Glutamate-cysteine ligase variants and methods of producing glutathione using the same

Abstract

The present invention relates to novel glutamate-cysteine ligase variants and methods of producing glutathione using the same.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a novel glutamate-cysteine ligase variant and a method for producing glutathione using the same. BACKGROUND

[0002] As an organosulfur compound that is ubiquitous in most cells, glutathione (GSH) is a tripeptide composed of three amino acids: glycine, glutamic acid, and cysteine.

[0003] Glutathione exists in vivo as reduced glutathione (GSH) and oxidized glutathione (GSSG). Reduced glutathione (GSH) is relatively high under normal conditions, is mainly distributed in liver and skin cells in the human body, and has the following important roles: antioxidant function of decomposing and removing active oxygen, detoxification function of removing xenobiotics such as toxic substances, and whitening function of inhibiting melanin production.

[0004] Since the production of glutathione gradually decreases as the aging process proceeds, and the decrease in the production of glutathione, which plays an important role in antioxidant and detoxification functions, promotes the accumulation of active oxygen, which is a major cause of aging, it is necessary to externally supply glutathione (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).

[0005] Glutathione having the above-described various functions is attracting attention as a substance in various fields such as drugs, health functional foods, and cosmetics, and is also used in the manufacture of seasoning ingredients as well as food and feed additives. Glutathione is known to have a great effect on the taste of rich raw materials and maintaining a rich flavor, and can be used as a kokumi taste enhancer alone or in combination with other substances. In general, kokumi substances are known to have a richer flavor than, for example, umami substances such as known nucleic acids and monosodium glutamate (MSG), and are produced through protein decomposition during maturation.

[0006] Although the demand for the use of glutathione in various fields is increasing, the market for glutathione has not been activated due to the high cost of industrial production of glutathione because the method of synthesizing enzymes thereof has not been commercialized due to high production costs, and the yield of the method of culturing microorganisms and extracting glutathione therefrom is low. SUMMARY

[0007] TECHNICAL PROBLEM

[0008] As a result of a great deal of efforts to solve the above problems, the present inventors developed a new glutamate-cysteine ligase variant and found that glutathione production capacity of a strain into which the new glutamate-cysteine ligase variant was introduced was significantly improved, thereby completing the present disclosure.

[0009] Technical Solution

[0010] The present disclosure provides a glutamate-cysteine ligase variant in which an amino acid corresponding to the 86th amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with a different amino acid.

[0011] The present disclosure provides a polynucleotide encoding the variant and a vector comprising the same.

[0012] The present disclosure provides a microorganism producing glutathione by including at least one of the following: the variant; the polynucleotide encoding the variant; and the vector comprising the polynucleotide.

[0013] The present disclosure provides a method of producing glutathione, which includes culturing a microorganism.

[0014] Advantageous Effects

[0015] The new glutamate-cysteine ligase variant of the present disclosure significantly increases glutathione production, and thus can be used to produce glutathione at a high yield. Since the yeast producing glutathione at a high yield, its dry product, extract, culture, and lysate, and the produced glutathione have antioxidant, detoxification, and immunity-enhancing effects, they can be effectively used in cosmetic compositions, food compositions, feed compositions, and pharmaceutical compositions, and the preparation thereof. 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 different descriptions and embodiments herein. In other words, all combinations of various components disclosed in the present disclosure are included in 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 of ordinary skill in the art will recognize or be able to ascertain only using routine experimentation many equivalents to the specific embodiments of the present disclosure. Such equivalents are intended to be included in the scope of the appended claims.

[0018] One aspect of the present disclosure provides a glutamate-cysteine ligase variant in which an amino acid corresponding to the 86th amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with a different amino acid.

[0019] The variant can be a glutamate-cysteine ligase variant including at least one amino acid substitution in the amino acid sequence of SEQ ID NO: 1, wherein the substitution includes substitution of the amino acid corresponding to position 86 from the N-terminus of SEQ ID NO: 1 with a different amino acid.

[0020] In detail, the variant can be a protein variant in which the amino acid at position 86 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with a different amino acid.

[0021] The "glutamate-cysteine ligase (GCL)" of the present disclosure is an enzyme also called "glutamate-cysteine ligase" or "gamma-glutamyl cysteine synthetase (GCS)". It is known that glutamate-cysteine ligase catalyzes the following reaction:

[0022]

[0023] In addition, the reaction catalyzed by glutamate-cysteine ligase is called the first step of glutathione synthesis.

[0024] The glutamate-cysteine ligase as a yeast-derived sequence can be a protein including the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0025] In the present disclosure, the amino acid sequence of SEQ ID NO: 1 is an amino acid sequence encoded by a gshl gene, and can also be called a "GSH1 protein" or "glutamate-cysteine ligase". The amino acid sequence constituting the glutamate-cysteine ligase of the present disclosure can be obtained from a known database of NCBI GenBank. For example, the amino acid sequence can be derived from Saccharomyces cerevisiae, but is not limited thereto, and can include any sequence having the same activity as the amino acid sequence without limitation.

[0026] In addition, although the glutamate-cysteine ligase is defined in the present disclosure as a protein including the amino acid sequence of SEQ ID NO: 1, it does not exclude mutations that can occur naturally or by adding a meaningless sequence upstream or downstream of the amino acid sequence of SEQ ID NO: 1, or naturally occurring mutations or silent mutations thereof, and it is obvious to a person of ordinary skill in the art that any protein having the same or equivalent activity as the protein including the amino acid sequence of SEQ ID NO: 1 belongs to the glutamate-cysteine ligase of the present disclosure.

[0027] For example, the glutamate-cysteine ligase of the present disclosure can be a protein including the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity thereto. Also, it is obvious that any protein having an amino acid sequence including deletion, modification, substitution, or addition of one or several amino acids is within the scope of the present disclosure, as long as the amino acid sequence retains the above homology or identity and an equivalent action to the action of the protein.

[0028] In other words, although the expressions "protein or polypeptide having an amino acid sequence of a predetermined SEQ ID NO:" and "protein or polypeptide including an amino acid sequence of a predetermined SEQ ID NO:" are used in the present disclosure, it is obvious that any protein having an amino acid sequence including deletion, modification, substitution, or addition of one or several amino acids can be used in the present disclosure, as long as the protein has the same or equivalent activity to the polypeptide consisting of the predetermined amino acid sequence. For example, it is obvious that "polypeptide including the amino acid sequence of SEQ ID NO: 1" belongs to "polypeptide consisting of the amino acid sequence of SEQ ID NO: 1", as long as the former has the same or equivalent activity to the latter.

[0029] As used herein, the term "variant" refers 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 property of the protein, and can be a glutamate-cysteine ligase variant in which the amino acid corresponding to the 86th amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid residue other than cysteine, for the purpose of the present disclosure. The variant is different from the sequence identified by substitution, deletion, or addition of several amino acids. Such variants can generally be identified by modifying one of the above-described amino acid sequences of the protein and 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 can include at least one portion, such as an N-terminal leader sequence or a transmembrane domain, which has been removed therefrom. Other variants can include a variant in which a portion has been removed from the N and / or C terminus of the mature protein. The term "variant" can also be used interchangeably with other terms such as modification, modified protein, modified polypeptide, mutant, mutagen, and divergent, and any term used to indicate change can also be used without limitation. For the purpose of the present disclosure, the variant can have enhanced activity compared to the wild type or unmodified protein, but is not limited thereto.

[0030] As used herein, the term "conservative substitution" refers to the substitution of one amino acid with a different amino acid having similar structural and / or chemical properties. The variant can have at least one conservative substitution while retaining at least one biological activity. Such amino acid substitution can typically occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues.

[0031] The variant can also include deletion or addition of an amino acid having minimal impact on the properties and secondary structure of the polypeptide. For example, the polypeptide can be conjugated at the N-terminus of the protein to a signal (or leader) sequence, which co-translationally or post-translationally directs the transfer of the protein. The polypeptide can also be conjugated to another sequence or linker to identify, purify, or synthesize the polypeptide.

[0032] In the present disclosure, "substitution with a different amino acid" is not specifically limited as long as the substituted amino acid is different from the amino acid before substitution. That is, substitution of the cysteine at the 86th amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with a different amino acid can also be expressed as "substitution of the amino acid at the 86th position with an amino acid other than cysteine". Meanwhile, in the present disclosure, it is apparent that unless the expression "substitution with a different amino acid" is given, the expression "substitution with a predetermined amino acid" means that the substituted amino acid is different from the amino acid before substitution.

[0033] The "glutamate-cysteine ligase variant" of the present disclosure can also be referred to as a "(variant) 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.

[0034] In the variant, at least one amino acid of the amino acid sequence of SEQ ID NO: 1 can be substituted with a different amino acid. Specifically, the variant can include substitution of the amino acid corresponding to the 86th position of the amino acid sequence of SEQ ID NO: 1 with an amino acid other than cysteine. The different amino acid can 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.

[0035] In the present disclosure, it is apparent that the "variant in which the 86th amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with a different amino acid" includes a variant in which the amino acid corresponding to the 86th position of the amino acid sequence of SEQ ID NO: 1 is substituted with a different amino acid, although the amino acid is located at a position other than the 86th position due to deletion / addition / insertion, etc. of amino acids at the N or C-terminus or in the middle of the amino acid sequence of SEQ ID NO: 1.

[0036] Further, although the variant in which the amino acid at position 86 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with a different amino acid is disclosed as an example of the glutamate-cysteine ligase variant of the present disclosure, the glutamate-cysteine ligase variant of the present disclosure is not limited to the variant of the amino acid sequence of SEQ ID NO: 1, and it is obvious that the variant in which the "amino acid corresponding to position 86 of the amino acid sequence of SEQ ID NO: 1" is substituted with a different amino acid in any amino acid sequence having glutamate-cysteine ligase activity is also within the scope of the glutamate-cysteine ligase variant of the present disclosure.

[0037] In any amino acid sequence, the "amino acid corresponding to position 86 of the amino acid sequence of SEQ ID NO: 1" can be identified by various sequence alignment methods well known in the art.

[0038] 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 of SEQ ID NO: 1 is substituted with a different amino acid can be a protein including the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity thereto, in which the amino acid corresponding to position 86 of SEQ ID NO: 1 is substituted with a different amino acid.

[0039] 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 of SEQ ID NO: 1 is substituted with an amino acid other than cysteine can include one of the amino acid sequences of SEQ ID NOs: 3 to 21. Specifically, the variant can consist essentially of one of the amino acid sequences of SEQ ID NOs: 3 to 21, more specifically, it can consist of one of the amino acid sequences of SEQ ID NOs: 3 to 21, but is not limited thereto.

[0040] Further, the variant can include one of the amino acid sequences of SEQ ID NOs: 3 to 21 or include the fixed 86th amino acid (i.e., the amino acid corresponding to position 86 of SEQ ID NOs: 3 to 21 is the same as the amino acid at position 86 of SEQ ID NOs: 3 to 21 in the amino acid sequence of the variant), and have an amino acid sequence having at least 80% homology or identity thereto, but is not limited thereto.

[0041] In particular, the variants of the present disclosure can include a polypeptide having one of the amino acid sequences of SEQ ID NOs: 3 to 21 and a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to one of the amino acid sequences of SEQ ID NOs: 3 to 21. Furthermore, it is obvious that any protein having an amino acid sequence including deletion, modification, substitution, or addition of one or several amino acids at positions other than the 86th position is within the scope of the present disclosure, as long as the protein maintains the above-mentioned homology or identity and the equivalent action to the variant.

[0042] As used herein, the term "homology" or "identity" refers to the degree of relatedness between two given amino acid sequences or base sequences and can be expressed as a percentage. The terms homology and identity can be used interchangeably in general.

[0043] The sequence homology or identity of a conservative polynucleotide or polypeptide can be determined by a standard alignment algorithm and can be used with the default gap penalty established by the program. Basically, under medium or high stringency conditions, homologous or identical sequences can hybridize to each other at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or entire length. In the hybridized polynucleotides, polynucleotides including degenerate codons instead of codons can also be considered.

[0044] Sequence homology, similarity or identity between two given polypeptides or polynucleotides can be determined using any known computer algorithm, for example by using the "FASTA" program with default parameters as described in, 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) as implemented in the European Molecular Biology Open Software Suite (EMBOSS) package (Rice et al., 2000, Trends Genet. 16:276-277) version 5.0.0 or later can be used to perform the determination (including the GCG program package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994; and CARILLO et al. (1988) SIAM J Applied Math 48:1073). For example, homology, similarity or identity can be determined using BLAST from the National Center for Biotechnology Information database or ClustalW.

[0045] Homology, similarity or identity between polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, for example, the program described by Needleman et al. (1970), J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, 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. Default parameters for the GAP program can include: (1) a binary comparison matrix (containing a value of 1 for identical matches, and 0 for non-identical matches) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as 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 instead); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0046] Further, sequence homology, similarity or identity between two given polynucleotides or polypeptides can be identified by comparing their sequences by Southern hybridization under defined stringent conditions, and defined stringent hybridization conditions are within the purview of the present technology and can be defined by methods well known to those of ordinary skill in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). Another aspect of the present disclosure provides polynucleotides encoding variants.

[0047] As used herein, the term "polynucleotide" refers to a polymer of nucleotides, in which the nucleotide monomers are connected to each other in a long chain form by a covalent bond, and generally refers to a DNA or RNA chain having a certain minimum length, more specifically a polynucleotide fragment encoding a variant.

[0048] The polynucleotide encoding a variant of the protein according to the present disclosure can include any nucleotide sequence encoding a glutamate-cysteine ligase variant having enhanced activity, but is not limited thereto.

[0049] The gene encoding the glutamate-cysteine ligase of the present disclosure can be a gshl gene. The gene can be derived from a yeast. Specifically, the gene can be derived from a microorganism belonging to the genus Saccharomyces, more specifically, from Saccharomyces cerevisiae. Specifically, the gene can be a gene encoding the amino acid sequence of SEQ ID NO: 1, more specifically, a sequence including the base sequence of SEQ ID NO: 2, but is not limited thereto.

[0050] The polynucleotide of the present disclosure can include various modifications made in the coding region, which do not provide for a change in the amino acid sequence of the polypeptide expressed from the coding region by codon degeneracy or in consideration of the preferred codon of a living organism in which the polypeptide is expressed. Specifically, any polynucleotide sequence encoding a variant of the protein in which the amino acid at the position corresponding to position 86 of SEQ ID NO: 1 is substituted with a different amino acid can be included without limitation. For example, the polynucleotide of the present disclosure can have a polynucleotide sequence encoding a variant of the protein of the present disclosure, specifically a protein comprising one of the amino acid sequences of SEQ ID NOs: 3 to 21 or a polypeptide having homology or identity thereto, without limitation. The homology or identity is as described above.

[0051] Further, the polynucleotide can include a nucleotide sequence that hybridizes under stringent conditions to a probe constructed using a known gene sequence, for example, a nucleotide sequence completely or partially complementary to the nucleotide sequence, to encode a variant of the protein in which the amino acid at position 86 of the amino acid sequence of SEQ ID NO: 1 is substituted with a different amino acid, without limitation.

[0052] The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. These conditions are disclosed in detail in known literature (for example, J. Sambrook et al.). For example, the conditions can include hybridization between genes having high homology, for example, homology of 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more, most specifically 99% or more, and no hybridization between genes having homology lower than the above homology, or hybridization once, specifically two or three times, under the conventional washing conditions of Southern hybridization of 60°C, 1xSSC, and 0.1% SDS, specifically 60°C, 0.1xSSC, 0.1% SDS, more specifically 68°C, 0.1xSSC, and 0.1% SDS. However, the stringent conditions are not limited thereto, and can be appropriately adjusted by those skilled in the art according to the intended purpose.

[0053] Hybridization requires that two polynucleotides have complementary sequences, but there can be base mismatches depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, for DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the present disclosure can include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments complementary to the entire sequence.

[0054] Specifically, polynucleotides having homology or identity can be detected using the above-mentioned hybridization conditions, including hybridization at a T m value of 55°C. In addition, the T m value can be, but is not limited to, 60°C, 63°C, or 65°C, and can be appropriately adjusted by those skilled in the art according to the intended purpose.

[0055] The appropriate degree of stringency of polynucleotide hybridization can depend on the length of the polynucleotide, and the degree of complementarity and its parameters are well known in the art.

[0056] Another aspect of the present disclosure provides a vector including a polynucleotide encoding a protein variant.

[0057] As used herein, the term "vector" refers to a DNA construct including a base sequence of a polynucleotide encoding a target protein, which is operably linked to a suitable regulatory sequence to express the target protein in a suitable host cell. The regulatory sequence can include a promoter allowing initiation of transcription, an operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating transcription and translation termination. After the vector is introduced into a suitable host cell, it can replicate or function independently of the host genome, and can be integrated into the genome itself.

[0058] 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 yeast expression, integrated yeast plasmid (YIp) and extrachromosomal plasmid vectors can be used. The extrachromosomal plasmid vector can include episomal yeast plasmid (YEp), replicative yeast plasmid (YRp), and yeast centromeric plasmid (YCp). In addition, artificial yeast chromosome (YAC) can also be used as a vector of the present disclosure. As specific examples, usable vectors can include pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZA, pGAPZB, pGAPZC, pGAPaA, pGAPaB, pGAPaC, pPIC3.5K, pPIC6A, pPIC6B, pPIC6C, pPIC6aA, pPIC6aB, pPIC6aC, pPIC9K, pYC2 / CT, pYD1 yeast display vector, pYES2, pYES2 / CT, pYES2 / NTA, pYES2 / NTB, pYES2 / NTC, pYES2 / CT, pYES2.1, pYES-DEST52, pTEF1 / Zeo, pFLD1, PichiaPinkTM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, pBY011, pSGP47, pSGP46, pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, 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.

[0059] For example, the polynucleotide encoding the target protein in the chromosome can be replaced with the mutated polynucleotide using a vector for chromosome insertion into the cell. The polynucleotide can be inserted into the chromosome by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker can be further included to confirm the chromosome insertion. The selection marker is used to select the cell transformed with the vector, that is, to confirm the insertion of the desired polynucleotide, and examples of the selection marker can include a marker providing a selectable phenotype, for example, drug resistance, nutritional requirement, resistance to a cytotoxic agent, or surface expression of a variant polypeptide. Only the cell expressing the selection marker can survive in an environment treated with a selection agent or show a different phenotype, and thus the transformed cell can be selected.

[0060] As used herein, the term "transformation" refers to a process of introducing a vector including a polynucleotide encoding a glutamate-cysteine ligase variant into a host cell so that a protein encoded by the polynucleotide is expressed in the host. The transformed polynucleotide can be in a form inserted into a chromosome of the host cell, or can be in a form located outside the chromosome, as long as the protein is expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the glutamate-cysteine ligase variant. The polynucleotide can be introduced into the host cell in any form, as long as the polynucleotide is introduced into the host cell and the protein is expressed therein. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic construct including all essential elements required for self-replication. The expression cassette can typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are operatively linked to the polynucleotide. The expression cassette can be in the form of a self-replicable expression vector. In addition, the polynucleotide can be introduced into the host cell in its original form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

[0061] In addition, as used herein, the term "operably linked" refers to a functional linkage between a polynucleotide sequence encoding a polypeptide of the present disclosure and a promoter sequence that initiates and mediates transcription of the polynucleotide sequence. The transformation method according to the present disclosure includes any method capable of introducing a vector into a host cell, and can be performed by a suitable standard technique known in the art according to the selection of 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 used, but the present disclosure is not limited thereto.

[0062] The present disclosure can provide a microorganism producing glutathione by including at least one of: a variant; a polynucleotide encoding the variant; and a vector including the polynucleotide.

[0063] The microorganism can be a microorganism expressing a variant or a microorganism into which a variant is introduced.

[0064] As used herein, the term "microorganism including a variant", "microorganism into which a variant is introduced", or "microorganism expressing a variant" can refer to a microorganism prepared by enhancing the ability of a microorganism naturally having low glutathione production ability to produce glutathione or by providing a parent strain incapable of producing glutathione with the ability to produce glutathione. Specifically, the microorganism can be a microorganism expressing a glutamate-cysteine ligase variant including at least one amino acid mutation in the amino acid sequence of SEQ ID NO: 1, and the amino acid mutation can include substitution of the amino acid corresponding to position 86 from the N-terminus with a different amino acid. In addition, the microorganism can be a microorganism expressing a glutamate-cysteine ligase variant in which the amino acid corresponding to position 86 of the amino acid sequence of SEQ ID NO: 1 is substituted with a different amino acid, but is not limited thereto.

[0065] The glutamate-cysteine ligase and variants thereof are as described above.

[0066] As used herein, "to be expressed / expressed" of a protein refers to a state in which a target protein is introduced into a microorganism or expressed in a microorganism. In the case where the protein exists in a microorganism, the activity of the protein is enhanced compared to the activity of its endogenous protein or the activity before modification. For the purpose of the present disclosure, the "target protein" can be the above-described glutamate-cysteine ligase variant.

[0067] Specifically, the term "introducing a protein" refers to providing a microorganism not having a specific protein with the activity of the protein or enhancing the activity of the protein compared to the inherent activity or the activity before modification of the protein. For example, introducing a protein can refer to introducing a polynucleotide encoding a specific protein into a chromosome or introducing a fragment or a vector including a polynucleotide encoding a specific protein into a microorganism, thereby expressing the activity of the protein. In addition, "activity enhancement" can refer to enhancement of the activity of a specific protein of a microorganism when compared to the inherent activity or the activity before modification. The term "inherent activity" refers to the activity of a specific protein that a parent strain has before transformation when the microorganism is transformed by natural or artificial genetic variation.

[0068] In detail, the activity enhancement according to the present disclosure can be performed by at least one of the methods including increasing the copy number of a gene encoding a protein variant, introducing a mutation into an expression regulatory sequence of a gene encoding a protein variant, replacing an expression regulatory sequence of a gene encoding a protein variant with a sequence having stronger activity, replacing a chromosomal gene encoding a wild-type protein with a gene encoding a protein variant, additionally introducing a mutation into a gene encoding a protein variant to enhance the activity of the protein variant, and introducing a protein variant into a microorganism, but is not limited thereto.

[0069] Although not specifically limited thereto, the increase in the copy number of a gene is performed in the form of a vector operably linked thereto or in the form of being integrated into a chromosome of a host cell. In detail, the method can be performed by introducing a vector into a host cell, which replicates and functions independently of the host and is operably linked to a polynucleotide encoding a protein of the present disclosure. Alternatively, the copy number of a gene can be increased by introducing a vector into a host cell, which inserts a polynucleotide into a chromosome of the host cell and is operably linked to the polynucleotide. The insertion of the polynucleotide into the chromosome can be performed by any method known in the art, for example, homologous recombination.

[0070] Next, the modification of the expression regulatory sequence can be performed to increase the expression of the polynucleotide by inducing a variation in the nucleotide sequence by deletion, insertion, non-conservative substitution, conservative substitution, or any combination thereof to further enhance the activity of the expression regulatory sequence, or by replacing the nucleotide sequence with a nucleotide sequence having stronger activity, but is not limited thereto. The expression regulatory sequence can include a promoter, an operator sequence, a ribosome binding site encoding sequence, and a sequence for regulating transcription and translation, but is not limited thereto.

[0071] A promoter stronger than the intrinsic promoter can be linked to the upstream of the polynucleotide expression unit, but is not limited thereto. For example, when the host cell is yeast, the available promoters can include a TEF1 promoter, a TEF2 promoter, a GAL10 promoter, a GAL1 promoter, an ADH1 promoter, an ADH2 promoter, a PHO5 promoter, a GAL1-10 promoter, a TDH3 promoter (GPD promoter), a TDH2 promoter, a TDH1 promoter, a PGK1 promoter, a PYK2 promoter, an ENO1 promoter, an ENO2 promoter, and a TPI1 promoter, but is not limited thereto. In addition, the modification of the polynucleotide sequence on the chromosome can be performed by inducing a variation in the expression regulatory sequence by deletion, insertion, non-conservative substitution, conservative substitution, or any combination thereof to further enhance the activity of the polynucleotide sequence, or by replacing the nucleotide sequence with a nucleotide sequence modified to have stronger activity, but is not limited thereto.

[0072] Generally, introduction and enhancement of protein activity can increase the activity or concentration of the corresponding protein by 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, or 500%, up to 1000% or 2000% compared to the activity or concentration of the wild type or unmodified microbial strain, but is not limited thereto.

[0073] As used herein, the term "unmodified microorganism" does not exclude a strain having mutations that can occur naturally in the microorganism, and can be a wild type strain, a microorganism not including a protein variant, or a microorganism not transformed with a vector including a polynucleotide encoding a protein variant.

[0074] In the present disclosure, the microorganism including the glutamate-cysteine ligase variant or the polynucleotide encoding the same can be a recombinant microorganism, for example, prepared by transforming the microorganism with a vector including the polynucleotide, but is not limited thereto. The recombinant microorganism can be a yeast, for example, a microorganism belonging to the genus Saccharomyces, specifically, Saccharomyces cerevisiae. For example, the microorganism can be a Saccharomyces cerevisiae strain having accession number KCCM12659P, but is not limited thereto.

[0075] As used herein, the term "glutathione" can be used interchangeably with "GSH" and refers to a tripeptide compound consisting of three amino acids: glutamic acid, cysteine, and glycine. Glutathione can be used as a raw material for drugs, health functional foods, seasoning ingredients, food and feed additives, cosmetics, etc., but is not limited thereto.

[0076] As used herein, the term "glutathione-producing microorganism" includes a microorganism modified by natural or artificial genetic modification, and can refer to a microorganism having a specific mechanism that is attenuated or enhanced via introduction of an exogenous gene or inactivation or enhancement of an endogenous gene by genetic modification in order to produce glutathione. For the purpose of the present disclosure, the glutathione-producing microorganism can refer to a microorganism including glutamate-cysteine ligase and capable of producing a large amount of target glutathione compared to a wild type or unmodified microorganism. The "glutathione-producing microorganism" can be used interchangeably with "microorganism having the ability to produce glutathione", "microorganism having the ability to produce glutathione", "glutathione-producing strain", "strain having the ability to produce glutathione", etc.

[0077] The glutathione-producing microorganism can be a recombinant microorganism. The recombinant microorganism is as described above.

[0078] The type of the glutathione-producing microorganism is not particularly limited as long as glutathione is produced therefrom, but can be a microorganism belonging to the genus Saccharomyces, specifically, Saccharomyces cerevisiae, but is not limited thereto.

[0079] The parent strain of the glutathione-producing microorganism including the variant includes no particular limitation as long as the strain has the ability to produce glutathione. The microorganism can include modifications for enhancing the biosynthetic pathway to increase glutathione production ability, releasing feedback inhibition, and inactivating genes that attenuate the degradation pathway or the biosynthetic pathway, and such modifications do not exclude modifications that occur naturally. However, the present disclosure is not limited thereto.

[0080] Another aspect of the present disclosure provides a method of producing glutathione, the method including culturing a microorganism. The microorganism and the glutathione are as described above. The glutathione can be accumulated in the strain by culturing the strain.

[0081] As for the culture medium or other culture conditions for culturing the strain of the present disclosure, any medium commonly used for culturing microorganisms belonging to the genus Saccharomyces can be used without limitation. Specifically, the strain of the present disclosure can be cultured in a general 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, and the like.

[0082] In the present disclosure, as the 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, rice bran, cassava, sugar cane bagasse, and corn steep liquor can be used, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugars) can be used, 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.

[0083] As the nitrogen source, 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 hydrolysate, fish or its degradation product, and defatted soybean meal or its degradation product can be used. These nitrogen sources can be used alone or in combination of at least two thereof.

[0084] As the phosphorus source, potassium dihydrogen phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto can be used. As the inorganic compound, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, and the like can be used.

[0085] The culture medium can further include amino acids, vitamins, and / or suitable precursors. Specifically, L-amino acids or the like can be added to the culture medium of the strain. Specifically, glycine, glutamic acid, and / or cysteine can be added to the culture medium, and if necessary, L-amino acids such as lysine can be further added thereto, but the present disclosure is not limited thereto.

[0086] The culture medium and precursors can be added to the culture in a batch or continuous process, but are not limited thereto.

[0087] In the present disclosure, during the culture process of the strain, a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be appropriately added to the culture to adjust the pH of the culture. In addition, to inhibit the formation of foam during the culture, a defoaming agent such as a fatty acid polyethylene glycol ester can also be added. Furthermore, oxygen or an oxygen-containing gas can be injected into the culture to maintain the culture under aerobic conditions, and nitrogen, hydrogen, or carbon dioxide gas can be injected into the culture to maintain the culture under anaerobic and microaerophilic conditions, without injecting any other gas for this purpose.

[0088] The temperature of the culture can be maintained at 25 to 40°C, more specifically at 28 to 37°C, but is not limited thereto. The culture can continue until a desired amount of a desired substance is obtained, specifically for 1 to 100 hours, but is not limited thereto.

[0089] The method of producing glutathione can further include an additional process after the culturing step. The additional process can be appropriately selected depending on the purpose of use of the glutathione.

[0090] Specifically, the method of producing glutathione can include recovering glutathione from at least one selected from the group consisting of a strain, a dried product, an extract, a culture product, and a lysate thereof after culturing the microorganism.

[0091] The method can further include lysing the strain before or simultaneously with the recovering step. The lysis of the strain can be performed by any method commonly used in the field to which the present disclosure pertains, for example, by heat treatment or by using a buffer solution for lysis, an ultrasonic device, and a French press. Also, the lysis step can include an enzymatic reaction by a cell wall lyase, a nuclease, a transnuclease, a protease, or the like, but is not limited thereto.

[0092] In view of the purpose of the present disclosure, dried yeast, yeast extract, yeast extract mixed powder, and pure glutathione each having a high glutathione content can be prepared by the method of producing glutathione. However, the present disclosure is not limited thereto, and these products can be appropriately prepared depending on the desired product.

[0093] In the present disclosure, dry yeast can be used interchangeably with "dried product of a strain". Dry yeast can be prepared by drying a yeast strain in which glutathione is accumulated, and in particular, it can be included in a feed composition, a food composition, etc., but is not limited thereto.

[0094] In the present disclosure, yeast extract can be used interchangeably with, for example, the term "extract of a strain". The extract of a strain can refer to a substance remaining after the cell wall is separated from the strain. In particular, the extract of a strain can refer to a remaining component other than the cell wall among components obtained by lysing cells. The extract of a strain includes glutathione and one or more other components selected from proteins, carbohydrates, nucleic acids, and fibers other than glutathione, but is not limited thereto.

[0095] The recovery step can be performed using any suitable method known in the art, and glutathione as a target substance can be recovered.

[0096] The recovery step can include a purification process. The purification process can be performed by isolating only glutathione from the strain. Through the purification process, pure glutathione can be prepared.

[0097] If necessary, the method of preparing glutathione can further include mixing an excipient with one selected from a strain, a dried product thereof, an extract, a culture, and a lysate thereof, and glutathione recovered therefrom. Through the mixing step, a yeast extract mixed powder can be prepared.

[0098] The excipient can be appropriately selected and used according to a desired 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, monobasic calcium phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, dextrin, sodium alginate, methyl cellulose, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methyl cellulose, propylene glycol, casein, calcium lactate, sodium carboxymethyl starch (primojel), and gum arabic, and in particular, it can include at least one component selected from starch, glucose, cellulose, lactose, dextrin, glycogen, D-mannitol, and maltodextrin, but is not limited thereto.

[0099] The excipient can include, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffering agent, a stabilizer, or an isotonic agent, but is not limited thereto.

[0100] Another aspect of the present disclosure provides use of a glutamate-cysteine ligase variant for the production of glutathione. Another aspect of the present disclosure provides use of a microorganism for the production of glutathione. The glutamate-cysteine ligase variant, the microorganism, and the glutathione are as described above.

[0101] Invention mode

[0102] Hereinafter, the present disclosure will be described in greater detail with reference to the following examples and experimental examples. However, the following examples and experimental examples are for illustration only, and the scope of the present disclosure is not limited thereto.

[0103] Example 1: Selection of glutathione-producing strain and confirmation of glutathione production ability

[0104] Strains were obtained from a yeast block containing a plurality of strains, and the characteristics thereof were improved to select a strain having a glutathione production ability.

[0105] Specifically, grain samples such as rice, barley, mung beans, and oats were collected from 20 regions of Hwaseong, Pyeongtaek, Yongin, etc. of Gyeonggi-do, Korea, crushed, kneaded, wrapped in cloth, pressed into shape, and fermented for 10 days while being wrapped in straw, and then slowly dried to make a yeast block.

[0106] The following experiment was performed to isolate a plurality of strains from the prepared yeast block. 45 mL of a saline solution was added to 5 g of the yeast block and crushed using a mixer. In order to isolate the yeast strains cleanly, the resulting product was diluted by serial dilution, spread on YPD agar (10 g / L of yeast extract, 20 g / L of bacteriological peptone, and 20 g / L of glucose per 1 L of distilled water), and cultured at 30°C for 48 hours. Then, the yeast colonies were streaked on YPD agar according to the colony morphology and microscopic verification. 25 mL of YPD broth was inoculated in a 250 mL conical flask, the cleanly isolated strain was inoculated thereon, and cultured in a shaking incubator at 30°C and 200 rpm for 48 hours. The strains were screened by identifying glutathione production.

[0107] In order to improve the primarily isolated strain, random mutation was induced in the isolated strain. Specifically, a strain confirmed to have a glutathione production ability was isolated from the yeast block and named as a CJ-37 strain. The CJ-37 strain was cultured in a solid medium and inoculated into broth to obtain a culture solution thereof, and the culture solution was exposed to ultraviolet light using an ultraviolet lamp. After the culture solution exposed to ultraviolet light was plated on a plate medium, only the mutant strain that had formed a colony was isolated, and glutathione production thereof was identified. As a result, among the mutant strains, a strain exhibiting the highest glutathione yield was selected as a glutathione-producing strain, named as a CJ-5 strain, and deposited at the Korean Culture Center of Microorganisms (KCCM) according to the Budapest Treaty on July 31, 2019, and assigned as Accession No. KCCM 12568P.

[0108] Example 2: Experiment for further improving glutathione production ability In order to further improve the glutathione production ability of the CJ-5 strain, mutation was induced in the following manner.

[0109] The CJ-5 strain was cultured in a solid medium and inoculated into a broth to obtain a culture solution, and the culture solution was exposed to ultraviolet light using an ultraviolet lamp. After the culture solution exposed to the ultraviolet light was plated on a plate medium, only a mutant strain having formed a colony was isolated. The strain exhibiting the highest glutathione production was isolated and designated as CC02-2490 strain, which was deposited in the Korean Culture Center of Microorganisms (KCCM) on January 17, 2020, according to the Budapest Treaty, and assigned accession number KCCM12659P. As a result of analyzing the base sequence of the glutathione biosynthesis gene gsh1, it was confirmed that the cysteine at the 86th amino acid of the GSH1 protein encoded by the gsh1 gene had been replaced with arginine in terms of enhancing the glutathione production capacity of the strain.

[0110] Example 3: Experiment of GSH1 C86 residue mutation

[0111] Based on the results of Example 2, considering that the amino acid at the 86th position of the GSH1 protein is important in glutathione production, mutant strains of S. cerevisiae CEN.PK2-1D and S. cerevisiae CJ-5 were prepared so as to express protein variants in which the cysteine at the 86th position of the GSH1 protein is replaced with different amino acids, and an increase in glutathione production was identified.

[0112] To prepare a strain in which the cysteine at position 86 of the GSH1 protein of S. cerevisiae is substituted with arginine, the plasmids pWAL100 and pWBR100 were used in reference to the paper by Lee TH et al. (J. Microbiol. Biotechnol. (2006), 16(6), 979-982). Specifically, polymerase chain reaction (PCR) was performed using genomic DNA of the CJ-5 strain as a template as follows. By performing PCR using primers SEQ ID NO: 22 and 23, a partial sequence of the N-terminal end of the GSH1 protein was obtained, which includes the N-terminal BamHI flanking sequence, the GSH1 ORF start codon, and the C86R mutation coding sequence, and by performing PCR using primers SEQ ID NO: 24 and 25, a partial sequence of the C-terminal end of the GSH1 protein was obtained, which includes the C-terminal XhoI flanking sequence, the GSH1 ORF stop codon, and the C86R mutation coding sequence. Subsequently, as a result of performing overlap PCR using the 2 sequences as templates, using primers SEQ ID NO: 22 and 25, a GSH1 ORF fragment was obtained, which contains a sequence encoding a GSH1 modified protein in which the 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.

[0113] In addition, by performing PCR using genomic DNA of the CJ-5 strain as a template and primers SEQ ID NO: 26 and 27, 500 bp downstream of the GSH1 ORF stop codon including the N-terminal Spel and C-terminal Ncol restriction enzyme sequences were obtained, and treated with SpeI and NcoI restriction enzymes. Subsequently, the resultant was cloned into pWBR100 treated with the same enzymes to prepare the pWBR100-GSH1 vector.

[0114] To prepare the final DNA fragment to be introduced into the yeast, a PCR product including the sequence encoding the arginine mutation and part of KlURA3 was obtained using the pWAL100-GSH1 (C86R) vector prepared as described above as a template and primers SEQ ID NO: 22 and 28, and a PCR product including part of KlURA3 and 500 bp downstream of the GSH1 stop codon was obtained using the pWBR100-GSH1 vector as a template and primers SEQ ID NO: 29 and 27. Saccharomyces cerevisiae CEN.PK2-1D and Saccharomyces cerevisiae CJ-5 were transformed with the same molar ratio of PCR products. The PCR was performed via denaturation at 95°C for 5 minutes, annealing at 53°C for 1 minute, and polymerization at 72°C for 1 minute / kb, and the transformation of the yeast was performed according to the lithium acetate method improved from the method disclosed in the paper from Geitz (Nucleic Acid Research, 20(6), 1425). Specifically, the yeast cells having an OD of 0.7 to 1.2 were washed twice with a lithium acetate / TE buffer and mixed with the PCR products and single-stranded DNA (Sigma D-7656). The cells were incubated at 30°C for 30 minutes and at 42°C for 15 minutes under static culture conditions in a lithium acetate / TE / 40% PEG buffer. Then, the cells were cultured in SC (2% glucose) agar plates not including uracil until colonies were visible to obtain strains in which the GSH1 C86R mutation encoding sequence and the KlURA3 gene were 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 including 0.1% 5-FOA to prepare Saccharomyces cerevisiae CEN.PK2-1D GSH1 C86R mutant strains and Saccharomyces cerevisiae CJ-5 GSH1 C86R mutant strains from which the uracil marker had been removed. Strains capable of expressing GSH1 modified proteins in which cysteine was substituted with 18 amino acids other than arginine were also prepared in the same manner, except that primer pair SEQ ID NO: 23 and 24, in which the sequence encoding the 86th arginine was replaced with a sequence encoding a different amino acid, was used.

[0115] Table 1

[0116]

[0117] After culturing the strains prepared as described above for 26 hours, the concentration of produced glutathione (GSH) was measured and is listed in Tables 2 and 3.

[0118] Table 2

[0119]

[0120] Table 3

[0121]

[0122] Based on the experimental results, it was confirmed that the glutathione production capacity obtained by substituting the cysteine at position 86 of the GSH1 protein with different amino acids increased up to 27% compared to the glutathione production capacity obtained by the wild-type GSH1 protein.

[0123] Based on this, it was confirmed that the GSH1 variant prepared by substituting the cysteine at position 86 of the GSH1 protein with different amino acids has significantly improved glutathione production capacity.

[0124] Example 4: Confirmation of the effect of another Cys residue on glutathione production

[0125] As a comparative example, another Cys residue of the GSH1 protein was modified, and the glutathione production capacity obtained by the modification was identified. The results are shown in Table 4 below.

[0126] Table 4

[0127]

[0128] Referring to the experimental results, the cysteine residue other than the cysteine at position 86 had no effect on increasing glutathione production, but rather decreased glutathione production.

[0129] Based on this, it was confirmed that not all cysteine residues present in the protein have an effect on increasing glutathione production. In addition, it was confirmed that the novel GSH1 variant developed in the present disclosure increases glutathione production.

[0130] Since the yeast producing glutathione at a high yield, its dried product, extract, culture, and lysate, and the produced glutathione have antioxidant, detoxification, and immunity-enhancing effects, they can be effectively used to prepare cosmetic compositions, food compositions, feed compositions, and pharmaceutical compositions.

[0131] The above description of the present application 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 idea and essential characteristics of the present application. Therefore, it is clear that the above-described embodiments are illustrative in all aspects, not limiting the present application. The various embodiments disclosed herein are not intended to be limiting, and the true scope and spirit are indicated by the appended claims. The present application is limited only by the clauses of the appended claims and the full scope of equivalents enjoyed by these claims.

[0132]

[0133] <110> CJ First Sugar Co., Ltd. <120> Variants of glutamate-cysteine ligase and methods of producing glutathione using the same <130> OPA20226 <150> KR 10-2020-0036456 <151> 2020-03-25 <160> 29 <170> KoPatentIn 3.0 <210> 1 <211> 678 <212> PRT <213> Saccharomyces cerevisiae <400> 1 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His Ile Arg Asp Glu Gly Ile Glu Gin Leu Leu Tyr 20 25 30 Ile Phe Gin 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 Tyr Gly Arg Tyr Met Leu Glu Ala Thr Pro Ala Ser Pro Tyr Leu Asn100 105 110 Tyr Val Gly Ser Tyr Val Glu Val Asn Met Gin Lys Arg Arg Ala He 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 He Asn He Lys Asp Pro Trp Asn His 165 170 175 Lys Asn Ala Ala Ser Arg Ser Leu Phe Leu Pro Asp Glu Val He Asn 180 185 190 Arg His Val Arg Phe Pro Asn Leu Thr Ala Ser He Arg Thr Arg Arg 195 200 205 Gly Glu Lys Val Cys Met Asn Val Pro Met Tyr Lys Asp He Ala Thr 210 215 220 Pro Glu Thr Asp Asp Ser He 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 He Tyr Met Asp 245 250 255 Ser Met Gly Phe Gly Met Gly Cys Ser Cys Leu Gin Val Thr Phe Gin 260 265 270 Ala Pro Asn Ile Asn Lys Ala Arg Tyr Leu Tyr Asp Ala Leu Val Asn 275 280 285 Phe Ala Pro Ile Met Leu Ala Phe Ser Ala Ala Ala Pro Ala Phe Lys 290 295 300 Gly Trp Leu Ala Asp Gln Asp Val Arg Trp Asn Val Ile Ser Gly Ala 305 310 315 320 Val Asp Asp Arg Thr Pro Lys Glu Arg Gly Val Ala Pro Leu Leu Pro 325 330 335 Lys Tyr Asn Lys Asn Gly Phe Gly Gly Ile Ala Lys Asp Val Gln Asp 340 345 350 Lys Val Leu Glu Ile Pro Lys Ser Arg Tyr Ser Ser Val Asp Leu Phe 355 360 365 Leu Gly Gly Ser Lys Phe Phe Asn Arg Thr Tyr Asn Asp Thr Asn Val 370 375 380 Pro Ile Asn Glu Lys Val Leu Gly Arg Leu Leu Glu Asn Asp Lys Ala 385 390 395 400 Pro Leu Asp Tyr Asp Leu Ala Lys His Phe Ala His Leu Tyr Ile Arg 405 410 415 Asp Pro Val Ser Thr Phe Glu Glu Leu Leu Asn Gln Asp Asn Lys Thr 420 425 430 Ser Ser Asn His Phe Glu Asn Ile Gln Ser Thr Asn Trp Gln Thr Leu 435 440 445 Arg Phe Lys Pro Pro Thr Gln Gln Ala Thr Pro Asp Lys Lys Asp Ser 450 455 460 Pro Gly Trp Arg Val Glu Phe Arg Pro Phe Glu Val Gln Leu Leu Asp 465 470 475 480 Phe Glu Asn Ala Ala Tyr Ser Val Leu Ile Tyr Leu Ile Val Asp Ser 485 490 495 Ile Leu Thr Phe Ser Asp Asn Ile Asn Ala Tyr Ile His Met Ser Lys 500 505 510 Val Trp Glu Asn Met Lys Ile Ala His His Arg Asp Ala Ile Leu Phe 515 520 525 Glu Lys Phe His Trp Lys Lys Ser Phe Arg Asn Asp Thr Asp Val Glu 530 535 540 Thr Glu Asp Tyr Ser Ile Ser Glu Ile Phe His Asn Pro Glu Asn Gly 545 550 555 560 Ile Phe Pro Gln Phe Val Thr Pro Ile Leu Cys Gln Lys Gly Phe Val 565 570 575 Thr Lys Asp Trp Lys Glu Leu Lys His Ser Ser Lys His Glu Arg Leu 580 585 590 Tyr Tyr Tyr Leu Lys Leu Ile Ser Asp Arg Ala Ser Gly Glu Leu Pro 595 600 605 Thr Thr Ala Lys Phe Phe Arg Asn Phe Val Leu Gln His Pro Asp Tyr 610 615 620 Lys His Asp Ser Lys Ile Ser Lys Ser Ile Asn Tyr Asp Leu Leu Ser 625 630 635 640 Thr Cys Asp Arg Leu Thr His Leu Asp Asp Ser Lys Gly Glu Leu Thr 645 650 655 Ser Phe Leu Gly Ala Glu Ile Ala Glu Tyr Val Lys Lys Asn Lys Pro 660 665 670 Ser Ile Glu Ser Lys Cys 675 <210> 2 <211> 2037 <212> DNA <213> Saccharomyces cerevisiae <400> 2 atgggactct tagctttggg cacgcctttg cagtggtttg agtctaggac gtacaatgaa 60 cacataaggg atgaaggtat cgagcagttg ttgtatattt tccaagctgc tggtaaaaga 120 gacaatgacc ctcttttttg gggagacgag cttgagtaca tggttgtaga ttttgatgat 180 aaggagagaa attctatgct cgacgtttgc catgacaaga tactcactga gcttaatatg 240 gaggattcgt ccctttgtga ggctaacgat gtgagttttc accctgagta tggccggtat 300 atgttagagg caacaccagc ttctccatat ttgaattacg tgggtagtta cgttgaggtt 360 aacatgcaaa aaagacgtgc cattgcagaa tataagctat ctgaatatgc gagacaagat 420 agtaaaaata acttgcatgt gggctccagg tctgtccctt tgacgctgac tgtcttcccg 480 aggatgggat gccccgactt tattaacatt aaggatccgt ggaatcataa aaatgccgct 540 tccaggtctc tgtttttacc cgatgaagtc attaacagac atgtcaggtt tcctaacttg 600 acagcatcca tcaggaccag gcgtggtgaa aaagtttgca tgaatgttcc catgtataaa 660 gatatagcta ctccagaaac ggatgactcc atctacgatc gagattggtt tttaccagaa 720 gacaaagagg cgaaactggc ttccaaaccg ggtttcattt atatggattc catgggtttt 780 ggcatgggct gttcgtgctt acaagtgacc tttcaggcac ccaatatcaa caaggcacgt 840 tacctgtacg atgcattagt gaattttgca cctataatgc tagccttctc tgccgctgcg 900 cctgctttta aaggttggct agccgaccaa gatgttcgtt ggaatgtgat atctggtgcg 960 gtggacgacc gtactccgaa ggaaagaggt gttgcgccat tactacccaa atacaacaag 1020 aacggatttg gaggcattgc caaagacgta caagataaag tccttgaaat accaaagtca 1080 agatatagtt cggttgatct tttcttgggt gggtcgaaat ttttcaatag gacttataac 1140 gacacaaatg tacctattaa tgaaaaagta ttaggacgac tactagagaa tgataaggcg 1200 ccactggact atgatcttgc taaacatttt gcgcatctct acataagaga tccagtatct 1260 acattcgaag aactgttgaa tcaggacaac aaaacgtctt caaatcactt tgaaaacatc 1320 caaagtacaa attggcagac attacgtttt aaacccccca cacaacaagc aaccccggac 1380 aaaaaggatt ctcctggttg gagagtggaa ttcagaccat ttgaagtgca actattagat 1440 tttgagaacg ctgcgtattc cgtgctcata tacttgattg tcgatagcat tttgaccttt 1500 tccgataata ttaacgcata tattcatatg tccaaagtat gggaaaatat gaagatagcc 1560 catcacagag atgctatcct atttgaaaaa tttcattgga aaaaatcatt tcgcaacgac 1620 accgatgtgg aaactgaaga ttattctata agcgagattt tccataatcc agagaatggt 1680 atatttcctc aatttgttac gccaatccta tgccaaaaag ggtttgtaac caaagattgg 1740 aaagaattaa agcattcttc caaacacgag agactatact attatttaaa gctaatttct 1800 gatagagcaa gcggtgaatt gccaacaaca gcaaaattct ttagaaattt tgtactacaa 1860 catccagatt acaaacatga ttcaaaaatt tcaaagtcga tcaattatga tttgctttct 1920 acgtgtgata gacttaccca tttagacgat tcaaaaggtg aattgacatc ctttttagga 1980 gctgaaattg cagaatatgt aaaaaaaaat aagccttcaa tagaaagcaa atgttaa 2037 <210> 3 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86A <400> 3 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gln Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His Ile Arg Asp Glu Gly Ile Glu Gln Leu Leu Tyr 20 25 30 Ile Phe Gin 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 Ala 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 Gin Lys Arg Arg Ala Ile 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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> 4 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86D <400> 4 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His Ile Arg Asp Glu Gly Ile Glu Gin Leu Leu Tyr 20 25 30 Ile Phe Gin 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 Asp 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 Gin Lys Arg Arg Ala He 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 He Asn He Lys Asp Pro Trp Asn His 165 170 175 Lys Asn Ala Ala Ser Arg Ser Leu Phe Leu Pro Asp Glu Val He Asn 180 185 190 Arg His Val Arg Phe Pro Asn Leu Thr Ala Ser He Arg Thr Arg Arg 195 200 205 Gly Glu Lys Val Cys Met Asn Val Pro Met Tyr Lys Asp He Ala Thr 210 215 220 Pro Glu Thr Asp Asp Ser He 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 He Tyr Met Asp 245 250 255 Ser Met Gly Phe Gly Met Gly Cys Ser Cys Leu Gin Val Thr Phe Gin 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 Gin Thr Leu 435 440 445 Arg Phe Lys Pro Pro Thr Gin Gin Ala Thr Pro Asp Lys Lys Asp Ser 450 455 460 Pro Gly Trp Arg Val Glu Phe Arg Pro Phe Glu Val Gin 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 Gin Phe Val Thr Pro Ile Leu Cys Gin 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> 5 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86E <400> 5 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 Gin 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 Glu 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 Gin Lys Arg Arg Ala Ile 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 lie 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 lie 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 lie Arg 405 410 415 Asp Pro Val Ser Thr Phe Glu Glu Leu Leu Asn Gin Asp Asn Lys Thr 420 425 430 Ser Ser Asn His Phe Glu Asn lie Gin Ser Thr Asn Trp Gin Thr Leu 435 440 445 Arg Phe Lys Pro Pro Thr Gin Gin Ala Thr Pro Asp Lys Lys Asp Ser 450 455 460 Pro Gly Trp Arg Val Glu Phe Arg Pro Phe Glu Val Gin Leu Leu Asp 465 470 475 480 Phe Glu Asn Ala Ala Tyr Ser Val Leu lie Tyr Leu lie Val Asp Ser 485 490 495 lie Leu Thr Phe Ser Asp Asn lie Asn Ala Tyr lie 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> 6 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86F <400> 6 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His Ile Arg Asp Glu Gly Ile Glu Gin Leu Leu Tyr 20 25 30 Ile Phe Gin 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 Phe 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 Gin Lys Arg Arg Ala Ile 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 Gin Val Thr Phe Gin 260 265 270 Ala Pro Asn lie Asn Lys Ala Arg Tyr Leu Tyr Asp Ala Leu Val Asn 275 280 285 Phe Ala Pro lie Met Leu Ala Phe Ser Ala Ala Ala Pro Ala Phe Lys 290 295 300 Gly Trp Leu Ala Asp Gin Asp Val Arg Trp Asn Val lie 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 lie Ala Lys Asp Val Gin Asp 340 345 350 Lys Val Leu Glu lie 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 lie 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 lie Arg 405 410 415 Asp Pro Val Ser Thr Phe Glu Glu Leu Leu Asn Gin Asp Asn Lys Thr 420 425 430 Ser Ser Asn His Phe Glu Asn lie Gin Ser Thr Asn Trp Gin Thr Leu 435 440 445 Arg Phe Lys Pro Pro Thr Gin Gin Ala Thr Pro Asp Lys Lys Asp Ser 450 455 460 Pro Gly Trp Arg Val Glu Phe Arg Pro Phe Glu Val Gin Leu Leu Asp 465 470 475 480 Phe Glu Asn Ala Ala Tyr Ser Val Leu lie Tyr Leu lie Val Asp Ser 485 490 495 Ile Leu Thr Phe Ser Asp Asn lie Asn Ala Tyr lie His Met Ser Lys 500 505 510 Val Trp Glu Asn Met Lys lie Ala His His Arg Asp Ala lie 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 lie Ser Glu lie Phe His Asn Pro Glu Asn Gly 545 550 555 560 Ile Phe Pro Gin Phe Val Thr Pro lie Leu Cys Gin 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> 7 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86G <400> 7 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 Gly 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 lie 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 lie 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 lie Arg 405 410 415 Asp Pro Val Ser Thr Phe Glu Glu Leu Leu Asn Gin Asp Asn Lys Thr 420 425 430 Ser Ser Asn His Phe Glu Asn lie Gin Ser Thr Asn Trp Gin Thr Leu 435 440 445 Arg Phe Lys Pro Pro Thr Gin Gin Ala Thr Pro Asp Lys Lys Asp Ser 450 455 460 Pro Gly Trp Arg Val Glu Phe Arg Pro Phe Glu Val Gin Leu Leu Asp 465 470 475 480 Phe Glu Asn Ala Ala Tyr Ser Val Leu lie Tyr Leu lie Val Asp Ser 485 490 495 lie Leu Thr Phe Ser Asp Asn lie Asn Ala Tyr lie 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> 8 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86H <400> 8 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 Gin Leu Leu Tyr 20 25 30 Ile Phe Gin 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 His 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 Gin Lys Arg Arg Ala Ile 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 Gin Val Thr Phe Gin 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 lie Gin Ser Thr Asn Trp Gin Thr Leu 435 440 445 Arg Phe Lys Pro Pro Thr Gin Gin Ala Thr Pro Asp Lys Lys Asp Ser 450 455 460 Pro Gly Trp Arg Val Glu Phe Arg Pro Phe Glu Val Gin Leu Leu Asp 465 470 475 480 Phe Glu Asn Ala Ala Tyr Ser Val Leu lie Tyr Leu lie Val Asp Ser 485 490 495 Ile Leu Thr Phe Ser Asp Asn lie Asn Ala Tyr lie His Met Ser Lys 500 505 510 Val Trp Glu Asn Met Lys lie Ala His His Arg Asp Ala lie 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 lie Ser Glu lie Phe His Asn Pro Glu Asn Gly 545 550 555 560 Ile Phe Pro Gin Phe Val Thr Pro lie Leu Cys Gin 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 lie Ser Asp Arg Ala Ser Gly Glu Leu Pro 595 600 605 Thr Thr Ala Lys Phe Phe Arg Asn Phe Val Leu Gin His Pro Asp Tyr 610 615 620 Lys His Asp Ser Lys lie Ser Lys Ser lie 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 lie Ala Glu Tyr Val Lys Lys Asn Lys Pro 660 665 670 Ser lie Glu Ser Lys Cys 675 <210> 9 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86I <400> 9 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His lie Arg Asp Glu Gly lie Glu Gin Leu Leu Tyr 20 25 30 lie Phe Gin 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 Ile 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 lie Ala His His Arg Asp Ala lie 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 lie Ser Glu lie Phe His Asn Pro Glu Asn Gly 545 550 555 560 lie Phe Pro Gin Phe Val Thr Pro lie Leu Cys Gin 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 lie Ser Asp Arg Ala Ser Gly Glu Leu Pro 595 600 605 Thr Thr Ala Lys Phe Phe Arg Asn Phe Val Leu Gin His Pro Asp Tyr 610 615 620 Lys His Asp Ser Lys lie Ser Lys Ser lie 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 lie Ala Glu Tyr Val Lys Lys Asn Lys Pro 660 665 670 Ser Ile Glu Ser Lys Cys 675 <210> 10 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86K <400> 10 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His Ile Arg Asp Glu Gly Ile Glu Gin Leu Leu Tyr 20 25 30 Ile Phe Gin 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 Lys 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 Gin Lys Arg Arg Ala Ile 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 Gin Val Thr Phe Gin 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 Gin Gin Ala Thr Pro Asp Lys Lys Asp Ser 450 455 460 Pro Gly Trp Arg Val Glu Phe Arg Pro Phe Glu Val Gin 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 Gin Phe Val Thr Pro Ile Leu Cys Gin 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 lie Ser Asp Arg Ala Ser Gly Glu Leu Pro 595 600 605 Thr Thr Ala Lys Phe Phe Arg Asn Phe Val Leu Gin His Pro Asp Tyr 610 615 620 Lys His Asp Ser Lys lie Ser Lys Ser lie 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 lie Ala Glu Tyr Val Lys Lys Asn Lys Pro 660 665 670 Ser lie Glu Ser Lys Cys 675 <210> 11 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86L <400> 11 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His lie Arg Asp Glu Gly lie Glu Gin Leu Leu Tyr 20 25 30 lie Phe Gin 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 Leu 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 lie Ala Thr 210 215 220 Pro Glu Thr Asp Asp Ser lie 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 lie Tyr Met Asp 245 250 255 Ser Met Gly Phe Gly Met Gly Cys Ser Cys Leu Gin Val Thr Phe Gin 260 265 270 Ala Pro Asn lie Asn Lys Ala Arg Tyr Leu Tyr Asp Ala Leu Val Asn 275 280 285 Phe Ala Pro lie Met Leu Ala Phe Ser Ala Ala Ala Pro Ala Phe Lys 290 295 300 Gly Trp Leu Ala Asp Gin Asp Val Arg Trp Asn Val lie 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 lie Ala Lys Asp Val Gin Asp 340 345 350 Lys Val Leu Glu lie 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> 12 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86M <400> 12 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His Ile Arg Asp Glu Gly Ile Glu Gin Leu Leu Tyr 20 25 30 Ile Phe Gin 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 Met 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 Gin Lys Arg Arg Ala Ile 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 Gin Val Thr Phe Gin 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 Gin Gin Ala Thr Pro Asp Lys Lys Asp Ser 450 455 460 Pro Gly Trp Arg Val Glu Phe Arg Pro Phe Glu Val Gin 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 Gin Phe Val Thr Pro Ile Leu Cys Gin 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 Gin His Pro Asp Tyr 610 615 620 Lys His Asp Ser Lys He Ser Lys Ser He 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 He Ala Glu Tyr Val Lys Lys Asn Lys Pro 660 665 670 Ser He Glu Ser Lys Cys 675 <210> 13 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86N <400> 13 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His He Arg Asp Glu Gly He Glu Gin Leu Leu Tyr 20 25 30 He Phe Gin 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 Asn 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 lie Ala Thr 210 215 220 Pro Glu Thr Asp Asp Ser lie 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 lie Tyr Met Asp 245 250 255 Ser Met Gly Phe Gly Met Gly Cys Ser Cys Leu Gin Val Thr Phe Gin 260 265 270 Ala Pro Asn lie Asn Lys Ala Arg Tyr Leu Tyr Asp Ala Leu Val Asn 275 280 285 Phe Ala Pro lie Met Leu Ala Phe Ser Ala Ala Ala Pro Ala Phe Lys 290 295 300 Gly Trp Leu Ala Asp Gin Asp Val Arg Trp Asn Val lie 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 lie Ala Lys Asp Val Gin Asp 340 345 350 Lys Val Leu Glu lie 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> 14 <211> 678 <212> PRT <213> Artificial sequence <220> <223> Saccharomyces cerevisiae GSH1 C86P <400> 14 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His Ile Arg Asp Glu Gly Ile Glu Gin Leu Leu Tyr 20 25 30 Ile Phe Gin 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 Pro 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 Gin Lys Arg Arg Ala Ile 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 lie Met Leu Ala Phe Ser Ala Ala Ala Pro Ala Phe Lys 290 295 300 Gly Trp Leu Ala Asp Gin Asp Val Arg Trp Asn Val lie 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 lie Ala Lys Asp Val Gin Asp 340 345 350 Lys Val Leu Glu lie 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 lie 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 lie Arg 405 410 415 Asp Pro Val Ser Thr Phe Glu Glu Leu Leu Asn Gin Asp Asn Lys Thr 420 425 430 Ser Ser Asn His Phe Glu Asn lie Gin Ser Thr Asn Trp Gin Thr Leu 435 440 445 Arg Phe Lys Pro Pro Thr Gin Gin Ala Thr Pro Asp Lys Lys Asp Ser 450 455 460 Pro Gly Trp Arg Val Glu Phe Arg Pro Phe Glu Val Gin 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 Gin Phe Val Thr Pro Ile Leu Cys Gin 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> 15 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86Q <400> 15 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 Gln 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> 16 <211> 678 <212> PRT <213> Artificial sequence <220> <223> Saccharomyces cerevisiae GSH1 C86R <400> 16 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His Ile Arg Asp Glu Gly Ile Glu Gin Leu Leu Tyr 20 25 30 Ile Phe Gin Ala Ala Gly Lys Arg Asp Asn Asp Pro Leu Phe Trp Gly 35 40 45 Asp Glu Leu Glu Tyr Met Val Val Asp Phe Asp Asp Lys Glu Arg Asn 50 55 60 Ser Met Leu Asp Val Cys His Asp Lys Ile Leu Thr Glu Leu Asn Met 65 70 75 80 Glu Asp Ser Ser Leu Arg Glu Ala Asn Asp Val Ser Phe His Pro Glu 85 90 95 Tyr Gly Arg Tyr Met Leu Glu Ala Thr Pro Ala Ser Pro Tyr Leu Asn 100 105 110 Tyr Val Gly Ser Tyr Val Glu Val Asn Met Gin Lys Arg Arg Ala Ile 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 Gin His Pro Asp Tyr 610 615 620 Lys His Asp Ser Lys lie Ser Lys Ser lie 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 lie Ala Glu Tyr Val Lys Lys Asn Lys Pro 660 665 670 Ser lie Glu Ser Lys Cys 675 <210> 17 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86S <400> 17 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His lie Arg Asp Glu Gly lie Glu Gin Leu Leu Tyr 20 25 30 lie Phe Gin 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 Ser 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> 18 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86T <400> 18 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His He Arg Asp Glu Gly He Glu Gin Leu Leu Tyr 20 25 30 He Phe Gin 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 He Leu Thr Glu Leu Asn Met 65 70 75 80 Glu Asp Ser Ser Leu Thr 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 Gin Lys Arg Arg Ala He 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 lie Ser Lys Ser lie 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 lie Ala Glu Tyr Val Lys Lys Asn Lys Pro 660 665 670 Ser lie Glu Ser Lys Cys 675 <210> 19 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86V <400> 19 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His lie Arg Asp Glu Gly lie Glu Gin Leu Leu Tyr 20 25 30 lie Phe Gin 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 lie Leu Thr Glu Leu Asn Met 65 70 75 80 Glu Asp Ser Ser Leu Val 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 Gin Lys Arg Arg Ala lie 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 lie Asn lie Lys Asp Pro Trp Asn His 165 170 175 Lys Asn Ala Ala Ser Arg Ser Leu Phe Leu Pro Asp Glu Val lie Asn 180 185 190 Arg His Val Arg Phe Pro Asn Leu Thr Ala Ser lie Arg Thr Arg Arg 195 200 205 Gly Glu Lys Val Cys Met Asn Val Pro Met Tyr Lys Asp lie Ala Thr 210 215 220 Pro Glu Thr Asp Asp Ser lie 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 lie Tyr Met Asp 245 250 255 Ser Met Gly Phe Gly Met Gly Cys Ser Cys Leu Gin Val Thr Phe Gin 260 265 270 Ala Pro Asn lie Asn Lys Ala Arg Tyr Leu Tyr Asp Ala Leu Val Asn 275 280 285 Phe Ala Pro lie Met Leu Ala Phe Ser Ala Ala Ala Pro Ala Phe Lys 290 295 300 Gly Trp Leu Ala Asp Gin Asp Val Arg Trp Asn Val lie 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 lie Ala Lys Asp Val Gin Asp 340 345 350 Lys Val Leu Glu lie 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> 20 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86W <400> 20 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His He Arg Asp Glu Gly He Glu Gin Leu Leu Tyr 20 25 30 He Phe Gin 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 He Leu Thr Glu Leu Asn Met 65 70 75 80 Glu Asp Ser Ser Leu Trp 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 Gin Lys Arg Arg Ala He 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 lie Ser Lys Ser lie 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 lie Ala Glu Tyr Val Lys Lys Asn Lys Pro 660 665 670 Ser lie Glu Ser Lys Cys 675 <210> 21 <211> 678 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GSH1 C86Y <400> 21 Met Gly Leu Leu Ala Leu Gly Thr Pro Leu Gin Trp Phe Glu Ser Arg 1 5 10 15 Thr Tyr Asn Glu His lie Arg Asp Glu Gly lie Glu Gin Leu Leu Tyr 20 25 30 lie Phe Gin 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 lie Leu Thr Glu Leu Asn Met 65 70 75 80 Glu Asp Ser Ser Leu Tyr 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 Gin Lys Arg Arg Ala lie 115 120 125 Ala Glu Tyr Lys Leu Ser Glu Tyr Ala Arg Gin 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 lie Asn lie Lys Asp Pro Trp Asn His 165 170 175 Lys Asn Ala Ala Ser Arg Ser Leu Phe Leu Pro Asp Glu Val lie Asn 180 185 190 Arg His Val Arg Phe Pro Asn Leu Thr Ala Ser lie Arg Thr Arg Arg 195 200 205 Gly Glu Lys Val Cys Met Asn Val Pro Met Tyr Lys Asp lie Ala Thr 210 215 220 Pro Glu Thr Asp Asp Ser lie 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 lie Tyr Met Asp 245 250 255 Ser Met Gly Phe Gly Met Gly Cys Ser Cys Leu Gin Val Thr Phe Gin 260 265 270 Ala Pro Asn lie Asn Lys Ala Arg Tyr Leu Tyr Asp Ala Leu Val Asn 275 280 285 Phe Ala Pro lie Met Leu Ala Phe Ser Ala Ala Ala Pro Ala Phe Lys 290 295 300 Gly Trp Leu Ala Asp Gin Asp Val Arg Trp Asn Val lie 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 lie Ala Lys Asp Val Gin Asp 340 345 350 Lys Val Leu Glu lie 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> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F_BamHI_GSH1 <400> 22 ggtaggatcc atgggactct tagctttggg cac 33 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> R_GSH1_C86R <400> 23 ttagcctccc taagggacga atcct 25 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> F_GSH1_C86R <400> 24 cgtcccttag ggaggctaac gatgt 25 <210> 25 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R_XhoI_GSH1 <400> 25 atgactcgag ttaacatttg ctttctattg aaggc 35 <210> 26 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F_SpeI_GSH1_DW <400> 26 tagaactagt actcctttta tttcggttgt gaa 33 <210> 27 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R_NcoI_GSH1_DW <400> 27 gctgccatgg gaatagtgtg aaccgataac tgtgt 35 <210> 28 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R_AL killer <400> 28 gagcaatgaa cccaataacg aaatctt 27 <210> 29 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> F_BR killer <400> 29 cttgacgttc gttcgactga tgag 24

Claims

1.A glutamate-cysteine ligase variant, wherein an amino acid at position 86 from the N-terminus of a glutamate-cysteine ligase consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, tyrosine, asparagine, glutamic acid, glutamine, aspartic acid, lysine, arginine, or histidine. 2.A polynucleotide encoding the glutamate-cysteine ligase variant of claim 1. 3.A vector comprising the polynucleotide of claim 2. 4.A microorganism producing glutathione, comprising at least one of: the glutamate-cysteine ligase variant of claim 1; a polynucleotide encoding the variant; and a vector comprising the polynucleotide. 5.The microorganism of claim 4, wherein the microorganism is a microorganism belonging to the genus Saccharomyces. 6.The microorganism of claim 4, wherein the microorganism is Saccharomyces cerevisiae. 7.The microorganism of claim 4, wherein the microorganism is a Saccharomyces cerevisiae strain deposited under accession number KCCM12659P. 8.A method of producing glutathione, the method comprising culturing a microorganism in a culture medium, the microorganism comprising at least one of: the glutamate-cysteine ligase variant of claim 1; a polynucleotide encoding the variant; and a vector comprising the polynucleotide. 9.The method of 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 of the microorganism, and a lysate of the microorganism.

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