JP4832153B2 - Recombinant microorganism - Google Patents

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, 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. In recent years, the development of microbial genetics and biotechnology has led to more efficient breeding of production microorganisms using genetic recombination techniques, and the development of host microorganisms for genetic recombination has been promoted. It has been. For example, a strain obtained by further improving a microbial strain recognized as safe and excellent as a host microorganism, such as Bacillus subtilis Marburg No.168 strain, has been developed.

In addition, the phr gene ( phrA , phrE , phrF , phrG , phrl , phrK) is a gene involved in the intercellular signal transduction mechanism required when Bacillus subtilis senses changes in the external growth environment and makes various responses. Any of the above-mentioned overexpression or inactivation of rap genes ( rapA , rapE , rapF , rapG , rapl , rapK ) promotes sporulation of Bacillus subtilis and is secreted during the sporulation process (AprE ) And a useful protein production technique using a transcription control region (promoter) of the gene has been reported (see Patent Document 1).

However, in such a technique, it is considered that the productivity of the protease inherent in the host cell is increased, and the effect is hardly exerted when the target product is susceptible to degradation by the protease. In addition, when a microorganism in which spore formation is promoted is used for production, it takes a lot of energy and time to sterilize the microorganism at the end of production. For this reason, it is thought that the further improvement is needed for the use to the actual production of the above microorganisms.
International Publication No. 03/070963 Pamphlet

  The present invention relates to a recombinant microorganism obtained by introducing a gene encoding a target protein or polypeptide into a host microorganism capable of improving the productivity of the protein or polypeptide, a target protein using the recombinant microorganism, It aims at providing the manufacturing method of polypeptide.

  The present inventors have eagerly searched for genes that act unnecessary or harmful to the production of useful proteins or polypeptides in various genes encoded on the microbial genome. Surprisingly, Bacillus subtilis grows. After deleting or inactivating a specific gene or a gene corresponding to the gene in a group of genes involved in a cell-cell information transmission mechanism for sensing and responding to environmental changes, It has been found that when a gene encoding a protein or polypeptide is introduced, the productivity of the target protein or polypeptide is improved as compared with that before deletion or inactivation.

That is, the present invention provides a deletion or inactivation of at least one gene of Bacillus subtilis genes phrA , phrE , phrG , phrI , phrK , rapC , comX , comP or comQ , or a gene corresponding to the gene. The present invention relates to a recombinant microorganism in which a gene encoding a target protein or polypeptide is introduced into a converted microorganism strain.

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

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

  In the present invention, the identity of amino acid sequences and nucleotide sequences is 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).

The parent microorganism for constructing the microorganism of the present invention may be any gene as long as it has a gene involved in an intercellular information transmission mechanism, specifically, a Bacillus subtilis gene shown in Table 1 or a gene corresponding to the gene. More preferred are microorganisms having a “Quorum Sensing” mechanism. These 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 preferred. Furthermore, Bacillus subtilis is particularly preferred 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.

Genes involved in the intercellular signal transduction mechanism scattered on the Bacillus subtilis genome include Rap / Phr genes, such as phrA (BG10653), phrC (BG11959), phrE (BG11521), phrF (BG11960), phrG (BG11465) ), PhrI (BG12645), phrK (BG12646), rapA (BG10652), rapB (BG11965), rapC (BG11966), rapD (BG11967), rapE (BG11299), rapF (BG11968), rapG (BG11466), rapH (BG11031) ), RapI (BG12119), rapJ (BG12663), rapK (BG12664), comX (BG11324), comP (BG10380), comQ (BG10379), etc., but the subject of deletion or inactivity in the present invention Among these genes, one of the Bacillus subtilis genes shown in Table 1 below or a gene corresponding to the gene is selected. The inventors have found that such genes are not directly involved in the production of the protein or polypeptide of interest, and are not necessary for the growth of microorganisms in normal industrial production media. It was.

  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/, updated March 10, 2004), based on genome data of Bacillus subtilis.

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.

Among the genes of the present invention, the phrA gene product is processed outside the cell, then taken into the cell as a pentapeptide, and controls the phosphorylation of phosphoroyl Spo0F that transmits a sporulation initiation signal And is involved in the transmission of sporulation initiation signals (Proc. Natl. Acad. Sci. USA, 94, 8612, 1997). Similarly, the phrG gene product is also taken into cells as a pentapeptide, and it is reported that it binds to RapG protein that binds to phosphorylated DegU protein and participates in the transcriptional control of alkaline protease (AprE) gene (Mol). Microbiol., 49, 1685, 2003). The rapC gene product has then been shown to bind to the ComA protein and inhibit phosphorylated ComA DNA binding (Mol. Microbiol., 49, 1509, 2003). Furthermore, the comX gene product is activated as a decapeptide by the comQ gene product, and this activated decapeptide activates (phosphorylates) the ComP protein, which is a comP gene product present in the cell membrane (J. Bacteriol., 184, 410, 2002), it has been reported that transfer of a phosphate group from an activated ComP protein to a ComA protein leads to induction of transcription of the ComA regulon, and a signal for acquiring competence is transmitted into the cell (Cell, 77, 195). , 1994).

  It is considered that by deleting or inactivating the above gene, it is possible to construct a host cell lacking various intercellular signal transduction mechanisms related to changes in the growth environment.

  Here, the number of genes to be deleted or inactivated may be one or more, and deletion or inactivation of genes other than those described above can be combined. Furthermore, in addition to the deletion of the above gene group, expression enhancement and function enhancement of other gene groups can be combined, and a greater effect is expected for improving productivity. 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 the deletion or inactivation of such a gene, in addition to the method of systematically deleting or inactivating the target gene shown in Table 1, random gene deletion or inactivation mutation was given. 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. 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 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. The gene deletion method in the present invention is not limited to the following.

  The deletion DNA fragment used in this method is a fragment in which a drug resistance marker gene fragment is inserted between the approximately 1.0 kb fragment adjacent to the upstream of the deletion target gene and the approximately 1.0 kb fragment adjacent to the downstream. . 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 3 types of PCR fragments prepared in the first round as a template, and performing the second round of PCR using the upstream primer of the upstream fragment and the downstream primer of the downstream fragment, the downstream end of the upstream fragment and the downstream fragment In the drug resistance marker gene sequence added to the upstream end, annealing occurs with the drug resistance marker gene fragment, and as a result of PCR amplification, a DNA fragment in which the drug resistance marker gene is inserted between the upstream fragment and the downstream fragment Can be obtained (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, or cells in which the drug resistance gene is inserted into the target gene can be separated by selection with a drug resistance marker (FIG. 1). That is, when a deletion (inactivation) DNA fragment prepared using the primer set shown in Table 2 was introduced, colonies growing on an agar medium containing chloramphenicol were isolated, and the genome was used as a template. The target gene on the genome may be replaced with the chloramphenicol resistance gene by the PCR method or the like, or it may be confirmed that the chloramphenicol resistance gene is inserted into the target gene.

Proteins and polypeptides produced using the above-mentioned host microorganism mutants are not particularly limited, and various industrial enzymes such as detergents, food processing, fiber processing, feed processing, cosmetics, pharmaceuticals, diagnostics, etc. And physiologically active 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 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 liquefied amylase derived from Bacillus bacteria is particularly preferable. 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. 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. 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.

In addition, the target protein or polypeptide gene is upstream and includes a control region related to transcription, translation and secretion of the gene, that is, a transcription initiation control region including a promoter and a transcription initiation site, a ribosome binding site and a start codon. It is desirable that one or more regions selected from the initiation region and the 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 Bacillus bacteria, It is desirable that the translation initiation region is a 0.6-1 kb region upstream of the cellulase gene to be bound to the target protein or polypeptide gene in an appropriate form. 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 transcription initiation regulatory region, translation initiation region, and secretory signal peptide region of the cellulase gene are appropriately bound 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 the identity of 70% or more, preferably 80% or more, more preferably 90% or more, further 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 is deleted is appropriately bound 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.

  The recombinant microorganism of the present invention can be obtained by incorporating a recombinant plasmid obtained by binding a DNA fragment containing the target protein or polypeptide gene and an appropriate plasmid vector into a host microorganism cell by a general transformation method. Obtainable. 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.

  Production of the target protein or polypeptide using the recombinant microorganism of the present invention is carried out by inoculating the strain in a medium containing an assimilable carbon source, nitrogen source and other essential components, and cultivating it by a normal microorganism culture method. Then, after completion of the culture, the protein or polypeptide may be collected and purified.

The following is a method for constructing a recombinant microorganism according to the present invention and a method for producing cellulase using the recombinant microorganism, with a focus on examples of construction of a recombinant Bacillus subtilis strain in which the phrA gene (BG10653) of Bacillus subtilis is inactivated. Will be described in detail.

Example 1
Using genomic DNA extracted from Bacillus subtilis 168 as a template and using each of the phrA-AF and phrA-A / CmR and phrA-B / CmF and phrA-BR primer sets shown in Table 2, phrA on the genome A 1.0 kb fragment adjacent to the upstream of the gene (A) and a 1.0 kb fragment adjacent to the downstream (B) were prepared. On the other hand, a 0.9 kb fragment (C) containing the chloramphenicol resistance gene was prepared using plasmid pC194 (J. Bacteriol., 158, 543, 1984) as a template and using the CmFW and CmRV primer sets shown in Table 2. . Next, the 3 fragments obtained (A), (B), and (C) were mixed to form a template, SOE-PCR was performed using phrA-AF and phrA-BR primers, and the 3 fragments were (A)-( The DNA fragments 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. Furthermore, genomic DNA is extracted from the obtained transformant, and the gene inactivation in which the chloramphenicol resistance gene is inserted into the structural gene (ORF) of the phrA gene by PCR using this as a template It was confirmed that it was a stock.

Example 2
On the other hand, deletion was carried out using the primer sets (gene name-AF and gene name-A / CmR, gene name-B / CmF and gene name-BR, CmFW and CmRV) shown in Table 2 in the same manner as in Example 1. DNA fragments are prepared for each phrE gene (BG11521), phrG gene (BG11465), phrI gene (BG12645), phrK gene (BG12646), rapC gene (BG11966), comX gene (BG11324), comP gene ( BG10380), comQ gene (BG10379) partially replaced with chloramphenicol resistance gene, or chloramphenicol resistance gene inserted into structural gene (ORF) Activated strain). Furthermore, it was confirmed by the same method as in Example 1 that the obtained mutant strain was the target gene deletion strain (inactivated strain).

Example 3 (Evaluation of cellulase productivity)
To each of the gene-deleted strains, gene-inactivated strains obtained in Examples 1 and 2, and 168 strains of Bacillus subtilis as a control, an alkaline cellulase gene derived from the Bacillus genus KSM-S237 strain (FERM BP-7875) (special A recombinant plasmid pHY-S237 in which a DNA fragment (3.1 kb) encoding Kai 2000-210081 was inserted at the Bam HI restriction enzyme cleavage point of the shuttle vector pHY300PLK was introduced by protoplast transformation. The resulting strain was subjected to shaking culture 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 3, when each gene-deleted strain and inactivated strain were used as the host, higher secretory production of alkaline cellulase was observed compared to the control 168 strain (wild type). It was.

Example 4 (Protease productivity evaluation)
To each of the gene deletion strains, gene inactivation strains, and Bacillus subtilis 168 strains obtained in Examples 1-2, an alkaline protease gene derived from Bacillus clausii KSM-K16 strain (FERM BP-3376) (Appl. Microbiol. Biotechnol., 43, 473, 1995) DNA fragment (1.3 kb) is a promoter region of an alkaline cellulase gene (JP 2000-210081) derived from Bacillus genus KSM-S237 strain (FERM BP-7875) the DNA fragment was ligated downstream of DNA encoding fragment (0.6 kb), a shuttle vector pHY300PLK the Bam HI- Bgl II recombinant plasmid pHYKAP inserted into the restriction enzyme cleavage point (S237p) was introduced at the protoplast transformation method . The transformant thus obtained is cultured under the conditions shown in Example 3, and after culturing, the alkaline protease activity of the culture supernatant obtained by removing the cells by centrifugation is measured and secreted outside the cells. The amount of alkaline protease produced was determined. As a result, as shown in Table 4, when each gene-deleted strain and inactivated strain were used as the host, higher secretion of alkaline protease was observed compared to the control 168 strain (wild type). It was.

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 (8)

The target protein or microbial strain in which at least one of the Bacillus subtilis genes phrA , phrI, phrK, comX, comP or comQ or any one of the genes corresponding to the gene is deleted or inactivated A recombinant microorganism into which a gene encoding a polypeptide is introduced.   The recombinant microorganism according to claim 1, wherein the microorganism is a Bacillus bacterium.   The recombinant microorganism according to claim 2, wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis.   The group according to any one of claims 1 to 3, wherein any one or more of 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. Replacement microorganism.   The recombinant microorganism according to claim 4, wherein three regions comprising a transcription initiation control region, a translation initiation control region, and a secretion signal region are combined.   The secretion 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. The recombinant microorganism described. 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: A DNA fragment comprising a base sequence of base numbers 1 to 696 of the cellulase gene or a base sequence having 80 % 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 any one of claims 4 to 6, which is a fragment.   The manufacturing method of the target protein or polypeptide using the recombinant microorganism of any one of Claims 1-7.

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