JP2006345860A - Recombinant bacillus bacterium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a recombinant Bacillus bacterium having improved productivity of a protein or a polypeptide; and to provide a method for producing the protein or the polypeptide by using the bacteria. <P>SOLUTION: The recombinant Bacillus bacterium is obtained by introducing a DNA fragment containing three regions comprising a transcription initiation-control region, a translation initiation-control region and a secretion signal region of an alkaline cellulase gene derived from the Bacillus bacterium, and a gene encoding the target protein or peptide to a Bacillus bacterium strain in which both of Bacillus subtilis genes spoOA and abrB, or genes corresponding to the genes are deleted or inactivated. The method for producing the protein or the polypeptide uses the recombinant Bacillus bacterium. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a recombinant Bacillus bacterium used for production of a useful protein or polypeptide, and a protein or polypeptide production method using the same.

  The industrial production of useful substances by microorganisms includes a wide variety of types including foods such as alcoholic beverages, miso and soy sauce, as well as amino acids, organic acids, nucleic acid-related substances, antibiotics, carbohydrates, lipids, proteins, etc. In addition, its application has been extended to a wide range of fields from foods, medicines, daily necessaries such as detergents and cosmetics to various chemical raw materials.

In industrial production of useful substances by such microorganisms, improvement of productivity is one of the important issues, and breeding of produced bacteria by genetic techniques such as mutation has been performed as a technique. In recent years, the development of microbial genetics and biotechnology has led to more efficient breeding of production microorganisms using genetic recombination techniques, and the development of host microorganisms for genetic recombination has been promoted. It has been. For example, a strain obtained by further improving a microbial strain recognized as safe and excellent as a host microorganism, such as Bacillus subtilis Marburg No.168 strain, has been developed.

Recently, a strain has been constructed that deletes or inactivates genes related to spore formation (for example, sigE , sigF, etc.) for certain microorganisms, resulting in improved productivity of proteins and polypeptides. (See Patent Document 1), and it has been reported that the productivity of γ-glutamyl transpeptidase is improved in a strain in which spo0A gene and abrB gene which are regulators of Bacillus subtilis sporulation initiation are inactivated. (Non-Patent Document 1)

On the other hand, for Bacillus bacteria such as Bacillus subtilis, if the host vector system does not have an appropriate transcription initiation control region, translation initiation control region, secretion signal region, normal expression of the target product is not performed, Alternatively, it is known that the target product remains in the cell and is not secreted normally. Furthermore, Bacillus subtilis is known to produce a proteolytic enzyme inside and outside the cell, and there is a problem that the target protein produced secreted is decomposed on the membrane or outside the cell.
JP 2003-47490 A Xu, K. et al., J. Bacteriol. 178, 4319 (1996)

  The present invention relates to providing a recombinant Bacillus bacterium having improved secretion productivity of a target protein or polypeptide, and a method for producing a protein or polypeptide using the same.

As a result of various studies on protein production methods using Bacillus bacteria, the present inventors have deleted or inactivated both the Bacillus subtilis genes spo0A and abrB , or the corresponding genes, from the genome. Then, by introducing a DNA fragment containing a gene encoding a protein or polypeptide that is the purpose of production and three regions consisting of a transcription initiation control region, a translation initiation control region and a secretion signal region of an alkaline cellulase gene derived from a Bacillus bacterium It has been found that the secretory productivity of the target protein or polypeptide is greatly improved and the target product can be obtained efficiently.

That is, the present invention provides a Bacillus bacterium gene spo0A and abrB , or a Bacillus bacterium strain in which each of the genes corresponding to the gene is deleted or inactivated, a transcription initiation control region of a Bacillus bacterium-derived alkaline cellulase gene, The present invention relates to a recombinant Bacillus bacterium obtained by introducing a DNA fragment containing a gene encoding a target protein or polypeptide and three regions consisting of a translation initiation control region and a secretory signal region.

  The present invention also relates to a method for producing a protein or polypeptide using the recombinant Bacillus bacterium.

  By using the recombinant Bacillus genus bacterium of the present invention, the secretion productivity of the target protein or polypeptide can be improved, and the target protein or polypeptide can be produced efficiently and in large quantities.

  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 Unit size to compare (ktup) set to 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

  In the present invention, the transcription initiation control region is a region containing a promoter and a transcription start site, and the ribosome binding site is a Shine-Dalgarno (SD) sequence (Proc. Natl. Acad. Sci. USA 74, 5463 (1974)).

The host Bacillus bacterium strain used for the recombinant Bacillus bacterium of the present invention includes the spo0A gene of Bacillus subtilis (Nature, 390, 249-256, (1997), and JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB, (http://bacillus.genome.ad.jp/, updated March 10, 2004), gene number BG10765) and abrB gene (Nature, 390, 249-256, (1997), and JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB, http://bacillus.genome.ad.jp/, updated on March 10, 2004) gene number BG10100), or the corresponding genes are deleted together Or it has been inactivated.
As such a host Bacillus genus bacterial strain, Bacillus subtilis (particularly Bacillus subtilis) has been disclosed from the viewpoint that whole genome information has been clarified and that genetic engineering and genomic engineering techniques have been established, and that it has the ability to secrete and produce proteins outside the cells. Bacillus subtilis ) is preferred.

The spo0A gene is a gene encoding Spo0A, which is a spore formation initiation regulator. Spo0A phosphorylated (Spo0A~P), the other to suppress the expression of abrB genes, including sigF gene or sigE gene encoding sporulation specific sigma factor involved in the regulation of expression of various genes Yes.

The abrB gene is a gene encoding a repressor AbrB. AbrB suppresses the expression of the sigH gene encoding sigma factor SigH specific for the sporulation phase, and is involved in the regulation of the expression of various genes including the aprE gene encoding the major extracellular alkaline protease.

  Information on transcriptional regulators of Bacillus subtilis such as Spo0A and AbrB is available on the Internet at DBTBS: database of transcriptional regulation in Bacillus subtilis (DBTBS release3.4) (http://dbtbs.hgc.jp/, January 14, 2005) In the same database, genes whose transcription is regulated by Spo0A or AbrB are shown (Makita Y, Nakao M, Ogasawara N, Nakai K., DBTBS: database of transcriptional regulation in Bacillus subtilis and its contribution to comparative genomics Nucleic Acids Res., 32, D75-77 (2004) (NAR online).

Examples of genes corresponding to these genes include the genes shown in the following (1) to (2), and are included in the genes to be deleted or inactivated in the present invention.
(1) It has the same function as the spo0A gene and abrB gene of Bacillus subtilis, and 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, in each gene and base sequence, A gene derived from another microorganism, preferably a bacterium belonging to the genus Bacillus , having an identity of preferably 98% or more.
(2) A gene having the same function as the spo0A gene and abrB gene of Bacillus subtilis and hybridizing under stringent conditions with DNA comprising a base sequence complementary to the base sequence of each gene.
Here, examples of the “stringent conditions” include a method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell., Cold Spring Harbor Laboratory Press]. For example, probe into a solution containing 6 × SSC (1 × SSC composition: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhart and 100 mg / mL herring sperm DNA In addition, there is a condition in which the temperature is kept constant at 65 ° C. for 8 to 16 hours for hybridization.

In the recombinant Bacillus bacterium of the present invention, the spo0A gene of Bacillus subtilis described above may be used as a gene to be deleted or inactivated as long as it can be expected to have a greater effect on the productivity improvement of the target protein or the like. It is also possible to combine the deletion or inactivation of genes other than the abrB gene or the genes corresponding to the gene, and in addition to the deletion of the gene, the expression enhancement and function of other genes It is also possible to combine enhancements.

  In the deletion or inactivation of a gene in the present invention, the target gene is inactivated by a method such as inserting another DNA fragment into the target gene or mutating the transcription / translation initiation control region of the gene. Preferably, it is more desirable to physically delete the target gene.

  As a procedure for the deletion or inactivation of such a gene, in addition to the method of systematically deleting or inactivating the above target gene, after giving a random gene deletion or inactivation mutation, Examples include a method for evaluating protein productivity and performing gene analysis by an appropriate method.

  In order to delete or inactivate a target gene, for example, there is a method of deleting or inactivating a target gene by homologous recombination, and several methods have already been reported (Mol. Gen. Genet., 223, 268, 1990, etc.). That is, a circular recombinant plasmid obtained by cloning a DNA fragment containing a part of the target gene into an appropriate plasmid vector is introduced into the parent bacterial cell, and the bacteria are obtained by homologous recombination in a part of the target gene. The target gene on the genome is divided and inactivated, the target gene inactivated by mutation such as base substitution or base insertion, or the target gene including the upstream and downstream regions of the target gene as shown in FIG. A linear DNA fragment, etc. that does not contain DNA is constructed by a method such as PCR, and this is incorporated into the parent bacterial cell, two places outside the mutation site in the target gene of the parent bacterial genome, or upstream of the target gene There is a method in which the target gene on the genome is replaced with a deleted or inactivated gene fragment by causing homologous recombination twice in the downstream. In addition, in the present specification, upstream and downstream of a gene is not a position from the replication start point, but upstream refers to a region continuing on the 5 ′ side of the start codon of the gene considered as a target, Indicates the region that continues 3 'of the stop codon of the gene being captured.

  In addition, random gene deletion or inactivation can be achieved by using homologous recombination similar to the above-described method using randomly cloned DNA fragments, or by irradiating parent bacteria with γ rays, etc. It can be implemented.

  Hereinafter, the deletion method by the double crossing method using the DNA fragment for deletion prepared by SOE (splicing by overlap extension) -PCR method (Gene, 77, 61, 1989) will be described. The gene deletion method in the present invention is not limited to the following.

  The deletion DNA fragment used in this method is about 0.1-3.0 kb, preferably about 0.4-3.0 kb, adjacent to the upstream of the gene to be deleted, and about 0.1-3.0 kb, preferably about 0.1-3.0 kb, preferably adjacent to the downstream. This is a fragment in which a drug resistance marker gene fragment is inserted between 0.4-3.0 kb fragments. First, an upstream fragment and a downstream fragment of a deletion target gene and three fragments of a drug resistance marker gene fragment are prepared by the first PCR. In this case, for example, upstream of the drug resistance marker gene is formed at the downstream end of the upstream fragment. A primer designed so that a 10-30 base pair sequence on the side and, on the contrary, a 10-30 base pair sequence downstream of the drug resistance marker gene is added to the upstream end of the downstream fragment is used (FIG. 1).

  Next, using the 3 types of PCR fragments prepared in the first round as a template, and performing the second round of PCR using the upstream primer of the upstream fragment and the downstream primer of the downstream fragment, the downstream end of the upstream fragment and the downstream fragment In the drug resistance marker gene sequence added to the upstream end, annealing occurs with the drug resistance marker gene fragment, and as a result of PCR amplification, a DNA fragment in which the drug resistance marker gene is inserted between the upstream fragment and the downstream fragment Can be obtained (FIG. 1).

  When a chloramphenicol resistance gene is used as a drug resistance marker gene, for example, a primer selected from those shown in Table 1 is used as a primer set as shown in Table 2, and for general PCR such as Pyrobest DNA polymerase (Takara Shuzo). Using an enzyme kit, etc., SOE-PCR is carried out under the usual conditions described in the book (PCR Protocols. Current Methods and Applications, Edited by BAWhite, Humana Press pp251, 1993, Gene, 77, 61, 1989). As a result, a DNA fragment for deletion of each gene is obtained.

  When the deletion DNA fragment thus obtained is introduced into the cell by a known method, the gene in the cell is located between the deletion DNA fragment and the homologous region upstream and downstream of the deletion target gene of the parent bacterial genome. Cells in which recombination has occurred and the target gene has been replaced with a drug resistance gene can be isolated by selection with a drug resistance marker (FIG. 1). That is, when a deletion (inactivation) DNA fragment prepared using the primer set shown in Table 2 was introduced, colonies growing on an agar medium containing chloramphenicol were isolated, and the genome was used as a template. What is necessary is just to confirm that the target gene on the genome is replaced with the chloramphenicol resistance gene by PCR method or the like. Specifically, as a method for introducing a DNA fragment for introduction into a cell, a competent cell transformation method (J. Bacterial. 93, 1925 (1967)), a protoplast transformation method (Mol. Gen. Genet. 168, 111 (1979)) or electroporation method (FEMS Microbiol. Lett. 55, 135 (1990)), etc., and competent cell transformation method is particularly preferred.

  The recombinant Bacillus bacterium of the present invention includes a Bacillus bacterium strain (Bacillus bacterium mutant) thus obtained, a transcription initiation control region, a translation initiation control region, and a secretion signal region of an alkaline cellulase gene derived from a Bacillus bacterium. And a DNA fragment containing a gene encoding the target protein or polypeptide. In other words, the target protein or polypeptide gene is bound in an appropriate manner with a DNA fragment containing three regions consisting of a transcription initiation control region, a translation initiation control region, and a secretion signal region of an alkaline cellulase gene derived from a Bacillus genus upstream. It is necessary to be.

Examples of transcription initiation control region, translation initiation control region, and secretion signal region of alkaline cellulase gene derived from Bacillus bacterium include, for example, Bacillus genus bacteria described in JP-A-2000-210081 and JP-A-4-190793 That is, the transcription initiation regulatory region, translation initiation regulatory region, and secretory signal region of the cellulase gene derived from the KSM-S237 strain (FERM BP-7875) and the KSM-64 strain (FERM BP-2886) can be mentioned. 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 Or a DNA fragment comprising a nucleotide sequence having the identity of 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 98% or more, or any of the above bases It is desirable that a DNA fragment consisting of a base sequence in which a part of the sequence is deleted, substituted, or added is appropriately bound to the structural gene of the target protein or polypeptide. Here, a DNA fragment consisting of a base sequence in which a part of the base sequence is deleted, substituted, or added is a part of the base sequence that is deleted, substituted, or added with a plurality of base sequences. It means a DNA fragment that retains functions related to gene transcription, translation, and secretion.

  The introduction of a DNA fragment containing the protein or polypeptide gene of interest into a host bacterium belonging to the genus Bacillus is performed by using a recombinant plasmid obtained by linking such a DNA fragment and an appropriate plasmid vector, a competent cell transformation method, or protoplast transformation. Or a general transformation method such as electroporation or the like, or a DNA fragment in which an appropriate homology region with the host Bacillus subtilis genome is bound to the DNA fragment. This can be done by direct integration. Examples of the method for direct integration into the host bacterial genome include a competent cell transformation method, a protoplast transformation method, an electroporation method, and the like, and a competent cell transformation method is particularly preferred.

Here, the target protein or polypeptide is not particularly limited, and various industrial enzymes such as detergents, food processing, fiber processing, feed processing, cosmetics, pharmaceuticals, diagnostics, and bioactive peptides Etc. are included. In addition, industrial enzymes are classified according to their functions: oxidoreductase, transferase, hydrolase, lyase, isomerase, and synthase (Ligase / Synthetase). ) And the like, and preferred examples include genes for hydrolases such as cellulase, α-amylase, and protease. Specifically, cellulases belonging to Family 5 are listed in the classification of polysaccharide hydrolases (Biochem. J., 280, 309, 1991), among which cellulases derived from microorganisms, particularly Bacillus bacteria. More specific examples include alkaline cellulase derived from Bacillus sp. KSM-S237 strain (FERMBP-7875) or Bacillus sp. KSM-64 strain (FERM BP-2886). as a more specific example, Bacillus bacteria-derived alkaline cellulase comprising the amino acid sequence shown in SEQ ID NO: 2, or, Bacillus bacteria-derived alkaline cellulase comprising the amino acid sequence shown in SEQ ID NO: 4, or the Examples include proteins having an identity of 70%, preferably 80%, more preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more, and an alkaline cellulase activity in the amino acid sequence with cellulase. .

Specific examples of α-amylase include α-amylase derived from microorganisms, and liquefied amylase derived from Bacillus bacteria is particularly preferable. More specific examples include α-amylase derived from Bacillus sp. KSM-K38 strain (FERM BP-6946). More specific examples include the amino acid sequence represented by SEQ ID NO: 6. Alkaline amylase derived from a bacterium belonging to the genus Bacillus or having an amino acid sequence of 70%, preferably 80%, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more in the amino acid sequence. And a protein having alkaline amylase activity. Specific examples of proteases include serine proteases and metalloproteases derived from microorganisms, in particular, Bacillus bacteria.

  Production of the target protein or polypeptide using the recombinant Bacillus bacterium of the present invention is carried out by inoculating the strain on a medium containing an anabolic carbon source, nitrogen source and other essential components, and subjecting it to a normal microbial culture method. After completion of the culture, the target protein or polypeptide may be collected and purified.

  Hereinafter, the method for constructing the recombinant Bacillus bacterium of the present invention and the method for producing alkaline cellulase using the same will be described in detail, with a focus on examples of constructing the recombinant Bacillus subtilis strain.

  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.

  Further, Bacillus subtilis was transformed as follows. That is, Bacillus subtilis strains are 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.

  Cellulase activity in the culture supernatant was determined by measuring the degradation activity of the synthetic substrate 4 mM p-nitrophenyl-β-D-cellotrioside (Seikagaku Corporation). That is, 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, Ltd.), and mixed. The amount of p-nitrophenol liberated when the reaction was carried out at ℃ was quantified by the change in absorbance (OD420nm) at 420nm. The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was defined as 1 U.

  Liquitec Amy EPS (Roche Diagnostics) was used to measure the amylase activity in the culture supernatant. In other words, 50 μL of a sample solution appropriately diluted with 1% NaCl-1 / 7.5 M phosphate buffer (pH 7.4 Wako Pure Chemical Industries) was added to 100 μL of R1 and R2 mixture (R1 (coupling enzyme): R2 (amylase substrate) ) = 5: 1 (Vol.)) Was added and mixed, and the amount of p-nitrophenol released when the reaction was performed at 30 ° C. was quantified by the change in absorbance (OD405 nm) at 405 nm. The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was defined as 1 U.

Example 1 Deletion of spo0A gene Genome extracted from Bacillus subtilis 168 strain using spo0A-260F and SPO-1-RX, SPO-2-F and SPO-2-RB primer sets shown in Table 1 Using DNA as a template, a 699 bp fragment (A) on the 5 ′ end side including the upstream of the spo0A gene and a 348 bp fragment (B) on the 3 ′ end side were prepared. In addition, restriction enzyme recognition sequences for Apa I, Xho I, Pst I, and Bam HI are added to the 5 ′ end of each primer. The obtained fragment (A) was treated with Apa I and Xho I, and (B) was treated with Pst I and Bam HI. On the other hand, the spectinomycin resistance gene region was excised from the Bam HI and Xho I restriction enzyme cleavage points of the plasmid pDG1727 (Gene, 167, 335, (1995)) (C). Next, pBluescript II SK (+) (Stratagene) was placed in the order of (A) (C) (B), and (A) was Apa I and Xho I, and (C) was Xho I and Pst I. , (B) were inserted at the Pst I and Bam HI restriction enzyme breakpoints, respectively. The resulting recombinant plasmid DNA was treated with the restriction enzyme Sca I to obtain linear DNA, which was used as a donor DNA for transformation (see FIG. 2). Using this DNA fragment, Bacillus subtilis 168 strain was transformed by a competent method, and colonies grown on LB agar medium containing spectinomycin (100 μg / mL) were isolated as transformants. The genome of the obtained transformant was extracted, and it was confirmed by PCR that the spo0A gene was deleted and replaced with a spectinomycin resistance gene. The constructed strain was named Δspo0A strain.

Example 2 Deletion of abrB gene Deletion of the abrB gene on the Bacillus subtilis genome was performed in the same manner as in the method shown in FIG. Using genomic DNA extracted from Bacillus subtilis 168 strain as a template and using the abrB-FW and abrB / Cm-R primer sets shown in Table 1, a 1.0 kb fragment adjacent to the downstream of the abrB gene in the genome (D) Was amplified by PCR. Further, using the genomic DNA as a template, a 1.0 kb fragment (E) adjacent to the upstream of the abrB gene in the genome was amplified by PCR using abrB / Cm-F and abrB-RV primer sets.
Furthermore, using plasmid pC194 (J. Bacteriol. 150 (2), 815 (1982)) as a template and the catf and catr primer sets shown in Table 1, a 0.85 kb fragment containing the chloramphenicol resistance gene (F) Was amplified by PCR.
Next, by mixing the obtained (D) (E) (F) 3 fragments into a template and performing SOE-PCR using the abrB-FW2 and abrB-RV2 primer sets shown in Table 1, 3 fragments are combined in the order of (D) (F) (E), and the 3 fragments are contained in the order of 1.0 kb fragment (D), chloramphenicol resistance gene (F), 1.0 kb fragment (E). A 2.8 kb DNA fragment (G) was obtained. The primer sets used for the preparation of DNA fragments (D) to (G) are shown in Table 2.
Furthermore, the Δspo0A strain constructed in Example 1 was transformed with the obtained DNA fragment by a competent cell transformation method. After transformation, colonies that grew on the LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants.
Genomic DNA of the obtained transformant was extracted, and it was confirmed by PCR that the abrB gene was deleted and replaced with the chloramphenicol resistance gene. As described above, a Δspo0A / abrB strain in which both spo0A gene and abrB gene were deleted was constructed. Further, by using the Bacillus subtilis 168 strain in place of the Δspo0A strain in the competent cell transformation, a ΔabrB strain in which only the abrB gene was deleted was constructed.

Example 3 Evaluation of Alkaline Cellulase Secretion Production of Bacillus subtilis Mutants Δspo0A / abrB strain obtained in Examples 1 and 2, Bacillus subtilis 168 strain as a control, Δspo0A strain obtained in Example 1, and Example In each of the ΔabrB strains obtained in No. 2, an alkaline cellulase gene (JP 2000-210081) fragment (3.1 kb) derived from a Bacillus sp. KSM-S237 strain (FERM BP-7875) is a shuttle vector. A recombinant plasmid pHY-S237 inserted at the Bam HI restriction enzyme breakpoint of pHY300PLK was introduced by protoplast transformation method. The resulting strain was shake-cultured overnight in 10 mL of LB medium at 37 ° C., and 0.05 mL of this culture was further added to 50 mL of 2 × L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline) and shaking culture at 30 ° C. The culture solution was collected over time, and the alkaline cellulase activity of the supernatant of the culture solution from which the cells were removed by centrifugation was measured, and the amount of alkaline cellulase secreted and produced outside the cells by the culture was determined. As a result, as shown in FIG. 3, when the Δspo0A / abrB strain was used as the host, significantly higher production of alkaline cellulase was observed compared to the case of using the 168 strain or ΔabrB strain as the host. . In addition, in Δspo0A / abrB strain, the time required for enzyme production has been shortened since there is no slowing of cellulase activity accumulation after 20 hours of culture seen in the other 3 strains, and it is almost the highest at 38 hours after culture. Reached activity. At this point, the cellulase production of 168 strains, Δspo0A strain, and ΔabrB strain is about 3,100 U / L, about 3,100 U / L, and 3,200 U / L, respectively, whereas the production amount of Δspo0A / abrB strain is 4,800. It was U / L. Thus, in the Δspo0A / abrB strain, a significant amount of enzyme production was possible in a shortened time, and it was shown that it is very advantageous for industrial protein production.

Example 4 Evaluation of Alkaline Amylase Secretion Production of Bacillus subtilis Mutant Strains Alkaline amylase productivity of the Δspo0A / abrB strain obtained in Examples 1 and 2 was evaluated as follows.
Using genomic DNA extracted from Bacillus sp. KSM-K38 strain (FERM BP-6946) as a template, using the primer set of K38matu-F2 (ALAA) and SP64K38-R (XbaI) shown in Table 1 PCR is performed to amplify a 1.5 kb DNA fragment (G) of the base sequence represented by SEQ ID NO: 5 encoding alkaline amylase (JP 2000-184882, Eur. J. Biochem., 268, 2974, 2001) did. In addition, PCR was performed using the S237ppp-F2 (BamHI) and S237ppp-R2 (ALAA) primer sets shown in Table 1 using the genomic DNA extracted from the Bacillus sp. KSM-S237 strain as a template. A 0.6 kb DNA fragment (H) containing a region encoding the transcription initiation control region, translation initiation control region, and secretory signal region of the cellulase gene (Japanese Patent Laid-Open No. 2000-210081) was amplified. Next, the obtained 2 fragments of (G) and (H) are mixed and used as a template, and SOE-PCR using the S237ppp-F2 (BamHI) and SP64K38-R (XbaI) primer sets shown in Table 1 is performed. Thus, a 2.1 kb DNA fragment was obtained in which the alkaline amylase gene was linked downstream of the transcription initiation control region, translation initiation control region, and secretory signal region of the alkaline cellulase gene. The obtained 2.2 kb DNA fragment was inserted into the Bam HI- Xba I restriction enzyme cleavage point of shuttle vector pHY300PLK (Yakult) to construct alkaline amylase productivity evaluation plasmid pHYK38 (S237ps).

  The alkaline amylase productivity evaluation plasmid pHYK38 (S237ps) was introduced into the Δspo0A / abrB strain obtained in Examples 1 and 2 and the Δspo0A strain obtained in Example 1 as a control by the protoplast transformation method. The resulting strain was cultured with shaking in 10 mL of LB medium overnight at 37 ° C., and 0.05 mL of this culture was further added to 50 mL of 2 × L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% Maltose, 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline), followed by shaking culture at 30 ° C. for 5 days. After culturing, the alkaline amylase activity of the culture supernatant after removing the cells by centrifugation was measured, and the amount of alkaline amylase secreted and produced outside the cells by the culture was determined. As a result, as shown in Table 3, when the Δspo0A / abrB strain was used as the host, a higher secretion of alkaline amylase was observed than when the Δspo0A strain was used as the host.

Example 5 Evaluation of culture turbidity (OD600nm) of Bacillus subtilis mutants In the same manner as in Example 3, strains Δspo0A / abrB, Bacillus subtilis 168, Δspo0A and ΔabrB transformed with plasmid pHY-S237 were cultured. . When the turbidity (OD600 nm) of the culture solution at that time was measured over time, as shown in FIG. 4, when the Δspo0A strain was used as the host, a marked decrease in turbidity was observed after about 40 hours of culture. It was. On the other hand, such a decrease in turbidity was not observed in the Δspo0A / abrB strain, and it was revealed that the decrease in turbidity caused by the spo0A gene deletion was suppressed by the deletion of the abrB gene. The decrease in turbidity observed when the Δspo0A strain is used as the host is thought to be due to rapid lysis of the bacterial cells, and the release of intracellular proteins by such lysis into the culture medium is effective for recovery and purification of the target protein. There was concern about adverse effects. This point was improved when the Δspo0A / abrB strain was used as a host, and it was shown that the mutant strain was particularly advantageous for industrially producing proteins.

Comparative Example Evaluation of Alkaline Cellulase Secretion Production without Using Alkaline Cellulase Gene Transcription Initiation Control Region Using the S237FWsh and S237RV primer sets shown in Table 4, the recombinant plasmid pHY-S237 as a template, and the transcription initiation control region A free alkaline cellulase gene fragment (2.8 kb) was prepared by PCR. The prepared 2.8 kb fragment was inserted into the SmaI restriction enzyme cleavage point of shuttle vector pHY300PLK to construct a recombinant plasmid pHY-S237sh. The amplification of a 1.9 kb fragment containing the shuttle vector-derived tetracycline resistance gene and the alkaline cellulase gene upstream region was confirmed by performing PCR using the obtained plasmid as a template and the TET4 and preRV primer sets shown in Table 4. did. That is, as shown in FIG. 5, the alkaline cellulase gene is located downstream of the tetracycline resistance gene on the recombinant plasmid pHY-S237sh, and transcription of the alkaline cellulase gene is performed using the transcription initiation control region of the tetracycline resistance gene. It was considered a thing.
The constructed recombinant plasmid pHY-S237sh was introduced into each of the Δspo0A / abrB strain, the Bacillus subtilis 168 strain, and the Δspo0A strain in the same manner as in Example 3 and cultured. As a result, as shown in FIG. 6, the Δspo0A strain showed a marked improvement in productivity compared to the wild-type 168 strain, but the Δspo0A / abrB strain had a large increase as observed in Example 3. There was no change. That is, the Δspo0A / abrB strain should be used in combination with the transcription initiation regulatory region of the alkaline cellulase gene (JP 2000-210081) derived from Bacillus sp. KSM-S237 strain (FERM BP-7875). It was shown that it exerts an effect on the improvement of protein or polypeptide productivity.

The preparation of a DNA fragment for gene deletion by SOE-PCR and a method for deleting a target gene (substitution with a drug resistance gene) using the DNA fragment are schematically shown. FIG. 2 schematically shows deletion of a target gene by a double crossover method using a gene disruption plasmid. It is the graph which showed the secretory production amount of alkaline cellulase. It is the graph which showed the time-dependent change of culture solution turbidity (OD600nm). FIG. 6 is a construction diagram of recombinant plasmid pHY-S237sh. It is the graph which showed the alkaline cellulase secretion production amount when not using the transcription | transfer start control region of an alkaline cellulase gene.

Claims (7)

Bacillus subtilis genes spo0A and abrB , or a Bacillus bacterium strain in which each of the corresponding genes is deleted or inactivated, a transcription initiation control region of a Bacillus bacterium-derived alkaline cellulase gene, a translation initiation control region, and A recombinant Bacillus bacterium obtained by introducing a DNA fragment containing three regions comprising a secretory signal region and a gene encoding a target protein or polypeptide.   The recombinant Bacillus bacterium according to claim 1, wherein the Bacillus bacterium is Bacillus subtilis. Three regions consisting of a transcription initiation control region, a translation initiation control region and a secretion signal region are derived from the gene of Bacillus sp. KSM-64 (FERM P-10482) or Bacillus sp. KSM-S237 (FERM P-16067). The recombinant Bacillus bacterium according to claim 1 or 2.   Three regions comprising a transcription initiation control region, a translation initiation control region, and a secretion signal region are derived from the base sequence of base numbers 1 to 659 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 1, and the base sequence represented by SEQ ID NO: 3. A DNA fragment comprising a base sequence of base numbers 1 to 696 of the cellulase gene or a base sequence having 70% or more identity with any of the base sequences, or a part of the base sequence deleted, substituted, or added The recombinant Bacillus bacterium according to claim 1 or 2, which is a DNA fragment comprising a base sequence.   The recombinant Bacillus bacterium according to any one of claims 1 to 4, wherein the target protein is cellulase or amylase.   A protein comprising a cellulase having the amino acid sequence represented by SEQ ID NO: 2 or 4, or a protein having 70% or more identity with the amino acid sequence and having an alkaline cellulase activity, wherein the amylase is represented by SEQ ID NO: 6. The recombinant Bacillus bacterium according to claim 5, which is a protein comprising a sequence, or a protein having 70% or more identity with the amino acid sequence and having an alkaline amylase activity.   A method for producing a target protein or polypeptide using the recombinant Bacillus bacterium according to any one of claims 1 to 6.