KR100545563B1 - A novel saccaromyces cerevisiae strain with enhanced recombinant protein production capability - Google Patents

Abstract

The present invention relates to a Saccharomyces cerevisiae strain with improved recombinant protein production capacity, and more particularly, provides gal1Δhxk2 Δ strain and Saccharomyces cerevisiae Y28G1H2UV74 having excellent recombinant protein production efficiency. Since the strains of the present invention do not use galactose as a carbon source, by using only galactose as an expression inducer and using glucose as a carbon source when expressing a recombinant protein using a GAL pro rotor, a useful host for an economical and convenient recombinant protein production system. Can be used as

Saccharomyces cerevisiae, recombinant protein, protein secretion, GAL promoter

Description

Saccharomyces cerevisiae mutant with improved recombinant protein production ability {A NOVEL SACCAROMYCES CEREVISIAE STRAIN WITH ENHANCED RECOMBINANT PROTEIN PRODUCTION CAPABILITY}

Figure 1 shows the pop-out cassette and homologous recombination mechanism for GAL1 gene or HXK2 gene disruption.

Figure 2a to c is a graph showing the recombinant albumin production rate of wild type GAL strain, gal1 Δ mutant and gal1 Δ hxk2 Δ mutant.

3A to 3C are graphs showing the yield of recombinant albumin- Timf -2 protein of wild type GAL strain, gal1 Δ mutant and gal1 Δ hxk2 Δ mutants, respectively.

4 shows a vector map of the pEU-Amyl vector.

5 is a graph showing the activity of amylase expressed and secreted in the Y28G1H2UV74 strain.

Figure 6 is a graph showing the growth rate, growth characteristics and albumin-Timf-2 fusion protein production rate of Y28G1H2UV74 mutants.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to Saccharomyces cerevisiae mutants with improved recombinant protein production ability, and more particularly, to Saccharomyces cerevisiae mutants having higher recombinant protein production efficiency than wild type.

[Private Technology]

Yeast, a unicellular eukaryote, has a gene transcription and translation system and protein secretory that are very similar to higher organisms, which has the advantage of releasing activated proteins through post-translational modification. In addition, since yeast has a very small amount of protein normally secreted out of cells, recombinant proteins secreted from yeast can be easily recovered and purified (Romanos et al., Yeast, 8: 423-488, 1992). In addition to baker's yeast Saccharomyces cerevisiae, which has been used recently, Genus, Pichia Genus, cleveromyces Genus and Yarois The development of recombinant protein production technology using a rare yeast strain having specific properties such as genus as a host cell is being actively promoted.

Saccharomyces cerevisiae are generally Recognized As Safe (GRAS) microorganisms that are not pathogenic to the human body and do not produce endotoxins, and among the various yeast systems, the host-vector system is currently well established. Efforts have been actively made to develop a recombinant protein expression system having excellent secretion expression rate using yeast as a host. Among the promoters used for gene expression in Saccharomyces cerevisiae, the GAL1, GAL2, GAL7 and GAL10 promoters , which are widely used for recombinant protein expression, are promoters of genes involved in galactose metabolism. The promoters are inhibited when glucose is present in the medium, but the expression is increased more than 1000 times by galactose when glucose is not present (Carlson, 1987, J. Bacteriol. 169: 4873-4877). However, galactose, which is used as an inducer of the GAL promoter, is also consumed as a carbon source, so galactose must be continuously supplied to induce continuous gene expression. Galactose is a substrate that is about 30 times more expensive than glucose, which is economical in mass culture. Becomes In addition, when galactose is continuously supplied, there is a problem that the protein metabolism is changed due to the lack of nitrogen source compared to the carbon source, thereby promoting the activity of proteolytic enzymes and degrading the secreted and expressed protein. There is also difficulty in determining the optimal concentration of galactose (Hovland et al., 1989, Gene, 83: 57-64).

With the introduction of the 501 mutation in galactose inhibit expression of glucose to the GAL promoter mothamyeonseo not use lactose are reduced saccharide - as a way for solving the above problems, the galactose mutant strains (gal1) can not metabolize lactose reg1 there are prepared three bars My process Levy jiae 334 strain (MATα pep4-3 prb1-1122 ura3-52 leu2-3112 gal1, reg1-501) (Hovland , etc., 1989, Gene, 83: 57-64 ). In this strain, which can use galactose as a gratuitous inducer, it has been reported that the expression of the GAL promoter is 1500-fold at a low galactose concentration of 0.02%. However, this Saccharomyces cerevisiae 334 strain was produced by a point mutation, so there is a possibility of reversion, and regl mutation causes growth to be much slower than wild type strains, especially high concentration cells. The disadvantage is that the culture is not good.

Therefore, while developing a large amount of recombinant protein using a GAL promoter without metabolizing galactose, development of a strain with improved cell growth rate is required.

Accordingly, an object of the present invention is to provide a Saccharomyces cerevisiae mutant strain capable of efficiently producing and secreting recombinant proteins.

It is also an object of the present invention to provide a Saccharomyces cerevisiae mutant strain wherein recombinant protein expression is not inhibited by glucose under conditions provided with glucose as a carbon source.

It is another object of the present invention to provide a Saccharomyces cerevisiae mutant strain having a physiological characteristic that produces little ethanol even in the presence of a large amount of glucose unlike wild-type strains.

In order to achieve the above object, the present invention provides a Saccharomyces cerevisiae mutant strain in which the GAL1 gene and the HXK2 gene are destroyed.

The present invention also provides Saccharomyces cerevisiae mutants deposited with KCTC 10568BP.

The present invention also provides a vector comprising a gene whose expression is regulated under a promoter selected from the group consisting of GAL1, GAL2, GAL7, and GAL10 promoters, according to claim 1 or 2 according to Saccharomyces cerevisiae Transform; And

(b) providing a recombinant protein production method comprising culturing the transformant in the presence of galactose to produce a recombinant protein.

Hereinafter, the present invention will be described in detail.

The present inventors were studying to develop a strain of Saccharomyces cerevisiae modified to produce a large amount of recombinant protein, and both GAL1 gene (GeneBank No. X76078) and HXK2 gene (GeneBank No.X67787) were destroyed. The mutant strain was excellent in recombinant protein production efficiency, and the strain mutated to UV also showed excellent recombinant protein production efficiency, thus completing the present invention.

Saccharomyces cerevisiae strains of the present invention are Saccharomyces cerevisiae strains of which the GAL1 gene and HXK2 gene are destroyed and Saccharomyces cerevisiae Y28G1H2UV74.

Saccharomyces cerevisiae strain (hereinafter referred to as “ gal1 Δ hxk2 Δ mutant strain ”) in which the GAL1 gene and the HXK2 gene were destroyed is a gene involved in galactose metabolism and a GAL1 gene activated in the presence of galactose and glucose phosphorylation. HXK2 gene, a gene involved in, has been destroyed. By gal1 △ △ mutant hxk2 by the method described in Figure 1 in my process three Levy jiae dielectric with the wild-type Saccharomyces remove the GAL1 and HXK2 gene, can be easily produced. Saccharomyces cerevisiae comprising genotype MATα pep4 :: HIS4 prb-Δ1.6R can1 his3-20 ura3-52 gal1 :: tc5 hxk2 :: tc5 prepared as an example in the present invention Love 2805 is a variant.

Saccharomyces cerevisiae Y28G1H2UV74 was selected as a strain having excellent recombinant protein expression efficiency after UV irradiation on the gal1 △ hxk2 △ mutant strain, and was assigned to the Gene Bank of Korea Research Institute of Biotechnology, Daejeon, Korea. It was deposited as Saccharomyces cerevisiae Y28G1H2UV74 and was assigned accession number KCTC 10568 BP.

According to the present invention, gal1 Δ hxk2 Δ strains and Saccharomyces cerevisiae Y28G1H2UV74 are conventional yeast cultures such as YPD medium (yeast extract 1%, peptone 2%, dextrose 2% and residual water), YPGal medium. (yeast extract 1%, peptone 2%, galatose 2% and residual water), YPDG complex medium (yeast extract 1%, peptone 2%, dextrose 2%, galactose 2%) and SC synthetic medium (nitrogen base w / o amino acids 0.67%, glucose 2%, required amino acids mixture) can be easily incubated in conventional yeast culture conditions, that is 24 to 72 hours at 25 to 32 ℃.

Since the gal1 Δ hxk2 Δ strain and Saccharomyces cerevisiae Y28G1H2UV74 of the present invention exhibit excellent recombinant protein production efficiency, it can be provided as a suitable host for heterologous protein production.

The vector that can be used to use the mutant strain as a host, may be a conventional yeast expression vector, preferably a promoter for regulating the expression of a heterologous gene is activated by galactose, that is, GAL1, GAL2, It is preferred that they are GAL7 and GAL10 promoters. In addition, a heterologous gene may be inserted into a vector derived from a 2 μm plasmid, which is a conventional yeast expression vector, and multicopy integration vectors (Lopes et al., 1989, Gene, 179: 199-206). Sakai et al., 1990, Appl.Microbiol.Biotechnol. 33: 302-306; Parehk et al., 1996, Biotechnol.Prog. 12: 16-21). Examples of the heterologous genes include hirudin, human growth hormone and albumin (Romanos et al., Yeast, 8: 423-488, 1992; Kang et al., 1998, J. Microbiol. Biotechnol. 8: 42-48; Kim et al. , J. Biotechnol. 2001, 85: 41-48).

Since the transformation method in yeast is a conventional transformation method, those skilled in the art to which the present invention pertains can easily implement the method. Transformants transformed with a gal1 Δ hxk2 Δ mutant and a vector containing a heterologous gene in Saccharomyces cerevisiae Y28G1H2UV74, respectively, can be grown in yeast culture and culture conditions, in particular 0.1 to for recombinant protein expression. In a medium comprising 2 (w / v)% yeast extract, 0.5 to 10 (w / v)% dextrose, 0.1 to 3 (w / v)% galactose and residual water (final 1 L) , The culture temperature may be incubated at 25 to 30 ℃, 4.5 to 6.5 pH conditions.

In one embodiment, in the present invention, pYHSA5 (Kang et al., 2000, Appl. Microbiol. Biotechnol. 253: 1-3), which is a vector expressing human albumin in gal1 Δ hxk2 Δ mutant strain and Saccharomyces cerevisiae Y28G1H2UV74, respectively. And a transformant was prepared by introducing pHSATIMP (Korean Patent Application No. 10-2002-0001057), which is a vector expressing an albumin-timf-2 fusion protein, and the transformant was introduced into wild-type Saccharomyces cerevisiae. Compared with the recombinant protein expression and secretion efficiency is remarkably excellent compared to the above, it was confirmed that the cell growth rate is high even under the conditions of providing glucose as a carbon source.

Thus, the gal1 Δ hxk2 Δ strain and Saccharomyces cerevisiae Y28G1H2UV74 of the present invention can be used as a host useful for recombinant protein expression, thereby establishing various protein expression systems.

Hereinafter, preferred examples are provided to aid in understanding the present invention. The following examples are provided only to more easily understand the present invention, but the protection scope of the present invention is not limited to the following examples.

Example 1 gal1Δ Mutant

1-1. gal1 △ mutant strain manufacturing

Saka access to my Celebi jiae 2805 (MATα pep4 :: HIS4 prb- 1.6R can1 his3-20 ura3-52) in GAL1 shredding cassette for the strain of even one so you can reuse the URA3 selection marker URA3 pop when needed - Out cassette was produced.

The URA3 pop-out cassette surrounded by N-terminal (350 bp) and C-terminal (250 bp) DNA fragments of the GAL1 gene obtained by PCR from genomic DNA of Saccharomyces cerevisiae was subjected to double homologous recombination. When introduced into the GAL1 gene region on the chromosome, about 1.3 kb of GAL1 gene was removed and a transformant ( gal1 :: URA3: tc5 ) was replaced with the URA3 pop-out cassette instead. This was first screened in selection medium lacking uracil, designated as gal1 :: URA3: tc5 transformant, and screened for colonies that survived by plating on 5'-Floacetic acid (5'-FOA) -added plates. yet how the URA3 selection marker is uracil auxotrophic mutants exiting the gal1 mutant strain △: was produced (S28G1 MATα pep4 :: HIS4 prb- 1.6R can1 his3-20 ura3-52 gal1 :: tc5).

In order to confirm the deletion of the GAL1 gene on the chromosome, PCR was performed on the genomic DNA of the mutant strains prepared using the primers used to obtain the GAL1 gene fragment. About 1.8 kb of GAL1 DNA fragments were obtained in the GAL1 strain, while about 2.4 kb DNA fragments were expected in the gal1 :: URA3: tc5 strain, and about 0.9 kb DNA fragments in the gal1 Δ mutant ( gal1 :: tc5 ) strain. It was confirmed that the GAL1 gene disruption was performed correctly.

1-2. Characterization of gal1 △ mutant strain

The growth of wild type GAL1 strain and gal1 Δ mutant S28G1 was observed in various media using glucose or galactose as a carbon source.

The gal1 △ mutant showed the same growth rate as the wild-type strain in YPD medium (yeast extract 1%, peptone 2%, dextrose 2%), which had a 2% glucose-based carbon source, but YPGal containing 2% galactose as a carbon source. The growth rate was significantly decreased in the medium (yeast extract 1%, peptone 2%, galatose 2%) compared to the GAL1 strain.

In addition to galactose, the YPGal medium contains nutrients that can be used as a carbon source in yeast extracts. Thus, in order to confirm galactose metabolism more precisely, a minimal medium having only galactose in the medium was prepared and tested. YNB (Yeast Nitrogen Base w / o Amino Acid) + uracil medium, supplemented with uracil, contain 2% glucose or 2% galactose as the carbon source and no carbon source as a control. The growth of the strains grown in YNB + uracil medium was compared. As a result, the growth rate of the GAL1 strain and the gal1 Δ mutant strain was the same in YNB + uracil + 2% glucose medium, but both strains did not grow in YNB + uracil medium without a carbon source. On the other hand, contrary to the expected growth of the GAL1 strain, the growth rate of the GAL1 strain was significantly lower than that of the gall △ strain in YNB + uracil + 2% galactose medium, which would not grow at all due to the lack of the supplied galactose, the only carbon source. It was observed that. In the minimal medium made with 99.9% purity galactose, gal1 Δ mutant strains were insignificant, but grown enough to form colonies.

Example 2: gal1 △ hxk2 △ Variant

2-1. gal1 △ hxk2 △ mutant strain production

The strains of the gal1 and HXK2 genes were mutated by disrupting the hexokinase gene ( HXK2 ) of the gal1 Δ mutant strain.

HXK2 fragmentation cassette ( hxk2 :: tc: URA3: tc5 ) of FIG. 1 was prepared , and then transformed into gal1 Δ mutant strains. The URA3 : tc5 pop-out cassette for this cassette was obtained from pTcUR3 (Kang et al., 1998, Appl. Microbiol. Biotechnol. 50; 187-192)), and the N-terminal portion was obtained from genomic DNA. primers are HXK2-NF (X) (SEQ ID NO: 1: 5'-CCC TCTAGA GAATACTGAACGCCATAG3 ' ) used to amplify the HXK2-NR (B) (SEQ ID NO: 2: 5'-CCC GGATCC TAGCGTACTTATTATGTG3' ) and, C- terminal primers used to amplify are HXK2-CF (B) is: ((SEQ ID NO: 3 5'CCC GGATCC TGCCCTTTTTCTACTTAT3) 5'CCC GAATTC CAACATTCCGGAATCTGAA3 SEQ ID NO: 4) and HXK2-CR (E) '. Whether the HXK2 fragmentation cassette was inserted into the HXK2 gene region through homologous recombination mechanism was confirmed by PCR using HXK2-NF (X) and HXK2-CR (E) once selected from Ura ⁺ transformants. . Then the smear on the seed plate acetoxy tikaek 5-flow in order to remove the URA3 selective marker gene URA3 selection marker is due to the uracil auxotrophic visible gal1 mutant strain △ △ hxk2 exception (S28GH2: MATα pep4 :: HIS4 prb-Δ1.6R can1 his3-20 ura3-52 gal1 :: tc5 hxk2 :: tc5 ) were selected. Status (pop-out) as to whether the URA3 selection marker was removed was confirmed by PCR using the HXK2-NF (X) and HXK2-CR (E).

2-2. gal1 △ hxk2 △ Mutant strain trait analysis

The characteristics of the gal1 △ hxk2 △ strain S28GH2 were compared with the wild-type strain and the gal1 △ strain.

Each strain was incubated in YPDG complex medium containing 2% glucose and 2% galactose (yeast extract 1%, peptone 2%, dextrose 2%, galactose 2%) and analyzed for ethanol production and cell growth. As a result, in the medium containing 2% glucose and 2% galactose, the gal1 △ hxk2 △ mutant strain, which cannot use galactose as a carbon source, formed fewer cells than the wild type strain which could use both glucose and galactose as a carbon source. , gal1 △ formed a more than 20% increased cells compared to the strain.

Since Saccharomyces cerevisiae ferments glucose to produce a lot of ethanol even under aerobic conditions, more sophisticated culture methods and a lot of attention are required at high concentrations. By the way, gal1 △ mutant strains produce much more ethanol than wild type strains, there is a problem that the cell formation yield is low and high concentration culture is not easy. However, the gal1 Δ hxk2 Δ mutant strain produced less ethanol and consumed ethanol faster than the gal1 Δ mutant strain.

2-3. Analysis of Recombinant Protein Production Efficiency of gal1 △ hxk2 △ Mutant I

recombinant protein production efficiency of the gal1 hxk2 △ △ mutants was compared with wild-type strain and △ gal1 mutant strain.

Albumin-producing strains were prepared by transforming each strain with pYHSA5 (Kang et al., 2000, Appl. Microbiol. Biotechnol. 253: 1-3), a vector expressing human albumin from the GAL promoter. Each strain was incubated in a 5 L fermenter (30 ° C., pH 5.5) using a YPDG complex medium containing 2% glucose and 2% galactose (yeast extract 1%, peptone 2%, dextrose 2%, galactose%).

Figure 2a to c is a graph showing the recombinant albumin production rate of wild type GAL strain, gal1 Δ mutant and gal1 Δ hxk2 Δ mutant. The wild-type GAL strain of FIG. 2A produces albumin at about 150 mg / L, and the gal1 Δ mutant strain of FIG. 2B produces about 130 mg / L, and in particular, the gal1 Δ hxk2 Δ mutant of FIG. 2C is about 220 mg / L. Albumin was produced. In addition, the gal1 △ hxk2 △ mutant showed a maximum protein secretion within a faster time, that is, 24 hours than the wild type strain or gal1 △ mutant strain. The above results are thought to be due to the decrease in glucose expression inhibition on the GAL promoter in the gal1 Δ hxk2 Δ mutant strain due to HKX2 gene disruption, which plays an important role in the regulation of glucose inhibition. Therefore, when the gal1 Δ hxk2 Δ mutant is cultured at a high concentration for recombinant protein expression, the gal1 Δ hxk2 Δ mutant is hardly affected by the expression suppression by the continuously added glucose, which is expected to be a very useful host.

2-4. Analysis of Recombinant Protein Production Efficiency of gal1 △ hxk2 △ Mutants II

In order to confirm that the gal1Δhxk2Δ mutant strain is more useful than the wild type strain or the gal1Δ mutant strain for expression of other recombinant proteins other than human albumin, the human Timph -2 protein fused with human albumin (HSA-TIMP-2) is expressed. Its production efficiency was analyzed.

Each strain was introduced with a pHSATIMP vector (Korea Patent Publication No. 2003-60384) expressing an albumin-timp-2 fusion protein from a GAL promoter to obtain transformants, and then, YPDG complex medium (yeast extract 1%, peptone 2). %, Dextrose 2%, galactose%) was incubated in a 5 L fermenter (30 ℃, pH 5.5).

3A to 3C are graphs showing recombinant albumin- timp -2 protein production rates of wild type GAL strain, gal1 Δ mutant and gal1 Δ hxk2 Δ mutants, respectively (◇: galactose, ◆: glucose, ○: HSA-TIMP-2, ●: cell dry weight, Δ: ethanol). As a result of analyzing the amount of HSA-TIMP-2 protein secreted into the culture, wild-type strain secreted albumin-timp-2 fusion protein at 21 mg / l, whereas gal1 Δ hxk2 Δ mutant strain secreted at about 24 mg / l. It was. Given that the gal1 △ hxk2 △ mutant strain cannot use galactose, the amount of produced cells is lower than that of the wild-type strain (12 g / L vs. 18 g / L), and the amount of protein produced per cell is gal1 △ hxk2 △ mutant strain. It can be seen that it is about 70% higher than the wild type strain.

Therefore, the gal1 Δ hxk2 Δ mutant of the present invention can culture the cells at high concentration under continuous glucose supply conditions and at the same time can express a large amount of recombinant protein from the GAL promoter. In addition, the strain can reduce the cost of recombinant protein production by using glucose rather than galactose as a carbon source.

Example 3: Preparation and Characterization of Y28G1H2UV74 Mutant

3-1. Y28G1H2UV74 Variation Manufacture

The gal1 △ hxk2 △ mutant strain is more useful than the wild-type strain or gal1 △ mutant strain, but in order to develop a strain having better characteristics than the strain, the ability to secrete protein after inducing random mutations in the gal1 △ hxk2 △ strain Improved strains were selected.

Amylose derived from rice was used as a reporter protein, and ultraviolet light was used as a mutagen. A pEU-Amyl vector expressing amylase (FIG. 4) from the GAL promoter was introduced into the gal1 Δ hxk2 Δ mutant to prepare a transformant that secreted amylase, and then the transformed strain 5 × 10 ^{2 was transferred} to 1% starch and 0.5 A uracil deficient synthetic (SC-URASG) plate medium containing% galactose was plated and irradiated with ultraviolet light. After incubation at 30 ° C. for 5 days, colonies were selected to form a clear ring larger than the gal1 Δ hxk2 Δ mutants by staining with an iodide solution (0.3% iodine, 0.6% potassium iodine). The larger transparent ring indicates more amylase secretion. However, it is unclear whether the increase in amylase secretion is due to mutation at the genome level or due to mutations on the plasmid encoding the reporter protein, so further experiments were conducted to confirm this.

After removing the pEU-Amyl plasmid from the first selected strains, new pEU-Amyl was introduced again. These strains were cultured in SC-URASG medium and stained with Iodine solution to re-examine the size of the clear ring. Most of the primary screening strains formed clear rings of the same size as the parent strain, but named Y28G1H2UV74. The strain formed a much larger clear ring than the gal1 △ hxk2 △ mutant strain. The secreted amylase activity was analyzed by DNS method in the Y28G1H2UV74 strain was cultured in liquid medium (1% yeast extract, 2% bacto-peptone, 0.1 M potassium phosphate buffer pH 6.7, 1% glucose, and 0.1% galactose).

Figure 5 shows the activity of amylase expressed and secreted in the Y28G1H2UV74 strain, "1" is wild type strain / pEY-Amyl, "2" is hxk2 Δ / pEY-Amyl strain, "3" gal1 Δ mutation / pEY-Amyl, "4" is gal1 Δ hxk2 Δ strain / pEY-Amyl, and "5" is Y28G1H2UV74 / pEY-Amyl strain. Y28G1H2UV74 strain of the present invention showed a secretion rate of about 7 times higher amylase than the control and gal1 Δ hxk2 Δ mutant strain.

 Said Y28G1H2UV74 strain was deposited with the Korea Biotechnology Research Institute Gene Bank in Yuseong-gu, Daejeon, Korea as December 12, 2003, and designated as Saccharomyces cerevisiae Y28G1H2UV74, and was given accession number KCTC 10568 BP.

As a result of introducing pHSATIMP plasmid expressing albumin-timp-2 fusion protein into Y28G1H2UV74 strain instead of pEU-Amyl plasmid, albumin-timf-2 fusion protein was also 1.5-2.0 or more than wild type strain and gal1 Δ hxk2 △ strain. It was confirmed that a lot of secretion.

3-2. Y28G1H2UV74 Mutant Characterization

PHSATIMP was introduced into the Y28G1H2UV74 mutant, which was incubated in a 5 L fermenter (30 ° C, pH 5.5) using YPDG complex medium (yeast extract 1%, peptone 2%, dextrose 2%, galactose%). Thereafter, the amount of albumin-Timf-2 fusion protein secreted in the culture was measured.

Figure 6 shows the growth rate, growth characteristics, and albumin-timp-2 fusion protein production rate of Y28G1H2UV74 mutant strain , Y28G1H2UV74 mutant strain growth rate and final cell concentration compared to the gal1 △ hxk2 △ mutant strain , but albumin-timf-2 The fusion protein expression rate was about 50%. In addition, compared with the wild type strain, Y28G1H2UV74 mutant protein was about 3 times higher than the same amount of bacteria. In addition, the Y28G1H2UV74 mutant was found to be lower in ethanol production than the gal1 △ hxk2 △ mutant strain , indicating that reducing ethanol production may increase recombinant protein production.

As mentioned above, the gal1 Δ hxk2 Δ strains and Saccharomyces cerevisiae Y28G1H2UV74 of the present invention exhibit significantly superior recombinant protein production efficiency compared to the wild type Saccharomyces cerevisiae strain. In addition, since the strains do not use galactose as a carbon source, the galactose is used only as an expression-inducing substance and glucose is used as a carbon source when producing recombinant proteins using a GAL prorotor. It can be used as a useful host.

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Claims (4)

Saccharomyces cerevisiae strains in which the GAL1 gene and HXK2 gene were destroyed. The method of claim 1, wherein the Saccharomyces cerevisiae genotype MATα pep4 :: HIS4 prb-Δ1.6R can1 his3-20 ura3-52 gal1 :: tc5 hxk2 :: tc5 Serenity. Saccharomyces cerevisiae strain deposited with KCTC 10568 BP. (a) transforming a Saccharomyces cerevisiae according to claim 1 or 2 with a vector comprising a gene whose expression is regulated under a promoter selected from the group consisting of GAL1, GAL2, GAL7 and GAL10 promoters; And (b) culturing the transformant in the presence of galactose to produce a recombinant protein.

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