Novel promoter and method for producing glutathione using the same

Abstract

Provided are a novel promoter, a vector comprising the same, a microorganism comprising the same, and a method for producing glutathione using the same.

Description

Technical Field

[0001] The present disclosure relates to a novel promoter, a vector comprising the same, a microorganism comprising the same, and a method for producing glutathione using the same. Background Art

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

[0003] Glutathione exists in the living body in the form of reduced glutathione (GSH) and oxidized glutathione (GSSG). Reduced glutathione (GSH), which is normally present in a relatively high proportion, is mainly distributed in the liver and skin cells of the human body and has important functions: antioxidant function that breaks down and removes reactive oxygen species, detoxification function that removes xenobiotic compounds such as toxic substances, and whitening function that inhibits melanin production (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).

[0004] Since the production of glutathione gradually decreases as the aging process progresses, and the reduced production of glutathione, which plays an important role in antioxidant and detoxification functions, promotes the accumulation of reactive oxygen species, which is the main cause of aging, it is necessary to supply glutathione from the outside.

[0005] Glutathione, with its various functions, has attracted attention as a substance in various fields, such as pharmaceuticals, health functional foods, and cosmetics. It is also used in the manufacture of flavoring ingredients and food and feed additives. Glutathione is known to have a significant effect on enriching the taste of raw materials and maintaining a rich flavor. It can be used alone or in combination with other substances as a kokumi flavor enhancer. In general, kokumi substances are known to have a richer flavor than umami substances, such as known nucleic acids and monosodium glutamate (MSG), and are known to be produced by protein decomposition during the ripening process.

[0006] Despite the growing demand for glutathione applications in various fields, its market has not been activated due to the high cost of industrial production of glutathione, as the enzymatic synthesis process has not been commercialized due to high costs, and the method of extracting glutathione from microorganisms provides low yields. Summary of the Invention

[0007] Technical issues

[0008] The present disclosure provides a novel promoter, a vector comprising the same, a microorganism comprising the same, and a method for producing glutathione using the same.

[0009] Technical Solution

[0010] The present disclosure provides a polynucleotide having promoter activity, wherein at least one nucleotide selected from nucleotides 92, 94, 102, 103, 249 and 251 of the polynucleotide sequence of SEQ ID NO: 1 or 2 is substituted with a different nucleotide.

[0011] The present disclosure provides a vector comprising a polynucleotide having promoter activity.

[0012] The present disclosure provides a microorganism belonging to the genus Saccharomyces sp., which includes one or more of the following: a polynucleotide having promoter activity and a gene encoding a target protein; and a vector including the same.

[0013] The present disclosure provides a method for producing glutathione, the method comprising culturing a microorganism in a culture medium.

[0014] Beneficial effects

[0015] The novel promoter sequences disclosed herein significantly increase the production of glutathione and can therefore be used to produce glutathione in high yields. DETAILED DESCRIPTION

[0016] The present disclosure will be described in detail. Meanwhile, each description and embodiment disclosed in the present disclosure can be applied to the different descriptions and embodiments herein. In other words, all combinations of the various components disclosed in the present disclosure are included within the scope of the present disclosure. In addition, the scope of the present disclosure should not be limited by the description provided below.

[0017] 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 this disclosure. Such equivalents are intended to be encompassed by the following claims.

[0018] One aspect of the present disclosure provides a polynucleotide having promoter activity, wherein at least one nucleotide selected from nucleotides 92, 94, 102, 103, 249 and 251 of the polynucleotide sequence of SEQ ID NO: 1 or 2 is substituted with a different nucleotide.

[0019] As used herein, the term "polynucleotide" refers to a DNA chain of a certain minimum length, as a nucleotide polymer in which nucleotide monomers are linked to each other by covalent bonds in the form of a long chain.

[0020] As used herein, the term "polynucleotide having promoter activity" refers to a DNA region near the transcription start site of a gene to be expressed (i.e., target gene), which includes sites where RNA polymerase, enhancers, etc. bind to express the target gene.

[0021] The polynucleotide having promoter activity disclosed herein can be used as a universal enhanced promoter.

[0022] For example, the polynucleotide can be used as a promoter capable of enhancing the expression of a polypeptide having glutamate-cysteine ​​ligase activity. In addition, the polynucleotide can be a polynucleotide for increasing the production or output of glutathione. The polynucleotide of the present disclosure can include any polynucleotide sequence having promoter activity.

[0023] In the present disclosure, the polynucleotide sequence of SEQ ID NO: 1 or 2 may be a sequence capable of serving as a promoter of glutamate-cysteine ​​ligase.

[0024] However, the polynucleotide sequence of SEQ ID NO: 1 or 2 is a representative polynucleotide sequence indicating the position of modification, and any polynucleotide sequence corresponding thereto and having promoter activity can also be included in the sequence allowing for modification introduction. For example, any polynucleotide sequence capable of acting as a promoter for a glutamate-cysteine ​​ligase or a polypeptide having equivalent activity thereto can be included without limitation within the scope of the modified sequence introduced therein. In such a sequence, when at least one nucleotide corresponding to nucleotides 92, 94, 102, 103, 249, and 251 of SEQ ID NO: 1 or 2 is substituted by a different nucleotide, a promoter having higher activity than the unsubstituted (unmodified) promoter sequence can be provided.

[0025] The nucleotide sequence of SEQ ID NO: 1 or 2 can be obtained from the known NCBI GenBank database. The sequence corresponding to SEQ ID NO: 1 or 2 and serving as a promoter for glutamate-cysteine ​​ligase can be derived from a microorganism belonging to the genus Saccharomyces, specifically Saccharomyces cerevisiae, but is not limited thereto, and may include, without limitation, any sequence having an activity equivalent to that of the polynucleotide.

[0026] In the present disclosure, the polynucleotide having promoter activity can be a polynucleotide sequence of SEQ ID NO: 1 or 2, or a polynucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity to SEQ ID NO: 1 or 2, wherein at least one nucleotide at a specific position or a corresponding position thereof is substituted with a different nucleotide. The polynucleotide sequence having homology or identity may not include a sequence having 100% identity, or may be a sequence having less than 100% identity.

[0027] Meanwhile, although “a polynucleotide having a polynucleotide sequence of a predetermined SEQ ID NO” and “a polynucleotide comprising a polynucleotide sequence of a predetermined SEQ ID NO” are used in the present disclosure, it is obvious that any polynucleotide having a polynucleotide sequence comprising a deletion, modification, substitution or addition of one or several nucleotides can also be used in the present disclosure, as long as the polynucleotide has the same or equivalent activity as a polynucleotide consisting of a polynucleotide sequence of a predetermined SEQ ID NO.

[0028] For example, it is obvious that any polynucleotide including the addition of a nonsense sequence within or at the end of the polynucleotide sequence of a predetermined SEQ ID NO or the deletion of a partial polynucleotide sequence within or at the end of the polynucleotide sequence of a predetermined SEQ ID NO may also be within the scope of the present disclosure, as long as the polynucleotide has the same or equivalent activity as the polynucleotide of the present disclosure.

[0029] Homology and identity refer to the degree of relatedness between two given polynucleotide sequences and can be expressed as a percentage.

[0030] The terms homology and identity are often used interchangeably.

[0031] The sequence homology or identity of conserved polynucleotides can be determined by standard comparison algorithms, and the default gap penalties (default gap penalties) set up by the program can be used in conjunction with it. Basically, homology or identity sequences can hybridize to each other under moderate or high stringency conditions over at least about 50%, 60%, 70%, 80% or 90% of the entire sequence or entire length. In the hybridized polynucleotides, it is also contemplated that degenerate codons may be used in place of codons.

[0032] Sequence homology, similarity or identity between two given polynucleotide sequences can be determined using any known computer algorithm, such as the "FASTA" program, by using the default parameters introduced by, for example, Pearson et al. (1988, Proc. Natl. Acad. Sci. USA 85:2444). Alternatively, the Needleman-Wunsch algorithm (1970, J. Mol. Biol. 48: 443-453) performed by the Needleman program of the European Molecular Biology Open Software Suite (EMBOSS) package (Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or higher) can be used to determine it (including the GCG program package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF 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, homology, similarity or identity can be determined using BLAST from the National Center for Biotechnology Information database or ClustalW.

[0033] Homology, similarity or identity between polynucleotides can be determined by comparing sequence information using a GAP computer program, such as the program introduced by Needleman et al. (1970), J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In short, the GAP program defines similarity as the number of similar aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The default parameters of the GAP program may include: (1) a binary comparison matrix (containing an identity value of 1 and a non-identity value of 0) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, which is disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, as used herein, the terms "homology" or "identity" refer to the relatedness between sequences.

[0034] In addition, polynucleotides can include, without limitation, any probes prepared from any known gene sequence, such as polynucleotide sequences that hybridize under stringent conditions to sequences that are fully or partially complementary to the above-mentioned polynucleotide sequences and have the same activity. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are disclosed in detail in known documents (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). For example, stringent conditions may include hybridization between genes having high homology or identity (e.g., homology or identity of 40% or more, specifically 70% or more, 80% or more, 85% or more, or 90% or more, more specifically 95% or more, even more specifically 97% or more, and most specifically 99% or more), but not hybridization between genes having lower homology or identity than the above homology or identity, or washing once, specifically twice or three times, under conventional washing conditions for Southern hybridization at a salt concentration and temperature of 60°C, 1×SSC and 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC and 0.1% SDS.

[0035] Hybridization requires that the two polynucleotides have complementary sequences, although base mismatches may occur depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between the bases of nucleotides that are capable of hybridizing to each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the present disclosure can include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0036] Specifically, polynucleotides having homology or identity can be detected using the above hybridization conditions, including a T of 55°C. m The hybridization process under the value. In addition, T m The 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 according to the intended purpose.

[0037] The degree of stringency appropriate for hybridization of polynucleotides may depend on the length of the polynucleotides and the degree of complementation, and its parameters are well known in the art (Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0038] The polynucleotides having promoter activity provided by the present disclosure can have enhanced promoter activity by substituting nucleotides at specific positions of the polynucleotide sequences having promoter activity.

[0039] In an embodiment, the polynucleotide with promoter activity of the present disclosure may include a polynucleotide with promoter activity in which at least one nucleotide in the nucleotide sequence of SEQ ID NO: 1 or 2 is replaced by a different nucleotide. Specifically, the polynucleotide may consist of a polynucleotide with promoter activity in which at least one nucleotide in the nucleotide sequence of SEQ ID NO: 1 or 2 is replaced by a different nucleotide. In the present disclosure, the polynucleotide with promoter activity can be used interchangeably with a "mutant promoter". A mutant promoter can be a promoter in which the nucleotides at one or more positions, two or more positions, three or more positions, four or more positions, five or more positions, or all six positions or their corresponding positions are replaced by different nucleotides.

[0040] In an embodiment, the polynucleotide having promoter activity can be a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 1 or 2, wherein at least one nucleotide selected from nucleotides 92, 94, 102, 103, 249 and 251 is substituted by a different nucleotide and has promoter activity.

[0041] There is no specific limitation on the term "different nucleotide", as long as the nucleotide after the substitution is different from the nucleotide before the substitution. For example, when the 92nd nucleotide in the polynucleotide sequence of SEQ ID NO: 1 includes thymine (T), the expression "the 92nd nucleotide of SEQ ID NO: 1 is replaced by a different nucleotide" means that thymine (T) is replaced by cytosine (C), adenine (A) or guanine (G) other than thymine (T). In the present disclosure, when a nucleotide is "substituted", unless otherwise specified, the nucleotide is replaced by another nucleotide different from the nucleotide before the substitution.

[0042] At the same time, those skilled in the art will determine the nucleotides at positions corresponding to nucleotides 92, 94, 102, 103, 249, and 251 of the polynucleotide sequence of SEQ ID NO: 1 or 2 of the present disclosure in any polynucleotide sequence by sequence alignment known in the art, and it will be apparent that, unless otherwise specified in the present disclosure, the expression "nucleotides at a specific position of a specific SEQ ID NO:" includes "nucleotides at corresponding positions thereof" in any polynucleotide sequence. Therefore, any polynucleotide sequence having promoter activity in which at least one nucleotide selected from the nucleotides corresponding to nucleotides 92, 94, 102, 103, 249, and 251 of the polynucleotide sequence of SEQ ID NO: 1 or 2 is substituted with a different nucleotide is within the scope of the present disclosure.

[0043] In the polynucleotide having promoter activity derived from Saccharomyces cerevisiae strains CEN KSD-Yc, YJM1450, YJM1401, YJM1307, and the strain with accession number KCCM12568P deposited with the Korean Culture Center of Microorganisms (KCCM) under the Budapest Treaty, that is, in SEQ ID NO: 2, "positions 92, 94, 102, 103, 249, and 251 of SEQ ID NO: 1 or 2" are located at 409 nt, 407 nt, 399 nt, 398 nt, 252 nt, and 250 nt upstream of the start codon ATG as a reference (0), respectively, and thus these positions can be described as positions -409, -407, -399, -398, -252, and -250 upstream of the ORF, respectively, according to methods commonly used in the art.

[0044] At the same time, with respect to the polynucleotide with promoter activity derived from Saccharomyces cerevisiae CEN.PK1-D, i.e., SEQ ID NO: 1, wherein the nucleotide at position 74 (i.e., -74) upstream of the ORF is deleted in the promoter sequence of Saccharomyces cerevisiae CEN KSD-Yc, "positions 92, 94, 102, 103, 249 and 251 of SEQ ID NO: 1 or 2" correspond to positions -408, -406, -398, -397, -251 and -249 upstream of the ORF, respectively.

[0045] In an embodiment, the polynucleotide having promoter activity of the present disclosure may be a polynucleotide in which at least one nucleotide selected from nucleotides 92, 94, 102, 103, 249 and 251 of the polynucleotide sequence of SEQ ID NO: 1 or 2 is substituted with a different nucleotide.

[0046] Specifically, in the present disclosure, the polynucleotide having promoter activity may be a polynucleotide in which two or more nucleotides at positions 92, 94, 102, 103, 249 and 251 of the polynucleotide sequence selected from SEQ ID NO: 1 or 2 are substituted with different nucleotides.

[0047] Specifically, in the present disclosure, the polynucleotide having promoter activity may be a polynucleotide in which four or more nucleotides selected from nucleotides 92, 94, 102, 103, 249 and 251 of the polynucleotide sequence of SEQ ID NO: 1 or 2 are substituted with different nucleotides.

[0048] Specifically, the polynucleotide may be a polynucleotide in which all six nucleotides among nucleotides 92, 94, 102, 103, 249 and 251 of the polynucleotide sequence selected from SEQ ID NO: 1 or 2 are substituted with different nucleotides.

[0049] In an embodiment of the present disclosure, the polynucleotide having promoter activity may be a polynucleotide in which nucleotides 249 and 251 of the polynucleotide sequence of SEQ ID NO: 1 or 2 are substituted with different nucleotides.

[0050] In an embodiment of the present disclosure, the polynucleotide having promoter activity may be a polynucleotide in which nucleotides 92, 94, 102, and 103 of the polynucleotide sequence of SEQ ID NO: 1 or 2 are substituted with different nucleotides.

[0051] However, the present disclosure is not limited thereto.

[0052] In an embodiment of the present disclosure, the polynucleotide having promoter activity may include, in the polynucleotide sequence of SEQ ID NO: 1 or 2:

[0053] The thymine (T) at position 92 is replaced by guanine (G), cytosine (C), or adenine (A);

[0054] The thymine (T) at position 94 is replaced by guanine (G), cytosine (C), or adenine (A);

[0055] Adenine (A) at position 102 is replaced by guanine (G), cytosine (C), or thymine (T);

[0056] Adenine (A) at position 103 is replaced by guanine (G), cytosine (C), or thymine (T);

[0057] The guanine (G) at position 249 is replaced by thymine (T), cytosine (C), or adenine (A);

[0058] The cytosine (C) at position 251 is substituted with thymine (T), guanine (G) or adenine (A); or any combination thereof.

[0059] In an embodiment, the thymine (T) at position 92 may be substituted with a cytosine (C). This may also be described as 92 (T→C) or -409 (T→C), and depending on the reference sequence, as -408 (T→C).

[0060] In an embodiment, the thymine (T) at position 94 may be substituted with a cytosine (C). This may also be described as 94 (T→C) or -407 (T→C), and depending on the reference sequence, as -406 (T→C).

[0061] In an embodiment, the adenine (A) at position 102 may be substituted with a cytosine (C). This may also be described as 102 (A→C) or -399 (A→C), and depending on the reference sequence, -398 (A→C).

[0062] In an embodiment, the adenine (A) at position 103 may be substituted with a thymine (T). This may also be described as 103 (A→T) or -398 (A→T), and according to the reference sequence, as -397 (A→T).

[0063] In an embodiment, the guanine (G) at position 249 may be substituted with an adenine (A). This may also be described as 249 (G→A) or -252 (G→A), and depending on the reference sequence, as -251 (G→A).

[0064] In an embodiment, the cytosine (C) at position 251 may be substituted with thymine (T). This may also be described as 251 (C→T) or -250 (C→T), and, depending on the reference sequence, as -249 (C→T).

[0065] In an embodiment of the present disclosure, the polynucleotide having promoter activity may include at least one of 92 (T→C), 94 (T→C), 102 (A→C), 103 (A→T), 249 (G→A) and 251 (C→T) substitutions.

[0066] In an embodiment of the present disclosure, the polynucleotide having promoter activity may include substitutions of 249 (G→A) and 251 (C→T).

[0067] In an embodiment of the present disclosure, the polynucleotide having promoter activity may include substitutions of 92 (T→C), 94 (T→C), 102 (A→C), and 103 (A→T).

[0068] In an embodiment of the present disclosure, the polynucleotide having promoter activity may include all substitutions in 92 (T→C), 94 (T→C), 102 (A→C), 103 (A→T), 249 (G→A) and 251 (C→T).

[0069] In an embodiment of the present disclosure, the polynucleotide having promoter activity may include at least one polynucleotide sequence selected from SEQ ID NOs: 3 to 32. Specifically, the polynucleotide may consist of one polynucleotide sequence selected from SEQ ID NOs: 3 to 32, but is not limited thereto.

[0070] As described above, although the expression "a polynucleotide having a nucleotide sequence of a specific SEQ ID NO:" or "a polynucleotide comprising a nucleotide sequence of a specific SEQ ID NO:" is used in the present disclosure, it is obvious that any polynucleotide having a nucleotide sequence comprising a deletion, modification, substitution or addition of one or several nucleotides can also be used in the present disclosure, as long as the polynucleotide has the same or equivalent activity as a polynucleotide consisting of the nucleotide sequence of the specific SEQ ID NO.

[0071] In addition, the present disclosure is not limited to the above-mentioned embodiments, and the polynucleotide sequence may include various modifications within the range that does not significantly impair the promoter activity.

[0072] The polynucleotide having promoter activity disclosed herein can be used as a promoter.

[0073] The promoter can be located in the 5' region of the mRNA transcription start site.

[0074] The promoter may have enhanced promoter activity compared to conventional promoters. That is, the promoter may not only increase the expression of the target gene in the host cell, but also increase the expression and / or activity of the protein encoded by the target gene. In view of the purposes of the present disclosure, the target gene for enhanced expression can be modified according to the product to be obtained, and the promoter can be used as a universal promoter to enhance the target gene.

[0075] "Target gene" refers to a gene whose expression is regulated by the promoter sequence of the present disclosure. The protein encoded by the target gene can be referred to as the "target protein", and the gene encoding the "target protein" can be referred to as the "target gene".

[0076] In addition, the polynucleotide encoding the target protein may have various modifications made in the coding region, provided that the amino acid sequence of the protein expressed by the coding region is not changed due to codon degeneracy or codon preference in consideration of the living organism in which the polynucleotide is expressed. The description of the polynucleotide sequence is provided above.

[0077] In an embodiment, the target protein may be a polypeptide having glutamate-cysteine ​​ligase activity. That is, the target gene of the promoter may be a gene encoding a polypeptide having glutamate-cysteine ​​ligase activity.

[0078] In the present disclosure, "glutamate-cysteine ​​ligase" is an enzyme also known as "glutamate-cysteine ​​ligase," "γ-glutamylcysteine ​​synthetase (GCS)," or "GSH1 protein."

[0079] Glutamate-cysteine ​​ligase is known to catalyze the following reaction:

[0080] L-glutamate + L-cysteine ​​+ ATP γ-glutamylcysteine+ADP+P i

[0081] Furthermore, the reaction catalyzed by glutamate-cysteine ​​ligase is known as the first step in glutathione synthesis.

[0082] The amino acid sequence constituting the glutamate-cysteine ​​ligase can be obtained from the known NCBI GenBank database. For example, the glutamate-cysteine ​​ligase can be derived from Saccharomyces cerevisiae. For example, the glutamate-cysteine ​​ligase can be a protein comprising the amino acid sequence of SEQ ID NO: 33, but can include, without limitation, any sequence having the same activity as this amino acid sequence.

[0083] In addition, the "polypeptide having glutamate-cysteine ​​ligase activity" of the present disclosure may include not only wild-type, unmodified or naturally occurring forms of glutamate-cysteine ​​ligase, but also variants having the same or enhanced glutamate-cysteine ​​ligase activity.

[0084] In the present disclosure, "modified (polypeptide)" having the same meaning as "variant" may refer to a protein obtained by conservative substitution and / or modification of at least one amino acid different from the sequence while retaining the function or properties of the protein. For example, the modified polypeptide may be a variant in which the amino acid at position 86 from the N-terminus of SEQ ID NO: 33 in the above-mentioned glutamate-cysteine ​​ligase is substituted with a different amino acid residue other than cysteine.

[0085] Variants differ from a defined sequence by substitutions, deletions, or additions of several amino acids. Such variants can be obtained by modifying one or more amino acids in the amino acid sequence of the protein described above and identified by evaluating the properties of the modified protein. That is, the ability of the variant can be enhanced relative to the native protein. In addition, some variants may include variants in which at least one portion, such as an N-terminal leader sequence or a transmembrane domain, has been removed. Other variants may include variants in which a portion has been removed from the N-terminus and / or C-terminus of the mature protein.

[0086] The term "variant" may also be used interchangeably with other terms such as modification, modified protein, modified polypeptide, mutant, mutein, and divergent, and any term for expressing a variation may also be used without limitation. For the purposes of this disclosure, a variant may have an enhanced activity compared to a wild-type or unmodified protein, but is not limited thereto.

[0087] As used herein, the term "conservative substitution" refers to the replacement of an amino acid with a different amino acid having similar structural and / or chemical properties. A variant can have at least one conservative substitution while retaining at least one biological activity. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues.

[0088] Variants can also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a polypeptide can be conjugated to a signal (or leader) sequence at the N-terminus of a protein that co-translationally or post-translationally directs the transfer of the protein. A polypeptide can also be conjugated to another sequence or linker to identify, purify, or synthesize the polypeptide.

[0089] In the present disclosure, the phrase "substituted with a different amino acid" is not particularly limited, as long as the amino acid after substitution is different from the amino acid before substitution. For example, if the amino acid cysteine ​​at position 86 from the N-terminus of the amino acid sequence of SEQ ID NO: 33 is replaced with an amino acid other than cysteine, this can also be expressed as "the amino acid at position 86 is replaced with a different amino acid." Furthermore, in the present disclosure, unless the phrase "substituted with a different amino acid" is used, the phrase "predetermined amino acid is substituted" means that the amino acid after substitution is different from the amino acid before substitution.

[0090] The "glutamate-cysteine ​​ligase variant" of the present disclosure may also be referred to as a "(modified) polypeptide having glutamate-cysteine ​​ligase activity" or a "GSH1 variant", which can increase glutathione production compared to the protein before modification, the wild-type polypeptide or the unmodified polypeptide, but is not limited thereto.

[0091] In the variant, at least one amino acid in the amino acid sequence of SEQ ID NO: 33 may be substituted with a different amino acid. Specifically, the variant may include a substitution of the amino acid at position 86 of the amino acid sequence corresponding to SEQ ID NO: 33 with a different amino acid. The different amino acid may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, tyrosine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, and histidine.

[0092] In an embodiment, the amino acid at position 86 of the amino acid sequence corresponding to SEQ ID NO: 33 may be substituted with arginine, but is not limited thereto.

[0093] In the present disclosure, it is obvious that “a variant in which the amino acid at position 86 from the N-terminus of the amino acid sequence of SEQ ID NO: 33 is substituted with a different amino acid” includes variants in which the amino acid corresponding to position 86 of the amino acid sequence of SEQ ID NO: 33 is substituted with a different amino acid, even if the amino acid is at a position other than (except) position 86 due to deletion / addition / addition of an amino acid at the N-terminus, C-terminus, or in the middle of the amino acid sequence of SEQ ID NO: 33. In addition, although a variant in which the amino acid at position 86 from the N-terminus of the amino acid sequence of SEQ ID NO: 33 is substituted with a different amino acid is disclosed as an example of a glutamate-cysteine ​​ligase variant of the present disclosure, it is obvious that the glutamate-cysteine ​​ligase variant of the present disclosure is not limited to variants of the amino acid sequence of SEQ ID NO: 33, and variants in which the amino acid at position 86 of the amino acid sequence of SEQ ID NO: 33 is substituted with a different amino acid in any amino acid sequence having glutamate-cysteine ​​ligase activity are also within the scope of the glutamate-cysteine ​​ligase variants of the present disclosure. In any amino acid sequence, the "amino acid corresponding to position 86 of the amino acid sequence of SEQ ID NO: 33" can be identified by various sequence alignment methods known in the art.

[0094] The glutamate-cysteine ​​ligase variant of the present disclosure, in which the amino acid at position 86 from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 33 is substituted with a different amino acid, can be a protein comprising the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity thereto. In addition, it is obvious that any protein having an amino acid sequence comprising one or more amino acid deletions, modifications, substitutions or additions at positions other than position 86 is also within the scope of the present disclosure, as long as the protein retains the above-mentioned homology or identity and an effect equivalent to that of the variant. Homology and identity are as described above.

[0095] The gene encoding the polypeptide having glutamate-cysteine ​​ligase activity of the present disclosure may be referred to as a "GSH1 gene."

[0096] The gene may be derived from yeast. Specifically, the gene may be derived from a microorganism belonging to the genus Saccharomyces, more specifically, from Saccharomyces cerevisiae. Specifically, the gene may be a gene encoding the amino acid sequence of SEQ ID NO: 33, but is not limited thereto.

[0097] In the present disclosure, the “GSH1 gene”, i.e., a polynucleotide encoding a polypeptide having glutamate-cysteine ​​ligase activity, may have various modifications made in the coding region as long as the amino acid sequence of the polypeptide expressed by the coding region is not changed due to codon degeneracy or codon preference in consideration of the living organism in which the polynucleotide is expressed.

[0098] Since the polypeptides having glutamate-cysteine ​​ligase activity disclosed herein also include variant sequences, any polynucleotide sequences encoding protein variants in which the amino acid at position 86 of the amino acid sequence of SEQ ID NO: 33 disclosed herein is substituted with a different amino acid can also be included without limitation.

[0099] In addition, the polynucleotide may include, without limitation, a polynucleotide sequence that hybridizes with a probe constructed using a known gene sequence, such as a polynucleotide sequence that is fully or partially complementary to the polynucleotide sequence under stringent conditions to encode a protein variant in which the amino acid at position 86 of the amino acid sequence corresponding to SEQ ID NO: 33 is substituted by a different amino acid.

[0100] Another aspect of the present disclosure provides a composition for expressing a gene, which comprises the polynucleotide having promoter activity of the present disclosure.

[0101] The composition for expressing a gene refers to a composition capable of expressing a gene using the polynucleotide having promoter activity disclosed herein.

[0102] For example, a composition for expressing a gene includes the polynucleotide having promoter activity of the present disclosure, and may further include any other components capable of operating the polynucleotide as a promoter.

[0103] In the composition for expressing a gene according to the present disclosure, the polynucleotide may be in the form of being contained in a vector, which can achieve expression of the gene operably linked thereto in a host cell.

[0104] Another aspect of the present disclosure provides a vector comprising a polynucleotide having promoter activity or the polynucleotide and a gene encoding a target protein.

[0105] In an embodiment, the target protein may be a polypeptide having glutamate-cysteine ​​ligase activity.

[0106] As used herein, the term "vector" refers to a DNA construct comprising a polynucleotide sequence encoding a target protein, which is operably linked to appropriate regulatory sequences for expressing the target protein in a suitable host cell.

[0107] For the purposes of the present disclosure, a regulatory sequence may include a polynucleotide having promoter activity disclosed herein.

[0108] At the same time, regulatory sequences may include a promoter for initiating transcription, an operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating transcription and translation termination. After the vector is introduced into a suitable host cell, it can replicate or work independently of the host genome, and can be integrated into the genome.

[0109] The vector used in the present disclosure is not particularly limited, and any vector known in the art can be used. As a vector for expression in yeast, an integrating yeast plasmid (Yip) and an extrachromosomal plasmid vector can be used.

[0110] Extrachromosomal plasmid vectors can include episomal yeast plasmids (Yep), replicating yeast plasmids (YRp), and yeast centromeric plasmids (YCp).

[0111] Furthermore, artificial yeast chromosomes (YACs) can also be used as vectors of the present disclosure.

[0112] As specific examples, available vectors may include pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPαA, pGAPαB, pGAPαC, pPIC3.5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6αA, pPIC6αB, pPIC6αC, 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 form), pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, pYJ405, and pYJ406, but are not limited thereto.

[0113] The insertion of polynucleotides in chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker can be further included to confirm chromosome insertion. Selection markers are used to select cells transformed through the vector, i.e., to identify the insertion of the desired nucleic acid molecule, and examples of selection markers can include markers that provide surface expression of selectable phenotypes such as drug resistance, nutritional requirements, cytotoxic agent resistance or variant polypeptides. Only cells expressing the selection marker can survive or show different phenotypes under the environment treated with the selection agent, and therefore transformed cells can be selected. For example, a vector for chromosome insertion can be used to replace wild-type polynucleotides with mutant polynucleotides by utilizing them.

[0114] As used herein, the term "transformation" refers to a process in which a vector including a polynucleotide encoding a target protein is inserted into a host cell so that the target protein can be expressed in the host cell.

[0115] The polynucleotide of conversion can be the form of inserting into the host cell chromosome or the form of being positioned at extrachromosomal, as long as protein is expressed in host cell.In addition, the polynucleotide of encoding target protein can comprise the DNA and RNA of encoding target protein.The polynucleotide of encoding target protein can be introduced into host cell in any form, as long as these polynucleotide are introduced into host cell and express protein therein.For example, the polynucleotide of encoding target protein can be introduced into host cell in the form of expression cassette, and this expression cassette is the gene construct comprising all basic elements required for self-replication.

[0116] The expression cassette may generally include a promoter operably linked to a polynucleotide encoding a target protein, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide encoding the target protein may be introduced into a host cell in its original form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

[0117] In addition, as used herein, the term "operably linked" means that the polynucleotide sequence encoding the target protein of the present disclosure is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide sequence.

[0118] For the purpose of the present disclosure, a promoter may be a polynucleotide having promoter activity of the present disclosure.

[0119] The transformation methods according to the present disclosure include any method capable of achieving the introduction of the vector into the host cell, and can be performed by suitable standard techniques known in the art selected according to the host cell. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method can be utilized, but the present disclosure is not limited thereto.

[0120] Another aspect of the present disclosure provides a microorganism comprising the polynucleotide having promoter activity of the present disclosure, a polynucleotide comprising the above polynucleotide and a gene encoding a target protein, or a vector comprising the same.

[0121] The target protein may be a polypeptide having glutamate-cysteine ​​ligase activity. The polynucleotide having promoter activity, the target protein, the polypeptide having glutamate-cysteine ​​ligase activity, and the vector disclosed herein are as described above.

[0122] The microorganism may be a yeast, particularly a microorganism belonging to the genus Saccharomyces, more particularly Saccharomyces cerevisiae.

[0123] The microorganism may be a microorganism expressing glutamate-cysteine ​​ligase, a microorganism expressing a polypeptide having glutamate-cysteine ​​ligase activity, or a microorganism into which a polypeptide having glutamate-cysteine ​​ligase activity is introduced, but is not limited thereto.

[0124] In an embodiment, the microorganism can be a microorganism having glutathione production ability, a microorganism prepared by enhancing glutathione production ability in a parent strain naturally having low glutathione production ability, or a microorganism prepared by providing glutathione production ability to a parent strain that cannot produce glutathione. In an embodiment, the microorganism can be a microorganism expressing a glutamate-cysteine ​​ligase variant, the variant comprising at least one amino acid variation in the amino acid sequence of SEQ ID NO: 33, and the amino acid variation can include substitution of the 86th amino acid from the N-terminus of SEQ ID NO: 33 with a different amino acid. However, the present disclosure is not limited thereto. The glutamate-cysteine ​​ligase variant is as described above.

[0125] As used herein, the term "protein to be expressed / expressed" means a state in which a target protein, such as glutamate-cysteine ​​ligase or a variant thereof, is introduced or modified to be expressed in a microorganism. When the protein is present in the microorganism, the activity of the protein is enhanced compared to the activity of its endogenous protein or the activity before modification.

[0126] Specifically, the term "introduction of a protein" refers to providing the activity of a specific protein to a microorganism that does not have the protein or enhancing the activity of the protein compared to the inherent activity or activity of the protein before modification. For example, introduction of a protein may refer to introducing a polynucleotide encoding the protein into a chromosome or introducing a vector comprising the polynucleotide encoding the specific protein into a microorganism to express the activity of the protein.

[0127] In addition, "enhancement of activity" can mean that the activity of a specific protein of a microorganism is enhanced compared to the inherent activity or activity before modification. The term "intrinsic activity" can refer to the activity of a specific protein possessed by the parent strain before transformation when a microorganism is transformed by natural or artificial genetic variation.

[0128] In view of the purpose of the present disclosure, the activity can be enhanced by using the polynucleotide sequence having promoter activity disclosed herein as the expression control sequence of the target protein. As described above, the target protein can be a wild-type or variant, and the expression control sequence can be the expression control sequence of a gene encoding a protein variant or the expression control sequence of a chromosomal gene encoding a wild-type protein.

[0129] In addition, any other method or combination of methods for enhancing activity can be used. For example, in addition to the method of using the polynucleotide sequence having promoter activity disclosed herein as the expression control sequence of the target protein, at least one method selected from the following can be used: a method of increasing the copy number of the gene encoding the target protein in the cell, a method of replacing the chromosomal gene encoding the wild-type protein with a gene encoding a protein variant, a method of introducing a mutation into the gene encoding the protein to enhance the activity of the protein variant, and a method of introducing a protein variant into a microorganism, but are not limited thereto.

[0130] By using the polynucleotide having promoter activity disclosed herein as an expression regulatory sequence for a target protein in a microorganism, the activity of the target protein can be enhanced.

[0131] For example, the activity or concentration of the protein can be generally increased by at least 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500% to at most 1000% or 2000% compared to the activity or concentration of the wild-type or unmodified microbial strain, but is not limited thereto.

[0132] As used herein, the term "unmodified microorganism" does not exclude strains having mutations that may occur naturally in microorganisms, and may be wild-type strains, microorganisms that do not include the polynucleotide having promoter activity of the present disclosure, or microorganisms that have not been transformed with a vector including the polynucleotide having promoter activity of the present disclosure.

[0133] The microorganism of the present disclosure may be a glutathione-producing microorganism.

[0134] As used herein, the term "glutathione" is used interchangeably with "GSH" and refers to a tripeptide compound composed of the following three amino acids: glutamic acid, cysteine, and glycine. Glutathione can be used as a raw material for pharmaceuticals, health functional foods, flavoring ingredients, food and feed additives, cosmetics, and the like, but is not limited thereto.

[0135] As used herein, the term "glutathione-producing microorganism" includes microorganisms in which natural or artificial genetic modifications have occurred, and may refer to microorganisms in which glutathione production is weakened or enhanced by a specific mechanism through the introduction of exogenous genes, or in which endogenous genes are enhanced or inactivated through genetic modification. For the purposes of the present disclosure, a glutathione-producing microorganism may refer to a microorganism that includes a polynucleotide having promoter activity disclosed herein and is capable of producing a large amount of target glutathione compared to a wild-type or unmodified microorganism.

[0136] The “glutathione-producing microorganism” may be used interchangeably with “glutathione-producing microorganism”, “microorganism having glutathione-producing ability”, “glutathione-producing strain”, “strain having glutathione-producing ability” and the like.

[0137] The microorganism producing glutathione can be a recombinant microorganism. The recombinant microorganism is as described above. The microorganism may include such mutations: enhancing the biosynthetic pathway to increase glutathione production capacity, releasing feedback inhibition, or inactivating genes that weaken the degradation pathway or biosynthetic pathway, and such mutations may be caused by artificial methods such as UV irradiation, and naturally occurring mutations are not excluded.

[0138] Another aspect of the present disclosure provides a method for producing glutathione, the method comprising culturing a microorganism in a culture medium. The microorganism and glutathione are as described above. Glutathione can be accumulated in the microorganism by strain cultivation.

[0139] Regarding the culture medium or other culture conditions for culturing the strains of the present invention, any culture medium commonly used for culturing microorganisms belonging to the genus Saccharomyces can be used without limitation, and specifically, the strains of the present invention can be cultured in a conventional culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin under aerobic or anaerobic conditions while adjusting the temperature, pH, etc.

[0140] In the present disclosure, as a carbon source, carbohydrates such as glucose, fructose, sucrose and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid and citric acid; and amino acids such as glutamic acid, methionine and lysine can be used, but are not limited thereto. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses (blackstrap molasses), rice bran, cassava, sugar cane bagasse (sugar cane bagasse) and corn steep liquor (corn steep liquor) can be used, as well as carbohydrates such as glucose and sterile pretreated molasses (i.e., molasses converted into reducing sugars), and any other carbon source in an appropriate amount can also be used without limitation. These carbon sources can be used alone or in combination of at least two thereof.

[0141] As nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, and defatted soybean meal or its degradation products can be used. These nitrogen sources can be used alone or in combination of at least two thereof.

[0142] As the phosphorus source, potassium dihydrogen phosphate, dipotassium phosphate, or their corresponding sodium salts can be used. As the inorganic compound, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. can be used.

[0143] The culture medium may further include amino acids, vitamins and / or suitable precursors. Specifically, L-amino acids and the like may be added to the culture medium of the strain. Specifically, glycine, glutamic acid and / or cysteine ​​may be added to the culture medium, and if necessary, L-amino acids such as lysine may be further added thereto, but the present disclosure is not limited thereto.

[0144] The culture medium and precursors may be added to the culture in a batch or continuous process, but are not limited thereto.

[0145] In the present disclosure, in the culturing process of bacterial strain, can suitably add compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid in culture, to regulate the pH of culture.In addition, can add defoamer such as fatty acid polyglycol ester, to suppress the foam formation during incubation.In addition, can oxygen or oxygen-containing gas be injected in the culture to keep the culture in aerobic condition, and can be injected in the culture with nitrogen, hydrogen or carbon dioxide gas to keep the culture in anaerobic and microaerobic condition, and not inject any other gas to it.

[0146] The temperature of the culture may be maintained at 25 to 40° C., more specifically 28 to 37° C., but not limited thereto. The culture may be continued until a desired amount of product is obtained, specifically 1 to 100 hours, but not limited thereto.

[0147] The method for producing glutathione may further include an additional process after the culturing step. The additional process may be appropriately selected according to the intended use of glutathione.

[0148] Specifically, the method for producing glutathione may further include recovering glutathione from at least one selected from the group consisting of a cultured microorganism, a dried product of a microorganism, an extract of a microorganism, a culture product of a microorganism, and a lysate of a microorganism after the culturing step.

[0149] The method may further include lysing the microorganism (strain) before or during the recovery step. The lysis of the strain can be performed by any method commonly used in the field of the present disclosure, such as by heat treatment or by using a lysis buffer solution, a sonicator, and a French press. In addition, the lysis step may include an enzymatic reaction performed by a cell wall lysing enzyme, a nuclease, a transnucleotidase, a protease, etc., but is not limited thereto.

[0150] In view of the purpose of the present disclosure, according to the method for producing glutathione, dry yeast, yeast extract and yeast extract mixed powder (all of which have high glutathione content) and pure glutathione can be prepared. However, the present disclosure is not limited thereto, and these products can be appropriately prepared according to the desired product.

[0151] In the present disclosure, dry yeast may be used interchangeably with "dried product of microorganism", "dried product of strain", etc. Dry yeast can be prepared by drying a yeast strain in which glutathione is accumulated, and specifically, it can be included in feed compositions, food compositions, etc., but is not limited thereto.

[0152] In the present disclosure, yeast extract may be used interchangeably with terms such as "microorganism extract" and "strain extract." A strain extract may refer to the material remaining after separating the cell wall from a strain. Specifically, a strain extract may refer to the components remaining after removing the cell wall from components obtained by lysing cells. A strain extract includes glutathione and one or more other components selected from proteins, carbohydrates, nucleic acids, and fibers in addition to glutathione, but is not limited thereto.

[0153] The recovery step can be performed using any suitable method known in the art, and glutathione can be recovered as a target substance.

[0154] The recovery step may include a purification process. The purification process can be performed by isolating only the glutathione from the strain. Through the purification process, pure glutathione can be prepared.

[0155] If necessary, the method for preparing glutathione may further comprise mixing an excipient with one selected from the group consisting of a strain, a dried product, an extract, a culture product, and a lysate thereof, and glutathione recovered therefrom. Through the mixing step, a yeast extract mixed powder may be prepared.

[0156] The excipient can be appropriately selected and used according to the intended use or form, and can be, 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 silica, hydroxypropyl starch, hydroxypropyl methylcellulose, propylene glycol, casein, calcium lactate, primojel and gum arabic, and specifically, it can include at least one component selected from starch, glucose, cellulose, lactose, dextrin, glycogen, D-mannitol and maltodextrin, but is not limited thereto.

[0157] Excipients may include, for example, but are not limited to, preservatives, humectants, dispersing agents, suspending agents, buffers, stabilizers, or isotonic agents.

[0158] Another aspect of the present disclosure provides use of a polynucleotide in which at least one nucleotide selected from nucleotides 92, 94, 102, 103, 249 and 251 of the polynucleotide sequence of SEQ ID NO: 1 or 2 is substituted with a different nucleotide as a promoter.

[0159] The polynucleotide is as described above.

[0160] Example

[0161] Hereinafter, the present disclosure will be described in more detail with reference to the following Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely presented to illustrate the present disclosure, and the scope of the present disclosure is not limited thereto.

[0162] Example 1: Obtaining CJ-5 strain with glutathione production ability

[0163] Strains were obtained from a yeast mass containing various strains, and their characteristics were improved to select strains having glutathione-producing ability.

[0164] Specifically, grain samples such as rice, barley, mung beans, and oats were collected from 20 regions in Gyeonggi-do, Republic of Korea, such as Hwaseong, Pyeongtaek, Yongin, etc., and then crushed, kneaded, wrapped in cloth, compacted into shapes, wrapped with straw to ferment for 10 days, and slowly dried to prepare yeast blocks.

[0165] The following experiment was carried out to separate various strains from the yeast mass prepared. 45mL of saline solution was added to the 5g yeast mass and pulverized using a mixer. In order to simply separate the yeast strains, the resulting mixture was diluted by serial dilution, spread on YPD agar (per 1L of distilled water, 10g / L yeast extract, 20g / L bacto peptone and 20g / L glucose), and cultivated at 30°C for 48 hours. Then, according to colony morphology and microscope verification, yeast colonies were streaked on YPD agar. 25mL of YPD broth was inoculated in a 250mL conical flask, and the strains isolated were inoculated thereon and cultivated at 30°C and 200rpm in a shaking incubator for 48 hours. The strains were screened by identifying glutathione production.

[0166] To enhance the primary isolated strain, random mutations were induced in the isolated strain. Specifically, a strain confirmed to have glutathione production ability was isolated from a yeast mass and named CJ-37. The CJ-37 strain was cultured on solid culture medium and inoculated into broth to obtain a culture solution, which was then exposed to UV light using a UV lamp. After the UV-exposed culture solution was plated onto a culture medium, only the mutant strains that had formed colonies were isolated and their glutathione production was assessed.

[0167] As a result, among the mutant strains, the strain that exhibited the maximum glutathione production was selected as a glutathione-producing strain and named CJ-5 strain, and was deposited in the Korea Center for the Culture of Microorganisms (KCCM) in accordance with the Budapest Treaty on July 31, 2019 and assigned the accession number KCCM12568P.

[0168] Example 2: Experimental development of modified sequences of GSH1 for additional enhancement of CJ-5 strain

[0169] Example 2-1: Inducing mutations and identifying modified sequences

[0170] Mutations were induced in the following manner to further improve the glutathione-producing ability of the CJ-5 strain.

[0171] The CJ-5 strain was cultured in a solid medium and inoculated into broth to obtain a culture solution, and the culture solution was exposed to UV light using a UV lamp. After the culture solution exposed to UV light was spread on a flat plate culture medium, only the mutant strains that had formed colonies were isolated. The strain with the highest glutathione production was isolated and named CC02-2490 strain, and was deposited in the Korea Center for the Culture of Microorganisms (KCCM) in accordance with the Budapest Treaty on January 17, 2020 and assigned accession number KCCM12659P. As a result of analyzing the base sequence of the glutathione biosynthesis gene GSH1, it was confirmed that the 86th amino acid cysteine ​​of the GSH1 protein encoded by the GSH1 gene had been replaced by arginine, with respect to the enhancement of the glutathione production capacity of the strain.

[0172] Example 2-2: Substitution experiment of C86 residue of GSH1 protein

[0173] Based on the results of Example 2-1, considering that amino acid 86 of the GSH1 protein is important in glutathione production, mutant strains of S. cerevisiae CEN.PK2-1D and S. cerevisiae CJ-5 strains were prepared to express protein variants in which cysteine ​​86 of the GSH1 protein was substituted with different amino acids, and an increase in glutathione production was assumed.

[0174] To prepare a strain in which the cysteine ​​at position 86 of the GSH1 protein of Saccharomyces cerevisiae was substituted with arginine, the pWAL100 and pWBR100 plasmids were used with reference to the publication of Lee TH, et al. (J. Microbiol. Biotechnol. (2006), 16(6), 979-982). Specifically, polymerase chain reaction (PCR) was performed as follows using genomic DNA of the CJ-5 strain as a template. By performing PCR using primers of SEQ ID NOs: 34 and 35, the N-terminal partial sequence of the GSH1 protein, including the N-terminal BamHI flanking sequence, the GSH1 ORF start codon, and the C86R mutation coding sequence, was obtained. By performing PCR using primers of SEQ ID NOs: 36 and 37, the C-terminal partial sequence of the GSH1 protein, including the C-terminal XhoI flanking sequence, the GSH1 ORF stop codon, and the C86R mutation coding sequence, was obtained. Subsequently, as a result of overlapping PCR using these two sequences as templates with primers of SEQ ID NOs: 34 and 37, a GSH1 ORF fragment was obtained, which includes a sequence encoding a GSH1 variant in which cysteine ​​at position 86 is substituted with arginine, as well as N-terminal BamHI and C-terminal XhoI restriction enzyme sequences. The ORF fragment was treated with BamHI and XhoI and then cloned into the pWAL100 vector treated with the same enzymes to prepare the pWAL100-GSH1(C86R) vector.

[0175] In addition, 500 bp downstream of the GSH1 ORF stop codon, including the N-terminal SpeI and C-terminal NcoI restriction enzyme sequences, were obtained by PCR using genomic DNA from the CJ-5 strain as a template and primers of SEQ ID NOs: 38 and 39. The fragment was then treated with SpeI and NcoI restriction enzymes. The resulting fragment was then cloned into pWBR100 treated with the same enzymes to create the pWBR100-GSH1 vector.

[0176] Prior to preparing the final DNA fragment to be introduced into yeast, a PCR product containing the sequence encoding the arginine mutation and a portion of KlURA3 was obtained using the pWAL100-GSH1(C86R) vector prepared as described above as a template and primers of SEQ ID NOs: 34 and 40. A PCR product containing a portion of KlURA3 and 500 bp downstream of the GSH1 stop codon was obtained using the pWBR100-GSH1 vector as a template and primers of SEQ ID NOs: 41 and 39. Saccharomyces cerevisiae CEN.PK2-1D and Saccharomyces cerevisiae CJ-5 were transformed with the same molar ratio of the PCR products. PCR was performed by denaturation at 95°C for 5 minutes, annealing at 53°C for 1 minute, and polymerization at 72°C for 1 minute per kilogram. Yeast transformation was performed based on a modified lithium acetate method according to the method disclosed in Geitz (Nucleic Acid Research, 20(6), 1425). Specifically, yeast cells with an OD of 0.7 to 1.2 were washed twice with lithium acetate / TE buffer and mixed with the PCR product and single-stranded DNA (Sigma D-7656). The mixture was cultured in lithium acetate / TE / 40% PEG buffer under static culture conditions at 30°C for 30 minutes and at 42°C for 15 minutes. The cells were then cultured on SC (2% glucose) agar plates excluding uracil until colonies were visible to obtain strains into which the GSH1 C86R mutation coding sequence and the KlURA3 gene had been introduced. Subsequently, to remove KlURA3, the strains were cultured overnight in 2 mL of YPD, diluted at a ratio of 1 / 100, and plated on SC (2% glucose) agar plates containing 0.1% 5-FOA to prepare Saccharomyces cerevisiae CEN.PK2-1D GSH1 C86R mutant strains and Saccharomyces cerevisiae CJ-5 GSH1 C86R mutant strains in which the uracil marker had been removed. Strains capable of expressing GSH1 variants in which cysteine ​​was substituted with 18 amino acids other than arginine were also prepared in the same manner, except that the primer pair of SEQ ID NOs: 35 and 36 was used, in which the sequence encoding arginine at position 86 was replaced with a sequence encoding a different amino acid.

[0177] Table 1

[0178]

[0179]

[0180] After culturing the strain prepared above for 26 h, the concentration of produced glutathione (GSH) was measured and listed in Tables 2 and 3.

[0181] Table 2 Saccharomyces cerevisiae CEN.PK2-1D

[0182]

[0183] Table 3 Saccharomyces cerevisiae CJ-5

[0184]

[0185] As a result of the experiment, it was confirmed that the glutathione-producing ability obtained by substituting cysteine ​​at position 86 of the GSH1 protein with a different amino acid was increased by up to 27% compared with the glutathione-producing ability obtained by the wild-type GSH1 protein.

[0186] Example 2-3: Inducing additional mutations to enhance glutathione production capacity and identifying modified sequences

[0187] Mutations were induced in the following manner to further enhance the glutathione production capacity of the CC02-2490 strain.

[0188] The CC02-2490 strain was cultured on solid culture medium and inoculated into broth to obtain a culture solution, which was then exposed to UV light using a UV lamp. After spreading the UV-exposed culture solution onto a plate, only the mutant strains that had formed colonies were isolated. The sequences of the GS1 coding region and its upstream region were analyzed for the strains with the greatest improvement in glutathione production.

[0189] Results confirmed mutations at positions -250 (C→T), -252 (G→A), -398 (A→T), -399 (A→C), -407 (T→C), and -409 (T→C) upstream of the GSH1 ORF sequence. This strain, designated CC02-2544, was deposited at the Korea Center for the Culture of Microorganisms (KCCM) under the Budapest Treaty on February 20, 2020, under the accession number KCCM12674P.

[0190] Example 2-4: Identification of glutathione production ability based on mutations in the GSH1 promoter

[0191] To compare the glutathione production capacity in the presence or absence of a mutation in the promoter, the glutathione production capacity of the CC02-2490 strain prepared in Example 2-1 and the CC02-2544 strain prepared in Example 2-3 was measured. The glutathione production capacity of the wild-type CEN.PK1-D strain and the CJ-5 strain was measured as a control and is shown in Table 4 below.

[0192] Table 4

[0193]

[0194] As a result of the experiment, it was confirmed that the glutathione production of the CC02-2544 strain was significantly increased by 508% compared to the wild-type CEN.PK1-D strain.

[0195] Additionally, CC02-2544 showed an increase in glutathione production of 128% or more compared to the parent strain CC02-2490.

[0196] As a result, it was confirmed that mutation of the promoter sequence of the GSH1 gene leads to increased glutathione production.

[0197] Example 3: Experiment of introducing mutation into GSH1 promoter of strain

[0198] In order to identify the effect of promoter mutation on strains, the promoter mutation confirmed in Example 2-3 was introduced into the wild-type strain, CJ-5 strain and CC02-2490 strain prepared in Example 2-1, and their glutathione production capacity was evaluated.

[0199] First, sequence alignment of the introduced mutation and identification of the GSH1 ORF upstream sequence (SEQ ID NO: 2) of the CJ-5 strain confirmed that the CJ-5 strain had the same GSH1 promoter sequence as the Saccharomyces cerevisiae strains CEN KSD-Yc, YJM1450, YJM1401, and YJM1307. However, when compared with the GSH1 ORF upstream sequence (SEQ ID NO: 1) of the wild-type strain (Saccharomyces cerevisiae CEN.PK1-D), it was confirmed that adenine was inserted at position -74 of the promoter of the CJ-5 strain.

[0200] Based on this, in the GSH1 ORF upstream sequence of S. cerevisiae CEN.PK1-D, the positions corresponding to positions −250, −252, −398, −399, −407, and −409 of the GSH1 ORF upstream sequence of the CJ-5 strain were determined by sequence alignment to be positions −249, −251, −397, −398, −406, and −408.

[0201] Subsequently, the above six types of mutations were introduced into the GSH1 ORF upstream region of each of the Saccharomyces cerevisiae strains CEN.PK1-D, CJ-5, and CC02-2490.

[0202] Specifically, to prepare strains in which the above-mentioned six types of mutations were introduced into the upstream region of GSH1 ORF, pWAL100 and pWBR100 plasmids were used with reference to the publication of Lee TH, et al. (J. Microbiol. Biotechnol. (2006), 16(6), 979-982).

[0203] After synthesizing a gene comprising the GSH1 ORF upstream region (including the six types of mutations) and a fragment of the GSH1 ORF region, PCR was performed using primers of SEQ ID NOs: 34 and 37 to obtain a fragment of the GSH1 ORF upstream region comprising an N-terminal BamHI restriction enzyme sequence, a C-terminal XhoI restriction enzyme sequence, and the six types of mutations. Subsequently, pWAL100 and this fragment were treated with BamHI and XhoI, and then ligated to prepare a plasmid.

[0204] In addition, 500 bp downstream of the GSH1 ORF stop codon, including the N-terminal SpeI and C-terminal NcoI restriction enzyme sequences, were obtained by PCR using genomic DNA of the CJ-5 strain as a template and primers of SEQ.ID NOs: 38 and 39. The residue was then treated with SpeI and NcoI restriction enzymes. The resulting residue was then ligated into pWBR100 treated with the same enzymes to prepare a plasmid.

[0205] Before preparing the final DNA fragment to be introduced into yeast, a PCR product including the following sequence was obtained using the pWAL plasmid prepared above as a template and primers of SEQ ID NOs: 34 and 40: The sequence includes the upstream region of the GSH1 ORF (which contains six types of mutations), the GSH1 ORF region, and a portion of KlURA3. A PCR product including a portion of KlURA3 and 500 bp downstream of the GSH1 ORF stop codon was obtained using the pWBR plasmid as a template and primers of SEQ ID NOs: 41 and 39. Saccharomyces cerevisiae CEN.PK2-1D, Saccharomyces cerevisiae CJ-5, and CC02-02490 strains were transformed with the same molar ratio of the PCR products. PCR was performed by denaturing at 95°C for 5 minutes, annealing at 53°C for 1 minute, and polymerizing at 72°C for 1 minute per kilogram, and yeast transformation was performed according to a modified lithium acetate method based on the method disclosed in the publication Geitz (Nucleic Acid Research, 20(6), 1425). Specifically, yeast cells with an OD of 0.7 to 1.2 were washed twice with lithium acetate / TE buffer. DNA and single-stranded DNA (Sigma D-7656) were mixed, and the mixture was cultured in a lithium acetate / TE / 40% PEG buffer at 30°C for 30 minutes and at 42°C for 15 minutes under static culture conditions. The cells were then cultured on SC (2% glucose) agar plates not containing uracil until colonies became visible, to obtain strains in which six types of mutations and the K1URA3 gene were introduced into the upstream region of the GSH1 ORF. Subsequently, in order to remove KlURA3, the strain was cultured overnight in 2 mL of YPD, diluted at a ratio of 1 / 100, and plated on an SC (2% glucose) agar plate including 0.1% 5-FOA to prepare a Saccharomyces cerevisiae CEN.PK2-1D-derived mutant strain having six types of mutations in the upstream region of GSH1 ORF, as well as a Saccharomyces cerevisiae CJ-5-derived mutant strain and a CC02-2490-derived mutant strain having six types of mutations in the upstream region of GSH1 ORF.

[0206] The GSH production of the strains is shown in Table 5 below.

[0207] Table 5

[0208]

[0209]

[0210] As a result of the experiment, it was confirmed that glutathione production in the same strain increased up to 141% according to the mutation of the promoter.

[0211] As a result, it was confirmed that mutation of the promoter sequence of the GSH1 gene can lead to increased glutathione production.

[0212] Example 4: Experiment of introducing mutation into GSH1 promoter (1)

[0213] Based on the results confirmed in the above Examples, experiments were conducted to identify whether introduction of mutations only at some positions would enhance glutathione-producing ability.

[0214] Specifically, mutations were introduced into two sites upstream of the GSH1 ORF, -250 and -252 (-250 (C→T) and -252 (G→A)), and four sites upstream of the GSH1 ORF, -398, -399, -407, and -409 (-398 (A→T), -399 (A→C), -407 (T→C), and -409 (T→C)), in the CEN.PK1-D, CJ-5, and CC02-2490 strains, respectively. (In the case of CEN.PK1-D, mutations were introduced into the corresponding positions -249, -251, -397, -398, -406, and -408, which will be described below.) The strain was prepared in the same manner as in Example 3, except that a gene including the GSH1 ORF upstream region (which included mutations at 2 sites or 4 sites) and a fragment of the GSH1 ORF region was synthesized, and then PCR was performed using primers of SEQ ID NOs: 34 and 37 to obtain a fragment of the GSH1 ORF upstream region including an N-terminal BamHI restriction enzyme sequence, a C-terminal XhoI restriction enzyme sequence, and mutations at 2 sites or 4 sites.

[0215] The experimental results are shown in Tables 6 to 8.

[0216] Table 6

[0217]

[0218] Table 7

[0219]

[0220] Table 8

[0221]

[0222] As a result of the experiment, it was confirmed that the glutathione production ability was enhanced by introducing mutations at 2 sites (-250 (C→T) and -252 (G→A)) or 4 sites (-398 (A→T), -399 (A→C), -407 (T→C) and -409 (T→C)) compared to a strain without a mutation in the promoter region, and glutathione production increased by up to 135%.

[0223] Based on this, it was confirmed that the glutathione-producing ability was significantly enhanced by the mutations introduced at only some of the positions of the promoter developed in the present disclosure and at all of the six positions initially confirmed.

[0224] Example 5: Experiment of introducing mutation into GSH1 promoter (2)

[0225] Based on the results confirmed in the above examples, experiments were conducted to identify whether introducing mutations at only some positions would enhance glutathione production capacity. Specifically, mutations were introduced into one site at position -250 (C→T) (mutation (1) at one site) or -252 (G→A) (mutation (2) at one site) in CEN.PK1-D and CJ-5 strains, and into two sites at these two positions simultaneously, and their glutathione production capacities were measured and shown in Tables 9 and 10.

[0226] To this end, a strain was prepared in the same manner as in Example 3, except that a gene including the GSH1 ORF upstream region (which includes mutations at one or two sites) and a fragment of the GSH1 ORF region was synthesized, and then PCR was performed using primers of SEQ ID NOs: 34 and 37 to obtain a fragment of the GSH1 ORF upstream region including an N-terminal BamHI restriction enzyme sequence, a C-terminal XhoI restriction enzyme sequence, and mutations at one or two sites.

[0227] Table 9

[0228]

[0229] Table 10

[0230]

[0231]

[0232] As a result of the experiment, it was confirmed that introduction of mutations into only some sites enhanced glutathione-producing ability.

[0233] Based on this, it was confirmed that glutathione-producing ability was significantly enhanced by introducing mutations in only some of the positions of the promoter developed in the present disclosure as well as in all of the six positions initially confirmed.

[0234] Example 6: Experiment of introducing mutation into GSH1 promoter (3)

[0235] Based on the results confirmed in the above examples, experiments were conducted to identify whether introducing mutations at only some positions would enhance glutathione-producing ability.

[0236] Specifically, mutations were introduced independently or in combination at four sites at positions -398, -399, -407, and -409 (-398 (A→T), -399 (A→C), -407 (T→C), and -409 (T→C)).

[0237] The mutations are as follows.

[0238] 1) Mutation at one site (1): GSH1 -398 (A→T)

[0239] 2) Mutation at one site (2): GSH1 -399 (A→C)

[0240] 3) Mutation at one site (3): GSH1 -407 (T→C)

[0241] 4) Mutation at one site (4): GSH1 -409 (T→C)

[0242] 5) Mutations at two sites (1): GSH1 -398 (A→T) and -399 (A→C)

[0243] 6) Mutations at two sites (2): GSH1 -398 (A→T) and -407 (T→C)

[0244] 7) Mutations at two sites (3): GSH1 -398 (A→T) and -409 (T→C)

[0245] 8) Mutations at two sites (4): GSH1 -399 (A→C) and -407 (T→C)

[0246] 9) Mutations at two sites (5): GSH1 -399 (A→C) and -409 (T→C)

[0247] 10) Mutations at two sites (6): GSH1 -407 (T→C) and -409 (T→C)

[0248] 11) Mutations at four sites: GSH1 -398 (A→T), -399 (A→C), -407 (T→C), and -409 (T→C)

[0249] To this end, strains were prepared in the same manner as in Example 3, except that a gene including the GSH1 ORF upstream region (including mutations at 1 site, 2 sites, or 4 sites) and a fragment of the GSH1 ORF region was synthesized, and PCR was performed using primers of SEQ ID NOs: 34 and 37 to obtain a fragment of the GSH1 ORF upstream region including an N-terminal BamHI restriction enzyme sequence, a C-terminal XhoI restriction enzyme sequence, and mutations at 1 site, 2 sites, or 4 sites.

[0250] The glutathione production capabilities of the strains were identified, and the results are shown in Tables 11 and 12 below.

[0251] Table 11

[0252]

[0253] Table 12

[0254]

[0255] As a result of the experiment, it was confirmed that glutathione production capacity was enhanced by introducing mutations in only some of the four sites and in all four sites, compared to strains without mutations in the promoter region. Specifically, in the following cases: 5) mutations at two sites (1): GSH1 -398 (A→T) and -399 (A→C); and 10) mutations at two sites (6): GSH1 -407 (T→C) and -409 (T→C), glutathione production capacity was significantly enhanced compared to strains without mutations in the promoter region.

[0256] Based on this, it was confirmed that glutathione-producing ability was significantly enhanced by mutations introduced at only some of the positions of the promoter developed in the present disclosure as well as all of the six positions initially confirmed.

[0257] The above description of the present disclosure is provided for illustrative purposes, and those skilled in the art will understand that various changes and modifications can be made without changing the technical concept and basic features of the present disclosure. Therefore, it is clear that the above embodiments are illustrative in all aspects and do not limit the present disclosure. The various embodiments disclosed herein are not intended to be restrictive, and the true scope and spirit are indicated by the appended claims. The present disclosure is limited only by the full scope of the appended claims and all equivalent forms of such claims.

[0258]

[0259]

[0260] <110> CJ CheilJedang Co., Ltd. <120> Novel promoter and method for producing glutathione using the same <130> OPA21012 <150> KR 10-2020-0041184 <151> 2020-04-03 <160> 41 <170> KoPatentIn 3.0 <210> 1 <211> 499 <212> DNA <213> unknown <220> <223> Saccharomyces cerevisiae CEN.PK-1D <400> 1 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctctttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 2 <211> 500 <212> DNA <213> Unknown <220> <223> Saccharomyces cerevisiae CJ-5 <400> 2 ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattgg ccatgctctg 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> 3 <211> 499 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CEN.PK-1D promoter-mutation-92 94 102 103 249 251 <400> 3 ctcttgaatg gcgacagcct attgccccag 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 gccaaattag tcatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctctttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 4 <211> 499 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CEN.PK-1D promoter-mutation-92 94 102 103 <400> 4 ctcttgaatg gcgacagcct attgccccag 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 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 5 <211> 499 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae CEN.PK-1D Promoter - Mutation - 249 251 <400> 5 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattag tcatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 6 <211> 499 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae CEN.PK-1D promoter - mutation - 102 103 <400> 6 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc cctcgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 7 <211> 499 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae CEN.PK-1D promoter - mutation - 92 102 <400> 7 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt cccttctgcc ccacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 8 <211> 499 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CEN.PK-1D promoter - mutation - 92 103 <400> 8 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt cccttctgcc catcgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 9 <211> 499 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae CEN.PK-1D promoter - mutation - 94 102 <400> 9 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcctctgcc ccacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 10 <211> 499 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae CEN.PK-1D Promoter - Mutation - 94 103 <400> 10 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcctctgcc catcgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 11 <211> 499 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CEN.PK-1D promoter - mutation - 92 94 <400> 11 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt cccctctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 12 <211> 499 <212> DNA <213> Artificial sequence <220> <223> S. cerevisiae CEN.PK-1D promoter - mutation - 249 <400> 12 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattag ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 13 <211> 499 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CEN.PK-1D promoter-mutation-251 <400> 13 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattgg tcatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctctttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 14 <211> 499 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CEN.PK-1D promoter-mutation-92 <400> 14 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt cccttctgcc caacgacggc tgccattagt 120 gcaattagcg tatcctgtac catactaatt cccttctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 atacatatag aagaataaa 499 <210> 15<210> 15 <211> 499<211> 499 <212> DNA\<212> DNA <213> 人工序列\<213> Artificial sequence <220>\<220> <223> 酿酒酵母CEN.PK-1D启动子-突变-94\ <223> Saccharomyces cerevisiae CEN.PK-1D promoter - mutation - 94 <400> 15 <400> 15 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcctctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 16 <211> 499 <212> DNA <213> Artificial Sequence <220> <223> S. cerevisiae CEN.PK-1D promoter - mutation - 102 <400> 16 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc ccacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 17 <211> 499 <212> DNA <213> Artificial sequence <220> <223> S. cerevisiae CEN.PK-1D promoter - mutation - 103 <400> 17 ctcttgaatg gcgacagcct attgccccag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc catcgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 gccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcctcttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaattga gcagatttag tatagggcta cattgtaggg tggtttagag tatcgaaaat 480 atacatatag aagaataaa 499 <210> 18 <211> 500 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CJ-5 promoter - mutation - 92 94 102 103 249 251 <400> 18 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> 19 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae CJ-5 Promoter - Mutation - 92 94 102 103 <400> 19 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 tccaaattgg ccatgctctg 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> 20 <211> 500 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CJ-5 promoter - mutation - 249 251 <400> 20 ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc caacgacggc 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> 21 <211> 500 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CJ-5 promoter - mutation - 102 103 <400> 21 ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc cctcgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattgg ccatgctctg 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> 22 <211> 500 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CJ-5 promoter - mutation - 92 102 <400> 22 ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt cccttctgcc ccacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattgg ccatgctctg 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> 23 <211> 500 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CJ-5 promoter-mutation-92 103 <400> twenty three ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt cccttctgcc catcgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattgg ccatgctctg tgcctccctt gacaaaggac atcttccctg tttataaacg 300 gcggcttacc aaaagttgaa gcttgttctt gcttctttatg agtggagcaa tcgattatat 360 tgaatcgttg tgctggagta gttggatctt tccacgtggt ctcgagtcac ttgtagaagc 420 tgaaaaattg agcaggttta gtatagggct acattgtagg gtggtttaga gtatcgaaaa 480 tatacatata gaagaataaa 500 <210> twenty four <211> 500 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CJ-5 promoter-mutation-94 102 <400> twenty four ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcctctgcc ccacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattgg ccatgctctg 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> 25 <211> 500 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CJ-5 promoter - mutation - 94 103 <400> 25 ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcctctgcc catcgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattgg ccatgctctg 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> 26 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae CJ-5 Promoter-Mutation-92 94 <400> 26 ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt cccctctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattgg ccatgctctg 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> 27 <211> 500 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CJ-5 promoter - mutation - 249 <400> 27 ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattag ccatgctctg 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> 28 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae CJ-5 Promoter - Mutation - 251 <400> 28 ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattgg 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[[ID=�]] <210> 29 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae CJ-5 Promoter-Mutation-92 <400> 29 ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt cccttctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattgg ccatgctctg 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> 30 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae CJ-5 Promoter - Mutation - 94 <400> 30 ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcctctgcc caacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattgg ccatgctctg 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> 31 <211> 500 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CJ-5 promoter - mutation - 102 <400> 31 ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc ccacgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180 tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattgg ccatgctctg 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> 32 <211> 500 <212> DNA <213> Artificial sequence <220> <223> Saccharomyces cerevisiae CJ-5 promoter - mutation - 103 <400> 32 ctcttgaatg gcgacagcct attgcctcag tgttccctca acaaccttgg tagttggagc 60 gcaattagcg tatcctgtac catactaatt ctcttctgcc catcgacggc tgccattagt 120 cagcatggcg cgcacgtgac tacaactgtg gctggaaacc ttttcgtcct ccccggtttt 180[[ID=第23行]] tcagtgagcc gactctacta caatgctttt tcatttttca ctcagaaaaa cctgcaattt 240 tccaaattgg ccatgctctg 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> 33 It should be noted that in the above translation, for the 7 - digit tags like , etc., they are preserved exactly as in the original. And for the text content within the tags, it is translated according to the general rules of English translation. If there are any specific requirements or corrections regarding this translation, please feel free to let me know.<211> 678 <212> PRT <213> Saccharomyces cerevisiae <400> 33 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> 34 <211> 33 <212> DNA <213> Artificial sequence <220> <223> F_BamHI_GSH1 <400> 34 ggtaggatcc atgggactct tagctttggg cac 33 <210> 35 <211> 25 <212> DNA <213> Artificial sequence <220> <223> R_GSH1_C86R <400> 35 ttagcctccc taagggacga atcct 25 <210> 36 <211> 25 <212> DNA <213> Artificial sequence <220> <223> F_GSH1_C86R <400> 36 cgtcccttag ggaggctaac gatgt 25 <210> 37 <211> 35 <212> DNA <213> Artificial sequence <220> <223> R_XhoI_GSH1 <400> 37 atgactcgag ttaacatttg ctttctattg aaggc 35 <210> 38 <211> 33 <212> DNA <213> Artificial sequence <220> <223> F_SpeI_GSH1_DW <400> 38 tagaactagt actcctttta tttcggttgt gaa 33 <210> 39 <211> 35 <212> DNA <213> Artificial sequence <220> <223> R_NcoI_GSH1_DW <400> 39 gctgccatgg gaatagtgtg aaccgataac tgtgt 35 <210> 40 <211> 27 <212> DNA <213> Artificial sequence <220> <223> R_AL Killer <400> 40 gagcaatgaa cccaataacg aaatctt 27 <210> 41 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> F_BR killer <400> 41 cttgacgttc gttcgactga tgag 24

Claims

1. A polynucleotide having promoter activity, wherein at least one nucleotide selected from nucleotides 92, 94, 102, 103, 249 and 251 of the polynucleotide sequence of SEQ ID NO: 1 or 2 is substituted by a different nucleotide, wherein the substitution is any one of the following combinations: (i) substitution of nucleotides 92 and 94; (ii) substitution of nucleotides 102 and 103; (iii) substitution of nucleotides 249 and 251; (iv) substitution of nucleotides 92, 94, 102 and 103; and (v) substitution of nucleotides 92, 94, 102, 103, 249 and 251, wherein the substitution of nucleotide 92 is to cytosine (C); The substitution of the 94th nucleotide is substituted with cytosine (C); The substitution of the nucleotide at position 102 is substituted with cytosine (C); The substitution of the nucleotide at position 103 is substituted with thymine (T); The substitution of nucleotide No. 249 is to adenine (A); and The substitution of the nucleotide at position 251 is to thymine (T). 2 . The polynucleotide according to claim 1 , wherein the polynucleotide having promoter activity is represented by a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 3-6, 11, 18-21 and 26. 3 .

3. A composition for expressing a gene, comprising the polynucleotide according to any one of claims 1 to 2.

4. A vector comprising the polynucleotide according to any one of claims 1 to 2 and a gene encoding a target protein. The vector according to claim 4 , wherein the target protein has glutamate-cysteine ​​ligase activity.

6. A microorganism belonging to the genus Saccharomyces sp., comprising the polynucleotide according to any one of claims 1 to 2, a polynucleotide comprising the polynucleotide and a gene encoding a target protein, or a vector comprising the same. The microorganism according to claim 6 , wherein the target protein is a polypeptide having glutamate-cysteine ​​ligase activity.

8. A method for producing glutathione, comprising culturing the microorganism according to claim 6 in a culture medium.

9. The method according to claim 8, further comprising recovering glutathione from at least one selected from the group consisting of: the cultured microorganism, a dried product of the microorganism, an extract of the microorganism, a culture product of the microorganism, and a lysate of the microorganism.

10. Use of a polynucleotide sequence as a promoter, wherein at least one nucleotide selected from nucleotides 92, 94, 102, 103, 249, and 251 of the polynucleotide sequence of SEQ ID NO: 1 or 2 is substituted by a different nucleotide, wherein the substitution is any one of the following combinations: (i) substitution of nucleotides 92 and 94; (ii) substitution of nucleotides 102 and 103; (iii) substitution of nucleotides 249 and 251; (iv) substitution of nucleotides 92, 94, 102 and 103; and (v) substitution of nucleotides 92, 94, 102, 103, 249 and 251, wherein the substitution of nucleotide 92 is to cytosine (C); The substitution of the 94th nucleotide is substituted with cytosine (C); The substitution of the nucleotide at position 102 is substituted with cytosine (C); The substitution of the nucleotide at position 103 is substituted with thymine (T); The substitution of nucleotide No. 249 is to adenine (A); and The substitution of the nucleotide at position 251 is to thymine (T).

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