JP5829802B2 - Recombinant Bacillus subtilis - Google Patents

Description

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

  Industrial production of useful substances by microorganisms includes brewing foods such as alcoholic beverages, miso and soy sauce, industrial production of amino acids, organic acids, nucleic acid-related substances, antibiotics, carbohydrates, lipids, proteins, etc. It is carried out in various ways. In addition, its use has been extended to a wide range of fields from foods to daily necessities such as medicines, detergents, cosmetics, and various chemical raw materials.

  One important issue in the industrial production of useful substances using microorganisms is to improve the productivity of the useful substances. Conventionally, in order to solve the problem, breeding of high-producing bacteria by genetic techniques such as mutation has been performed. Particularly recently, with the development of microbial genetics and biotechnology, more efficient breeding of high-producing bacteria using genetic recombination technology 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. For example, Bacillus subtilis Marburg No. Strains obtained by further improving microbial strains recognized as safe and excellent as host microorganisms, such as 168 strains, have been developed.

  In Bacillus subtilis, maltose is incorporated into a cell after being incorporated into a cell by MalP, which is one of phosphoenolpyruvate-dependent sugar transporters (PTS: Phosphoenol pyruvate-dependent phosphate system). 1) Phosphorylated and converted to maltose 6-phosphate. Furthermore, maltose 6-phosphate is decomposed into glucose and glucose 6-phosphate by MalA having 6-phospho-α-glucosidase activity and metabolized in the glycolytic system. It is known that the gene malA encoding MalA constitutes the malA-glvR-malP operon (Non-patent Document 2).

  In the past patent application (Patent Document 1), the applicant of the present invention is a strain in which Bacillus subtilis 168 strain (wild strain) is used as a parent strain and malP (the name at that time is glvC) and glvR are deleted in the enzyme cellulase and α-amylase. It has been reported that the production volume will improve. However, it has not been clarified about the influence on the enzyme production amount by deletion of the malA gene.

Japanese Patent Laid-Open No. 2005-151983 JP 2007-130013 A

J. et al. bacteriol, 188, 11, p. 3911-3922, 2006 J. et al. bacteriol, 183, 17, p. 5110-5121, 2001

  The present invention relates to a recombinant Bacillus subtilis capable of improving the productivity of a protein or polypeptide, and further to a method for producing a protein or polypeptide using the recombinant Bacillus subtilis.

  The present inventors use Bacillus subtilis obtained by deleting from the genome a gene group that acts unnecessary or harmful for the production of useful proteins or polypeptides, and deletes or inactivates the malA gene of the host. By introducing a gene encoding a target heterologous protein or polypeptide, production of the heterologous protein or polypeptide was found to be significantly improved, and the present invention was completed.

That is, the present invention relates to the following (1) to (9).
(1) profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA- It has a genomic structure in which the yurT region, the cgeE-ypmQ region, the yeeK-yesX region, the pdp-rocR region, the ycxB-sipU region, the SKIN-Pro7 region, the sbo-ywhH region, the yybP-yyaJ region and the yncM-fosB region are deleted. A recombinant Bacillus subtilis produced by introducing a gene encoding a heterologous protein or polypeptide into a Bacillus subtilis mutant, wherein the malA gene has been deleted or inactivated. Bacillus subtilis.
(2) The recombinant Bacillus subtilis according to (1) above, wherein the Bacillus subtilis mutant has a genome structure in which one or more of the following regions are further deleted:
ybbU-ybfI region, ydjM-cotA region, yefA-yesX region, yfiB-yfiX region, yhcE-yhcU region, yhaU-yhaL region, yjbX-yjlB region, xkdA-ykcC region, bpr-ylgD region, fpr-ylgA region, ynfF-ppsA region, yoxC-yobO region, spoVAF-spoIIAA region, spoIIIAH-yqhV region, ytvB-ytqB region, yteA-ytaB region, yuaJ-yugO region, yusJ-mrgA region, gerAA-yvrM region, gerAA-yvR region araE-yveK region, yvdE-yvcP region, gerBA-ywsC region, ywrK-ywqM region, spoIIID-ywoB region, slp-ylaF region, licH-sigY Pass, yqeF-yrhK region, yuzE-yukJ region and yncM-yndN region.
(3) The above (1), wherein one or more regions selected from a transcription initiation control region, a translation initiation control region and a secretion signal region are operably linked upstream of a gene encoding a heterologous protein or polypeptide. Or the recombinant Bacillus subtilis according to (2).
(4) From the above (1) to (3), three regions comprising a transcription initiation control region, a translation initiation control region and a secretion signal region are operably linked upstream of a gene encoding a heterologous protein or polypeptide. The recombinant Bacillus subtilis according to any one of (1).
(5) The secretory signal region is derived from a cellulase gene of a Bacillus genus bacterium, and the transcription initiation control region and the translation initiation control region are derived from a 0.6-1 kb region upstream of the cellulase gene. The recombinant Bacillus subtilis according to (3) or (4) above.
(6) The three regions comprising the transcription initiation control region, the translation initiation control region, and the secretion signal region are the base sequence of base numbers 1 to 659 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 3, or the base sequence The recombinant Bacillus subtilis according to (4) above, which is a DNA fragment consisting of a base sequence having 70% or more identity with any one, or a DNA fragment consisting of a base sequence from which a part of the base sequence has been deleted.
(7) The recombinant Bacillus subtilis according to any one of (1) to (6) above, wherein the gene encoding the heterologous protein or polypeptide is an alkaline cellulase gene of a Bacillus bacterium.
(8) The alkaline cellulase gene is a cellulase gene comprising the base sequence represented by SEQ ID NO: 3, or a DNA fragment comprising a base sequence having 70% or more identity with the base sequence, or a part of the base sequence. The recombinant Bacillus subtilis according to (7) above, which is a DNA fragment consisting of a deleted, substituted or added nucleotide sequence.
(9) A method for producing a heterologous protein or polypeptide using the recombinant Bacillus subtilis according to any one of (1) to (8) above.

  If the recombinant Bacillus subtilis of the present invention is used, the target protein or polypeptide can be efficiently mass-produced.

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 (it replaces with a drug resistance gene).

  The recombinant Bacillus subtilis of the present invention uses a Bacillus subtilis mutant strain with a portion of its genome deleted as a parent strain, deletes or inactivates the malA gene, and introduces a gene encoding a heterologous protein or polypeptide Is produced.

  Bacillus subtilis is an aerobic Gram-positive rod and is a kind of eubacteria that form spores. Bacillus subtilis is a useful microorganism for the present invention because it has been clarified in whole genome information, has established genetic engineering and genomic engineering techniques, and has the ability to secrete and produce proteins and extracellularly.

  Bacillus subtilis used in the present invention includes large regions of its genome, that is, the profile6 (yoaV-yobO) region, the profile1 (ybbU-ybdE) region, the profile4 (yjcM-yjdJ) region, the PBSX (ykdA-xlyA) region, and the property5 ( ynxB-dut) region, profile3 (ydiM-ydjC) region, spb (yodU-ypqP) region, pks (pksA-ymaC) region, skin (spoIVCB-spoIIIC) region, pps (ppsE-ppsA) region, profile2 (ydcL) ydeJ) region, ydcL-ydeK-ydhU region, yisB-yitD region, yunA-yurT region, cgeE-ypmQ region, yeK-yesX region, pdp-r A strain constructed by deleting the cR region, ycxB-sipU region, SKIN-Pro7 (spoIVCB-yraK) region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region is used. Bacteria Bacillus subtilis Marburg No. 168 (Bacillus subtilis strain 168) is a wild strain, and Bacillus subtilis MGB874 strain constructed by deleting the large region can be mentioned (Patent Document 2). These deletion regions can also be represented as regions sandwiched by a pair of oligonucleotide sets shown in Table 1.

  The complete base sequence and gene of Bacillus subtilis 168 have been reported and published on the Internet (Nature, 390, 249-256, 1997 and BSORF Bacillus subtilis Genome Database [http://bacillus.genome.jp/ GenBank: AL009126.2 [http://www.ncbi.nlm.nih.gov/nuccore/386680335]). Those skilled in the art will know the genomic information of Bacillus subtilis 168 strains obtained from these sources, for example GenBank: AL009126.2 [http: // www. ncbi. nlm. nih. gov / nuccore / 38688035], the gene regions shown in Table 2 above to be deleted from the genome of the MGB874 strain can be found. Here, the gene region to be deleted is a natural or artificially induced deletion, substitution, insertion, or addition in one or more bases relative to the public base sequence of the Bacillus subtilis 168 gene. It may have a base sequence that may contain such mutations. The range of the number of bases that can be mutated is not particularly limited as long as the correspondence with the genome of Bacillus subtilis 168 can be recognized. For example, the identity of the base sequence of the genome region of Bacillus subtilis 168 is 50% or more. Preferably 60% identity, more preferably 70% identity, more preferably 80% identity, even more preferably 90% identity, still more preferably 95% identity. The range which has is mentioned. The “addition” includes addition of a base to one end and both ends of a base sequence.

  The Bacillus subtilis mutant strain according to the present invention may have a genomic structure in which the genomic region is further deleted from the genomic region of the Bacillus subtilis mutant strain MGB874. Specifically, the Bacillus subtilis mutant is a genome in which at least one of the gene regions shown in Table 2 on the genome of Bacillus subtilis 168 is further deleted from the genome region of Bacillus subtilis mutant MGB874. It may have a structure. Examples of the deletion region include a gene region shown in Table 2 and a region corresponding to the region.

  The MalA protein, which is the gene product of the malA gene (BSORF Gene Entry No .: BG11839, EC3.2.1.122) to be deleted in the present invention, has α-glucosidase activity, decomposes maltose and converts it into glucose. To do. The malA gene constitutes the malA-glvR-malP operon together with the malP gene and the glvR gene, and is involved in maltose metabolic activity.

  A combination with the deletion or inactivation of genes other than the malA gene is expected to have a greater effect on improving the productivity of heterologous proteins or polypeptides.

  The recombinant Bacillus subtilis from which the malA gene according to the present invention has been deleted or inactivated, for example, a method already reported (Mol. Gen. Genet., Etc.) such as a method of deleting or inactivating a target gene by homologous recombination. 223, 268 (1990), etc.).

  In addition, for deletion or inactivation of random genes, a method of causing homologous recombination similar to the above method using a randomly cloned DNA fragment, or irradiation of parental microorganisms with γ rays, etc. It can be implemented.

  In the present invention, the object of the present invention can be achieved even if the target gene is inactivated by a method of inserting another DNA fragment into the malA gene or a method of mutating the transcription / translation initiation region of the gene. More preferably, the target gene is physically deleted. Moreover, although the number of genes to be deleted or inactivated may be one, deletion of two or more genes may be combined. Furthermore, the construction of the recombinant Bacillus subtilis of the present invention can be combined with the deletion or inactivation of genes other than those involved in permeation of maltose, which is expected to have a greater effect on productivity improvement.

  As a specific example of deletion or inactivation of the malA gene in the present invention, two deletion DNA fragments prepared by SOE (splicing by overlap extension) -PCR method (Gene, 77, 61, (1989)) are used. Although the deletion method by the double crossing method will be described, the gene deletion method in the present invention is not limited to this method.

  The deletion DNA fragment used in this method has a drug resistance marker gene fragment inserted between the approximately 0.2 to 3 kb fragment adjacent to the upstream of the gene to be deleted and the approximately 0.2 to 3 kb fragment adjacent to the downstream. It is a fragment constructed by First, an upstream fragment and a downstream fragment of a gene to be deleted and three fragments of a drug resistance marker gene fragment are prepared by the first PCR. In this case, for example, at the upstream end of the drug resistance marker gene at the downstream end of the upstream fragment, A primer designed to add a 10 to 30 base pair sequence, and conversely, a downstream 10 to 30 base pair sequence of the drug resistance marker gene is used at the upstream end of the downstream fragment (FIG. 1).

  Next, using the three 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 with the drug resistance marker gene fragment occurs, and as a result of PCR amplification, a DNA fragment in which the drug resistance marker gene is inserted is inserted between the upstream fragment and the downstream fragment. Is obtained (FIG. 1).

  Using the primer set shown in Table 3 and an appropriate template DNA, using a general PCR enzyme kit such as Pyrobest DNA Polymerase (Takara Shuzo), etc., “PCR Protocols. Current Methods and Applications”, Edited by B. et al. A. DNA fragments for deletion of each gene can be obtained by performing SOE-PCR under the normal conditions shown in White, Humana Press, pp251 (1993), Gene, 77, 61, (1989) and the like.

  When the DNA fragment for deletion thus obtained is introduced into the cell by a competent method or the like, gene recombination in the cell is carried out in the region homologous to the DNA fragment for deletion upstream and downstream of the gene to be deleted. The resulting cells in which the gene to be deleted 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 DNA fragment prepared using the primer set shown in Table 3 is introduced, a colony growing on an agar medium containing a drug is isolated, and the target gene is deleted and replaced with a drug resistance gene. It may be confirmed by a PCR method using a genome as a template.

  Next, the recombinant Bacillus subtilis of the present invention can be obtained by introducing a gene encoding the desired heterologous protein or polypeptide into the Bacillus subtilis mutant strain according to the present invention thus obtained.

  When producing a heterologous protein or polypeptide using the recombinant Bacillus subtilis, a control region that controls transcription, translation, and secretion of the gene is linked in an appropriate form upstream of the gene of the target protein or polypeptide. It is desirable to let them. Examples of such a control region include 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 one or more regions selected from a secretory signal peptide region. In particular, it is preferable that three regions comprising 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, and transcription initiation It is desirable that the control region and the translation initiation control region are upstream regions of 0.6 to 1 kb in length starting from the start codon of the cellulase gene and are operably linked to a heterologous protein or polypeptide gene. For example, bacteria belonging to the genus Bacillus described in JP 2000-210081, JP 4-190793, etc., that is, KSM-S237 strain (FERM BP-7875), KSM-64 strain (FERM BP- It is desirable that the transcription initiation regulatory region, translation initiation region and secretory signal peptide region of the cellulase gene derived from 2886) be operably linked to the structural gene of the target protein or polypeptide. More specifically, the three regions comprising the transcription initiation control region, the translation initiation control region and the secretion signal region are the base sequences of base numbers 1 to 659 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 3, or A nucleotide sequence having the identity of 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more with any one of the nucleotide sequences, and having a function relating to gene transcription, translation and secretion It is desirable that a DNA fragment consisting of or a DNA fragment consisting of a base sequence from which a part of the base sequence is deleted is operably linked to the heterologous protein or polypeptide 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. Here, operably linked means that the control sequence functions and the expression of the target protein encoded by the coding sequence can be controlled. Refers to the arrangement of the coding sequence. As a specific example, it means that a promoter and a target gene adjacent thereto are arranged in a state where the gene can be expressed along the direction of the promoter. Here, the stringent conditions include, for example, conditions described in [Molecular cloning-a Laboratory manual 2nd edition (Sambrook et al., 1989)]. For example, 6 × SSC (1 × SSC composition: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhardt's solution and a solution containing 100 mg / mL herring sperm DNA Among them, conditions for incubation with a probe at 65 ° C. for 8 to 16 hours to hybridize can be mentioned.

  In order to express a gene encoding a heterologous protein or polypeptide in a cell, an expression plasmid in which a DNA fragment containing the gene is inserted into an appropriate vector is constructed, and the expression plasmid is introduced into a host for transformation. There is a need to. The expression vector is preferably a vector capable of autonomous replication in Bacillus subtilis, and examples thereof include the shuttle vector pHY300PLK, but are not particularly limited.

  A polypeptide generally refers to a polymer of linearly linked amino acids, and a protein generally refers to one or more polypeptides of 50 or more amino acids, although these terms are interchangeable in this application. It shall be used for

  Examples of the heterologous protein or polypeptide produced using the recombinant Bacillus subtilis of the present invention include, for example, various industrial enzymes used for foods, pharmaceuticals, cosmetics, washings, textiles, and testing, and physiological activities. Peptide etc. are mentioned. In addition, the functions of industrial enzymes are classified into oxidoreductase (oxidoreductase), transferase (transferase), hydrolase (hydrolase), elimination enzyme (lyase), isomerase (isomerase), synthetic enzyme (ligase / Synthase) and the like, preferably, hydrolases such as cellulase, α-amylase and protease, more preferably cellulase.

  More specific examples include alkaline cellulase derived from Bacillus bacterium KSM-S237 (FERM BP-7875) encoded by the nucleotide sequence represented by SEQ ID NO: 3, and Bacillus bacterium comprising the amino acid sequence represented by SEQ ID NO: 119. Examples include alkaline cellulase derived from KSM-64 strain (FERM BP-2886), and alkaline cellulase having an amino acid sequence in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 4 or 119 are deleted, substituted or added. Furthermore, cellulases comprising an amino acid sequence having 70%, preferably 80%, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more identity with the amino acid sequence. .

  Moreover, specific examples of α-amylase include α-amylase derived from microorganisms. In particular, liquefied amylase derived from Bacillus bacteria, and Bacillus bacteria KSM-K38 strain comprising the amino acid sequence represented by SEQ ID NO: 120 ( FERM BP-6946) -derived alkaline amylase, 70%, preferably 80%, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more of the amino acid sequence. And a protein having an amylase activity consisting of an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 120. Specific examples of the protease include serine proteases derived from microorganisms, particularly bacteria belonging to the genus Bacillus, metal proteases, and Bacillus clausii strain KSM-K16 (FERM BP-3376) comprising the amino acid sequence represented by SEQ ID NO: 121. ) Or an amino acid sequence having 70%, preferably 80%, more preferably 90% or more, still more preferably 95% or more, and even more preferably 98% or more identity with the amino acid sequence. Examples thereof include a protein having activity and a protein having protease activity comprising an amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 121.

  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)). More specifically, using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development), the unit size to compare (ktup) is set to 2 for analysis.

  Another aspect of the present invention relates to a method for producing a heterologous protein or polypeptide using the aforementioned recombinant Bacillus subtilis.

  The recombinant Bacillus subtilis of the present invention can be transformed into a Bacillus subtilis mutant strain that becomes a host by a general transformation method using a recombinant plasmid obtained by binding a DNA fragment containing the above heterologous protein or polypeptide gene and an appropriate plasmid vector. It is obtained by making it take in. Alternatively, the recombinant Bacillus subtilis of the present invention may be obtained by directly integrating the DNA fragment into the host genome using a DNA fragment in which an appropriate homologous region with the host genome is bound.

  Production of a heterologous protein or polypeptide using the above-mentioned recombinant Bacillus subtilis can be achieved by introducing a strain obtained by introducing the heterologous protein or polypeptide gene into a Bacillus subtilis mutant as a host as described above, for example, Inoculation can be performed by inoculating a medium containing a carbon source, a nitrogen source, and other essential components. In principle, the culture method may be a general method for culturing microorganisms, and is usually preferably performed under aerobic conditions such as shaking culture by liquid culture and aeration and agitation culture. After completion of the culture, the culture solution is centrifuged, and the desired heterologous protein or polypeptide can be obtained from the resulting supernatant or bacterial body by appropriately combining ammonium sulfate precipitation, chromatography, etc., and extraction and purification according to a conventional method. it can.

  In the following examples, a method for constructing the recombinant Bacillus subtilis of the present invention and a method for producing a protein using the recombinant Bacillus subtilis will be described, but the present invention is not limited to the following examples.

  The construction of a recombinant Bacillus subtilis strain from which the malA gene of Bacillus subtilis MGB874 strain has been deleted and the method for producing cellulase using the recombinant Bacillus subtilis will be specifically described below.

  Polymerase chain reaction (PCR) for DNA fragment amplification in the following examples is performed by GeneAmpl PCR System (Applied Biosystems) using Pyrobest DNA polymerase (Takara Bio) and attached reagents. It went by. The PCR reaction solution was prepared by adding 1 μL of appropriately diluted template DNA, 20 pmol each of sense and antisense primers, and 2.5 U of Pyrobest DNA polymerase, and further bringing the total reaction solution volume to 50 μL. PCR reaction conditions are 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), repeated 30 cycles. Then, the reaction was carried out at 72 ° C. for 5 minutes.

  Further, in the following examples, upstream and downstream of a gene do not refer to the position from the replication start point, but upstream refers to a region continuing on the 5 ′ side of the start codon of the target gene in each operation / step. On the other hand, the downstream refers to the region continuing 3 ′ of the stop codon of the gene of interest in each operation / step.

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

  Transformation of Bacillus subtilis was performed by the competent cell method (J. Bacteriol. 93, 1925 (1967)). That is, Bacillus subtilis strains were treated with 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. until the value of growth (OD600) is about 1. After shaking culture, after shaking culture, a part of the culture solution 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% glucose, 0.01% casamino acid (Difco), 5 mM magnesium sulfate, 0.4 μM manganese chloride, was inoculated into 5 [mu] g / mL tryptophan), by shaking culture until the value of the further degree of growth (OD600) of about 0.4, was prepared competent cells of B. subtilis strains.

  Next, 5 μL of a solution containing various DNA fragments (such as SOE-PCR reaction solution) 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, The entire amount was smeared on LB agar medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) containing the drug. After static culture at 37 ° C., the grown colonies were isolated as transformants. The genome of the obtained transformant was extracted and PCR was performed using this as a template to confirm that the target genome structure was altered.

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

(Example 1: Construction of malA gene deletion strain)
The malA gene (SEQ ID NO: 1) is a gene located at the beginning of the malA-glvR-malP operon, and when this gene is deleted, it does not affect the expression of glvR and malP, which are downstream genes of malA. The deletion strain must be constructed in Therefore, in the present invention, a malA gene-deficient strain was constructed by a method of replacing the structural gene of malA with the ORF (open reading frame) of the neomycin resistance gene. Using genomic DNA extracted from Bacillus subtilis MGB874 strain as a template, malAFW1 and malA / neof2. R and malA / neor2. Using each primer set of F and malA RV, a 1.0 kbp fragment adjacent to the upstream of the malA gene on the genome (A) and a 1.0 kbp fragment adjacent to the downstream (B) were prepared. Further, a neomycin resistance gene (C) was prepared using a plasmid pUB110 (Plasmid 15: 93-103. (1986)) having a neomycin resistance gene as a template and using the primer set of neof2 and neoor2 shown in Table 3. Next, the obtained 3 fragments of (A), (B) and (C) were mixed and used as a template, PCR was performed using primers of malA FW2 and malA RV2, and 3 fragments were converted into (A)-(C )-(B) in order, and a DNA fragment for gene deletion was obtained (see FIG. 1). Using this DNA fragment, the Bacillus subtilis strain 168 and MGB874 strain were transformed by the competent cell method described above, and colonies grown on an LB agar medium containing neomycin (10 mg / L) and maltose (1 g / L) Was isolated as a transformant. Since the expression of malA-glvR-malP operon is induced in the presence of maltose (J. bacteriol., 183, 17, 5110, (2001)), neomycin resistance gene is expressed in the transformant, and LB agar containing neomycin It can also grow on the medium.

  Genomic DNA was extracted from the obtained transformant, and PCR was performed using this as a template. As a result, it was confirmed that the malA gene was replaced with a neomycin resistance gene and a malA gene deletion strain was obtained. Among the malA-deficient strains obtained as described above, a strain constructed with 168 strains of Bacillus subtilis as the parent strain was designated as 168ΔmalA strain, and a strain constructed with the Bacillus subtilis MGB874 strain as the parent strain was designated as 874ΔmalA strain.

(Example 2: Cellulase productivity evaluation)
The alkaline cellulase gene was introduced into the 168ΔmalA strain, 874ΔmalA strain obtained in Example 1, and the parent strains, Bacillus subtilis 168 strain and MGB874 strain, as controls. Specifically, a DNA fragment (3.1 kb; SEQ ID NO: 3) encoding an S237 cellulase gene (see Japanese Patent Laid-Open No. 2000-210081) (SEQ ID NO: 3) derived from the Bacillus genus KSM-S237 strain (FERM BP-7875) 3 base numbers 13 to 3124) as a template, primer Egl-S237. F and primer Egl-S237. PCR was performed using the R primer set to construct a recombinant plasmid pHY-S237 inserted into the BamHI restriction enzyme cleavage point of the shuttle vector pHY300PLK and introduced into each strain by protoplast transformation.

  The obtained strain was cultured with shaking in 5 mL of LB medium at 30 ° C. for 15 hours, and further 0.6 mL of this culture solution was added to 30 mL of 2 × L-maltose medium (2% tryptone, 1% yeast extract, 1% chloride). Sodium, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline), and cultured with shaking at 30 ° C. for 3 days. At this time, the culture was performed three times for the purpose of calculating the measurement error. After culturing, the alkaline cellulase activity of the culture supernatant from which the cells were removed by centrifugation was measured, and the amount of alkaline cellulase secreted and produced outside the cells was calculated. Cellulase activity was measured by adding 0.4 mM p-nitrophenyl-β-D-cellotrioside (Seikagaku Corporation) to 50 μL of a sample solution appropriately diluted with 1 / 7.5 M phosphate buffer (pH 7.4, manufactured by Wako Pure Chemical Industries, Ltd.). The amount of p-nitrophenol released when reacted at 30 ° C. was quantified by the change in absorbance at 420 nm (OD 420 nm). Cellulase activity was defined as the amount of enzyme that liberates 1 μmol of p-nitrophenol per minute as 1 U. Comparison of productivity was performed based on a relative value with the productivity of the parent strain as 100% (Table 5: The values in the table indicate mean value ± standard deviation (N = 3)). As a result, when the MGB874 strain was used as the parent strain, a higher alkaline cellulase secretion production improvement effect was confirmed by malA deletion compared to the case where 168 strain was used as the parent strain.

Claims (9)

  profile6 region, profile1 region, profile4 region, PBSX region, profile5 region, profile3 region, spb region, pks region, skin region, pps region, profile2 region, ydcL-ydeK-ydhU region, ysB-yitD region, yunA-yurT region Bacillus subtilis mutation having genomic structure in which cgeE-ypmQ region, yeeK-yesX region, pdp-rocR region, ycxB-sipU region, SKIN-Pro7 region, sbo-ywhH region, yybP-yyaJ region and yncM-fosB region are deleted A recombinant Bacillus subtilis produced by introducing a gene encoding a heterologous protein or polypeptide into a strain, wherein the malA gene has been deleted or inactivated The recombinant Bacillus subtilis according to claim 1, wherein the Bacillus subtilis mutant has a genomic structure in which one or more of the following regions are further deleted:
ybbU-ybfI region, ydjM-cotA region, yefA-yesX region, yfiB-yfiX region, yhcE-yhcU region, yhaU-yhaL region, yjbX-yjlB region, xkdA-ykcC region, bpr-ylgD region, fpr-ylgA region, ynfF-ppsA region, yoxC-yobO region, spoVAF-spoIIAA region, spoIIIAH-yqhV region, ytvB-ytqB region, yteA-ytaB region, yuaJ-yugO region, yusJ-mrgA region, gerAA-yvrM region, gerAA-yvR region araE-yveK region, yvdE-yvcP region, gerBA-ywsC region, ywrK-ywqM region, spoIIID-ywoB region, slp-ylaF region, licH-sigY Pass, yqeF-yrhK region, yuzE-yukJ region and yncM-yndN region.   The one or more regions selected from a transcription initiation control region, a translation initiation control region, and a secretion signal region are operably linked upstream of a gene encoding a heterologous protein or polypeptide. Recombinant Bacillus subtilis.   The region of any one of claims 1 to 3, wherein three regions comprising a transcription initiation control region, a translation initiation control region, and a secretion signal region are operably linked upstream of a gene encoding a heterologous protein or polypeptide. The recombinant Bacillus subtilis described.   The secretory signal region is derived from a cellulase gene of a Bacillus bacterium, and the transcription initiation control region and the translation initiation control region are derived from a 0.6-1 kb region upstream of the cellulase gene. 5. The recombinant Bacillus subtilis according to 3 or 4. The three regions comprising the transcription initiation control region, the translation initiation control region, and the secretion signal region are either the base sequence of base numbers 1 to 659 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 3, or the base sequence and 90 % or more of a DNA fragment comprising the nucleotide sequence identity with the recombinant Bacillus subtilis according to claim 5, wherein.   The recombinant Bacillus subtilis according to any one of claims 1 to 6, wherein the gene encoding the heterologous protein or polypeptide is an alkaline cellulase gene of a Bacillus bacterium. The alkaline cellulase gene, cellulase gene consisting of the nucleotide sequence represented by SEQ ID NO: 3, or a DNA fragment consisting of a nucleotide sequence having the nucleotide sequence identity of 90% or more, recombinant Bacillus of claim 7, wherein Fungus.   A method for producing a heterologous protein or polypeptide using the recombinant Bacillus subtilis according to any one of claims 1 to 8.

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