KR20200084220A - Novel cell-wall lysis mutant strains of yeast and method for producing glutathione using the same - Google Patents

Abstract

The present invention relates to conditional cell-wall lysis mutant strains of yeast and a glutathione extracellular secretion production method using the same based on the regulation of expression of OCH1 and CHS3 genes involved in cell wall biosynthesis of Saccharomyces cerevisiae. OCH1 is a gene that encodes α-1,6-mannosyl transferase, an enzyme involved in the initiation of biosynthesis of the outer chain of the N-sugar chain attached to the yeast cell wall glycoprotein, and CHS3 is a gene that encodes chitin biosynthetic enzyme III, which is involved in the synthesis of most of the cell wall chitin. Instead of gene disruption that inhibits cell growth, strains that inhibit the expression of OCH1 and CHS3 genes were developed by using a promoter that inhibits activity under certain conditions, such as a MET3 promoter whose activity is inhibited by methionine. These strains showed a conditional cell wall lysis phenotype that was tightly regulated with or without methionine. The present inventors have observed that the conditional cell wall lysis mutant strains of yeast achieve remarkably improved glutathione extracellular production efficiency, thereby suggesting a high possibility of application as glutathione extracellular secretion-producing strains with high industrial economics.

Description

Novel cell-wall lysis mutant strains of yeast and method for producing glutathione using the same}

The present invention is conditionally based on a new cell wall degradation mutant yeast strain, and relates to a glutathione production method using the same, and more particularly, OCH1 and CHS3 gene expression involved in cell wall biosynthesis of my process three Levy jiae (Saccharomyces cerevisiae) in Saccharomyces Cell wall decomposition mutant yeast strains and relates to a method for producing glutathione extracellular secretion using the same.

Traditional yeast Saccharomyces Saccharomyces cerevisiae ) has been widely used as a host for the production of recombinant proteins and metabolites with industrial potential. The cell wall of yeast is involved in several processes such as budding, mating, aggregation and sporulation, and is a key function in stabilizing intracellular osmotic conditions, protecting against stress and maintaining cell morphology To perform. However, the thick and hard yeast cell wall is a major obstacle to the efficient recovery of products such as separation and purification. In order to recover intracellular products, cell wall crushing through physical, chemical, or enzymatic crushing methods is required. These methods cause problems such as loss of products and an increase in production cost. For some recombinant proteins, using a protein secretion pathway to release from the host cell is an alternative approach that does not require cell wall crushing. However, protein secretion production also faces some limitations such as low secretion efficiency, abnormal post-translational fertilization, or retention within the cell wall. In particular, large and complex recombinant proteins, such as virus-like particles (VLPs) and highly mannosylated proteins, are difficult to release effectively through the secretory pathway. Accordingly, there is a need to develop an inexpensive and effective method of crushing yeast cell walls to efficiently release intracellular contents from yeast and enhance secretion efficiency.

Glutathione (GSH) is the most common organic sulfur compound in cells and is present in high concentrations (0.2-10 mM) in most animal, plant and microbial cells. Glycine (glutine), glutamate (glutamate), and cysteine (cysteine) are three amino acid bound peptides (tripeptide). In cells, there are two types of reduced glutathione (GSH) and oxidized glutathione (GSSG). Reduced glutathione (GSH), which is present in a relatively high proportion, is mainly distributed in the liver and skin cells of the human body, and also plays an important role in the antioxidant function of inhibiting the production of melanin pigment and decomposing and removing free radicals. In addition, it affects the immune system and contributes to the enhancement of immunity, as well as the removal of exogenous compounds such as toxic substances. Thanks to these functions and effects, glutathione has been commercialized in various ways. Although it is basically produced in the body, as aging progresses, the amount of glutathione gradually decreases, and the decrease in the amount of glutathione that plays an important role in antioxidant and detoxification causes accumulation of free radicals, the main cause of aging, and further promotes aging. Therefore, the need to supply glutathione is emerging from the outside, and thanks to this need, glutathione is being used in various fields such as the pharmaceutical, food, and cosmetic industries. Glutathione is mainly used in the form of whitening creams, injections, nutrients, etc. As glutathione-containing foods that directly ingest glutathione-producing yeast are also released, the glutathione market has a potential for growth due to its functions and needs.

Previously, glutathione was produced by extraction or chemical synthesis from living organisms. Currently, mass production of glutathione by enzymes or microorganisms involved in the glutathione biosynthetic pathway is being performed. As a microorganism for the production of glutathione, yeast, which contains high glutathione in cells and recognized as a safe microorganism for food production, has been widely used. In particular, Saccharomyces cerevisiae ) and Candida utilis are typical. In the wild-type strains of these species, the concentration of glutathione is already quite high and varied to about 0.1-1% of the dry weight. In addition, fermentation using yeast capable of high-density and rapid growth in a cheap culture medium has high economic efficiency, and unlike bacteria, there is an advantage that there is no risk of endotoxin in the final product.

Glutathione biosynthesis is achieved by two stages of continuous ATP-consuming reactions in cells. That is, γ-GC is formed from glutamic acid and cysteine by γ-glutamylcysteine (γ-GC) synthetase (γ-GCS), which is encoded by the GSH1 gene, and GSH2 The glutathione synthase (GS) encoded from the gene produces glutathione from γ-GC and glycine. Since the activity of γ-GCS increases the amount of glutathione, the final product, it is subject to feedback inhibition by glutathione, so the final production amount of glutathione remains constant. An enzymatic glutathione production method using permeated cells has been developed as a strategy to eliminate the suppression of feedback by glutathione accumulated in cells. However, the enzymatic glutathione production method requires an external supply of ATP and substrate amino acids, thereby increasing the overall production cost. Therefore, the extracellular glutathione fermentation process has been proposed as an attractive way to achieve higher glutathione yields. The production method of discharging glutathione out of the cell has both the advantages of the general intracellular fermentation process and the enzymatic method because there is no space restriction and the demand for the substrate amino acid. Furthermore, by releasing the accumulated glutathione in the cell, it is possible to increase the total production of glutathione avoiding the effect of suppression of feedback, and it is also commercially advantageous because it can simplify the process of extracting glutathione from cells.

U.S. registered patent US 8,574,871 B2 (Registered on Nov. 2013)

An object of the present invention is to provide a promoter whose activity is inhibited by adding methionine Saccharomyces by using Saccharomyces cerevisiae ) is to provide a cell wall decomposition variant Saccharomyces cerevisiae strain capable of regulating the expression of genes involved in cell wall biosynthesis and a method for producing glutathione extracellular secretion using the strain.

In order to solve the above problems, the present invention is a cell wall degradation variant Saccharomyces cerevisiae strain in which the promoter gene of the cell wall biosynthesis gene in the Saccharomyces cerevisiae strain is replaced with a promoter gene capable of regulating expression. Provides

In addition, the present invention is a Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) cell wall degradation mutation Saccharo comprising the step of substituting the promoter gene of the cell wall biosynthesis gene with a MET3 promoter gene whose expression is inhibited by methionine Provided is a method for manufacturing a strain of Myces cerevisiae.

In addition, the present invention provides a method for producing glutathione (glutathione) comprising culturing the cell wall decomposition mutation Saccharomyces cerevisiae strain in a medium to which methionine is added.

The present invention relates to a novel cell wall degradation mutant strain and a method for producing glutathione using the same, in the present invention, a promoter whose activity is inhibited under specific conditions is used. Used in the OCH1 and CHS3 gene expression involved in cell wall biosynthesis thereby inhibiting the cell wall was produced in the conditional mutant yeast strain decomposition it is strictly regulated in certain conditions. The cell wall mutant yeast strains show a markedly improved glutathione extracellular production efficiency effect, and thus can be usefully utilized in the production of glutathione extracellular secretion with high industrial economy.

1 shows a production process of a conditional mutant cell wall with adjusting the OCH1 CHS3 and expressed in Saccharomyces cerevisiae. (a) a schematic view schematic diagram structure of the yeast cell wall and OCH1 and CHS3 action position. (b) a schematic view control gene expression cassette by making a schematic view and methionine to replace the OCH1 promoter and CHS3 a MET3 promoter.
Figure 2 shows a schematic diagram of the conditional cell wall degradation mutant strain production. (a) Schematic diagram of the vector used to construct the strain. (b) Schematic of the single and double mutant yeast strains and the OCH1 CHS3 expression control by the MET3 promoter.
Figure 3 shows the results of the conditional cell wall degradation mutant strain growth and osmolysis analysis. (a) Growth curves of yeast strains cultured in SD medium with or without 2 mM methionine. (b) Comparative analysis of osmotic dissolution efficiency of yeast strains. The lysis efficiency unit is the A ₂₆₀ unit value of the supernatant after osmotic shock per cell of A ₆₀₀ units before osmotic lysis.
Figure 4 shows the results of the growth analysis of conditional cell wall degradation mutant strains under various cell wall weakening conditions. (a) 30 μg/ml hygromycin B (HgB) with or without methionine, 7 mg/ml Calcofluor White (CFW) added medium and growth at various temperatures. (b) Recovery growth in medium supplemented with 1 M Sorbitol.
Figure 5 shows the results of comparative analysis of glutathione productivity of conditional cell wall degradation mutant strains. (a) Intracellular and (b) extracellular glutathione production analysis.

Thus, the inventors of the present invention, OCH1, which encodes a protein involved in the initiation of the outer chain biosynthesis of the N- sugar chain of the yeast Saccharomyces cerevisiae cell wall CHS3 involved in synthesizing most of the genes and cell wall chitin A series of conditional cell wall degradation mutant strains were developed by regulating the expression of genes, and the possibility of glutathione extracellular production was investigated using these strains. By showing the present invention in which an OCH1 and CHS3 cell wall degradation mutant strains are severely considerably said Genie conditional cell wall decomposition phenotype regulated enhanced glutathione extracellular production efficiency based on the gene expression developed by an outer secretion producing high economic efficiency glutathione cells It suggests the possibility of high utilization as a strain.

The present invention is Saccharomyces Saccharomyces cerevisiae ) provides a strain for cell wall degradation, Saccharomyces cerevisiae , in which the promoter gene of the cell wall biosynthesis gene in the strain is replaced with a promoter gene capable of regulating expression.

Preferably, the promoter gene capable of regulating expression may be a MET3 promoter gene whose expression is inhibited by methionine, but is not limited thereto.

Preferably, the cell wall biosynthesis gene is α-1,6- dehydratase only nosil transferase (α-1,6-mannosyltransferase; OCH1 ) and chitin biosynthesis enzyme gene III (chitin synthase III; CHS3) selected from the group consisting of gene It may be any one or more genes, but is not limited thereto. The OCH1 gene described in the Examples of the present invention is NCBI accession no. 852485, the CHS3 gene is NCBI accession no. 852311.

Preferably, the MET3 promoter gene may be represented by SEQ ID NO: 1, but is not limited thereto.

More preferably, the cell wall degradation mutant Saccharomyces as MY process Levy jiae three strains that the OCH1 promoter of the gene is substituted to the MET3 promoter gene, a P _MET3 -OCH1 strain described in the examples of the invention, the accession number of KCTC 13710BP It may be a deposited strain.

More preferably, the cell wall degradation mutant Saccharomyces as MY process Levy jiae three strains that the promoter of the gene CHS3 substituted by MET3 gene promoter, a P _MET3 -CHS3 strain described in the examples of the invention, the accession number of KCTC 13709BP It may be a deposited strain.

More preferably, the cell wall degradation mutant Saccharomyces as MY process Levy jiae three strains that the promoter of the promoter of the gene, and OCH1 gene CHS3 substituted by MET3 promoter gene, P _MET3 -OCH1 according to an embodiment of the present invention / P _MET3 As a -CHS3 strain, it may be a strain deposited with accession number KCTC 13711BP.

In addition, the present invention is Saccharomyces Saccharomyces cerevisiae ) provides a method for producing a cell wall degradation variant Saccharomyces cerevisiae strain comprising replacing the promoter gene of the cell wall biosynthesis gene in the strain with a MET3 promoter gene whose expression is suppressed by methionine.

Preferably, the cell wall biosynthesis gene is α-1,6- dehydratase only nosil transferase (α-1,6-mannosyltransferase; OCH1 ) and chitin biosynthesis enzyme gene III (chitin synthase III; CHS3) selected from the group consisting of gene It may be any one or more genes, but is not limited thereto.

Preferably, the MET3 promoter gene may be represented by SEQ ID NO: 1, but is not limited thereto.

More preferably, the cell wall degradation mutation Saccharomyces cerevisiae strain may be a strain deposited with accession number KCTC 13709BP, accession number KCTC 13710BP or accession number KCTC 13711BP.

In addition, the present invention provides a method for producing glutathione (glutathione) comprising culturing the cell wall decomposition mutation Saccharomyces cerevisiae strain in a medium to which methionine is added.

Preferably, the cell wall degradation mutation Saccharomyces cerevisiae strain may secrete glutathione extracellular, but is not limited thereto.

Hereinafter, examples will be described in detail to help understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example 1> New Cell wall degradation mutant strain production

The yeast cell wall is composed of cell wall mannoprotein, β-1,3- and β-1,6-glucans, and chitin. Most yeast cell wall mannoproteins are covalently bound to the mannose polymer and play an important role in cell wall integrity and regulate cell wall porosity. Yeast cell wall mannose proteins are usually attached with N -/ O -sugar chains. In particular, S. cerevisiae N- sugar chains have a very long sugar chain outer chain, S. cerevisiae. OCH1 The gene product is α-1,6-mannosyl transferase located in Golgi and is involved in initiating oligosaccharide chain synthesis by binding the first α(1,6)-mannose to the N- oligosaccharide attached to the protein. . The β-glucan layer is mostly composed of β-1,3-glucan (80-90%) and contains a small amount of β-1,6-glucan (10-18%). β-1,3-glucan is located on the outer surface, and β-1,6-glucan connects the components of the inner and outer walls, or the glycosyl phosphatidyl inositol (GPI)-mannoprotein. Chitin, a polymer in which 100 to 190 N-acetylglucosamines (GlcNAc) are bound to β-1,4, is present in a trace component of the outer wall, but it plays a large role in the mechanical strength and elasticity of the cell wall by combining with glucan polymer. S. cerevisiae The chitin biosynthesis enzyme III, a product of the CHS3 gene, plays an important role in most cell wall chitin and chitin ring biosynthesis (FIG. 1A).

The inventors have in MET3 promoter whose expression is inhibited by the CHS3 and OCH1 gene, respectively of a promoter (promoter) portion for adjustable conditional cell wall degradation mutant yeast strains produced in accordance with whether or not the methionine present CHS3, OCH1 expression in methionine (methionine) Substituted (FIG. 1B). For the substitution of the OCH1 promoter, PCR is performed using the corresponding primer set 5'-OCH1-SacI-fw and 5'-OCH1-NotI-rv (Table 1) for nucleotide sequence information corresponding to the OCH1 promoter -242 bp and -1 bp points. in the OCH1 5 'UTR DNA fragment the SacI / NotI handle by inserting the pBluSKP-pScMET3 vector with the MET3 promoter, and, OCH1 ATG N-OCH1-EcoRI -fw and ORF 297bp N-OCH1-SalI of the branch containing the amplifier Using the -rv primer (Table 1), the N segment of the OCH1 gene amplified by PCR was inserted after EcoRI/SalI treatment to _prepare pB-P _MET3 -OCH1 (Table 2). Then, pop-out capable URA3 (Kang et al., 2000) was amplified by PCR using ScURA3pp-NotI-fw and ScURA3pp-NotI-rv primers in pTcUR3 (Table 2), followed by NotI treatment and then pB-P _MET3 -OCH1 _Was inserted into pB-P _MET3- OCH1-URA3 (Table 2) (FIG. 2A). The prepared pB-P _MET3 -OCH1-URA3 was cut with SacI/SalI to transform the prepared cassette into S. cerevisiae BY4742 strain by LiAC/PEG method, and then Ura ⁺ was selected from SC-URA medium to select P _MET3 -OCH1 strain Was prepared (FIG. 2B). Construction of the CHS3 conditional expression vector was also performed in the same way. CHS3 5 'UTR fragment by using two kinds of primer sets 5'-CHS3-SacI-fw / 5'-CHS3-NotI-rv and N-CHS3-EcoI-fw / N-CHS3-SalI-rv ( Table 1) The pB-P _MET3 -CHS3 (Table 2) was prepared by amplifying the N and N sections by PCR and inserting them into the pBluSKP-pScMET3 vector, and pop-out URA3 (Kang et al., 2000) was ScURA3pp from pTcUR3 (Table 2). PB-P _MET3 -CHS3-URA3 (Table 2) was prepared by amplifying by PCR using the -NotI-fw/ScURA3pp-NotI-rv primer and inserting it into the NotI position (Figure 2A). The prepared pB-P _MET3 -CHS3-URA3 was subjected to SacI/SalI treatment to transform the prepared cassette into S. cerevisiae BY4742, P _MET3 -OCH1, and och1 Δ strains, and then screened in SC-URA medium to select P _MET3 -CHS3, P _MET3 -OCH1 / P _MET3 -CHS3, och1 Δ / P _MET3 -CHS3 (Table 3) strains were prepared (Fig. 2b). Each produced strain popped out the URA3 gene by culturing in 5'-FOA medium (Table 3).

Primer Sequence(5’-3’) Discription 5'-OCH1-SacI-fw C GAGCTC GAGCTGAATAATACTTCTTCAACC Amplification of 5’OCH1 flanking fragment 5'-OCH1-NotI-rv ATAAGAAT GCGGCCGC GTCCAAAAAGGCGCGTTTAG Amplification of 5’OCH1 flanking fragment N-OCH1-EcoRI-fw G GAATTC TCTAGGAAGTTGTCCCACCT Amplification of N-OCH1 flanking fragment N-OCH1-SalI-rv ACGC GTCGAC CACCCTTTGCGGGATGGG Amplification of N-OCH1 flanking fragment 5'-CHS3-SacI-fw GAC GAGCTC CTGTGTCACAAAGCTCTTGTATTG Amplification of 5’CHS3 flanking fragment 5'-CHS3-NotI-rv GCA GCGGCCGC ATTGAATATGCTTCAAGAAATATCC Amplification of 5’CHS3 flanking fragment N-CHS3-EcoI-fw GCA GAATTCA CCGGCTTGAATGGAGATG Amplification of N-CHS3 flanking fragment N-CHS3-SalI-rv GCA GTCGAC GCTTCTCACTAGGCCTTTGG Amplification of N-CHS3 flanking fragment ScURA3pp-NotI-fw ATTT GCGGCCGC GATCCACAGGACGGGTGTG Amplification of ScURA3 ScURA3pp-NotI-rv ATTT GCGGCCGC ATCGTCCATTCCGACAGC Amplification of ScURA3

* Restriction enzyme recognition sites are underlined.

Plasmid Description Reference pBluSKP-pScMET3 pBluSKP containing the ScMET3 promoter (Yoo et al. 2015) pTcUR3 pBluSKP containing the Tc:ScURA3:Tc blaster cassette (Kang et al. 2000) pB-P _MET3 -OCH1 pBluSKP containing the ScMET3 promoter and the 5'UTR/5'ORF OCH1 The present invention pB-P _MET3 -CHS3 pBluSKP containing the ScMET3 promoter and the 5'UTR/5'ORF CHS3 The present invention pB-P _MET3 -OCH1-URA3 pBluSKP-P _MET3 -OCH1-Tc:ScURA3:Tc The present invention pB-P _MET3 -CHS3-URA3 pBluSKP-P _MET3 -CHS3-Tc:ScURA3:Tc The present invention

S. cerevisiae strain Genotype Reference BY4742 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 Open Biosystems BY4742 och1 △ MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 och1 △ Open Biosystems P _MET3 - OCH1 BY4742 expressing OCH1 under the control of P _MET3 The present invention P _MET3 - CHS3 BY4742 expressing CHS3 under the control of P _MET3 The present invention P _MET3 -OCH1/P _MET3 -CHS3 BY4742 expressing CHS3 and OCH1 under the control of P _MET3 The present invention och1 △/ P _MET3 - CHS3 BY4742 och1 △ expressing CHS3 under the control of P _MET3 The present invention

< Example 2> New cell wall degradation mutation physiological characteristics

The growth pattern according to the presence or absence of methionine addition of the P _MET3 -OCH1, P _MET3 -CHS3, P _MET3 -OCH1/P _MET3 -CHS3, och1 Δ, och1 Δ/P _MET3 -CHS3 strains _{prepared in} Example 1 was S. cerevisiae BY4742 Comparative analysis with wild-type strains. Recombinant strains produced when cultured in minimal medium (SD) without methionine showed significantly similar growth as wild-type strains, and growth was not significantly inhibited, but when cultured in 2 mM methionine-added medium (SD+Met), P _MET3 -OCH1 , P _MET3 -CHS3, P _MET3 -OCH1 / P _MET3 -CHS3 strains were markedly reduced growth (Fig. 3a). In addition, in order to confirm the degree of cell lysis due to osmotic shock (osmotic shock), the strain cultured up to a log-growth phase was incubated in 1/10 volume of distilled water for 1 hour and then used as a TOMY mixer 24. After mixing for 10 minutes and centrifuging to obtain a sample, OD ₂₆₀ values were obtained using the spectrum. As a result of the spectrum measurement, it was observed that the cell decomposition of the P _MET3 -OCH1, P _MET3 -CHS3, and P _MET3 -OCH1/P _MET3 -CHS3 strains cultured in the methionine-added medium markedly increased, resulting in an increase in the A _260/ A ₆₀₀ value. As the incubation time increased, it became more pronounced. On the other hand, och1 Δ, Δ och1 / P _MET3 -CHS3 strain with OCH1 gene defects have been observed are taking place to some extent in the cell decomposition culture conditions without a methionine (Fig. 3b). Therefore, the condition _{P MET3 -OCH1, P MET3 -CHS3,} P MET3 -OCH1 / P MET3 -CHS3 strains produced in this invention is the expression of the OCH1, CHS3 is suppressed in accordance with the presence or absence of methionine is a cell decomposition progressive decomposition cell mutation Yeast was confirmed. In addition, the cell wall weakening material hygromycin B, YPD medium treated with CFW (Calcofluor White) and 25 °C (low temperature), 37 °C (high temperature) strain culture under the results of culturing results P _MET3 -OCH1, P _MET3 -CHS3, It was confirmed that growth was significantly inhibited when 2 mM methionine was added to the P _MET3- OCH1/P _MET3- CHS3 strain (FIG. 4A). It was confirmed that the growth inhibition was almost recovered when 1M sorbitol was added to reduce the osmotic shock (FIG. 4B). These results demonstrate that the P _MET3 -OCH1, P _MET3 -CHS3, and P _MET3 -OCH1/P _MET3 -CHS3 strains _prepared in the present invention are conditional cell wall degradation mutants with defects in cell wall stability.

< Example 3> Cell wall decomposition using yeast Glutathione Secretion production

The glutathione extracellular production capacity was analyzed in order to evaluate the potential as a useful metabolite secretion production host of the conditional cell wall degradation mutant strains developed in the present invention. The experimental method is as follows. Yeast cells were cultured at 30°C in a minimal medium (SD) of 50 ml with glucose added as a carbon source. The cell culture was centrifuged at 5,000 rpm and 4°C for 10 minutes to separate the cell pellet of OD ₆₀₀ 100 and the culture supernatant. The total amount of glutathione combined with the reduced form and the oxidized form is colorimetric method in a total reaction solution of 100 μl containing Ellman's reagent (5,5-dithio-bis-(2-nitrobenzoic) acid), glutathione reductase, and NADPH. ) Was measured using the standard recycling assay. Cell lysates were obtained by crushing glass beads or osmotic lysis from cell pellets. The culture supernatant was neutralized with 0.5 M NaOH after protein removal with 5% TCA (2,2,2-trichloroacetic acid).

For comparison of intracellular and extracellular glutathione amounts, the amount of glutathione was measured from cell lysates obtained by crushing glass beads or by osmolysis and the culture supernatant (FIG. 5). Intracellular glutathione secretion by osmotic lysis was significantly higher than most wild type strains by about 4-9 times in most conditional cell wall degradation mutants. However, glutathione secretion efficiency by osmotic dissolution was much lower even in conditional mutant strains, up to about 20% of glutathione secretion efficiency by glass bead crushing (FIG. 5A). Nevertheless, it was observed that most of the conditional mutant strains showed a significantly improved extracellular glutathione level, which was increased up to 4.5 times that of the wild type strain (FIG. 5B ). In particular, the conditional mutant strain _{P MET3 - OCH1 / P MET3 -} CHS3 and och1 Δ / P _MET3 - is the a glutathione of about 40% of the total glutathione cells, including in and extracellular form in CHS3 secreted as a product extracellular noteworthy . Therefore, the conditional cell wall degradation mutant strains developed in the present invention are useful as hosts for glutathione extracellular mass production, which is emerging as an effective alternative to the current extraction method from yeast cells in terms of productivity improvement and ease of separation and purification. Can be utilized.

Since the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that this specific technology is only a preferred embodiment, whereby the scope of the present invention is not limited. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology KCTC13709BP 20181112 Korea Research Institute of Bioscience and Biotechnology KCTC13710BP 20181112 Korea Research Institute of Bioscience and Biotechnology KCTC13711BP 20181112

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Novel cell-wall lysis mutant strains of yeast and method for producing glutathione using the same <130> ADP-2018-0350 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 631 <212> DNA <213> Saccharomyces cerevisiae <400> 1 gtataaatca cggaagggat gatttataag aaaaatgaat actattacac ttcatttacc 60 accctctgat ctagattttc caacgatatg tacgtagtgg tataaggtga gggggtccac 120 agatataaca tcgtttaatt tagtactaac agagactttt gtcacaacta catataagtg 180 tacaaatata gtacagatat gacacacttg tagcgccaac gcgcatccta cggattgctg 240 acagaaaaaa aggtcacgtg accagaaaag tcacgtgtaa ttttgtaact caccgcattc 300 tagcggtccc tgtcgtgcac actgcactca acaccataaa ccttagcaac ctccaaagga 360 aatcaccgta taacaaagcc acagttttac aacttagtct cttatgaagt tacttaccaa 420 tgagaaatag aggctctttc tcgagaaata tgaatatgga tatatatata tatatatata 480 tatatatata tatatatgta aacttggttc ttttttagct tgtgatctct agcttgggtc 540 tctctctgtc gtaacagttg tgatatcgtt tcttaacaat tgaaaaggaa ctaagaaagt 600 ataataataa caagaataaa gtataattaa c 631

Claims (13)

Saccharomyces cerevisiae ) A cell wall degradation variant Saccharomyces cerevisiae strain in which the promoter gene of the cell wall biosynthesis gene in the strain is replaced with a promoter gene capable of expression regulation. The cell wall degradation mutation Saccharomyces cerevisiae strain according to claim 1, wherein the promoter gene capable of regulating expression is a MET3 promoter gene whose expression is suppressed by methionine. The method of claim 1, wherein the cell wall biosynthesis gene is α-1,6- dehydratase only nosil transferase (α-1,6-mannosyltransferase; OCH1 ); the group consisting of (CHS3 chitin synthase III) gene and gene III chitin biosynthesis enzyme Cell wall degradation mutation Saccharomyces cerevisiae strain, characterized in that any one or more genes selected from. According to claim 2, wherein the MET3 promoter gene is a cell wall degradation variant Saccharomyces cerevisiae strain, characterized in that represented by SEQ ID NO: 1. The method of claim 3, wherein a cell wall decomposition variation saccharide to the cell wall degradation mutant Saccharomyces as MY process three Levy jiae strain is that the promoter of the OCH1 gene is substituted by MET3 promoter gene, characterized in that the strain deposited as accession number of KCTC 13710BP Myces cerevisiae strain. The method of claim 3, wherein a cell wall decomposition variation saccharide to the cell wall degradation mutant Saccharomyces as MY process three Levy jiae strain is that the promoter of CHS3 gene substituted by MET3 promoter gene, characterized in that the strain deposited as accession number of KCTC 13709BP Myces cerevisiae strain. The method of claim 3, wherein the cell wall decomposition mutation Saccharomyces cerevisiae strain is a strain in which the promoter of the OCH1 gene and the promoter of the CHS3 gene are replaced with the MET3 promoter gene, and is a strain deposited with accession number KCTC 13711BP. Cell wall degradation mutation Saccharomyces cerevisiae strain. Saccharomyces cerevisiae ) cell wall degradation mutation Saccharomyces cerevisiae strain production method comprising substituting the promoter gene of the cell wall biosynthesis gene in the strain with a MET3 promoter gene whose expression is suppressed by methionine. The method of claim 8 wherein said cell wall synthesis gene is α-1,6- dehydratase only nosil transferase (α-1,6-mannosyltransferase; OCH1 ); the group consisting of (CHS3 chitin synthase III) gene and gene III chitin biosynthesis enzyme Cell wall degradation mutation characterized in that any one or more genes selected from Saccharomyces cerevisiae strain manufacturing method. The method of claim 8, wherein the MET3 promoter gene is a cell wall degradation variant Saccharomyces cerevisiae strain, characterized in that represented by SEQ ID NO: 1. 10. The method of claim 9, wherein the cell wall decomposition mutation Saccharomyces cerevisiae strain is a cell wall degradation variant Saccharomyces three, characterized in that the strain deposited with accession number KCTC 13709BP, accession number KCTC 13710BP or accession number KCTC 13711BP Method of manufacturing a strain of Levisiae. A method for producing glutathione, comprising culturing a cell wall degradation variant Saccharomyces cerevisiae strain according to any one of claims 1 to 7 in a medium to which methionine is added. The method for producing glutathione according to claim 12, wherein the cell wall decomposition mutation Saccharomyces cerevisiae strain secretes glutathione extracellularly.

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