JP5739114B2 - Recombinant microorganism - Google Patents

Description

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

  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 use has been extended to a wide range of fields from daily necessities such as foods, medicines, 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, and using these genomic information, improved strains have been developed.
However, despite the above efforts, production efficiency is not always satisfactory.

On the other hand, the cell wall of Gram-positive bacteria including Bacillus subtilis has a skeleton composed of peptidoglycan. Peptidoglycan is a complex of polysaccharides covalently cross-linked by peptide chains, and an anionic polymer such as teichoic acid or teicuronic acid is covalently bonded to this polysaccharide. In the cell surface layer of Gram-positive bacteria, the cell wall having such a relatively high charge plays a role in changing the local environment.
Cell surface negative charges are also considered to be important for efficient folding of secreted proteins. Efficient protein folding often requires metals such as Fe ³⁺ and Ca ²⁺ , and the localization of these metals to the cell surface has been reported to involve the negative charge of anionic polymers (non- Patent Document 3).
Furthermore, production of enzymes such as α-amylase is improved in a mutant strain lacking a gene group encoding an enzyme group (DltA-E) that alanylates C2 in the glycerol phosphate skeleton of teichoic acid. There are reports (Non-Patent Document 4 and Patent Document 1).

Special Table 2002-520017

Kunst, F., et al., The complete genome sequence of the gram-positive bacterium Bacillus subtilis, Nature, 1997, 390 (6657): p. 249-56 Blattner, F.R., et al., The complete genome sequence of Escherichia coli K-12, Science, 1997. 277 (5331): p. 1453-62 Hughes, A.H., I.C. Hancock, and J.H. Baddiley, The function of teichoic acids in cation control in bacterial membranes, Biochem. J., 1973. 132 (1): p. 83-93 Hyyrylainen, H.L., et al., D-Alanine substitution of teichoic acids as a modulator of protein folding and stability at the cytoplasmic membrane / cell wall interface of Bacillus subtilis, J. et al. Biol. Chem., 2000. 275 (35): p. 26696-703

  An object of the present invention is to provide a recombinant microorganism having improved productivity of protein or polypeptide. Another object of the present invention is to provide a method for producing the recombinant microorganism. Another object of the present invention is to provide a method for producing a target protein or polypeptide using the recombinant microorganism. Furthermore, this invention makes it a subject to provide the method of improving the productivity of protein or polypeptide in microorganisms.

  In view of the above problems, the inventors have conducted intensive studies focusing on the relationship between the charge on the cell surface and protein production in order to obtain microorganisms with improved protein or polypeptide productivity. In order to construct a microorganism in which the amount of charge on the cell surface was artificially modified, an attempt was made to change the amount of anionic polymer present in the cell wall of the microorganism. As a result, it was found that the amount of teicuronic acid, which is a kind of anionic polymer on the cell surface, is reduced in the microorganism by suppressing the expression of the teicuronic acid synthase-related gene of the microorganism. Furthermore, when this microorganism was used for protein production, it was found that the amount of protein produced was significantly improved compared to the parent strain (wild strain). The present invention has been made based on these findings.

The present invention includes a group consisting of tuaA gene, tuaB gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene, which are genes related to teicronate synthase of Bacillus subtilis, and 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 microorganism constructed so that expression of any one or more selected genes is suppressed.
The present invention also relates to a method for producing a target protein or polypeptide using the recombinant microorganism.
The present invention also relates to a method for producing the recombinant microorganism.
Furthermore, the present invention relates to the expression of any one or more genes selected from the group consisting of a gene encoding the protein or polypeptide of interest and the teicronate synthase-related gene and a gene corresponding to the gene. The present invention relates to a method for improving the productivity of a target protein or polypeptide, characterized in that the protein or polypeptide is produced using a recombinant microorganism that has been genetically constructed so as to be suppressed.

According to the present invention, it is possible to provide a recombinant microorganism in which the productivity of the target protein or polypeptide is further improved. Moreover, according to this invention, the manufacturing method of the said recombinant microorganism can be provided. Furthermore, according to the present invention, a method for producing a protein or polypeptide using the recombinant microorganism can be provided.
By using the microorganism and the production method of the present invention, the target protein or polypeptide can be efficiently produced with high productivity, and the time and cost required for production of the substance can be reduced. In particular, the microorganism of the present invention can be suitably used for the production of a protein or polypeptide that decreases in productivity in a cssS gene-deficient strain of Bacillus subtilis.

It schematically shows a method for preparing a DNA fragment for deletion (for deletion) by the SOE-PCR method and a method for deleting a target gene (substituting a drug resistance gene) using the DNA fragment. Relative values when S237 cellulase, M-protease, K38 amylase and KP-43 protease production in the strain lacking the cssS gene (ΔcssS strain) is defined as 100 for the production of each protein in the wild strain (168 strain) It is shown by. In addition, the numerical value on a graph shows the average value which performed independent culture | cultivation 3 times by a relative value, and an error bar shows a standard deviation (N = 3). S237 cellulase, M-protease, K38 amylase, and KP-43 protease production by the strain lacking the tua operon (ΔtuaA-H strain), relative to the production in the wild strain (168 strain) as 100 It is shown. In addition, the numerical value on a graph shows the average value which performed independent culture | cultivation 3 times by a relative value, and an error bar shows a standard deviation (N = 3).

The recombinant microorganism of the present invention comprises tuaA gene, tuaB gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene, and genes corresponding to the genes related to teicronate synthase of Bacillus subtilis. A gene in which a gene encoding a target protein or polypeptide is introduced into a modified microorganism constructed by genetically modifying a parent microorganism so that expression of any one or more genes selected from the group consisting of is there. In this recombinant microorganism, the expression of the teicuronic acid synthase-related gene or the gene corresponding to the gene is suppressed, and as a result, the amount of teicuronic acid on the cell surface is reduced compared to the parent microorganism, regardless of the amount of phosphate in the medium. Furthermore, it has a feature that the productivity of the protein or the like is greatly improved as compared with the parental microorganism into which the target protein or the like is introduced.

In the present invention, the Bacillus subtilis teicuronic acid synthase-related gene refers to a gene involved in the biosynthesis of teicuronic acid, which is a kind of anionic polymer on the cell surface, specifically, the Bacillus subtilis tuaA gene, tuaB Gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene. These eight genes constitute the teicronate operon (also referred to as tua operon, teicronate synthase related gene group) (Soldo B., Lazarevic V., Pagni M., Karamata D., Teichuronic acid operon of Bacillus subtilis). 168. Mol. Microbiol., 1999, 31 (3), p. 795-805).
Expression of teicronate synthase-related genes is usually induced in phosphate starvation conditions. In Bacillus subtilis and the like, in general, in a state where the medium is rich in phosphoric acid (for example, in the logarithmic growth phase), teichoic acid is preferentially synthesized as an anionic polymer on the cell surface layer and the amount of teicuronic acid is low. In a state where phosphoric acid is depleted (for example, after the stationary phase), teicuronic acid is mainly synthesized. In addition, it has been reported that the amount of uronic acid in the cell wall decreases in mutants in which the genes related to teichronate synthase of Bacillus subtilis are inactivated (Soldo B., Lazarevic V., Pagni M., Karamata D. Teichuronic acid operon of Bacillus subtilis 168. See Mol Microbiol, 1999, 31 (3), p. 795-805). However, the above document does not show any knowledge about the protein productivity of the mutant strain.

In the present invention, the tuaA gene, tuaB gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene, which are genes related to teicronate synthase of Bacillus subtilis, are genes having the following base sequences, respectively. .
tuaA gene: gene consisting of the base sequence shown in SEQ ID NO: 1 (the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 9)
tuaB gene: gene consisting of the base sequence shown in SEQ ID NO: 2 (the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 10)
tuaC gene: gene consisting of the base sequence shown in SEQ ID NO: 3 (the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 11)
tuaD gene: gene consisting of the base sequence shown in SEQ ID NO: 4 (the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 12)
tuaE gene: gene consisting of the base sequence shown in SEQ ID NO: 5 (the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 13)
tuaF gene: gene consisting of the base sequence shown in SEQ ID NO: 6 (the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 14)
tuaG gene: gene consisting of the base sequence shown in SEQ ID NO: 7 (the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 15)
tuaH gene: gene consisting of the base sequence shown in SEQ ID NO: 8 (the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 16)
Table 1 shows the function and the like of each gene that is a gene related to teicronate synthase of Bacillus subtilis. The names of the genes and gene regions of Bacillus subtilis described in Table 1 and the present specification are reported in Nature, 390, 249-256, (1997). JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB ) And published on the Internet (http://bacillus.genome.ad.jp/, updated on March 10, 2004).

  In the present invention, “a gene corresponding to a teicuronate synthase-related gene of Bacillus subtilis” refers to a gene that is functionally equivalent to the above-described eight genes for teicuronate synthase of Bacillus subtilis. Examples of the gene corresponding to the teicuronate synthase-related gene of Bacillus subtilis include any of the following genes (1) to (4).

(1) It consists of a base sequence having 90% or more, preferably 95% or more, more preferably 99% or more identity with the base sequence represented by any of SEQ ID NOs: 1 to 8, and SEQ ID NOs: 9 to 16 A gene comprising a DNA encoding a protein functionally equivalent to a protein comprising an amino acid sequence represented by any one of them.
In the present invention, proteins functionally equivalent to the protein consisting of the amino acid sequence represented by any of SEQ ID NOs: 9 to 16 are encoded by the above eight genes that are genes related to teicuronic acid synthase of Bacillus subtilis. It refers to a protein that has substantially the same function as a protein and is involved in teicuronic acid synthesis in the cell surface. Specifically, the functions described in Table 1 are listed as functions of the above eight genes that are teicuronate synthase-related genes of Bacillus subtilis and proteins encoded by the genes.
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 Unit size to compare (ktup) set to 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

(2) An amino acid sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 1 to 8 and that is represented by any of SEQ ID NOs: 9 to 16 A gene comprising DNA encoding a protein functionally equivalent to a protein comprising
Here, as the “stringent conditions”, for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W., et al. Russell., Cold Spring Harbor Laboratory Press], for example, 6 × SSC (composition of 1 × SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% Examples include conditions in which a solution containing SDS, 5 × Denhart and 100 mg / mL herring sperm DNA is incubated with a probe at 65 ° C. for 8 to 16 hours and hybridized.

  (3) It consists of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 99% or more identity with the amino acid sequence represented by any of SEQ ID NOs: 9 to 16, and SEQ ID NOs: 9 to 16 A gene comprising a DNA encoding a protein functionally equivalent to a protein comprising an amino acid sequence represented by any one of them.

(4) In the amino acid sequence represented by any of SEQ ID NOs: 9 to 16, the amino acid sequence comprises one to several amino acids deleted, substituted, inserted and / or added, and any of SEQ ID NOs: 9 to 16 A gene consisting of DNA encoding a protein functionally equivalent to the protein consisting of the amino acid sequence shown in.
Here, 1 to several amino acids are deleted, substituted, inserted and / or added is preferably 1 to 10 amino acids, more preferably 1 to 5 amino acids, and still more preferably 1 to 2 amino acids. The addition includes addition of 1 to several amino acids at both ends.

As "gene corresponding to Teikuron synthase-related gene of Bacillus subtilis", specifically, Bacillus licheniformis (Bacillus licheniformis), Bacillus anthracis (Bacillus anthracis), Bacillus cereus (Bacillus cereus), Bacillus thuringiensis Jen cis (Bacillus thuringiensis ), or in O-Sha Roh Bacillus Iheenshisu (Oceanobacillus iheyensis), etc., mainly the tuaA homologous gene was identified by genomic analysis, TuaB homologous gene, TUAC homologous gene, tuaD homologous gene, TuaE homologous gene, TuaF homologous gene, TuaG homologous gene And tuaH homologous genes, which are preferably used in the present invention.
Hereinafter, the teicuronic acid synthase-related gene of Bacillus subtilis and the gene corresponding to the gene are collectively referred to as a teicuronic acid synthase-related gene.

The parent microorganism for constructing the microorganism of the present invention is not particularly limited as long as it has either a teicronate synthase-related gene of Bacillus subtilis or a gene corresponding to the gene. These parent microorganisms may be wild-type or mutated. Specific examples of the parent microorganism used in the present invention include fungi belonging to the genus Bacillus , fungi belonging to the genus Clostridium , yeast, and the like. Of these, fungi 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.

The recombinant microorganism of the present invention is constructed such that these parent microorganisms are genetically modified so that the expression of at least one teicronate synthase-related gene possessed by the parent microorganism is suppressed. In particular, the expression of all genes that make up the teicronate synthase related gene group (tua operon) of Bacillus subtilis, ie, tuaA gene, tuaB gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene It is preferably constructed so that expression is suppressed or the expression of all genes corresponding to the gene group is suppressed.
As a technique for suppressing the expression of a teicronate synthase-related gene, a normal technique can be used. For example, a method for modifying or deleting or inactivating the gene itself, a method for repressing or reducing transcription by modifying a transcriptional regulatory region such as a transcription promoter region, or a method for repressing or reducing translation by modifying a translational regulatory region Examples thereof include a method and a method of reducing or inactivating the function of the translated protein. In particular, a method in which a gene is physically deleted or inactivated is most preferable because expression of the gene of interest is completely lost.

As a technique for deleting or inactivating a gene, for example, a method by homologous recombination can be used. That is, a target gene into which an inactivating mutation has been introduced by base substitution or base insertion, or a linear DNA fragment that contains the outer region of the target gene but does not contain 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. Can be replaced with a deleted or inactivated gene fragment. Alternatively, 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 can also be inactivated by disrupting the target gene on the genome.
When Bacillus subtilis is used as a parent microorganism for constructing the microorganism of the present invention, a method for deleting or inactivating a target gene by homologous recombination is described in Mol. Gen. Genet., 223, 268, 1990 can be used.
In addition, as a method for introducing a mutation into a gene, a known method such as the Kunkel method or the Gapped duplex method, or a method equivalent thereto can be employed. For example, using a mutagenesis kit (for example, Mutant-K (TAKARA) or Mutant-G (TAKARA)) using site-directed mutagenesis, or TAKARA LA PCR in vitro Mutagenesis Mutation may be introduced using a series kit.
In the present specification, upstream / downstream of a gene is not a position from the replication start point, but upstream refers to a region continuing on the 5 ′ side of the gene or region considered as a target, whereas downstream is The region following the 3 ′ side of the gene or region captured as the target is shown.

Hereinafter, as an example of a method for deleting a gene by homologous recombination, double crossover using a deletion DNA fragment prepared by SOE (splicing by overlap extension) -PCR method (Gene, 77, 61, 1989) The deletion method by the method will be described with reference to FIG.
First, in the first PCR, using a host genomic DNA (eg, bacterial genomic DNA) as a template, a 5 ′ region adjacent to a deletion target gene (eg, teicronate synthase-related gene group (tua operon)) is selected. Specific primers for a DNA fragment (A fragment: for example, 0.1 to 3 kbp) and a DNA fragment (C fragment: for example, 0.1 to 3 kbp) including the 3 ′ region adjacent to the deletion target gene Amplify and prepare by PCR using the set. Separately, a DNA fragment (B fragment) containing a drug resistance gene is amplified and prepared by PCR using a specific primer set. At this time, in the PCR for A fragment amplification, a primer designed so as to add a homologous sequence (for example, 10 to 30 bases) of the 5 ′ end of the B fragment to the 3 ′ end of the A fragment is used. Similarly, in PCR for C fragment amplification, a primer designed to add a homologous sequence (eg, 10 to 30 bases) at the 3 ′ end of the B fragment to the 5 ′ end of the C fragment is used.
Any drug resistance gene may be used as long as it can appropriately select and isolate transformants obtained by the following homologous recombination, and can be used for selection using commonly used antibiotics. If it is, it will not specifically limit. Specific examples include drug resistance marker genes such as kanamycin resistance gene, chloramphenicol resistance gene, neomycin resistance gene, spectinomycin resistance gene, tetracycline resistance gene, and blasticidin S resistance gene.

Next, using the three types of PCR products (PCR products A, B, and C) prepared in the first PCR as templates, using the forward primer used for PCR amplification of the A fragment and the reverse primer used for PCR amplification of the C fragment The second PCR (SOE (splicing by overlap extension) -PCR (Gene, 77, 61, (1989))) is performed between the 3 ′ end of PCR product A and the 5 ′ end of PCR product B. Annealing occurs, and similarly, an annealing occurs between the 3 ′ end of PCR product B and the 5 ′ end of PCR product C. As a result of PCR amplification, PCR product D is ligated in the order of A fragment-B fragment-C fragment. (D fragment) can be obtained.
The PCR reaction carried out here is carried out using a general PCR enzyme kit such as Pyrobest DNA Polymerase (Takara Shuzo) with the primer set shown in Table 6 of the examples described later, for example (PCR Protocols. Current Methods and Applications, Edited by BAWhite, Humana Press pp251, 1993, Gene, 77, 61, 1989) and the like.

The obtained PCR product D is introduced into a host (for example, a bacterium) by an ordinary method. Between the homologous region upstream and downstream of the gene to be deleted, double crossover homologous recombination occurs between the introduced PCR product D and the host genomic DNA. As a result, a transformant (recombinant microorganism) in which the drug resistance gene in PCR product D is incorporated into the deletion target gene site of the host genomic DNA and the deletion target gene in the host genomic DNA is deleted is obtained. Can do. A transformant from which the gene to be deleted is deleted can be selected and isolated using a drug resistance gene newly incorporated into the host genomic DNA (drug resistance gene derived from PCR product D) as an index. Selection by drug resistance marker, for example, when a kanamycin resistance gene is introduced, colonies that grow on an agar medium containing kanamycin are isolated, and then introduced into the genome by PCR using the genome as a template This can be done by selecting.
As a method for introducing the PCR product D (DNA fragment for deletion) into the host microorganism, a usual method can be used, for example, competent cell transformation method (J. Bacterial. 93, 1925 (1967)), Examples include protoplast transformation method (Mol. Gen. Genet. 168, 111 (1979)), electroporation method (FEMS Microbiol. Lett. 55, 135 (1990)), and the like. In the present invention, the competent cell transformation method is preferred.

  In addition to the above, as a method for gene deletion or inactivation, there may be mentioned a method in which random gene deletion or inactivation mutation is given and then protein productivity evaluation and gene analysis are performed by an appropriate method. It is done. Specifically, random gene deletion or non-deletion by homologous recombination similar to the above-described method using randomly cloned DNA fragments, or by irradiating the parent bacteria with γ rays, etc. Activation is possible.

  As a method for suppressing gene transcription, for example, the transcription promoter region of the teicronate synthase-related gene group (tua operon) is replaced with a transcription-inducible promoter, and then transformed. And a method of culturing in the above. Specifically, a host microorganism is transformed using a vector or expression cassette having a transcription-inducible promoter sequence according to a conventional method, and the promoter sequence is incorporated upstream of a teicronate synthase-related gene group (tua operon). Is preferred. Here, the transcription-inducible promoter means a promoter having a function of enhancing the expression of a gene located downstream on the condition of the presence of a predetermined substance. The transcription-inducible promoter that can be used in the present invention is not particularly limited, and examples thereof include, for example, Pspac promoter (Proc. Natl. Acad. Sci. USA 81, 439-443. (1984)), zyrose-inducible promoter (Gene 181 , 71-76. (1996)), arabinose-inducible promoter (Microbiology 154, 2562-2570. (2008)), ECF sigma factor specific promoter (J. Helmann and C. Moran Jr., “Bacillus subtilis and its closest relatives” . "ASM Press, 2002, pp289-312).

The transformation method is not particularly limited as long as it is a method capable of introducing a target promoter region into a microorganism. For example, a method using calcium ions, a general competent cell transformation method (J. Bacterial. 93, 1925 (1967)), a protoplast transformation method (Mol. Gen. Genet. 168, 111 (1979)), electro Polation method (FEMS Microbiol. Lett. 55, 135 (1990)) or LP transformation method (T. Akamatsu and J. Sekiguchi, Archives of Microbiology, 1987, 146, p.353-357; Taguchi, Bioscience, Biotechnology, and Biochemistry, 2001, 65, 4, p.823-829) can be used.
Selection of a transformant introduced with a target DNA fragment can be performed by using a selection marker or the like. For example, a drug resistance gene derived from a vector or expression cassette is introduced into the host genome together with the target DNA fragment at the time of transformation, and as a result, the drug resistance acquired by the transformant can be used as an indicator. Examples of the drug resistance marker include the aforementioned drug resistance genes. The introduction of the target DNA fragment can also be confirmed by PCR method using a genome as a template.

  Examples of the method for suppressing the translation of teicronate synthase-related gene include a method using so-called antisense RNA. Specifically, the gene that transcribes an antisense RNA against the mRNA of a teicuronate synthase-related gene is incorporated into bacterial genomic DNA, and the antisense RNA is overexpressed, thereby translating the mRNA of the teicuronate synthase-related gene. Is suppressed.

  The microorganism of the present invention can be obtained by introducing a gene encoding a target protein or polypeptide into a microorganism constructed so that the expression of at least one teicronate synthase-related gene is suppressed by the method described above. Can be produced. Here, the “target protein or polypeptide” refers to a protein or polypeptide whose production or purification is one of the purposes. In addition, in the “microorganism having a gene encoding the target protein or polypeptide”, the gene includes not only a gene originally possessed by the microorganism but also a gene that the microorganism originally does not have, that is, a foreign gene. is there.

  The target protein or polypeptide used in the present invention is not particularly limited, and includes various industrial enzymes such as detergents, foods, fiber treatments, feed treatments, cosmetics, pharmaceuticals, diagnostics, bioactive peptides, etc. Is included. In addition, the functions of industrial enzymes are classified into oxidoreductase (Oxidoreductase), transferase (Transferase), hydrolase (Hydrolase), eliminase (Lyase), isomerase (Isomerase), and synthetic enzyme (Ligase / Synthetase). ) And the like.

Among them, the microorganism of the present invention preferably uses a protein or polypeptide whose productivity decreases in a cssS gene-deficient strain of Bacillus subtilis as the target protein or polypeptide. Here, the cssS gene of Bacillus subtilis is a gene encoding a membrane protein CssS involved in a protein secretion stress response system (two-component control system CssRS). The base sequence of the Bacillus subtilis cssS gene is shown in SEQ ID NO: 53, and the amino acid sequence of the Bacillus subtilis CssS protein is shown in SEQ ID NO: 54, respectively.
In Bacillus subtilis, secreted proteins are synthesized intracellularly and secreted extracellularly, and are folded at the cell surface by the folding promoting protein PrsA. At this time, proteins that are not normally folded (misfolded proteins) may accumulate on the cell surface. The CssRS control system controls the degradation of this misfolded protein. The membrane protein CssS functions as a sensor that recognizes misfolded proteins accumulated on the cell surface in the control system. The membrane protein CssS activates CssR that is a transcriptional activator of the htrA gene and the htrB gene via the phosphate relay system. The active CssR transcribes the htrA gene and the htrB gene to produce HtrA and HtrB, which are serine proteases responsible for degrading misfolded proteins present on the cell surface. (Hyyrylainen, HL, A. Bolhuis, E. Darmon, L. Muukkonen, P. Koski, M. Vitikainen, M. Sarvas, Z. Pragai, S. Bron, J. M. van Dijl, and V. P. Kontinen, 2001, A novel two-component regulatory system in Bacillus subtilis for the survival of severe secretion stress, Mol Microbiol 41, p. 1159-1172 and Darmon, E., D. Noone, A. Masson, S. Bron , O. P. Kuipers, K. M. Devine, and J. M. van Dijl, 2002, A novel class of heat and secretion stress-responsive genes is controlled by the autoregulated CssRS two-component system of Bacillus subtilis, J Bacteriol 184, p.5661-5571)

In the microorganism of the present invention, as shown in the examples described later, productivity of various proteins is improved. Among them, the target protein or polypeptide to be introduced into the microorganism is a Bacillus subtilis cssS gene-deficient strain. In the case of a protein or polypeptide whose productivity is reduced in, high productivity is exhibited.
Several examples of heterologous protein productivity studies using strains lacking the CssSR regulatory system have already been reported (Hyyrylainen, HL, A. Bolhuis, E. Darmon, L. Muukkonen, P. Koski, M. Vitikainen). , M. Sarvas, Z. Pragai, S. Bron, J. M. van Dijl, and V. P. Kontinen, 2001, A novel two-component regulatory system in Bacillus subtilis for the survival of severe secretion stress, Mol. 41, p. 1159-1172, and Vitikainen, M., H. L. Hyryryinen, A. Kivimaki, V. P. Kontinen, and M. Sarvas, 2005, Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering. cell components affecting post-translocational protein folding and degradation, J Appl Microbiol 99, p.363-375). According to these literatures, it has been reported that the productivity of α-amylases AmyS and AmyL and pneumolysin is decreased in strains lacking the CssSR control system, while the productivity of penicillinase and polygalacturonase is not affected. Yes. From these results, it is considered that proteins that have a negative effect on productivity, such as the former, are easily misfolded and are difficult to secrete, and proteins that do not affect productivity, such as the latter, are easily folded. .

In the present invention, the protein or polypeptide productivity is lowered in cssS gene-deficient strain of Bacillus subtilis, to the case where a 1 to a protein or polypeptide production in wild-type, in cssS gene-deficient strain of Bacillus subtilis It refers to a protein or polypeptide that produces a relative production value of the protein or polypeptide of less than 0.9. Specific examples of the protein or polypeptide whose productivity is reduced in the cssS gene-deficient strain of Bacillus subtilis include the following hydrolases such as protease and amylase.

  Specific examples of the protease include serine proteases derived from fungi belonging to the genus Bacillus, metal proteases, particularly serine proteases belonging to oxidatively stable alkaline proteases (OSPs) and the like (Extremophiles 8, 229-235. (2004)). As a more specific example, a protease derived from Bacillus genus KSM-KP43 (FERM BP-6532) consisting of the amino acid sequence represented by SEQ ID NO: 26, or 70%, preferably 80%, more preferably the amino acid sequence. A protease comprising an amino acid sequence having 90% or more, more preferably 95% or more, particularly preferably 98% or more, or one or several amino acids deleted or substituted in the amino acid sequence represented by SEQ ID NO: 26; Examples include alkaline protease consisting of an inserted and / or added amino acid sequence.

  Specific examples of α-amylase include α-amylase derived from microorganisms, and liquefied amylase derived from fungi belonging to the genus Bacillus is particularly preferable. As a more specific example, alkaline amylase derived from the Bacillus genus KSM-K38 strain (FERM BP-6946) comprising the amino acid sequence represented by SEQ ID NO: 24, or 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, or one or several amino acids deleted or substituted in the amino acid sequence shown in SEQ ID NO: 24 , Alkaline amylase consisting of an inserted and / or added amino acid sequence.

The target protein or target polypeptide gene introduced into the microorganism of the present invention is upstream of the control region involved in transcription, translation, and secretion of the gene, that is, the transcription initiation control region including the promoter and transcription start site, and ribosome binding. It is desirable that at least one region selected from a translation initiation control region including a site and an initiation codon and a secretory signal peptide region be 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 fungus belonging to the genus Bacillus, It is desirable that the control region and the translation initiation control region are 0.6 to 1 kb upstream of the cellulase gene and appropriately bound to the target protein or polypeptide gene.
For example, fungi belonging to the genus Bacillus described in JP 2000-210081, JP 4-190793, etc., that is, KSM-S237 strain (FERM BP-7875, SEQ ID NOs: 17 and 18), KSM-64 The cellulase gene derived from the strain (FERM BP-2886, SEQ ID NOs: 19 and 20) and the transcription initiation control region, the translation initiation control region, and the signal peptide region for secretion of the cellulase gene are appropriately combined with the structural gene of the target protein or polypeptide. It is desirable that they are combined. More specifically, it is desirable that a DNA fragment having any one of the following base sequences (a) to (c) is appropriately bound to the structural gene of the target protein or target polypeptide.
(A) the base sequence of base numbers 1 to 659 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 17 or the base sequence of base numbers 1 to 696 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 19 (b) 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 with respect to any one of the base sequences (a) ( c) A base sequence in which a part of the base sequence of (a) is deleted, substituted, inserted and / or added. Here, a part of the base sequence is deleted, substituted, inserted and / or added. A DNA fragment consisting of a base sequence is a DNA fragment in which a part of the base sequence has been deleted, substituted, inserted and / or added, but which retains functions related to gene transcription, translation and secretion. Means.

Such a gene encoding a target protein or polypeptide can be introduced by, for example, introduction by a vector or insertion into a genome.
When introduced by a vector, upstream of it, “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 translation initiation control region including a ribosome binding site and an initiation codon, and A vector containing a gene encoding a target protein or polypeptide in which one or more regions selected from a secretory signal peptide region are linked in an appropriate form is transformed into a competent cell transformation method, protoplast transformation method, or electro It may be introduced by an appropriate transformation method such as a poration method. Here, the vector is not particularly limited as long as it is an appropriate carrier nucleic acid molecule for introducing a target gene into a host, allowing the gene to propagate and express, and not only a plasmid but also an artificial such as YAC or BAC. Examples include vectors using chromosome and transposon, and cosmids. Examples of plasmids include pUB110 and pHY300PLK.

  Further, for example, a method by homologous recombination may be used for insertion into the genome. That is, a DNA fragment in which a part of a chromosomal region that causes introduction into a gene encoding a target protein or polypeptide is combined is introduced into a microbial cell, and homologous recombination occurs in a part of the chromosomal region. Can be integrated into the genome. Here, the chromosomal region causing the introduction is not particularly limited, but a non-essential gene region or a non-gene region upstream of the non-essential gene region is preferable.

By the above method, the microorganism of the present invention can be constructed.
The microorganism of the present invention produces a target protein or polypeptide as compared to a microorganism (wild type, parental microorganism) that has not been modified with respect to the expression of a teicronate synthase-related gene, as shown in the Examples below. The characteristics are greatly improved. In particular, the productivity of the microorganism of the present invention is greatly improved when a protein or polypeptide that decreases in productivity in a cssS gene-deficient strain of Bacillus subtilis is introduced as the target protein or polypeptide.
By producing the target protein or polypeptide using the microorganism of the present invention, the target protein or polypeptide can be efficiently produced with high productivity, and the time and cost required for production of the substance can be reduced. can do.

The production of the target protein or polypeptide using the recombinant microorganism of the present invention is performed by inoculating the strain with 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. For example, the secreted and produced protein can be isolated and purified from the culture supernatant and the like by culturing under the medium and conditions shown in the Examples described later.
The components and composition of the medium are not particularly limited, but it is more preferable to use a medium containing maltose or soluble starch or glucose as a carbon source.
For collection and purification of proteins and polypeptides, for example, separation of recombinant microorganisms by centrifugation or filtration, precipitation by adding salts such as ammonium sulfate of proteins and polypeptides in the supernatant or filtrate, organic solvents such as ethanol Can be carried out by using a method such as precipitation by adding sucrose, concentration or desalting using an ultrafiltration membrane or the like, purification using various chromatographies such as ion exchange or gel filtration.

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

  For the polymerase chain reaction (PCR) for DNA fragment amplification in the following examples, GeneAmp PCR System (Applied Biosystems) is used, and DNA amplification is performed using Pyrobest DNA Polymerase (Takara Bio) and attached reagents. went. The PCR reaction solution composition was 1 μl of appropriately diluted template DNA, 20 pmol each of sense primer and antisense primer, and 2.5 U of Pyrobest DNA Polymerase, so that the total reaction solution volume was 50 μl. PCR reaction conditions were 98 ° C. for 10 seconds, 55 ° C. for 30 seconds and 72 ° C. for 1 to 5 minutes (adjusted according to the target amplification product, 1 minute per 1 kb). After repeating, the reaction was carried out at 72 ° C. for 5 minutes.

Further, in the following examples, the upstream and downstream of a gene is not a position from the replication start point, and the upstream is a region continuing on the 5 ′ side of the start codon of the gene captured as a target in each operation / step. On the other hand, “downstream” indicates a region continuing 3 ′ of the stop codon of the gene taken as a target in each operation / step.
Furthermore, the names of the genes and gene regions in the following examples are reported in Nature, 390, 249-256, (1997), and published on the Internet at 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.

The transformation of Bacillus subtilis was performed by the competent cell method (J. Bacteriol., 93, 1925 (1967)). That is, Bacillus subtilis strains were treated with SPI medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50. The cells were cultured with shaking at 37 ° C. in% glucose, 0.02% casamino acid (Difco), 5 mM magnesium sulfate, 0.25 μM manganese chloride, 50 μg / ml tryptophan until the value of growth (OD600) was about 1. After shaking culture, a portion of the culture broth was added to 9 times the amount of SPII medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% tricitrate citrate). Sodium dihydrate, 0.50% glucose, 0.01% casamino acid (Difco), 5 mM magnesium sulfate, 0.40 μM manganese chloride, 5 μg / ml tryptophan), and the growth (OD600) value is further increased. A competent cell of Bacillus subtilis strain was prepared by shaking culture to about 0.4.
Next, 5 μ of a solution containing various DNA fragments (such as SOE-PCR reaction solution) is added to 100 μl of the prepared competent cell suspension (culture medium in SPII medium), and after shaking culture at 37 ° C. for 1 hour, an appropriate drug is obtained. LB agar medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) containing the whole amount was smeared. After stationary culture at 37 ° C., the grown colonies were isolated as transformants. The genome of the obtained transformant was extracted, and it was confirmed that the intended genomic structure was altered by PCR using this as a template.

  The plasmid expressing the target protein was introduced into the host microorganism by the protoplast transformation method (Mol. Gen. Genet. 168, 111 (1979)). For protein production by recombinant microorganisms, LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl), 2 × L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate).

Test Example 1 Construction of Bacillus subtilis cssS gene deletion strain (ΔcssS strain) The Bacillus subtilis cssS gene deletion strain (ΔcssS strain) was constructed. As shown below, deletion strains were constructed by the double crossover method using DNA fragments prepared by SOE (splicing by overlap extension) -PCR (Gene, 77, 61, (1989)) ( (See FIG. 1). The ΔcssS strain is a secretory stress sensitive strain.
Using genomic DNA extracted from Bacillus subtilis 168 strain as a template, using the cssS.FW primer and cssS / Cm.R primer shown in Table 2, and the respective cssS / Cm.F primer and cssS.RV primer set, A 1000 bp fragment (A) on the 5 ′ end side containing a promoter of the cssS gene and a 1000 bp fragment (B) on the 3 ′ end side were prepared. On the other hand, the chloramphenicol resistance gene is shown in Table 2, using the plasmid pCBB31 having the chloramphenicol (Cm) resistance gene of plasmid pC194 (J. Bacteriol. 150 (2), 815 (1982)) as a template. Using the CmF and CmR primer sets, a chloramphenicol resistance gene region was prepared by PCR (C). The obtained DNA fragments of (A), (B) and (C) were mixed to form a template, and (A)-(C)-was obtained by SOE-PCR using cssS.FW2 primer and cssS.RV2 primer. The DNA fragments for gene deletion were obtained by binding in the order of (B).
168 strains of Bacillus subtilis were transformed by the competent cell transformation method using the prepared DNA fragment. Thereafter, colonies that grew on the LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants. The genome of the obtained transformant was extracted, and it was confirmed by PCR that the cssS gene was deleted and replaced with the chloramphenicol resistance gene.
In the manner as described above, a strain lacking the cssS gene of Bacillus subtilis was constructed and named ΔcssS strain.

Test Example 2 Measurement of protein production by ΔcssS strain About ΔcssS strain obtained in Test Example 1 and parent strain Bacillus subtilis 168 strain (wild strain), the productivity of S237 cellulase, M-protease, K38 amylase and KP-43 protease Measured and evaluated.

1. Measurement of S237 Cellulase Productivity The S237 cellulase gene derived from the Bacillus genus KSM-S237 strain (FERM BP-7875) was added to the ΔcssS strain and the parental strain Bacillus subtilis 168 obtained in Test Example 1 (JP 2000-210081). The recombinant plasmid pHY-S237 in which a DNA fragment (3.1 kb; base numbers 13 to 3124 of SEQ ID NO: 17) encoding (SEQ ID NO: 17) is inserted at the Bam HI restriction enzyme cleavage point of shuttle vector pHY300PLK Was introduced by the protoplast transformation method. 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 added to 30 mL of 2 × L-maltose medium (2% tryptone, 1% yeast extract, 1% sodium chloride). 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline), followed by shaking culture at 30 ° C. for 3 days. After culturing, the cellulase activity of the culture supernatant after removing the cells was measured by centrifugation, and the amount of S237 cellulase secreted and produced outside the cells was evaluated.
For the measurement of cellulase activity, 0.4 mM p-nitrophenyl-β-D-cellotrioside (Seikagaku Corporation) was added to 50 μl of a sample solution appropriately diluted with 1 / 7.5 M phosphate buffer (pH 7.4, Wako Pure Chemical Industries). Was mixed, and the amount of p-nitrophenol released upon reaction at 30 ° C. was quantified by the change in absorbance at 420 nm (OD 420 nm). The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was defined as 1 U.

2. Measurement of M-protease productivity When constructing DNA for protein expression, the S237pKAPpp-F primer and KAPter shown in Table 3 were used with the genomic DNA extracted from Bacillus clausii strain KSM-K16 (FERM BP-3376) as a template. PCR is performed using a primer set of -R (BglII) primer, and the signal sequence of M-protease in the base sequence (SEQ ID NO: 21) of the gene encoding M-protease (alkaline protease, see Patent No. 3026111), A 1.2-kb DNA fragment (base numbers 163 to 1387 of SEQ ID NO: 21) containing a prosequence and a mature enzyme region was amplified. In addition, using genomic DNA extracted from Bacillus sp. KSM-S237 strain (FERM BP-7875) as a template, the primer set of S237ppp-F2 (BamHI) primer and S237pKAPpp-R primer shown in Table 3 was used. PCR was performed, and a 0.6 kb DNA fragment encoding the transcription initiation control region and the translation initiation control promoter region of the S237 cellulase gene (Japanese Patent Laid-Open No. 2000-210081) represented by SEQ ID NO: 17 (base of SEQ ID NO: 17) Numbers 13-572) were amplified. Subsequently, S237 cellulase was prepared by performing SOE-PCR using the S237ppp-F2 (BamHI) primer and the KAPter-R (BglII) primer set shown in Table 3 by mixing the obtained two fragments as a template. A 1.8 kb DNA fragment was obtained in which the M-protease signal sequence, prosequence and mature enzyme region were linked downstream of the transcription initiation control region and translation initiation control promoter region of the gene. The resulting DNA fragment of 1.8kb was inserted into the Bam HI- Bgl II restriction enzyme cleavage point of a shuttle vector pHY300PLK (Yakult), was constructed M- protease productivity evaluation plasmid pHYKAP (S237p).

The constructed plasmid pHYKAP (S237p) was introduced into the ΔcssS strain obtained in Test Example 1 and the parent strain Bacillus subtilis 168 by a protoplast transformation method. The obtained recombinant strain was cultured with shaking in 5 mL of LB medium at 37 ° C. overnight. 0.06 mL of this culture solution 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) And incubating with shaking at 30 ° C. for 3 days. After the culture, the protease activity of the culture supernatant after removing the cells by centrifugation was measured, and the amount of M-protease secreted and produced outside the cells by the culture was evaluated.
The activity of protease in the culture supernatant was measured as follows. 75 mM borate-KCl buffer containing 7.5 mM Succinyl-L-Alanyl-L-Alanyl-L-Alanine p-Nitroanilide (STANA Peptide Institute) as a substrate in 50 μL of culture supernatant appropriately diluted with ₂ mM CaCl ₂ solution 100 μL of (pH 10.5) was added and mixed, and the amount of p-nitroaniline released when the reaction was carried out at 30 ° C. was quantified by the change in absorbance at 420 nm (OD 420 nm). The amount of enzyme that liberates 1 μmol of p-nitroaniline per minute was defined as 1 U.

3. Measurement of K38 amylase productivity In the construction of DNA for expression, genomic DNA extracted from Bacillus sp. KSM-K38 strain (FERM BP-6946) was used as a template and K38matu-F2 (ALAA) shown in Table 4 PCR is performed using a primer set of SP64K38-R (XbaI), and the alkaline amylase AmyK38 represented by SEQ ID NO: 23 (JP 2000-184882 A, Eur. J. Biochem., 268, 2974, 2001) is encoded. A 1.5 kb DNA fragment containing the region (base numbers 1 to 1531 of SEQ ID NO: 23) was amplified. In addition, using the genomic DNA extracted from Bacillus sp. KSM-S237 strain (FERM BP-7875) as a template, the S237ppp-F2 (BamHI) and S237ppp-R2 (ALAA) primer sets shown in Table 4 were used. PCR is performed, and a 0.6 kb DNA fragment containing the transcription initiation control region, translation initiation control promoter region and secretory signal sequence coding region of the alkaline cellulase gene shown in SEQ ID NO: 17 (Japanese Patent Laid-Open No. 2000-210081) (Base numbers 13 to 572 of SEQ ID NO: 17) was amplified. Next, the obtained two fragments were mixed to serve as a template, and SOE-PCR using the S237ppp-F2 (BamHI) and SP64K38-R (XbaI) primer sets shown in Table 4 was performed. A 2.1 kb DNA fragment was obtained in which a gene encoding a mature alkaline amylase was linked downstream of a region encoding a transcription initiation control region, a translation initiation control promoter region, and a secretory signal sequence. The obtained 2.1 kb DNA fragment was inserted into the Bam HI- Xba I restriction enzyme cleavage point of shuttle vector pHY300PLK (Yakult) to construct alkaline amylase productivity evaluation plasmid pHYK38 (S237ps).

  Further, the constructed plasmid pHYK38 (S237ps) was introduced into the ΔcssS strain and the parent strain Bacillus subtilis 168 by a protoplast transformation method. The recombinant strain obtained in this manner was shaken and cultured overnight in 5 mL of LB medium at 37 ° C., and 0.05 mL of this culture was inoculated into 30 mL of 2 × L-maltose medium and shaken at 30 ° C. for 3 days. Culture was performed. After culturing, the amylase activity of the culture supernatant after removing the cells by centrifugation was measured, and the amount of amylase secreted and produced outside the cells by the culture was determined.

  Liquitec Amy EPS (Roche Diagnostics) was used to measure the amylase activity in the culture supernatant. Specifically, 50 μL of a sample solution appropriately diluted with 1% NaCl-1 / 7.5 M phosphate buffer (pH 7.4, Wako Pure Chemical Industries, Ltd.) was added to 100 μL of an R1 / R2 mixed solution (R1 (coupling enzyme ): R2 (amylase substrate) = 5: 1 (Vol.)) Was added and mixed, and the amount of p-nitrophenol released when the reaction was carried out at 30 ° C. was quantified by the change in absorbance at 405 nm (OD405 nm). . The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was defined as 1 U.

4). Measurement of KP-43 Protease Productivity When constructing DNA for expression, S237pKP43m-F and KP43m shown in Table 5 were used with the genomic DNA extracted from Bacillus sp. KSM-KP43 strain (FERM BP-6532) as a template. PCR was performed using a primer set of -R (XbaI), and alkaline protease KP-43 shown in SEQ ID NO: 25 (hereinafter simply referred to as “KP-43 protease”) (International Publication No. 99/18218 pamphlet: GenBank accession no AB051423), a 2.0 kb DNA fragment (base numbers 1 to 2015 of SEQ ID NO: 25) containing the signal sequence, pro sequence and mature enzyme region was amplified. Using genomic DNA extracted from Bacillus sp. KSM-S237 strain (FERM BP-7875) as a template, PCR was performed using the S237ppp-F2 (BamHI) and S237pKP43m-R primer sets shown in Table 5. A 0.6 kb DNA fragment (base number of SEQ ID NO: 17) containing the transcription initiation control region of the alkaline cellulase gene shown in SEQ ID NO: 17 (Japanese Patent Laid-Open No. 2000-210081) and the region encoding the translation start control promoter region 13-572) were amplified. Next, the obtained two fragments were mixed to serve as a template, and SOE-PCR using the S237ppp-F2 (BamHI) and KP43m-R (XbaI) primer sets shown in Table 5 was performed. A 2.6 kb DNA fragment was obtained in which a region encoding the signal sequence, pro sequence and mature enzyme region of KP-43 protease was linked downstream of the transcription initiation control region and the translation initiation control promoter region. The obtained 2.6 kb DNA fragment was inserted into the Bam HI- Xba I restriction enzyme cleavage point of shuttle vector pHY300PLK (Yakult) to construct a plasmid pHYKP43 (S237p) for evaluating KP-43 protease productivity.

  Furthermore, the constructed plasmid pHYKP43 was introduced into the ΔcssS strain and the parent strain Bacillus subtilis 168 by protoplast transformation. The recombinant strain thus obtained was shaken and cultured overnight in 5 mL of LB medium at 37 ° C., and 0.05 mL of this culture solution was inoculated into 30 mL of 2 × L-maltose medium and shaken at 30 ° C. for 3 days. Culture was performed. After culturing, the protease activity of the culture supernatant after removing the cells by centrifugation was measured, and the amount of KP-43 protease secreted and produced outside the cells by the culture was determined.

The activity of protease in the culture supernatant was measured as follows. Add 100 μL of 3 mM Glutamyl-L-Alanyl-L-Alanyl-L-Proyl-Leucine p-Nitroanilide (AAPL Peptide Institute) to 50 μL of the culture supernatant appropriately diluted with ₂ mM CaCl ₂ solution, and mix at 30 ° C. The amount of p-nitroaniline liberated when the reaction was performed was quantified by the change in absorbance at 420 nm (OD 420 nm). The amount of enzyme that liberates 1 μmol of p-nitroaniline per minute was defined as 1 U.

  The measurement results of S237 cellulase, M-protease, K38 amylase, and KP-43 protease activity are shown in FIG. In addition, the value shown by FIG. 2 shows an average value by a relative value, and an error bar shows a standard deviation. Values are calculated by culturing each in triplicate.

  As is clear from FIG. 2, in the transformant in which the S237 cellulase or M-protease expression vector was introduced into the ΔcssS strain, the production amount of the protein in the transformant introduced into the 168 strain (parent strain) was set as 100. S237 cellulase activity or M-protease activity was high, indicating that the productivity of S237 cellulase and M-protease was improved. On the other hand, in the transformant introduced with the K38 amylase or KP-43 protease expression vector in the ΔcssS strain, the K38 amylase activity or the KP-43 protease activity was lower than the transformant introduced into the 168 strain (parent strain). It was shown that the productivity of K38 amylase and KP-43 protease was decreased. From these results, in Bacillus subtilis, S237 cellulase and M-protease are relatively easily secreted enzyme proteins, whereas K38 amylase and KP-43 protease are difficult-to-secretive enzymes that stress the host when secreted. It is considered to be protein.

Example 1. Construction of recombinant microorganisms Construction Teikuron synthase-related genes of ΔtuaA-H strain deletions (TuAAs gene, TuaB gene, TUAC gene, tuaD gene, TuaE gene, TuaF gene, tua operon of eight genes tuaG gene and tuaH gene) A mutant strain was constructed. Construction of the mutant strain was performed by the double crossing method using a DNA fragment prepared by the SOE-PCR method in the same manner as the construction of the ΔcssS strain described above (see FIG. 1).
Using the genomic DNA extracted from Bacillus subtilis 168 as a template, and using the tuaA-177FrU primer and tuaA + 357RKm primer shown in Table 6, and the tuaH506FKm-2 primer and tuaH + 1059R primer set, the tuaA gene promoter 5 'end side 534 bp fragment (A) and 3' end side 553 bp fragment (B) were prepared. On the other hand, for the kanamycin resistance gene, the kanamycin resistance gene region was excised from the Eco RI restriction enzyme cleavage point of plasmid pDG873 (Gene, 167, 335, (1995)) and inserted into the Eco RI restriction enzyme cleavage point of pUC119 (TAKARA). PUCKm was constructed. Using the pUCKm DNA as a template, the kanamycin resistance gene region was amplified using the PB-M13-20 primer set and the PB-M13Rev primer set shown in Table 6 (3). The obtained DNA fragments of (A), (B) and (C) were mixed to form a template, and (A)-(C)-was obtained by SOE-PCR using tuaA-177FrU primer and tuaH + 1059R primer. The DNA fragments for gene deletion were obtained by binding in the order of (B).
168 strains of Bacillus subtilis were transformed by the competent cell transformation method using the prepared DNA fragment. Thereafter, colonies that grew on the LB agar medium containing kanamycin (10 μg / ml) were isolated as transformants. The genome of the resulting transformant was extracted, and the tuaA gene, tuaB gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene (tua operon) were deleted by PCR, and the kanamycin resistance gene was It was confirmed that it was replaced.
As described above, a strain lacking the tua operon of Bacillus subtilis was constructed and named ΔtuaA-H strain.

2. Measurement of the amount of teicuronic acid on the cell surface The cellulase gene was introduced into the obtained ΔtuaA-H strain and the parent strain (Bacillus subtilis 168 strain). Specifically, a DNA fragment (3.1 kb; SEQ ID NO: 17) encoding the S237 cellulase gene (see JP 2000-210081 A) (SEQ ID NO: 17) derived from the Bacillus genus KSM-S237 strain (FERM BP-7875) PCR using the Egl-S237.F primer and Egl-S237.R primer set shown in Table 7 using the base number 13 to 3124) of 17 as a template, and the Bam HI restriction enzyme cleavage point of shuttle vector pHY300PLK The recombinant plasmid pHY-S237 inserted into was constructed and introduced into each strain by the protoplast transformation method.

Cell walls were prepared from each strain into which the cellulase gene was introduced by the following method.
Each strain was cultured with shaking in 5 ml of LB medium at 30 ° C. for 15 hours. 0.6 mL of this culture solution was added to 30 mL of 2 × L-maltose medium (2% tryptone, 1% yeast extract, 1% sodium chloride, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline. ) And cultured with shaking at 30 ° C. for about 40 hours (OD600 nm = 20 to 30). The cells were collected by centrifugation (10000 rpm, 10 min, 20 ° C.) so that the total culture turbidity was about OD1500, and the cells were suspended in 20 ml of 3M LiCl. The bacterial suspension was boiled for 10 minutes, 0.1 mm size glass beads were added, homogenized (NISSEI Ace Homogenizer AM-8), and disruption of the bacterial cells was confirmed by microscopic observation. Thereafter, about 0.5 L of ion exchange water was added, the supernatant was centrifuged (KUBOTA KR-2000c rotor: RA-6, 3000 rpm, 5 min, 4 ° C.), and the resulting supernatant was further centrifuged (KUBOTA KR -2000c rotor: RA-3, 12,000 rpm, 10 min, 4 ° C). After suspending the precipitate in 20 ml of ion exchanged water, 20 ml of 8% SDS was added and the mixture was boiled for 10 minutes. Thereafter, the mixture is further centrifuged (KUBOTA KR-2000c rotor: RA-3, 12,000 rpm, 10 min, room temperature), the precipitate is suspended in 10 ml of 1M NaCl, and centrifuged (KUBOTA KR-2000c rotor: RA-3, 12). , 1,000 rpm, 10 min, room temperature). Suspension and centrifugation operations were repeated about 5 times under the above conditions to wash the precipitate. Further, the precipitate was subjected to substitution treatment about 5 times with 10 ml of ion exchange water, and then suspended in 2 ml of ion exchange water. This cell wall suspension was freeze-dried to obtain a powdered cell wall.

The amount of teicuronic acid contained in the powdered cell walls of each strain obtained was determined as follows.
Teicuronic acid is a polymer composed of N-acetylgalactosamine and glucuronic acid (Poly (GalNAc-GlcA)). Therefore, using the m-hydroxydiphenyl-sulfuric acid method (Anal. Biochem. 54, 484-489 (1973)) with the amount of uronic acid (glucuronic acid) as an index, ΔtuaA-H strain and Bacillus subtilis 168 strain (parent strain) ) Was determined. The results are shown in Table 8.

As can be seen from Table 8, the amounts of teicuronic acid per mg of cell wall of Bacillus subtilis 168 and ΔtuaA-H in the stationary phase were 0.24 μmol and 0.16 μmol, respectively.
Expression of teicronate synthase-related genes is induced in phosphate starvation. Since phosphorus is depleted in the stationary phase under the condition of this medium, it is considered that teicuronic acid synthase-related gene is expressed and teicuronic acid is synthesized in 168 strain. On the other hand, the amount of teicuronic acid in the cell wall of the ΔtuaA-H strain was reduced to about 60% compared to the 168 strain. From this, it is clear that ΔtuaA-H strain is more suppressed in the expression of teicronate synthase-related genes than the wild strain.

Example 2 Measurement of protein productivity In the same manner as in Test Example 2, the productivity of S237 cellulase, M-protease, K38 amylase and KP-43 protease was obtained using the ΔtuaA-H strain and the parent strain Bacillus subtilis 168 strain (wild strain) obtained in Measured and evaluated.
The measurement results of S237 cellulase, M-protease, K38 amylase, and KP-43 protease activity are shown in FIG. In addition, the value shown by FIG. 3 shows the relative value of an average value, and an error bar shows a standard deviation. Values are calculated by culturing each in triplicate.

As is apparent from FIG. 3, when the ΔtuaA-H strain was used as the host, the production amount of K38 amylase or KP secreted outside the bacterial cells was compared with the production amount of the control 168 strain (parent strain) as 100. -43 Protease production increased. Specifically, the production amount in the ΔtuaA-H strain was 1.14 times for K38 amylase and 1.38 times for KP-43 protease, compared to 168 strain (wild strain). On the other hand, in the transformant in which S237 cellulase or M-protease was introduced into the ΔtuaA-H strain, the productivity of these proteins was lower than that in the 168 strain.
From these results, it was found that mutant microorganisms that were genetically modified to suppress the expression of teicuronate synthase-related genes were useful as protein production hosts. In particular, the microorganisms of the present invention showed excellent productivity in the production of proteins such as K38 amylase and KP-43 protease, the productivity of which is reduced in the Bacillus subtilis cssS gene-deficient strain. From this, it is considered that the microorganism of the present invention can be suitably used for the production of a protein or polypeptide that is easily stressed during secretion.

Claims (11)

All of tuaA gene, tuaB gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene, or all genes corresponding to the gene , which are genes related to teicronate synthase of Bacillus subtilis the gene construct Bacillus (Bacillus) fungi belonging to the genus as current suppression, met recombinant microorganism productivity was introduced a gene encoding a protein or polypeptide decreases in cssS gene-deficient strain of Bacillus subtilis A recombinant microorganism, wherein the protein or polypeptide is amylase, pneumolysin, or a protease comprising the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having 90% or more identity with the sequence . The recombinant microorganism according to claim 1, wherein one or more genes selected from the group consisting of the teicuronic acid synthase-related gene and a gene corresponding to the gene are deleted. The amylase is a K38 amylases having the amino acid sequence shown in SEQ ID NO: 24, a set of claim 1, wherein said protease is KP-43 protease comprising the amino acid sequence shown in SEQ ID NO: 26 Replacement microorganism. One or more regions of a transcription initiation control region, a translation initiation control region, or a signal region for secretion are bound upstream of a gene encoding a protein or polypeptide that decreases in productivity in the cssS gene-deficient strain of Bacillus subtilis . The recombinant microorganism according to any one of claims 1 to 3 , wherein: 3 regions consisting of a transcription initiation control region, a translation initiation control region and a secretion signal region are linked upstream of a gene encoding a protein or polypeptide whose productivity is reduced in the cssS gene-deficient strain of Bacillus subtilis. The recombinant microorganism according to any one of claims 1 to 4 . The secretion signal region is derived from a cellulase gene of a fungus 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 according to claim 4 or 5 , characterized in that. The transcriptional initiation regulatory region, the translation initiation regulatory region and a 3 region consisting secretion signal region, claims 4-6, characterized in that a DNA fragment comprising any one of the following nucleotide sequence (a) ~ (c) The recombinant microorganism according to any one of the above.
(A) the base sequence of base numbers 1 to 659 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 17 or the base sequence of base numbers 1 to 696 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 19 (b) (C) A part of the nucleotide sequence of any one of the nucleotide sequences (c) and (a) having 90 % or more identity to any nucleotide sequence of (a) has been deleted, substituted, inserted and / or added. Base sequence The recombinant microorganism according to any one of claims 1 to 7 , wherein the fungus belonging to the genus Bacillus is Bacillus subtilis . An amylase, pneumolysin, or SEQ ID NO: 26, which is a protein or polypeptide that decreases in productivity in the cssS gene-deficient strain of Bacillus subtilis using the recombinant microorganism according to any one of claims 1 to 8. Or a method for producing a protease comprising an amino acid sequence having 90% or more identity with the amino acid sequence . All of tuaA gene, tuaB gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene, or all genes corresponding to the gene , which are genes related to teicronate synthase of Bacillus subtilis genetically build belonging fungi Bacillus (Bacillus) genus to be current suppression, and is a protein or polypeptide to reduced productivity in cssS gene-deficient strain of Bacillus subtilis, amylase, pneumolysin, or SEQ ID NO: 26 A method for producing a recombinant microorganism, comprising introducing a gene encoding a protease comprising the amino acid sequence shown or an amino acid sequence having 90% or more identity with the amino acid sequence . Using the recombinant microorganism according to any one of claims 1 to 8 , amylase, pneumolysin, or SEQ ID NO: 26, which is a protein or polypeptide that decreases in productivity in a cssS gene-deficient strain of Bacillus subtilis The production of a protein or polypeptide with reduced productivity in a cssS gene-deficient strain of Bacillus subtilis characterized by producing a protease comprising the amino acid sequence shown or an amino acid sequence having 90% or more identity with the amino acid sequence. How to improve.

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