JP2006211934A - Recombinant microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a recombinant microorganism obtained by introducing a gene encoding protein or polypeptide in host microorganism enabling increase of productivity of protein or polypeptide, and to provide a method for producing protein or polypeptide by using the recombinant microorganism. <P>SOLUTION: The recombinant microorganism is obtained by introducing a gene encoding dissimilar protein or polypeptide into a microorganism stain whose one or more genes of Bacillus subtilis genes ytcG, ypuH, yjbI, clpX, ykrB, ywgA, galE, yqfF, yghT, ylbO, ytqI, yjlC, yllB, yqeK, yjbH, ybbR, yrzD, ymcA, ylbG, yhzC, ykzF, ykoA, yebD and argH or a gene corresponding to the above-mentioned genes are deleted or inactivated. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

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

  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, pharmaceuticals, detergents, daily necessaries such as 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.

However, microorganisms originally have a wide variety of genes to cope with various environmental changes in nature, and in industrial production where limited production media and stable culture conditions are used, proteins or There are many gene expressions that are unnecessary for the production of polypeptides, and despite the above efforts, the production efficiency is not necessarily high.
Nature, 390,249,1997 Science, 277,1453,1997

  The present invention relates to a recombinant microorganism obtained by introducing a gene encoding a protein or polypeptide into a host microorganism capable of improving the productivity of the protein or polypeptide, and a protein or polypeptide using the recombinant microorganism. The object is to provide a manufacturing method.

  The inventors of the present invention have earnestly focused on a group of genes that are unnecessary or harmful to the production of useful proteins or polypeptides in various genes encoded on the microbial genome, particularly those that have unknown gene functions. After searching, a specific gene of a microorganism such as Bacillus subtilis is deleted or inactivated from the genome, and then a target protein or polypeptide is produced by introducing a gene encoding the target protein or polypeptide. It has been found that the sex is improved compared to before deletion or inactivation.

That is, the present invention is Bacillus subtilis genes ytcG, ypuH, yjbI, clpX, ykrB, ywgA, galE, yqfF, yqhT, ylbO, ytqI, yjlC, yllB, yqeK, yjbH, ybbR, yrzD, ymcA, ylbG, yhzC, ykzF, A group in which a gene encoding a heterologous protein or polypeptide is introduced into a microorganism strain in which any one of ykoA , yebD and argH , or any one of the genes corresponding to the gene is deleted or inactivated A replacement microorganism is provided.

  The present invention also provides a method for producing a protein or polypeptide using the recombinant microorganism.

  Since the microorganism of the present invention has a gene that is unnecessary or harmful to the production of the target protein or polypeptide has been deleted or inactivated, it suppresses waste of medium components such as energy loss, carbon source, and nitrogen source, and The target product can be efficiently produced by prolonging the production period of the protein or polypeptide because the unnecessary gene ceases to function.

  In the present invention, the identity of the amino acid sequence and the base sequence is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985). Specifically, it is calculated by performing an analysis with a unit size to compare (ktup) of 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

As a parent microorganism for constructing the microorganism of the present invention, a gene unnecessary for the production of a useful protein or polypeptide, specifically, a Bacillus subtilis gene shown in Table 1 or a gene corresponding to the gene is included. It may be wild type or mutated. Specifically, and Bacillus (Bacillus) bacteria such as Bacillus subtilis, Clostridium (Clostridium bacteria, or yeast, and among these bacteria belonging to the genus Bacillus are preferred. Furthermore, the whole genome information is revealed, genetic engineering, Bacillus subtilis is particularly preferred from the standpoint of the establishment of genome engineering technology and the ability to secrete and produce proteins outside the cells.

  Examples of the target protein or polypeptide produced using the microorganism of the present invention include proteins useful for foods, pharmaceuticals, cosmetics, detergents, fiber treatments, medical tests, etc. And polypeptides.

  In the present invention, the gene to be deleted or inactivated is any of the genes shown in Table 1 below present on the Bacillus subtilis genome and genes of microorganisms other than Bacillus subtilis corresponding to the gene. . It has been found by the present inventors that such a gene group is not directly involved in the production of the target protein or polypeptide and is unnecessary for the growth of microorganisms in a normal industrial production medium.

The names, numbers, functions, etc. of each gene in the table were reported by Kunst et al. (Nature, 390, 249-256, 1997) and published online on the JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) (http: //bacillus.genome.ad.jp/, March 2004
The data are based on Bacillus subtilis genome data updated on May 10).

Moreover, it has the same function as each gene of Bacillus subtilis shown in Table 1, or 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% in each gene and base sequence of Table 1. % Or more, particularly preferably 98% or more of the genes derived from other microorganisms, preferably derived from bacteria belonging to the genus Bacillus , are considered to be genes corresponding to the genes listed in Table 1, and are deleted in the present invention. Included in the gene to be inactivated. The nucleotide sequence identity is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985).

The gene list in Table 1 includes genes with known functions such as galE , argH , clpX , ykrB and ypuH in addition to genes with unknown functions.

Among these genes whose functions are known, deletion of galE (UDP-glucose 4-epimerase) or argH (argininosuccinate lyase) genes is expected to change sugar or amino acid metabolism in Bacillus subtilis cells. In addition, ypuH (or ypuG and smc genes predicted to have similar functions in the Bacillus subtilis genome) have been reported to be involved in the distribution of chromosomal DNA (EMBO J., 21, 3108, 2002). .

In addition, the clpX gene is a regulator of SigH regulon transcription activation (Mol. Microbiology, 37, 885, 2000), degradation of misfolded proteins (J. Bacteriol., 182, 3259, 2000), and late competence genes. It is known that it is involved in the regulation of the comK gene (J. Biochem., 133, 295, 2003). However, it has not been known so far that the deletion of these genes having known functions contributes to the improvement of protein or polypeptide productivity, and has been clarified by the inventors' research.

On the other hand, for the yqfF , yqhT and ylbO genes in the Bacillus subtilis gene list, the gene functions are all estimated in the Bacillus subtilis genome data (BSORF DB), but literature information on these genes has not been published. First, it is determined that the gene is classified as a function unknown gene as in the case of the function unknown genes other than those shown in Table 1.

  Deletion or inactivation of a gene selected from such a gene group does not cause expression of a gene that is unnecessary or harmful to the production of the protein or polypeptide. An improvement in the productivity of the protein or polypeptide is achieved.

  The number of genes to be deleted or inactivated may be 1 or more, more preferably 3 or more, and further preferably 5 or more. Furthermore, the construction of the microorganism of the present invention can be combined with deletion or inactivation of a gene group other than those described above and the enhancement of expression and function of genes other than those described above, which has a greater effect on productivity improvement. There is expected. In addition, the present invention can inactivate a target gene by a method such as inserting another DNA fragment into the target gene or giving a mutation to the transcription / translation initiation region of the gene. It is more desirable to physically delete the target gene.

  As a procedure for deletion or inactivation of gene groups, random deletion or inactivation mutation was given in addition to the method of systematically deleting or inactivating the target genes shown in Table 1. Thereafter, a method for evaluating protein productivity and performing gene analysis by an appropriate method may be mentioned.

  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. It is constructed by the method, and this is incorporated into the parent microbial cell, and two places outside the mutation site in the target gene of the parent microbial genome, or two places outside the target gene (preferably, one place each on the upstream side and downstream side) ), It is possible to replace the target gene on the genome with a deleted or inactivated gene fragment.

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

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

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

  The deletion DNA fragment used in this method is an approximately 0.5-3, preferably 0.5-1 kb fragment adjacent upstream of the gene to be deleted, and an approximately 0.5-3 kb, preferably 0.5-1 kb fragment adjacent downstream. A fragment into which a drug resistance marker gene fragment is inserted. First, an upstream fragment and a downstream fragment of a deletion target gene and three fragments of a drug resistance marker gene fragment are prepared by the first PCR. In this case, for example, upstream of the drug resistance marker gene is formed at the downstream end of the upstream fragment. A primer designed so that a 10-30 base pair sequence on the side and, on the contrary, a 10-30 base pair sequence downstream of the drug resistance marker gene is added to the upstream end of the downstream fragment is used (FIG. 1).

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

  When using a chloramphenicol resistance gene as a drug resistance marker gene, for example, using the primer set shown in Table 2, using a general PCR enzyme kit such as Pyrobest DNA polymerase (Takara Shuzo), etc. Protocols. Current Methods and Applications, Edited by BAWhite, Humana Press pp251, 1993, Gene, 77, 61, 1989) etc., by performing SOE-PCR under the usual conditions, etc., DNA fragments for deletion of each gene Is obtained.

  When the DNA fragment for deletion thus obtained is introduced into a cell by a competent method or the like, gene recombination occurs in the cell in the homologous region upstream and downstream of the identical deletion target gene, Cells in which the target gene is 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 2 was introduced, colonies growing on an agar medium containing chloramphenicol were isolated, and PCR was performed using the genome as a template. What is necessary is just to confirm that the target gene on the genome is replaced with the chloramphenicol resistance gene.

  Next, any of the Bacillus subtilis genes shown in Table 1 or one or more genes selected from the genes corresponding to the genes are deleted or inactivated, and the target protein or By introducing a gene encoding a polypeptide, the recombinant microorganism of the present invention can be obtained.

The target protein or polypeptide gene is not particularly limited, and includes various industrial enzymes such as detergents, foods, fiber treatments, feed treatments, cosmetics, pharmaceuticals, and diagnostic agents, and bioactive peptides. In addition, industrial enzymes are classified according to their functions: oxidoreductase, transferase, hydrolase, lyase, isomerase, and synthase (Ligase / Synthetase). ) And the like, and preferred examples include genes for hydrolases such as cellulase, α-amylase, and protease. Specifically, cellulases belonging to Family 5 are listed in the classification of polysaccharide hydrolases (Biochem. J., 280, 309, 1991), among which cellulases derived from microorganisms, particularly Bacillus bacteria. As a more specific example, an alkaline cellulase derived from a bacterium belonging to the genus Bacillus consisting of an amino acid sequence encoded by the nucleotide sequence of nucleotide numbers 573 to 3044 represented by SEQ ID NO: 1, or nucleotide numbers 610 to 3075 represented by SEQ ID NO: 3 Alkaline cellulase derived from Bacillus genus bacteria consisting of the amino acid sequence encoded by A cellulase consisting of an amino acid sequence having 98% or more identity can be mentioned.

Specific examples of α-amylase include α-amylase derived from microorganisms, and liquefied amylase derived from Bacillus bacteria is particularly preferable. As a more specific example, an alkaline amylase derived from a Bacillus bacterium comprising the amino acid sequence encoded by the base sequence of base numbers 162 to 1664 represented by SEQ ID NO: 5, or 70%, preferably 80%, An amylase having an amino acid sequence having an identity of 90% or more, more preferably 95% or more, particularly preferably 98% or more is more preferable. The amino acid sequence identity is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985). Specific examples of proteases include serine proteases and metalloproteases derived from microorganisms, in particular, Bacillus bacteria.

In addition, the target protein or polypeptide gene is upstream of the regulatory region involved in transcription, translation, and secretion of the gene, ie, the transcription initiation control region including the promoter and transcription initiation site, the ribosome binding site, and the initiation of translation including the initiation codon. It is desirable that at least one region selected from the region and the secretory signal peptide region is bound in an appropriate form. In particular, it is preferable that three regions consisting of a transcription initiation control region, a translation initiation control region and a secretion signal region are combined, and the secretion signal peptide region is derived from a cellulase gene of a bacterium belonging to the genus Bacillus, It is desirable that the translation initiation region that is 0.6-1 kb upstream from the cellulase gene is appropriately bound to the target protein or polypeptide gene. For example, the bacterium 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-2886) It is desirable that the cellulase gene, the transcription initiation control region, the translation initiation region, and the secretory signal peptide region of the cellulase gene are appropriately combined with 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 by SEQ ID NO: 1, the base sequence of base numbers 1 to 696 of the base sequence shown by SEQ ID NO: 3, or the base sequence 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more of a DNA fragment comprising a nucleotide sequence having identity or any of the above nucleotide sequences It is desirable that a DNA fragment consisting of a partially deleted base sequence is appropriately bound to the structural gene of the target protein or polypeptide. Here, a DNA fragment composed of a base sequence from which a part of the base sequence has been deleted means that a part of the base sequence has been deleted, but has functions related to gene transcription, translation and secretion. It means a retained DNA fragment.

  The recombinant microorganism of the present invention is obtained by incorporating a recombinant plasmid in which a DNA fragment containing the above target protein or polypeptide gene and an appropriate plasmid vector are combined into a host microorganism cell by a general transformation method. be able to. The recombinant microorganism of the present invention can also be obtained by using a DNA fragment in which an appropriate homologous region with the host microorganism genome is bound to the DNA fragment and directly integrating it into the host microorganism genome.

  In producing the target protein or polypeptide using the recombinant microorganism of the present invention, the strain is inoculated into a medium containing an assimilable carbon source, nitrogen source, and other essential components, and cultured by a normal microorganism culture method. After completion of the culture, the protein or polypeptide may be collected and purified.

  From the above, using any one of the Bacillus subtilis genes shown in Table 1, or a host microorganism mutant strain in which one or more genes selected from the genes corresponding to the gene are deleted or inactivated, and the mutant strain A recombinant microorganism can be constructed, and by using this, a useful protein or polypeptide can be produced efficiently. In addition, the deletion of the gene corresponding to the gene and the deletion or inactivation of other genes may be combined, the deletion of the gene corresponding to the gene and the enhancement of expression or function of the other gene. By combining these, recombinant microorganisms capable of efficiently producing useful proteins or polypeptides can be constructed.

In the following, focusing on the examples of the construction of a recombinant Bacillus subtilis strain lacking the ytcG gene (BG13833) of Bacillus subtilis, the method for constructing the recombinant microorganism of the invention, and the cellulase and amylase using the recombinant microorganism The production method will be specifically described.

Example 1
Using the genomic DNA extracted from Bacillus subtilis 168 as a template and using the ytcG-AF and ytcG-A / CmR and ytcG-B / CmF and ytcG-BR primer sets shown in Table 2, ytcG on the genome A 0.5 kb fragment (A) adjacent to the upstream of the gene and a 0.5 kb fragment (B) adjacent to the downstream were prepared. On the other hand, a 0.9 kb fragment containing a chloramphenicol resistance gene (C) using plasmid pC194 (J. Bacteriol., 158, 543, 1984) as a template and using the ytcG-CmF and ytcG-CmR primer sets shown in Table 2. ) Was prepared. Next, the obtained (A), (B), and (C) 3 fragments were mixed to form a template, SOE-PCR was performed using ytcG-AF and ytcG-BR primers, and the 3 fragments were converted to (A)-( The DNA fragments were bound in the order of C)-(B) to obtain a 1.9 kb DNA fragment (see FIG. 1). Using this DNA fragment, Bacillus subtilis 168 strain was transformed by a competent method, and colonies grown on LB agar medium containing chloramphenicol were isolated as transformants. Furthermore, the genomic DNA was extracted from the obtained transformant, and the ytcG gene was replaced with the chloramphenicol resistance gene by PCR using this as a template. confirmed.

Example 2
Similarly to Example 1, using each gene-AF and each gene-A / CmR shown in Table 2, and each gene-B / CmF and each gene-BR primer set, ypuH , yjbI on the genome , clpX, ykrB, ywgA, galE , yqfF, yqhT, ylbO, ytqI, yjlC and YllB 0.5kb fragment immediately upstream of each gene (a), and 0.5kb fragments adjacent to the downstream (B) was prepared. On the other hand, using plasmid pC194 (J. Bacteriol., 158, 543, 1984) as a template and using the gene-CmF and gene-CmR primer sets shown in Table 2, a 0.9 kb fragment containing the chloramphenicol resistance gene (C) was prepared. Next, the 3 fragments obtained (A), (B), and (C) were mixed to form a template, and SOE-PCR was performed using primers for each gene-AF and each gene-BR. -(C)-(B) were combined in this order to obtain a 1.9 kb DNA fragment (see FIG. 1). Using this DNA fragment, Bacillus subtilis 168 strain was transformed by a competent method, and colonies grown on LB agar medium containing chloramphenicol were isolated as transformants. Furthermore, genomic DNA of the obtained transformant was prepared, and it was confirmed by PCR using this as a template that the target gene was a gene deletion strain in which the chloramphenicol resistance gene was replaced.

Example 3
Using genomic DNA extracted from Bacillus subtilis 168 strain as a template, using each gene-AF and each gene-A / CmR and each gene-B / CmF and each gene-BR primer set shown in Table 3, YqeK , yjbH , ybbR , yrzD , ymcA , ylbG , yhzC , ykzF , ykoA , yebD and argH genes adjacent to the upstream of the 1.0 kb fragment (A), and the downstream 1.0 kb fragment to be deleted on the genome (B) was prepared respectively. Next, a 0.9 kb fragment (C) containing a chloramphenicol resistance gene was prepared using the plasmid pC194 as a template and the CmFW and CmRV primer sets shown in Table 3. Further, the obtained 3 fragments of (A), (B) and (C) were mixed to form a template, and SOE-PCR using each gene-AF and each gene-BR primer set was used to obtain 3 fragments as (A)- They were bound in the order of (C)-(B) to obtain a 2.9 kb DNA fragment (see FIG. 1). Using this DNA fragment, Bacillus subtilis 168 strain was transformed by a competent method, and colonies grown on LB agar medium containing chloramphenicol were isolated as transformants. Genomic DNA was extracted from the obtained transformant, and it was confirmed by PCR using this as a template that the target gene was a gene deletion strain substituted with a chloramphenicol resistance gene.

Example 4 (Evaluation of cellulase productivity)
Alkaline cellulase gene derived from Bacillus genus KSM-S237 strain (FERM BP-7875) to each gene deletion strain obtained in Examples 1 to 3 and Bacillus subtilis 168 strain as a control (JP 2000-210081) The recombinant plasmid pHY-S237 in which a DNA fragment (3.1 kb) encoding the publication was inserted into the shuttle vector pHY300PLK at the Bam HI restriction site was introduced by protoplast transformation. The obtained strain was shaken and 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, 7.5ppm manganese sulfate 4-5 hydrate, 15ppm tetracycline) and shake culture at 30 ° C for 3 days. 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. As a result, as shown in Table 4, when each gene-deficient strain was used as a host, a higher secretory production of alkaline cellulase was observed compared to the control 168 strain (wild type).

Example 5 (Amylase productivity evaluation strain)
Each gene deletion strain obtained in Examples 1 to 3, and Bacillus subtilis 168 strain as a control, shown in SEQ ID NO: 5 downstream of the promoter region and signal sequence region of the alkaline cellulase gene shown in SEQ ID NO: 1. Bacillus genus KSM-K38 strain (FERM BP-6946) -derived alkaline amylase gene (JP 2000-184882, Eur. J. Biochem., 268, 2974, 2001) mature enzyme region (Asp1-Gln480) the encoding DNA fragment (1.5 kb) the added was a DNA fragment (2.1 kb), introducing a recombinant plasmid pHY-K38 were inserted into Bgl II- Xba I restriction enzyme cleavage point of a shuttle vector pHY300PLK by protoplast transformation method did. The obtained strain was shaken and 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, 7.5ppm manganese sulfate 4-5 hydrate, 15ppm tetracycline) and shaking culture at 30 ° C for 7 days. After culturing, the alkaline amylase activity of the culture supernatant after removing the cells by centrifugation was measured, and the amount of alkaline amylase secreted and produced outside the cells was determined. As a result, as shown in Table 5, when each gene-deficient strain was used as a host, higher secretion of alkaline amylase was observed compared to the control 168 strain (wild type).

The preparation of a DNA fragment for gene deletion by SOE-PCR and a method for deleting a target gene (substitution with a drug resistance gene) using the DNA fragment are schematically shown.

Claims (7)

Bacillus subtilis genes ytcG, ypuH, yjbI, clpX, ykrB, ywgA, galE, yqfF, yqhT, ylbO, ytqI, yjlC, yllB, yqeK, yjbH, ybbR, yrzD, ymcA, ylbG, yhzC, ykzF, ykoA, yebD and argH Or a recombinant microorganism in which a gene encoding a heterologous protein or polypeptide is introduced into a microorganism strain in which any one or more of the genes corresponding to the gene is deleted or inactivated.   The recombinant microorganism according to claim 1, wherein the microorganism is Bacillus subtilis or other Bacillus bacterium.   The recombinant microorganism according to claim 1 or 2, wherein at least one region selected from a transcription initiation control region, a translation initiation control region and a secretion signal region is linked upstream of a gene encoding a heterologous protein or polypeptide.   The recombinant microorganism according to claim 3, wherein three regions comprising a transcription initiation control region, a translation initiation control region, and a secretion signal region are combined.   The recombinant microorganism according to claim 3 or 4, 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 are derived from a 0.6-1 kb region upstream of the cellulase gene. .   Three regions comprising a transcription initiation control region, a translation initiation control region, and a secretion signal region are derived from the base sequence of base numbers 1 to 659 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 1, and the base sequence represented by SEQ ID NO: 3. A DNA fragment comprising a base sequence of base numbers 1 to 696 of the cellulase gene or a base sequence having 70% or more identity with any of the base sequences, or a DNA comprising a base sequence from which a part of the base sequence has been deleted The recombinant microorganism according to claim 4, which is a fragment.   A method for producing a protein or polypeptide using the recombinant microorganism according to any one of claims 1 to 6.