ-Glu-Abu and/or the yeast extract may be added in an amount of γ-Glu-Abu, for example, in an amount of 1 ppm (w/w) or more and 100 ppm (w/w) or more, based on the food or beverage or the raw material thereof. Or add 1% (w/w) or more. In addition, the γ-Glu-Abu and/or the yeast extract may be added in an amount of γ-Glu-Abu, for example, 100% (w/w) or less and 10% (w/). w) below, or below 1% (w/w).

[Examples]

The invention is further illustrated by the following examples.

<Example 1> Construction of a high-performance strain of the GAP1 gene

In this example, in order to evaluate the effect of Gap1 on the extracellular excretion of γ-Glu-Abu by the total amino acid permease, the yeast containing γ-Glu-Abu Based on the strain (M006Y ura3-), a yeast strain having a high expression of the wild-type GAP1 gene and the cell membrane normalized localized GAP1 gene was constructed.

(1) Construction of a plant containing γ-Glu-Abu

By enhancing γ-Glu-Abu production, the related branched-chain amino acid transferase gene (BAT1), the serine deaminase gene (CHA1), and the γ-glutamine-based cysteine synthesis In the expression of the enzyme gene (GSH1), a yeast strain (M006Y ura3- strain) containing γ-Glu-Abu was constructed. The sequence is as follows.

As a parent strain, Saccharomyces cerevisiae AG1 ura3- strain (JP-A-2012-213376) was used. The AG1 ura3- strain is a γ-glutamate-based cysteine synthase gene (GSH1)-expressing strain of Saccharomyces cerevisiae Y006 strain (FERM BP-11299). The URA3 gene defect of AG1 ura3- strain indicates uracil requirement.

The production of AG1 ura3- is based on the method of Sofyanovich et al. (Olga A. Sofyanovich et al: A New Method for Repeated "Self-Cloning" Promoter Replacement in Saccharomyces cerevisiae. Mol. Biotechnol., 48, 218-227 (2011)). The promoter of the branched amino acid aminotransferase gene (BAT1) is substituted with a promoter region (hereinafter also referred to as pTDH3) constituting a glyceraldehyde 3 phosphate dehydrogenase gene (TDH3) which expresses a promoter. The sequence is as follows. The primer (5'-GCCAGGCGGTTGATACTTTGTGCAGATTTCATACCGGCTGTCGCTATTATTACTGATGAATTGGCTCTCTTTTTGTTTAATCTTAACCCAACTGCACAGA-3') having the sequence number 1 of the upstream sequence of BAT1 at the 5' end, and the sequence number 2 having the sequence of the partial ORF starting from the start codon of the BAT1 gene The primer (5'-TTGGATGCATCTAATGGGGCACCAGTAGCGAGTGTTCTGATGGAGAATTTCCCCAACTTCAAGGAATGTCTCTGCAACATTTGTTTATGTGTGTATTTC-3') was subjected to PCR using the pUC19-URA3-pTDH3-URA3 plastid (described in the report of Sofyanovich et al.) as a template to obtain a DNA fragment having URA3 sandwiched between TDH3 promoters. . The PCR conditions were thermal denaturation (94 ° C, 10 sec), adhesion (60 ° C, 10 sec), elongation (72 ° C, 4 min), 25 cycles. The AG1 ura3- strain was transformed with this DNA fragment and applied to an SD plate medium containing no uracil. A strain in which the BAT1 promoter was substituted with pTDH3-URA3-pTDH3 was obtained from the grown transform. The preparation method of the SD medium is shown below. The SD plate culture medium was prepared by adding Bacto-agar to the SD medium and making it 2%.

[SD medium composition]

The SD medium was prepared by mixing autoclaved sterilized glucose and filter-sterilized YNB in the above-described composition, and diluting it with autoclave-sterilized MilliQ water.

10 x YNB is prepared as follows. Dissolve 17g of Difco Yeast Nitrogen Base w/o Amino Acids relative to MilliQ Water 1L And Ammonium Sulfate and 50 g of ammonium sulfate were adjusted to pH 5.2 and sterilized by a filter. In order to prevent decomposition of vitamins, keep them in a cold place.

Since the URA3 gene-deficient strain exhibits 5-fluoroorotic acid (5-FOA) resistance, a strain containing 5-FOA can be used to select a strain in which the URA3 selection marker is removed. Therefore, the BAT1 promoter was substituted with the strain of pTDH3-URA3-pTDH3, and cultured in an SD medium supplemented with uracil for one night, and an appropriate amount was applied to a 5-FOA plate medium. From the growing colony, URA3 was removed by homologous recombination between the introduced two TDH3 promoters, and the promoter of BAT1 was replaced with the TDH3 promoter (AG1 pTDH3-BAT1 ura3- strain). Further, the 5-FOA plate medium was prepared in the following manner.

[5-FOA plate medium composition]

Mixing yeast growth medium bags SD/-Ura Broth (Clontech), Uracil, 5-FOA, and 10 x YNB, and diluting with MilliQ water to 500 mL and sterilizing by filter, and Bacto-agar with MilliQ The water is diluted to 500 mL and sterilized by autoclaving Prepare 5-FOA plate medium.

Subsequently, the promoter of the serine acid deaminase gene (CHA1) of the AG1 pTDH1-BAT1 ura3- strain obtained by the above method was replaced by the method of Sofyanovich et al. to form pTDH3 which constitutes a promoter. Strain. The sequence is as follows. Furthermore, the AG1 pTDH3-BAT1 ura3- strain is a defective strain of the URA3 gene, and shows uracil requirement. The primer (5'-GAGTACTAATCACCGCGAACGGAAACTAATGAGTCCTCTGCGCGGAGACATGATTCCGCATGGGCGGCTCCTGTTAAGCCTCTTAACCCAACTGCACAGA-3') having the sequence number 3 of the upstream sequence of CHA1 at the 5' end, and the primer of SEQ ID NO: 4 having the sequence of the ORF from the start of the start codon of the CHA1 gene ( 5'-TATTTCAAGAAAAATTGTGCAGAAGCCTTTCCGGGGAAGAATTGACGTAATAATGGTGTTTTATTGTAGACTATCGACATTTGTTTATGTGTGTATTATT-3-3), PCR was carried out using pUC19-URA3-pTDH3-URA3 plastid as a template to obtain a DNA fragment having URA3 sandwiched between TDH3 promoters. The PCR conditions were thermal denaturation (94 ° C, 10 sec), adhesion (60 ° C, 10 sec), elongation (72 ° C, 4 mi), 25 cycles. This DNA fragment was transformed into AG1 pTDH1-BAT1 ura3- strain and plated on uracil-free SD plate medium. From the growing transformant, the strain in which the CHA1 promoter was substituted with pTDH3-URA3-pTDH3 was obtained. Further, the CHA1 promoter was replaced with a strain of pTDH3-URA3-pTDH3, and cultured in SD medium supplemented with uracil. In the evening, an appropriate amount was applied to 5-FOA plate medium. From the growing colony, URA3 was removed by homologous recombination between the introduced two TDH3 promoters, and the promoter of CHA1 was replaced with the TDH3 promoter (AG1 pTDH3-BAT1 pTDH3-CHA1 ura3- strain). Hereinafter, this strain is referred to as M006Y ura3- strain. The M006Y ura3- strain showed uracil requirement.

(2) Production of 1 copy plastid for high performance of GAP1 gene

Yeast uses 1 copy of plastid, pUC19 (invitrogen) as the main skeleton, in order to autonomously replicate in yeast cells, the origin of replication and the centromere of budding yeast are colonized, and The strain was produced by using the URA3 gene as a selection marker.

(2-1) Import of selection mark URA3

First, the gene shown in SEQ ID NO: 5 with the Aat II restriction enzyme recognition sequence and the primer shown in SEQ ID NO: 6 with the Eco RI restriction enzyme recognition sequence were used as the gene of the wild type Saccharomyces cerevisiae Y006 strain (FERM BP-11299). The DNA was subjected to PCR as a template to obtain a DNA fragment having the URA3 gene. The PCR conditions were denaturation (94 ° C, 10 sec), adhesion (50 ° C, 10 sec), elongation (72 ° C, 4 min), 25 cycles. Next, the DNA fragment was colonized at the Aat II- Eco RI site of pUC19 to obtain a plastid pUC19-URA3. For reference, the base sequence of the URA3 gene of Saccharomyces cerevisiae S288C is shown in SEQ ID NO: 16.

(2-2) Import of copy origin (ARS)

Using the primer shown in SEQ ID NO: 7 with the Kpn I restriction enzyme recognition sequence and the primer shown in SEQ ID NO: 8 with the Eco RI restriction enzyme recognition sequence, the genomic DNA of the wild type Saccharomyces cerevisiae Y006 strain (FERM BP-11299) was used. PCR was carried out for the template to obtain a DNA fragment having ARS. The PCR conditions were denaturation (94 ° C, 10 sec), adhesion (60 ° C, 10 sec), elongation (72 ° C, 4 min), 25 cycles. Next, the DNA fragment was colonized at the Kpn I- Eco RI site of pUC19-URA3 to obtain a plastid pUC19-URA3-ARS. For reference, the base sequence of the ARS of Saccharomyces cerevisiae S288C is shown in SEQ ID NO: 17.

(2-3) Introduction of the centromere (CEN)

Using the primers shown in SEQ ID NO: 9 and SEQ ID NO: 10 having the Kpn I restriction enzyme recognition sequence, PCR was carried out using the genomic DNA of the wild type Saccharomyces cerevisiae Y006 strain (FERM BP-11299) as a template to obtain a DNA fragment having CEN. The PCR conditions were denaturation (94 ° C, 10 sec), adhesion (60 ° C, 10 sec), elongation (72 ° C, 4 min), 25 cycles. Subsequently, the DNA fragment was cloned into the Kpn I- Kpn I site of pUC19-URA3-ARS to obtain a 1-copy plastid pUC19-URA3-ARS-CEN (hereinafter also referred to as "YCp"). For reference, the base sequence of CEN of Saccharomyces cerevisiae S288C is shown in SEQ ID NO: 18.

(3) Production of high-quality plastids of wild-type GAP1 gene

The wild-type GAP1 gene is a high-performance plastid, and the TDH3 promoter which constitutes a high-performance promoter is transformed into a YCp plastid, and a wild-type GAP1 gene is further planted downstream of the promoter to produce a plasty .

(3-1) Introduction of TDH3 promoter

First, the gene shown in SEQ ID NO: 11 having the Sma I restriction enzyme recognition sequence and the primer shown in SEQ ID NO: 12 having the Bam HI restriction enzyme recognition sequence were used as the gene of the wild type Saccharomyces cerevisiae Y006 strain (FERM BP-11299). The DNA was subjected to PCR as a template to obtain a DNA fragment having a TDH3 promoter. The PCR conditions were denaturation (94 ° C, 10 sec), adhesion (60 ° C, 10 sec), elongation (72 ° C, 4 min), 25 cycles. Next, the DNA fragment was colonized at the Sma I- Bam HI site of YCp to obtain a plastid YCp-TDH3p.

(3-2) Production of high-quality plastids of wild-type GAP1 gene

Using the primer shown in SEQ ID NO: 13 with the Bam HI restriction enzyme recognition sequence and the primer shown in SEQ ID NO: 14 with the Sph I restriction enzyme recognition sequence, the genomic DNA of the wild type Saccharomyces cerevisiae Y006 strain (FERM BP-11299) was used. PCR was carried out for the template to obtain a DNA fragment having the wild type GAP1 gene. The PCR conditions were denaturation (94 ° C, 10 sec), adhesion (60 ° C, 10 sec), elongation (72 ° C, 4 min), 25 cycles. Next, the DNA fragment was colonized at the Bam HI- Sph I site of YCp-TDH3p to obtain a plastid YCp-GAP1.

(4) Production of high-performance plastids of GAP1 gene in normalized cell membrane

The Gap1 protein is ubiquitinated by the amount of amino acid inside and outside the cell, and is decomposed in the vacuole (Non-Patent Document 1). On the other hand, the quaternary acid of the 9th and 16th positions is substituted with the variant Gap1 protein of arginine, and does not have a ubiquitination site, and thus is not decomposed by ubiquitination. Therefore, the variant Gap1 protein is localized to the cell membrane in a normal state regardless of the concentration of the substrate inside and outside the cell (Non-Patent Document 2). Therefore, the high-performance plastids of the variant GAP1 gene (hereinafter also referred to as " gap1K9, 16R ") in which the quaternary acid of the ninth and the 16th position is substituted with the mutated Gap1 protein of arginine are as follows. The sequence was produced as a high-performance plastid of the GAP1 gene in the normal state of the cell membrane.

First, a primer having a Bam HI restriction enzyme recognition sequence and a GAP1 sequence in which the quaternic acid substituted with arginine is substituted into the 9th and 16th positions is used, and the Sph I restriction enzyme recognition sequence is used. The primer shown in SEQ ID NO: 14 was subjected to PCR using the genomic DNA of the wild type Saccharomyces cerevisiae Y006 strain (FERM BP-11299) as a template to obtain a DNA fragment having the variant GAP1 gene ( gap1K9, 16R ). The PCR conditions were denaturation (94 ° C, 10 sec), adhesion (60 ° C, 10 sec), elongation (72 ° C, 4 min), 25 cycles. Next, the DNA fragment was colonized at the Bam HI- Sph I site of YCp-TDH3p to obtain a plastid YCp-gap1K9, 16R. For reference, when the genomic DNA of Saccharomyces cerevisiae S288C is used as a template, the nucleotide sequence of the variant GAP1 gene ( gap1K9, 16R ) which can be obtained using the primers of SEQ ID Nos. 14 and 15 is shown in SEQ ID NO:21.

(5) Construction of high-performance strains of GAP1 gene

The M006Y ura3- strain was transformed with the prepared YCp, YCp-GAP1, and YCp-gap1K9, 16R, and applied to a uracil-free SD plate medium. From the grown transform, M006Y YCp-GAP1 strain and M006Y YCp-gap1K9, 16R strains each expressing the wild type GAP1 gene and the variant GAP1 gene ( gap1K9, 16R ), respectively, were obtained. Further, M006Y YCp strain was obtained as a control strain.

<Example 2> Analysis of γ-Glu-Abu concentration in vitro of high expression strain of GAP1 gene

In the present example, the concentration of γ-Glu-Abu in vitro of γ-Glu-Abu- derived and mutated high- yield wild-type GAP1 gene and variant GAP1 gene ( gap1K9, 16R ) was evaluated. The effect of Gap1 on extracellular excretion of γ-Glu-Abu.

(1) Cultivation and sampling

The plants prepared in Example 1 were each placed in a 500 mL volumetric flask with 50 mL of SD medium on the amount of the inoculum loop, and cultured at 30 ° C and 120 rpm for 24 hours.

The absorbance of the obtained culture solution was measured, and 70 mL of a 500 mL volumetric flask was placed in such a manner that the OD600 became 0.02. The bacteria were incubated on SD medium and shaken at 30 ° C and 120 rpm for about 16 to 20 hours (formal culture). Further, the absorbance was measured using a DU640 SPECTROPHTOMETER of BECKMAN COULTER. The culture solution was sampled at a time point when the OD600 became 1.8 and 2.5. The supernatant was recovered from the culture solution by centrifugation for measurement of the concentration of γ-Glu-Abu.

The concentration of γ-Glu-Abu in the supernatant was derivatized with AQC by the AQC reagent according to the well-known method (Example 1 of WO2011/129462) by LC- MS/MS measurement. The change point of the measurement conditions described in Example 1 of WO2011/129462 is as follows.

Change point 1: Using a standard solution of 5 μM 3-methyl-His-d2 (Sigma, stable isotope identification) and 5 μM Gly-d2 (Sigma, stable isotope identification) as a sample derivative 5 μM internal standard substance solution used. In the mass spectrometry of γ-Glu-Abu, Gly-d2 was used as an internal standard substance among these.

Change point 2: In the mass spectrometry of γ-Glu-Abu, the selected ion in the first mass spectrometer was set to 403.4, and the selected ion in the second mass spectrometer was set to 171.1. Further, in the case of γ-Glu-Abu quantification, when an admixture peak is rarely observed by the sample, the selected ion of the second mass spectrometer is changed to 145.2 or 104.1 to be quantified.

As a result of measuring the concentration of γ-Glu-Abu, as shown in Fig. 1, even if the wild-type GAP1 gene is highly expressed, the effect on the γ-Glu-Abu concentration in vitro is not observed, but the high-performance cell membrane normal localization type is observed. The GAP1 gene gap1K9 , 16R , the concentration of γ-Glu-Abu in vitro increased.

As described above, the Gap1 protein is known to be ubiquitinated according to the amount of amino acid inside and outside the cell, and is decomposed in the vacuole (Non-Patent Document 1). Therefore, even if the wild type GAP1 gene is highly expressed in the strain in which the γ-Glu-Abu production pathway is enhanced, the Gap1 protein is not localized in the cell membrane and is decomposed. Therefore, in order to achieve both the enhancement of the production pathway of γ-Glu-Abu and the promotion of excretion, the high expression of the normalized localized GAP1 gene of the cell membrane is effective.

<Example 3> Construction of a high-performance strain of YGL114w gene and a disrupted strain of YGL114w gene

In the present example, in order to evaluate the effect of the YGL114w protein on the extracellular excretion of γ-Glu-Abu, a high-performance strain of the YGL114w gene was constructed based on a yeast strain (M006Y ura3-) containing γ-Glu-Abu. And the disrupted strain of the YGL114w gene.

(1) Construction of high-performance strains of YGL114w gene

Using the primer shown in SEQ ID NO: 32 with the Bam HI restriction enzyme recognition sequence and the primer shown in SEQ ID NO: 33 with the Sph I restriction enzyme recognition sequence, the genomic DNA of the wild type Saccharomyces cerevisiae Y006 strain (FERM BP-11299) was used. PCR was carried out for the template to obtain a DNA fragment having the YGL114w gene. The PCR conditions were denaturation (94 ° C, 10 sec), adhesion (60 ° C, 10 sec), elongation (72 ° C, 4 min), 25 cycles. Next, the DNA fragment was colonized at the Bam HI- Sph I site of YCp-TDH3p to obtain a plastid YCp-YGL114w. The M006Y ura3- strain was transformed with the prepared YCp-YGL114w and applied to a uracil-free SD plate medium. From the grown transform, the M006Y YCp-YGL114w strain with high expression of the YGL114w gene was obtained.

(2) Construction of the disrupted strain of YGL114w gene

First, the M006Y ura3- strain was complemented by the URA3 gene based on the general method (JP-A-2012-213376) to obtain the M006Y strain. Next, the nucleotide sequence numbering of SEQ ID NO 80 using the additional YGL114w gene 80 bases upstream of the start codon of the primer 34 and the additional YGL114w gene termination codon downstream of the primer 35 to DNA M006Y genome of strain as a template, amplifying containing URA3 A DNA fragment of a gene. The conditions of PCR were thermal denaturation (94 ° C, 10 sec), adhesion (50 ° C, 10 sec), elongation (72 ° C, 2 min), 25 cycles. The obtained DNA fragment was transformed into M006Y ura3- strain and applied to uracil-free SD medium. The M006Y YGL114wΔ strain lacking the YGL114w gene was obtained from the grown transform.

<Example 4> Analysis of γ-Glu-Abu content in the strain of the high-performance strain of YGL114w gene and the disrupted strain of YGL114w gene

(1) Cultivation and sampling

The strain prepared in Example 3 and the control strain (M006Y YCp strain) were each placed in a 500 mL volumetric flask containing 50 mL of SD medium on the amount of the inoculum loop, and cultured at 30 ° C and 120 rpm for 24 hours.

The absorbance of the obtained culture solution was measured and became OD600. In a method of 0.02, 70 mL of SD medium was placed in a 500 mL volumetric flask, and the cells were incubated at 30 ° C and 120 rpm for about 16 to 20 hours (formal culture). Further, the absorbance was measured using a DU640 SPECTROPHTOMETER of BECKMAN COULTER. The culture solution was sampled at a time point when the OD600 became 1.8.

A culture solution in which 40 ODunit (the cell contained in 1 mL of the culture solution having an OD600 of 1 mL was defined as 1 ODunit) was fractionated, and the supernatant was removed as much as possible by centrifugation to take the cells. The cells were suspended in 45 mL of milliQ water. The cells were again collected by centrifugation and resuspended in 45 mL of milliQ water. By repeating this operation three times, the medium components were completely removed from the cells. The obtained washed cells were suspended in about 1.5 mL of milliQ water and heated at 70 ° C for 10 minutes. This step extracts the extract components contained in the cells. The separation of the extract-containing components is then separated from the bacterial residue by centrifugation.

A 10 kDa centrifugal filtration membrane (MILLIPORE: Amicon Ultra-0.5 mL 10K (model number UFC501096)) was used to remove cell debris from the distinction containing the extract components, and the obtained filtrate was used as an analytical sample.

The γ-Glu-Abu concentration in the sample was measured in the same manner as in Example 2.

As a result of measuring the concentration of γ-Glu-Abu, as shown in Fig. 2, in the M006Y YCp-YGL114w strain of the high-performance strain of the YGL114w gene, the concentration of γ-Glu-Abu in the cells was decreased (Fig. 2A). On the other hand, in the M006Y YGL114wΔ strain of the disrupted strain of the YGL114w gene, the concentration of γ-Glu-Abu in the cells was increased (Fig. 2B).

Although the YGL114w protein was previously predicted to be a peptide transport protein, the actual function is unknown. From the above results, it was confirmed that the YGL114w protein is a cell extracellular protein of γ-Glu-Abu. Therefore, the high expression of the YGL114w gene is effective for the extracellular excretion of γ-Glu-Abu.

[Industrial availability]

By the present invention, localization of cells inside and outside the γ-Glu-Abu in yeast can be controlled. Thus, the present invention is directed to the manufacture of γ-Glu-Abu or a yeast extract containing γ-Glu-Abu.

<Description of sequence list>

Sequence number 1~15: introduction

SEQ ID NO:16: Saccharomyces cerevisiae S288C URA3 gene base sequence

SEQ ID NO: 17: Base sequence of the origin of replication (ARS) of Saccharomyces cerevisiae S288C

SEQ ID NO: 18: Base sequence of the centromere (CEN) of Saccharomyces cerevisiae S288C

SEQ ID NO: 19: Saccharomyces cerevisiae S288C base sequence of wild type GAP1 gene

SEQ ID NO: 20: Saccharomyces cerevisiae S288C amino acid sequence of wild type Gap1 protein

SEQ ID NO:21: Base sequence of the variant GAP1 gene (gap1K9, 16R) of Saccharomyces cerevisiae S288C

SEQ ID NO: 22: Base sequence of the CHA1 gene of Saccharomyces cerevisiae S288C

SEQ ID NO:23: Amino acid sequence of Cha1 protein of Saccharomyces cerevisiae S288C

SEQ ID NO:24: Base sequence of the ILV1 gene of Saccharomyces cerevisiae S288C

SEQ ID NO: 25: Saccharomyces cerevisiae S288C amino acid sequence of Ilv1 protein

SEQ ID NO:26: Base sequence of BAT1 gene of Saccharomyces cerevisiae S288C

SEQ ID NO:27: Saccharomyces cerevisiae S288C amino acid sequence of Bat1 protein

SEQ ID NO: 28: Base sequence of the GSH1 gene of Saccharomyces cerevisiae S288C

SEQ ID NO: 29: Saccharomyces cerevisiae S288C Gsh1 protein amino acid sequence

SEQ ID NO: 30: Base sequence of YGL114W gene of Saccharomyces cerevisiae S288C

SEQ ID NO: 31: Saccharomyces cerevisiae S288C YGL114W protein amino acid sequence

Serial number 32~35: introduction

<110> Ajinomoto Co., Ltd.

<120> Method for producing γ-Glu-Abu and method for producing yeast extract containing γ-Glu-Abu

<130> D600-14369

<150> JP2014-008584

<151> 2014-01-21

<160> 35

<170> PatentIn version 3.5

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Claims (16)

A method for producing γ-Glu-Abu, which comprises culturing a yeast having a γ-Glu-Abu production capacity in a medium, causing γ-Glu-Abu to accumulate in the medium, and taking from the medium γ-Glu-Abu, and the aforementioned yeast has the properties of either or both of the following (1) to (2): (1) changed by retaining the variant GAP1 gene; (2) encoded by the YGL114w gene The manner in which the activity of the protein increases increases. The method of claim 1, wherein the variant GAP1 gene is a gene having the following variation of (A) and (B): (A) the quaternary acid residue at position 9 of the wild-type Gap1 protein is substituted with Variation of other amino acid residues; (B) The amino acid residue at position 16 of the wild-type Gap1 protein is substituted with a variation of other amino acid residues. The method of claim 2, wherein the quaternary acid residues of the 9th and 16th positions are substituted with arginine residues. The method of claim 2 or 3, wherein the wild type Gap1 protein is a protein selected from the group consisting of (a) to (c): (a) comprising the amino acid sequence of SEQ ID NO: 20. a protein; (b) an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 20, comprising a substitution, deletion, insertion, or additional amino acid sequence of one or more amino acid residues, and having an amino acid transport Protein active protein; (c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 20 and having amino acid transport protein activity. The method of any one of claims 1 to 4, wherein the activity of the protein encoded by the YGL114w gene is increased by increasing the expression of the YGL114w gene. The method of claim 5, wherein the expression of the YGL114w gene is increased by increasing the copy number of the gene and/or changing the expression regulatory sequence of the gene. A method for producing a yeast extract containing γ-Glu-Abu, which comprises preparing a yeast extract by using yeast having a γ-Glu-Abu accumulation ability as a raw material, and the aforementioned yeast has the following ( 3) Properties: (3) The manner of the activity of the protein encoded by the YGL114w gene is decreased. The method of claim 7, wherein the activity of the protein encoded by the YGL114w gene is decreased by decreasing the expression of the YGL114w gene or by disrupting the gene. The method of any one of claims 1 to 8, wherein the YGL114w gene is selected from the group consisting of the following groups (A) to (E): (A) encoding an amino group represented by SEQ ID NO: 31 Acid-sequence protein DNA; (B) a DNA encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 31, comprising a substitution, deletion, insertion, or additional amino acid sequence of one or more amino acid residues, Yeast γ-Glu-Abu when the activity of yeast is increased in activity, the γ-Glu-Abu production of yeast is improved compared to non-modified strains, and/or the activity is decreased in yeast. The cumulative amount is improved compared to the non-modified strain; (C) the DNA encoding the protein comprising an amino acid having 90% or more identity to the amino acid sequence shown in SEQ ID NO:31. Yeast γ-Glu when the sequence has an increased activity in yeast, the γ-Glu-Abu production of yeast is improved compared to the non-modified strain, and/or the activity is decreased in yeast. -Abu cumulative amount is improved compared to non-modified strain; (D) DNA comprising the base sequence shown in SEQ ID NO: 30; (E) complementary sequence to the nucleotide sequence shown in SEQ ID NO: 30 Or a probe formulated from the complementary sequence will hybridize under stringent conditions and encode a protein DNA, which has the property of increasing the activity of yeast, increasing the γ-Glu-Abu production of yeast compared to non-modified strains, and/or reducing the activity in yeast, γ of yeast The -Glu-Abu accumulation has improved properties compared to non-modified strains. The method according to any one of claims 1 to 9, wherein the yeast system is further modified such that it is selected from the group consisting of γ-glutamine-based cysteine synthetase, α-butyroic acid synthase, and aminotransferase The activity of one or more enzymes in a group is increased. The method of claim 10, wherein the aforementioned α-butyric acid synthase Is an enzyme encoded by the CHA1 gene. The method of claim 10 or 11, wherein the aforementioned aminotransferase is an enzyme encoded by the BAT1 gene. The method of any one of claims 1 to 12, wherein the yeast is a yeast of the genus Saccharomyces or a yeast of Candida. The method of claim 13, wherein the yeast is Saccharomyces cerevisiae or Candida utilis. A yeast having the properties of any one of the following (1) to (4) and having γ-Glu-Abu productivity and/or γ-Glu-Abu accumulation ability: (1) to retain the variant GAP1 The manner of the gene is changed; (2) is changed in such a manner that the activity of the protein encoded by the YGL114w gene is increased; (3) is changed in such a manner that the activity of the protein encoded by the YGL114w gene is decreased; (4) the above (1) and (2) A combination of properties. The yeast according to claim 15 which is further selected from the group consisting of γ-glutamine-based cysteine synthetase, α-butyric acid synthase, and aminotransferase or The manner in which the activity of the above enzymes increases increases.