KR20090016364A - A method for producing a recombinant protein using a gal80 deficient yeast strain - Google Patents

Abstract

A GAL80 deficient yeast strain is provided to improve the expression rate and cell growth as compared to the conventional GAL1 deficient strain and eliminate need of galactose which is essential expensive substrate in the cultivation of GAL deficient strain, thereby producing the recombinant protein with low cost and high concentration cell culture. The method for producing a recombinant protein comprises the steps of: crushing the GAL80 gene of yeast; transforming the GAL80 gene crushed yeast strain with the recombinant expression vector encoding the foreign protein; and culturing the transformed yeast stain, wherein the yeast is Saccharomyces cerevisiae.

Description

A method for producing a Recombinant protein using a GAL80 Deficient Yeast Strain

The present invention relates to a method for producing a recombinant protein using a mutant strain lacking the GAL80 gene of Saccharomyces cerevisiae.

       As human health improves, the demand for high-purity proteinaceous medicines for the treatment of intractable diseases has soared, and the proportion of medicinal recombinant proteins in the health-related biotechnology sector is increasing. Early medicinal protein production was isolated from human tissues and blood, but this has turned into a relatively safe recombinant drug due to the possibility of secondary infection or residual disease-related factors. For this reason, animal cell culture, which has been suggested as an alternative, is a method that can replace it due to the presence of disease factors. As a result, the genetic and physiological characteristics of traditional microorganisms are well known. It is used for mass production.

     Among them, a protein expression secretion system using yeast has a number of advantages in recombinant protein production. Yeast is a unicellular eukaryote that can be easily cultivated at low cost and can be modified after synthesis such as glycosylation, unlike prokaryotic expression systems such as E. coli. Suitable for In addition, unlike expression systems such as Escherichia coli, soluble expression is possible in almost all cases, which do not form aggregates, and the protein secretion pathway is well developed to secrete and express recombinant proteins, resulting in complicated downstream processing of the final product. (Romanos et al., Yeast 8, 423-488 (1992)).

   Yeast Saccharomyces cerevisiae, in particular, offers additional benefits as a generally proven GRAS (Generally Regarded as Safe) organism, and has a long history of research. Also recently, genome sequencing has been completed and can be studied using the latest techniques such as microarray analysis, which is one of the most used systems for recombinant protein expression.

      The GAL1, GAL10 and GAL7 gene promoters involved in the use of galactose in Saccharomyces cerevisiae strains have been widely used in the production of recombinant proteins as the most potent promoters in Saccharomyces cerevisiae strains. In particular, these promoters have been widely used for inducing expression of recombinant proteins because they have a mechanism of inhibiting transcription by glucose and rapidly inducing transcription by galactose to induce expression of about 1000-fold or more.

       Despite these advantages, however, a large amount of galactose is consumed in using Saccharomyces cerevisiae as a host cell using a wild-type mutant strain as a host cell. Galactose must be present at a certain concentration as an inducer because galactose is also continuously metabolized and consumed as a carbon source. In addition, galactose is 30-100 times more expensive than glucose and has been very expensive to be used for mass fermentation. In addition, if 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, and the activity of proteolytic enzymes is promoted, resulting in the degradation of secreted and expressed proteins. There is also difficulty in determining the concentration. In order to partially solve this problem, a method using GAL1 deficient mutants has been developed (Kang HA et al., Biotechnol. Bioeng. 89, 619-629 (2005)), and galactokinase, the first process of galactose metabolism. When using a mutant strain that does not metabolize galactose by deleting the GAL1 gene encoding), galactose cannot be used as a carbon source, but only as an inducer, and thus a very small amount of galactose can continuously induce the expression of proteins.

       However, in the case of GAL1 deficiency strains, galactose is not consumed, but galactose above a certain concentration still needs to exist as an inducer, so there is still a problem in large-scale fermentation of recombinant proteins such as cheap industrial enzymes.

       In order to fundamentally solve this problem, a system in which the GAL gene can be expressed is most preferable even when galactose is not present as an inducer. Recently, a study on the variant strains of galactose metabolic pathway genes in Saccharomyces cerevisiae has been suggested. To understand this, the mechanism of GAL gene expression is as follows (Verma et al., Biotechnol. Appl. Biochem. 39, 89-97 (2004); Ostergaard et al., FEMS Yeast Research 1, 47-55 (2001)).

        The expression of the GAL gene is regulated by three regulatory proteins, Gal4p, Gal80p and Gal3p. The GAL gene is expressed only when Gal4p binds to an upstream activation sequence (UAS) at the promoter region of the GAL gene. If galactose is not present, Gal80p interacts with Gal4p and the binding of Gal4p is inhibited. On the other hand, when galactose is present, Gal3p releases Gal80p's inhibition of Gal4p, allowing the GAL gene to be expressed. On the other hand, when glucose is present, it inhibits the transcription of the GAL gene by galactose, which is caused by the decrease in the concentration of Gal4p, which stops the transcription of the GAL gene. In addition, MIG1p binds to the GAL4 upstream repression sequence (URS), thereby interfering with transcription and inhibiting the transcription of the GAL gene. Thus, the state of the GAL gene is regulated by altering the efficacy of Gal4p (when glucose is present), or the transcriptional capacity of Gal4p by Gal80p.

        In the wild, the GAL gene exists in three states. When glucose is present, the GAL switch is turned off by GAL4 inhibition by MIG1p and GAL gene inhibition by Gal80p. In non-induced non-inhibiting conditions, such as in the presence of glycerol and lactate, Gal80p interacts with Gal4p, which is bound to DNA, and becomes inhibited. In the presence of galactose, the activated Gal3p sequesters Gal80p in the cytosol, allowing the migration of Gal80p from the nucleus of the cytoplasm to the cytoplasm. In GAL80 deficient variants there are only two states, which are off when glucose is present and expressed when glucose is absent. Thus, galactose is not essential as an inducer (Verma et al., Biotechnol. Appl. Biochem. 39, 89-97 (2004); Ostergaard et al., FEMS Yeast Research 1, 47-55 (2001)).

       As such, much research has been conducted on the regulation of GAL genes involved in galactose metabolism and understanding of GAL switches (Sil AK et al., Prot. Expr. Purif. 18, 2020-212 (2000)). In addition, GAL80-deficient strains were used to smoothly use galactose contained in the raw materials during alcohol fermentation or beer fermentation without glucose inhibition (Ostergaard S. et al., Nat. Biotechnol. 18, 1283-1286 (2000) However, there have been no studies on how or to what extent they are useful for the production of recombinant proteins compared to the use of host cells in actual production of recombinant proteins. Research reports investigating the effects of various deficiencies on the expression of the GAL1 gene (Stagoj et al., FEMS Microbiology Letters 244, 105-110 (2005)) suggest that the GAL1 promoter in 28 strains of deficiency of 28 genes involved in galactose metabolism. The expression level of the GFP gene linked to the fluorescent protein was examined by measuring the fluorescence intensity. The highest expression increase was 2.4-fold higher than that of the control, and only 1.3-fold increase was observed in GAL80-deficient strains, indicating that cell growth was twice as good as GAL80-deficient strains. GAL1 deficiency was reported to be significantly higher in GAL1 deficiency than in GAL80 deficiency, while GAL3, GAL4 and MTH1 deficiency did not show any protein expression and growth of GAL7, GAL10 and GAL3 deficiency in galactose medium. However, there has been no detailed study of the applicability of these mutants to the actual production of recombinant proteins, particularly if they can be stably operated without the addition of galactose in large-scale cultivation to produce industrial enzymes that require mass production.

     The present inventors have conducted research to produce recombinant proteins using mutant strains and GAL promoters in Saccharomyces cerevisiae, and efficiently use recombinant strains and GAL gene promoters in Saccharomyces cerevisiaes without adding galactose. While studying how to produce proteins, it was found that the expression rate of recombinant proteins in GAL80 deficient strains was higher than in GAL1 deficient strains. In addition, GAL80-deficient strains provide better expression and cell growth than GAL1-deficient strains, and do not require a certain amount of galactose required by GAL1-deficient strains, and expression systems capable of high concentration cell culture and high-efficiency recombinant protein production in fed-batch cultures. By completing the present invention, the present invention was completed.

      It was also confirmed that expression was inhibited in the presence of glucose and that the expression occurred when glucose was consumed when no additional MIG1 deficiency was introduced into the GAL80 mutant strain. However, the concentration of glucose inhibited the expression under the conditions of normal fermentation and fed-batch culture. By focusing on maintaining a sufficiently low concentration, it was confirmed that high-efficiency expression can be achieved by using a mutant strain containing only GAL80 deficiency without introducing MIG1 deficiency, thereby establishing a late expression system capable of expressing a target protein at a late stage of fermentation. The present invention has been completed.

The present invention aims to produce recombinant proteins using GAL80 deficient strains of Saccharomyces cerevisiae and to establish an expression system for industrial application thereof.

GAL80 deficient strains of Saccharomyces cerevisiae were prepared to develop an industrially applicable recombinant protein producer, which confirmed its usefulness in recombinant protein production when compared to the host previously used for recombinant protein. In addition, the present invention was completed by constructing an expression system capable of producing highly efficient recombinant protein by applying it to fed-batch culture.

       Accordingly, the present invention provides, as a first aspect, a yeast variant that can induce the expression of recombinant protein with high efficiency by deficiency of the GAL80 gene.

       The yeast is preferably Saccharomyces cerevisiae, but the present invention is not limited thereto. That is, Saccharomyces cerevisiae and Saccharomyces cerevisiae and galactose metabolic pathways and regulatory genes can be used if the homogeneous yeast.

In a preferred embodiment of the present invention, a variant strain (Y2805 Δ gal 80) lacking its GAL80 gene was prepared using S805 in the Saccharomyces cerevisiae, which was sold by the National Institutes of Health, and it was prepared in Yuseong-gu, Daegu, Korea. It was deposited on July 30, 2007 with accession number KCTC 11162BP to the Korean Collection for Type Cultures (KCTC) in the Korea Research Institute of Bioscience and Biotechnology. The base sequence of the GAL80 gene was obtained through SGD (Saccharomyces Genome Database), and used after amplification using the primers of SEQ ID NOs: 2-5. Since the disruption of the GAL80 gene using a gene pop-out technique that is well known in the art it is shown in FIG. The mutant strain for expressing GAL promoter prepared as described above is shown in Table 1. As shown in Table 1, in the present Example, in addition to the mutant strain deficient in the GAL80 gene, mutant strains additionally deficient in MIG1p, MIG2p and MIG1p and GAL6 genes involved in glucose metabolism were prepared to compare growth rates and activities. In addition to producing the mutant (△ GAL1) which lack the GAL1 gene as the known control group was compared to activity. As will be described later, according to the present invention, the mutant strain lacking the GAL80 gene ( Δ GAL180) shows much higher growth rate and recombinant protein expression in the galactose-free medium than the mutant strain lacking the GAL1 gene. It allows the production of recombinant proteins at high concentrations without the need for The use of such mutant strains that does not require galactose is particularly advantageous in large volume culture system applications, as described in detail in the Examples herein, the present invention provides for high concentration culture potential and high recombination of the mutant strains in such mass culture processes. Protein expression was confirmed.

In a second aspect of the present invention, there is provided a recombinant protein secreting mutant strain using the Δ GAL80 mutant strain as a host and a method for producing the same. In particular, the mutant strain is characterized in that it can secrete and express recombinant protein at a high concentration in a medium without galactose. Such mutants can be readily prepared by introducing a gene encoding a foreign protein in △ GAL80 mutant according to the first aspect of the present invention. Specifically, it can be easily produced by disrupting the GAL80 gene of the yeast mutant strain and transforming the recombinant expression vector encoding the foreign protein in the mutant strain. The transformation may be performed by appropriately selecting various techniques well known to those skilled in the art.

In a specific embodiment of the present invention Candida antetique ( Candida) antarctica) was prepared in the yeast mutant strain which express, secrete the mutant △ lipase protein GalB14 by introducing the GAL80 mutant recombinant expression vector comprising a gene encoding a mutant protein of the CalB14 protein lipase B (CALB) of origin. The CalB14 mutant lipase protein is a protein previously developed and patented by the present inventors (Korean Patent No. 475133) and its amino acid sequence is shown in SEQ ID NO: 1. CalB14 expression vector was transformed into each of the mutant strains shown in Table 1 by the lithium / acetate (lithium / acetate) method, it was cultured in YPD liquid medium to measure CalB activity. As a result, it was confirmed that the ΔGal1 and ΔGal80 mutants had high CalB14 expression efficiency (FIG. 3). However, when cultured in a medium without galactose, the ΔGal80 mutant strain according to the present invention showed higher CalB14 expression efficiency than the ΔGal1 mutant strain. On the other hand, mutant strains additionally deficient in MIG1p, MIG2p and MIG1p and Gal6, which are involved in glucose metabolism, showed little CalB14 expression activity.

      In still another aspect of the present invention, there is provided a method for producing a recombinant protein, in particular, a method for mass production of a recombinant protein using the ΔGal80 mutant strain.

      Previous studies showed that ΔGal1 showed a higher expression rate than ΔGal80 when the expression of recombinant proteins ΔGal1 and ΔGal80 was measured, but cell growth was superior to ΔGal80, and the expression rate per unit cell was ΔGal1. This was reported to be higher than ΔGal80. However, this study was conducted in a medium composition in which galactose, a high cost substrate, is present and has a disadvantage in that it has a high cost when applied to mass culture.

In a specific embodiment of the present invention, a large-scale fed-batch culture using the medium composition shown in Tables 2 and 3, ΔGal80 mutant strain on the amount of cells and lipase activity after culture in the medium without galactose, which is a high cost substrate It was confirmed that compared to △ Gal1 mutant strain increased about 59% in 5L culture, about 66% in 300L culture (Fig. 4-7). As described above, ΔGal80 mutant strains in the medium without galactose show 1.5 times higher CalB14 expression rate than ΔGal1 mutants in various fermenters, and according to the present invention, ΔGal80 is the best mutant strain for producing recombinant genes. Mutant strains were selected. In addition, by performing a mass production test in 2,500L fermenter using this, the possibility of mass culture of the mutant strain according to the present invention was verified (Fig. 8).

       As described above, the GAL80 deficient strain according to the present invention has a higher expression rate and cell growth than the conventional GAL1 deficient strain and does not require galactose, which is an expensive substrate essential for culturing the GAL1 deficient strain, and thus produces high concentration cell culture and high efficiency recombinant protein at low cost. This is possible. Therefore, the present invention can be usefully used for the production of recombinant protein in a number of industries, such as the animal feed manufacturing, baking industry, wool, wine industry, wastewater treatment, as well as the medical field.

Hereinafter, an Example demonstrates this invention. These examples are intended to specifically illustrate the present invention, thereby not limiting the scope of the present invention.

( Example )

One. GAL  Genetic Deficiency Manufacturing

       The parent strain for the production of mutant strains deficient in galactose metabolism-related genes 2805 (Y2805) (Sohn JH et al., J Microbiol. Biotechnol. 1, 266-273 (1991) (Choi ES) obtained from the National Institutes of Health. Et al., Appl.Microbiol.Biotechnol 42, 587-594 (1994)) In order to prepare GAL80 deficient variants, gal80 open reading frame (ORF) sequences were obtained from the Saccharomyces Genome Database (SGD) and PCR was used. GAL80 deficient variants were prepared using the gene pop-out technique well known in the art (Johnston M. et al., Meth. Enzymol. 350, 290-315 (2002)) as shown in FIG. 1.

      Specifically, genomic DNA of Saccharomyces cerevisiae was used as a template to obtain DNA fragments about 350bp upstream and 350bp downstream of the GAL80 gene ORF (SEQ ID NO: 2: 5'-atg gac tac aac aag aga tc). -3 ') and DH2 (SEQ ID NO: 3'5'-ggc gac cac acc cgt cct gtg caa ggg ccc att cta cga a-3') to amplify upstream DNA fragments by PCR (polymerase chain reaction) In the same way, the downstream DNA fragments were prepared by primers DH3 (SEQ ID NO: 5'-gcg atg ctg tcg gaa tgg acc agt gga act aga gca aacg-3 ') and DH4 (SEQ ID NO: 5: 5'-tta taa act ata atg). cga ga-3 '). The amplified DNA bands were identified by ethidium bromide staining on agarose gels and purified using a gel purification kit (Bioneer). To connect the purified upstream DNA fragments and downstream fragments to the pop-out casettes containing the URA3 gene, synthetic extension PCR was performed through primers DH1 and u200r, and primers u200f and DH4 were used to connect the downstream DNA fragments. Lithium / acetate method (Hill J. et al., Nucleic Acids Res. 16, 5971 (1991)) was used to transform the obtained DNA fragment into the Y2805 wild strain (wt). Colonies formed in the UD solid medium were transferred to the solid medium again, and then confirmed by PCR using primers DH1 and u200r to confirm that the clones were transformed correctly.

       The clones were incubated for about 1 hour in YPD liquid medium, a high nutrient medium without selective pressure, and then colonized with URA3 gene in 5-FOA (5-fluoro-orotic acid) solid medium. After transferring to the solid medium was confirmed by PCR using primers DH1 and DH4. At this time, in order to prevent an incorrect result by primer-jumping, the extension time was reacted for more than 3 minutes.

        Variants Y2805Δgal80Δmig1, GAL80, MIG1 and GAL6 genes simultaneously lacking the Y2805Δgal1, GAL80, and MIG1 genes that lack the GAL1 gene, and the Y2805Δgal80Δmig1Δgal6, GAL80, MIG1, and MIG2 genes. A co-deficient variant strain Y2805Δgal80Δmig1Δmig2 was prepared. Their genotypes are shown in Table 1 below.

Strain genotype Y2805wt Y2805Δgal1 Y2805Δgal80 Y2805Δgal80Δmig1 Y2805Δgal80Δmig2 Y2805Δgal80Δmig1Δgal6 MATa pep4 :: HIS3 prb -1.6R can1 his3 -20 ura3 -52 gal1 :: Tc190 gal80 :: Tc190 gal80 :: Tc190 mig1 :: Tc190 gal80 :: Tc190 mig2 :: Tc190 gal80 :: Tc190 mig1 :: Tc190 gal6 :: Tc190

2. Growth of Host Cells

    Destroying the activity of certain genes in yeast often leads to slow cell growth. In order to overexpress recombinant proteins, the growth of host cells is very important. Therefore, the growth of the modified mutant strains prepared in this study was measured first. Each strain was inoculated in 3 ml YPD liquid medium and grown for 1 day, and then 25 ml YPD was put in a 250 ml flask and inoculated so that the absorbance (O.D.600) of each strain was 1.0. The result of dilution with one-hundredth so as to fall within the effective measurement range of the spectrometer is shown in FIG. 2.

As can be seen from the results, the Y2805Δ gal80 Δ mig2 strain was relatively lag behind the other mutant strains and was excluded from the host to be used in this experiment. Δgal1 has less growth than the Y2805 wild strain (wt), but Δ gal80 , Δ gal80 Δ mig1 and Δ gal80 Δ mig1 Δ gal6 Was not significantly different from Y2805 wt in growth rate.

3. Confirmation of Recombinant Protein Expression Using Deficiency Mutants

       In the expression of recombinant protein, we used CalB14 having an amino acid sequence of SEQ ID NO: 1, which is a variant of the industrial enzyme Candida Antartica lipase B gene (CalB), which is highly useful for chemical synthesis, which has been recently patented by the inventors (Korean Patent No. 475133). Reference).

The gene coding for the CalB14 protein was cloned into a yeast expression vector having a secretion factor of GAL10 promoter and TFP3, and the vector pYGT3-CalB14 (see Korean Patent No. 697310) using the lithium / acetate (lithium / acetate) method. Y2805 wt, Y2805Δ gal1 , Y2805Δ gal80 , Y2805Δ gal80 Δ mig1 and Y2805Δ g al 80 Δ mig1 Δ gal6 mutants were transformed. For these cultures, all mutants were inoculated in 3 ml of YPD and grown for 1 day, and then inoculated in a 250 ml flask in a 25 ml medium so that the value measured at OD600 was 1.0. The medium used was 2% glucose (all Δgal80 mutants), or 1% glucose and 1% galactose (Y2805 wt), or 2% glucose and 0.3% galactose (Δgal1 mutant) based on YP (2% yeast extract and 1% peptone). ) Was used. At 24, 36 and 48 hours after inoculation of the cells into each medium, the OD600 of each strain was measured and CalB14 activity was measured by pNPP as follows (FIG. 3).

      The lipase activity was measured using p-nitrophenyl palmitate (pNPP). 10 μl of 10 mM pNPP, 40 μl ethyl alcohol and 950 μl 50 mM pH 7.5 Tris buffer were added to 20 ml of enzyme solution, which was diluted properly, and the absorbance was measured at 405 nm for 10 minutes. One unit of lipase activity was defined as the activity of an enzyme that liberates 1 μmole of pNP groups per minute.

Y2805 wt was grown at a very high rate, and it was believed that galactose-induced expression occurred rapidly in the early stage of rapid depletion of glucose due to the rapid depletion of glucose and the increase of total cell mass. In contrast, the Δgal1 and Δ gal80 mutants showed slightly slower growth than the wt mutants, but did not show a significant difference.In the flask culture, fermenter was used in subsequent experiments because glucose consumption was relatively slow and pH control was difficult to distinguish. The expression patterns of the two mutants were compared. CalB activity was hardly observed in the Δ gal80 Δ m i g1 mutant and Δ gal80 Δ mig1 Δ gal6 .

4. Δ gal1  Mutant Lipase Calb  production

The mutant strain lacking the GAL1 gene was used for fed-batch culture using a 5L fermenter for mass production of lipase CalB14. Δ gal1 mutant strain transformed with the expression vector described in Example 3 was used and the composition of the medium is shown in Table 2. The initial glucose concentration was 20 g / L, and after all the glucose was depleted and the metabolite by-product ethanol was consumed, the feed medium containing glucose was fed to the fermenter in a stepwise feeding method until the end of the culture. When the cell concentration reached 30 OD (20 hours), 7.5 g of the inducer galactose was temporarily supplied to maintain 0.3% of the total culture solution. As a result of the culture, the concentration of the cells increased to 64 OD (dry weight, 15.4 g / L) at the end of the culture and the lipase activity increased to 6,868 U / L (FIG. 4).

5. △ gal80  Mutant Lipase Calb  production

Carried out using a 5L fermenter of △ gal80 mutant strain transformed with the lipase expression vector technology seen in Example 3 to induce mass production of the lipase via a fed-batch culture. The medium composition was the same as that used when the Δ gal1 mutant was used, but galactose was not added as an inducer. The initial glucose concentration was 20 g / L, and after all the glucose was depleted and ethanol, a metabolite by-product, was consumed, the glucose-containing feed medium was fed to the end of the culture stepwise culture method. As a result of the culture, the concentration of the cells was increased to 80 OD (dry weight, 19.2g / L) at the end of the culture, lipase activity was increased to 10,951 U / L (Fig. 5). This value was confirmed by △ increased about 59% than the value of al gal1 mutant △ g 80 mutants is also high recombinant protein productivity than the △ gal1 mutant does not need an expensive substrate, galactose FIG.

6. △ gal1 Mutational strain  On the 300 liter fermenter scale Lipase  production

The △ △ gal1 mutant strain and mutant 80 g al, as described above in order to mass-producing recombinant CalB14 was used. Δ gal80 in a 5L fermenter in the previous experiment Because the mutant Bar confirmed that mass production of lipase without any addition of galactose, but in large-scale fermenters may not be stable lipase expression and mutant △ gal1 △ gal80 Mutant strains were used to compare the stable expression of lipase in each fermenter of larger scale. In order to industrialize this mutant strain, fermenters over 300L scale were used for cheap industrial medium.

Based on the results of the 5L fermenter obtained in Example 4 and the stirring time and oxygen transfer rate measured to scale up to 300L fermenter, the recombinant Δ gal1 mutant was scaled up in the 300L fermenter. The medium composition used is shown in Table 3 and all of the mediums were used for the cheap industrial medium. The culture conditions were 30 ^° C, pH 5.5. Initially, the stirring speed was increased step by step, starting with 100 rpm and the air flow amount was 0.5 vvm.

The spawn culture was incubated for 24 hours at a temperature of 30 ^° C., pH 5.5, with a working volume of 20L in a 50L fermenter. In the main culture 300L fermenter 120L working volume was used. After seeding inoculation, such as 5 L fermentation of Example 4, the initial glucose was depleted and the metabolite byproduct ethanol was consumed, and then the glucose-containing feed medium was fed in a stepwise manner until the end of the culture. When the cell concentration reached 40 OD in culture (22 hours), 450 g of the inducer galactose was temporarily supplied to maintain 0.3% of the total culture solution. As a result of the culture, the concentration of the cells increased to 112 OD (dry cell weight, 26.9 g / L) at the end of the culture, and the activity of CalB14 increased to 11,016 U / L (FIG. 6). Cells increased more than two times compared to 5L fermentation, and lipase expression more than doubled.

7. △ gal80  On a 300 liter fermenter scale using mutants Lipase  production

Cultures were performed in 300 L fermenters using recombinant Δ gal80 mutants. The medium composition and spawn culture used and the culture conditions of 300L were the same as 300L fermentation using Δ gal1 mutants. Galactose was not added to induce the expression of lipase in 300L culture only.

After spawn inoculation, the initial glucose was depleted and the metabolite by-product ethanol was consumed, and then the feed medium ¹ containing glucose was fed in a stepwise manner until the end of the culture. Culture results, the concentration of cells was increased to 136 OD (dry weight, 32.6g / L) on the end of the incubation CalB activity was increased to 16,632 U / L, also when compared to the 5L fermentation in analogy to △ gal1 mutant CalB Lipase activity increased 66% (FIG. 7).

8. In 2,500L fermenter Lipase Calb  production

As described above, when △ gal80 mutant was used without galactose addition, CalB activity was produced 1.5 times higher than △ gal1 mutant in both 5L fermenter and experimental scale 300L fermenter. Therefore, △ gal80 mutant was finally selected as the best mutant strain and calB mass production test in 2,500L fermenter was performed.

The medium composition used was the same as in the 300 L fermentation and all of the medium used cheap industrial medium. The culture conditions were 30 ^° C, pH 5.5. Initially, the stirring speed was increased stepwise starting at 80 rpm and the air flow rate was 0.5 vvm.

The spawn culture was incubated for 17 hours at a temperature of 30 ^° C., pH 5.5 with a working volume of 150L in a 300L fermenter. In the main culture of 2,500 L fermenter, 1,000 L working volume was used. As in the 5L and 300L fermentation of the previous section, the initial glucose was depleted after the seed inoculation and the metabolite by-product ethanol was consumed, and then the glucose-containing feed medium was fed in a stepwise manner until the end of the culture. As a result of the culture, the concentration of the cells was increased to 106 OD (dry weight, 25.4g / L) at the end of the culture and CalB activity increased to 19,438 U / L (Fig. 8).

1 is a schematic diagram illustrating the gene disruption process of the yeast gene (Saccharomyces cerevisiae 2085) for producing a GAL80 deficient strain.

Figure 2 shows the growth of the improved mutant strain by measuring the absorbance of each mutant strain after culturing the modified mutant strain produced by the gene destruction process in YPD liquid medium for one week.

FIG. 3 is a diagram comparing CalB14 secretion by culturing various deficient mutants and pNPP.

Figure 4 is a diagram showing the absorbance and activity of 5L fed-batch culture for the production of CalB lipase using the △ GAL1 mutant strain and the resulting mutant strain.

Figure 5 is a diagram showing the absorbance and activity of 5L fed-batch culture and mutant strains for the production of CalB lipase using △ GAL80 mutants.

Figure 6 is a diagram showing the absorbance and activity of 300L fed-batch culture and mutant strains for the production of CalB lipase using Δ GAL1 mutants.

Figure 7 is a diagram showing the absorbance and activity of 300L fed-batch culture for the production of CalB lipase using the Δ GAL80 mutant strain and thus the mutant strain.

8 is a figure showing the glucose feeding rate and the stirring rate, and the absorbance of the culture when mutant, secretion of lipase activity, and specific activity for the mass production in the CalB 2,500L fermenter by selecting a mutant △ GAL80 proven through the culture to be.

<110> Korea Research Institure of Bioscience & Biotechnology <120> A method for Producing a Recombinant Protein using a GAL80          Deficient Yeast Strain <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 317 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of CalB mutant <400> 1 Leu Pro Ser Gly Ser Asp Pro Ala Phe Ser Gln Pro Lys Ser Val Leu   1 5 10 15 Asp Ala Gly Leu Thr Cys Gln Gly Ala Ser Pro Ser Ser Val Ser Lys              20 25 30 Pro Ile Leu Leu Val Pro Gly Thr Gly Thr Thr Gly Pro Gln Ser Phe          35 40 45 Asp Ser Asn Trp Ile Pro Leu Ser Ala Gln Leu Gly Tyr Thr Pro Cys      50 55 60 Trp Ile Ser Pro Pro Pro Phe Met Leu Asn Asp Thr Gln Val Asn Thr  65 70 75 80 Glu Tyr Met Val Asn Ala Ile Thr Thr Leu Tyr Ala Gly Ser Gly Asn                  85 90 95 Asn Lys Leu Pro Val Leu Thr Trp Ser Gln Gly Gly Leu Val Ala Gln             100 105 110 Trp Gly Leu Thr Phe Phe Pro Ser Ile Arg Ser Lys Val Asp Arg Leu         115 120 125 Met Ala Phe Ala Pro Asp Tyr Lys Gly Thr Val Leu Ala Gly Pro Leu     130 135 140 Asp Ala Leu Ala Val Ser Ala Pro Ser Val Trp Gln Gln Thr Thr Gly 145 150 155 160 Ser Ala Leu Thr Thr Ala Leu Arg Asn Ala Gly Gly Leu Thr Gln Ile                 165 170 175 Val Pro Thr Thr Asn Leu Tyr Ser Ala Thr Asp Glu Ile Val Gln Pro             180 185 190 Gln Val Ser Asn Ser Pro Leu Asp Ser Ser Tyr Leu Phe Asn Gly Lys         195 200 205 Asn Val Gln Ala Gln Ala Val Cys Gly Pro Gln Phe Val Ile Asp His     210 215 220 Ala Gly Ser Leu Thr Ser Gln Phe Ser Tyr Val Val Gly Arg Ser Ala 225 230 235 240 Leu Arg Ser Thr Thr Gly Gln Ala Arg Ser Ala Asp Tyr Gly Ile Thr                 245 250 255 Asp Cys Asn Pro Leu Pro Ala Asn Asp Leu Thr Pro Glu Gln Lys Val             260 265 270 Ala Ala Ala Ala Leu Pro Ala Pro Ala Ala Ala Ala Ile Val Ala Gly         275 280 285 Pro Lys Gln Asn Cys Glu Pro Asp Leu Met Pro Tyr Ala Arg Pro Phe     290 295 300 Ala Val Gly Lys Arg Thr Cys Ser Gly Ile Val Thr Pro 305 310 315 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DH1 forward primer for upstream DNA amplification <400> 2 atggactaca acaagagatc 20 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DH2 reverse primer for upstream DNA amplification <400> 3 ggcgaccaca cccgtcctgt gcaagggccc attctacgaa 40 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> DH3 forward primer for down stream DNA amplificaton <400> 4 gcgatgctgt cggaatggac cagtggaact agagcaaacg 40 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DH4 reverse primer for downstream DNA amplification <400> 5 ttataaacta taatgcgaga 20  

Claims (12)

      a) disrupting the GAL80 gene of yeast,      b) a method of producing a mutant strain expressing the recombinant foreign protein with high efficiency in a galactose-free medium, comprising transforming the recombinant expression vector encoding the foreign protein into a yeast mutant strain in which the GAL80 gene is disrupted. The method of claim 1, wherein the yeast MIG1 gene is not disrupted.  2. The method of claim 1, wherein said yeast is Saccharomyces cerevisiae or Saccharomyces cerevisiae and yeast whose galactose metabolic pathways and regulatory genes are homologous. 4. The method according to claim 3, wherein said yeast is Saccharomyces cerevisiae 2805 (Y2805).        a) disrupting the GAL80 gene in Saccharomyces cerevisiae,       b) introducing a gene encoding a recombinant protein into a mutant strain in which the GAL80 gene is disrupted c) A method of producing a recombinant protein comprising culturing a mutant strain into which the gene encoding the recombinant protein is introduced. The method for producing a recombinant protein according to claim 5, wherein the mutant strain in which the GAL80 gene is smashed is a mutant strain in Saccharomyces cerbizi of accession number KCTC 11162BP. The method for producing a recombinant protein according to claim 5 or 6, wherein the recombinant protein is a lipase B protein derived from Candida anthracica or a variant thereof. The method of claim 7, wherein the recombinant protein is a mutant lipase protein having an amino acid sequence represented by SEQ ID NO: 1. The method of claim 5 or 6, wherein the recombinant protein comprising the step of culturing the mutant strain using a bioreactor (bioreactor). 10. The method of claim 9, wherein the culture is a fed-batch culture. The method of claim 5, wherein the MIG1 gene of the mutant strain is not deleted so that the recombinant protein is produced at a stop period after glucose is consumed. Variant to Saccharomyces cerbizi of accession number KCTC 11162BP, in which the GAL80 gene was disrupted.

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