Superoxide dismutase 1 mutant and method for producing glutathione or its derivatives using the same

Abstract

The present application relates to a novel superoxide dismutase 1 mutant and a method for producing glutathione or a derivative thereof using the same.

Description

[Technical field]

[0001] The present application relates to a superoxide dismutase 1 mutant and a method for producing glutathione or a derivative thereof using the mutant. [Background technology]

[0002] Glutathione (GSH) is the most common organosulfur compound present in cells and is a tripeptide consisting of three amino acids: glycine, glutamate, and cysteine.

[0003] Glutathione exists in two forms in the body: reduced glutathione (GSH) and oxidized glutathione (GSSG). Reduced glutathione (GSH), which is present in relatively high proportions under normal circumstances, is mainly distributed in the liver and skin cells of the human body and plays important roles such as antioxidant function that breaks down and removes active oxygen, detoxification function that removes exogenous compounds including toxic substances, and whitening function that suppresses the production of melanin pigment.

[0004] Glutathione, which has such various functions, is in the limelight as a material in various fields such as pharmaceuticals, functional foods, and cosmetics, and is also used in the manufacture of flavor materials and food and feed additives. Glutathione has a large effect of improving the taste of the original product and maintaining the taste durability, and is known to be used as a kokumi flavor enhancer by using it alone or in combination with other substances. Kokumi materials are usually richer than conventional umami materials such as nucleic acids and MSG, and are known to be generated by decomposition and maturation of proteins. Glutathione can also be converted into other γ-glutamyl peptides (Non-Patent Document 1). γ-glutamyl peptides are low molecular weight compounds that have a γ-carboxy group of glutamic acid at the N-terminus of the molecule, and are known to be widely used as kokumi flavor enhancers.

[0005] However, despite the increasing demand for glutathione and its derivatives, which can be used in such a variety of fields, the enzymatic synthesis process has not yet been commercialized due to the high production costs, and the industrial production of glutathione and its derivatives is very costly, so the market is not sufficiently active. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Korean Patent No. 10-2222210 [Non-patent literature]

[0007] [Non-Patent Document 1] Sofyanovich OA et al,(2019) Multiple pathways for the formation of the γ-glutamyl peptides γ-glutamyl-valine and γ-glutamyl-valylglycine in Saccharomyces cerevisiae. PLoS ONE 14 (5): e0216622 [Non-Patent Document 2] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-Patent Document 3] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-Patent Document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-Patent Document 5] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) [Non-Patent Document 6] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990)

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

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Non-licensed Document 15

[0008] The problem to be solved by the present application is to provide a superoxide dismutase 1 mutant and a method for producing glutathione or a derivative thereof using the mutant. [Means for solving the problem]

[0009] The present application provides a superoxide dismutase 1 mutant in which the amino acid corresponding to the 37th position from the N-terminus of the amino acid sequence of SEQ ID NO:1 is substituted with another amino acid.

[0010] The present application provides polynucleotides encoding the mutants and vectors containing the same.

[0011] The present application provides a microorganism comprising at least one of the variants and a polynucleotide encoding the variant.

[0012] The present application provides a method for producing glutathione or a derivative thereof, comprising the step of culturing said microorganism. Effect of the Invention

[0013] The novel superoxide dismutase 1 mutant of the present application is useful for hyper-production of glutathione or a derivative thereof. [Brief description of the drawings]

[0014] [Figure 1] The results show that a superoxide dismutase 1 mutant was introduced into a Saccharomyces cerevisiae strain and the glutathione production level was confirmed. [Diagram 2] The results show that a superoxide dismutase 1 mutant was introduced into a Saccharomyces cerevisiae strain and the glutathione production level was confirmed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Each description and embodiment disclosed in this application applies to each of the other descriptions and embodiments, i.e., all combinations of the various elements disclosed in this application are included in this application, and the application is not limited to the specific description below.

[0016] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein which equivalents are intended to be encompassed by this application.

[0017] Furthermore, many papers and patent documents are referred to throughout this specification, and citations are provided thereto, the disclosures of which are incorporated herein by reference in their entirety in order to more clearly describe the state of the art to which the present invention pertains and the contents of the present invention.

[0018] One aspect of the present application provides a superoxide dismutase 1 (SOD1) mutant comprising an amino acid substitution in a protein having superoxide dismutase 1 activity, the substitution comprising a substitution of the amino acid corresponding to the 37th position from the N-terminus of SEQ ID NO:1 with another amino acid.

[0019] The mutant may be a protein mutant in which the amino acid corresponding to the 37th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0020] In the present application, "superoxide dismutase 1" is an enzyme also known as "SOD1" or "superoxide dismutase [Cu-Zn]." Superoxide dismutase is known to catalyze the following reaction.

[0021] 2O2 - +2H + →H2O2+O2

[0022] In the present application, the amino acid sequence of superoxide dismutase 1 is an amino acid sequence encoded by the sod1 gene, and is also referred to as "SOD1 protein." The amino acid sequence constituting superoxide dismutase 1 in the present application can be obtained from GenBank of NCBI, a publicly known database. The superoxide dismutase 1 is a protein containing the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. As an example, the superoxide dismutase 1 is derived from Saccharomyces cerevisiae, and as another example, the amino acid corresponding to the 37th position in the amino acid sequence of SEQ ID NO: 1 is glycine. However, the superoxide dismutase 1 is not limited thereto, and any sequence having the same superoxide dismutase 1 activity as the amino acid sequence may be used.

[0023] As a specific example, the superoxide dismutase 1 of the present application may be a protein containing 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 more homology or identity thereto. Needless to say, proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, or added are also included in the proteins to be mutated in the present application, so long as the amino acid sequence has such homology or identity and exhibits efficacy equivalent to that of the protein.

[0024] Furthermore, even if the present application defines superoxide dismutase 1 as a protein containing the amino acid sequence of SEQ ID NO: 1 as an example, this does not exclude meaningless addition of sequences before or after the amino acid sequence of SEQ ID NO: 1, naturally occurring mutations, or silent mutations thereof, and it goes without saying that any substance having the same or corresponding activity as a protein consisting of the amino acid sequence of SEQ ID NO: 1 is included in the superoxide dismutase 1 of the present application.

[0025] In other words, even if the present application describes "a protein or polypeptide having an amino acid sequence represented by a specific SEQ ID NO:" or "a protein or polypeptide containing an amino acid sequence represented by a specific SEQ ID NO:," it goes without saying that a protein having an amino acid sequence in which part of the sequence has been deleted, modified, substituted or added can also be used in the present application, so long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the SEQ ID NO.

[0026] By "variant" or "modified polypeptide" in the present application is meant a protein that differs from the recited sequences by conservative substitution and / or modification of at least one amino acid, but which maintains the functions or properties of said protein.

[0027] The variant differs from the identified sequence by substitution, deletion or addition of several amino acids. Such variants can generally be identified by modifying at least one amino acid in the amino acid sequence of the protein and evaluating the properties of the modified protein. That is, the ability of the variant may be improved, unchanged or decreased compared to the native protein. Some variants also include mutant polypeptides in which at least one portion, such as an N-terminal leader sequence or a transmembrane domain, has been deleted. Other variants also include variants in which a portion has been deleted from the N- and / or C-terminus of a mature protein. The term "variant" or "mutant polypeptide" is used interchangeably with terms such as mutation, modification, mutated protein, and mutation (in English, modification, modified protein, mutant, mutein, divergent, variant, etc.), but may be any term that means a mutation.

[0028] In the present application, a "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. The variant has, for example, at least one conservative substitution while still retaining at least one biological activity. Such amino acid substitutions can generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues.

[0029] For example, among amino acids having electrically charged side chains, positively charged (basic) amino acids include arginine, lysine, and histidine, 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, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0030] Variants may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be linked to an N-terminal signal (or leader) sequence of a protein involved in co-translationally or post-translationally protein transfer. The polypeptide may also be linked to other sequences or linkers that allow the polypeptide to be identified, purified or synthesized.

[0031] In one embodiment, the mutant of the present application may be a superoxide dismutase 1 mutant in which the amino acid corresponding to the 37th position from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid, or a mutant polypeptide having superoxide dismutase 1 activity. The mutant of the present application is also called a "superoxide dismutase 1 mutant," "(mutant) polypeptide having superoxide dismutase 1 activity," or "SOD1 mutant," and is capable of increasing glutathione production compared to the pre-mutation protein, natural wild-type polypeptide, or unmodified polypeptide, but is not limited to these.

[0032] In any of the above-mentioned embodiments, the mutant may be a superoxide dismutase 1 mutant in which the amino acid corresponding to the 37th position from the N-terminus of the amino acid sequence of SEQ ID NO:1 is replaced with another amino acid.

[0033] In the present application, "substitution with another amino acid" may be any amino acid different from the amino acid before substitution. It goes without saying that "a specific amino acid is substituted" in the present application means that the amino acid before substitution is substituted with an amino acid different from the amino acid before substitution, even if it is not specified that the amino acid is substituted with another amino acid.

[0034] In any of the above-mentioned embodiments, the "other amino acid" may be an amino acid other than glycine. Specifically, the other amino acid is an amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine, but is not limited thereto.

[0035] By "corresponding position" in this application is meant the amino acid residue at the recited position in a protein or polypeptide, or an amino acid residue similar, identical or corresponding to the recited residue in a protein or polypeptide. By "corresponding region" in this application is generally meant a similar or corresponding position in a related or comparison protein.

[0036] In the present application, specific numbering is used for amino acid residue positions in the proteins used in the present application. For example, positions corresponding to amino acid residue positions in the proteins of the present application can be renumbered by aligning the polypeptide sequences of the proteins to be compared with those of the proteins of the present application.

[0037] The superoxide dismutase 1 mutant in which the amino acid corresponding to the 37th position from the N-terminus of the amino acid sequence of SEQ ID NO:1 in the present application has been replaced with another amino acid may be a protein in which the amino acid corresponding to the 37th position of SEQ ID NO:1 has been replaced with another amino acid in 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 more homology or identity thereto.

[0038] Such variants include, but are not limited to, variants having 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology or identity to SEQ ID NO:1, and variants having less than 100% homology or identity to SEQ ID NO:1.

[0039] The superoxide dismutase 1 mutant in which the amino acid corresponding to the 37th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 of the present application is substituted with another amino acid may comprise any of the amino acid sequences of SEQ ID NO: 12 to 30. Specifically, it is one that is substantially composed of any of the amino acid sequences of SEQ ID NO: 12 to 30, and more specifically, it is one that is composed of any of the amino acid sequences of SEQ ID NO: 12 to 30, but is not limited thereto.

[0040] Furthermore, the mutant includes an amino acid sequence of any one of SEQ ID NOs: 12 to 30, or the amino acid sequence in which the 37th amino acid is fixed (i.e., in the amino acid sequence of the mutant, the amino acid corresponding to the 37th position in the amino acid sequence of SEQ ID NOs: 12 to 30 is identical to the amino acid at the 37th position in the amino acid sequence of SEQ ID NOs: 12 to 30), and includes an amino acid sequence having 80% or more homology or identity thereto, but is not limited to these.

[0041] Specifically, the mutant of the present application may include an amino acid sequence of any one of SEQ ID NOs: 12 to 30, and a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity with an amino acid sequence of any one of SEQ ID NOs: 12 to 30, in which the amino acid corresponding to the 37th position in the amino acid sequence of any one of SEQ ID NOs: 12 to 30 is an amino acid other than glycine. Furthermore, it goes without saying that the present application also includes a protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added other than at the 37th position, so long as the amino acid sequence has such homology or identity and exhibits efficacy corresponding to the mutant.

[0042] By "homology" or "identity" in this application is meant the degree of relatedness between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0043] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to at least about 50%, 60%, 70%, 80%, or 90% or more of the entire sequence or length under moderate or high stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides having common codons or codons that take into account codon degeneracy in the polynucleotide.

[0044] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in, for example, Non-Patent Document 2. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 4), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) (version 5.0.0 or later), which includes the GCG program package (Non-Patent Document 5), BLASTP, BLASTN, FASTA (Non-Patent Documents 6, 7 and 8). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0045] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in, for example, J. Biol. 1999, 143:1311-1354 (2001). Briefly, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a binary comparison matrix (identity takes a value of 1 and non-identity takes a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0046] Furthermore, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be ascertained by comparing the sequences in a Southern hybridization experiment under defined stringent conditions, and appropriate defined hybridization conditions are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., Non-Patent Documents 12, 13).

[0047] Another aspect of the present application provides a polynucleotide encoding said variant.

[0048] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and refers to a DNA or RNA chain longer than a certain length.

[0049] The gene encoding superoxide dismutase 1 of the present application may be the sod1 gene.

[0050] The gene may be derived from yeast. Specifically, it may be from the genus Saccharomyces, and more specifically, it may be derived from Saccharomyces cerevisiae. More specifically, it may be any gene derived from Saccharomyces cerevisiae that encodes a polypeptide having superoxide dismutase 1 activity, and in one embodiment, it is a gene encoding the amino acid sequence of SEQ ID NO: 1, and in one embodiment, it includes the base sequence of SEQ ID NO: 2 or SEQ ID NO: 3, but is not limited thereto.

[0051] A polynucleotide encoding the superoxide dismutase 1 variant of the present application may be any polynucleotide that encodes the superoxide dismutase 1 variant of the present application and a polypeptide having activity corresponding thereto.

[0052] Polynucleotides encoding superoxide dismutase 1 and its variants of the present application may have various modifications in the coding region, taking into account codon degeneracy or preferred codons in the organism in which the polypeptide is expressed, without changing the amino acid sequence of the polypeptide.

[0053] Specifically, the polynucleotide encoding the superoxide dismutase 1 mutant of the present application may be any polynucleotide sequence that encodes a protein mutant in which the amino acid corresponding to the 37th position in the amino acid sequence of SEQ ID NO:1 is replaced with another amino acid.

[0054] For example, a polynucleotide encoding a protein variant of the present application is a polynucleotide sequence encoding the protein variant of the present application, specifically, any of the amino acid sequences of SEQ ID NOs: 12 to 30, or a polypeptide having homology or identity thereto, in which the sequence encoding the amino acid corresponding to the 37th position in any of the amino acid sequences of SEQ ID NOs: 12 to 30 encodes an amino acid other than glycine, but is not limited thereto. The homology or identity is as described above.

[0055] Furthermore, the polynucleotide encoding the protein mutant of the present application may be any sequence that encodes a protein mutant in which the amino acid corresponding to the 37th position in the amino acid sequence of SEQ ID NO:1 is substituted with another amino acid by hybridizing under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or part of the base sequence.

[0056] The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in literature (e.g., Non-Patent Document 12). For example, the conditions include conditions under which polynucleotides having high homology or identity, 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and even more specifically 99% or more, hybridize with each other, and polynucleotides having lower homology or identity than that do not hybridize with each other, or conditions under which washing is performed once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS, which are washing conditions for normal southern hybridization.

[0057] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0058] Specifically, polynucleotides having homology or identity can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0059] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, and variables are known in the art (see Non-Patent Document 14).

[0060] Yet another aspect of the present application provides a vector comprising a polynucleotide encoding said protein variant.

[0061] In this application, the term "vector" refers to a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. The expression control region includes a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and function independently of the host genome, and is integrated into the genome itself.

[0062] For example, a polynucleotide encoding a target protein in a chromosome can be replaced with a mutated polynucleotide by a vector for intracellular chromosome introduction. The polynucleotide can be inserted into a chromosome by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming whether or not the target nucleic acid molecule has been inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether or not the target nucleic acid molecule has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein is used. In an environment treated with a selection agent, only cells expressing the selection marker survive or show a different phenotype, so that transformed cells can be selected.

[0063] The vector used in the present application is not particularly limited, and any vector known in the art may be used. The yeast expression vector may be either a yeast integrative yeast plasmid (YIp) or an extrachromosomal plasmid vector. The extrachromosomal plasmid vector includes a yeast episomal yeast plasmid (YEp), a yeast replicative yeast plasmid (YRp), and a yeast centromere plasmid (YCp). Yeast artificial chromosomes (YACs) may also be used as vectors in the present application.As specific examples, vectors that can be used 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, pPIC6α A, pPIC6α B, pPIC6α C, pPIC9K, pYC2 / CT, pYD1 Yeast Display Vector, 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, P TEF-MF, pBY011, pSGP47, pSGP46, pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7, pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron A Forms include, but are not limited to, pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, pYJ405 and pYJ406.

[0064] In the present application, "transformation" means expressing a protein encoded by a polynucleotide in a host cell by introducing a vector containing a polynucleotide encoding a target protein into a host cell or a microorganism. The transformed polynucleotide may be any polynucleotide that is expressed in a host cell, regardless of whether it is inserted into the chromosome of the host cell or located extrachromosomally. The polynucleotide may include DNA or RNA that encodes the target protein. The polynucleotide may be introduced in any form as long as it is introduced into the host cell and expressed. For example, the polynucleotide is introduced into the host cell in the form of an expression cassette, which is a genetic structure that contains all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0065] Furthermore, the term "operably linked" means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target polypeptide of the present application.

[0066] The method for transforming the vector of the present application may be any method for introducing a nucleic acid into a cell, and may be carried out by selecting a standard technique suitable for the host cell as known in the art, such as, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, etc.

[0067] The present application provides a microorganism comprising at least one polynucleotide encoding superoxide dismutase 1, and said variants.

[0068] In the present application, the term "microorganism" includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and is a concept that includes all microorganisms in which a specific mechanism has been weakened or strengthened by, for example, inserting an exogenous gene or strengthening or weakening the activity of an endogenous gene.

[0069] In one embodiment, the microorganism may be a microorganism that produces glutathione. In this application, the term "glutathione-producing microorganism" is used interchangeably with "glutathione-producing microorganism," "microorganism having glutathione-producing ability," "glutathione-producing strain," "strain having glutathione-producing ability," and the like.

[0070] In any of the above embodiments, the microorganism may be a microorganism that produces a glutathione derivative.

[0071] In any of the above-mentioned embodiments, the microorganism of the present application may comprise a vector comprising a polynucleotide encoding superoxide dismutase 1.

[0072] In this application, "glutathione" is used interchangeably with "glutathione" and "GSH" and means a tripeptide consisting of three amino acids, glutamic acid, cysteine, and glycine. Glutathione is used as a raw material for pharmaceuticals, functional foods, flavoring materials, food and feed additives, cosmetics, etc., but is not limited thereto.

[0073] In the present application, a "glutathione derivative" refers to any substance that can be converted from glutathione as a starting material.

[0074] In the present application, glutathione or a derivative thereof is also referred to as “γ-glutamyl peptide.” γ-glutamyl peptide refers to a low molecular weight compound having a γ-carboxy group of glutamic acid at the N-terminus of the molecule.

[0075] Specifically, the γ-glutamyl peptide is a compound represented by general formula 1.

[0076] [General formula 1] γ-Glu-XY

[0077] In general formula 1, X is cysteine, glutamic acid, tyrosine, methionine, valine, tryptophan, aspartic acid, isoleucine, leucine, asparagine, glutamine, histidine, lysine, ornithine, arginine, S-methylcysteine, or S-ethylcysteine, and Y is glycine, alanine, leucine, isoleucine, serine, threonine, methionine, cysteine, aspartic acid, asparagine, glutamine, lysine, ornithine, arginine, phenylalanine, tyrosine, proline, hydroxyproline, α-aminobutyric acid, or absent.

[0078] In one embodiment, the γ-glutamyl peptide may be selected from γ-Glu-Cys-Gly (glutathione), γ-Glu-Glu (γ-EF), γ-Glu-Tyr (γ-EY), γ-Glu-Met (γ-EM), γ-Glu-Val (γ-EV), γ-Glu-Trp (γ-EW), γ-Glu-Asp (γ-ED), γ-Glu-Ile (γ-EI), γ-Glu-Leu (γ-EL), γ-Glu-Cys-Gly (γ-ECG), γ-Glu-Val-Gly (γ-EVG), γ-glutamyl-S-methyl-cysteine ​​(γ-ES-methyl-C), γ-glutamyl-S-ethenyl-cysteine ​​(γ-ES-ethenyl-C), and γ-glutamyl-cysteinyl-β-alanine (γ-EC-β-A).

[0079] In any of the above embodiments, the gamma-glutamyl peptide may be, but is not limited to, glutathione.

[0080] The microorganism in the present application means a microorganism that contains superoxide dismutase 1 and produces the target glutathione in excess compared to a wild-type or unmodified microorganism. The type of the microorganism is not particularly limited as long as it produces glutathione, and is a microorganism of the genus Saccharomyces, specifically Saccharomyces cerevisiae, but is not limited thereto.

[0081] The parent strain of the glutathione-producing microorganism containing the mutant may be any strain that produces glutathione. The microorganism may further include a mutation such as a mutation that enhances the biosynthetic pathway to improve glutathione production ability, a mutation that removes feedback inhibition, or a mutation that weakens the degradation pathway or the biosynthetic pathway by inactivating a gene, and the mutation does not exclude natural mutations.

[0082] In the present application, "enhancement" of a polypeptide activity means improving the activity of the polypeptide compared to its endogenous activity. The enhancement is used interchangeably with activation, up-regulation, overexpression, increase, etc. Here, activation, enhancement, up-regulation, overexpression, and increase all include the display of an activity that was not originally present, and the improvement of activity compared to the endogenous activity or activity before modification. The "endogenous activity" means the activity of a specific polypeptide originally possessed by a parent strain or an unmodified microorganism before the trait change when the trait is changed due to genetic mutation caused by natural or artificial factors. This is used interchangeably with "activity before modification." The "enhancement," "up-regulation," "overexpression," or "improvement" of a polypeptide activity compared to its endogenous activity means the improvement of the activity and / or concentration (expression level) of a specific polypeptide originally possessed by a parent strain or an unmodified microorganism before the trait change.

[0083] The enhancement may be achieved by introducing a foreign polypeptide, or by enhancing the activity and / or increasing the concentration (expression amount) of an endogenous polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed by an increase in the level of activity of the polypeptide, the expression amount, or the amount of a product produced from the polypeptide.

[0084] To enhance the activity of the polypeptide, various methods well known in the art can be applied, and any method can be used as long as it can enhance the activity of the target polypeptide compared to the unmodified microorganism. Specifically, the method is a conventional method in molecular biology that uses genetic engineering and / or protein engineering well known to those skilled in the art, but is not limited to these (e.g., Non-Patent Documents 15, 16, etc.).

[0085] Specifically, the enhancement of the polypeptide activity of the present application is carried out by 1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide, 2) replacing the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence having stronger activity, 3) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide, 4) modifying the amino acid sequence of the polypeptide so as to enhance the polypeptide activity, 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide activity), 6) introducing a foreign polypeptide exhibiting the polypeptide activity or a foreign polynucleotide encoding the same, 7) optimizing the codons of the polynucleotide encoding the polypeptide, 8) analyzing the tertiary structure of the polypeptide and selecting and modifying or chemically modifying the exposed portion, or 9) a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

[0086] More specifically, the above 1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide is carried out by introducing into a host cell a vector to which a polynucleotide encoding the polypeptide is operably linked, the vector replicating and functioning independently of the host. Alternatively, the above may be carried out by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The above introduction into a chromosome is carried out by introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell, but is not limited thereto. The above vector is as described above.

[0087] The expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide is replaced with a sequence having a stronger activity, for example, by generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so that the activity of the expression regulatory region is further enhanced, or by replacing the expression regulatory region with a sequence having a higher activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, and the like. For example, the expression regulatory region is replaced with a strong promoter, but is not limited to this.

[0088] Examples of known promoters for eukaryotes include the promoters for translation elongation factor 1 (TEF1), glycerol-3-phosphate dehydrogenase 1 (GPD1), 3-phosphoglycerate kinase, or other glycolic 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; examples of other yeast promoters that are inducible promoters with the added advantage of transcription controlled depending on growth conditions include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, and phosphoglucose isomerase. Examples of suitable promoters include those for maltose, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization; if the host cell is yeast, promoters that may be used include, but are not limited to, the TEF1 promoter, TEF2 promoter, GAL10 promoter, GAL1 promoter, ADH1 promoter, ADH2 promoter, PHO5 promoter, GAL1-10 promoter, TDH3 promoter (GPD promoter), TDH2 promoter, TDH1 promoter, PGK1 promoter, PYK2 promoter, ENO1 promoter, ENO2 promoter, and TPI1 promoter; suitable vectors and promoters for use in yeast expression are further described in EP 073657, but are not limited to these. Yeast enhancers are also useful with yeast promoters.

[0089] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide (3) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher expression rate of the polypeptide compared to the endogenous start codon, but is not limited to this.

[0090] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above can be performed by, but is not limited to, generating a sequence mutation in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to enhance the activity of the polypeptide, or by replacing the amino acid sequence or polynucleotide sequence with an improved amino acid sequence or polynucleotide sequence having a higher activity or an improved activity. Specifically, the replacement can be performed by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. Here, the vector used may further include a selection marker for checking whether or not it has been inserted into a chromosome. The selection marker is as described above.

[0091] The above 6) introduction of a foreign polynucleotide exhibiting the activity of a polypeptide is carried out by introducing into a host cell a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the above polypeptide. The foreign polynucleotide may be of any origin or sequence as long as it exhibits the same / similar activity as the above polypeptide. The introduction can be carried out by a person skilled in the art by appropriately selecting a known transformation method, and the introduced polynucleotide is expressed in the host cell as described above, thereby producing a polypeptide and improving its activity.

[0092] 7) Optimizing the codons of a polynucleotide encoding a polypeptide can be carried out by optimizing the codons of an endogenous polynucleotide so as to increase transcription or translation in a host cell, or by optimizing the codons of an exogenous polynucleotide so as to perform optimized transcription or translation in a host cell.

[0093] 8) Analyzing the tertiary structure of a polypeptide and selecting and altering or chemically modifying an exposed portion thereof can be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, determining candidates for template proteins according to the degree of sequence similarity, confirming the structure based on the candidates, and selecting and altering or modifying the exposed portion to be altered or chemically modified.

[0094] Such enhancement of polypeptide activity can be achieved by, but is not limited to, improving the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or by increasing the amount of a product produced from the polypeptide.

[0095] In the microorganism of the present application, modification of a part or the whole of a polynucleotide can be induced by (a) homologous recombination using a vector for chromosomal introduction in the microorganism, or genome editing using genetic scissors (engineered nuclease, e.g., CRISPR-Cas9), and / or (b) light such as ultraviolet light or radiation and / or chemical treatment, but is not limited thereto. Methods for modifying a part or the whole of the gene include methods using DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene is introduced into the microorganism to cause homologous recombination, thereby deleting a part or the whole of the gene. The introduced nucleotide sequence or vector may contain, but is not limited to, a dominant selection marker.

[0096] In the present application, the term "attenuation" of a polypeptide refers to a concept that includes all of the following: a decrease in activity compared to the endogenous activity, or the absence of activity. The term "attenuation" is also used interchangeably with other terms such as inactivation, deficiency, down-regulation, decrease, reduction, and attenuation.

[0097] The weakening includes the reduction or elimination of the activity of the polypeptide itself compared to the activity of the polypeptide originally possessed by the microorganism due to a mutation in the polynucleotide encoding the polypeptide, the reduction in the overall level and / or concentration (expression amount) of the polypeptide activity in the cell compared to the natural strain due to inhibition of gene expression of the encoding polynucleotide or inhibition of translation into the polypeptide, the complete absence of expression of the polynucleotide, and the absence of the polypeptide activity even if the polynucleotide is expressed. The "endogenous activity" refers to the activity of a specific polypeptide originally possessed by a parent strain, a wild type, or an unmodified microorganism before the trait change when the trait is changed due to genetic mutation caused by natural or artificial factors. This is used interchangeably with "activity before modification." The activity of a polypeptide being "inactivated," "deficient," "reduced," "down-regulated," "decreased," or "attenuated" compared to the endogenous activity means that the activity of a specific polypeptide is reduced compared to the activity of a specific polypeptide originally possessed by a parent strain or an unmodified microorganism before the trait change.

[0098] Such reduction in the activity of a polypeptide can be achieved by applying various methods well known in the art, including, but not limited to, those described above (eg, Non-Patent Documents 16 and 17).

[0099] Specifically, the attenuation of a polypeptide activity in the present application can be achieved by 1) deleting all or a part of a gene encoding the polypeptide, 2) modifying an expression regulatory region (or an expression regulatory sequence) so as to reduce the expression of a gene encoding the polypeptide, 3) modifying an amino acid sequence constituting the polypeptide so as to delete or weaken the activity of the polypeptide (e.g., deleting / substituting / adding one or more amino acids in the amino acid sequence), or 4) modifying a gene sequence encoding the polypeptide so as to delete or weaken the activity of the polypeptide (e.g., modifying the gene sequence encoding the polypeptide so as to encode a polypeptide modified so as to delete or weaken the activity of the polypeptide). The modification can be carried out by, but is not limited to, one or more of the following: deleting / substituting / adding one or more nucleic acid bases in the nucleic acid base sequence of a gene; 5) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding a polypeptide; 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding a polypeptide; 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding a polypeptide so that a secondary structure that makes ribosome attachment impossible is formed; 8) adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide so that it is reverse transcribed (reverse transcription engineering, RTE); or 9) a combination of two or more selected from 1) to 8) above.

[0100] For example, 1) deleting a part or the whole of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in a chromosome, or by replacing it with a polynucleotide lacking some nucleotides or a marker gene.

[0101] In addition, the expression regulatory region (or expression regulatory sequence) may be modified by generating a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting a sequence having a lower activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0102] Furthermore, the base sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide can be modified, for example, by substituting a base sequence encoding another start codon that has a lower polypeptide expression rate compared to the endogenous start codon, but is not limited to this.

[0103] Furthermore, the modification of the amino acid sequence or polynucleotide sequence of 3) and 4) above can be performed by, but is not limited to, generating sequence mutations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to weaken the activity of the polypeptide, or by replacing the sequence with an improved amino acid sequence or polynucleotide sequence having a lower activity, or an improved amino acid sequence or polynucleotide sequence having no activity. For example, gene expression can be inhibited or weakened by, but is not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon.

[0104] 6) Introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcription product encoding the polypeptide can be seen in, for example, Non-Patent Document 18.

[0105] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide so that a secondary structure is formed that makes ribosome attachment impossible may be achieved by making mRNA translation impossible or slowing down the rate.

[0106] The above 8) adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide so as to reverse transcribe the promoter (reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide to reduce activity.

[0107] In any of the above-mentioned embodiments, the microorganism of the present application may contain a mutation in the expression regulatory region of glutamate-cysteine ​​ligase (GSH1) that improves glutathione production ability. The mutation may be at least one mutation selected from -250 (C→T), -252 (G→A), -398 (A→T), -399 (A→C), -407 (T→C), and -409 (T→C) upstream of the GSH1 ORF.

[0108] In any of the above-mentioned Examples, the microorganism comprises a mutation in which the 86th amino acid and / or the 653rd amino acid of glutamate-cysteine ​​ligase (GSH1) is substituted with another amino acid. Specifically, the microorganism comprises a mutation in which the 86th amino acid is substituted with arginine and the 653rd amino acid is substituted with methionine. However, the present invention is not limited thereto.

[0109] In any of the above embodiments, the microorganism may include, but is not limited to, a mutation that enhances activity of glutamate-cysteine ​​ligase (GSH1).

[0110] A microorganism containing at least one of the superoxide dismutase 1 mutants and polynucleotides encoding the mutants of the present application is a "microorganism expressing superoxide dismutase 1 in which the amino acid corresponding to the 37th position in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid," but is not limited thereto.

[0111] The superoxide dismutase 1 and its mutants are as described above.

[0112] In this application, "causing / making" a protein to be expressed refers to a state in which a target protein is introduced into a microorganism or modified so as to be expressed in the microorganism.

[0113] The microorganism expressing the protein variant of the present application may be a microorganism that has been modified to express the protein variant of the present application. Thus, a further aspect of the present application provides a method for producing a microorganism expressing the protein variant of the present application.

[0114] In this application, the term "strain before modification" or "microorganism before modification" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed due to genetic mutation caused by natural or artificial factors. The term "strain before modification" or "microorganism before modification" is used interchangeably with "non-mutated strain," "non-modified strain," "non-mutated microorganism," "non-modified microorganism," or "reference microorganism."

[0115] In the present application, a microorganism comprising a polynucleotide containing or encoding said superoxide dismutase 1 mutant, or a vector containing said polynucleotide, may be a recombinant microorganism, and said recombination may be achieved by genetic modification, such as transformation.

[0116] For example, but not limited to, a recombinant microorganism produced by transformation with a vector containing the polynucleotide. The recombinant microorganism is a yeast, for example, a microorganism of the genus Saccharomyces, specifically, but not limited to, Saccharomyces cerevisiae.

[0117] Yet another aspect of the present application provides a method for producing glutathione or a derivative thereof, the method comprising culturing the microorganism. The microorganism, glutathione, or a derivative thereof is as described above.

[0118] The medium and other culture conditions used for culturing the strain of the present application may be any that are used for culturing a typical Saccharomyces microorganism. Specifically, the strain of the present application can be cultured in a typical medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, etc., under aerobic or anaerobic conditions, with adjustment of temperature, pH, etc.

[0119] The carbon source in the present application includes, but is not limited to, carbohydrates such as glucose, fructose, sucrose, maltose, sugar alcohols such as mannitol, sorbitol, organic acids such as pyruvic acid, lactic acid, citric acid, amino acids such as glutamic acid, methionine, lysine, etc. Also, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, corn steeping liquid, etc., carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugars), and any other suitable carbon source can be used. These carbon sources can be used alone or in combination of two or more.

[0120] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., or an organic nitrogen source such as amino acid, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more kinds, but are not limited thereto.

[0121] The phosphorus source may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a sodium-containing salt equivalent thereto, etc. The inorganic compound may be sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc.

[0122] In addition, the medium may contain amino acids, vitamins and / or suitable precursors. Specifically, the culture medium for the strain may contain L-amino acids. More specifically, glycine, glutamic acid and / or cysteine ​​may be added, and L-amino acids such as lysine may be further added as needed, but are not necessarily limited thereto.

[0123] The medium or precursors can be added to the culture in a batch or continuous manner, including but not limited to the above.

[0124] During the cultivation of the strains in the present application, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the culture in a suitable manner. Also, during the cultivation, foam formation can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, oxygen or oxygen-containing gas can be injected into the culture to maintain the aerobic state of the culture, and no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected to maintain the anaerobic and microaerobic state.

[0125] The temperature of the culture is 25° C. to 40° C., more specifically, but not limited to, 28° C. to 37° C. The culture period is continued until a desired amount of useful substance is produced, specifically, but not limited to, 1 hour to 100 hours.

[0126] The method may further include an additional step after the culturing step. The additional step is appropriately selected depending on the application of glutathione or a derivative thereof.

[0127] Specifically, the method for producing glutathione or a derivative thereof may include a step of recovering the glutathione or a derivative thereof accumulated in the bacterial cells by the culturing step, and may include, for example, a step of recovering glutathione or a derivative thereof from at least one substance selected from the strain, its dried product, extract, culture, and crushed product after the culturing step. As another example, the method for producing a glutathione derivative may include a step of converting the glutathione into a derivative.

[0128] The method may further comprise a step of lysing the strain prior to or simultaneously with the recovery step. Lysis of the strain may be carried out using a method commonly used in the technical field to which the present application pertains, such as a lysis buffer, a sonicator, heat treatment, a French press, etc. The lysis step may include, but is not limited to, enzyme reactions using cell wall degrading enzymes, nucleases, nuclease transferases, proteases, etc.

[0129] According to the production method of the present application, dry yeast, yeast extract, yeast extract mix powder, pure glutathione or its derivatives containing high contents of glutathione or its derivatives can be produced, but the production method is not limited to these, and products can be produced appropriately depending on the desired product.

[0130] In this application, dry yeast is used interchangeably with "dried strain", etc. The dry yeast can be produced by drying yeast cells that have accumulated glutathione, and is specifically included in feed compositions, food compositions, etc., but is not limited thereto.

[0131] In this application, yeast extract is used interchangeably with "strain extract" and the like. The strain extract means a substance remaining after separating the cell wall from the cells of the strain. Specifically, it means a component remaining after removing the cell wall from a component obtained by lysing the cells. The strain extract contains glutathione or a derivative thereof, and other components include at least one of proteins, carbohydrates, nucleic acids, and fibers, but is not limited thereto.

[0132] In the recovery step, the target substance, glutathione or a derivative thereof, can be recovered by any suitable method known in the art.

[0133] The recovery step may include a purification step. The purification step may be a step of isolating only glutathione or a derivative thereof from the strain and purifying it. By the purification step, pure glutathione can be produced.

[0134] If necessary, the method for producing glutathione may further include a step of mixing the obtained strain, its dried product, extract, culture, or crushed product, and glutathione recovered therefrom with an excipient after the culturing step. This mixing step can produce yeast extract mix powder. This also applies to glutathione derivatives.

[0135] The excipient can be appropriately selected depending on the intended use and form, and is, for example, 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 silicon dioxide, hydroxypropyl starch, hydroxypropylmethylcellulose, propylene glycol, casein, calcium lactate, primogel, and gum arabic, and specifically, at least one component selected from starch, glucose, cellulose, lactose, dextrin, glycogen, D-mannitol, and maltodextrin, but is not limited thereto.

[0136] The excipients include, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonic agents, and the like, but are not limited thereto. EXAMPLES

[0137] The present application will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present application, and the present application is not limited to these examples and experimental examples. EXAMPLES

[0138] Improvement of glutathione-producing strains In order to further improve the glutathione-producing ability of the CJ-5 strain (Patent Document 1), which is a glutathione-producing strain deposited under the accession number KCCM12568P, mutations were induced in the strain as follows.

[0139] The CJ-5 strain was cultivated in solid medium, then inoculated into broth to obtain a culture solution, and the bacteria were irradiated with UV light using a UV lamp.The UV-irradiated culture solution was then spread onto a plate medium, and only the mutant strains that formed colonies were isolated and collected, and the strain with the greatest improvement in glutathione production was isolated.

[0140] Examination of the genome of the isolated strain revealed that mutations occurred at -250 (C → T), -252 (G → A), -398 (A → T), -399 (A → C), -407 (T → C), and -409 (T → C) upstream of the GSH1 ORF, and that the 86th amino acid, cysteine, of the GSH1 protein encoded by the gsh1 gene had been replaced by arginine, and the 653rd amino acid (glycine) had been replaced by methionine.

[0141] The strain was named CC02-2816 and deposited at the Korea Microorganism Collection, a depository institution under the Budapest Treaty, on December 8, 2020 under the accession number KCCM12891P. EXAMPLES

[0142] Further strain improvement experiments In order to further improve the glutathione-producing ability of the CC02-2816 strain of Example 1, mutations were induced in the strain in the following manner.

[0143] The CC02-2816 strain was cultivated on solid medium, then inoculated into broth to obtain a culture solution, which was then irradiated with UV light using a UV lamp.The UV-irradiated culture solution was then spread onto a plate medium, and only the mutant strains that formed colonies were isolated and collected, and their base sequences were analyzed.

[0144] As a result of the experiment, it was confirmed that the 37th amino acid (glycine) of SOD1 (SEQ ID NO: 1), a protein encoded by sod1, which encodes superoxide dismutase 1, in the strain with a 26% increased glutathione content, was replaced with arginine. EXAMPLES

[0145] Mutation experiment of the 37th residue of SOD1 protein From the results of Example 2, it was determined that the 37th position of the SOD1 protein is important for glutathione production, and wild-type S. cerevisiae CEN.PK2-1D and the CC02-2816 strain of Example 1 were prepared to express mutant proteins in which the 37th amino acid of the SOD1 protein was replaced with another amino acid, and it was confirmed whether the amount of glutathione produced increased.

[0146] To prepare a strain of Saccharomyces cerevisiae yeast in which the 37th amino acid of the SOD1 protein was replaced with arginine, we used the pWAL100 and pWBR100 plasmids, referring to the disclosure in Non-Patent Document 19.

[0147] Specifically, PCR was performed using the genomic DNA of the CJ-5 strain as a template as follows: PCR was performed using primers of SEQ ID NO: 4 and SEQ ID NO: 6 to obtain a partial sequence of the SOD1 N-terminal including the N-terminal BamHI flanking sequence, the start codon of the SOD1 ORF, and the G37R mutant coding sequence, and primers of SEQ ID NO: 5 and SEQ ID NO: 7 to obtain a partial sequence of the SOD1 C-terminal including the C-terminal XhoI flanking sequence, the stop codon of the SOD1 ORF, and the G37R mutant coding sequence. Then, overlap PCR was performed using these two sequences as templates and SEQ ID NO: 4 and SEQ ID NO: 7 to obtain an SOD1 ORF fragment including the coding sequence of the SOD1 mutant protein in which the 37th amino acid was replaced with arginine, the N-terminal BamHI, and the C-terminal XhoI restriction enzyme sequence. The ORF fragment was digested with BamHI and XhoI and then cloned into pWAL100 vector treated with the same enzymes to prepare pWAL100-SOD1(G37R) vector.

[0148] In addition, PCR was performed using SEQ ID NO:8 and SEQ ID NO:9 with the genomic DNA of the CJ-5 strain as a template to obtain a 500 bp fragment after the termination codon of the SOD1 ORF containing the N-terminal SpeI and C-terminal NcoI restriction enzyme sequences, which was then treated with SpeI and NcoI restriction enzymes. The fragment was then cloned into pWBR100 treated with the same restriction enzymes to prepare the pWBR100-SOD1 vector.

[0149] In order to prepare the DNA fragment to be finally introduced into yeast, the pWAL100-SOD1(G37R) vector prepared as described above was used as a template, and a PCR product containing the arginine mutation coding sequence and a part of KlURA3 was obtained using primers of SEQ ID NO: 4 and SEQ ID NO: 10. A PCR product containing a part of KlURA3 and 500 bp after the stop codon of SOD1 was obtained using primers of SEQ ID NO: 11 and SEQ ID NO: 9 with the pWBR100-SOD1 vector as a template, and then each PCR product was transformed into S. cerevisiae CEN.PK2-1D and S. cerevisiae CC02-2816 at the same molar ratio. PCR was performed under the conditions of 95°C for 5 minutes for the heat denaturation process, 53°C for 1 minute for the binding process, and 72°C for 1 minute per 1 kb for the polymerization process. For yeast transformation, the lithium acetate method modified from Non-Patent Document 20 was used. Specifically, yeast cells with an OD between 0.7 and 1.2 were washed twice with lithium acetate / TE buffer, then the PCR product and single stranded DNA (Sigma D-7656) were mixed and cultured in lithium acetate / TE / 40% PEG buffer at 30°C for 30 minutes and at 42°C for 15 minutes. The cells were then cultured on a uracil-free SC (2% glucose) agar plate until colonies were formed, and a strain carrying the SOD1 G37R mutant coding sequence and the KlURA3 gene was obtained. Then, to remove KlURA3, each strain was cultured overnight in 2 ml of YPD, then diluted 1 / 100 and spread on a SC (2% glucose) agar plate containing 0.1% 5-FOA to prepare S. cerevisiae CEN.PK2-1D SOD1 G37R mutant strain and S. cerevisiae CC02-2816 SOD G37R mutant strain from which the uracil marker was removed. Strains expressing SOD1 mutant proteins substituted with other types of amino acids other than arginine were also prepared in the same manner, except that a primer pair in which the 37th arginine coding sequence on the primer sequences of SEQ ID NO:5 and SEQ ID NO:6 was replaced with a sequence coding for another amino acid was used.

[0150] [Table 1]

[0151] The above-prepared strains were cultured for 26 hours, and the concentrations and contents of glutathione (GSH) produced were measured. The results are shown in Tables 2, 3, and FIGS. 1 and 2.

[0152] [Table 2]

[0153] [Table 3]

[0154] These results confirm that glutathione production ability is improved by replacing the 37th amino acid of SOD1 protein with another amino acid, and therefore demonstrate that the SOD1 protein mutant provided in the present application is useful for producing γ-glutamyl peptides.

[0155] From the above description, a person skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present application should be interpreted as including all modifications and variations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

[0156] [Table 4]

Claims

1. A superoxide dismutase 1 (SOD1) mutant having superoxide dismutase 1 activity, The variant has an identity of 90% to 100% with the amino acid sequence of SEQ ID NO:1, The amino acid corresponding to the 37th position from the N-terminus of the amino acid sequence of SEQ ID NO:1 is replaced with another amino acid, the amino acid at the 37th position from the N-terminus of the amino acid sequence of SEQ ID NO:1 is glycine; and The other amino acid is an amino acid selected from alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine. Mutant.

2. The mutant according to claim 1, comprising an amino acid sequence of any one of SEQ ID NOs: 12 to 30.

3. A polynucleotide encoding the superoxide dismutase 1 mutant of claim 1 or 2.

4. A microorganism comprising at least one of the superoxide dismutase 1 mutants according to claim 1 or 2 and a polynucleotide encoding said mutant.

5. The microorganism according to claim 4 , which produces glutathione or a derivative thereof.

6. The microorganism according to claim 4 , wherein the microorganism is a Saccharomyces sp.

7. The microorganism of claim 6, wherein the microorganism is Saccharomyces cerevisiae.

8. A method for producing glutathione or a derivative thereof, comprising the step of culturing in a medium a microorganism containing at least one of the superoxide dismutase 1 mutants according to claim 1 or 2 and a polynucleotide encoding said mutant.

9. The method for producing glutathione or a derivative thereof according to claim 8, further comprising a step of recovering glutathione or a derivative thereof from at least one substance selected from the cultured microorganism, a dried product of the microorganism, an extract of the microorganism, a culture of the microorganism, and a disrupted product of the microorganism.

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