JP4509743B2 - 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 range of types including foods such as alcoholic beverages, miso and soy sauce, and amino acids, organic acids, nucleic acid-related substances, antibiotics, carbohydrates, lipids, and proteins. 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.

  However, microorganisms originally have a wide variety of genes for coping with environmental changes in nature, and are necessarily efficient in industrial production of proteins and the like that use a limited production medium. It was a situation that could not be said.

  The present invention finds a host microorganism capable of improving the productivity of a protein or polypeptide, introduces a recombinant microorganism obtained by introducing a gene encoding the protein or polypeptide into the host microorganism, and further provides a protein or a protein using the recombinant microorganism. It aims at providing the manufacturing method of polypeptide.

  The present inventors have eagerly searched for a group of genes that act unnecessary or harmful to the production of useful proteins or polypeptides in various genes encoded on the microbial genome. After deleting or inactivating a specific gene involved in permeation of maltose used as a source or a gene corresponding to the gene from the genome, the gene encoding the target protein or polypeptide is introduced and the target protein is introduced Or when the polypeptide was produced, it discovered that the productivity improved compared with before deletion or inactivation.

That is, the present invention relates to a microbial mutant in which one or more genes involved in maltose membrane permeation, in particular, any one or more genes of glvR or glvC or a gene corresponding to the gene are deleted or inactivated. Furthermore, the present invention provides a recombinant microorganism into which a gene encoding a heterologous protein or polypeptide is introduced, and a method for producing a protein or polypeptide using the recombinant microorganism.

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

In the present invention, the identity of the amino acid sequence and the base sequence is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). More specifically, it is calculated by performing a unit size to compare (ktup) of 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 permeation of maltose, specifically a Bacillus subtilis gene shown in Table 1 or a gene corresponding to the gene. When culturing using a medium containing maltose or maltooligosaccharide as a main carbon source, a microorganism having another maltose permeation system not involving the gene is desirable. These may be wild-type or mutated. Specifically, and Bacillus (Bacillus) bacteria such as Bacillus subtilis, Clostridium (Clostridium) bacteria, or yeast, and among these Bacillus bacterium is preferable. 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 protein and secretory production are possible.

  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.

  The gene group to be deleted or inactivated in the present invention is a gene involved in permeation of maltose and is selected from any of the Bacillus subtilis genes shown in Table 1 or a gene corresponding to the gene. It is what is done.

The name, number, function, etc. of each gene in the table were reported in Nature, 390, 249-256, (1997) and published on the Internet at JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) (http: //Bacillus.genome.ad.jp/, updated June 17, 2003), 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. %, Particularly preferably 98% or more of the genes derived from other microorganisms, preferably from Bacillus bacteria, 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.

Such a gene is a so-called phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS, J. Mol. Microbiol. Biotechnol., 4, 37, which is involved in membrane permeation when maltose in a medium is taken into cells. (2002)). More specifically, a gene corresponding glvR gene, or they encode positive regulators of glv operon containing glvC gene, and glvC gene encoding maltose-specific transmission enzyme IICB of PTS. It is expected that deletion or inactivation of any of these genes will reduce the cell's ability to permeate maltose membranes. Surprisingly, an enzyme using a medium containing maltose as the main carbon source For the first time by the present inventors, it is possible to achieve a significant improvement in protein productivity compared to the case of using a normal host by using a host microorganism in which the gene is deleted or inactivated in production culture. It was found.
In addition, ptsH (BG10200) and ptsI gene (BG10201) are known to be involved in the incorporation of maltose into cells by PTS, but glcT is a regulatory gene essential for the expression of the pts operon containing these two genes. Since it is possible to improve protein productivity by deleting or inactivating the gene (BG12593), it is considered that deletion or inactivation of either ptsH or ptsI is also effective for improving productivity. It is done.

  In the present invention, it can also be achieved by inactivating the target gene by a method such as inserting another DNA fragment into the above gene or giving a mutation to the transcription / translation initiation region of the gene. It is more desirable to physically delete the target gene. Further, the number of genes to be deleted or inactivated may be one or more, and two or more deletions may be combined. Furthermore, the construction of the microorganism of the present invention can be combined with the deletion or inactivation of genes other than those involved in permeation of maltose, which is expected to have a greater effect on productivity.

  As a procedure for gene deletion or inactivation, in addition to the method of systematically deleting or inactivating the target genes shown in Table 1, random gene deletion or inactivation mutations are applied, The target gene group can also be deleted or inactivated by evaluating protein productivity and performing gene analysis by various methods.

  For example, a method by homologous recombination may be used to delete or inactivate the target gene. That is, a circular recombinant plasmid obtained by cloning a DNA fragment containing a part of the target gene into an appropriate plasmid is incorporated into the parent microorganism cell, and homologous recombination in a partial region of the target gene is performed. It is possible to disrupt and inactivate target genes on the genome. Alternatively, a target gene into which an inactivating mutation has been introduced by base substitution or base insertion, or a linear DNA fragment containing the outer region of the target gene but not including the target gene is constructed by a method such as PCR. Of the target gene on the genome by causing two homologous recombination to occur in the two regions outside the target gene mutation site of the parent microorganism genome or two regions outside the target gene. It is also possible to replace with a deleted or inactivated gene fragment.

In particular, when Bacillus subtilis is used as a parent microorganism for constructing the microorganism of the present invention, there have already been several reports on methods for deleting or inactivating target genes 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, for deletion or inactivation of random genes, a method of causing homologous recombination similar to the above method using a randomly cloned DNA fragment, or irradiation of parental microorganisms with γ rays, etc. It can be implemented.

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

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. Fragment. 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 10-30 base pair sequence, and conversely, a downstream 10-30 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 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 a primer set shown in Table 2 and an appropriate template DNA, using a general PCR enzyme kit such as Pyrobest DNA Polymerase (Takara Shuzo), etc. By performing SOE-PCR under the usual conditions shown in the book (PCR Protocols. Current Methods and Applications, Edited by BAWhite, Humana Press, pp251 (1993), Gene, 77, 61, (1989), etc.) A DNA fragment for deletion of each gene is obtained.

  When the DNA fragment for deletion thus obtained is introduced into the cell by a competent method or the like, gene recombination occurs in the cell in the homologous region upstream and downstream of the identical gene to be deleted, and the target 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 the target gene was deleted and chloramphenic The replacement with the call resistance gene may be confirmed by a PCR method using the genome as a template.

  Next, a target protein or a target microorganism mutant in which one or more genes selected from the genes corresponding to Bacillus subtilis shown in Table 1 or a gene corresponding to the gene are deleted or inactivated 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, fibers, feeds, chemicals, medicines, diagnostics, bioactive peptides, and the like. In addition, the functions of industrial enzymes are classified into oxidoreductase, transferase,
It includes hydrolase (Hydrolase), elimination enzyme (Lyase), isomerase (Isomerase), synthetic enzyme (Ligase / Synthetase), etc., but preferably hydrolase such as cellulase, α-amylase, protease, etc. Gene. Specifically, cellulases belonging to Family 5 are listed in the classification of polysaccharide hydrolases (Biochem. J., 280, 309 (1991)), and among them, cellulases derived from microorganisms, particularly from Bacillus bacteria. As a more specific example, an alkaline cellulase derived from a Bacillus bacterium comprising the amino acid sequence represented by SEQ ID NO: 2 or 4, or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added Furthermore, 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. A cellulase consisting of

  Specific examples of α-amylase include α-amylase derived from microorganisms, and liquefied amylase derived from bacteria belonging to the genus Bacillus is particularly preferable. As a more specific example, an alkaline amylase derived from a Bacillus bacterium comprising the amino acid sequence represented by SEQ ID NO: 22, or 70%, preferably 80%, more preferably 90% or more, and still more preferably 95% with the amino acid sequence. As mentioned above, amylase consisting of an amino acid sequence having an identity of 98% or more is particularly preferred. The amino acid sequence identity is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specific examples of proteases include serine proteases, metal proteases, and the like derived from microorganisms, particularly from 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 in the 0.6-1 kb region upstream of the cellulase gene is 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 an 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 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. The components and composition of the medium are not particularly limited, but preferably better results can be obtained by using a medium containing maltose or malto-oligosaccharide as a carbon source.

  As described above, any one 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 mutant strains are used. Thus, a recombinant microorganism can be constructed, and by using this, a useful protein or polypeptide can be efficiently produced.

Examples of construction of a recombinant Bacillus subtilis strain from which the Bacillus subtilis glvC gene (BG11848) or glvR gene (BG11847) has been deleted and the production method of cellulase or α-amylase using the recombinant microorganism are specifically described below. To do.

Example 1
Using genomic DNA extracted from Bacillus subtilis strain 168 as a template, and using each primer set of glvC- AF and glvC- A / CmR and glvC- B / CmF and glvC- BR shown in Table 2, glvC on the genome A 0.5 kb fragment adjacent to the upstream of the gene (A) and a 0.5 kb fragment adjacent to the downstream (B) were prepared, respectively. On the other hand, using plasmid pC194 (J. Bacteriol. 150 (2), 815 (1982)) as a template and the glvC-A / CmF and glvC-B / CmR primer sets shown in Table 2, resistance to chloramphenicol A 0.9 kb fragment (C) containing the gene was prepared. Next, 3 fragments obtained by mixing the obtained (A), (B), and (C) 3 fragments as a template and performing SOE-PCR using the primers glvC- AF and glvC- BR in Table 2 were obtained. A) A DNA fragment of 1.9 kb was obtained by binding in the order of (C) and (B) (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. The genome of the obtained transformant was extracted, and it was confirmed that the glvC gene was deleted by PCR and replaced with a chloramphenicol resistance gene.

Example 2
As in Example 1, using the primer sets glvR-AF and glvR-A / CmR and glvR-B / CmF and glvR-BR shown in Table 2, 0 adjacent to the upstream of the glvR gene on the genome. A 0.6 kb fragment (A) and a downstream adjacent 0.6 kb fragment (B) were prepared, respectively. On the other hand, Table 2 shows a recombinant plasmid pCBB31 in which the chloramphenicol resistance gene of plasmid pC194 (J. Bacteriol. 150 (2), 815 (1982)) was inserted into the plasmid pUC18 at the XbaI-BamHI breakpoint. A 0.9 kb fragment (C) containing a chloramphenicol resistance gene was prepared using the primer sets glvR-A / CmF and glvR-B / CmR. Next, 3 fragments obtained by mixing the obtained 3 fragments (A), (B) and (C) as a template and performing SOE-PCR using the primers glvR- AF and glvR- BR in Table 2 were obtained. A) (C) and (B) were combined in this order to obtain a 2.2 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. The genome of the obtained transformant was extracted, and it was confirmed that the glvR gene was deleted by PCR and replaced with a chloramphenicol resistance gene. Similarly, using the glcT-AF and glcT-A / CmR and gclT-B / CmF and glcT-BR primer sets, isolate the transformants in which the glcT gene has been deleted and replaced with chloramphenicol. did.

Example 3
Each of the gene-deleted strains obtained in Examples 1 and 2 and Bacillus subtilis strain 168 as a control, an alkaline cellulase gene derived from Bacillus sp. KSM-S237 strain (JP 2000-210081, sequence) Number 1) Fragment (3.1 kb) is shuttle vector pHY30
Recombinant plasmid pHY-S237 inserted into the 0PLK BamHI restriction site was introduced by protoplast transformation. The resulting strain was shaken and cultured in 5 mL of LB medium at 30 ° C. overnight, and 0.03 mL of this culture was added to 30 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), followed by shaking culture at 30 ° C. for 3 days. After the cultivation, 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 by the cultivation was determined. As a result, as shown in Table 3, in all cases where the spore-forming gene deleted strain was used, high secretory production of alkaline cellulase was observed as compared to the control 168 strain (wild type).

Example 4
Each gene-deleted strain obtained in Example 1-3, and Bacillus subtilis 168 strain as a control, an alkaline amylase gene derived from Bacillus sp. KSM-K38 strain (JP 2000-184882, Eur J. Biochem., 268, 2974 (2001)), the promoter region and signal sequence of the alkaline cellulase gene shown in SEQ ID NO: 3 upstream of the DNA fragment (1.5 kb) encoding the mature enzyme region (Asp1-Gln480). Recombinant plasmid pHSP-K38 in which a 2.1 kb fragment (SEQ ID NO: 21) formed by ligating an upstream 0.6 kb fragment containing a part of the region was inserted into the BglII-XbaI restriction enzyme cleavage site of shuttle vector pHY300PLK, It was introduced by the protoplast transformation method. The resulting strain was shaken and cultured in 5 mL of LB medium at 30 ° C. overnight, and 0.6 mL of this culture was further added to 30 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), followed by shaking culture at 30 ° C. for 3 to 6 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 by the culture was determined. As a result, as shown in Table 4, when each gene-deleted strain was used as a host, higher secretion of alkaline amylase was observed compared to the control 168 strain (wild type).

FIG. 2 schematically shows 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.

Claims (8)

  A Bacillus subtilis having a maltose permeation system not involving glvR and glvC, wherein one or more genes selected from glvR and glvC involved in membrane permeation of the maltose are deleted or inactivated, A recombinant microorganism into which a gene encoding a polypeptide is introduced.   The recombinant microorganism according to claim 1, wherein the heterologous protein or polypeptide is a hydrolase selected from cellulase, α-amylase and protease.   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 combination according to claim 3 or 4, wherein 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. Replacement microorganism. 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. claim Ri Do either from the nucleotide sequence having 95% identity or more nucleotide sequences or the nucleotide sequence of nucleotide numbers 1 to 696 of cellulase gene, transcription, translation, and DNA fragments having the control functions related to secretion comprising 5. The recombinant microorganism according to 5.   A method for producing a protein or polypeptide using the recombinant microorganism according to any one of claims 1 to 6.   A method for producing a protein or polypeptide comprising culturing the recombinant microorganism according to any one of claims 1 to 6 in a medium containing maltose.