KR100485055B1 - Gamma-glutamylcysteine synthetase and gene coding for the same - Google Patents

Abstract

The present invention relates to enzymes involved in glutathione biosynthesis and genes encoding the same, and more specifically gamma-glutamylcysteine synthase and enzymes involved in glutathione biosynthesis isolated from methanolentic yeast Hanshenula polymorpha. It's about genes. When the gene of the present invention is introduced into a glutathione biosynthetic deficiency strain, the glutathione biosynthesis function can restore and increase the heavy metal resistance and the cell proliferation function, so that heavy metal adsorption using glutathione, production of coenzyme, liver function enhancer and detoxicant It can be usefully used for production.

Description

Gamma-glutamylcysteine synthetase and gene coding for the same

The present invention relates to glutathione biosynthesis genes, and more particularly, to gamma-glutamylcysteine synthase, which restores heavy metal resistance and cell proliferation function as well as glutathione biosynthesis function when introduced into a glutathione biosynthetic function-deficient strain.

Glutathione (GSH) is the most thiol-based compound in aerobic prokaryotic and eukaryotic cells and is an important physiological function that is involved in the oxidative stress response and the defense against the toxicity of biological foreign substances such as heavy metals. . Glutathione, consisting of gamma-glutamyl-L-cysteineglycin, is composed of gamma-glutamylcysteine synthase (γ-) using L-glutamic acid, L-cysteine and glycine in cells. It is produced by the chain action of glutamylcysteine synthetase (hereinafter abbreviated 'γ-GCS') and glutathione synthetase (abbreviated as 'GS'). The two genes encoding glutathione biosynthesis-related enzymes γ-GCS and GS are yeast as well as in E. coli, plants, mice and the human body (Griffith and Mulcahy, Advances in Enzymology and Related Areas of Molecular Biology , 1999 , 73, 209-267). Isolated from Saccharomyces cerevisiae and Schizosaccharomyces pombe (Ohtake and Yabuuchi, Yeast , 1991 , 7, 953; Al-Lahham et al ., Yeast , 1999 , 15, 385). In Saccharomyces cerevisiae, the expression of the GSH1 gene encoding γ-GCS, the first step in the glutathione biosynthetic pathway and involved in the rate-determining step, is associated with reactive oxygen groups (H ₂ O ₂ ). Is controlled by the transcription factors Yap1, Skn7, Met-4, Met-31, Met-32 and Cbf1 (Wu et al ., Mol. Cell). Biol., 1994 , 14, 5832; Lee et al ., J. Biol. Chem., 1999 , 274, 16040; Dormer et al ., J. Biol. Chem ., 2000 , 275, 32611).

Glutathione plays an essential role in methanol metabolism. Methanol metabolism shows that methanol is converted from peroxysomes to formaldehyde and hydrogen peroxide (Sahm H., Adv. Biochem. Eng., 1977 , 6, 103), and the converted formaldehyde goes into the cytoplasm and is oxidized to formic acid and dehydrogenated. The dehydrogenase reaction finally results in carbon dioxide. In order to oxidize formaldehyde, the reaction proceeds by NAD ⁺ -dependent dehydrogenase using glutathione as a cofactor. In addition, Candida boidinii incubated in methanol has a significant concentration with CbPmp20 protein, a glutathione peroxidase that is involved in the reaction of peroxysomes to alkyl hydroperoxides produced on the membrane surface. Reduced glutathione was observed at the peroxysome site (Sakai et al ., Microbiology, 2002 , 148, 2697). These studies report that glutathione is essential for methanol metabolism by being involved in detoxification of various metabolites involved in the oxidation of methanol. Indeed, methanol is known to increase the content of glutathione in cells, particularly in formaldehyde dehydrogenase-deficient strains (Sibirny et al ., Arch. Microbiol., 1990 , 154, 566). Therefore, methanolized yeasts using methanol as the sole carbon source are expected to be a good source of glutathione.

In addition to microorganisms such as yeast, glutathione is widely distributed in the liver of animals, and has important physiological effects such as respiratory system, redox reaction, detoxification, enzyme reactivation, and coenzyme in vivo. In addition, as a cell protector against oxidative damage caused by toxic compounds, reactive enzyme derivatives and radiation, it generally plays an important role in inactivating substances that damage cells.

As mentioned above, glutathione having various functions is not only useful as a therapeutic agent for enhancing liver function or detoxification, but also can be used as a food additive for health supplements such as sports drinks, so that the demand for mass production of glutathione is gradually increased. (Yin et al ., Chinese J. Biotech., 1998 , 14, 85). Conventionally, in order to produce glutathione, a method of culturing yeast by a fermentation industrial production method and extracting glutathione from the cultured yeast has been widely used. For example, in culturing yeast, a method of adding or mixing three amino acids, L-cysteine, L-glutamic acid and glycine, constituting glutathione (Japanese Patent Nos. 48092579 and 53094089) and L-cystine There is a method of adding, and a method of adding methionine and its derivatives. Alternatively, in cultured yeast, a method using a methionine analog resistant strain, which is a mutant strain, a method using a methionine or a methionine-lysine requesting strain, or a method using an L or D or L-ethionine sensitive strain has already been reported. Patents 4596775 and 4582801). Recently, due to the development of genetic recombination technology, studies have been conducted to increase the productivity of glutathione by introducing multiple genes of enzymes involved in glutathione biosynthesis into E. coli or yeast. For example, when non-enzymatic activity of glutathione synthesis system was increased up to 40-fold when E. coli-derived glutathione biosynthesis genes, gshI and gshII , were inserted into E. coli by multiple copy plasmids (Watanabe et al ., Appl. Microbiol) Biotech ., 1986 , 24, 375; Japanese Patent No. 20316900). In addition, when gshI and gshII were inserted into multiple copy plasmids and introduced into Saccharomyces cerevisiae, glutathione production increased by about 30% in cells (Japanese Patent No. 61052299 and European Patent No. 0300168).

Meanwhile, glutathione biosynthesis genes have been usefully used for the purpose of developing recombinant strains having improved resistance and accumulation to heavy metals by utilizing glutathione's high heavy metal adsorption capacity. In one example, E. coli encoding γ-GCS and GS Origin Brassica juncea seedlings overexpressing the genes gshI and gshII have increased resistance to cadmium (Cd) compared to wild seedlings, phytokelatin, gamma-glutamylcysteine, glutathione and total nonprotein types. Thiol concentrations were increased (Zhu et al . Plant Physiology , 1999 , 121, 1169; Zhu et al ., Plant Physiology , 1999 , 119, 73). Furthermore, expression of glutathione related genes is sensitively regulated and induced depending on the presence of toxic substances such as heavy metals (Dormer et al . J. Biol. Chem ., 2000 , 275, 32611). Therefore, using a promoter derived from genes encoding γ-GCS and GS as a promoter for expression of a reporter protein such as a luminescent protein or a fluorescent protein may be useful for the development of a recombinant strain for developing a biosensor for detecting toxic substances. (International Publication No. W09008826).

In the traditional yeast Saccharomyces cerevisiae and the cleavage yeast Schizocaromyces pombe , gsh mutants lacking GSH1 or GCS1 , a gene encoding γ-GCS that acts at the first stage of glutathione biosynthesis, It did not grow on synthetic medium without addition, and it showed remarkably enhanced resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and increased sensitivity to cadmium ions (Kistler et al ., Mutat) . Res ., 1986 , 173, 117; Glaeser et a l., Curr. Genet ., 1991 , 19, 207). In addition, the present inventors , while resistant to MNNG in the methanolic yeast Hansenula polymorpha ( aka Pichia angusta ), while sensitive to cadmium ions and methanol, glutathione based on methanol or multiple carbon sources as a substrate Mutant strains that cannot grow in deficient media have been isolated (Ubiyvovk et al ., Microbiology , 1999 , 68, 26). The gsh1 and gsh2 mutants of Hanshenula polymorpha , named ' gsh1-2 leu1-1 ' and ' gsh2-1 leu1-1 ', had very low total intracellular glutathione content and no γ-GCS activity compared to wild-type strains. GS activity was similar to wild type strains. The gsh2 mutant was very sensitive to cadmium, while the gsh1 mutant was very sensitive to chromium.

Therefore, the present inventors are involved in glutathione biosynthesis from mutants (Ubiyvovk et al ., Microbiology , 1999 , 68, 26) isolated from the inventors for mass production of glutathione having important physiological function and heavy metal adsorption function in vivo. Cloning an important gene and introducing it into a glutathione deficient strain resulted in an increase in the biosynthesis of glutathione and the recovery of resistance to heavy metals.

An object of the present invention is to produce a large amount of glutathione using a gamma-glutamylcysteine synthetase, a gene encoding the enzyme, an expression vector containing the gene, a transformant transducing the vector, and the transformant. To provide a way.

In order to achieve the above object, the present invention provides a genomic DNA comprising a promoter and gene of a gamma-glutamylcysteine synthetase derived from Hansenula polymorpha.

The present invention also provides a promoter of gamma-glutamylcysteine synthase derived from Hanshenula polymorpha.

The present invention also provides gamma-glutamylcysteine synthase derived from Hanshenula polymorpha.

The present invention also provides a gene encoding the gamma-glutamylcysteine synthase.

The present invention also provides an expression vector comprising the gene.

The present invention also provides a transformant in which the vector is introduced into a host cell.

Hereinafter, the present invention will be described in detail.

The present invention provides a genomic DNA comprising a promoter and gene of a gamma-glutamylcysteine synthetase derived from Hanshenula polymorpha.

The genomic DNA described in SEQ ID NO: 1 of the present invention has a function of restoring glutathione biosynthesis of Hanshenula polymorpha mutant strain ( gsh2 ) lacking gamma-glutamylcysteine synthetase activity. have. The DNA contains a promoter of gamma-glutamylcysteine synthase and a gene encoding gamma-glutamylcysteine synthase (GenBank, Accession No. AF435121). The gene region encoding gamma-glutamylcysteine synthase from Nos. 1 to 450 is a promoter region of gamma-glutamylcysteine synthase among 4195 nucleotide sequences of the gene, and 451 to 2403 is an open reading frame (ORF). And the remaining 2436 to 3140 and 2691 to 3146 are unknown protein coding gene sites (ORFs).

The present invention also provides a promoter of gamma-glutamylcysteine synthase derived from Hanshenula polymorpha.

Promoter of the present invention consists of the sequence from 1 to 450 of the nucleotide sequence described in SEQ ID NO: 1 , to induce the expression of gamma-glutamylcysteine synthase related to glutathione biosynthesis to increase the biosynthesis of glutathione As a promoter that plays a role, it may be a nucleotide sequence shown in SEQ ID NO: 2 or some nucleotide sequence thereof. In addition, since expression of glutathione-related genes is sensitively regulated and induced according to the presence or absence of toxic substances such as heavy metals, an expression vector including a gene encoding a reporter protein, such as a luminescent protein or a fluorescent protein, together with a promoter of the present invention is used. The recombinant strain prepared and transduced is useful and easily used for detecting toxic substances.

The present invention also provides gamma-glutamylcysteine synthase derived from Hanshenula polymorpha.

The present inventors have identified three protein coding sequences in the nucleotide sequence shown in SEQ ID NO: 1 having the function of restoring glutathione biosynthesis of a Hanshenula polymorpha mutant strain lacking glutathione biosynthesis due to a lack of gamma-glutamylcysteine synthase activity. After confirming that the gene sites (451 to 2403, 2436 to 3140, and 2691 to 3146) existed, an expression vector containing these genes was prepared and transformed using the transformants. Glutathione biosynthesis ability was investigated. As a result, it was found that only the transformants prepared with the gene containing the protein coding gene region of 451 to 2403 increased glutathione biosynthesis ability (see Table 2 ). Thus, as a result of performing a homology analysis of the sequence to analyze the protein encoding the protein coding gene region of the 451 to 2403, the sequence is Gcs1p (GenBank Accession No. AAG43412) of Candida albicans ( C. albicans ) 53% identity and 69% similarity to the gene and 47% identity and 61% similarity to the ScGsh1p gene of Saccharomyces cerevisiae. In addition, it showed 46% identity and 63% similarity to the SpGcs1p gene of Schizocarmyosis pombe, and 45% identity and 63% similarity to HsGCSh gene of human ( Homo sapiens ), and other gamma-glu It can be seen that the similarity with tamil cysteine synthase was also very high (see Fig. 2 ). Thus, the present inventors confirmed that the protein is gamma-glutamylcysteine synthase. The gamma-glutamylcysteine synthetase of the present invention has an amino acid sequence set forth in SEQ ID NO: 3 encoded from a protein coding gene sequence of Nos. 451 to 2403 of the nucleotide sequence set forth in SEQ ID NO: 1 above.

The present invention also provides a gene encoding the gamma-glutamylcysteine synthase.

The gene of the present invention is a gene encoding the enzyme described in SEQ ID NO: 3 which has been identified as a gamma-glutamylcysteine synthase of Hanshenula polymorpha, and preferably has a nucleotide sequence described in SEQ ID NO: 4 . The gene sequence of the present invention described in SEQ ID NO: 4 has a 5 'contiguous region sequence of about 450 bp and a 3' contiguous sequence of about 1792 bp, and typical TATA box, CAAT box, and polyadenylation sequences are observed. It doesn't work. But, A sequence presumed to be the CDEI-binding region (GCACG), known as the binding site of Cbf1, a protein that regulates the expression of the GSH1 gene in Saccharomyces cerevisiae at 416 bp upstream from the protein translation initiation codon, is observed ( FIG. 2 ). Therefore, it can be seen that the gene of the present invention is regulated by Cbf1 homologous protein depending on the presence of reactive oxygen groups or heavy metal cadmium.

In addition, the genes of the invention, like gamma-glutamylcysteine synthetases from other organisms, are conserved in the GWRVEFRPME motif set forth in SEQ ID NO: 5 , which relates to the specific interaction with glutathione commonly found in glutathione-binding proteins. Lysine and arginine residues, and have a well-conserved cysteine residue in the MGFGMGXXCLQ region as set forth in SEQ ID NO: 6 , presumably the active center of the enzyme (see FIG. 2 ).

From the above results, it can be seen that the gene of the present invention encodes γ-GCS which acts on the most important first-step response in the glutathione biosynthetic pathway as well as the glutathione requirement complementary function.

The present invention also provides an expression vector comprising the gene.

The expression vector of the present invention is an expression vector prepared to produce gamma-glutamylcysteine synthase, and is an expression vector capable of increasing the biosynthesis of glutathione. In a preferred embodiment of the present invention, the vector prepared by inserting the gene described in SEQ ID NO: 4 into the pYT3 vector was named 'pG24'.

The present invention also provides a transformant in which the vector is introduced into a host cell.

The host cell is preferably selected from the group consisting of bacteria, yeast, E. coli, fungi, plant cells and animal cells. In a preferred embodiment of the present invention, a transformant in which the pG24 expression vector was introduced into a Hanshenula polymorpha strain lacking glutathione biosynthetic enzyme was prepared and named 'Gsh ⁺ Leu ⁺ (pG24)'. In addition, as a result of examining the response to the glutathione biosynthesis ability and the heavy metal resistance of the transformant, it was confirmed that the glutathione biosynthesis ability is increased and the heavy metal resistance is increased (see Table 2 ).

The present inventors prepared a transformant transformed with the pG24 expression vector to E. coli DH5α, and deposited it on 8 November 2002 to the Korea Biotechnology Research Institute Gene Bank (Accession Number KCTC 10370BP).

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Cloning of Glutathione Biosynthesis Gene from Hanshenula Polymorpha

The present inventors have a resistance to M-N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) from the methanolic yeast Hansenula polymorpha strain, but are sensitive to cadmium ions and methanol, Glutathione deficient media has isolated gsh1 ( gsh1-2 leu1-1 ) and gsh2 ( gsh2-1 leu1-1 ) strains that cannot grow (Ubiyvovk et al ., Microbiology , 1999 , 68, 26). The gsh1 and gsh2 mutants have an extremely low total glutathione content and no gamma-glutamylcysteine synthetase (hereinafter, referred to as 'γ-GCS') activity, but are glutathione synthetase, or less than wild type strains. The activity of 'GS') was similar to that of the wild type strain, and the gsh2 mutant was very sensitive to cadmium, while the gsh1 mutant was very sensitive to chromium. Thus, the present inventors attempted to clone genes involved in glutathione biosynthesis from the gsh mutant strain.

<1-1> Screening for transformants that restore glutathione requirements

The present inventors century Cronulla poly Maurepas - genomic DNA library of E. coli shuttle plasmid, pYT3 (GenBank Accession No. AF347016) Hanse Cronulla poly Maurepas leu2 strain CBS4732 (Centraalbureau voor Schimmelcultures, Delft, Netherland ) in (Tan et al, J. Cell Biol ., 1995 , 128, 307) were cloned using the cloned vector and the conventional electrical methods (Faber) for the gins1-2 leu2 (495-145L) and gsh2-1 leu2 (495-40L) strains et al ., Curr Genet , 1994 , 25, 305). The transduced Hanshenula polymorpha strain was glutathione-free synthetic medium containing glucose (10 g / l glucose, 1 μg / l biotin, 0.2 mg / l thiamine hydrochloride, trace element, 20 mg / ml leucine). After incubation in (Sibirny et al ., Arch. Microbiol , 1990 , 154, 566), transformants that could grow without glutathione were selected.

As a result, the Gsh mutants had a function of complementing the glutathione requirements, thereby selecting a transformant that grew without glutathione. The plasmid was isolated from the transformant, digested with BamH I, and confirmed by electrophoresis. As a result, DNAs of about 7.5 kb and 9.6 kb were confirmed, and the vectors into which the DNA fragments were inserted were respectively 'pG1' and 'pG2'. The transformants transfected with the vector growing in glutathione free medium were named 'Gsh ⁺ Leu ⁺ (pG1)' and 'Gsh ⁺ Leu ⁺ (pG2)', respectively.

<1-2> Analysis of glutathione biosynthesis and resistance to heavy metals of transformants

The present inventors used the wild type strain NCYC495 leu1-1 (National Collection of Yeast Clustures, Food Reaearch Institute, Norwich, UK), gsh mutation to analyze whether the transformants selected in Example <1-1> biosynthesis of glutathione. Transformants Gsh ⁺ Leu ⁺ (pG1) and Gsh ⁺ Leu ⁺ (pG2) with strains gsh1-2 leu1-1, gsh2-1 leu1-1 and glutathione gene were added to the semisynthetic medium (1) Culture medium in% glycerol, 0.05% yeast extract, 20 mg / l leucine, and then 1% glycerol added synthetic medium (glycerol 1%, biotin 1 ug / l, thiamine hydrochloride 0.2 mg / l, trace element, louis) Shin 20 mg / ml) (Sibirny et al ., Arch. Microbiol , 1990 , 154, 566) for 14 hours. After the incubation, the total glutathione content (nmol) contained in the protein (mg) was measured from the cell lysate by the method of Brehe and Burch, Anal. Biochem ., 1976 , 74, 189. In addition, the activity of gamma-glutamylcysteine synthase (γ-GCS), which is an enzyme involved in the biosynthesis of glutathione, and glutathione syntase (hereinafter, abbreviated as 'GS') Γ-GCS contained in protein (mg) for 30 minutes according to the method of Ubibok (Ubiyvovk et al ., Microbiology , 1999 , 68, 26) from cells in the mid- log phase cultured in semi-synthetic medium to analyze The content of GS (nmol) was measured. In addition, yeast cells were cultured for 3 days in a solid synthetic medium containing 1% glucose and heavy metals such as cadmium (0.05 mM) or chromium (0.4 mM) in order to analyze resistance to heavy metals, and then yeast growth was observed and analyzed. .

As a result, the transformants Gsh ⁺ Leu ⁺ (pG1) and Gsh ⁺ Leu ⁺ (pG2) have increased glutathione biosynthesis and wild-type growth ability in synthetic media based on methanol or multiple carbon sources (glucose and methanol) as substrates. In addition, the resistance to cadmium (Cd) and chromium (Cr) ions appeared to be similar to that of wild-type strains, and was more sensitive to MNNG than the untransformed gsh1 and gsh2 mutants themselves ( Table 1 ).

Comparison of Physiological Characteristics of gsh Variants and Gsh + Transformants Strain Total glutathione content Growth degree Growth degree Glucose Methanol Glucose + chromium Glucose + cadmium NCYC495 leu1-1 202 +++ ++ + ++ gsh1-2 leu1-1 <1.0 - - - - gsh2-1 leu1-1 <1.0 - - - - Gsh ⁺ Leu ⁺ (pG1) 83 ++ + ++ ++ Gsh ⁺ Leu ⁺ (pG2) 87 ++ + ++ ++

+++: strongest growth, ++: moderate growth,

+: Slight growth,-: no growth

Thus, insert DNAs of pG1 and pG2 vectors introduced into Gsh ⁺ Leu ⁺ (pG1) and Gsh ⁺ Leu ⁺ (pG2) transformants were analyzed ( Molecular cloning : a laboratory manual, 2nd edn. Cold Spring harbor laboratory press, Cold spring harbor, NY, 1989 ).

As a result, the 7.5 kb DNA fragment cleaved with BamH I of the pG1 vector contained four protein coding sites but was not related to the glutathione synthesis gene. Next, the 9.6 kb DNA fragment cleaved with BamH I of the pG2 vector was further digested with BamH I or Xba I, resulting in 3.9 kb ( Bam HI), 4.0 kb ( Bam HI), 3.3 kb ( Xba I) and 4.2 kb. It can be seen that it is cleaved into a small DNA fragment of ( Xba I). Thus, the vectors prepared by inserting each of the DNA fragments into the pYT3 vector were named as pG21, pG22, pG23 and pG24, respectively ( Fig. 1 ). The vector was transformed back into gsh2 mutant strains, so that each transformant transformed into 'Gsh ⁺ Leu ⁺ (pG21)', 'Gsh ⁺ Leu ⁺ (pG22)', 'Gsh ⁺ Leu ⁺ (pG23)' and It was named 'Gsh ⁺ Leu ⁺ (pG24)'. As a result of analyzing the transformants for glutathione biosynthesis and heavy metal resistance in the same manner as above, it was confirmed that only Gsh ⁺ Leu ⁺ (pG24) transformants biosynthesized glutathione as wild type ( Table 2 ).

Comparison of Transformant Phenotypes with gsh2 Variants and pG2 Family Vectors Strain Total glutathione content Growth degree Growth degree Glucose Methanol Glucose + chromium Glucose + cadmium NCYC495 leu1-1 202 +++ ++ + ++ gsh2-1 leu1-1 <1.0 - - - - Gsh ⁺ Leu ⁺ (pG21) <1.0 - - - - Gsh ⁺ Leu ⁺ (pG22) <1.0 - - - - Gsh ⁺ Leu ⁺ (pG23) <1.0 - - - - Gsh ⁺ Leu ⁺ (pG24) 87 ++ + ++ ++

+++: strongest growth, ++: moderate growth,

+: Slight growth,-: no growth

Example 2 Analysis of Glutathione Biosynthesis Gene

The present inventors isolated pG24 plasmid DNA from Gsh ⁺ Leu ⁺ (pG24) transformants confirmed that the glutathione biosynthetic ability was returned to the wild type in Example 1 using a standard yeast gene method, and agarose gel electrophoresis was performed. The size of the isolated DNA was confirmed ( Molecular cloning : a laboratory manual, 2nd edn. Cold Spring harbor laboratory press, Cold spring harbor, NY, 1989 ). DNA sequencing of the identified DNA was performed using the GPS-1 genome priming system (New England Biolabs, Beverly, MA, USA) and the BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, USA). ) Was performed.

As a result, it was confirmed that the inserted DNA fragment of the pG24 vector was the nucleotide sequence described in SEQ ID NO: 1 in total 4195 bp. There were three open reading frames (ORFs) of 593 bp, 456 bp and 1953 bp in the total DNA. PG22 vector comprising the three protein-coding region of 593 bp and 456 bp in size small protein coding region of was not not return a glutathione deficiency capacity of gsh2 mutants, the similarity with the reported protein is very low so far (Table 2 ). In contrast, the pG24 vector, which contains the largest protein coding region that encodes 650 amino acids at 1953 bp, had the ability to convert gsh2 mutants into traits with glutathione synthesis, and the gene encoding the protein is Candida albican. ( C. albicans ) Gcs1p (GenBank Accession No. AAG43412) gene showed 53% identity and 69% similarity. It also shows 47% identity and 61% similarity to the ScGsh1p gene of S. cerevisiae and 46% identity to the SpGcs1p gene of Sch. Pombe . It showed 63% similarity, 45% identity and 63% similarity with HsGCSh gene of human ( Homo sapiens ), and showed significantly high similarity with other γ-GCS ( FIG. 2 ). Thus, the present inventors assumed that the 4195 bp pG24 plasmid DNA was a gene encoding Hanshenula polymorpha γ-GCS and named it as 'HpGSH2' and deposited it in GenBank (Accession No. AF435121).

<Example 3> HpGSH2 Compositional Characterization of Genes

The present inventors performed the protein coding region, protein sequence homology analysis and sequence alignment analysis using the nucleotide sequence shown in SEQ ID NO: 1 to analyze the detailed components present in the nucleotide sequence of the HpGSH2 gene isolated in Example 2 . Specifically, an open reading frame (ORF) was analyzed using ORF Finder, a graph analysis program of the National Center for Biotechnology Information, Bethesda, MD, USA. Protein sequence homology analysis was performed using the BLAST server, and sequence alignment analysis was performed using the MultAlin program (Corpet, F. Nucleic Acids Res ., 198 8, 16, 10881).

As a result, the HpGSH2 gene described in SEQ ID NO: 1 has a 5 'contiguous region sequence of about 450 bp (from 1 to 450) and a 3' contiguous sequence of about 1792 bp (from 2401 to 4195). Typical TATA boxes, CAAT boxes and polyadenylation sequences were not observed. But, Sequence estimated to be the CDEI-binding region (GCACG) known as the binding site of Cbf1, a protein that regulates expression of the GSH1 gene in Saccharomyces cerevisiae at 416 bp upstream from the protein translation initiation codon (Nos. 31 to 35). Times) were observed ( FIG. 2 ). Through this, the expression of the Hansenula polymorpha gene HpGSH2 is regulated by Cbf1 homologous protein depending on the presence of reactive oxygen groups or heavy metal cadmium.

Also, like gamma-glutamylcysteine synthetases from other organisms, the Hansenula polymorph HpGsh2p is a GWRVEFRPME motif described by SEQ ID NO: 5 which is involved in the specific interaction with glutathione commonly found in glutathione-binding proteins. ( Numbers 457 to 466 of the amino acid sequence set forth in SEQ ID NO: 3 ) and conserved lysine or arginine residues (amino acid residues 459 and 503 of the amino acid sequence set forth in SEQ ID NO: 3 ) (Coblenz and Wolf, Yeast, 1995 , 11, 1171), having a well-conserved cysteine residue (251nd to 261th of the amino acid sequence set forth in SEQ ID NO: 3) in the MGFGMGXXCLQ region described as SEQ ID NO: 6 , presumed to be the active center of the enzyme. 2 ). Thus, the structural features, together with the glutathione complimentary function of gsh2 mutants, suggest that the HpGSH2 gene encodes γ-GCS, which acts as the most important first-stage response in the glutathione biosynthetic pathway.

<Example 4> HpGSH2  Copy number and structure analysis of genes

Copy number and structure of HpGSH2 gene from Hanshenula polymorpha wild type strain (CBS4732 leu2 ), gsh 2 variant (gsh2-1 leu1-1) and plasmid pG24 introduced gsh2 transformant (Gsh ⁺ Leu ⁺ (pG24)) Southern blot was performed to obtain information about. Specifically, a 2.0 kb HpGSH2 fragment ( A of FIG. 3 ) obtained by cutting the HpGSH2 gene with Bam HI was prepared to prepare a DNA probe. Chromosomes were isolated from each of the cells and digested with restriction enzyme Pst I, followed by Southern blot analysis using a DNA probe prepared above ( Molecular cloning : a laboratory manual, 2nd edn. Cold Spring harbor laboratory press, Cold spring harbor, NY, 1989 ).

As a result, the hybridization signal was found in the 4.0 kb Pst I fragment corresponding to a single chromosome copy of the HpGSH2 gene in the wild type strain, and in the 6.5 kb Pst I fragment instead of the 4.0 kb Pst I fragment in the untransformed gsh2 strain. Hybridization signal appeared. In addition, the gsh2 transformant (Gsh ⁺ Leu ⁺ (pG24)) into which the plasmid pG24 was introduced was subjected to two hybridizations by Pst I fragment (4.0 kb) from pG24 introduced and Pst I fragment (6.5 kb) from chromosomal DNA. The signal appeared and the 4.0 kb fragment showed a darker signal than the 6.5 kb fragment ( B in FIG. 3 ). The results show that the gsh2 mutation will be located in one of the two Pst I sites surrounding the HpGSH2 ORF, and the introduction of the wild-type HpGSH2 gene complements the defective phenotype of the gsh2 mutant strain. It was found that multiple HpGSH2 genes were introduced into the transformants. Therefore, γ-GCS complementary ability of HpGSH2 gene for a deficient phenotype and is a HpGSH2 gene obtained by the present invention encoding a γ-GCS of a century Cronulla poly Maurepas, resistance to heavy metals of gsh2 transformant increased (Table 1 and Table 2 ) was found to be induced by multiple introduction of the HpGSH2 gene.

The present inventors prepared a transformant transformed with the pG24 expression vector to E. coli DH5α, and deposited it on 8 November 2002 to the Korea Biotechnology Research Institute Gene Bank (Accession Number KCTC 10370BP).

As described above, when the glutathione biosynthesis gene isolated from the Hanshenula polymorpho strain of the present invention is introduced into the glutathione biosynthetic defect mutant strain, glutathione biosynthesis function can be restored or increased in addition to glutathione biosynthesis function, and thus glutathione can be used. It can be usefully used as heavy metal adsorption, production of coenzyme, liver function enhancer and detoxicant.

1 is a map showing a cleavage site by a restriction enzyme of a 9.6 kb DNA fragment inserted into a pG2 plasmid,

Figure 2 is a homologous analysis of the nucleotide sequence of Hanshenula polymorpha HpGSH2 gene and other glutathione biosynthetic enzyme gene,

ScGsh1p: Saccharomyces cerevisiae CaGcs1p: Candida albicans

SpGcs1p: Ski Research Caromyces Pombe and HsGCSh: Human

Figure 3 is a probe DNA (A) prepared for Southern blot analysis for copy number analysis of HpGSH2 gene and electrophoresis picture (B) Southern blot analysis of the gene of wild strain or transformant using the probe .

Lane 1: DNA size marker,

Lane 2: gsh2-1 leu1-1 mutant,

Lanes 3 to 7: Gsh ⁺ Leu ⁺ (pG24) strains, and

Lane 8: NCYC495 leu1-1 wild-type strain.

<110> Korea Research Institute of Bioscience and Biotechnology Institute of Cell Biology <120> Gamma-glutamylcysteine synthetase and gene coding for the same <130> 2p-11-22 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 4195 <212> DNA <213> Hansenula polymorpha <220> <221> gene (222) (1) .. (4195) <223> gamma-glutamylcysteine synthetase complete cds and unknown gene <400> 1 tctagaatca gcctccacat aagccagcga gcacgggcct gcctgcgtct gcacaaggta 60 tcctgcagtc gtcgggctgg cagtgttacc gcttgtctgc aagttggggc ccagaacaag 120 ctcggatcct gcctccagcg taaattggtc tccgggaagt aacgatagac atggcaggta 180 gcaaaaaaaa aaaaaatgga gcggaaaaaa ttgtacagat cagacgcgtc gatcgacgcc 240 cactcgctga taaccaagca ccgcatgtag tcattatttg cttgatcacc cgaaatcaag 300 tgagtgtacg acaactaccc aaacatgtgg acagcggcca cccgcgcctg aatgcggatt 360 tgttagccga ctttaaaaat gcagcattcg cttgtacgtg ctgacaaata attgagaaag 420 gaaaaacttt cctgaaaaac cttatcgacc atgggtctgc tctctctggg tacgcctcta 480 cattggaacg actcccgtct ctatgctgat catgtgcgca caaacggtat cattcagctg 540 ataaactgtt tcaacgcggc acgagaccgc aagaatgacc cgtttttgtg gggagacgaa 600 gtggaatata tgctggtgaa gctggacagg gagcacaaag ttgctcgtct tgccatcaac 660 aaggaccatc ttttgaaaga ccttggcgag aatggctcat ccttcgaggt ggcggtcagt 720 aataatgtgg tgttccaccc cgagtacggc agatacatga tagaagcaac tccgctccgg 780 ccctacgatg ggacgaaagt ggaagagttt gaatatgttg agcataacat gagaactaga 840 cggcagattg ccacacagga gcttggagaa gaagatatgt tgccattgac actcacttct 900 tttccgcgaa tgggcgttga gatttttaca tatccggccg ccgagcctaa cggtgaagcg 960 tccaagtctc tcttccttcc ggacgagatc atcaaccgcc acttccgttt tccaacgctg 1020 acagccaaca ttcgtcgcag aagggaccag aaggtgagta tcaacatccc tctgtacaaa 1080 gacgagaaaa ctgtggcttc aggaattgat ccgtccattc ctcgaaggaa cttgttcccg 1140 catcacgacg aagaaccatt tttgggggcc gccaagaagg gctacatata catggactcg 1200 atgggctttg gcatgggctc gtcgtgtctg caagtaacga tgcaggcccc agatgtggct 1260 catgctagat tcctgtacga ctctctggtc aacatcgctc cattgatgct ggctgtgaca 1320 gctgcggctc cgattttccg gggccatctt gctgatcagg acgttagatg gaatgtgatt 1380 gccgcagcag ttgatgatcg gaccccgtat gagcgcggag agccgccatt gaagggtcac 1440 aaagagagag ggaacacttt ggacacagca gagcttgtgc gcataccaaa gtcacggtat 1500 gactcggtgg accagtacct gggtgacttg gattcttccg gcagattctc ctatttcaga 1560 aaagagtaca atgacatcga atctccgaaa aatagcaagg tttacgaaaa acttgtttcc 1620 aatgggtttg acgaaacact ggcgggccat ttcgcacacc tgttcatccg ggaccctatc 1680 gtcattttca ccgaatccat agagcaggac aacaccgtcg agaccgacca tttcgaaaac 1740 atccagtcta caaactggca gacactgcgt ttcaagcctc caaaacagtc tgctgtgccg 1800 gaaaggcacg atgttccggg gtggagagtt gagctccggc caatggaaat ttccttgaca 1860 gatttcgaaa acgctgcgta tgcaaacttt tccgtcctgc tctcgctagc ggtgctcaaa 1920 tatagaccaa acttctacct ccctatttcg tatgtggagg ctaatatgaa gacggcgcat 1980 cgccggaatg ccatagtgga gcagacattc cacttccgaa ccaatgtgtg ggaggcagga 2040 gaggcaattg tggagcagct gagtcttgaa gaggttttcc acggatccgg ctctttcgag 2100 ggacttctat ctctagttta ccgatacatc aaagagacat ggggagaacc tcttcccgca 2160 aagcttgagg cgtacctcaa gctggtgagc tttagagctt cggcaaaaat cccatccaca 2220 gcgcaataca tacggaattt tgtcgtgagt catccagatt acaagaagga cagtattgtc 2280 agcgaccaaa taaactacga cctgctggag atggcggcca aactgtccac atacgatcac 2340 ggcctggtga ctgacttctt cgggcccgaa ctgggtcaat ggttaattga taatggttac 2400 tagataaaaa acataaacca tacagctgcg caaacctagt tctcgttttc gctttcggac 2460 tcagactgag agtcgaaggc ttccttgtgg acttcctcct gagccttagc tggtctcttg 2520 tgcttctgtc tgcgtccctt ctcgaccttc tcgaccttct cttcctcgtc ctttggctcc 2580 tcgtcttttg gctccttctc caacttctcc ttcgaatctt ccttaaggtg cttgtcctgt 2640 ttcgtcttgg agaactcgcc tcccttgtgg cccttcaact tcattccctt atgatgtggt 2700 ctgtcgtgag ccttgtgctc ctcgatttca aactcgtgtt cggtctctct gagttcacct 2760 tcagcaccct tgatgctctt gtggcccttg tgcatctcgt gagccttcag catgccgccc 2820 ttcttgtcca tcttgcagtc cttgaatggc ttgacctcat cttccatctc gtgcatgcct 2880 tcgaatcccg gtggagggcg gtgtgctccg tggaatccgt gggcatgtgg gtggtgtgga 2940 tgggtcaagg acttgacacc gtggacaacc ggaccggact gcaaaaccag acaggcaacg 3000 aagaacaaaa acactcccaa gccgagtttg gcaatctttt tgttggtctc atatctgctc 3060 ttgatcttgt agagtttctc gacctcctcg cgaggcaaag tgacgtcggt gtattctggc 3120 aattctgttt tagaagacat ggttaacaac aaggaaaaat tgcacgctat ataaatatca 3180 ctcgataagc caccaaacta agcatattcc aaaacgggct tcccaaacgc atttgaatca 3240 ccgaataaaa tttttggcaa agcccgatga cataatatta caatccgagg ctgacaaacg 3300 cgcttggcaa ggcaggaggg ttcggatggg acctaagcaa gtgtgtgagc gcctcatagg 3360 gctatcaccg ataagtcttc gtccaatcgc acatctgagc attttcagaa gtaagctgct 3420 gtgacactgt gtgactactt gactatttga cttgggtaca aggcccctaa actaagtaag 3480 cagcagtctt ccagactcct ttctgcatat aggcaatctt atacgaaact agcacacttt 3540 tttgcatata tggttggtgt acagatgtcg aatattattt ttcaagattt ttccccagtc 3600 gattgacttt ccttatgtat cgaccctcaa ctggtctgct caatcagcgt tcagtgccgt 3660 tatttgcccc tcgcttttat tcatcagctt gcagaaaata tgggacagcg acacagcaaa 3720 atccggattt tctttcccca ccagaacctc aaaattggta cgcacttcta accaaaccac 3780 cttcccaaaa taaacatcgc ttagccaatt tccaaaagga ggtcatcgaa ggtctccgat 3840 cgccgtcata cgattccttc aactcgctct tcatcaagcc ctacagacga cacagagatt 3900 tggcgcatgt cacaaactcc aagagcgccc tcccccatgt tcctagttcc tacgatgctc 3960 cagaagcatt gcgcaaacaa ctgaaacgca ggcccttacc ctttcaccta tcccatcgac 4020 tttcgacata tctgcagaag aagagcctgc gtgatatagg ttccatctat tttgaaatcc 4080 ctgcaccacg agcagctagt ttttccatac ttcagctgga gcagcttcta tcgctgattt 4140 taagcaccaa gcgagccaat attgagattg tgaatgtggc gagacgtatt ctaga 4195 <210> 2 <211> 450 <212> DNA <213> Hansenula polymorpha <220> <221> promoter (222) (1) .. (450) Gamma-glutamylcysteine synthetase <400> 2 tctagaatca gcctccacat aagccagcga gcacgggcct gcctgcgtct gcacaaggta 60 tcctgcagtc gtcgggctgg cagtgttacc gcttgtctgc aagttggggc ccagaacaag 120 ctcggatcct gcctccagcg taaattggtc tccgggaagt aacgatagac atggcaggta 180 gcaaaaaaaa aaaaaatgga gcggaaaaaa ttgtacagat cagacgcgtc gatcgacgcc 240 cactcgctga taaccaagca ccgcatgtag tcattatttg cttgatcacc cgaaatcaag 300 tgagtgtacg acaactaccc aaacatgtgg acagcggcca cccgcgcctg aatgcggatt 360 tgttagccga ctttaaaaat gcagcattcg cttgtacgtg ctgacaaata attgagaaag 420 gaaaaacttt cctgaaaaac cttatcgacc 450 <210> 3 <211> 650 <212> PRT <213> Hansenula polymorpha <220> <221> PEPTIDE (222) (1) .. (650) Gamma-glutamylcysteine synthetase <400> 3 Met Gly Leu Leu Ser Leu Gly Thr Pro Leu His Trp Asn Asp Ser Arg 1 5 10 15 Leu Tyr Ala Asp His Val Arg Thr Asn Gly Ile Ile Gln Leu Ile Asn 20 25 30 Cys Phe Asn Ala Ala Arg Asp Arg Lys Asn Asp Pro Phe Leu Trp Gly 35 40 45 Asp Glu Val Glu Tyr Met Leu Val Lys Leu Asp Arg Glu His Lys Val 50 55 60 Ala Arg Leu Ala Ile Asn Lys Asp His Leu Leu Lys Asp Leu Gly Glu 65 70 75 80 Asn Gly Ser Ser Phe Glu Val Ala Val Ser Asn Asn Val Val Phe His 85 90 95 Pro Glu Tyr Gly Arg Tyr Met Ile Glu Ala Thr Pro Leu Arg Pro Tyr 100 105 110 Asp Gly Thr Lys Val Glu Glu Phe Glu Tyr Val Glu His Asn Met Arg 115 120 125 Thr Arg Arg Gln Ile Ala Thr Gln Glu Leu Gly Glu Glu Asp Met Leu 130 135 140 Pro Leu Thr Leu Thr Ser Phe Pro Arg Met Gly Val Glu Ile Phe Thr 145 150 155 160 Tyr Pro Ala Ala Glu Pro Asn Gly Glu Ala Ser Lys Ser Leu Phe Leu 165 170 175 Pro Asp Glu Ile Ile Asn Arg His Phe Arg Phe Pro Thr Leu Thr Ala 180 185 190 Asn Ile Arg Arg Arg Arg Asp Gln Lys Val Ser Ile Asn Ile Pro Leu 195 200 205 Tyr Lys Asp Glu Lys Thr Val Ala Ser Gly Ile Asp Pro Ser Ile Pro 210 215 220 Arg Arg Asn Leu Phe Pro His His Asp Glu Glu Pro Phe Leu Gly Ala 225 230 235 240 Ala Lys Lys Gly Tyr Ile Tyr Met Asp Ser Met Gly Phe Gly Met Gly 245 250 255 Ser Ser Cys Leu Gln Val Thr Met Gln Ala Pro Asp Val Ala His Ala 260 265 270 Arg Phe Leu Tyr Asp Ser Leu Val Asn Ile Ala Pro Leu Met Leu Ala 275 280 285 Val Thr Ala Ala Ala Pro Ile Phe Arg Gly His Leu Ala Asp Gln Asp 290 295 300 Val Arg Trp Asn Val Ile Ala Ala Ala Val Asp Asp Arg Thr Pro Tyr 305 310 315 320 Glu Arg Gly Glu Pro Pro Leu Lys Gly His Lys Glu Arg Gly Asn Thr 325 330 335 Leu Asp Thr Ala Glu Leu Val Arg Ile Pro Lys Ser Arg Tyr Asp Ser 340 345 350 Val Asp Gln Tyr Leu Gly Asp Leu Asp Ser Ser Gly Arg Phe Ser Tyr 355 360 365 Phe Arg Lys Glu Tyr Asn Asp Ile Glu Ser Pro Lys Asn Ser Lys Val 370 375 380 Tyr Glu Lys Leu Val Ser Asn Gly Phe Asp Glu Thr Leu Ala Gly His 385 390 395 400 Phe Ala His Leu Phe Ile Arg Asp Pro Ile Val Ile Phe Thr Glu Ser 405 410 415 Ile Glu Gln Asp Asn Thr Val Glu Thr Asp His Phe Glu Asn Ile Gln 420 425 430 Ser Thr Asn Trp Gln Thr Leu Arg Phe Lys Pro Pro Lys Gln Ser Ala 435 440 445 Val Pro Glu Arg His Asp Val Pro Gly Trp Arg Val Glu Leu Arg Pro 450 455 460 Met Glu Ile Ser Leu Thr Asp Phe Glu Asn Ala Ala Tyr Ala Asn Phe 465 470 475 480 Ser Val Leu Leu Ser Leu Ala Val Leu Lys Tyr Arg Pro Asn Phe Tyr 485 490 495 Leu Pro Ile Ser Tyr Val Glu Ala Asn Met Lys Thr Ala His Arg Arg 500 505 510 Asn Ala Ile Val Glu Gln Thr Phe His Phe Arg Thr Asn Val Trp Glu 515 520 525 Ala Gly Glu Ala Ile Val Glu Gln Leu Ser Leu Glu Glu Val Phe His 530 535 540 Gly Ser Gly Ser Phe Glu Gly Leu Leu Ser Leu Val Tyr Arg Tyr Ile 545 550 555 560 Lys Glu Thr Trp Gly Glu Pro Leu Pro Ala Lys Leu Glu Ala Tyr Leu 565 570 575 Lys Leu Val Ser Phe Arg Ala Ser Ala Lys Ile Pro Ser Thr Ala Gln 580 585 590 Tyr Ile Arg Asn Phe Val Val Ser His Pro Asp Tyr Lys Lys Asp Ser 595 600 605 Ile Val Ser Asp Gln Ile Asn Tyr Asp Leu Leu Glu Met Ala Ala Lys 610 615 620 Leu Ser Thr Tyr Asp His Gly Leu Val Thr Asp Phe Phe Gly Pro Glu 625 630 635 640 Leu Gly Gln Trp Leu Ile Asp Asn Gly Tyr 645 650 <210> 4 <211> 1950 <212> DNA <213> Hansenula polymorpha <220> <221> gene (222) (1) .. (1950) <223> gamma-glutamylcysteine synthetase ORF <400> 4 atgggtctgc tctctctggg tacgcctcta cattggaacg actcccgtct ctatgctgat 60 catgtgcgca caaacggtat cattcagctg ataaactgtt tcaacgcggc acgagaccgc 120 aagaatgacc cgtttttgtg gggagacgaa gtggaatata tgctggtgaa gctggacagg 180 gagcacaaag ttgctcgtct tgccatcaac aaggaccatc ttttgaaaga ccttggcgag 240 aatggctcat ccttcgaggt ggcggtcagt aataatgtgg tgttccaccc cgagtacggc 300 agatacatga tagaagcaac tccgctccgg ccctacgatg ggacgaaagt ggaagagttt 360 gaatatgttg agcataacat gagaactaga cggcagattg ccacacagga gcttggagaa 420 gaagatatgt tgccattgac actcacttct tttccgcgaa tgggcgttga gatttttaca 480 tatccggccg ccgagcctaa cggtgaagcg tccaagtctc tcttccttcc ggacgagatc 540 atcaaccgcc acttccgttt tccaacgctg acagccaaca ttcgtcgcag aagggaccag 600 aaggtgagta tcaacatccc tctgtacaaa gacgagaaaa ctgtggcttc aggaattgat 660 ccgtccattc ctcgaaggaa cttgttcccg catcacgacg aagaaccatt tttgggggcc 720 gccaagaagg gctacatata catggactcg atgggctttg gcatgggctc gtcgtgtctg 780 caagtaacga tgcaggcccc agatgtggct catgctagat tcctgtacga ctctctggtc 840 aacatcgctc cattgatgct ggctgtgaca gctgcggctc cgattttccg gggccatctt 900 gctgatcagg acgttagatg gaatgtgatt gccgcagcag ttgatgatcg gaccccgtat 960 gagcgcggag agccgccatt gaagggtcac aaagagagag ggaacacttt ggacacagca 1020 gagcttgtgc gcataccaaa gtcacggtat gactcggtgg accagtacct gggtgacttg 1080 gattcttccg gcagattctc ctatttcaga aaagagtaca atgacatcga atctccgaaa 1140 aatagcaagg tttacgaaaa acttgtttcc aatgggtttg acgaaacact ggcgggccat 1200 ttcgcacacc tgttcatccg ggaccctatc gtcattttca ccgaatccat agagcaggac 1260 aacaccgtcg agaccgacca tttcgaaaac atccagtcta caaactggca gacactgcgt 1320 ttcaagcctc caaaacagtc tgctgtgccg gaaaggcacg atgttccggg gtggagagtt 1380 gagctccggc caatggaaat ttccttgaca gatttcgaaa acgctgcgta tgcaaacttt 1440 tccgtcctgc tctcgctagc ggtgctcaaa tatagaccaa acttctacct ccctatttcg 1500 tatgtggagg ctaatatgaa gacggcgcat cgccggaatg ccatagtgga gcagacattc 1560 cacttccgaa ccaatgtgtg ggaggcagga gaggcaattg tggagcagct gagtcttgaa 1620 gaggttttcc acggatccgg ctctttcgag ggacttctat ctctagttta ccgatacatc 1680 aaagagacat ggggagaacc tcttcccgca aagcttgagg cgtacctcaa gctggtgagc 1740 tttagagctt cggcaaaaat cccatccaca gcgcaataca tacggaattt tgtcgtgagt 1800 catccagatt acaagaagga cagtattgtc agcgaccaaa taaactacga cctgctggag 1860 atggcggcca aactgtccac atacgatcac ggcctggtga ctgacttctt cgggcccgaa 1920 ctgggtcaat ggttaattga taatggttac 1950 <210> 5 <211> 10 <212> PRT <213> Hansenula polymorpha <400> 5 Gly Trp Arg Val Glu Phe Arg Pro Met Glu 1 5 10 <210> 6 <211> 11 <212> PRT <213> Hansenula polymorpha <400> 6 Met Gly Phe Gly Met Gly Xaa Xaa Cys Leu Gln 1 5 10

Claims (9)

A genomic DNA from Nos. 1 to 2403 of SEQ ID NO: 1 constituting a promoter and gene of gamma-glutamylcysteine synthase derived from Hanshenula polymorpha. A promoter of gamma-glutamylcysteine synthetase derived from Hansenula polymorpha as set forth in SEQ ID NO: 2. A gamma-glutamylcysteine synthetase derived from Hansenula polymorpha as set forth in SEQ ID NO: 3. The gene encoding the synthetase of claim 3. The gene of claim 4, wherein the gene is set forth in SEQ ID NO: 4. 6. An expression vector comprising the gene of claim 4. The expression vector of claim 6, wherein the expression vector is pG24. A transformant transformed with the expression vector of claim 6 into a host cell. The transformant according to claim 8, wherein the pG24 expression vector is transfected into E. coli DH5α (Accession Number KCTC 10370BP).