JP5513759B2 - Method for producing protein or polypeptide - Google Patents

Description

  The present invention relates to a method for producing a useful protein or polypeptide, and a recombinant microorganism used in the production method.

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

In industrial production of useful substances by such microorganisms, improvement of productivity is one of the important issues, and breeding of produced bacteria by genetic techniques such as mutation has been performed as a technique. Particularly recently, with the development of microbial genetics and biotechnology, more efficient breeding of production bacteria using genetic recombination techniques has been carried out. Furthermore, in response to the rapid development of genome analysis technology in recent years, attempts have been made to decode genome information of target microorganisms and actively apply them to industry. Industrially useful host microorganisms whose genome information has been disclosed include Bacillus subtilis Marburg No. 168 (Non-patent Document 1), Escherichia coli K-12 MG1655 (Non-patent Document 2), Corynebacterium Corynebacterium glutamicum ATCC132032 and the like are mentioned, and strains with improved information have been developed using these genomic information.

The stringent response was first discovered as a phenomenon in which protein synthesis is suppressed in response to amino acid starvation, etc., but now it is a phenomenon that involves changes in various physiological activities such as cessation of cell division and inhibition of DNA replication. I know that there is. The molecules that play a central role in the stringency response are guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp). Since these two molecules are not functionally different, they are generally expressed as (p) ppGpp.
(p) It is known that an enzyme called RelA is involved in the synthesis of ppGpp. RelA exists on the ribosome and monitors the amino acid supply status through tRNA that is not aminoacylated. That is, when tRNA that is not aminoacylated increases due to amino acid starvation and binds to ribosome, RelA protein is activated and (p) ppGpp is synthesized from ATP and GDP / GTP. The synthesized (p) ppGpp regulates the activity of many enzymes related to transcription, translation and DNA replication as a global regulator. In E. coli, in addition to RelA that synthesizes (p) ppGpp, there is an enzyme called SpoT that mainly degrades (p) ppGpp, and the concentration of (p) ppGpp in the cell can be controlled by these two enzymes. It is known (Non-patent Document 3).

  In Bacillus subtilis, only the existence of RelA involved in both synthesis and degradation of (p) ppGpp was previously known, but recently, Nanamiya et al. Discovered new (p) ppGpp synthases, YwaC and YjbM. (Non-Patent Document 4). Therefore, it was found that in Bacillus subtilis, there are three enzymes, (p) ppGpp synthases YwaC and YjbM, and RelA that functions in both synthesis and degradation. However, it is unclear how these enzymes work together to regulate (p) ppGpp synthesis and control protein synthesis in stringent responses.

Nature, 390 (6657), 249-256, 1997 Science, 277 (5331), 1453-1462, 1997 J. Biol. Chem., 266 (9), 5980-5990, 1991 Mol. Microbiol., 67 (2), 291-304, 2008

  The present invention relates to a method for producing a target protein or polypeptide more efficiently and a recombinant microorganism used in the method.

The present inventors searched for genes that affect the production of useful proteins or polypeptides in various genes encoded on the microbial genome. In Bacillus subtilis, the gene for the enzyme RelA ( relA ) Was deleted or inactivated, the productivity of the target protein or polypeptide was found to be improved as compared to that before deletion or inactivation of the gene. In addition to the above-mentioned relA , the present inventors have further lost or inactivated the genes of the enzymes YwaC and YjbM ( ywaC and yjbM ) that are also related to stringent responses, and the productivity of the target protein or polypeptide Has been found to improve synergistically.

That is, the present invention provides the following.
(1) Culturing a recombinant microorganism obtained by deleting or inactivating the following gene and introducing a gene encoding the target protein or polypeptide, and then obtaining the target protein or polypeptide from the obtained culture A method for producing a protein or polypeptide comprising:
1) Bacillus subtilis gene relA or a gene corresponding to the gene; or 2) Bacillus subtilis gene relA or a gene corresponding to the gene, and a Bacillus subtilis gene ywaC or a gene corresponding to the gene and / or a Bacillus subtilis gene yjbM or the gene A gene corresponding to a gene.
(2) The production method according to (1), wherein one or more of a transcription initiation control region, a translation initiation control region, and a signal region for secretion are linked upstream of a gene encoding a target protein or polypeptide.
(3) The production method according to (2), wherein three regions comprising a transcription initiation control region, a translation initiation control region, and a secretory signal region are combined.
(4) The upstream signal region of 0.6 to 1 kb in length, wherein the secretory signal region is derived from a cellulase gene of a bacterium belonging to the genus Bacillus , and the transcription initiation control region and the translation initiation control region start from the initiation codon of the cellulase gene The production method according to (2) or (3), wherein the production method is derived.
(5) The three regions comprising the transcription initiation control region, the translation initiation control region, and the secretion signal region are represented by the base sequence of base numbers 1 to 659 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 1 and SEQ ID NO: 3. A nucleotide sequence of the cellulase gene consisting of a base sequence, a base fragment of base numbers 1 to 696, a DNA fragment comprising a base sequence having 70% or more identity with the base sequence, or a base sequence in which a part of the base sequence is deleted The production method according to any one of (2) to (4), which is a DNA fragment comprising:
(6) the microorganism is a Bacillus (Bacillus) bacteria (1) The method of producing according to any one of the - (5).
(7) The production method according to (6), wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis .
(8) The production method according to any one of (1) to (7), wherein the gene encoding the target protein or polypeptide is a cellulase gene.
(9) A recombinant microorganism used in the production method according to any one of (1) to (8).

  By the method for producing a protein or polypeptide using the recombinant microorganism of the present invention, the productivity of the target protein or polypeptide can be improved.

1 schematically shows a method for preparing a DNA fragment for gene deletion by SOE-PCR and a method for deleting a target gene (substitution with a drug resistance gene) using the DNA fragment.

Examples of genes that are deleted or inactivated in the present invention include the Bacillus subtilis gene relA . In addition to relA , the Bacillus subtilis gene ywaC or yjbM can also be subject to deletion or inactivation in the present invention. Table 1 below shows the gene numbers of relA , ywaC and yjbM . These are published in JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) (http://bacillus.genome.ad.jp/, updated on January 18, 2006).

70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, even more preferably 98% or more in identity with each of the genes described above, preferably A gene having a similar stringent response-related function (for example, (p) ppGpp synthesis or degradation) to each gene is considered to be a gene corresponding to the gene, and is included in the gene to be deleted or inactivated in the present invention. It is.
Amino acid sequence and base sequence identity can be calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it is calculated by performing an analysis with Unit size to compare (ktup) set to 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

Therefore, as a parent microorganism for constructing the recombinant microorganism used in the production method of the present invention, those having the gene relA of Bacillus subtilis or a gene corresponding to the gene, preferably the gene ywaC of Bacillus subtilis and Examples include one or both of yjbM , or those having a gene corresponding to the gene. These parent microorganisms may be wild type or mutated. Specific examples include bacteria belonging to the genus Bacillus, bacteria belonging to the genus Clostridium , yeast, etc. Among them, bacteria belonging to the genus Bacillus are preferable. 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 it has the ability to secrete and produce proteins outside the cells.

In order to produce a recombinant microorganism used in the production method of the present invention, the gene relA or a gene corresponding thereto is deleted or inactivated in the parent microorganism. Preferably, in addition to relA, further genes ywaC or gene equivalent thereto, and genes yjbM or either or both deletions or inactivation of a gene corresponding thereto. More preferably, all of relA or its corresponding gene, ywaC or its corresponding gene, and yjbM or its corresponding gene are deleted or inactivated. Furthermore, it is also possible to combine the mutation of the above gene group with modification for enhancing expression and function of other gene group (for example, prsA gene etc.) that can contribute to improvement of the productivity of the target protein or polypeptide. is there.

Examples of the microorganism in which the gene is deleted or inactivated include naturally occurring mutant strains and artificially created mutant strains. Examples of mutant strains of relA , ywaC and yjbM include RIK900, RIK908, RIK909, RIK913, RIK1001, and RIK1003 described in Mol. Microbiol., 67 (2), 291-304, 2008.

Artificial deletion or inactivation of a gene can be accomplished by removing part or all of the target gene from the genome or replacing it with another gene, inserting another DNA fragment into the gene, It can be performed by a method such as giving a mutation to the transcription / translation initiation region. Preferably, the gene is physically deleted. More specifically, a method for systematically deleting or inactivating relA , ywaC or yjbM , or a gene corresponding to the gene (hereinafter, target gene), and random gene deletion or inactivation mutation And a method of selecting a desired mutation by evaluating protein productivity and analyzing a gene by an appropriate method.

  In order to delete or inactivate the target gene, for example, a method by homologous recombination may be used. That is, a circular recombinant plasmid obtained by cloning a DNA fragment containing a part of the target gene into an appropriate plasmid vector is introduced into the parent microbial cell, and the parent gene is homologously recombined in a part of the target gene. It is possible to disrupt and inactivate target genes on the microbial genome. Alternatively, a target 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 target gene as shown in FIG. Constructed by the method, this is incorporated into the parent microbial cells to cause two crossover homologous recombination at two locations outside the mutation site in the target gene of the parent microbial genome, or upstream and downstream of the target gene Thus, it is possible to inactivate the target gene on the genome by deleting or replacing it with another gene fragment.

  For example, when Bacillus subtilis is used as a parent microorganism for constructing the microorganism used in the method of the present invention, there are already several reports on methods for deleting or inactivating a target gene by homologous recombination ( Mol. Gen. Genet., 223, 268, 1990, etc.), the microorganisms for the present invention can be obtained by repeating these methods.

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

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

  The deletion DNA fragment is, for example, a fragment in which a drug resistance marker gene fragment is inserted between an approximately 1.0 kb fragment adjacent to the upstream of the gene to be deleted and an approximately 1.0 kb fragment adjacent to the downstream. 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, and at this time, for example, upstream of the drug resistance marker gene at the downstream end of the upstream fragment. A primer designed so that a 10-30 base pair sequence on the side and, on the contrary, a 10-30 base pair sequence downstream of the drug resistance marker gene is added to the upstream end of the downstream fragment is used (FIG. 1).

  Next, using the 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 between the upstream fragment and the downstream fragment is obtained. (FIG. 1). The above PCR is shown in a book (PCR Protocols. Current Methods and Applications, Edited by BA White, Humana Press pp251, 1993, Gene, 77, 61, 1989) using a commercially available enzyme kit for PCR. It can be performed under normal conditions.

  When the DNA fragment for gene deletion thus obtained is introduced into a microbial cell by a conventional method such as a competent method, recombination in the cell occurs in the upstream and downstream homologous regions of the corresponding gene to be deleted. A cell in which the target gene is replaced with a drug resistance gene or a cell in which the drug resistance gene is inserted into the target gene can be generated (FIG. 1). This is separated by selection with a drug resistance marker. In other words, after culturing the gene-introduced microorganism on an agar medium containing the above drug and separating the colonies that grow, the target gene on the genome is replaced with the drug resistance gene by PCR method using the genome as a template. You should confirm.

  A DNA fragment containing the target protein or polypeptide gene is introduced into a microorganism in which the target gene thus obtained has been deleted or inactivated to produce the recombinant microorganism of the present invention. The DNA fragment is introduced by incorporating a recombinant plasmid combined with an appropriate plasmid vector into the microorganism using a general transformation method. The recombinant microorganism of the present invention can also be obtained by using a DNA fragment in which an appropriate homologous region with the genome of the microorganism is bound to the DNA fragment and directly integrating the DNA fragment into the genome of the microorganism.

  The target protein or target polypeptide to be introduced into the recombinant microorganism of the present invention is not particularly limited, and examples thereof include various industrial enzymes such as detergents, foods, fibers, feeds, chemicals, medicines, and diagnostics, and bioactive peptides. Can be mentioned. Industrial enzymes are preferred. As industrial enzymes, oxidoreductase (Oxidoreductase), transferase (Transferase), hydrolase (Hydrolase), elimination enzyme (Lyase), isomerase (Isomerase), synthase (Ligase / Synthetase) and the like. Preferable examples include hydrolases such as cellulase, α-amylase and protease, and transferases such as chloramphenicol acetyltransferase (CAT), and more preferable examples include cellulase.

Examples of the cellulase include cellulases belonging to Family 5 in the classification of polysaccharide hydrolase (Biochem. J., 280, 309, 1991), and among them, cellulases derived from microorganisms, particularly Bacillus bacteria. As a more specific example, an alkaline cellulase derived from the Bacillus bacterium strain KSM-S237 (FERM BP-7875) comprising the amino acid sequence represented by SEQ ID NO: 2 or a Bacillus bacterium comprising the amino acid sequence represented by SEQ ID NO: 4 Alkaline cellulase derived from KSM-64 strain (FERM BP-2886) or the amino acid sequence and 70%, preferably 80%, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more. A cellulase consisting of an amino acid sequence having identity is exemplified.

Specific examples of α-amylase include α-amylase derived from microorganisms, and particularly liquefied amylase derived from bacteria belonging to the genus Bacillus . More specific examples include alkaline amylase derived from Bacillus genus KSM-K38 strain (FERM BP-6946) consisting of the amino acid sequence represented by SEQ ID NO: 6, and 70%, preferably 80%, more preferably the amino acid sequence. Is an amylase consisting of an amino acid sequence having 90% or more, more preferably 95% or more, particularly preferably 98% or more.

Specific examples of proteases include serine proteases, metal proteases, and the like derived from microorganisms, in particular, Bacillus bacteria. More specific examples include alkaline protease derived from Bacillus clausii KSM-K16 strain (FERM BP-3376) comprising the amino acid sequence represented by SEQ ID NO: 8, and 70%, preferably 80% of the amino acid sequence. More preferred is a protease comprising an amino acid sequence having an identity of 90% or more, more preferably 95% or more, particularly preferably 98% or more.

Chloramphenicol acetyltransferase (CAT) is an enzyme that transfers the acetyl group of acetyl-CoA to the 3-position of chloramphenicol. Specifically, CAT derived from Staphylococcus aureus consisting of the amino acid sequence represented by SEQ ID NO: 10, and 70%, preferably 80%, more preferably 90% or more, still more preferably 95% or more, particularly preferably the amino acid sequence. Include proteins having 98% or more identity and having chloramphenicol acetyltransferase activity.

The target protein or polypeptide gene to be introduced into the recombinant microorganism used in the method of the present invention is upstream of the regulatory region involved in transcription, translation, and secretion of the gene, that is, a promoter and a transcription initiation site. It is desirable that one or more regions selected from a control region, a translation initiation region including a ribosome binding site and an initiation codon, and a secretory signal peptide region are bound in a proper 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 , and transcription It is desirable that the start region and the translation start region are upstream regions having a length of 0.6 to 1 kb starting from the start codon of the cellulase gene and are operably linked to the target protein or polypeptide gene. 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, translation initiation region, and secretory signal peptide region of the cellulase gene derived from 2886) be appropriately operably linked to the structural gene of the target protein or polypeptide.

  More specifically, the base sequence of base numbers 1 to 659 of the base sequence shown in SEQ ID NO: 1, the base sequence of base numbers 1 to 696 of the cellulase gene consisting of the base sequence shown in SEQ ID NO: 3, and the base sequence Or a DNA fragment comprising a nucleotide sequence having an identity of 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, or any of the above bases It is desirable that a DNA fragment consisting of a base sequence from which a part of the sequence has been deleted is operably linked to the structural gene of the target protein or polypeptide. 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.

  Production of the target protein or polypeptide by the method of the present invention is carried out by inoculating the above-mentioned recombinant microorganism into a medium containing an assimilable carbon source, nitrogen source and other essential components, and culturing by a normal microorganism culture method. After completion of the culture, the target protein or polypeptide may be collected and purified. A person skilled in the art appropriately selects the composition of the medium used for the culture and the culture conditions, and the procedure for collecting and purifying the target protein or polypeptide according to the type of microorganism to be used and the type of target protein or polypeptide. be able to.

As shown in Examples below, the productivity of the target protein or polypeptide in the method of the present invention using a recombinant microorganism lacking the relA gene alone or in combination with ywaC or yjbM Compared to the case of using microorganisms that had not been deleted or inactivated, this was improved. Further, when all of relA , ywaC and yjbM were deleted, productivity was dramatically improved.

  The following examples illustrate the invention in more detail.

Evaluation of alkaline cellulase secretion production
(p) Heterologous strains lacking ppGpp synthesis-related genes using RIK900, RIK908, RIK909, RIK913, RIK1001, RIK1003 (Mol. Microbiol., 67 (2), 291-304, 2008) and Bacillus subtilis 168 strain as a control Protein productivity was evaluated. The evaluation was carried out as follows using the productivity of alkaline cellulase derived from Bacillus bacteria consisting of the amino acid sequence represented by SEQ ID NO: 2 as an index. That is, an alkaline cellulase gene (JP-A 2000-210081) fragment (3.1 kb) derived from Bacillus sp. KSM-S237 strain (FERM BP-7875) represented by SEQ ID NO: 1 was subjected to polymerase chain reaction (PCR). ). In this PCR reaction, GeneAmp PCR System (Applied Biosystems) was used, and DNA amplification was carried out using Pyrobest DNA Polymerase (Takara Bio) and attached reagents. PCR reaction solution composition was 1 μL of appropriately diluted template DNA (genomic DNA of KSM-S237 strain), 237UB1 (5′-TGCGGATCCAACAGGCTTATATTTAGAGGAAATTTC: SEQ ID NO: 11), and 237DB1 (5′-TTGCGGATCCAACAACTCTGTGTCCAGTTATGCAAG: SEQ ID NO: 12) primers, respectively. 20 pmol and 2.5 U of Pyrobest DNA Polymerase were added to make the total reaction volume 50 μL. PCR reaction conditions were carried out by repeating the temperature change in three steps of 98 ° C. for 10 seconds, 55 ° C. for 30 seconds and 72 ° C. for 3 minutes 30 times, and then reacting at 72 ° C. for 5 minutes. The amplified DNA fragment was Bam HI restriction enzyme treatment, the recombinant plasmid pHY-S237 was inserted into Bam HI restriction enzyme cleavage point of a shuttle vector pHY300PLK, protoplast transformation method (Mol. Gen. Genet. 168, 111 (1979) ) To each strain. The recombinant strain obtained in this way was subjected to shaking culture overnight at 37 ° C. in 10 mL of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl), 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) is inoculated and cultured at 30 ° C for 3 days. went. 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 production of alkaline cellulase secreted per cell.
For the measurement of cellulase activity, add 50 μL of 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 Wako Pure Chemical Industries). The amount of p-nitrophenol liberated when mixed and reacted at 30 ° C. was quantified by the change in absorbance (OD420 nm) at 420 nm. The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was defined as 1 U.
The results of the alkaline cellulase activity measurement are shown in Table 2. When a single strain of ywaC and yjbM was used as the host, the secretion production of alkaline cellulase was not improved compared to the case of the control strain 168 (wild type), but relA alone and relA ywaC and relA When a double-deficient strain of yjbM was used, the secretion production of alkaline cellulase was improved by 127%, 121% and 126%, respectively. In addition, it was found that the secretory production of alkaline cellulase was improved by 237% when a triple-deficient strain of relA ywaC yjbM was used.

Claims (12)

A recombinant Bacillus bacterium obtained by deleting or inactivating the following gene and introducing a gene encoding the target protein or polypeptide is cultured, and the target protein or polypeptide is obtained from the obtained culture. A method for producing a protein or polypeptide comprising collecting the protein or polypeptide:
Bacillus subtilis gene relA or a gene having 90% or more identity in the nucleotide sequence and a stringent response-related function,
Bacillus subtilis gene ywaC or a gene having 90% or more identity in the base sequence, and a gene having a stringent response-related function, and Bacillus subtilis gene yjbM or having a nucleotide sequence of 90% or more in the base sequence, And a gene having a stringent response-related function. The production method according to claim 1, wherein three regions comprising a transcription initiation control region, a translation initiation control region, and a secretion signal region are linked upstream of a gene encoding a target protein or polypeptide. Secretion signal region are those derived from a cellulase gene of Bacillus bacteria, transcriptional initiation regulatory region and translational initiation regulatory region is derived from the upstream region from the start codon begins length 0.6~1kb of the cellulase genes The production method according to claim 2 . Three regions comprising a transcription initiation control region, a translation initiation control region, and a secretion signal region are derived from the base sequence of base numbers 1 to 659 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 1, and the base sequence represented by SEQ ID NO: 3. A base sequence of base numbers 1 to 696 of the cellulase gene or a base sequence having 90% or more identity with any of the base sequences and functions as a transcription initiation control region, a translation initiation control region and a secretion signal region The production method according to claim 2 or 3 , wherein the production method is a DNA fragment. The method of producing according to any one of claims 1 to 4 Bacillus bacterium is Bacillus subtilis. The production method according to any one of claims 1 to 5 , wherein the gene encoding the target protein or polypeptide is a cellulase gene. A recombinant Bacillus bacterium obtained by deleting or inactivating the following gene and introducing a gene encoding a target protein or polypeptide:
Bacillus subtilis gene relA or a gene having 90% or more identity in the nucleotide sequence and a stringent response-related function,
Bacillus subtilis gene ywaC or a gene having 90% or more identity in the base sequence, and a gene having a stringent response-related function, and Bacillus subtilis gene yjbM or having a nucleotide sequence of 90% or more in the base sequence, And a gene having a stringent response-related function. The recombinant Bacillus bacterium according to claim 7, wherein three regions comprising a transcription initiation control region, a translation initiation control region, and a signal region for secretion are linked upstream of a gene encoding a target 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 an upstream region having a length of 0.6 to 1 kb starting from the initiation codon of the cellulase gene. Item 9. A recombinant Bacillus bacterium according to Item 8. Three regions comprising a transcription initiation control region, a translation initiation control region, and a secretion signal region are derived from the base sequence of base numbers 1 to 659 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 1, and the base sequence represented by SEQ ID NO: 3. A base sequence of base numbers 1 to 696 of the cellulase gene or a base sequence having 90% or more identity with any of the base sequences and functions as a transcription initiation control region, a translation initiation control region and a secretion signal region The recombinant Bacillus bacterium according to claim 8 or 9, which is a DNA fragment. The recombinant Bacillus bacterium according to any one of claims 7 to 10, wherein the Bacillus bacterium is Bacillus subtilis. The recombinant Bacillus bacterium according to any one of claims 7 to 11, wherein the gene encoding the target protein or polypeptide is a cellulase gene.