TWI726484B - Mutant yeast and method for producing protein using the same - Google Patents

Abstract

An object of the present invention is to find out a peptidase or a protease involved in degradation of cutinase-like enzymes, and efficiently produce a cutinase-like enzyme by suppressing the activity of the peptidase or the protease.

The present invention provides a yeast in which the activity of at least one peptidase or protease involved in the degradation of PaE is suppressed by introducing a mutation into the gene of the peptidase or protease.

Description

Mutant yeast and method for producing protein using the mutant yeast

The present invention relates to a mutant yeast and a method for producing a protein using the mutant yeast.

Cutinase-like enzymes such as esterase (PaE) produced by Pseudozyma antarctica, which can rapidly decompose biodegradable plastics, are isolated from the vegetative yeast or filamentous fungus. Identification. Research is currently underway to increase the production of cutinase-like enzymes by optimizing the culture conditions of the bacteria that produce this enzyme, selecting high-producing strains, and introducing high-performance gene cassettes (gene cassette), etc. (Non-Patent Document 1 ).

On the other hand, it is generally known that the enzyme system produced by the bacteria is decomposed by the protease derived from the bacteria (Patent Document 1). However, a protease that decomposes cutinase-like enzymes is currently unknown.

[Prior Technical Literature]

[Patent Literature]

[Patent Document 1] International Publication No. 02/101038

[Non-Patent Literature]

[Non-Patent Document 1] Watanabe et al., Applied Microbiology and Biotechnology, Vol. 100, No. 7, pp. 3207-3217, 2016.

In the culture solution of the yeast that produces and secretes PaE, the degradation product of the PaE accumulates as the culture continues. In order to utilize the PaE industry, such decomposition must be suppressed. The purpose of the present invention is to find a peptidase or protease involved in the decomposition of PaE, and to efficiently produce PaE by inhibiting the activity of the peptidase or protease.

The inventors of the present invention, as a result of careful examination to solve the above-mentioned problems, discovered proteases involved in the decomposition of PaE. Then, it was unexpectedly discovered that if the activities of these proteases are inhibited, in addition to the degradation of PaE, the degradation of other proteins is also inhibited, and the present invention is ended. That is, the present invention provides the following mutant yeast, a method for producing protein using the mutant yeast, and a method for producing protein using yeast.

[1] A yeast in which at least one peptidase or protease gene involved in the degradation of esterase (PaE) produced by Pseudozyma antarctica is introduced with mutations, thereby making the peptide The activity of the enzyme or protease is inhibited.

[2] The yeast according to [1], wherein at least one base of the gene is deleted or replaced with another base, or at least one base is inserted into the gene.

[3] The yeast according to [1] or [2] above, wherein all of the aforementioned genes are deleted.

[4] The yeast described in any one of [1] to [3] above, wherein the peptidase or protease system comprises one or more selected from PAN1_016f05330 (SEQ ID NO 1), PAN1_004r02266 (SEQ ID NO 2), PAN1_003r01856 Serial number 3), PAN1_003r01668 (serial number 4), PAN1_001f00846 (serial number 5), PAN1_019f05845 (serial number 6), PAN1_024f06685 (serial number 7), PAN1_012r04564 (serial number 8), PAN1_010r04227 (serial number 9), PAN1_013 r04779 serial number 10) An enzyme encoded by the gene of Candida antarctica or its homolog or otholog in the group consisting of PAN1_023r06563 (SEQ ID NO: 11).

[5] The yeast according to any one of [1] to [4] above, which is a yeast of the genus Pseudozyma.

[6] The yeast according to any one of [1] to [5] above, which produces the above PaE.

[7] The yeast according to any one of [1] to [6] above, which produces a recombinant protein.

[8] A method for producing protein using the yeast described in any one of [1] to [7] above.

[9] The production method according to [8] above, wherein the protein is PaE.

[10] A method for producing protein using yeast, comprising the step of inhibiting the activity of at least one peptidase or protease involved in the decomposition of PaE in the yeast.

According to the present invention, by introducing mutations into at least one peptidase or protease gene involved in the decomposition of PaE, a yeast in which the activity of the peptidase or protease is inhibited can be provided. By using such mutant yeast, not only can the decomposition of PaE in the culture solution be inhibited, but also the decomposition of other proteins can unexpectedly be inhibited. Also, when yeast is used to make protein, if the participation in PaE is inhibited The gene of at least one peptidase or protease can not only inhibit the decomposition of PaE in the culture solution, but also inhibit the decomposition of other proteins. Therefore, protein can be efficiently produced.

Figure 1 shows an electrophoresis photograph of PaE and its degradation products in the culture supernatant.

Figure 2 shows a photograph of PaE and its degradation products in the culture supernatant of each gene disrupted strain analyzed by the Western blot method.

Figure 3 shows the results of the ProA gene complementation experiment.

Figure 4 shows the Western blot analysis results of PaE and its decomposition products cultivated by different methods.

Figure 5 shows a graph of lipase activity in the culture supernatant of each gene disrupted strain.

Figure 6 shows a photograph of a gel representing the gelatin decomposition activity in the culture supernatant of each gene disrupted strain.

Figure 7 shows a graph showing changes in the PaE activity of the culture solution and the weight of the dried cells as the culture time elapses.

Figure 8 shows an electrophoresis photograph of PaE and its degradation products in the culture medium.

Figure 9 shows a graph showing changes in the residual viability of PaE and the weight of dry cells in the culture medium with the standing time.

Figure 10 shows an electrophoresis photograph of PaE and its degradation products in the culture solution after standing.

Figure 11 shows a graph of the residual viability of PaE in the culture supernatant and the weight of the dried cells with the standing time.

Figure 12 shows an electrophoresis photograph of PaE and its degradation products in the culture supernatant after standing.

Hereinafter, the present invention will be explained in further detail.

The yeast of the present invention is characterized in that the gene of at least one peptidase or protease involved in the decomposition of PaE is introduced with a mutation, thereby inhibiting the activity of the peptidase or protease. The "yeast" described in this specification means a fungus that exhibits unicellularity, and it may be a basidiomycete yeast or a zymocystis yeast. In a certain aspect, the aforementioned yeast may also be Candida, Rhodotorula, Cryptococcus, Moesziomyces, Pichia , Saccharomyces (Saccharomyces), or Schizosaccaromyces (Schizosaccaromyces) yeast. In addition, the aforementioned yeast can also produce PaE and/or other recombinant proteins. The yeast that produces the aforementioned PaE can be the one that originally expresses PaE, or the one that expresses PaE after transformation.

The aforementioned PaE is derived from Candida antarctica (recently changed in classification, also known as Moesziomyces antarcticus), which is derived from the basidiomycete yeast that is common in plants. It is an isolated species that decomposes biodegradable plastics. Cutinase enzyme. Specifically, the aforementioned PaE system can be obtained from the GB-4(0) strain of P.antactica (yeast deposited in the Patent Microorganism Deposit Center of the Independent Administrative Agency for Product Evaluation Technology; deposit number NITE P- 02392), GB-4(1)W strain, GB-4(0)-HPM7 strain (yeast deposited at the Patent Microorganism Deposit Center of the Technical Infrastructure for Product Evaluation of Independent Administrative Legal Person; deposit number NITE P-02238) and OMM62-2 Strain (Yeast deposited at the Patent Microorganism Deposit Center of the Independent Administrative Agency for Product Evaluation Technology; deposit number NITE P-02239) and so on. In addition, although cutinase-like enzymes In its amino acid sequence, it has a lipase cassette common to cutinase, but it does not necessarily have the cutinase activity like cutinase, and the amino acid sequence of the entire protein is not highly similar to cutinase. Regarding substrate specificity, cutinase has high decomposition activity of short-chain fatty acid esters, and low decomposition activity of long-chain fatty acid esters. In contrast, cutinase-like enzymes are more effective for short-chain fatty acid esters and long-chain fatty acid esters. Either can be fully decomposed. Therefore, cutinase and cutinase-like enzymes can be regarded as different kinds of enzymes with different characteristics.

At least one peptidase or protease involved in the decomposition of the aforementioned PaE can be produced under the conditions for the production of the aforementioned PaE. Therefore, for example, candidates for introducing the aforementioned mutation target gene can be analyzed by transcriptome analysis. And proteome analysis. The transcriptome analysis and proteome analysis system respectively compare the mRNA or protein expressed under the conditions in which the amount of PaE production increases, and the mRNA or protein expressed under these conditions. Alternatively, from the genome of Candida antarctica that originally expresses PaE, orthologs of the peptidase or protease of the genomic analysis of Pseudomonas cerevisiae may be used as candidates for the introduction of the aforementioned mutation target gene. Furthermore, the missing variants of each candidate gene are also produced by conventional methods, and the PaE and its decomposition products produced by the variants are confirmed by Western blotting method, etc., by which at least one species participating in the aforementioned PaE can be identified. Decomposed peptidase or protease. In addition, the peptidase or protease identified in this way may be the one that directly decomposes the aforementioned PaE, or the peptidase or protease that cleaves the precursor of the peptidase or protease that directly decomposes the aforementioned PaE to activate it .

In a certain aspect, at least one peptidase or protease involved in the decomposition of PaE may include one or more genes selected from the following group of Candida antarctica genes or their homologs or orthologs are encoded The enzymes:

PAN1_016f05330 (serial number 1)

PAN1_004r02266 (serial number 2)

PAN1_003r01856 (serial number 3)

PAN1_003r01668 (serial number 4)

PAN1_001f00846 (serial number 5)

PAN1_019f05845 (serial number 6)

PAN1_024f06685 (serial number 7)

PAN1_012r04564 (serial number 8)

PAN1_010r04227 (serial number 9)

PAN1_013r04779 (serial number 10)

PAN1_023r06563 (serial number 11)

The specific base sequence of each gene is as follows.

<PAN1_016f05330>

(序列編號1)

(Serial number 1)

<PAN1_004r02266>

(序列編號2)

(Serial number 2)

<PAN1_003r01856>

(序列編號3)

(Serial number 3)

<PAN1_003r01668>

(序列編號4)

(Serial number 4)

<PAN1_001f00846>

(序列編號5)

(Serial number 5)

<PAN1_019f05845>

(序列編號6)

(Serial number 6)

<PAN1_024f06685>

(序列編號7)

(Serial number 7)

<PAN1_012r04564>

(序列編號8)

(Serial number 8)

<PAN1_010r04227>

(序列編號9)

(Serial number 9)

<PAN1_013r04779>

(序列編號10)

(Serial number 10)

<PAN1_023r06563>

(序列編號11)

(Serial number 11)

The mutation introduced into at least one peptidase or protease gene involved in the decomposition of the aforementioned PaE is not particularly limited as long as it can inhibit the activity of the peptidase or protease. For example, it may be at least one base of the aforementioned gene. Deletion of base, placement of at least 1 base of the aforementioned gene Replacement, or insertion of at least one base in the aforementioned gene, etc. In a certain aspect, all the aforementioned genes in the yeast of the present invention are deleted.

In other aspects, the present invention also relates to a method for producing protein using the aforementioned yeast. As for the aforementioned protein, various proteins can be used, but PaE is preferred. When producing proteins other than the aforementioned PaE, the gene to be mutated can be selected that is optimal for improving the stability of the produced protein. For example, when the aforementioned protein is lipase A, the aforementioned mutation target gene may include PAN1_001f00846 (SEQ ID NO: 5) and/or PAN1_010r04227 (SEQ ID NO: 9) or homologs or orthologs thereof. In addition, when inhibiting the activity of the peptidase or protease encoded by PAN1_004r02266 (SEQ ID NO: 2) or its homologs or orthologs, the degradation of proteins can be broadly inhibited, and therefore the stability of various proteins can be expected to be improved.

Furthermore, in other aspects, the present invention also relates to a method for producing protein using yeast, and the method is characterized by including: inhibiting at least one peptidase or protease involved in the decomposition of PaE in the aforementioned yeast, or the same gene. The step of the activity of the peptidase or protease encoded by the source or ortholog. Regarding such inhibitory means, although the means commonly used in the technical field can be used without particular limitation, for example, it can also be the gene of the aforementioned peptidase or protease or its homologs or orthologs. The mutation is introduced to inhibit the activity, or the activity is inhibited by using antibodies or inhibitors against the peptidase or protease encoded by the aforementioned peptidase or protease or homologs or orthologs of the genes thereof. In addition, the activity can also be suppressed by controlling culture, separation/purification, and storage conditions such as pH.

Hereinafter, the present invention will be explained in detail through examples, but the scope of the present invention is not limited by these examples.

[Example]

[Example 1] Confirmation of PaE degradation products produced in yeast culture and decrease in enzyme activity

The GB-4(0)-X14 strain (if necessary, refer to Non-Patent Document 1), which is a PaE high-producing mutant of Candida antarctica, was cultured in the presence of xylose in accordance with a conventional method. The culture supernatant containing the recombinant PaE was recovered by centrifugation and allowed to stand at room temperature. The culture supernatant immediately after culture and the culture supernatant after cryopreservation were electrophoresed by SDS polyacrylamide gel, and the gel was stained with Coomassie brilliant blue (CBB). In addition, the PaE activity of the culture supernatant immediately after culture and the culture supernatant after cryopreservation was measured.

As a result, under the PaE-derived band, the PaE-derived band was confirmed, and the band of PaE-derived product became thicker as the number of days after culture passed (Figure 1). In addition, as shown in Table 1 below, it was confirmed that the PaE activity of the culture supernatant decreased with the passage of days after the culture. In addition, the determination of PaE activity was performed in accordance with the previous method. That is, in 20 mM Tris-HCl buffer (pH 6.8), the absorbance (OD660) becomes about 0.65, and the polybutylene succinate-adipate copolymer (polybutylene succinate- co-adipate) (PBSA) emulsion suspension. Add 1.9 mL of this aqueous suspension to a test tube with a diameter of 13 mm, appropriately dilute the above-mentioned culture supernatant and add to 100 μL, shake at 30° C. and 180 rpm for 15 minutes, and measure the absorbance of the aqueous suspension. In terms of the unit of enzyme activity, the amount of enzyme that reduces the absorbance by 1.0 per minute is defined as 1U.

[Example 2] Exploration of peptidase or protease genes involved in the decomposition of PaE

The Candida antarctica GB-4(0) strain was cultured in the presence of xylose or glucose, and the transcriptome of Candida antarctica was analyzed over time using a DNA array prepared based on the genome information of Candida antarctica. When cultured in the presence of xylose, the PaE gene was strongly expressed at the beginning of the culture. In contrast, compared with the culture in the presence of glucose, genes that are characteristically expressed when cultured in the presence of xylose and whose expression levels gradually increase in the later stages of the culture are selected as candidates for genes involved in the breakdown of PaE.

In addition, the putative protease gene in the genome of Candida antarctica predicted from the genome information of Saccharomyces cerevisiae may be selected as a candidate for the gene involved in the decomposition of PaE.

Furthermore, the proteome analysis of the culture supernatant is performed. Compared with the culture in the presence of glucose, the protein that is characteristically produced during the culture in the presence of xylose and the amount of production gradually increases in the later period of the culture is selected, and the gene encoding the protein is selected It is a candidate for genes involved in the breakdown of PaE.

Table 2 below shows the candidate genes of peptidase or protease involved in the breakdown of PaE that were selected by transcriptome analysis, putative protease gene search, and proteome analysis.

[Example 3] Effect of Disruption of Candidate Genes on PaE Degradation Products

In Candida antarctica GB-4(0) strain, any of the genes described in Table 2 was disrupted by conventional methods. In short, the nourseothricin resistance gene is used as a drug resistance marker, a gene disrupted fragment inserted between the target genes is produced, and the gene disrupted fragment is homologously recombined and introduced into the cell. Thus, a gene disrupted strain was produced. Confirm whether the gene is damaged by colony PCR.

Each gene disrupted strain produced was cultured in a 300 mL Erlenmeyer flask containing a medium containing xylose for 5 days (30°C, 200 rpm). The bacterial cells were removed from each culture solution by centrifugation (5000 rpm, 5 minutes), and the culture supernatant was obtained, which was allowed to stand at 25°C for 8 days. The PaE and its decomposition products in each culture supernatant were analyzed according to the Western blot method. In the Western blot method, multiple anti-PaE antibodies and HRP-anti-rabbit IgG antibodies are used, and by chemiluminescence using ECL Prime (manufactured by GE Healthcare), PaE and its decomposition products transferred to the film are detected (exposure Time 60 minutes). The culture supernatant (WT) of wild-type Candida antarctica GB-4(0) strain and crude purified PaE were used as a control group.

The representative results of 2 to 4 experiments are shown in Figure 2, and the signal intensities derived from the decomposition products of PaE are summarized in Table 3 below.

++++: In each experiment, a stronger signal than the wild type is detected

+++: More than half of the experiments have detected a stronger signal than the wild type, and other experiments have detected the same or weaker signal

++: The same level of signal as the wild type was detected in more than half of the experiments, while weaker signals were detected in other experiments

+: Weak signals are detected in each experiment

-: No signal was detected in each experiment

Each gene disrupted strain produces PaE and secretes it into the culture supernatant. In the disrupted strains of candidate gene 3 or 10, the signal derived from the degradation product of PaE was observed to have an intensity equal to or higher than that of the wild type. On the other hand, among the disrupted strains of candidate genes 1, 2, 4 to 9, or 11 to 13, compared with the wild type, the signal derived from the degradation product of PaE was reduced. In particular, the disrupted strains of candidate gene 1 and the candidate In the gene 9 disrupted strain, almost no signal derived from the degradation product of PaE could be confirmed. Therefore, it is known that candidate genes 1, 2, 4 to 9, or 11 to 13 are involved in the decomposition of PaE, and therefore, these gene-disrupted strains are useful in the production of PaE. In addition, regarding the disrupted strain of gene 12, although abnormal proliferation was observed under the culture conditions this time, the culture conditions were not optimized, so it was not used in subsequent experiments.

[Example 4] Complementation experiment of ProA gene

For wild-type Candida antarctica GB-4(0) strain or a disrupted strain (ΔProA) of candidate gene 1 (ProA gene), introduce a complementary gene fragment (pUXV1-neo: ProA) or a control gene fragment containing the ProA gene (pUXV1-neo), to make a transformation. Using these transformants, the culture supernatant was prepared by the method described in Example 3. After the culture solution containing each culture supernatant and the cells before centrifugation was allowed to stand at 25°C for 7 days, the PaE decomposition product was detected by the Western blot method. The results are shown in Figure 3.

In the culture supernatant of the transformant in which the control gene fragment was introduced into the ProA gene disrupted strain (ΔProA), it was almost impossible to confirm the signal derived from the PaE degradation product. However, in the transformant in which the complementary gene fragment containing the ProA gene was introduced In the culture supernatant, the signal derived from PaE decomposition products increased. In addition, compared to the culture supernatant of the transform introduced with the control gene fragment, the culture supernatant of the transform in which the complementary gene fragment containing the ProA gene was introduced into the wild-type strain was derived from PaE decomposition The signal of things increases. Therefore, it was confirmed that the reduction of PaE decomposition products caused by the destruction of the ProA gene can be compensated by the introduction of the ProA gene.

[Example 5] Effect of gene disruption when cultured in a jar

Since the gene profile expressed by different culture conditions may change at the same time, the influence of gene disruption on PaE decomposition products was also confirmed in the culture in a jar larger than the culture scale in the flask of Example 3. Specifically, a disrupted strain of candidate gene 1 (ProA gene) or candidate gene 9 (ProB gene) was cultured in a 5L jar fermenter containing a medium containing xylose for 3 days (20°C, 423rpm) . The PaE and its decomposition products in each culture supernatant were analyzed by Western blotting method. The results are shown in Figure 4.

Even in the case of using jar culture to produce PaE, compared to the culture supernatant of the wild-type strain, the signal derived from the degradation product of PaE is cultivated in either the ProA gene disrupted strain or the ProB gene disrupted strain. All were reduced in the supernatant. On the other hand, when the culture supernatant of the ProA gene disrupted strain is compared with the culture supernatant of the ProB gene disrupted strain, the signal derived from the PaE degradation product is less in the culture supernatant of the latter than in the culture supernatant of the former Liquid, the system cannot be detected at all. Therefore, it suggests the possibility that the ProB gene disrupted strain is particularly useful when producing PaE on a large scale.

[Example 6] Lipase activity of each gene disrupted strain

It is known that lipase A is contained in the culture supernatant of Candida antarctica. Check whether the gene-disrupted strain selected using the decomposition of PaE as an indicator affects the activity of lipase A. Specifically, the lipase activity was measured using the lipase kit S (manufactured by DS Pharma Biomedical Co., Ltd.) using the culture supernatant of each gene disrupted strain prepared according to the method of Example 3. The results are shown in Figure 5.

Compared with the culture supernatant of the wild-type strain, the lipase activity is increased in the culture supernatant of the disrupted strain of candidate genes 2 to 4, 6, 8 to 11, or 13. Among the disrupted strains of candidate gene 6 or 11, lipase activity was particularly high. Therefore, it is suggested that the degradation of lipase A is inhibited in these gene disrupted strains.

[Example 7] Gelatin degradation activity of the culture supernatant of each gene disrupted strain

Since gelatin decomposition activity is regarded as an indicator of general protease activity, if the gelatin decomposition activity is low, it is believed that it is beneficial to the stable production of various proteins. In order to evaluate whether the gene disrupted strains selected using the degradation of PaE as an indicator are also useful for the production of other proteins, the disrupted strains of candidate genes 1 to 7 were used as representative examples to evaluate the gelatin degradation activity of the culture supernatants. Specifically, the culture supernatant and sample buffer (62.5mM Tris-HCl (pH6.8), 2% SDS, 10% glycerol, 0.002% BPB ) Mix and supply to a gel containing 0.1% gelatin for SDS-PAGE. Next, the gel after swimming was treated with 2.5% Triton X-100, and then immersed in 0.1M Tris-HCl. After incubating this at 37°C for 16 hours, it was stained with CBB. The results are shown in Figure 6.

If the gelatin in the gel is decomposed, it can be seen that the gel becomes white because its part is not stained by CBB. However, in the culture supernatant of the disrupted strain of candidate gene 2, the gelatin is not decomposed and the gel is not white. That is, in the culture supernatant of these gene disruptions, the activity of protease is usually suppressed. The same result can be obtained when the pH of the gelatin gel is adjusted to 5.2 or 9.8. Therefore, it is suggested that the gene-disrupted strain selected using the decomposition of PaE as an index is also useful for the production of other proteins.

It can be seen from the above that the use of mutant yeast can not only inhibit the decomposition of PaE in the culture solution, but unexpectedly, it can also inhibit the decomposition of other proteins. The mutant yeast is based on the gene of at least one peptidase or protease involved in the decomposition of PaE. Introduce variation, and inhibit the activity of the peptidase or protease. Therefore, protein can be efficiently produced.

[Example 8] Stability of PaE in culture

The expression cassette containing the xylanase promoter and PaE gene derived from Candida antarctica GB4(0) strain and the neomycin resistance gene as a marker was introduced into wild-type Candida antarctica GB4(0) The PaE high-production mutant strain (XG8 strain) or its ProB gene disrupted strain (ΔproB strain) was prepared by straining the above-mentioned PaE high-production mutant strain (XG8 strain) or its ProB gene disrupted strain (ΔproB strain) in the presence of xylose Next, according to the usual method, culture in a 3L jar fermenter. After 24 hours, 48 hours, and 72 hours after the start of the culture, each culture solution was collected, and the weight of the dried cells was measured. In addition, the subsequent PaE activity measurement method is performed as follows. That is, the PBSA emulsion was suspended in 25 mM HEPES buffer (pH 7.3±0.1) so that the absorbance (OD _{660) became about 0.55.} Add 1.995 mL of aqueous suspension to a test tube with a diameter of 10 mm, appropriately dilute the culture supernatant to 5 μL, shake at 30° C. and 150 rpm for 15 minutes, and measure the absorbance of the aqueous suspension. In terms of the unit of enzyme activity, the amount of enzyme that reduces the absorbance by 1.0 per minute is defined as 1U. Next, each culture supernatant was subjected to electrophoresis by SDS polyacrylamide gel, and the gel was stained by CBB.

As a result, there was no difference between the XG8 strain and the ΔproB strain regarding the dry cell weight, but the PaE activity was higher in the culture supernatant of the ΔproB strain (Figure 7). This situation is consistent with the results of CBB staining. That is, in the culture supernatant of the XG8 strain, under the PaE-derived band, a band derived from PaE decomposed product can be confirmed, and the band of PaE decomposed product becomes thicker as the number of days after culture elapses. However, in the culture supernatant of the ΔproB strain, almost no PaE degradation product was confirmed (Figure 8).

[Example 9] Stability of PaE in cell suspension

When the enzyme is industrially manufactured in a large capacity (for example, a 300L tank), it takes more than one day after the completion of the culture until all the culture solution is filtered by centrifugation. In the meantime, the culture medium was left for more than 24 hours in a state of being incorporated into the bacteria. At this time, it enters the lysis, and the PaE in the culture supernatant may be decomposed by protease. Therefore, review whether the destruction of the ProB gene is effective for the maintenance of PaE after the end of the culture. Specifically, the XG8 strain or the ΔproB strain was cultured in the same manner as in Example 8 for 72 hours, and 1 mL of each culture solution was dispensed into low-adhesion microtubes. These tubes are mixed upside down immediately after being dispensed (stand at 0 hours) or after standing at 25°C for 4, 8, 12, 24, 36, or 48 hours, and centrifuged at 15,000 rpm for 10 minutes. The recovered culture supernatant was immediately stored at -30°C, and the remaining bacterial system was dried at 105°C for 6 hours, and then its weight (dry cell weight) was measured. In addition, the PaE activity of each culture supernatant was measured by the method described in Example 8, and the residual viability (%) was determined based on the PaE activity of the culture supernatant immediately after the dispensing. Next, each culture supernatant was subjected to electrophoresis by SDS polyacrylamide gel, and the gel was stained by CBB.

As a result, the weight of the dried cells of both the XG8 strain and the ΔproB strain was reduced, and it was confirmed that lysis occurred during standing. On the other hand, in the culture supernatant of the XG8 strain, PaE activity decreased as the standing time passed, and decreased to 55.1% after 48 hours. On the other hand, in ΔproB In the culture supernatant of the strain, there was almost no decrease in PaE activity (Figure 9). This is consistent with the result of CBB staining. That is, in the culture supernatant of the XG8 strain, the bands derived from PaE decomposition products were confirmed under the PaE-derived bands. As the standing time passed, the bands of PaE decomposition products became thicker. In the culture supernatant of the ΔproB strain, almost no PaE degradation product was confirmed (Figure 10).

[Example 10] Stability of PaE in the culture filtrate

The XG8 strain or the ΔproB strain was cultured in the same manner as in Example 8 for 72 hours, and 700 mL of the culture solution immediately after the culture was centrifuged at 8,000 rpm for 10 minutes. In addition, 700 mL of each culture solution was transferred to two 500-mL Erlenmeyer flasks, and after standing at 25°C for 24 hours, each was centrifuged at 8,000 rpm for 10 minutes. Each recovered culture supernatant was filtered through a 0.45 μm-hole Stericup, and each 18 mL was injected into 3 vials with a volume of 20 mL. Immediately after dispense (0 hour standing) or after standing at 25°C for 1, 3, 5, 7, 11, or 14 days, 1 mL of each vial was sampled. The PaE activity of each culture supernatant was measured by the method described in Example 8, and the residual viability (%) was determined based on the PaE activity of the culture supernatant immediately after the injection. Next, each culture supernatant was subjected to electrophoresis by SDS polyacrylamide gel, and the gel was stained by CBB.

As a result, in either of the XG8 strain and the ΔproB strain, the PaE activity of the culture supernatant recovered immediately after the culture was maintained at least 80% during the subsequent standing period, but the PaE activity recovered after 24 hours of culture In the culture supernatant of the XG8 strain, PaE activity decreased as the standing time passed, and decreased to about 30% after 14 days (Figure 11). It is presumed that between the end of the culture and the recovery of the culture supernatant, the peptidase or protease that decomposes PaE is released from the lysed bacterial cells and the PaE in the culture supernatant is decomposed. On the other hand, in the culture supernatant of the ΔproB strain, there was almost no decrease in PaE activity (Figure 11). This situation is the same as the result of CBB staining To. That is, in the culture supernatant of the XG8 strain, under the PaE-derived band, the PaE-derived band was confirmed. As the standing time passed, the PaE-derived band became thicker, but, In the culture supernatant of the ΔproB strain, almost no PaE degradation product was confirmed (Figure 12).

According to the test results of Examples 8 to 10, the destruction of the ProB gene not only inhibits the decomposition of PaE during the culture, but is also effective in inhibiting the decomposition of PaE accompanying the lysis after the culture. Therefore, if a mutant yeast that inhibits the activity of the peptidase or protease by introducing mutations into at least one peptidase or protease gene involved in the decomposition of PaE is used, a large-capacity industrial manufacturing process that requires time is required. Medium, it can also produce protein efficiently.

<110> National Institute of Advanced Injustrial Science and Technology) National Agriculture and Food Industry Technology Research Institute (National Agriculture and Food Research Organization)

<120> Mutant yeast and method for producing protein using the mutant yeast

<150> JP 2018-212755

<151> 2018-11-13

<160> 11

<170> PatentIn version 3.5

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Claims (9)

A yeast in which at least one peptidase or protease gene involved in the degradation of esterase (PaE) produced by Pseudozyma antarctica is introduced with mutations, thereby making the peptidase or protease The activity of the aforementioned peptidase or protease system includes one or more selected from PAN1_016f05330 (sequence number 1), PAN1_004r02266 (sequence number 2), PAN1_003r01856 (sequence number 3), PAN1_003r01668 (sequence number 4), PAN1_001f00846 (sequence number Serial number 5), PAN1_019f05845 (serial number 6), PAN1_024f06685 (serial number 7), PAN1_012r04564 (serial number 8), PAN1_010r04227 (serial number 9), PAN1_013r04779 (serial number 10) and PAN1_023r06563 (serial number 11) An enzyme encoded by the gene of Candida antarctica or its homolog or ortholog. The yeast described in item 1 of the scope of patent application, wherein at least one base of the aforementioned gene is deleted or replaced with another base, or at least one base is inserted into the aforementioned gene. The yeast described in item 1 or 2 of the scope of patent application, wherein all of the aforementioned genes are deleted. The yeast described in item 1 or 2 of the scope of patent application is a yeast of the genus Candida. The yeast described in item 1 or 2 of the scope of patent application produces the aforementioned PaE. For example, the yeast described in item 1 or 2 of the scope of patent application produces recombinant protein. A protein manufacturing method uses the yeast described in any one of items 1 to 6 in the scope of the patent application. The manufacturing method described in item 7 of the scope of patent application, wherein the aforementioned protein is PaE. A protein production method that uses yeast to produce protein. The aforementioned production method includes the step of inhibiting the activity of at least one peptidase or protease involved in the decomposition of PaE in the aforementioned yeast, and the aforementioned peptidase or protease system contains more than one Selected from PAN1_016f05330 (serial number 1), PAN1_004r02266 (serial number 2), PAN1_003r01856 (serial number 3), PAN1_003r01668 (serial number 4), PAN1_001f00846 (serial number 5), PAN1_019f05845 (serial number 6), PAN1_0247f06685 (serial number 6) ), PAN1_012r04564 (sequence number 8), PAN1_010r04227 (sequence number 9), PAN1_013r04779 (sequence number 10) and PAN1_023r06563 (sequence number 11) gene of Candida antarctica or its homologs (homolog) Or an enzyme encoded by an ortholog.

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