JP2009038980A - Microorganism strain and method for producing objective substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve secretion productivity in such a microorganism as used as a host for producing a substance. <P>SOLUTION: Provided is a microorganism in which at least one of a nuclease-related gene of Bacillus subtilis and a gene corresponding to the gene is deleted or inactivated. Especially, the nuclease-related gene is preferably an endogenous nuclease-related gene. The endogenous nuclease-related gene includes addA gene, addB gene, sbcD gene, xseA gene, xseB gene and nucB gene. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a microbial strain having improved secretory productivity of a substance and a method for producing a target substance using the microbial strain.

  Bacillus subtilis is not only widely used in molecular biology studies as a model of Gram-positive bacteria, but is also widely used in the fermentation industry, the pharmaceutical industry, and the like as a bacterium that produces various enzymes such as amylase and protease. Although the entire base sequence of the Bacillus subtilis genome has already been determined by the Japan-European Joint Genome Project, the functional identification of about 4100 genes existing in the Bacillus subtilis genome has not been completed.

  To date, about 4100 types of disrupted strains of genes present in the Bacillus subtilis genome have been exhaustively 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 involved in biosynthesis or secretion of surfactin alone are lacking. Lost or inactivated strains have been constructed (see Patent Literature 1, Patent Literature 2, Patent Literature 3, Patent Literature 4, Patent Literature 5, Patent Literature 6, Patent Literature 7, and Patent Literature 8). However, the improvement of protein productivity by these strains is not sufficient, and until now, useful knowledge about elemental technologies for improving the productivity of various proteins in microorganisms has not been obtained.

K. Kobayashi et al., Proc. Natl. Acad. Sci. USA., 100, 4678-4683, 2003 JP-A-58-190390 JP 61-1381 WO89 / 04866 pamphlet Japanese National Patent Publication No. 11-509096 Patent No. 3210315 Special table 2001-527401 Special table 2002-520017 Special table 2001-503641

  Therefore, in view of the above situation, the present invention provides a microorganism strain having improved secretion productivity in a microorganism that is a host for substance production, and a method for producing a target substance using the microorganism strain. Objective.

  As a result of intensive studies by the present inventors in order to achieve the above-described object, secretion productivity is improved when a nuclease-related gene in Bacillus subtilis or a gene corresponding to the gene is deleted or inactivated. As a result, the present invention has been completed.

That is, the microbial strain according to the present invention is one in which at least one or more of nuclease-related genes possessed by Bacillus subtilis or genes corresponding to the genes are deleted or inactivated. In particular, the nuclease-related gene is preferably an endogenous nuclease-related gene. Examples of endogenous nuclease-related genes include addA gene, addB gene, sbcD gene, xseA gene, xseB gene and nucB gene.

The microbial strain according to the present invention is particularly preferably a recombinant derived from Bacillus bacteria. More preferably the Bacillus (Bacillus) the bacterium is Bacillus subtilis (Bacillus subtilis).

Furthermore, it is preferable that the microorganism strain according to the present invention has a gene encoding a target protein or polypeptide, or has a vector having the gene. In particular, at least one region selected from the group consisting of a transcription initiation control region, a translation initiation control region and a secretion signal region is more preferably bound to the upstream of the gene. Examples of the signal region for secretion include a region derived from a cellulase gene of a bacterium belonging to the genus Bacillus , and examples of the transcription initiation control region and the translation initiation control region include upstream regions of the cellulase gene. The region comprising the transcription initiation control region, the translation initiation control region, and the secretion signal region is preferably the following polynucleotide (a), (b), (c) or (d).
(A) a polynucleotide comprising the base sequence of base numbers 1 to 659 in the base sequence represented by SEQ ID NO: 1 (b) a polynucleotide comprising the base sequence of base numbers 1 to 696 in the base sequence represented by SEQ ID NO: 3 (c) ) A polynucleotide having 70% or more identity with the polynucleotide of (a) or (b) (d) in the polynucleotide of (a) or (b), one or more bases are deleted, substituted, Added or inserted polynucleotide

  In particular, the microbial strain according to the present invention has improved secretory productivity, and secretory productivity means the production amount per cell or the total secretory production amount.

  On the other hand, the method for producing a target substance according to the present invention comprises culturing the microorganism strain according to the present invention and producing the target substance outside the cells. Examples of the target substance include proteins and polypeptides.

  In the microbial strain according to the present invention, secretion productivity is greatly improved as compared with a normal wild strain. Therefore, according to the microorganism strain according to the present invention, it is possible to construct a production system in which the productivity of the target substance is greatly improved. In addition, the method for producing a target substance according to the present invention can greatly improve the secretion productivity as compared with the case of using a normal wild strain. Therefore, according to the method for producing a target substance according to the present invention, it is possible to greatly reduce the cost in the production system of the target substance.

Hereinafter, the present invention will be described in detail with reference to the drawings.
In the microbial strain according to the present invention, at least one or more of the nuclease-related genes possessed by Bacillus subtilis or the genes corresponding to the genes (hereinafter collectively referred to as nuclease-related genes) compared to the wild type are deleted or It has technical features such as being inactivated. Here, the microbial strain according to the present invention includes a recombinant strain in which a nuclease-related gene is deleted or inactivated from a wild-type microorganism by a genetic recombination method, and the nuclease-related gene is deleted without using a genetic recombination method. Or a naturally occurring mutant that has been inactivated.

Although it does not specifically limit as a wild strain of the said recombinant strain, Bacillus ( Bacillus ) genus bacteria, Clostridium ( Clostridium ) genus bacteria, yeast, etc. are mentioned. Of these, bacteria belonging to the genus Bacillus are preferred. The Bacillus (Bacillus) bacteria, e.g., Bacillus subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis), Bacillus anthracis (Bacillus anthracis), Bacillus cereus (Bacillus cereus), Bacillus thuringiensis Jen cis (Bacillus thuringiensis), Bacillus clausii (Bacillus clausii), Bacillus halodurans (Bacillus halodurans), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), mention may be made of Bacillus brevis (Bacillus brevis) and the like. As the Clostridium (Clostridium) bacteria, Clostridium acetobutylicum, Clostridium perfringens, Clostridium tetani, Clostridium novyi, Clostridium thermocellum, Clostridium difficile, Clostridium bi Fel Men wardrobe, Clostridium para Putri Fi cam (Clostridium paraputrificum), Clostridium hydroxy Benzoicum ( clostridium hydroxybenzoicum ), etc. can be mentioned. Moreover, as the yeast, Saccharomyces cerevisiae, Schizosaccharomyces Pont base (Schizosaccharomyces pombe), Pichia pastoris (Pichia pastoris), mention may be made of Kluyveromyces lactis (Kluyveromyces lactis) or the like.

Of these, microbial strains of the present invention, a Bacillus subtilis (Bacillus subtilis) 1 or more nuclease-related gene recombinant Bacillus subtilis or Bacillus subtilis was deleted or inactivated to (Bacillus subtilis) as a wild strain as the wild strain It is preferably a Bacillus subtilis mutant in which one or more nuclease-related genes have been deleted or inactivated. In particular, the microorganism strain according to the present invention is preferably derived from Bacillus subtilis 168 strain. In addition, the microbial strain according to the present invention may be derived from a mutant strain having a genome structure in which a specific region or one or more genes are deleted with the Bacillus subtilis 168 strain as a wild strain. Mutants derived from Bacillus subtilis 168 include 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-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 (spoIVCB-yraK) region, sbo-ywhH Mutants (referred to as Bacillus subtilis MGB874 strain) from which the region, yybP-yyaJ region and yncM-fosB region have been deleted can also be used. Note that these regions can be rephrased as regions sandwiched between a pair of oligonucleotide sets shown in Table 1.

  In the present invention, the nuclease-related gene means a gene encoding a protein having a nuclease activity and a gene encoding a subunit of the protein. Here, the nuclease activity may be either deoxyribonuclease activity or ribonuclease activity, and may be either endo-type or exo-type. The nuclease activity may be a nuclease activity for either a single-stranded polynucleotide or a double-stranded polynucleotide. The nuclease activity may be either ATP-dependent or ATP-independent. Furthermore, the nuclease activity may be activity inside or outside the cell.

In particular, the nuclease-related gene is preferably an endogenous nuclease-related gene. The endogenous nuclease-related gene means a nuclease-related gene originally possessed by a microorganism and excludes a phage-derived nuclease-related gene. For example, examples of endogenous nuclease-related genes in Bacillus subtilis include addA gene, addB gene, sbcD gene, xseA gene, xseB gene and nucB gene. Note gene numbers addA genes are BG10466, is known to encode a subunit A of ATP-dependent DNase. Gene number addB genes are BG10465, it is known that encoding subunit B of the ATP-dependent DNase. gene number of sbcD gene is BG10467, is known to be a sbcD homolog of exonuclease. Gene number xseA genes are BG11712, it is known that encoding the large subunit of exonuclease deoxy nuclease VII. Gene number xseB genes are BG11713, it is known that encoding the small subunit of exonuclease deoxy nuclease VII. Gene number nucB genes are BG11237, is known to encode a specific extracellular nucleases during sporulation.

In addition, the gene corresponding to the nuclease-related gene of Bacillus subtilis is a gene having high homology to the nuclease-related gene specified in Bacillus subtilis as described above, and has the same function as that of Bacillus subtilis. It means a gene having a function. Specifically, the nuclease-related protein in Bacillus subtilis described above, preferably 70% or more, 80% or more, or 90% with respect to the amino acid sequences of addA gene, addB gene, sbcD gene, xseA gene, xseB gene and nucB gene %, Preferably 95% or more, more preferably 96% or more, particularly preferably 97% or more, more particularly preferably 98% or more, and most preferably 99% or more. A gene encoding a nuclease-related protein derived from a microorganism other than Bacillus subtilis having

  Further, the microorganism strain according to the present invention may be one in which one of the nuclease-related genes as described above is deleted or inactivated, or a plurality of nuclease-related genes are deleted or inactivated. It may be what you did. The method for deleting or inactivating the nuclease-related gene described above is not particularly limited, and conventionally known gene recombination methods can be applied. Deletion or inactivation of a nuclease-related gene means that the target nuclease-related gene is deleted from the genome, another DNA fragment is inserted into the target nuclease-related gene, or the transcription / translation initiation region of the gene This also includes the case of giving mutations. In addition, the microorganism strain according to the present invention may be one in which the target nuclease-related gene is intentionally deleted or inactivated, or the result of randomly giving a gene deletion or inactivating mutation. The nuclease-related gene described above may be deleted or inactivated.

  In order to delete or inactivate the above-mentioned nuclease-related gene, for example, a homologous recombination method can be applied. That is, a circular recombinant plasmid obtained by cloning a DNA fragment containing a part of a target nuclease-related gene to be deleted into an appropriate plasmid vector is taken into the parent microorganism cell, and a partial region of the nuclease-related gene It is possible to disrupt and inactivate nuclease-related genes on the parental microbial genome by homologous recombination.

  In particular, when Bacillus subtilis is used as a parental microorganism for constructing the microorganism strain according to the present invention, there have already been several reports on methods for deleting or inactivating a target gene by homologous recombination (Mol). .Gen.Genet., 223,268,1990, etc.), the microorganism strain according to the present invention can be produced by repeating such a method.

  As an example of a method for deleting a nuclease-related gene, more specifically, double using a deletion DNA fragment prepared by SOE (splicing by overlap extension) -PCR method (Gene, 77, 61, 1989). The intersection method will be described with reference to FIG. The deletion DNA fragment is a fragment in which a drug resistance marker gene fragment is inserted between the approximately 1.0 kb fragment adjacent to the upstream of the deletion target gene and the approximately 1.0 kb fragment adjacent to the downstream of the deletion target gene. It is. 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 downstream side of the drug resistance marker gene is added to the upstream end of the downstream fragment, while a 10-30 base pair sequence on the side is used.

  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.

  When using a chloramphenicol resistance gene as a drug resistance marker gene, use a general PCR enzyme kit such as Pyrobest DNA Polymerase (Takara Shuzo). , Humana Press pp251, 1993, Gene, 77, 61, 1989), etc., DNA fragments for deletion of each gene can be obtained by performing SOE-PCR under the usual conditions.

  When the DNA fragment for gene deletion thus obtained is introduced into the cell by a competent method or the like, gene recombination occurs in the cell in the homologous region upstream and downstream of the identical deletion target gene. The cells in which the target gene is replaced with the drug resistance gene, or the cells in which the drug resistance gene is inserted into the target gene can be separated by selection with a drug resistance marker. That is, if a colony that grows on an agar medium containing chloramphenicol is isolated and it is confirmed that the target gene on the genome has been replaced with the chloramphenicol resistance gene by PCR using the genome as a template good.

  By applying the SOE-PCR method as described above, the above-mentioned nuclease-related gene can be deleted from the Bacillus subtilis genome. A nuclease-related gene inactivated by mutation such as base substitution or base insertion, or a linear DNA fragment containing the upstream and downstream regions of the target gene but not the deletion target gene as shown in FIG. Is constructed by a method such as PCR, and is incorporated into the parent microbial cell, and the two homologous groups crossing twice at the two positions outside the mutation site in the gene of the parent microbial genome, or upstream and downstream of the target gene. It is also possible to replace the target gene on the genome with a deleted or inactivated gene fragment by causing replacement. In addition, random gene deletion or inactivation can be achieved by a method of causing homologous recombination similar to the above method using a randomly cloned DNA fragment, or by irradiating a parental microorganism with γ rays, etc. It is also possible to delete or inactivate the above-mentioned nuclease-related genes.

  In addition, as described above, inactivation methods of nuclease-related genes include methods of expressing antisense RNA for nuclease-related genes, methods of expressing ribozymes that specifically cleave nuclease-related gene mRNA, and RNA interference. Examples thereof include a method for suppressing the expression of a nuclease-related gene and a method for adding a substance that specifically inhibits a protein encoded by a nuclease-related gene.

  The microorganism strain according to the present invention configured as described above has an effect that the protein and / or peptide secretion productivity is superior to the wild type. Here, secretory productivity means the production amount per cell or the total secretory production amount when a microorganism strain is cultured. That is, the microbial strain according to the present invention is superior in secretion production amount of the target protein or peptide per cell compared to the wild type and / or has a secretion production amount to the culture medium in comparison with the wild type. Is excellent. Here, the target protein and peptide for secretory production are not limited in any way.

When secretory production of a predetermined protein or peptide using the microorganism strain according to the present invention, the gene encoding the protein is introduced into the microorganism strain so that the gene can be expressed. The target protein may be a secretory protein that inherently has a signal sequence, or may be a secreted protein that is artificially added with a signal sequence. Further, upstream of the region encoding the protein, a control region involved in transcription, translation, and secretion of the gene, that is, a transcription initiation control region including a promoter and a transcription initiation site, a translation initiation region including a ribosome binding site and an initiation codon, and It is desirable that at least one region selected from the 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 , It is preferable that the start region and the translation start region are 0.6-1 kb region upstream of the cellulase gene and are linked to the target protein or polypeptide gene in an appropriate form. For example, Bacillus (Bacillus) bacteria as described in 2000-210081 and JP 4-190793 Patent Publication JP, i.e. 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. SEQ ID NO: 1 shows the cellulase gene derived from the KSM-S237 strain and its upstream region, and SEQ ID NO: 2 shows the amino acid sequence of the cellulase encoded by the cellulase gene. SEQ ID NO: 3 shows the cellulase gene derived from the KSM-64 strain and the base sequence of the upstream region, and SEQ ID NO: 4 shows the amino acid sequence of the cellulase encoded by the cellulase gene.

  More specifically, the base sequence of base numbers 1 to 659 of the cellulase gene consisting 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, It is preferable to have 70% or more, 80% or more, or 90% or more homology to the base sequence, more preferably 95% or more, still more preferably 96% or more, particularly preferably 97% or more, more particularly Preferably, a DNA fragment consisting of a base sequence having 98% or more, most preferably 99% or more identity, or a DNA fragment consisting of a base sequence from which any one of the above base sequences has been deleted is a target protein or poly It is desirable that it is properly linked to the structural gene of the peptide.

  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.

  Furthermore, the gene encoding the target protein thus configured may be integrated into the genome of the microorganism strain according to the present invention, or may be retained in the microorganism strain in the form of an expression vector. In any case, by culturing the microorganism strain, the gene encoding the target protein or peptide is expressed and secreted into the medium. Then, the protein or peptide can be obtained in large quantities by separating and purifying the target protein or peptide from the medium according to a conventional method. In particular, since the microorganism strain according to the present invention is superior in secretory productivity to the culture medium as compared with the wild type, the target protein or peptide can be produced in large quantities, and the production cost can be greatly reduced.

  The target protein or target polypeptide produced using the microbial strain of the present invention is not particularly limited. For example, industrial enzymes used in various industries such as detergents, foods, fibers, feeds, chemicals, medicines, and diagnostics Examples thereof include physiologically active peptides, and industrial enzymes are particularly preferable. Industrial enzymes can be classified according to their functions: oxidoreductase, transferase, hydrolase, lyase, isomerase, and synthase (Ligase / Synthetase) and the like.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

[Example 1]
In this example, Bacillus subtilis strains lacking the addA gene, addB gene, sbcD gene, xseA gene, xseB gene, yncB gene, yorK gene or yokF gene from Bacillus subtilis 168 strain were prepared as follows. Table 2 summarizes the names of primers and their base sequences used in the following procedures.

  First, using the genomic DNA extracted from Bacillus subtilis 168 strain as a template, each of “gene name-FW” and “gene name / CmR”, and “gene name / CmF” and “gene name-RV” shown in Table 2 were used. Using the primer set, a 1.0 kb fragment adjacent to the upstream of the gene on the genome (A) and a 1.0 kb fragment adjacent to the downstream (B) were prepared. On the other hand, Table 2 shows a recombinant plasmid pCBB31 in which the chloramphenicol resistance gene of plasmid pC194 (J. Bacteriol. 150 (2), 815 (1982)) was inserted at the XbaI-BamHI breakpoint of plasmid pUC18. Using the CmF and CmR primer sets, a 1 kb fragment (C) containing a chloramphenicol resistance gene was prepared. Next, 3 fragments obtained (A), (B) and (C) are mixed and used as a template, and SOE-PCR using primers “gene name-FW2” and “gene name-RV2” in Table 2 is performed. Thus, the 3 fragments were combined in the order of (A), (C), and (B) to obtain a DNA fragment of 3.0 kb (see FIG. 1). Using this DNA fragment, Bacillus subtilis 168 strain was transformed by a competent method, and colonies grown on LB agar medium containing chloramphenicol were isolated as transformants. The genome of the obtained transformant was extracted, and it was confirmed by PCR that the target gene was deleted and replaced with a chloramphenicol resistance gene.

  On the other hand, a deletion strain of the nucB gene was prepared in the same manner except that the spectinomycin resistance gene was used in place of the chloramphenicol resistance gene in the procedure described above. That is, using genomic DNA extracted from Bacillus subtilis 168 as a template, and using each of the nucB-FW and nucB / SpR and nucB / SpF and nucB-RV primer sets shown in Table 2, the nucB gene on the genome A 1.0 kb fragment adjacent to the upstream (A) and a 1.0 kb fragment adjacent to the downstream (B) were prepared. On the other hand, using the spectinomycin resistance gene of plasmid pDG1727 (Gene. 167: 335-336 (1995)) as a template and using the SpF and SpR primer sets shown in Table 2, 1.2 kb containing the spectinomycin resistance gene is used. Fragment (C) was prepared. Next, the 3 fragments obtained by mixing the obtained 3 fragments (A), (B), and (C) as a template, and performing SOE-PCR using the primers nucBFW2 and nucB-RV2 in the table were converted into 3 fragments (A) ( C) The DNA fragments were bound in the order of (B) to obtain a DNA fragment of 3.0 kb (see FIG. 1). Using this DNA fragment, Bacillus subtilis 168 strain was transformed by a competent method, and colonies grown on LB agar medium containing spectinomycin were isolated as transformants. The genome of the obtained transformant was extracted, and it was confirmed by PCR that the nucB gene was deleted and replaced with a spectinomycin resistance gene.

As described above, in Example 1, a Bacillus subtilis strain lacking the addA gene, addB gene, sbcD gene, xseA gene, xseB gene, yncB gene, yorK gene or yokF gene could be prepared from 168 strains of Bacillus subtilis.

[Example 2]
In this example, a Bacillus subtilis strain lacking the addA gene, addB gene, sbcD gene, xseA gene or xseB gene was prepared from the Bacillus subtilis MGB874 strain. Specifically, as in Example 1, transformation of Bacillus subtilis MGB874 strain was performed by a competent method using a DNA fragment for deleting a gene for deletion, and transformation was performed using the same drug resistance as an index. The body was isolated, and it was confirmed by PCR that the gene for deletion was deleted and replaced with a drug resistance gene.
As described above, in Example 2, a Bacillus subtilis strain lacking the addA gene, addB gene, sbcD gene, xseA gene or xseB gene could be produced from the Bacillus subtilis MGB874 strain.

Example 3
Each gene-deficient strain obtained in Example 1 and Bacillus subtilis strain 168 as a control are encoded with an alkaline cellulase gene derived from Bacillus sp. KSM-S237 strain (Japanese Patent Laid-Open No. 2000-210081). The recombinant plasmid pHY-S237 in which the DNA fragment (3.1 kb) to be inserted was inserted into the BamHI restriction enzyme cleavage point of the shuttle vector pHY300PLK was introduced by the protoplast transformation method. The resulting strain was subjected to shaking culture in 10 mL of LB medium at 30 ° C. overnight, 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 3 days. After culturing, the OD600 of the culture solution is measured as the cell density, and then the alkaline cellulase activity of the supernatant of the culture solution from which the cells have been removed by centrifugation is measured, and the alkaline cellulase secreted and produced outside the cells by culturing. And the amount of alkaline cellulase secretion produced per cell. The results are shown in Table 3.

As shown in Table 3, when a Bacillus subtilis strain lacking the addA gene, addB gene, sbcD gene, xseA gene, xseB gene or nucB gene was used as a host, the control Bacillus subtilis strain 168 (wild type) Compared with the case of using, higher production of alkaline cellulase and higher production of alkaline cellulase per cell were observed. On the other hand, when a Bacillus subtilis strain lacking the yncB gene, yorK gene or yokF gene is used, the amount of alkaline cellulase secreted and the equivalent amount of alkaline cellulase per cell equivalent to that of the control Bacillus subtilis 168 strain (wild type) Secretory production was observed.

Example 4
Recombinant plasmid pHY-S237 was introduced into each gene deletion strain obtained in Example 2 and MGB874 strain as a control in the same manner as in Example 3 by the protoplast transformation method. The resulting strain was cultured in the same manner as in Example 3, and the amount of alkaline cellulase secreted and produced outside the cells and the amount of alkaline cellulase secreted and produced per cell were determined. The results are shown in Table 4.

As shown in Table 4, when a Bacillus subtilis strain lacking the addA gene, addB gene or nucB gene was used as the host, a higher production amount of alkaline cellulase per cell was observed compared to the control MGB874 strain. It was. When a Bacillus subtilis strain lacking the sbcD gene, xseA gene, xseB gene or nucB gene is used as the host, MGB8 as a control is used.
A higher production amount of alkaline cellulase was observed compared to the case of 74 strains.

It is a schematic diagram for demonstrating an example (SOE-PCR method) of deleting a predetermined area | region from the genome of Bacillus subtilis.

Claims (12)

  A microbial strain in which at least one or more of nuclease-related genes possessed by Bacillus subtilis or genes corresponding to the genes are deleted or inactivated, thereby improving secretion productivity.   2. The microorganism strain according to claim 1, wherein the nuclease-related gene or a gene corresponding to the gene is an endogenous nuclease-related gene or a gene corresponding to the gene. The endogenous nuclease-related gene, or a gene corresponding to the gene, addA gene, addB gene, sbcD gene, xseA gene, xseB gene and nucB gene, or a gene corresponding to the gene 2. The microorganism strain according to 2. The microorganism strain according to any one of claims 1 to 3, which is a recombinant derived from a Bacillus bacterium. The genus Bacillus (Bacillus) bacteria microbial strain according to claim 4, characterized in that the Bacillus subtilis (Bacillus subtilis).   The microorganism strain according to any one of claims 1 to 5, which has a gene encoding a target protein or polypeptide, or has a vector having the gene.   The microbial strain according to claim 6, wherein at least one region selected from the group consisting of a transcription initiation control region, a translation initiation control region, and a secretion signal region is bound upstream of the gene. The secretion for signal region is derived from a cellulase gene of Bacillus (Bacillus) bacteria, the transcriptional initiation regulatory region and the translational initiation control region of claim 7, wherein it is derived from the region upstream of the cellulase gene Microbial strain. The region comprising the transcription initiation control region, the translation initiation control region, and the secretion signal region is the following polynucleotide (a), (b), (c), or (d): 6. The microorganism strain according to 6.
(A) a polynucleotide comprising the base sequence of base numbers 1 to 659 in the base sequence represented by SEQ ID NO: 1 (b) a polynucleotide comprising the base sequence of base numbers 1 to 696 in the base sequence represented by SEQ ID NO: 3 (c) ) A polynucleotide having 70% or more identity with the polynucleotide of (a) or (b) (d) in the polynucleotide of (a) or (b), one or more bases are deleted, substituted, Added or inserted polynucleotide   The microbial strain according to any one of claims 1 to 9, wherein the secretory productivity is a production amount per cell or a total secretory production amount.   A method for producing a target substance, comprising culturing the microorganism strain according to any one of claims 1 to 10 and producing the target substance outside the cells.   The production method, wherein the target substance is a protein or a polypeptide.

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