JP5841749B2 - Recombinant microorganism - Google Patents

Description

  The present invention relates to a method for enhancing expression of a gene encoding a useful protein or polypeptide, and a method for producing a protein or polypeptide using the method.

  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.

  In recent years, about 4100 types of disrupted strains of genes present in the Bacillus subtilis genome have been extensively studied, and it has been reported that 271 genes are essential for growth (Non-patent Document 1). In addition, the present applicant has exhaustively analyzed gene-disrupted strains of Bacillus subtilis, and has succeeded in creating mutant strains excellent in productivity of various enzymes by deleting a large region of the Bacillus subtilis genome (patents). Reference 1).

  Glutamine and glutamic acid are considered to be extremely important compounds in nitrogen metabolism because they become nitrogen donors to all biological substances. In general, the glutamine synthesis pathway is known to be catalyzed by glutamine synthase, and GlnA plays a role in Bacillus subtilis (Non-patent Document 2).

  GlnA is encoded by the second gene of the glnRA operon, and the expression of this operon is suppressed by the transcriptional repression factor GlnR encoded by the first gene glnR of the glnRA operon (Non-patent Document 3). GlnR is a factor that suppresses transcription of the glnRA operon under conditions where the nitrogen source is abundant. On the other hand, under conditions where the nitrogen source is depleted, it is known that the global regulator TnrA in nitrogen metabolism of Bacillus subtilis functions as a transcriptional repressor of the glnRA operon.

  In addition, according to a report by Fisher et al., It has been found that the expression level of glnA is improved in a nitrogen-deficient condition and a nitrogen-depleted condition in a strain lacking a gene encoding GlnR and TnrA, respectively (non-patent document). Reference 4).

  There have been many reports on the functional modification of organisms by modifying the glutamine synthetase gene. For example, a method of reducing the odor of ammonia by highly expressing a glutamine synthetase gene of Bacillus natto (Patent Document 2), a negative regulator of glutamine synthetase gene (glutamine synthetase adenylyltransferase (GlnE protein) and glutamine synthetase regulation) A method of increasing the production of glutamine by deleting protein PII) (Patent Document 3), a method of increasing glutamine production by mutating the glutamine synthetase gene of coryneform bacteria (Patent Document 4), A method for eliminating growth inhibition by alanine / serine by introducing a glutamine synthetase gene (Patent Document 5), A method for increasing the growth rate and dry weight by highly expressing a glutamine synthase gene in plants (Patent Document 6) , Glutami for transformation of eukaryotic cells (CHO cells, etc.) Synthase gene utilizing (singles transformants corrective glutamine metabolism deficiency as markers) method (Patent Document 7) have been reported.

  However, there is no report on the relationship between the production of a recombinant protein or peptide by a gene recombinant and the expression level of glutamine synthase.

JP 2007-130013 A JP 2007-143467 A International Publication No. 2006/001380 Pamphlet International Publication No. 2007/074857 Pamphlet JP 2006-508633 A JP 2010-68805 A Special table 2009-504160

K. Kobayashi et al., Proc. Natl. Acad. Sci. USA, 100, 4678-4683, 2003 Gene, 32, 427 (1984) Mol. Microbiol. , 32, 223 (1999) J. et al. Bacteriol. , 188, 2578 (2006)

  The present invention relates to a method for enhancing expression of a gene encoding a useful protein or polypeptide, and a method for producing a protein or polypeptide using the method.

  The present inventors have found that production of a heterologous protein or polypeptide is markedly improved by increasing the expression level of the glnA gene in a host microorganism into which a gene encoding a heterologous protein or polypeptide is introduced. The present invention has been completed.

That is, the present invention relates to the following (1) to (14).
(1) A method for enhancing expression of a gene encoding a heterologous protein or polypeptide, comprising enhancing the expression of the glnA gene in a host microorganism into which the gene encoding the heterologous protein or polypeptide is introduced.
(2) The method according to (1) above, wherein the glnA gene expression is enhanced by deletion or inactivation of a transcriptional repression factor of the glnA gene.
(3) The method according to (2) above, wherein the transcription repressing factor is GlnR and / or TnrA.
(4) The gene expression enhancing method according to any one of (1) to (3), wherein the host microorganism is a bacterium belonging to the genus Bacillus.
(5) The method for enhancing gene expression according to (4), wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis.
(6) A method for producing a heterologous protein or polypeptide using a host microorganism into which a gene encoding a heterologous protein or heterologous polypeptide is introduced, characterized by enhancing the expression of the glnA gene. A method for producing a protein or heterologous polypeptide.
(7) The production method according to (6), wherein the glnA gene expression is enhanced by deletion or inactivation of a transcriptional repression factor of the glnA gene.
(8) The production method according to (7), wherein the transcription repressing factor is GlnR and / or TnrA.
(9) The above (6), 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. To (8). The production method according to any one of (8).
(10) From the above (6) to (8), 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 manufacturing method as described in any one of.
(11) The secretion 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. The method according to (9) or (10).
(12) 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 consisting of the base sequence represented by SEQ ID NO: 4, or the base sequence The production method according to the above (10), 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 is deleted.
(13) The production method according to any one of (6) to (12), wherein the host microorganism is a bacterium belonging to the genus Bacillus.
(14) The production method according to (13), wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis.

  Use of the recombinant Bacillus subtilis of the present invention makes it possible to efficiently mass-produce the target heterologous protein or polypeptide.

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 figure which shows the alkaline cellulase activity of the culture supernatant after culture | cultivation of 168 (DELTA) glnR, 168 (DELTA) tnrA strain | stump | stock, 168 (DELTA) glnR (DELTA) tnrA strain | stump | stock, and the parent strain Bacillus subtilis 168 strain | stump | stock.

  Examples of the host microorganism used in the present invention include Gram-positive bacteria such as Bacillus bacteria and Clostridium bacteria, with Bacillus bacteria being preferred. Furthermore, Bacillus subtilis is more preferable from the viewpoint that whole genome information has been clarified, genetic engineering and genome engineering techniques have been established, and that the protein has the ability to secrete and produce proteins outside the cells.

  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.

  The Bacillus subtilis used in the present invention is not particularly limited, but specifically, from the viewpoint of enhancing the expression level of a gene encoding a heterologous protein or heterologous polypeptide, specifically, Bacillus subtilis Marburg No. 168 (Bacillus subtilis 168 strain) or Bacillus subtilis MGB874 strain constructed by deleting a large region of the genome based on the strain (Patent Document 1).

  The glnA gene is a gene encoding glutamine synthase (GlnA), which is an enzyme that synthesizes glutamine from glutamic acid and ammonia using the energy of ATP. The glnA gene has been registered and published in Genbank, and for example, the glnA gene of Bacillus subtilis is Genbank Accession No. It is registered and published as NC_000964.

  In the present invention, the glnA gene having the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence are 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably 98% or more. And a gene encoding a protein having glutamine synthesis activity from glutamic acid is also considered to be a gene corresponding to the glnA gene, and is included in the glnA gene whose expression is enhanced in the present invention. In addition, the identity of an amino acid sequence and a base sequence can be calculated by Lipman-Pearson method (Science, 227, 1435 (1985)). Specifically, it is calculated by performing an analysis by setting Unit size to compare (ktup) as 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

  In the present invention, the method of enhancing the expression of glnA gene may be performed by increasing the copy number of glnA gene in the host microorganism cell. It may be performed by deletion or inactivation. In the latter case, the transcriptional repressor to be deleted or inactivated includes GlnR and TnrA, either one of which may be deleted or inactivated, or both of which may be deleted or inactivated. Good.

  GlnR is a factor that suppresses transcription of the glnRA operon under conditions where the nitrogen source is abundant, while TnrA is a global regulator that suppresses transcription of the glnRA operon under conditions where the nitrogen source is depleted. Therefore, it is preferable to delete or inactivate the glnR gene encoding GlnR under conditions rich in nitrogen sources to enhance the expression of the glnA gene, while under conditions depleted of nitrogen sources, it encodes TnrA. Preferably, the expression of the glnA gene is enhanced by deleting or inactivating the tnrA gene.

  In the present invention, the glnR gene having the base sequence of SEQ ID NO: 2 and the base sequence are 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and still more preferably 98% or more. The gene encoding a protein that is a factor that suppresses the transcription of the glnRA operon under the condition that the nitrogen source is abundant is also considered to be a gene corresponding to the glnR gene. Included in genes of transcriptional repressors that are deleted or inactivated to enhance expression.

  In the present invention, the tnrA gene having the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence are 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably 98% or more. And a gene that encodes a protein that is a global regulator that suppresses transcription of the glnRA operon under conditions where the nitrogen source is depleted, is also considered to be a gene corresponding to the tnrA gene. Included in genes of transcriptional repressors that are deleted or inactivated to enhance expression.

  As a specific example of deletion or inactivation of glnR and / or tnrA gene in the present invention, a deletion DNA fragment prepared by SOE (splicing by overlap extension) -PCR method (Gene, 77, 61 (1989)) Although the deletion method by the double crossing method used 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 1 or 2 and an appropriate template DNA, using a general PCR enzyme kit such as Pyrobest DNA Polymerase (Takara Shuzo), “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 1 or 2 is introduced, a colony that grows on an agar medium containing the drug is isolated, and the target gene is deleted and the drug resistance gene and The substitution may be confirmed by a PCR method using the genome as a template.

  A gene encoding a target heterologous protein or polypeptide is introduced into the host microorganism with enhanced glnA gene expression obtained as described above, and the gene encoding the heterologous protein or polypeptide is expressed. In this case, the expression of the gene encoding the heterologous protein or polypeptide is enhanced as compared with a host microorganism in which the expression of the glnA gene is not enhanced.

  A polypeptide generally refers to a polymer of amino acids linked in a straight chain, and a protein generally refers to one or more polypeptides composed of 50 or more amino acids. It shall be used interchangeably.

  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. As the expression vector, when Bacillus subtilis is used as the host microorganism, a vector capable of autonomous replication in Bacillus subtilis is preferable, and examples thereof include the shuttle vector pHY300PLK, but are not particularly limited. Alternatively, a recombinant Bacillus subtilis may be obtained by using a DNA fragment obtained by binding an appropriate homologous region with the host genome to the DNA fragment and directly integrating the DNA fragment into the host genome.

  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 consisting of the transcription initiation control region, the translation initiation control region and the secretion signal region are base sequences of base numbers 1 to 659 of the cellulase gene consisting of the base sequence represented by SEQ ID NO: 4, 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.

  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) having the amino acid sequence represented by SEQ ID NO: 5, and Bacillus bacterium KSM- having the amino acid sequence represented by SEQ ID NO: 6. Alkaline cellulase derived from 64 strains (FERM BP-2886), alkaline cellulase having an amino acid sequence in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 5 or 6 are deleted, substituted or added, Furthermore, a cellulase 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.

  Further, specific examples of α-amylase include microorganism-derived α-amylase, and in particular, liquefied amylase derived from Bacillus bacteria, and Bacillus bacteria KSM-K38 strain comprising the amino acid sequence represented by SEQ ID NO: 7 ( 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: 7. Specific examples of the protease include serine proteases derived from microorganisms, particularly bacteria derived from the genus Bacillus, metal proteases, and Bacillus clausii strain KSM-K16 (FERM BP-3376) comprising the amino acid sequence represented by SEQ ID NO: 8. ) 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, a protein having a 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: 8.

  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, the method of the present invention will be described, but the present invention is not limited to the following examples.

  Hereinafter, a method for constructing the recombinant microorganism of the present invention and a method for producing a protein using the recombinant microorganism will be specifically described.

  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 each of sense primer and antisense primer, and 2.5 U of Pyrobest DNA Polymerase to make the total reaction solution volume 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. The standard is 1 minute per 1 kb). After repeating, the reaction was carried out at 72 ° C. for 5 minutes.

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

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

  Transformation of Bacillus subtilis was performed by a 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. The cells were cultured with shaking at 37 ° C. in% glucose, 0.02% casamino acid (Difco), 5 mM magnesium sulfate, 0.25 μM manganese chloride, 50 μg / ml tryptophan until the growth degree (OD600) was 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% tricitrate citrate). Sodium dihydrate, 0.50% glucose, 0.01% casamino acid (Difco), 5 mM magnesium sulfate, 0.40 μM manganese chloride, 5 μg / ml tryptophan), and the growth degree (OD600) is A competent cell of Bacillus subtilis strain was prepared by shaking culture to about 0.4.

  Next, 5 μ 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, 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 stationary culture at 37 ° C., the grown colonies were isolated as transformants. The genome of the obtained transformant was extracted, and it was confirmed that the intended genomic structure was altered by PCR using this as a template.

  The plasmid expressing the protein of interest was introduced into the host microorganism by the protoplast transformation method (Mol. Gen. Genet., 168, 111 (1979)). For protein production by recombinant microorganisms, LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl), 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 a glnR-deficient strain glnR (SEQ ID NO: 1) is a gene located at the beginning of the glnR-glnA operon. When this gene is deleted, glnA (SEQ ID NO: 1) is a downstream gene of glnR. The deletion strain must be constructed in a manner that does not affect the expression of 2). Therefore, in this study, deletion strains were constructed by replacing the structural gene of glnR with the ORF (open reading frame) of the neomycin resistance gene. 1. Genomic DNA extracted from Bacillus subtilis strain 168 is used as a template, and each primer set of glnRFW1 and glnR / NmR and glnR / NmF and glnRRV shown in Table 1 is used to be adjacent to the upstream of the glnR gene on the genome. A 0 kbp fragment (A) and a downstream 1.0 kbp fragment (B) were prepared, respectively. Further, a neomycin resistance gene (C) was prepared using the plasmid pUB110 (Plasmid 15: 93-103. (1986)) having a neomycin resistance gene as a template and using the primer set of neof2 and neor2 shown in Table 1. Next, the 3 fragments obtained (A), (B), and (C) were mixed to form a template, PCR was performed using the primers for glnRFW2 and glnRRV2, and the 3 fragments were (A)-(C)-(B) The DNA fragments for gene deletion were obtained (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 neomycin (10 mg / L) were isolated as transformants. Genomic DNA was extracted from the resulting transformant, and it was confirmed that the glnR gene was replaced with the neomycin resistance gene by PCR using this as a template, resulting in a glnR gene deletion strain. The glnR deletion strain obtained as described above was designated as 168ΔglnR strain.

Example 2: Construction of tnrA deletion strain Using genomic DNA extracted from Bacillus subtilis 168 strain as a template, and using each of tnrAFW1 and tnrA / spR and tnrA / sp F and tnrARV primer sets shown in Table 2, A 1.0 kbp fragment (A) adjacent to the upstream of the above tnrA gene (SEQ ID NO: 3) and a 1.0 kbp fragment (B) adjacent to the downstream were respectively prepared. Further, a spectinomycin resistance gene (C) was prepared using the plasmid pDG1727 (Gene, 167, 335, (1995)) having a spectinomycin resistance gene as a template and using the spf and spr primer sets shown in Table 2. did. Next, the obtained (A), (B) and (C) 3 fragments were mixed and used as a template, PCR was performed using primers of tnrAFW2 and tnrARV2, and the 3 fragments were (A)-(C)-(B) The DNA fragments for gene deletion were obtained (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 (0.5 μg / mL) were isolated as transformants. Genomic DNA was extracted from the obtained transformant, and it was confirmed that the tnrA gene was replaced with a spectinomycin-resistant gene by PCR using this as a template, resulting in a tnrA gene-deficient strain. The tnrA deletion strain obtained as described above was designated as 168ΔtnrA strain.

Example 3 Construction of glnR, tnrA double deletion strain Using the transformation DNA obtained in Example 2, transformation of the 168ΔglnR strain obtained in Example 1 was carried out by a competent method, and spectinomycin ( Colonies that grew on the LB agar medium containing 0.5 μg / mL) were isolated as transformants. Genomic DNA was extracted from the obtained transformant, and it was confirmed that the tnrA gene was replaced with a spectinomycin-resistant gene by PCR using this as a template, resulting in a tnrA gene-deficient strain. The mutant strain obtained as described above was designated as 168ΔglnRΔtnrA strain.

Example 4 Cellulase Productivity Evaluation An alkaline cellulase gene was introduced into the 168ΔglnR, 168ΔtnrA strain, 168ΔglnRΔtnrA strain and the parent strain Bacillus subtilis 168 obtained in Example 1. Specifically, a DNA fragment (3.1 kb; SEQ ID NO: 4) encoding an S237 cellulase gene (see Japanese Patent Laid-Open No. 2000-210081) (SEQ ID NO: 4) derived from Bacillus genus KSM-S237 strain (FERM BP-7875) 4 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 shake-cultured in 5 mL of LB medium at 30 ° C. for 15 hours, and 0.6 mL of this culture solution was further added to 30 mL of 2 × L-maltose medium (2% tryptone, 1% yeast extract, 1% Sodium chloride, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline) was inoculated and subjected to shaking culture at 30 ° C. for 3 days (in this case, for the purpose of calculating measurement errors) The culture is performed 3 times). After culturing, the alkaline cellulase activity of the culture supernatant after removing the cells by centrifugation was measured, and the amount of the alkaline cellulase secreted and produced outside the cells was determined. For the measurement of cellulase activity, 0.4 mM p-nitrophenyl-β-D-cellotrioside (Seikagaku Corporation) 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). Was mixed, and the amount of p-nitrophenol released when the reaction was carried out at 30 ° C. was quantified by the change in absorbance at 420 nm (OD 420 nm). The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was set to 1 U, and productivity comparison was made based on relative values with the productivity of the parent strain being 100% (Table 4, values in the table are average values ± standard) Deviation (N = 3)). As a result, 168ΔglnR strain lacking glnR showed higher secretion of alkaline cellulase than the parent strain (FIG. 2).

Claims (6)

In Bacillus subtilis introduced with a gene encoding a heterologous protein or polypeptide, the gene encoding the heterologous protein or polypeptide is characterized by enhancing the expression of the glnA gene by deletion or inactivation of the glnR gene A method for enhancing gene expression. A method for producing a heterologous protein or polypeptide using Bacillus subtilis into which a gene encoding a heterologous protein or polypeptide is introduced, wherein the expression of the glnA gene is enhanced by deletion or inactivation of the glnR gene A method for producing the heterologous protein or polypeptide, characterized in that The production method according to claim 2 , wherein at least one region selected from a transcription initiation control region, a translation initiation control region and a secretion signal region is operably linked upstream of a gene encoding a heterologous protein or polypeptide. . The production method according to claim 2 , 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 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. 3. The production method according to 3 or 4 . The transcriptional initiation regulatory region, the translation initiation regulatory region and 3 regions consisting of secretion signal region, the nucleotide sequence of the nucleotide numbers 1 to 659 of cellulase gene comprising the nucleotide sequence represented by SEQ ID NO: 4, or the nucleotide sequence and 90% more a DNA fragment comprising the nucleotide sequence identity with, the production method according to claim 4 or 5, wherein

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