JP5520498B2 - Method for producing protein or polypeptide - Google Patents

Description

  The present invention relates to a method for producing a protein or polypeptide.

  In industrial production of useful substances by microorganisms, improvement of productivity is one of the important issues, and breeding of producing bacteria by genetic techniques such as mutation has been carried out as a technique. Particularly recently, with the development of microbial genetics and biotechnology, more efficient breeding of production bacteria using genetic recombination techniques has been carried out. Furthermore, in response to the rapid development of genome analysis technology in recent years, attempts have been made to decode genome information of target microorganisms and actively apply them to industry.

In recent years, as for Bacillus subtilis, about 4100 kinds of disrupted strains of genes present in the Bacillus subtilis genome have been comprehensively studied, and it has been pointed out that 271 genes are essential for growth (Non-patent Document 1). . In addition, genes related to early sporulation such as Bacillus subtilis, protease genes, genes related to D-alanine addition to teichoic acid in the cell wall or cell membrane, and genes related to biosynthesis or secretion of surfactin alone are lacking. Lost or inactivated strains have been constructed (see Patent Documents 1 to 8).
In addition, the present applicant has comprehensively analyzed gene-disrupted strains derived from Bacillus subtilis, successfully deleted a large region of the Bacillus subtilis genome, and found mutant strains excellent in productivity of various enzymes. (Patent Document 9).

  However, in industrial production using a limited production medium and stable culture conditions, it is considered that there are genes necessary or unnecessary for the production of proteins or polypeptides, and further research is required.

On the other hand, RocG exists in the most downstream of the arginine degradation pathway and has a catabolic glutamate dehydrogenase activity of degrading glutamate into ammonia and 2-oxoglutarate, and also has a function of suppressing the expression of glutamate synthase GltAB. However, it is known that it is an important protein that controls the TCA cycle and glutamate synthesis pathway in a complex manner (see FIG. 2).
However, there is no report that such RocG is involved in the productivity of proteins and polypeptides.

JP-A-58-190390 JP 61-1381 WO89 / 04866 pamphlet Japanese National Patent Publication No. 11-509096 Japanese Patent No. 3210315 Special table 2001-527401 Special Table 2002-520017 Special table 2001-503641 Japanese Unexamined Patent Publication No. 2007-130013

K. Kobayashi et al., Proc. Natl. Acad. Sci. USA., 100, 4678-4683, 2003 B. R. Belitsky, A. L. Sonenshein, J Bacteriol 179, 1035 (Feb, 1997)

  The present invention relates to providing an efficient method for producing a protein or polypeptide using recombinant Bacillus subtilis.

The present inventors used Bacillus subtilis Marburg No.168 (Bacillus subtilis 168 strain) as a wild strain, and Bacillus subtilis mutant MGB874 strain (Patent Document 9) prepared by deleting a total of 874 kbp of the genome. When the productivity of protein or polypeptide was examined, cellulase productivity was significantly reduced in the R-533 strain in which the gene rocG encoding glutamate dehydrogenase was deleted from the MGB874 strain, but the expression of the rocG gene was reduced. It was found that the cellulase productivity was significantly improved when induced gently compared to the case of using the MGB874 strain.

That is, the present invention relates to the following 1) to 8).
1) A method for producing a protein or polypeptide using recombinant Bacillus subtilis into which a gene encoding a heterologous protein or polypeptide is introduced, characterized in that the expression level of RocG in the host Bacillus subtilis is regulated. A method for producing a protein or polypeptide.
2) The production method of 1) above, wherein the expression level of RocG is regulated by inducing transcription.
3) RocG transcription is induced by transforming the Bacillus subtilis mutant MGB874, which has been deleted from the genome of the Bacillus subtilis gene rocG, with the rocG gene linked to the Pspac promoter and transforming the transformant into isopropyl-β- The production method of the above 2), which is carried out by culturing in the presence of thiogalactopyranoside.
4) The above-mentioned 1), wherein the recombinant Bacillus subtilis has one or more regions selected from a transcription initiation control region, a translation initiation control region and a secretory signal region upstream of a gene encoding a heterologous protein or polypeptide. ~ 3) production method.
5) The production method of 4) above, wherein three regions comprising a transcription initiation control region, a translation initiation control region, and a secretory signal region are combined.
6) The secretory signal region is derived from a cellulase gene of a bacterium belonging to the genus Bacillus, and the transcription initiation control region and the translation initiation control region are derived from an upstream region having a length of 0.6 to 1 kb starting from the start codon of the cellulase gene. 4. The production method of 4) or 5) above.
7) Three regions consisting of a transcription initiation control region, a translation initiation control region, and a secretion signal region are either the base sequence of base numbers 1 to 659 of the cellulase gene consisting of the base sequence represented by SEQ ID NO: 1 or the base sequence and 70 5) The production method of 5) above, which is a DNA fragment consisting of a base sequence having at least% identity, or a DNA fragment consisting of a base sequence from which a part of the base sequence has been deleted.
8) The manufacturing method of said 1) -7) whose protein or polypeptide is a cellulase.

  According to the present invention, the target protein or polypeptide can be efficiently produced with high production, and industrial production of useful proteins, particularly cellulases, can be realized.

The figure which showed typically the method of preparing the DNA fragment for gene deletion by SOE-PCR, and deleting the target gene using the said DNA fragment (substitution with a drug resistance gene). The figure which showed the role of RocG typically.

The Bacillus subtilis mutant strain MGB874 in the present invention is a wild strain of Bacillus subtilis Marburg No.168 (168 strains of Bacillus subtilis ), that is, a large region of its genome, ie, prophage6 (yoaV-yobO) region, prophage1 (ybbU- ybdE) region, prophage4 (yjcM-yjdJ) region, PBSX (ykdA-xlyA) region, prophage5 (ynxB-dut) region, prophage3 (ydiM-ydjC) region, spb (yodU-ypqP) region, pks (pksA-ymaC) Region, skin (spoIVCB-spoIIIC) region, pps (ppsE-ppsA) region, prophage2 (ydcL-ydeJ) region, ydcL-ydeK-ydhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeeK- The yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 (spoIVCB-yraK) region, sbo-ywhH region, yybP-yyaJ region, and yncM-fosB region are deleted (Patent Document 9) ). The deletion region shown in Table 1 can be rephrased as a region sandwiched between a pair of oligonucleotide sets shown in the same table.

  In the method for producing a protein or polypeptide of the present invention, the expression level of RocG of the host Bacillus subtilis is regulated. Here, RocG is present in the most downstream of the arginine degradation pathway, and glutamic acid is mixed with ammonia. In addition to having a catabolic glutamate dehydrogenase activity of degrading into -oxoglutarate, it also has a function of suppressing the expression of glutamate synthase GltAB by interacting with the transcription factor GltC (Non-patent Document 2) It is an important protein that controls the TCA cycle and glutamate synthesis pathway, which are the major metabolic pathways of cells.

The regulation of the expression level of RocG is performed by, for example, inducing transcription of RocG.
Transcription induction is achieved by transforming a host Bacillus subtilis strain with a promoter that has the function of enhancing the expression of a gene located downstream on the condition of the presence of a specific substance, that is, a rocG gene linked to a transcription-inducible promoter. It is carried out by culturing in the presence of the substance.
Examples of such transcription-inducible promoters include lac promoter, Pspac promoter, syllose-inducible promoter, arabinose-inducible promoter, ECF sigma factor-specific promoter (J. Helmann and C. Moran Jr., “Bacillus subtilis and its closest relatives.”). ASM Press, 2002, pp289-312), promoters induced by two components of Mg and Cu, etc. Among them, Pspac promoter (Proc. Natl) controlled by lacl which is a repressor of lac operon. Acad. Sci. USA 81, 439-443. (1984)).
Examples of the expression inducer of Pspac promoter include methyl β-D-galactoside, methyl 1-thio-β-D-galactoside, n-propyl 1-thio-β-D-galactoside, isopropyl β-D-galactoside, isopropyl- β-D-thiogalactopyranoside, n-butyl β-D-galactoside, n-butyl 1-thio-β-D-galactoside, benzyl β-D-galactoside, benzyl 1-thio-β-D-galactoside, 2-phenylethyl β-D-galactoside, 2-phenylethyl 1-thio-β-D-galactoside, p-aminophenyl β-D-galactoside, p-aminophenyl 1-thio-β-D-galactoside, O— Nitrophenyl 1-thio-β-D-galactoside, p-nitrophenyl 1-thio-β-D-galactoside, D-fucose, g Kutosu, 6-fluoro-6-deoxy -D- galactose, although melibiose and thio allolactose etc., of isopropyl-beta-D-thiogalactopyranoside (IPTG) are preferred.

  Specifically, for example, a Bacillus subtilis mutant strain MGB874 (R-533 strain) in which the Bacillus subtilis gene rocG has been deleted from its genome is used using a plasmid pAPNC-rocG containing the rocG gene linked to the Pspac promoter. Thus, a strain (R-716 strain) capable of controlling the expression of RocG with isopropyl-β-thiogalactopyranoside (IPTG) is constructed and used as a host.

  The method for deleting the rocG gene from the genome of the MGB874 strain is not particularly limited, and for example, the following method shown in FIG. 1 can be applied. The deletion DNA fragment used in the present method includes an approximately 0.1 to 3 kb fragment adjacent to the upstream of the deletion target region (referred to as upstream fragment), a fragment containing a drug resistance gene marker, and approximately 0.1 to 3 kb adjacent to the downstream. A DNA fragment in which fragments (referred to as downstream fragments) are linked.

  For example, when a chloramphenicol resistance gene is used as a drug resistance gene marker, rocG-FW1, rocG / Cm-R, and rocG / Cm- shown in Table 3 below are used as a template with genomic DNA extracted from Bacillus subtilis 168 strain. Using the F and rocG-RV primer sets, a fragment adjacent to the upstream of the rocG gene on the genome (A) and a 1.0 kbp fragment adjacent to the downstream (B) are prepared. Furthermore, the chloramphenicol resistance gene (C) of plasmid pC194 (J. Bacteriol. 150 (2), 815 (1982)) is amplified using the CmFW and CmRV primer sets shown in Table 1.

Next, the obtained 3 fragments of (A), (B) and (C) were mixed and used as a template, PCR was performed using rocG-FW2 and rocG-RV2 primers, and the 3 fragments were converted to (A)-(C )-(B) to obtain a DNA fragment for gene deletion (see FIG. 1).
Using this DNA fragment, the MGB874 strain is transformed by a competent method, and colonies grown on an LB agar medium containing chloramphenicol (10 μg / mL) are isolated as transformants.

  By extracting the genomic DNA of the transformant and using this as a template DNA, it can be confirmed that the rocG gene is replaced with the chloramphenicol resistance gene and the target gene is deleted. Genomic DNA can be extracted by the method of Saito & Miura (Saito & Miura Biochem. Biophys. Acta. 72, 619-629 (1963)) or a commercially available genomic DNA extraction kit such as UltraClean Microbial DNA. Isolation Kit (MO BIO Laboratories, Inc.) can be used.

From the thus obtained R-533 strain, a strain capable of inducing transcription of RocG (R-716 strain; (R-533 aprE :: Pspac-rocG spec) is constructed. The case of inducing expression of the rocG gene using the Pspac promoter whose expression can be controlled with IPTG will be described as an example.
First, for the purpose of constructing plasmid DNA used for transformation, restriction enzyme sites SalI / SacI were inserted at both ends using Bacillus subtilis genome as a template and primers rocG.f.SalI and rocG.f.SacI shown in Table 4 below. The rocG gene fragment contained in is amplified by PCR. Furthermore, the SalI of plasmid pAPNC213 (Microbiology 148, 3539-3552) containing the Pspac promoter (Proc. Natl. Acad. Sci. USA 81, 439-443. (1984)) capable of controlling expression of the amplified gene fragment with IPTG. Plasmid pAPNC-rocG for transformation is constructed by inserting into the / SacI site. pAPNC-rocG has a rocG gene (Psapc-rocG) linked to the Pspac promoter and a spectinomycin resistance gene inserted into the aprE gene from Bacillus subtilis 168 strain, and Pspac-rocG is transformed into the aprE site on the genome by transformation. A plasmid into which a gene can be inserted. After sequencing using the primers rocG.f.1 to rocG.f.3 and rocG.r.1 shown in Table 4 and confirming that the plasmid was constructed without mutation, R-533 was prepared by a competent method. The strain is transformed, and colonies that grow on the LB agar medium containing spectinomycin (0.5 μg / mL) are isolated as transformants. Furthermore, genomic DNA is extracted from the obtained transformant, and it is confirmed by PCR using this as a template that the Pspac-rocG gene and the spectinomycin resistance gene have been inserted into the aprE gene site. As described above, a strain (R-716 strain) capable of controlling rocG gene expression by IPTG can be constructed.

Using the thus obtained Bacillus subtilis mutant (R-716 strain) as a host, introducing a gene encoding a heterologous protein or polypeptide into a recombinant Bacillus subtilis for producing the protein or polypeptide, By culturing this in the presence of an expression inducer such as IPTG, the target protein or polypeptide is produced under the induction of RocG transcription.
Here, recombinant Bacillus subtilis takes a recombinant plasmid in which a DNA fragment containing a gene encoding a heterologous protein or polypeptide and an appropriate plasmid vector are combined into a cell line R-716 by a general transformation method. Can be produced. Alternatively, a recombinant Bacillus subtilis can be obtained by directly integrating the DNA fragment containing a gene encoding the heterologous protein or polypeptide and an appropriate homologous region with the Bacillus subtilis genome into the R-716 strain genome. Can be obtained.

  The target heterologous protein or polypeptide is not particularly limited, and includes various industrial enzymes such as detergents, foods, textiles, feeds, cosmetics, pharmaceuticals, diagnostics, bioactive peptides, etc. . In addition, industrial enzymes are classified according to their functions: oxidoreductase, transferase, hydrolase, lyase, isomerase, and synthase (Ligase / Synthetase). ) And the like, preferably hydrolyzing enzymes such as cellulase, α-amylase and protease, more preferably cellulase.

As cellulase, for example, classification of polysaccharide hydrolase (Biochem. J., 280, 309, 1991)
Among them, cellulases belonging to family 5 are mentioned, and among them, cellulases derived from microorganisms, in particular, Bacillus bacteria are mentioned. As a more specific example, an alkaline cellulase derived from the Bacillus bacterium strain KSM-S237 (FERM BP-7875) comprising the amino acid sequence represented by SEQ ID NO: 2 or a Bacillus bacterium comprising the amino acid sequence represented by SEQ ID NO: 4 Alkaline cellulase derived from KSM-64 strain (FERM BP-2886) or the amino acid sequence and 70%, preferably 80%, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more. Examples include cellulases composed of amino acid sequences having identity, or alkaline cellulases composed of amino acid sequences in which one or several amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.

  Specific examples of α-amylase include α-amylase derived from microorganisms, and particularly liquefied amylase derived from bacteria belonging to the genus Bacillus. As a more specific example, alkaline amylase derived from the Bacillus genus bacterium KSM-K38 strain (FERM BP-6946) consisting of the amino acid sequence represented by SEQ ID NO: 5, or 70%, preferably 80%, more preferably the amino acid sequence. Is an amylase consisting of an amino acid sequence having 90% or more, more preferably 95% or more, particularly preferably 98% or more, or a substitution or deletion of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 5 Alternatively, alkaline amylase consisting of an added amino acid sequence can be mentioned.

  Specific examples of proteases include serine proteases, metal proteases, and the like derived from microorganisms, in particular, Bacillus bacteria. As a more specific example, an alkaline protease derived from Bacillus clausii KSM-K16 strain (FERM BP-3376) consisting of the amino acid sequence shown in SEQ ID NO: 6, or 70%, preferably 80% with the amino acid sequence. More preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, a protease comprising an amino acid sequence, or one or several amino acids in the amino acid sequence shown in SEQ ID NO: 6 And alkaline protease consisting of a deleted or added amino acid sequence.

In the present invention, the identity of the amino acid sequence and the base sequence is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985). Specifically, it is calculated by performing an analysis with a unit size to compare (ktup) of 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).
The amino acid sequence in which one or several amino acids have been deleted, substituted or added is preferably an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted or added. Addition of one to several amino acids to is included.

  The gene of the heterologous protein or polypeptide introduced into the host Bacillus subtilis is a control region involved in transcription, translation, and secretion of the gene upstream thereof, that is, a transcription initiation control region including a promoter and a transcription initiation site, a ribosome binding site It is desirable that at least one region selected from a translation initiation region including a start codon and a secretory signal peptide region is bound in an appropriate form. In particular, it is preferable that three regions consisting of a transcription initiation control region, a translation initiation control region and a secretion signal region are combined, and the secretion signal peptide region is derived from a cellulase gene of a bacterium belonging to the genus Bacillus. That the start control region and the translation start control region are upstream regions of 0.6 to 1 kb in length starting from the start codon of the cellulase gene are appropriately bound to the target protein or polypeptide gene desirable. For example, bacteria belonging to the genus Bacillus described in JP 2000-210081, JP 4-190793, and the like, that is, KSM-S237 strain (FERM BP-7875), KSM-64 strain (FERM BP- It is desirable that the transcription initiation regulatory region, the translation initiation region, and the secretory signal peptide region of the cellulase gene derived from 2886) are appropriately combined with the structural gene of the target protein or polypeptide. More specifically, the base sequence of base numbers 1 to 659 of the base sequence shown in SEQ ID NO: 1, the base sequence of base numbers 1 to 696 of the cellulase gene consisting of the base sequence shown in SEQ ID NO: 3, and the base sequence DNA fragment comprising a base sequence having 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 98% or more, or any one of the above bases A DNA fragment comprising a nucleotide sequence that hybridizes to a DNA comprising a sequence under stringent conditions and has a function related to transcription, translation, or secretion of a gene, or a nucleotide sequence in which any one of the above nucleotide sequences is deleted, It is desirable that the target gene or polypeptide is appropriately bound to the structural gene. Here, a DNA fragment consisting of a base sequence from which a part of the base sequence has been deleted is a part of the base sequence that has been deleted, but retains functions related to gene transcription, translation, and secretion. Means a DNA fragment. The stringent conditions referred to herein include, for example, the conditions described in [Molecular cloning-a Laboratory manual 2nd edition (Sambrook et al., 1989)]. For example, 6XSSC (composition of 1XSSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5X Denhart and 100 mg / mL herring sperm DNA together with the probe at 65 ° C. for 8 to 16 hours Examples of the conditions include constant temperature and hybridization.

Culturing can be performed by inoculating the transformed strain into a medium containing an assimilable carbon source, nitrogen source, and other essential components, and using an ordinary microorganism culture method. The addition of IPTG is performed at the start of culture or at the initial stage of culture, and is preferably performed in the range of 4 μM to 15 μM, more preferably in the range of 6 μM to 10 μM from the viewpoint of protein or polypeptide productivity.
After completion of the culture, the target protein or polypeptide can be obtained by collecting the protein or polypeptide and purifying it appropriately.
Thus, as shown in the Examples below, according to the method of the present invention, the productivity of the target protein or polypeptide is significantly improved as compared with the case where the Bacillus subtilis mutant MGB874 strain is produced as a host. The

  Below, the construction method of the recombinant microorganism used for the method of this invention and the manufacturing method of a cellulase using the said recombinant microorganism are demonstrated concretely.

  For the polymerase chain reaction (PCR) for DNA fragment amplification in the following examples, GeneAmp PCR System (Applied Biosystems) is used, and DNA amplification is performed using Pyrobest DNA Polymerase (Takara Bio) and attached reagents. went. The PCR reaction solution composition was 1 μL of appropriately diluted template DNA, 20 pmol of sense and antisense primers and 2.5 U of Pyrobest DNA Polymerase, respectively, and the total amount of the reaction solution was 50 μL. PCR reaction conditions were 98 ° C for 10 seconds, 55 ° C for 30 seconds and 72 ° C for 1 to 5 minutes (adjusted according to the target amplification product, 1 minute per 1 kb). After repeating, the reaction was carried out at 72 ° C. for 5 minutes.

  Further, in the following examples, upstream and downstream of a gene are not positions from the replication start point, but upstream is a region continuing on the 5 ′ side of the start codon of the gene that is regarded as a target in each operation / step. On the other hand, “downstream” indicates a region continuing 3 ′ of the stop codon of the gene taken as a target in each operation / step.

  Furthermore, the names of the genes and gene regions in the following examples are reported in Nature, 390, 249-256, (1997), and published on the Internet at JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) (http: //bacillus.genome.ad.jp/, updated March 10, 2004), based on genome data of Bacillus subtilis.

  Bacillus subtilis was transformed by the competent cell method (J. Bacteriol. 93, 1925 (1967)). That is, the Bacillus subtilis strain is SPI medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% glucose, 0.02% casamino acid (Difco) , 5 mM magnesium sulfate, 0.25 μM manganese chloride, 50 μg / ml tryptophan) at 37 ° C. with shaking until the degree of growth (OD600) is about 1. After shaking culture, a portion of the culture broth was added to 9 times the amount of SPII medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% Inoculated with glucose, 0.01% casamino acid (Difco), 5mM magnesium sulfate, 0.40μM manganese chloride, 5μg / ml tryptophan) A competent cell was prepared.

  Next, 5 μL of a solution containing various DNA fragments (SOE-PCR reaction solution, etc.) is added to 100 μL of the prepared competent cell suspension (culture medium in SPII medium), and after shaking culture at 37 ° C. for 1 hour, an appropriate drug is obtained. LB agar medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) containing the whole amount was smeared. After static culture at 37 ° C., the grown colonies were isolated as transformants. The genome of the obtained transformant was extracted, and it was confirmed that the intended genomic structure was altered by PCR using this as a template.

  The plasmid expressing the target protein was introduced into the host microorganism by the protoplast transformation method (Mol. Gen. Genet. 168, 111 (1979)). LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl), 2xL-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose for protein production by recombinant microorganisms 7.5 ppm manganese sulfate 4-5 hydrate) was used.

Example 1 Construction of strain (1) Construction of R-169 strain (MGB874ΔaprE :: spec)
First, aprE-deficient DNA fragments were amplified using genomic DNA extracted from Bacillus subtilis strain 168 as a template, aprE-FW1 and aprE / Sp-R, and aprE / Sp-F and aprE-RV shown in Table 2. A 1.0 kbp fragment adjacent to the upstream of the aprE gene on the genome (A) and a 1.0 kbp fragment adjacent to the downstream (B) were prepared using each of the primer sets, and the spf and spr shown in Table 1 were further prepared. The spectinomycin resistance gene (C) of the plasmid pDG1727 (Gene, 167, 335, (1995)) was amplified using the primer set. The obtained 3 fragments of (A), (B), and (C) were mixed to form a template, PCR was performed using aprE-FW2 and aprE-RV2 primers, and the 3 fragments were (A)-(C)-( The DNA fragments for gene deletion were obtained by binding in the order of B) (see FIG. 1). Using this DNA fragment, transformation of MGB874 strain (Biotech. Appl. Biochem. 46: 169-178 2007) (DNA research 15, 73-81 2008) was performed by a competent method, and spectinomycin (0.5 μg / The colonies that grew on the LB agar medium containing mL) were isolated as transformants. Further, genomic DNA was extracted from the obtained transformant, and it was confirmed that the aprE gene was replaced with the spectinomycin resistance gene by PCR using this as a template, and the target gene was deleted. As described above, an aprE deletion strain (R-169 strain) was prepared.

(2) Construction of R-533 strain (MGB874ΔrocG :: cat)
The procedure was the same as that used to construct R-169 strain. First, genomic DNA extracted from Bacillus subtilis 168 strain was used as a template, and using the rocG-FW1 and rocG / Cm-R and rocG / Cm-F and rocG-RV primer sets shown in Table 3, A 1.0 kbp fragment adjacent to the upstream of the rocG gene (A) and a 1.0 kbp fragment adjacent to the downstream (B) were prepared, respectively, and further, using the CmFW and CmRV primer sets shown in Table 1, the plasmid pC194 The chloramphenicol resistance gene (C) of (J. Bacteriol. 150 (2), 815 (1982)) was amplified. Next, the obtained 3 fragments of (A), (B) and (C) were mixed and used as a template, PCR was performed using rocG-FW2 and rocG-RV2 primers, and the 3 fragments were converted to (A)-(C )-(B) in order, and a DNA fragment for gene deletion was obtained (see FIG. 1). Using this DNA fragment, the MGB874 strain was transformed by a competent method, and colonies grown on LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants. Further, genomic DNA was extracted from the obtained transformant, and it was confirmed that the rocG gene was replaced with the chloramphenicol resistance gene by PCR using this as a template, and the target gene was deleted. A rocG deletion strain (R-533 strain) was prepared as described above.

(3) Construction of R-716 strain (R-533 aprE :: Pspac-rocG spec) First, for the purpose of constructing plasmid DNA used for transformation, primer rocG. Shown in Table 4 using Bacillus subtilis genome as a template. Using f.SalI and rocG.f.SacI, a rocG gene fragment containing restriction enzyme sites SalI / SacI at both ends was amplified by PCR. Furthermore, the plasmid pAPNC213 (Microbiology 148, containing the Pspac promoter (Proc. Natl. Acad. Sci. USA 81, 439-443. (1984)) capable of controlling expression of the amplified gene fragment with IPTG (isopropyl sD-thiogalactopyranoside). 3539-3552) was inserted into the SalI / SacI site to construct a transformation plasmid pAPNC-rocG. pAPNC-rocG has a rocG gene (Psapc-rocG) linked to the Pspac promoter and a spectinomycin resistance gene inserted into the aprE gene from Bacillus subtilis 168 strain, and Pspac-rocG is transformed into the aprE site on the genome by transformation. A plasmid into which a gene can be inserted. After sequencing using the primers rocG.f.1 to rocG.f.3 and rocG.r.1 shown in Table 4 and confirming that the plasmid was constructed without mutation, R-533 was prepared by a competent method. The strain was transformed, and colonies grown on LB agar medium containing spectinomycin (0.5 μg / mL) were isolated as transformants. Further, genomic DNA was extracted from the obtained transformant, and it was confirmed by PCR using this as a template that the Pspac-rocG gene and the spectinomycin resistance gene were inserted into the aprE gene site. A strain (R-716 strain) capable of controlling rocG expression by IPTG was constructed as described above.

Example 2 (Evaluation of protein productivity of each mutant)
The protein productivity of the R-169 strain, R-533 strain, and R-716 strain obtained in Example 1 was evaluated using the productivity of alkaline cellulase consisting of the amino acid sequence represented by SEQ ID NO: 1 as an index.
Regarding the evaluation of alkaline cellulase productivity, first, the R-169 strain, R-533 strain, and R-716 strain obtained in Example 1 were derived from the Bacillus genus bacteria KSM-S237 strain (FERM BP-7875). A recombinant plasmid pHY-S237 in which a DNA fragment (3.1 kb) encoding an alkaline cellulase gene (Japanese Patent Laid-Open No. 2000-210081) was inserted at the BamHI restriction enzyme cleavage point of the shuttle vector pHY300PLK was introduced by protoplast transformation. . The obtained strain was shaken and cultured in 5 mL of LB medium at 30 ° C. for 15 hours, and 0.6 mL of this culture solution was further added to 30 mL of 2 × L-maltose medium (2% tryptone, 1% yeast extract, 1% sodium chloride, 7.5% maltose, 7.5ppm manganese sulfate 4-5 hydrate, 15ppm tetracycline) and shake culture at 30 ° C for 3 days. When evaluating the productivity of the R-716 strain, IPTG was appropriately added at the start of culture for the purpose of inducing transcription of rocG. Further, the culture was performed three times each in consideration of measurement errors. After culturing, the alkaline cellulase activity of the culture supernatant after removing the cells by centrifugation was measured, and the amount of the alkaline cellulase secreted and produced outside the cells was determined. For the measurement of cellulase activity, 50 μL of 0.4 mM p-nitrophenyl-β-D-cellotrioside (Seikagaku) was added to 50 μL of a sample solution appropriately diluted with 1 / 7.5 M phosphate buffer (pH 7.4 Wako Pure Chemical Industries). The amount of p-nitrophenol liberated when mixed and reacted at 30 ° C. was quantified by the change in absorbance at 420 nm (OD420 nm). The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was defined as 1 U, and the productivity comparison was made by relative values with the productivity of the MGB874 strain as 100% (Table 5).

  From the results of productivity evaluation of the R-533 strain, RocG deletion caused a significant decrease in productivity, while in the R-716 strain, when IPTG was added 4 μM to 15 μM and rocG was expressed slowly, compared to the MGB874 strain High productivity was demonstrated. Since the productivity of the R-169 strain is equivalent to that of the MGB874 strain, it can be determined that the deletion of the aprE gene generated when the R-716 strain is constructed does not affect the increase in productivity.

Claims (4)

A method for producing a protein or polypeptide using a recombinant Bacillus subtilis introduced with a gene encoding a heterologous protein or polypeptide, wherein the Bacillus subtilis mutant MGB874 has a Bacillus subtilis gene rocG deleted from its genome. strains of Bacillus mutant strain transformed with a host B. subtilis rocG gene linked to Pspac promoter, the recombinant Bacillus subtilis introduced a gene encoding a heterologous protein or polypeptide in the host, 6Myuemu～10myuM concentration of isopropyl -A method for producing a protein or polypeptide, comprising culturing in the presence of β-thiogalactopyranoside. A recombinant Bacillus subtilis is obtained by linking three regions comprising a transcription initiation control region, a translation initiation control region and a secretion signal region upstream of a gene encoding a heterologous protein or polypeptide , wherein the secretion signal region is a Bacillus ( 2. A Bacillus) bacterium belonging to a cellulase gene, wherein the transcription initiation control region and the translation initiation control region are derived from an upstream region of 0.6 to 1 kb in length starting from the initiation codon of the cellulase gene. Manufacturing method. Transcriptional initiation regulatory region, the translation initiation regulatory region and 3 regions consisting of secretion signal region has a nucleotide sequence identity of 90% or more of the nucleotide numbers 1 to 659 of cellulase gene comprising the nucleotide sequence represented by SEQ ID NO: 1 The production method according to claim 2, which is a DNA fragment consisting of a base sequence. The production method according to any one of claims 1 to 3 , wherein the protein or polypeptide is cellulase.

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