RU2821273C1 - Variant of glutamate cysteine ligase and method of producing glutathione using same - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a polypeptide involved in production of glutathione, in which the amino acid corresponding to position 653 from the N-end of the amino acid sequence SEQ ID NO: 1 is replaced by methionine. Also disclosed is a polynucleotide encoding said polypeptide, an expression vector containing said polynucleotide, a microorganism for producing glutathione, containing any one or more of said polypeptides, a polynucleotide coding said polypeptide, or an expression vector containing said polynucleotide. Also disclosed is a method of producing glutathione.

EFFECT: invention enables to produce glutathione with high output.

12 cl, 6 tbl, 3 ex

Description

1. Field of the invention

The present disclosure relates to a new variant of glutamate cysteine ligase and a method for producing glutathione using it.

2. Description of related area

Glutathione (GSH) is an organic sulfur compound most commonly found in cells, and it is found in the form of a tripeptide that combines three amino acids: glycine, glutamate cysteine.

In the body, glutathione exists in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSG). Reduced glutathione (GSH), which under normal conditions exists in a relatively high percentage, is mainly distributed in the liver and skin cells of the human body and has important roles such as the antioxidant function of decomposing and removing oxygen radicals, the detoxification function of removing exogenous compounds such as as toxic substances, whitening function - inhibition of melanin pigment production, etc.

Since the production of glutathione gradually decreases with age, the decrease in the production of glutathione, which has an important role in antioxidant function and detoxification function, stimulates the accumulation of oxygen radicals, which is one of the main causes of aging, and therefore external supply of glutathione is necessary (Sipes IG et al. , The role of glutathione in the toxicity of xenobiotic compounds: metabolic activation of 1,2-dibromoethane by glutathione, Adv Exp Med Biol. 1986;197:457-67.).

With such a variety of functions, glutathione has attracted much attention as a substance in various fields such as pharmaceuticals, healthy functional food, cosmetics, etc., and it is also used in the preparation of flavoring agents, food and feed additives. Glutathione is known to have a significant effect on enhancing the flavor of raw materials and maintaining a rich flavor, and glutathione can be used as a flavor enhancer for kokumi by using it alone or in combination with other substances. Generally, kokumi substances have a richer flavor than existing umami substances such as nucleic acids, MSG (monosodium glutamate), etc., and are known to be produced through the decomposition and aging of proteins.

However, despite the increasing demand for glutathione, which can be used in various applications as described above, the enzymatic synthesis method has not yet been commercialized due to high production costs, and the market has not been significantly activated due to the fact that for industrial Glutathione production requires significant costs.

The present inventors have discovered that a microorganism introduced with a newly developed variant of glutamate cyste inligase is capable of producing glutathione in high yield, thereby realizing the present disclosure.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a variant of glutamate cysteine ligase in which the amino acid corresponding to position 653 from the N-terminus of the amino acid sequence SEQ ID NO: 1 is replaced by methionine in a protein having glutamate cysteine ligase activity.

Another object of the present invention is to provide a polynucleotide encoding a given variant and a vector comprising the polynucleotide.

It is yet another object of the present invention to provide a glutathione-producing microorganism by including any one or more options; a polynucleotide encoding this variant; and a vector including the polynucleotide.

Yet another object of the present invention is to provide a method for producing glutathione, which includes the step of cultivating the microorganism.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present disclosure will be described in detail as follows. Meanwhile, each description and embodiment disclosed herein may also apply to other descriptions and embodiments. That is, all combinations of different elements disclosed herein fall within the scope of the present specification.

Moreover, the scope of the present disclosure is not limited to the specific description described below.

In addition, those skilled in the art will be aware of, or will be able to determine by no more than conventional experimentation, many equivalents to specific embodiments of the disclosure described herein. Moreover, these equivalents should be interpreted as falling within the scope of this disclosure.

In one aspect of the present disclosure, a glutamate cysteine ligase variant may be provided comprising an amino acid substitution in a protein having glutamate cysteine ligase activity, wherein the substitution comprises a methionine substitution of the amino acid corresponding to position 653 from the N-terminus of SEQ ID NO: 1.

This variant may be a protein variant in which glycine, which is the amino acid corresponding to position 653 from the N-terminus of the glutamate cysteine ligase amino acid sequence SEQ ID NO: 1, is replaced by methionine.

"Glutamate cysteine ligase (GCL)" as used herein is an enzyme also referred to as "glutamate cysteine ligase" or "gamma-glutamylcysteine synthetase (GCS)". This glutamate cysteine ligase is known to catalyze the following reaction:

Additionally, the reaction catalyzed by glutamate cysteine ligase is known as the first step in glutathione synthesis.

In the present disclosure, the amino acid sequence of glutamate cysteine ligase is the amino acid sequence encoded by the gsh1 gene, and may also be referred to as “GSH1 protein” or “glutamate cysteine ligase”. The amino acid sequence constituting the glutamate cyste inligase of the present disclosure can be obtained from NCBI (National Center for Biotechnology Information) GenBank, which is a well-known database. The glutamate cysteine ligase may be a protein comprising, but not limited to, the amino acid sequence of SEQ ID NO: 1. As another example, glutamate cysteine ligase may be derived from Saccharomyces cerevisiae, and, as yet another example, the amino acid corresponding to position 653 in the amino acid sequence SEQ ID NO: 1 of Saccharomyces may be glycine. However, the glutamate cysteine ligase is not limited to these and may include any sequence without limitation, as long as it is a sequence having the same glutamate cysteine ligase activity as the amino acid sequence.

As a specific example, the glutamate cysteine ligase of the present disclosure may be a protein comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80%, 85%, 90%, 95%, 96 %, 97%, 98%, or 99%, or greater homology or identity with it. It is further understood that proteins having amino acid sequences in which certain sequences are deleted, modified, replaced or added are also included within the scope of the protein that is the target of the modifications of the present disclosure, provided that the amino acid sequences have such homology or identity and demonstrate efficacy consistent with that of the protein above.

Further, as an example of a glutamate cysteine ligase, the present disclosure has been defined as a protein comprising the amino acid sequence of SEQ ID NO: 1, but does not exclude a nonsense sequence upstream or downstream of the amino acid sequence of SEQ ID NO: 1, a naturally occurring mutation, or a silent mutation thereof mutation, and those skilled in the art will appreciate that proteins are also included within the scope of the glutamate cysteine ligase of the present disclosure, provided that they have an activity identical to or corresponding to that of a protein consisting of the amino acid sequence of SEQ ID NO: 1.

That is, in the present disclosure, although the expression of “a protein or polypeptide having the amino acid sequence described by a specific SEQ ID NO”, “a protein or polypeptide comprising the amino acid sequence described by a specific SEQ ID NO” is used, it is obvious that in the present disclosure any protein comprising an amino acid sequence in which certain sequences have been deleted, modified, replaced or added may be used, provided that the protein or polypeptide has an activity identical to or equivalent to that of a polypeptide consisting of the amino acid sequence corresponding to SEQ ID NO.

The term "variant" or "modified polypeptide" as used herein refers to a protein in which one or more amino acids differ from the amino acids of the listed sequence by conservative substitution and/or modification, but the functions or properties of the protein are maintained . With respect to the purposes of the present disclosure, this variant may be a glutamate cysteine ligase variant in which the amino acid corresponding to position 653 from the N terminus of SEQ ID NO: 1 is replaced by methionine in the glutamate cysteine ligase described above or a modified polypeptide having glutamate cysteine ligase activity . With respect to a variant of the present disclosure, a “glutamate cysteine ligase variant” may also be described as a “(modified) polypeptide having glutamate cysteine ligase activity” or a “GSH1 variant.”

This variant differs from sequences that are identified by substitution, deletion, or addition of multiple amino acids. Such a variant can typically be identified by modifying one or more amino acids of the amino acid sequence of the protein and evaluating the properties of the modified protein. In other words, the ability of a given variant may increase, remain the same, or decrease compared to that of the native protein. In addition, a variant may include a modified polypeptide in which one or more than one portion, such as an N-terminal leader sequence or transmembrane domain, is removed. Other variants may include variants in which a portion of the N- and/or C-terminus of the mature protein is removed. The term "variant" or "modified polypeptide" may be used interchangeably with, and is not limited to, terms such as modification, modified protein, mutant, mutein, divergent, variant, etc., provided that it is used to mean modification .

With respect to the objectives of the present disclosure, this embodiment may have increased activity of the modified protein compared to, but is not limited to, the natural wild-type or unmodified protein, or may increase the amount of glutathione produced compared to the pre-modified protein or the natural wild-type or modified polypeptide. .

The term "conservative substitution" as used herein refers to the replacement of an amino acid with another amino acid having similar structural and/or chemical properties. A given variant may have, for example, one or more conservative substitutions while retaining one or more biological activities. This amino acid substitution can generally occur on the basis of similarity in polarity, electrical charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues.

For example, among electrically charged amino acids, positively charged (basic) amino acids include arginine, lysine and histidine, and negatively charged (acidic) amino acids include glutamic acid and aspartic acid; and amino acids having uncharged side chains include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline, serine, threonine, cysteine, tyrosine, asparagine and glutamine.

In addition, this variant may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of a protein that is involved in co-translational or post-translational protein transfer. In addition, a given polypeptide may also be conjugated to another sequence or linker to identify, purify, or synthesize the polypeptide.

In one embodiment, the variant may be a glutamate cysteine ligase variant in which the amino acid corresponding to position 653 from the N-terminus of the amino acid sequence SEQ ID NO: 1 is replaced with methionine. In one embodiment, this variant may be a variant in which the glycine corresponding to position 653 in the amino acid sequence of SEQ ID NO: 1 is replaced by, but is not limited to, methionine.

The term "replacement with another amino acid" as used herein is not limited, provided that the replaced amino acid is different from the amino acid before the substitution. Meanwhile, in the present disclosure, when the expression “a specific amino acid is replaced,” it is obvious that the amino acid is replaced by an amino acid different from the amino acid before the replacement, although it is not even specifically indicated that the amino acid is replaced by another amino acid. The term "corresponding position" as used herein refers to an amino acid residue at a position listed in a protein or polypeptide, or an amino acid residue similar, identical or homologous to a residue listed in a protein or polypeptide. The term "corresponding region" as used herein generally refers to a similar or corresponding position in a related protein or reference protein.

In the present disclosure, specific numbering may be used for amino acid residue positions in the proteins used in the present disclosure. For example, by aligning the target protein to be compared with the polypeptide sequence of the protein of the present disclosure, it is possible to renumber the position corresponding to the position of the amino acid residue of the protein of the present disclosure.

A variant of the glutamate cysteine ligase of the present disclosure in which the amino acid corresponding to position 653 from the N-terminus of the amino acid sequence SEQ ID NO: 1 is replaced by methionine may be a protein in which the amino acid corresponding to position 653 of SEQ ID NO: 1 is replaced by methionine in amino acid sequence SEQ ID NO: 1, or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% -ny, or greater homology or identity with it. Such a variant may be a variant having an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% greater or greater homology or identity to SEQ ID NO: 1, and having less than 100% homology or identity to, but not limited to, SEQ ID NO: 1.

A glutamate cysteine ligase variant of the present disclosure in which the amino acid corresponding to position 653 from the N-terminus of the amino acid sequence SEQ ID NO: 1 is replaced by methionine may comprise the amino acid sequence SEQ ID NO: 3. In particular, it may consist substantially of the amino acid sequence of SEQ ID NO: 3 and, more specifically, may consist of any amino acid sequence of SEQ ID NO: 3, but is not limited to it.

In addition, the variant may include the amino acid sequence of SEQ ID NO: 3 or may include an amino acid sequence having 80% or greater homology or identity with SEQ ID NO: 3, provided that the amino acid at position 653 in this amino acid sequence is fixed (ie, in the amino acid sequence of this variant, the amino acid corresponding to position 653 of SEQ ID NO: 3 is the same as the amino acid at position 653 of SEQ ID NO: 3), but is not limited to them.

In particular, a variant of the present disclosure may include a polypeptide having SEQ ID NO: 3 or having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity with the amino acid sequence of SEQ ID NO: 3. It is further understood that variants having amino acid sequences in which certain sequences are deleted, modified, replaced or added other than position 653 are also included in the scope of the present disclosure provided that the amino acid sequences have such homology or identity and demonstrate potency consistent with that of the variant.

The term "homology" or "identity" as used herein means the correspondence between two given amino acid or base sequences and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.

The homology or sequence identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms and can share a default gap penalty set by the program being used. Substantially homologous or identical sequences are typically capable of hybridizing to all or at least 50%, 60%, 70%, 80% or 90% of the entire length under moderately to highly stringent conditions. It will be appreciated that hybridization also includes hybridization of a polynucleotide with a polynucleotide comprising a common codon or codon, given the degeneracy of the codon.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program, for example using default parameters as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443^153), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277 (version 5.0.0 or later) (including the GCG software package ((Devereux, J., et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 and [CARILLO et al.](1988) SIAM J Applied Math 48: 1073). For example, National Center for Biotechnology Information BLAST or National Center for Biotechnology Information BLAST can be used to determine homology, similarity, or identity. ClustalW.

Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, a GAP computer program such as Needleman et al. (1970), J Mol Biol. 48:443, as announced, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In conclusion, a GAP program can be defined as the value obtained by dividing the number of similarly aligned characters (namely nucleotides or amino acids) by the total number of characters in the shorter of the two sequences. Default parameters for the GAP program may include (1) a binary comparison matrix (including values of 1 for identity and 0 for non-identity) and a weighted comparison matrix Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 (or EDNAFULL substitution matrix (EMBOSS version NCBI NUC4.4)), as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each space and an additional penalty of 0.10 for each character in each space (or a space opening penalty of 10, a space expansion penalty of 0.5); and (3) no penalty for trailing spaces.

Additionally, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing these sequences through Southern hybridization experiments under certain stringent conditions, and suitable hybridization conditions to be determined may be within the given scope of the technology and may be determined in a manner well known to one of ordinary skill in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al. ., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

A glutamate cysteine ligase variant of the present disclosure in which the amino acid corresponding to position 653 from the N-terminus of the amino acid sequence SEQ ID NO: 1 is replaced by methionine may further include a change in which the amino acid corresponding to position 86 from the N-terminus of the amino acid sequence SEQ ID NO: 1, replaced by another amino acid.

In particular, this variant may include a change in which the cysteine corresponding to position 86 is replaced by another amino acid, for example, arginine.

For example, a variant may include, but is not limited to, the amino acid sequence of SEQ ID NO: 13.

According to another aspect of the present disclosure, a polynucleotide encoding a given variant may be provided.

The term "polynucleotide" as used herein refers to a strand of DNA or RNA having a specific length, or moreover, to a polymer of nucleotides in which the nucleotide monomers are linked into a long chain by covalent bonds.

The gene encoding the glutamate cysteine ligase of the present disclosure may be the gsh1 gene.

This gene may come from yeast. In particular, it may be derived from the genus Saccharomyces and, more specifically, it may be derived from Saccharomyces cerevisiae. In particular, the gene may include any gene without limitation, provided that it encodes a polypeptide having glutamate cysteine ligase activity derived from Saccharomyces cerevisiae, and in one embodiment, the gene may be a gene encoding the amino acid sequence SEQ ID NO: 1 and in one embodiment, the gene may include, but is not limited to, the nucleotide sequence of SEQ ID NO: 2.

A polynucleotide encoding a protein variant of the present disclosure may include any polynucleotide, without limitation, so long as it is a polynucleotide encoding a glutamate cysteine ligase variant of the present disclosure and a polypeptide having an activity corresponding thereto.

In a polynucleotide encoding a glutamate cyste inligase of the present disclosure and encoding a variant thereof, various modifications may be made in the coding region, provided that the amino acid sequence of the polypeptide is not altered to account for codon degeneracy or codon preference in the organisms that are intended to express it. polypeptide.

In particular, the polynucleotide encoding a protein variant of the present disclosure may include any polynucleotide sequence, without limitation, provided that it is a polynucleotide sequence encoding a protein variant in which the amino acid corresponding to position 653 in the amino acid sequence SEQ ID NO: 1, replaced by methionine. For example, a polynucleotide encoding a protein variant of the present disclosure may be a polynucleotide sequence encoding a protein variant of the present disclosure, particularly a protein comprising the amino acid sequence of SEQ ID NO: 3, or a polypeptide having homology or identity thereto, but not limiting ourselves to them. This homology or identity is the same as described above.

In addition, a polynucleotide encoding a protein variant of the present disclosure may include a probe that may be derived from a sequence of a known gene, e.g., a sequence without limitation, provided that it is a sequence that hybridizes to a complementary sequence to all or part of that polynucleotide sequence under stringent conditions encoding a protein variant in which the amino acid corresponding to position 653 in the amino acid sequence SEQ ID NO: 1 is replaced by methionine.

The term "stringent conditions" means conditions that ensure specific hybridization between polynucleotides. These conditions are specifically described in documents (eg, J. Sambrook et al., 1989, supra). For example, stringent conditions may include conditions in which polynucleotides having high homology or identity, e.g., 40% or greater, particularly 90% or greater, more particularly 95% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, more particularly 99% or greater homology or identity hybridize with each other, and polynucleotides having less homology or identity, than the above homology or identity do not hybridize with each other, or conventional Southern hybridization wash conditions in which the wash is carried out once, in particular two to three times at a salt concentration and temperature equivalent to 60°C, 1× SSC (sodium citrate and chloride solution), 0.1% SDS, in particular at 60°C, 1× SSC, 0.1% SDS, more particularly at 68°C, 0.1× SSC, 0 .1% SDS.

Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are allowed, depending on the stringency of hybridization. The term "complementarity" is used to describe the relationship between nucleotide bases that are capable of hybridizing with each other. For example, in relation to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, a polynucleotide of the present disclosure may also include substantially similar nucleic acid sequences, as well as isolated nucleic acid fragments that are complementary to the entire sequence.

In particular, a polynucleotide having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and the conditions described above. Moreover, this Tm value may be, but is not limited to, 60°C, 63°C, or 65°C, and may be suitably adjusted by those skilled in the art according to purpose.

The appropriate hybridization stringency of a given polynucleotide depends on the length and level of complementarity of the polynucleotide, and these variables are well known in the art (see Sambrook et al., 1989, supra, 9.50-9.51, 11.7-11.8).

According to yet another aspect of the present disclosure, a vector may be provided including a polynucleotide encoding a given protein variant.

The term “vector” as used herein refers to a DNA construct comprising a polynucleotide sequence encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) such that the polypeptide of interest can be expressed in suitable owner. This expression control region may include a promoter capable of initiating transcription, any operator sequence for providing transcriptional control, a sequence encoding a suitable ribosome binding site for the mRNA, and a sequence controlling the termination of transcription and translation. The vector may be transformed into a suitable host cell and then replicate or function independently of the host genome, or may integrate into the genome itself.

For example, a polynucleotide encoding a protein of interest on a chromosome can be replaced by a mutated polynucleotide by means of a vector for intracellular insertion into the chromosome. Insertion of a given polynucleotide into a chromosome can be accomplished by any method known in the art, such as, but not limited to, homologous recombination. The vector may further include a selectable marker to enable identification of the chromosomal insertion. The selectable marker serves to select cells transformed with vectors, i.e. to achieve identification of the insert of a nucleic acid molecule of interest, and markers that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides, can be used. In media treated with a selection agent, only cells expressing the selectable marker survive or exhibit other phenotypic traits, and thus transformed cells can be selected.

The vector used in the present disclosure is not particularly limited, and any vector known in the art can be used. Yeast expression vectors include both yeast integrating plasmid (Yip) and extrachromosomal plasmid-based vectors. The extrachromosomal plasmid-based vector may include a yeast episomal plasmid (YEp), a yeast replicative plasmid (YRp), and a yeast centromeric plasmid (YCp). Additionally, yeast artificial chromosomes (YACs) can also be used as vectors of the present disclosure. As a specific example, useful vectors may include pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPα A, pGAPα B, pGAPα C, pPIC3. 5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6a A, pPIC6a B, pPIC6α C, pPIC9K, pYC2/CT, yeast display vector pYD1, pYES2, pYES2/CT, pYES2/NT A, pYES2/NT B, pYES2/NT C, pYES2/CT, pYES2.1, pYES-DEST52, pTEF1/Zeo, pFLD1, PichiaPinkTM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, pBY011, pSGP47, PSGP46, PSGP36, PSGP40, ZM552, PAG303GAL-CCDB, PAG414GAL-CCDB, PAS404, PBRIDGE, PGAD-GH, PGBK T7, PHIS-2, POBD2, PRS408, PRS410, PRS418, PRS4 20, PRS428, Form of yeast Micron A , pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, pYJ405 and pYJ406, but are not limited to them.

The term "transformation" as used herein means that a vector comprising a polynucleotide encoding a target protein is introduced into a host cell or microorganism such that the protein encoded by the polynucleotide can be expressed in the host cell . The transformed polynucleotide may be localized by insertion into a host cell chromosome or may be located off a given chromosome, provided that it can be expressed in the host cell. In addition, a given polynucleotide includes DNA and RNA encoding the protein of interest. The polynucleotide may be administered in any form as long as it can be introduced into a host cell and then expressed. For example, a given polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a genetic construct containing all the elements required for autonomous expression. The expression cassette typically may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. This expression cassette may be in the form of an expression vector capable of autonomous replication. In addition, the polynucleotide may be introduced into a host cell in its own form and may be operably linked to, but not limited to, a sequence required for expression in the host cell.

In addition, the term “operably linked” as used herein means that a gene sequence is operably linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the polypeptide of interest herein.

The vector transformation method of the present disclosure includes any method of introducing a nucleic acid into a cell, and can be carried out by selecting an appropriate standard technique, as known in the art, according to the host cell. For example, the method may include electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE (diethylaminoethyl)-dextran method, cationic liposome method, with lithium acetate-DMSO (dimethyl sulfoxide), etc., but not limited to them.

According to the present disclosure, a glutathione-producing microorganism may be provided by including any one or more than one variant, a polynucleotide encoding the variant; and a vector including the polynucleotide.

The term "microorganism" as used herein includes all wild type microorganisms or naturally or artificially genetically modified microorganisms, and the idea is to include all microorganisms in which a specific mechanism is weakened or enhanced due to the insertion of a foreign gene or enhancement activity, or inactivation of an endogenous gene. As used herein, the microorganism may include any microorganism, without limitation, so long as it is a microorganism into which the glutamate cysteine ligase variant of the present disclosure is introduced or incorporated.

A given microorganism, for example, is a cell or microorganism that is transformed by a gene encoding a target protein, or a vector incorporating it, and expresses the target protein. For purposes of the present disclosure, the host cell or microorganism is any microorganism so long as it is capable of producing glutathione through the inclusion of a glutamate cysteine ligase variant.

The term “glutathione” as used herein is used interchangeably with “GSH” and refers to a tripeptide consisting of three amino acids: glutamate, cysteine and glycine. Glutathione can be used as a raw material in, but not limited to, pharmaceuticals, healthy functional foods, flavorings, foods, feed additives, cosmetics, etc.

In the present disclosure, the term "glutathione-producing microorganism" includes all microorganisms in which a genetic change occurs naturally or artificially, and is a microorganism in which a specific mechanism is weakened or enhanced due to the insertion of a foreign gene or the upregulation or inactivation of an endogenous gene. , and may be a microorganism in which genetic changes have occurred or activity has been enhanced to produce the desired glutathione. For purposes of the present disclosure, the term “glutathione-producing microorganism” may refer to a microorganism that includes a glutamate cysteine ligase to produce an excess amount of the desired glutathione compared to a wild-type microorganism or an unmodified microorganism. The term "glutathione-producing microorganism" may be used interchangeably with terms such as "glutathione-producing microorganism", "microorganism having the ability to produce glutathione", "glutathione-producing strain", "strain having the ability to produce glutathione", etc.

The type of glutathione-producing microorganism is not particularly limited as long as the microorganism is capable of producing glutathione, but it may be a microorganism of the Saccharomyces genus, particularly but not limited to Saccharomyces cerevisiae.

The parent strain of the glutathione-producing microorganism including this variant is not particularly limited as long as it is capable of producing glutathione. The microorganism may further include changes such as enhancement of the biosynthetic pathway to increase the ability to produce glutathione, reduction of feedback inhibition, inactivation of genes that weaken the degradation pathway or biosynthetic pathway, not excluding changes found in nature. In one embodiment, the microorganism may include a change that increases the ability to produce glutathione in the control of glutamate cysteine ligase expression. The change may be any one or more than one change selected from -250(C→T), -252(G→A), -398(A→T), -399(A→C), -407( T→C) and -409(T→C) upstream of, but not limited to, the GSH1 ORF.

A microorganism including any one or more than one embodiment of the present disclosure; a polynucleotide encoding this variant; and the vector comprising the polynucleotide may be a microorganism expressing a glutamate cysteine ligase variant in which the amino acid corresponding to position 653 in the amino acid sequence of SEQ ID NO: 1 is replaced by, but is not limited to, methionine.

This glutamate cysteine ligase and its variant are as described above.

The term protein "to be expressed/expressed" as used herein means a state in which a target protein is introduced into a microorganism or is modified for expression in a microorganism. When the target protein is a protein present in microorganisms, it means a state in which its activity is enhanced relative to endogenous activity or activity before modification.

A microorganism expressing a protein variant of the present disclosure may be a microorganism modified to express a protein variant of the present disclosure, and therefore, according to yet another aspect of the present disclosure, a method for producing a microorganism expressing a protein variant of the present disclosure is provided.

As used herein, the term “introducing a protein” refers to demonstrating the activity of a particular protein that the microorganism does not initially possess, or demonstrating enhanced activity compared to the endogenous activity of the corresponding protein or activity before modification. For example, introduction of a protein may refer to the introduction of a particular protein, the introduction of a polynucleotide encoding a particular protein into the chromosome of a microorganism, or the introduction of a vector comprising a polynucleotide encoding a particular protein into a microorganism, thereby expressing its activity.

The term "enhancing" the activity of a polypeptide or protein as used herein means that the activity of the polypeptide or protein is increased relative to its endogenous activity. The term "amplification" can be used interchangeably with terms such as up-regulation, overexpression, increase, etc. Here, enhancement may include both demonstrating activity that was not originally possessed and demonstrating improved activity compared to endogenous activity or activity before modification. This "endogenous activity" means the activity of a specific polypeptide or protein that was originally possessed by the parent strain before the trait changed, or by an unmodified microorganism when the trait is modified by genetic change due to natural or man-made factors. This can be used interchangeably with the phrase "activity before modification". The fact that the activity of a polypeptide or protein is “enhanced” or “increased” relative to endogenous activity means that the activity of the polypeptide or protein is improved relative to the activity of the specific polypeptide or protein originally possessed by the parent strain before the trait change or by the unmodified microorganism .

This “enhancement of activity” can be achieved by introducing a foreign polypeptide or protein, or enhancing the endogenous activity of the polypeptide or protein, in particular by enhancing the endogenous activity of the polypeptide or protein. Increased activity of a given polypeptide or protein can be demonstrated by an increase in the activity level and expression level of the corresponding polypeptide or protein, or the amount of product produced from the corresponding protein.

Various methods well known in the art can be used to enhance the activity of a polypeptide or protein, and this method is not limited to, provided that the activity of the polypeptide or protein of interest can be enhanced relative to the activity of the microorganism before modification. In particular, genetic engineering and/or protein engineering, well known to those skilled in the art, which are traditional methods of molecular biology, can be used, but this method is not limited to them (Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol.2. 116, Sambrook et al. Molecular Cloning 2012, etc.).

In particular, a method of enhancing the activity of a polypeptide or protein using genetic engineering can be carried out, for example, as follows:

1) an increase in the number of intracellular copies of a gene or polynucleotide encoding a polypeptide or protein;

2) replacement of the regulatory region of gene expression on the chromosome encoding the polypeptide or protein with a sequence demonstrating strong activity;

3) modification of the start codon of a polypeptide or protein, or the base sequence encoding the 5'-UTR (untranslated) region;

4) modification of a polynucleotide sequence on a chromosome to enhance the activity of a polypeptide or protein;

5) introduction of a foreign polynucleotide demonstrating the activity of a polypeptide or protein, or a variant of this polynucleotide with optimized codons; or

6) a combination of the above methods, but not limited to them.

This method of enhancing the activity of a polypeptide or protein using a protein engineering method can be carried out, for example, by, but not limited to, modifying or chemically modifying the exposed region selected by analyzing the three-dimensional structure of the polypeptide or protein.

(1) Increasing the intracellular copy number of a gene or polynucleotide encoding a given polypeptide or protein can be accomplished by any method well known in the art, for example, by introducing a vector that replicates and functions independently of the host cell and is linked in a functional manner to the gene or a polynucleotide encoding the corresponding polypeptide or protein into a host cell. Alternatively, it can be carried out by introducing a vector that is functionally linked to a given gene and is capable of inserting a given gene or polynucleotide into, but not limited to, the chromosome of a host cell, into the host cell.

(2) Replacement of a gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding a given polypeptide or protein with a sequence exhibiting potent activity can be accomplished by any method known in the art, for example, by introducing a mutation into the sequence through a deletion, insertion, non-conservative or conservative substitution of a nucleotide sequence, or any combination thereof, or by substitution of a sequence with a nucleotide sequence with stronger activity to further enhance the activity of a given expression regulatory region. This expression regulatory region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence for regulating transcription and translation termination. This method may specifically be carried out by binding to a stronger heterologous promoter instead of, but not limited to, an endogenous promoter.

Examples of known promoters for eukaryotes include prototors for translation elongation factor 1 (TEF1), glycerol-3-phosphate dehydrogenase 1 (GPD1), 3-phosphoglycerate kinase, or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase, and examples of other yeast promoters that are inducible promoters with the added benefit of transcription controlled by growth conditions include promoters for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase and enzymes responsible for the utilization of maltose and galactose, and when the host cell is yeast, available promoters may include TEF1 promoter, TEF2 promoter, GAL10 promoter, GAL1 promoter, ADH1 promoter, ADH2 promoter, PH05 promoter, GAL1-10 promoter, TDH3 promoter (GPD promoter), TDH2 promoter, TDH1 promoter, PGK1 promoter, PYK2 promoter, ENO1 promoter, ENO2 promoter and TRI promoter, and vectors and promoters suitable for use in yeast expression are further described in EP 073657, but are not limited to them. Yeast enhancers can also be used advantageously, but not limited to, with yeast promoters.

(3) Modification of the start codon of the polypeptide or protein, or the base sequence encoding the 5'-UTR region, can be accomplished by any method known in the art, for example, by replacing the endogenous start codon of the polypeptide or protein with another start codon with a higher expression level of the polypeptide or protein compared to, but not limited to, endogenous initiation.

(4) Modification of a polynucleotide sequence on a chromosome to enhance the activity of the polypeptide or protein can be accomplished by any method known in the art, for example, by inducing a modification on an expression control sequence through deletion, insertion, non-conservative or conservative substitution, or any combination thereof to further enhance activity a given polynucleotide sequence, or by replacing the given sequence with a polynucleotide sequence modified to have greater activity. This substitution may be, but is not limited to, a specific insertion of a gene into a chromosome via homologous recombination.

The vector used herein may further include a selectable marker to detect a chromosomal insertion. This selectable marker is as described above.

(5) Administration of a foreign polynucleotide exhibiting the activity of a given polypeptide or protein can be accomplished by any method known in the art, for example, by introducing a foreign polynucleotide encoding a polypeptide or protein having identical/similar activity to that of the polypeptide or protein, or by introducing into the host cell of its codon-optimized polynucleotide variant. The origin or sequence of the foreign polynucleotide is not particularly limited as long as the foreign polynucleotide exhibits identical/similar activity to that of the polypeptide or protein. In addition, a polynucleotide with optimized codons may be introduced into a host cell to optimize transcription and translation of the introduced foreign polynucleotide in the host cell. This administration can be accomplished by any known transformation method suitably selected by those of ordinary skill in the art. As the introduced polynucleotide is expressed in the host cell, the polypeptide or protein is produced, thereby increasing its activity.

Finally, (6) the combination of the above methods can be carried out by applying one or more than one method (1) to (5) in combination.

An increase in the activity of a polypeptide or protein, as described above, may be an increase in the activity or concentration of the corresponding polypeptide or protein compared to the activity or concentration of the polypeptide or protein expressed in the wild-type microorganism or the microorganism before modification, or an increase in the amount of product obtained from the corresponding polypeptide or protein, but not limited to them.

The term "pre-modification strain" or "pre-modification microorganism" as used herein does not exclude strains containing mutations naturally occurring in microorganisms, and it may refer to the wild-type strain itself or a natural-type strain or strain before transformation by genetic modification due to natural or artificial factors. The term "pre-modification strain" or "pre-modification microorganism" may be used interchangeably with the term "unmutated strain", "unmodified strain", "unmutated microorganism", "unmodified microorganism" or "reference microorganism".

A microorganism comprising a glutamate cysteine ligase variant, or comprising a polynucleotide encoding the variant, or a vector comprising the polynucleotide, as used herein, may be a recombinant microorganism, or recombination may be achieved by genetic modification such as transformation.

For example, the microorganism may be, but is not limited to, a recombinant microorganism obtained by transformation using a vector including the polynucleotide. The recombinant microorganism may be a yeast, and an example may be a microorganism of the genus Saccharomyces, particularly but not limited to Saccharomyces cerevisiae.

According to yet another aspect of the present disclosure, there is provided a method for producing glutathione, comprising the step of culturing the microorganism. This microorganism and glutathione are as described above.

The medium and other culture conditions used for cultivating the strain of the present disclosure can be any medium without particular limitation, provided that it is a medium commonly used for cultivating microorganisms of the genus Saccharomyces. In particular, the strain of the present disclosure can be cultivated in a conventional medium containing proper carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and/or vitamins, etc. when monitoring temperature, pH, etc. under aerobic or anaerobic conditions.

In the present disclosure, these carbon sources include carbohydrates such as glucose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc.; and the like, but are not limited to them. In addition, natural organic nutrients such as starch hydrolysate, molasses, raw molasses, rice bran, cassava, sugar cane residue and liquid corn extract can be used. In particular, carbohydrates such as glucose and sterilized pre-processed molasses (namely, molasses converted to reducing sugar) can be used, and suitable amounts of other carbon sources can be used in various ways without limitation. These carbon sources can be used alone or in combination of two or more of them.

As nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc. can be used; and organic nitrogen sources such as amino acids, peptone, NZ-amine, meat extract, yeast extract, malt extract, liquid corn extract, casein hydrolysate, fish or its decomposition products and defatted soybean meal or its decomposition products, etc. These nitrogen sources may be used alone or in combination of, but not limited to, two or more of them.

Sources of phosphorus may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium containing salts. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, calcium carbonate, etc. can be used.

In addition to these compounds, amino acids, vitamins and/or suitable precursors may be contained. In particular, L-amino acids, etc. can be added to the culture medium of the strain. In particular, glycine, glutamate and/or cysteine, etc. can be added, and if necessary, L-amino acids such as lysine, etc. can be further added, but not necessarily limited to them.

These media or precursors can be added to the culture in batches or continuously, but are not limited to them.

In the present disclosure, during the cultivation of a given strain, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture in the correct manner. During culture, foaming can be suppressed by the use of an antifoam agent such as a fatty acid polyglycol ester. In addition, oxygen or oxygen-containing gas may be injected into the culture in order to maintain an aerobic state of the environment, or no gas may be injected, or nitrogen, hydrogen or carbon dioxide gas may be injected in order to maintain anaerobic and microaerobic states.

The culture temperature may be maintained between 25°C and 40°C, more specifically, but not limited to, 28°C to 37°C. Cultivation may continue until the desired amount of product is obtained, in particular, but not limited to, within 1 hour to 100 hours.

The method for producing glutathione may further include an additional process after the cultivation step. This additional process can be suitably selected according to the use of glutathione.

In particular, the method for producing glutathione may include a step of collecting glutathione that accumulates in cells through a culture step, for example, a step of collecting glutathione from one or more materials selected from a strain, a dry product, an extract, a culture and a lysate thereof, after the culture step. .

The method may further include the step of lysing the strain before or simultaneously with the collection step. Lysis of a given strain can be accomplished by a method commonly used in the field to which the present disclosure relates, for example, using a lysis buffer, a sonicator, a heat treatment and a coffee maker, etc. Additionally, this lysis step may involve enzymatic reactions such as, but not limited to, cell wall degrading enzymes, nucleic acid degrading enzymes, nucleic acid transferases, and protein degradation enzymes.

With respect to the objects of the present disclosure, dry yeast, yeast extract, mixed yeast extract powder, or pure glutathione having a high glutathione content can be produced by, but not limited to, the glutathione production method, and can be suitably produced according to the desired product.

The term "dry yeast" as used here can be used interchangeably with the term "dry strain", etc. Dry yeast can be obtained by drying a yeast strain in which glutathione accumulates, and in particular can be included in, but not limited to, a feed composition, food composition, etc.

The term "yeast extract" as used herein may be used interchangeably with the term "strain extract", etc. Strain extract may refer to the substance that remains after the cell wall is separated from the cell body of the strain. In particular, the strain extract may refer to the remaining components, excluding the cell wall, obtained by lysis of the cell body. A given strain extract may include glutathione and may include, as components other than glutathione, one or more than one protein, carbohydrate, nucleic acid, and fiber components, but is not limited to.

The collection step can be carried out using a suitable method known in the art, thereby collecting glutathione, which is the desired substance.

The collection stage may include a purification process. This purification process may be the process of separating only pure glutathione from the strain. Through the purification method, pure glutathione can be obtained.

As necessary, the method for producing glutathione may further include the step of mixing an excipient with a substance selected from the strain obtained after the cultivation step, the dry product, the extract, the culture and its lysate, and the glutathione collected therefrom. Through this mixing step, a mixed yeast extract powder can be obtained.

The excipient may be suitably selected for use according to its intended use or form and, for example, may be selected from starch, glucose, cellulose, lactose, glycogen, D-mannitol, sorbitol, lactitol, maltodextrin, calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, dextrin, sodium alginate, methylcellulose, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose, propylene glycol, casein, calcium lactate, Primogel, gum acacia and in particular one or more than one a component selected from, but not limited to, starch, glucose, cellulose, lactose, dextrin, glycogen, D-mannitol and maltodextrin.

This excipient may include, for example, but is not limited to, a preservative, humectant, dispersing agent, suspending agent, buffering agent, stabilizer or isotonic agent, etc.

According to yet another aspect of the present disclosure, the use of a variant of the present disclosure in the production of glutathione is provided.

According to yet another aspect of the present disclosure, there is provided the use of a microorganism including a variant of the glutamate cysteine ligase of the present disclosure in the production of glutathione.

The variant, polynucleotide and microorganism are as described above.

Below, the present invention will be described in more detail with reference to Examples and Experimental Example. However, these Examples and the Experimental Example serve only to illustrate the present disclosure, and the scope of the present disclosure is not intended to be limited by these Examples and the Experimental Example.

Example 1: Selection and Improvement of a Glutathione Producing Strain

Example 1-1: Selection of a Glutathione Producing Strain

Strains were obtained from Nuruk starter containing different strains, and strains with the ability to produce glutathione were selected, improving these strains.

In detail, samples of grains such as rice, barley, mung bean, oats, etc. were taken from a total of 20 regions including Yongin, Icheon, Pyeongtaek, Hwaseong, Gyeonggi Province of the Republic of Korea, and then ground, kneaded, wrapped in cloth , squeezed tightly, wrapped in straw, followed by fermentation for 10 days. These samples were then slowly dried to obtain nuruk.

In order to isolate different strains from the resulting nuruk, experiments were carried out as follows. 45 ml of saline solution was added to 5 g of nuruk and crushed using a mixer. For pure separation of yeast strains, serial dilution was carried out, followed by distribution on YPD (yeast peptone dextrose) agar (10 g/l yeast extract, 20 g/l bactopeptone, 20 g/l glucose, based on 1 liter of distilled water) and incubation at 30°C for 48 hours. Yeast colonies were then streaked onto YPD agar by colony morphology and microscopic inspection. 25 ml of YPD broth was dosed into a 250 ml Erlenmeyer flask, the pure separated strain was inoculated and cultured with shaking (30°C, 200 rpm) for 48 hours to confirm glutathione production, and the strain was screened.

To improve the initially isolated strains, random mutations were induced in these isolated strains. In detail, among the yeasts isolated from nuruk, a strain was isolated that was confirmed to produce glutathione and was called strain CJ-37. This CJ-37 strain was cultured on a solid medium, inoculated into a broth to obtain a culture medium, and the cells were irradiated with UV (ultraviolet) light using a UV lamp. The UV-irradiated culture medium was then transferred to plate medium to obtain only colony-forming mutant strains and their glutathione production was tested.

As a result, from the mutant strains, the strain demonstrating the highest production of glutathione was selected as the glutathione-producing strain, named strain CJ-5 and deposited in the Korean Microbial Cultivation Center (KCMC), an international depository body operating under the Budapest Agreement, on July 31, 2019 ., and assigned it accession number KCCM12568P.

Example 1-2: Experiment for Additional Improvement for Increased Glutathione Production Capacity

In order to further improve the glutathione production capacity of strain CJ-5, mutations were induced as follows.

Strain CJ-5 was cultured on solid medium, inoculated into broth to obtain culture medium, and the cells were irradiated with UV using a UV lamp. The UV-irradiated culture medium was then transferred to plate medium to obtain only colony-forming mutant strains, the strain showing the most improved glutathione production ability was isolated, named strain CC02-2490, and deposited in the Korea Center for Microbial Cultivation (KCCM) - International depository authority operating under the Budapest Agreement, January 17, 2020, and assigned accession number KCCM12659P.

As a result of the analysis of the nucleotide sequence of the glutathione biosynthesis gene gsh1, in connection with the increased ability to produce glutathione in strain CC02-2490, it was confirmed that cysteine, which is the 86th amino acid of the GSH1 protein (SEQ ID NO: 1), encoded by the gsh1 gene, is replaced by arginine .

Example 1-3: Experiment for Further Improvement to Increase Glutathione Production Capacity

In order to further improve the glutathione production capacity of strain CC02-2490, mutations were induced as follows.

Strain CC02-2490 was cultured on solid medium, inoculated into broth to obtain culture medium, and these cells were irradiated with UV using a UV lamp. The UV-irradiated culture medium was then transferred to plate medium to obtain only colony-forming mutant strains, the strain exhibiting the most improved glutathione production ability was isolated, named strain CC02-2544, and deposited in the Korean Microbial Cultivation Center (KCMC) - International depository authority operating in accordance with the Budapest Agreement, February 20, 2020, and assigned it access number KSSM 12674R.

As a result of the analysis of the nucleotide sequence of the glutathione biosynthesis gene gsh1 in connection with the increase in the ability to produce glutathione of strain CC02-2544, it was confirmed that changes occurred -250(C→T), -252(G→A), -398(A→T ), -399(A→C), -407(T→C), -409(T→C) above ORF GSH1 (SEQIDNO: 12).

Example 2: experiment to further improve strain CC02-2544 to increase the ability to produce glutathione

In order to further improve the glutathione production capacity of strain CC02-2544, mutations were induced as follows.

Strain CC02-2544 was cultured on solid medium, inoculated into broth to obtain culture medium, and these cells were irradiated with UV using a UV lamp. The UV-irradiated culture medium was then transferred to plate medium to obtain only colony-forming mutant strains, followed by nucleotide sequence analysis.

This experiment confirmed that methionine was replaced by the 653rd amino acid (glycine) of GSH1, which is a protein encoded by the glutathione biosynthesis gene gsh1 of the strain, which exhibits a 27% increase in glutathione content. This strain was named CC02-2816 and was deposited in the Korean Microbial Cultivation Center (KCMC), the international depositary body operating under the Budapest Agreement, on December 8, 2020, and assigned accession number KCM12891R.

Example 3: Experiment to change residue G653 of GSH1

From the above results of Example 2, it was determined that position 653 of the GSH1 protein would be important for glutathione production, and variant strains of Saccharomyces cerevisiae (S. cerevisiae) CEN.PK2-1D and CC02-2544 were prepared to express protein variants in which the amino acid position 653 of the GSH1 protein was replaced with another amino acid, and the intention was to test whether glutathione production was increased or not. On the other hand, as mentioned above, strain CC02-2544 is a strain having the changes -250(C→T), -252(G→A), -398(A→T), -399(A→C), -407(T→C), -409(T→C) upstream of the GSH1 ORF in the GSH1 C86R strain variant. In the corresponding strain, an additional amino acid change was introduced at position 653 of the GSH1 protein.

To obtain a strain in which the amino acid at position 653 of the GSH1 protein of the yeast Saccharomyces cerevisiae was replaced by methionine, plasmids pWAL100 and pWBR100 were used with reference to the content disclosed in the literature: Lee TH, et al. (J. Microbiol. Biotechnol. (2006), 16(6), 979 982). In detail, PCR (polymerase chain reaction) was performed as follows using the genomic DNA of strain CJ-5 as a template. PCR was performed using primers SEQ ID NO: 4 and SEQ ID NO: 5 to obtain a portion of the N-terminal sequence of GSH1, including the N-terminal flanking sequence BamHI, the start codon ORE of GSH1 and the intermediary encoding change G653M, and using primers SEQ ID NO: 6 and SEQ ID NO: 7 to obtain part of the C-terminal sequence of GSH1, including the C-terminal flanking sequence XhoI, the stop codon of the GSH1 ORF, and the intermediary encoding the G653M change. PCR was then performed with overlapping primers using these two sequences as templates and SEQ ID NO: 4 and SEQ ID NO: 7, resulting in a GSH1 ORF fragment including a sequence encoding a variant of the GSH1 protein in which the amino acid at position 653 was replaced by methionine, the N-terminal restriction enzyme sequence BamHI and the C-terminal restriction enzyme sequence XhoI. This ORF fragment was digested with BamHI and XhoI, and then cloned into the pWAL100 vector, which was treated with the same enzymes, to obtain the pWAL100-GSH1(G653M) vector.

In addition, PCR was performed using the genomic DNA of strain CJ-5 as a template and SEQ ID NO: 8 and SEQ ID NO: 9 to obtain a 500 bp fragment. after the stop codon of the GSH1 ORF containing the N-terminal restriction enzyme sequence SpeI and the C-terminal restriction enzyme sequence NcoI. This fragment was then cloned into pWBR100, which was treated with the same restriction enzymes, to obtain the pWBR100-GSH1 vector.

Finally, to obtain the DNA fragment to be introduced into the yeast, a PCR product containing the sequence encoding the methionine variant and part of KIURA3 was obtained using the previously obtained vector pWAL100-GSH1 (G653M) as a template and primers SEQ ID NO: 4 and SEQ ID NO: 10, and a PCR product containing part of KIURA3 and 500 bp. after the stop codon, GSH1 was prepared using the vector pWBR100-GSH1 as a template and primers SEQ ID NO: 11 and SEQ ID NO: 9, and then each PCR product was transformed into S. cerevisiae CEN.PK2-1D and S. cerevisiae CC02- 2544 in the same molar ratio. PCR was performed under conditions of heat denaturation at 95°C for 5 minutes, annealing at 53°C for 1 minute, and polymerization at 72°C for 1 minute per 1 kb. Yeast transformation was carried out using the lithium acetate method modified from Geitz's thesis (Nucleic Acid Research, 20(6), 1425). In detail, yeast cells at OD (optical density) of 0.7 ~ 1.2 were washed twice with lithium acetate/TE buffer, and then PCR products and single-stranded DNA (Sigma D-7656) were mixed together and incubated in lithium acetate buffer /TE/40% PEG (polyethylene glycol) for 30 minutes at 30°C and 42°C for 15 minutes, and then these cells were cultured on an SC agar plate (2% glucose) without uracil until colonies became visible, and a strain was obtained into which the coding sequence of the G653M GSH1 change and the KIURA3 gene were introduced. Then, to remove KIURA3, each strain was incubated overnight in 2 ml YPD, diluted 1/100 and spread on an SC agar plate (2% glucose) containing 0.1% 5-FOA to obtain a variant strain S. cerevisiae CEN.PK2-1D GSH1 G653M and the variant strain S. cerevisiae CC02-2544 GSH1 G653M, from which the uracil marker was removed. A strain was also obtained capable of expressing a variant of the GSH1 protein in which substitutions were made with amino acid types other than methionine in the same manner, except using a pair of primers in which the sequence encoding methionine at position 653 on the primer sequences SEQ ID NO: 5 and SEQ ID NO: 6, has been replaced by a sequence encoding a different amino acid.

The results of measuring the concentration and content of glutathione (GSH) produced by culturing each of the resulting strains for 26 hours are shown in Table 2 and Table 3. Strain CC02-2544 was further introduced with G653M GSH1 variation (SEQ ID NO: 13). As a result, the GSH concentration increased by 77 mg/L - from 471.5 mg/L to 548.5 mg/L, and as a result of introducing the G653M GSH1 change (SEQ ID NO: 3) into strain CEN.PK-1D, the GSH concentration increased by 58 mg/l from 42 mg/l to 100 mg/l.

Example 3-1: Introduction of G653 GSH1 Change into Strain CC02-2544

Example 3-2: Introduction of the G653 GSH1 change into strain CEN.PK-1D

From this it can be seen that the GSH1 variant, in which the glycine at position 653 of the GSH1 protein is replaced by methionine, significantly increases the ability to produce glutathione.

This confirmed that the new GSH1 variant developed in this disclosure exhibits increased glutathione production. In addition, the yeast exhibiting high glutathione production through the inclusion of the GSH1 variant of the present disclosure, its dry product, its extract, its culture, its lysate and the glutathione produced have antioxidant effects, detoxifying effects and immune enhancing effects and thus they can also usefully used in cosmetic compositions, food compositions, feed compositions, pharmaceutical compositions and their preparation.

Reference example: experiment on the replacement of the C86 residue of the GSH1 protein

Variants of the strain Saccharomyces cerevisiae (S. cerevisiae) CEN.PK2-1D and Saccharomyces cerevisiae (S. cerevisiae) CJ-5 were obtained to express a protein variant in which the amino acid cysteine at position 86 of the GSH1 protein is replaced by another amino acid. The intention was to test whether glutathione production increases.

To obtain a strain in which the cysteine at position 86 of the Saccharomyces cerevisiae GSH1 protein was replaced by arginine, plasmids pWAL100 and pWBR100 were used with reference to the content disclosed in the literature by Lee TH, et al. (J. Microbiol. Biotechnol. (2006), 16(6), 979 982). In detail, PCR was performed as follows using genomic DNA of strain CJ-5 as a template. PCR was performed using primers SEQ ID NO: 4 and SEQ ID NO: 14 to obtain a portion of the N-terminal sequence of GSH1, including the N-terminal flanking sequence BamHI, the start codon ORE of GSH1 and the sequence encoding the C86R change, and using primers SEQ ID NO: 15 and SEQ ID NO: 7 to obtain a portion of the C-terminal sequence of GSH1, including the C-terminal flanking sequence of XhoI, the stop codon of the GSH1 ORF, and the sequence encoding the C86R change. PCR was then performed with overlapping primers using these two sequences as templates and SEQ ID NO: 4 and SEQ ID NO: 7, resulting in a GSH1 ORF fragment including a sequence encoding a variant of the GSH1 protein in which a cysteine is at position 86 was replaced by arginine, the N-terminal restriction enzyme sequence BamHI and the C-terminal restriction enzyme sequence XhoI. This ORF fragment was digested with BamHI and XhoI, and then cloned into the pWAL100 vector, which was treated with the same enzymes, to obtain the pWAL100-GSHl(C86R) vector.

In addition, PCR was performed using the genomic DNA of strain CJ-5 as a template and SEQ ID NO: 8 and SEQ ID NO: 9 to obtain a 500 bp fragment. after the stop codon of the GSH1 ORF containing the N-terminal restriction enzyme sequence SpeI and the C-terminal restriction enzyme sequence NcoI, which was then treated with the restriction enzymes SpeI and NcoI. This fragment was then cloned into pWBR100, which was treated with the same restriction enzymes, to obtain the pWBR100-GSHl vector.

Finally, to obtain a DNA fragment to be introduced into yeast, a PCR product was obtained containing the sequence encoding the arginine change and part of KIURA3, using the previously obtained vector pWBR100-GSH1(C86R) as a template and primers SEQ ID NO: 4 and SEQ ID NO: 10, and obtained a PCR product containing part of KIURA3 and 500 bp. after the stop codon of GSH1, using the vector pWBR100-GSHl as a template and primers SEQ ID NO: 11 and SEQ ID NO: 9, and then each PCR product was transformed into S. cerevisiae CEN.PK2-1D and S. cerevisiae CJ- 5 at the same molar ratio. PCR was performed under conditions of heat denaturation at 95°C for 5 minutes, annealing at 53°C for 1 minute, and polymerization at 72°C for 1 minute per 1 kb. Yeast transformation was carried out using the lithium acetate method modified from Geitz's thesis (Nucleic Acid Research, 20(6), 1425). In detail, yeast cells at an OD of 0.7-1.2 were washed twice with lithium acetate/TE buffer, and then PCR products and single-stranded DNA (Sigma D-7656) were mixed together and incubated in lithium acetate/TE/40 buffer. % PEG for 30 minutes at 30°C and 42°C for 15 minutes, and then these cells were subjected to stationary culture on an SC agar plate (2% glucose) without uracil until colonies became visible, and a strain was obtained, in which the coding sequence for the C86R GSH1 change and the KIURA3 gene were introduced. Then, to remove KIURA3, each strain was incubated overnight in 2 ml YPD, diluted 1/100 and spread on an SC agar plate (2% glucose) containing 0.1% 5-FOA to obtain a variant strain S. cerevisiae CEN.PK2-1D GSH1 C86R and variant strain S. cerevisiae CJ-5 GSH1 C86R, from which the uracil marker was removed. A strain was also obtained capable of expressing a variant of the GSH1 protein in which substitutions were made with amino acid types other than arginine in the same manner, except using a pair of primers in which the sequence encoding arginine at position 86 on the primer sequences SEQ ID NO: 14 and SEQ ID NO: 15 has been replaced by a sequence encoding a different amino acid.

The results of measuring the concentration of glutathione (GSH) produced by culturing each of the resulting strains for 26 hours are shown in Table 5 and Table 6.

■

As a result of this experiment, it was confirmed that when replacing the cysteine at position 86 of the GSH1 protein with another amino acid, the ability to produce glutathione increases compared to the ability of the wild-type GSH1 protein.

This indicates that the GSH1 variant, in which the cysteine at position 86 of the GSH1 protein is replaced by another amino acid, has a significantly increased ability to produce glutathione.

Based on the above description, those skilled in the art will appreciate that the present disclosure may be applied in various specific forms without altering its technical nature or essential characteristics. In this regard, it is to be understood that the above embodiment is not limiting, but is illustrative in all aspects. The scope of this disclosure is determined by the appended claims and not by the description preceding them, and it is therefore intended that all changes and modifications that fall within the scope of the claims, or equivalents thereto, are therefore covered by the scope of the claims.

Invention effect

The novel glutamate cysteine ligase variant of the present disclosure significantly increases glutathione production and thus can be usefully used for high glutathione production. As described, yeasts exhibiting high glutathione production, their dry product, their extract, their culture, their lysate and the glutathione produced have antioxidant effects, detoxification effects and immune enhancing effects, and thus they can also be usefully used in cosmetic compositions , food compositions, feed compositions, pharmaceutical compositions and their preparation.

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LIST OF SEQUENCES

<110>CJ CheilJedang Corporation

<120> VARIANT OF GLUTAMATE-CYSTEINE LIGASE AND METHOD FOR OBTAINING GLUTATHIONE

USING IT

<130>OPA21295

<150> KR10-2021-0000361

<151> 2021-01-04

<160> 15

<170> KoPatentIn 3.0

<210> 1

<211> 678

<212>PRT

<213> Saccharomyces cerevisiae

<400> 1

Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gln Trp Phe Glu Ser Arg

1 5 10 15

Thr Tyr Asn Glu His Ile Arg Asp Glu Gly Ile Glu Gln Leu Leu Tyr

20 25 30

Ile Phe Gln Ala Ala Gly Lys Arg Asp Asn Asp Pro Leu Phe Trp Gly

35 40 45

Asp Glu Leu Glu Tyr Met Val Val Asp Phe Asp Asp Lys Glu Arg Asn

50 55 60

Ser Met Leu Asp Val Cys His Asp Lys Ile Leu Thr Glu Leu Asn Met

65 70 75 80

Glu Asp Ser Ser Leu Cys Glu Ala Asn Asp Val Ser Phe His Pro Glu

85 90 95

Tyr Gly Arg Tyr Met Leu Glu Ala Thr Pro Ala Ser Pro Tyr Leu Asn

100 105 110

Tyr Val Gly Ser Tyr Val Glu Val Asn Met Gln Lys Arg Arg Ala Ile

115 120 125

Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gln Asp Ser Lys Asn Asn

130 135 140

Leu His Val Gly Ser Arg Ser Val Pro Leu Thr Leu Thr Val Phe Pro

145 150 155 160

Arg Met Gly Cys Pro Asp Phe Ile Asn Ile Lys Asp Pro Trp Asn His

165 170 175

Lys Asn Ala Ala Ser Arg Ser Leu Phe Leu Pro Asp Glu Val Ile Asn

180 185 190

Arg His Val Arg Phe Pro Asn Leu Thr Ala Ser Ile Arg Thr Arg Arg

195 200 205

Gly Glu Lys Val Cys Met Asn Val Pro Met Tyr Lys Asp Ile Ala Thr

210 215 220

Pro Glu Thr Asp Asp Ser Ile Tyr Asp Arg Asp Trp Phe Leu Pro Glu

225 230 235 240

Asp Lys Glu Ala Lys Leu Ala Ser Lys Pro Gly Phe Ile Tyr Met Asp

245 250 255

Ser Met Gly Phe Gly Met Gly Cys Ser Cys Leu Gln Val Thr Phe Gln

260 265 270

Ala Pro Asn Ile Asn Lys Ala Arg Tyr Leu Tyr Asp Ala Leu Val Asn

275 280 285

Phe Ala Pro Ile Met Leu Ala Phe Ser Ala Ala Ala Pro Ala Phe Lys

290 295 300

Gly Trp Leu Ala Asp Gln Asp Val Arg Trp Asn Val Ile Ser Gly Ala

305 310 315 320

Val Asp Asp Arg Thr Pro Lys Glu Arg Gly Val Ala Pro Leu Leu Pro

325 330 335

Lys Tyr Asn Lys Asn Gly Phe Gly Gly Ile Ala Lys Asp Val Gln Asp

340 345 350

Lys Val Leu Glu Ile Pro Lys Ser Arg Tyr Ser Ser Val Asp Leu Phe

355 360 365

Leu Gly Gly Ser Lys Phe Phe Asn Arg Thr Tyr Asn Asp Thr Asn Val

370 375 380

Pro Ile Asn Glu Lys Val Leu Gly Arg Leu Leu Glu Asn Asp Lys Ala

385 390 395 400

Pro Leu Asp Tyr Asp Leu Ala Lys His Phe Ala His Leu Tyr Ile Arg

405 410 415

Asp Pro Val Ser Thr Phe Glu Glu Leu Leu Asn Gln Asp Asn Lys Thr

420 425 430

Ser Ser Asn His Phe Glu Asn Ile Gln Ser Thr Asn Trp Gln Thr Leu

435 440 445

Arg Phe Lys Pro Pro Thr Gln Gln Ala Thr Pro Asp Lys Lys Asp Ser

450 455 460

Pro Gly Trp Arg Val Glu Phe Arg Pro Phe Glu Val Gln Leu Leu Asp

465 470 475 480

Phe Glu Asn Ala Ala Tyr Ser Val Leu Ile Tyr Leu Ile Val Asp Ser

485 490 495

Ile Leu Thr Phe Ser Asp Asn Ile Asn Ala Tyr Ile His Met Ser Lys

500 505 510

Val Trp Glu Asn Met Lys Ile Ala His His Arg Asp Ala Ile Leu Phe

515 520 525

Glu Lys Phe His Trp Lys Lys Ser Phe Arg Asn Asp Thr Asp Val Glu

530 535 540

Thr Glu Asp Tyr Ser Ile Ser Glu Ile Phe His Asn Pro Glu Asn Gly

545 550 555 560

Ile Phe Pro Gln Phe Val Thr Pro Ile Leu Cys Gln Lys Gly Phe Val

565 570 575

Thr Lys Asp Trp Lys Glu Leu Lys His Ser Ser Lys His Glu Arg Leu

580 585 590

Tyr Tyr Tyr Leu Lys Leu Ile Ser Asp Arg Ala Ser Gly Glu Leu Pro

595 600 605

Thr Thr Ala Lys Phe Phe Arg Asn Phe Val Leu Gln His Pro Asp Tyr

610 615 620

Lys His Asp Ser Lys Ile Ser Lys Ser Ile Asn Tyr Asp Leu Leu Ser

625 630 635 640

Thr Cys Asp Arg Leu Thr His Leu Asp Asp Ser Lys Gly Glu Leu Thr

645 650 655

Ser Phe Leu Gly Ala Glu Ile Ala Glu Tyr Val Lys Lys Asn Lys Pro

660 665 670

Ser Ile Glu Ser Lys Cys

675

<210> 2

<211> 2037

<212> DNA

<213> Saccharomyces cerevisiae

<400> 2

atgggactct tagctttggg cacgcctttg cagtggtttg agtctaggac gtacaatgaa

60

cacataaggg atgaaggtat cgagcagttg ttgtatattt tccaagctgc tggtaaaaga

120

gacaatgacc ctcttttttg gggagacgag cttgagtaca tggttgtaga ttttgatgat

180

aaggagagaa attctatgct cgacgtttgc catgacaaga tactcactga gcttaatatg

240

gaggattcgt ccctttgtga ggctaacgat gtgagttttc accctgagta tggccggtat

300

atgttagagg caacaccagc ttctccatat ttgaattacg tgggtagtta cgttgaggtt

360

aacatgcaaa aaagacgtgc cattgcagaa tataagctat ctgaatatgc gagacaagat

420

agtaaaaata acttgcatgt gggctccagg tctgtccctt tgacgctgac tgtcttcccg

480

aggatgggat gccccgactt tattaacatt aaggatccgt ggaatcataa aaatgccgct

540

tccaggtctc tgtttttacc cgatgaagtc attaacagac atgtcaggtt tcctaacttg

600

acagcatcca tcaggaccag gcgtggtgaa aaagtttgca tgaatgttcc catgtataaa

660

gatatagcta ctccagaaac ggatgactcc atctacgatc gagattggtt tttaccagaa

720

gacaaagagg cgaaactggc ttccaaaccg ggtttcattt atatggattc catgggtttt

780

ggcatgggct gttcgtgctt acaagtgacc tttcaggcac ccaatatcaa caaggcacgt

840

tacctgtacg atgcattagt gaattttgca cctataatgc tagccttctc tgccgctgcg

900

cctgctttta aaggttggct agccgaccaa gatgttcgtt ggaatgtgat atctggtgcg

960

gtggacgacc gtactccgaa ggaaagaggt gttgcgccat tactacccaa atacaacaag

1020

aacggatttg gaggcattgc caaagacgta caagataaag tccttgaaat accaaagtca

1080

agatatagtt cggttgatct tttcttgggt gggtcgaaat ttttcaatag gacttataac

1140

gacacaaatg tacctattaa tgaaaaagta ttaggacgac tactagagaa tgataaggcg

1200

ccactggact atgatcttgc taaacatttt gcgcatctct acataagaga tccagtatct

1260

acattcgaag aactgttgaa tcaggacaac aaaacgtctt caaatcactt tgaaaacatc

1320

caaagtacaa attggcagac attacgtttt aaacccccca cacaacaagc aaccccggac

1380

aaaaaggatt ctcctggttg gagagtggaa ttcagaccat ttgaagtgca actattagat

1440

tttgagaacg ctgcgtattc cgtgctcata tacttgattg tcgatagcat tttgaccttt

1500

tccgataata ttaacgcata tattcatatg tccaaagtat gggaaaatat gaagatagcc

1560

catcacagag atgctatcct atttgaaaaa tttcattgga aaaaatcatt tcgcaacgac

1620

accgatgtgg aaactgaaga ttattctata agcgagattt tccataatcc agagaatggt

1680

atatttcctc aatttgttac gccaatccta tgccaaaaag ggtttgtaac caaagattgg

1740

aaagaattaa agcattcttc caaacacgag agactatact attatttaaa gctaatttct

1800

gatagagcaa gcggtgaatt gccaacaaca gcaaaattct ttagaaattt tgtactacaa

1860

catccagatt acaaacatga ttcaaaaatt tcaaagtcga tcaattatga tttgctttct

1920

acgtgtgata gacttaccca tttagacgat tcaaaaggtg aattgacatc ctttttagga

1980

gctgaaattg cagaatatgt aaaaaaaaat aagccttcaa tagaaagcaa atgttaa

2037

<210> 3

<211> 678

<212>PRT

<213> Artificial Sequence

<220>

<223> GSH1_G653M

<400> 3

Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gln Trp Phe Glu Ser Arg

1 5 10 15

Thr Tyr Asn Glu His Ile Arg Asp Glu Gly Ile Glu Gln Leu Leu Tyr

20 25 30

Ile Phe Gln Ala Ala Gly Lys Arg Asp Asn Asp Pro Leu Phe Trp Gly

35 40 45

Asp Glu Leu Glu Tyr Met Val Val Asp Phe Asp Asp Lys Glu Arg Asn

50 55 60

Ser Met Leu Asp Val Cys His Asp Lys Ile Leu Thr Glu Leu Asn Met

65 70 75 80

Glu Asp Ser Ser Leu Cys Glu Ala Asn Asp Val Ser Phe His Pro Glu

85 90 95

Tyr Gly Arg Tyr Met Leu Glu Ala Thr Pro Ala Ser Pro Tyr Leu Asn

100 105 110

Tyr Val Gly Ser Tyr Val Glu Val Asn Met Gln Lys Arg Arg Ala Ile

115 120 125

Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gln Asp Ser Lys Asn Asn

130 135 140

Leu His Val Gly Ser Arg Ser Val Pro Leu Thr Leu Thr Val Phe Pro

145 150 155 160

Arg Met Gly Cys Pro Asp Phe Ile Asn Ile Lys Asp Pro Trp Asn His

165 170 175

Lys Asn Ala Ala Ser Arg Ser Leu Phe Leu Pro Asp Glu Val Ile Asn

180 185 190

Arg His Val Arg Phe Pro Asn Leu Thr Ala Ser Ile Arg Thr Arg Arg

195 200 205

Gly Glu Lys Val Cys Met Asn Val Pro Met Tyr Lys Asp Ile Ala Thr

210 215 220

Pro Glu Thr Asp Asp Ser Ile Tyr Asp Arg Asp Trp Phe Leu Pro Glu

225 230 235 240

Asp Lys Glu Ala Lys Leu Ala Ser Lys Pro Gly Phe Ile Tyr Met Asp

245 250 255

Ser Met Gly Phe Gly Met Gly Cys Ser Cys Leu Gln Val Thr Phe Gln

260 265 270

Ala Pro Asn Ile Asn Lys Ala Arg Tyr Leu Tyr Asp Ala Leu Val Asn

275 280 285

Phe Ala Pro Ile Met Leu Ala Phe Ser Ala Ala Ala Pro Ala Phe Lys

290 295 300

Gly Trp Leu Ala Asp Gln Asp Val Arg Trp Asn Val Ile Ser Gly Ala

305 310 315 320

Val Asp Asp Arg Thr Pro Lys Glu Arg Gly Val Ala Pro Leu Leu Pro

325 330 335

Lys Tyr Asn Lys Asn Gly Phe Gly Gly Ile Ala Lys Asp Val Gln Asp

340 345 350

Lys Val Leu Glu Ile Pro Lys Ser Arg Tyr Ser Ser Val Asp Leu Phe

355 360 365

Leu Gly Gly Ser Lys Phe Phe Asn Arg Thr Tyr Asn Asp Thr Asn Val

370 375 380

Pro Ile Asn Glu Lys Val Leu Gly Arg Leu Leu Glu Asn Asp Lys Ala

385 390 395 400

Pro Leu Asp Tyr Asp Leu Ala Lys His Phe Ala His Leu Tyr Ile Arg

405 410 415

Asp Pro Val Ser Thr Phe Glu Glu Leu Leu Asn Gln Asp Asn Lys Thr

420 425 430

Ser Ser Asn His Phe Glu Asn Ile Gln Ser Thr Asn Trp Gln Thr Leu

435 440 445

Arg Phe Lys Pro Pro Thr Gln Gln Ala Thr Pro Asp Lys Lys Asp Ser

450 455 460

Pro Gly Trp Arg Val Glu Phe Arg Pro Phe Glu Val Gln Leu Leu Asp

465 470 475 480

Phe Glu Asn Ala Ala Tyr Ser Val Leu Ile Tyr Leu Ile Val Asp Ser

485 490 495

Ile Leu Thr Phe Ser Asp Asn Ile Asn Ala Tyr Ile His Met Ser Lys

500 505 510

Val Trp Glu Asn Met Lys Ile Ala His His Arg Asp Ala Ile Leu Phe

515 520 525

Glu Lys Phe His Trp Lys Lys Ser Phe Arg Asn Asp Thr Asp Val Glu

530 535 540

Thr Glu Asp Tyr Ser Ile Ser Glu Ile Phe His Asn Pro Glu Asn Gly

545 550 555 560

Ile Phe Pro Gln Phe Val Thr Pro Ile Leu Cys Gln Lys Gly Phe Val

565 570 575

Thr Lys Asp Trp Lys Glu Leu Lys His Ser Ser Lys His Glu Arg Leu

580 585 590

Tyr Tyr Tyr Leu Lys Leu Ile Ser Asp Arg Ala Ser Gly Glu Leu Pro

595 600 605

Thr Thr Ala Lys Phe Phe Arg Asn Phe Val Leu Gln His Pro Asp Tyr

610 615 620

Lys His Asp Ser Lys Ile Ser Lys Ser Ile Asn Tyr Asp Leu Leu Ser

625 630 635 640

Thr Cys Asp Arg Leu Thr His Leu Asp Asp Ser Lys Met Glu Leu Thr

645 650 655

Ser Phe Leu Gly Ala Glu Ile Ala Glu Tyr Val Lys Lys Asn Lys Pro

660 665 670

Ser Ile Glu Ser Lys Cys

675

<210> 4

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> F_BamHI_GSH1

<400> 4

ggtaggatcc atgggactct tagctttggg cac

33

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> R_GSH1_G653M

<400> 5

gtcaattcca tttttgaatc gtcca

25

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> F_GSH1_G653M

<400> 6

attcaaaaat ggaattgaca tcctt

25

<210> 7

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> R_XhoI_GSH1

<400> 7

atgactcgag ttaacatttg ctttctattg aaggc

35

<210> 8

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> F_SpeI_GSH1_DW

<400> 8

tagaactagt actcctttta tttcggttgt gaa

33

<210> 9

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> R_NcoI_GSH1_DW

<400> 9

gctgccatgg gaatagtgtg aaccgataac tgtgt

35

<210> 10

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> R_AL killer

<400> 10

gagcaatgaa cccaataacg aaatctt

27

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> F_BR killer

<400> 11

cttgacgttc gttcgactga tgag

24

<210> 12

<211> 500

<212> DNA

<213> Artificial Sequence

<220>

<223>CJ-5 promoter of S. cerevisiae

<400> 12

ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc

60

gcaattagcg tatcctgtac catactaatt cccctctgcc cctcgacggc tgccattagt

120

cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt

180

tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt

240

tccaaattag tcatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg

300

gcggcttacc aaaagttgaa gcttgttctt gcttcttatg agtggagcaa tcgattatat

360

tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc

420

tgaaaaattg agcaggttta gtatagggct acattgtagg gtggtttaga gtatcgaaaa

480

tatacatata gaagaataaa

500

<210> 13

<211> 678

<212>PRT

<213> Artificial Sequence

<220>

<223> GSH1_G653M+C86R

<400> 13

Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gln Trp Phe Glu Ser Arg

1 5 10 15

Thr Tyr Asn Glu His Ile Arg Asp Glu Gly Ile Glu Gln Leu Leu Tyr

20 25 30

Ile Phe Gln Ala Ala Gly Lys Arg Asp Asn Asp Pro Leu Phe Trp Gly

35 40 45

Asp Glu Leu Glu Tyr Met Val Val Asp Phe Asp Asp Lys Glu Arg Asn

50 55 60

Ser Met Leu Asp Val Cys His Asp Lys Ile Leu Thr Glu Leu Asn Met

65 70 75 80

Glu Asp Ser Ser Leu Arg Glu Ala Asn Asp Val Ser Phe His Pro Glu

85 90 95

Tyr Gly Arg Tyr Met Leu Glu Ala Thr Pro Ala Ser Pro Tyr Leu Asn

100 105 110

Tyr Val Gly Ser Tyr Val Glu Val Asn Met Gln Lys Arg Arg Ala Ile

115 120 125

Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gln Asp Ser Lys Asn Asn

130 135 140

Leu His Val Gly Ser Arg Ser Val Pro Leu Thr Leu Thr Val Phe Pro

145 150 155 160

Arg Met Gly Cys Pro Asp Phe Ile Asn Ile Lys Asp Pro Trp Asn His

165 170 175

Lys Asn Ala Ala Ser Arg Ser Leu Phe Leu Pro Asp Glu Val Ile Asn

180 185 190

Arg His Val Arg Phe Pro Asn Leu Thr Ala Ser Ile Arg Thr Arg Arg

195 200 205

Gly Glu Lys Val Cys Met Asn Val Pro Met Tyr Lys Asp Ile Ala Thr

210 215 220

Pro Glu Thr Asp Asp Ser Ile Tyr Asp Arg Asp Trp Phe Leu Pro Glu

225 230 235 240

Asp Lys Glu Ala Lys Leu Ala Ser Lys Pro Gly Phe Ile Tyr Met Asp

245 250 255

Ser Met Gly Phe Gly Met Gly Cys Ser Cys Leu Gln Val Thr Phe Gln

260 265 270

Ala Pro Asn Ile Asn Lys Ala Arg Tyr Leu Tyr Asp Ala Leu Val Asn

275 280 285

Phe Ala Pro Ile Met Leu Ala Phe Ser Ala Ala Ala Pro Ala Phe Lys

290 295 300

Gly Trp Leu Ala Asp Gln Asp Val Arg Trp Asn Val Ile Ser Gly Ala

305 310 315 320

Val Asp Asp Arg Thr Pro Lys Glu Arg Gly Val Ala Pro Leu Leu Pro

325 330 335

Lys Tyr Asn Lys Asn Gly Phe Gly Gly Ile Ala Lys Asp Val Gln Asp

340 345 350

Lys Val Leu Glu Ile Pro Lys Ser Arg Tyr Ser Ser Val Asp Leu Phe

355 360 365

Leu Gly Gly Ser Lys Phe Phe Asn Arg Thr Tyr Asn Asp Thr Asn Val

370 375 380

Pro Ile Asn Glu Lys Val Leu Gly Arg Leu Leu Glu Asn Asp Lys Ala

385 390 395 400

Pro Leu Asp Tyr Asp Leu Ala Lys His Phe Ala His Leu Tyr Ile Arg

405 410 415

Asp Pro Val Ser Thr Phe Glu Glu Leu Leu Asn Gln Asp Asn Lys Thr

420 425 430

Ser Ser Asn His Phe Glu Asn Ile Gln Ser Thr Asn Trp Gln Thr Leu

435 440 445

Arg Phe Lys Pro Pro Thr Gln Gln Ala Thr Pro Asp Lys Lys Asp Ser

450 455 460

Pro Gly Trp Arg Val Glu Phe Arg Pro Phe Glu Val Gln Leu Leu Asp

465 470 475 480

Phe Glu Asn Ala Ala Tyr Ser Val Leu Ile Tyr Leu Ile Val Asp Ser

485 490 495

Ile Leu Thr Phe Ser Asp Asn Ile Asn Ala Tyr Ile His Met Ser Lys

500 505 510

Val Trp Glu Asn Met Lys Ile Ala His His Arg Asp Ala Ile Leu Phe

515 520 525

Glu Lys Phe His Trp Lys Lys Ser Phe Arg Asn Asp Thr Asp Val Glu

530 535 540

Thr Glu Asp Tyr Ser Ile Ser Glu Ile Phe His Asn Pro Glu Asn Gly

545 550 555 560

Ile Phe Pro Gln Phe Val Thr Pro Ile Leu Cys Gln Lys Gly Phe Val

565 570 575

Thr Lys Asp Trp Lys Glu Leu Lys His Ser Ser Lys His Glu Arg Leu

580 585 590

Tyr Tyr Tyr Leu Lys Leu Ile Ser Asp Arg Ala Ser Gly Glu Leu Pro

595 600 605

Thr Thr Ala Lys Phe Phe Arg Asn Phe Val Leu Gln His Pro Asp Tyr

610 615 620

Lys His Asp Ser Lys Ile Ser Lys Ser Ile Asn Tyr Asp Leu Leu Ser

625 630 635 640

Thr Cys Asp Arg Leu Thr His Leu Asp Asp Ser Lys Met Glu Leu Thr

645 650 655

Ser Phe Leu Gly Ala Glu Ile Ala Glu Tyr Val Lys Lys Asn Lys Pro

660 665 670

Ser Ile Glu Ser Lys Cys

675

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> R_GSH1_C86R

<400> 14

ttagcctccc taagggacga atcct

25

<210> 15

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> F_GSH1_C86R

<400> 15

cgtcccttag ggaggctaac gatgt

25

<---

Claims (12)

1. A polypeptide involved in the production of glutathione, in which the amino acid corresponding to position 653 from the N-terminus of the amino acid sequence SEQ ID NO: 1 is replaced by methionine. 2. The polypeptide according to claim 1, wherein the amino acid corresponding to position 653 is glycine. 3. The polypeptide according to claim 1, wherein the polypeptide consists of the amino acid sequence SEQ ID NO: 3. 4. The polypeptide according to claim 1, where the polypeptide has an additional replacement of the amino acid corresponding to position 86 with another amino acid. 5. The polypeptide of claim 4, wherein the polypeptide consists of the amino acid sequence SEQ ID NO: 13. 6. A polynucleotide encoding a polypeptide according to any one of claims. 1-5. 7. An expression vector containing a polynucleotide according to claim 6. 8. A microorganism for producing glutathione, containing any one or more than one polypeptide according to any one of paragraphs. 1-5; a polynucleotide encoding a given polypeptide; and an expression vector including the polynucleotide. 9. A microorganism for producing glutathione according to claim 8, where the microorganism is a microorganism of the genus Saccharomyces . 10. A microorganism for producing glutathione according to claim 8, where the microorganism is Saccharomyces cerevisiae . 11. A method for producing glutathione, comprising the step of cultivating in a microorganism medium comprising any one or more than one polypeptide according to any one of claims. 1-5; a polynucleotide encoding this variant; and an expression vector including the polynucleotide. 12. The method of claim 11, further comprising the step of collecting glutathione from one or more of a material selected from a cultured microorganism, a dried microorganism product, an extract of a microorganism, a culture of a microorganism, and a lysate of a microorganism.