JP2011234685A - Recombinant microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a recombinant microorganism improved in the productivity of a protein or polypeptide, to provide a method for producing the recombinant microorganism, to provide a method for producing the objective protein or polypeptide with the recombinant microorganism, and to provide a method for improving the productivity of the protein or polypeptide.SOLUTION: There are provided the recombinant microorganism prepared by transferring a gene encoding the objective protein or polypeptide to a microorganism whose gene is built so that the expression of a teichuronic acid-synthetase-related gene group comprising the tuaA gene, tuaB gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene of Bacillus subtilis, or genes corresponding to the gene group are reinforced; the method for producing the recombinant microorganism; the method for producing the objective protein or polypeptide with the recombinant microorganism; and the method for improving the productivity of the objective protein or polypeptide.

Description

  The present invention relates to a recombinant microorganism used for producing 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 the protein productivity in order to obtain microorganisms with further 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 has been found that the amount of teicuronic acid, which is a kind of anionic polymer on the cell surface, increases in the microorganism by constantly expressing the teicuronate synthase-related gene group possessed by 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.

In the present invention, the expression of a teicronate synthase-related gene group consisting of a Bacillus subtilis tuaA gene, tuaB gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene or a gene corresponding to the gene group is expressed. The present invention relates to a recombinant microorganism in which a gene encoding a target protein or polypeptide is introduced into a microorganism that has been genetically constructed to be enhanced.
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 a recombinant engineered so that the gene encoding the target protein or polypeptide is introduced and the expression of the teicronate synthase-related gene group or the gene corresponding to the gene group is enhanced. The present invention relates to a method for improving the productivity of a target protein or polypeptide, which comprises producing the protein or polypeptide using a microorganism.

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 whose productivity does not decrease in the cssS gene-deficient strain of Bacillus subtilis.

1 schematically shows a method for preparing a DNA fragment for introduction by the SOE-PCR method and a method for constructing a gene so that a target gene is constitutively expressed by transforming a parent strain using the DNA fragment. The preparation method of the DNA fragment for introduction | transduction by SOE-PCR method and the method of deleting a target gene using the said DNA fragment (it replaces with a drug resistance gene) are shown typically. 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 with enhanced tua operon expression (PrplU-tuaA-H strain), when the production in the wild strain (168 strain) is 100 It is a relative value. 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 corresponds to a teicronate synthase-related gene group consisting of tuaA gene, tuaB gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene derived from Bacillus subtilis or the gene group 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 the gene to be enhanced is enhanced. As a result of the enhanced expression of teicuronate synthase-related genes in this recombinant microorganism, the amount of teicuronic acid on the cell surface is increased regardless of the amount of phosphate in the medium compared to the parent microorganism, Compared with the parent microorganism into which the target protein or the like has been introduced, the productivity of the protein or the like is greatly improved.

In the present invention, the teicuronic acid synthase-related gene group of Bacillus subtilis refers to a series of genes involved in biosynthesis of teicuronic acid, which is a kind of anionic polymer on the cell surface. Specifically, the Teicronate synthase-related gene group derived from Bacillus subtilis consists of tuaA gene, tuaB gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene. These eight genes constitute the teicronate operon (tua operon) (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 (tua operon) 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 and the amount of teicuronic acid is low. In the state depleted of phosphoric acid (for example, after the stationary phase), teicuronic acid is mainly synthesized.
So far, mutant strains in which the promoter region of teicuronic acid expressed upon phosphate depletion is replaced with the IPTG-inducible promoter Pspac have been reported. It has been reported that this mutant synthesizes taicuronic acid on the cell surface even when phosphate is not depleted by addition of IPTG (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 amount of teicuronic acid on the cell surface of this mutant strain is about half of the amount of teicuronic acid in the wild strain when phosphate is depleted, even when induced by IPTG. Furthermore, the above-mentioned document does not show any knowledge regarding 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 constituting the teicronate synthase-related gene group of Bacillus subtilis are genes each comprising the following base sequences: Say.
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 functions and the like of each gene constituting the teicronate synthase-related gene group 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 group of Bacillus subtilis” refers to a gene functionally equivalent to the teicuronate synthase-related gene group of Bacillus subtilis. Examples of genes corresponding to the teicronate synthase-related gene group 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, a protein functionally equivalent to the protein consisting of the amino acid sequence represented by any of SEQ ID NOs: 9 to 16 is encoded by the above eight genes constituting the teicronate synthase-related gene group of Bacillus subtilis. The protein which has substantially the same function as the protein to be used and is involved in the synthesis of teicronate in the cell surface layer. Specifically, the functions described in Table 1 are listed as functions of the above eight genes constituting the teicronate synthase-related genes of Bacillus subtilis and the 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 one of SEQ ID NOs: 9 to 16, the amino acid sequence consists of an amino acid sequence in which 1 to several amino acids are 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 genes in 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 Examples thereof include genes and tuaH homologous genes, which are preferably used in the present invention.
Hereinafter, `` Teicronate synthase related gene group consisting of tuaA gene, tuaB gene, tuaC gene, tuaD gene, tuaE gene, tuaF gene, tuaG gene and tuaH gene of Bacillus subtilis or a gene corresponding to the gene group '' It is also called teicronate synthase-related gene group or teicronate operon (tua operon).

The parent microorganism for constructing the microorganism of the present invention is not particularly limited as long as it has a teicronate synthase-related gene group 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 so that these parent microorganisms are genetically modified to enhance the expression of teicronate synthase-related genes.
Examples of methods for enhancing the expression of genes related to teicronate synthase include, for example, a method of introducing a transcription initiation control region such as a promoter that is constitutively expressed in the parent microorganism, Examples thereof include a method for introducing a mutation that increases the activity of the acting transcription factor PhoP, a method for introducing a teicronate synthase-related gene into a plasmid, and retaining it in a cell at a high copy number. Among them, in the present invention, it is preferable to constitutively express a teicronate synthase-related gene group by introducing a transcription initiation control region having a function in the parent microorganism upstream of the teicronate operon genome. By adopting such a configuration, it is possible to constantly express a teicronate synthase-related gene group that is originally expressed in a phosphate-starved state. In the present invention, the constitutive expression of teicuronate synthase-related genes is constitutively expressed regardless of the amount of phosphate present in the growth environment (for example, in the medium). Is expressed.

In the present invention, the transcription initiation control region that functions in a microorganism is a transcription initiation control region that functions in a host microorganism (parental microorganism) and can constitutively express teicronate synthase-related genes. There is no particular limitation. Preferably, a transcription initiation control region located upstream of the Bacillus subtilis rplU gene, or a spoVG gene, rpsG gene, fusA gene, tuf gene, rplX gene, malA gene, ykrS gene having a high expression level as a gene corresponding to the gene. , YkrT gene, rplS gene, cspD gene, hbs gene, ypfD gene, sodA gene, yqeY gene, rpsU gene, rpmA gene, ysxB gene, icd gene, hag gene, yydF gene It is done. In the present invention, the transcription initiation control region refers to a region including a promoter and a transcription initiation site.
Examples of the transcription initiation control region derived from the Bacillus subtilis rplU gene include DNA consisting of the base sequence shown in SEQ ID NO: 17, or DNA consisting of a base sequence homologous to the sequence and having a function as a transcription initiation control region. . Examples of the base sequence homologous to the base sequence shown in SEQ ID NO: 17 include, for example, a base sequence having 90% or more, preferably 95% or more, more preferably 99% or more identity with the base sequence shown in SEQ ID NO: 17, Alternatively, a base sequence consisting of DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 17 can be mentioned. Here, the method for calculating the identity of the base sequence and the stringent conditions are the same as those described above.

  A transcription initiation control region that functions in these microorganisms is introduced upstream of the genome of a teicronate synthase-related gene group. In the present invention, it is preferable to insert the transcription initiation control region upstream of the tecronate synthase-related gene group, with the transcription initiation control region and the ribosome binding site originally retained. The upstream in the genome into which the transcription initiation control region is introduced is not particularly limited as long as it is upstream of the transcription initiation region and the ribosome binding site inherent in the teicronate synthase-related gene group, but within the adjacent 2000 base pairs. A region is preferred, a region within 500 base pairs is more preferred, a region within 100 base pairs is more preferred, and a region within 50 base pairs is particularly preferred. The ribosome binding site is a site corresponding to the Shine-Dalgarno (SD) sequence (Proc. Natl. Acad. Sci. USA 74, 5463 (1974)) that forms a translation initiation control region together with the initiation codon.

  The introduction of the transcription initiation control region can be performed, for example, using a usual method by homologous recombination. Specifically, a DNA fragment and a drug resistance gene fragment containing the transcription initiation control region inherent to the teicronate synthase-related gene group and the upstream region of the ribosome binding site upstream of the DNA fragment containing the transcription initiation control region, On the other hand, a DNA fragment containing a part of the transcription initiation control region and ribosome binding site and the structural gene region of the teicronate synthase-related gene group downstream is subjected to SOE (splicing by overlap extension) -PCR (Gene, 77, 61, 1989). In this way, a DNA fragment containing the original transcription initiation control region and the upstream region from the ribosome binding site of the teicronate synthase-related gene group, a drug resistance gene fragment, a DNA fragment containing the transcription initiation control region, and teicuronic acid synthase A DNA fragment in which the original transcription initiation control region and the ribosome binding site of the related gene group and a part of the structural gene region are joined in this order can be obtained (see FIG. 1).

Next, by incorporating such a DNA fragment into the parent microbial cell by a normal method, a region upstream of the original transcription initiation control region of the tecronate synthase-related gene group of the parent microbial genome, and tecronate synthase-related Double crossover homologous recombination occurs in two places, the transcription initiation region of the gene group and the region including a part of the ribosome binding site and the structural gene region (see FIG. 1). As a result, a transformant in which the transcription initiation control region is inserted upstream of the original transcription initiation control region and the ribosome binding site can be isolated using the drug resistance gene as an index. As a result, the original transcription initiation control region of the teicronate synthase-related gene group on the genome and the transcription initiation control region introduced into the upstream region of the ribosome binding site are genetically stably maintained.
Specifically, as a usual method for introducing a DNA fragment for introduction into a host microorganism, a competent cell transformation method (J. Bacterial. 93, 1925 (1967)), a protoplast transformation method (Mol. Gen. Genet. 168). 111 (1979)) or electroporation (FEMS Microbiol. Lett. 55, 135 (1990)), etc., and the competent cell transformation method is preferred in the present invention.
Further, in the present specification, upstream / downstream of a gene is not a position from the replication start point, and upstream indicates a region continuing on the 5 ′ side of the gene or region considered as a target, while downstream is The region following the 3 ′ side of the gene or region captured as the target is shown.
In particular, using Bacillus subtilis as a host of the microorganism of the present invention, when inserting the transcription initiation control region of Bacillus subtilis rplU gene upstream of the original transcription initiation control region of the teicuronate synthase-related genes and the ribosome binding site, Introduction by homologous recombination is described in Mol. Gen. Genet., 223, 268, 1990 can be used.

Hereafter, the DNA fragment prepared by SOE (splicing by overlap extension) -PCR method (Gene, 77, 61, 1989) was used, and the transcription initiation control region was transferred to the parent microorganism genome by double crossover homologous recombination. A method for introducing the gene into the upstream of the synthase-related gene group will be specifically described.
The introduction DNA fragment used is about 0.1 to 3, preferably 0.4 to 3 kb fragment (hereinafter referred to as fragment (1)) adjacent to the upstream of the introduction site on the parent microorganism genome, and about 0 adjacent to the downstream. .1 to 3, preferably 0.4 to 3 kb fragment (hereinafter referred to as fragment (2)), a drug resistance marker gene fragment (hereinafter referred to as fragment (3)), a fragment containing the transcription initiation control region (hereinafter referred to as “fragment”) The DNA fragment into which the fragment (4) is inserted First, four fragments of the fragment (1) to the fragment (4) are prepared by the first PCR, for example, at the downstream end of the fragment (1). 10-30 base pair sequence upstream of (3), 10-30 base pair sequence downstream of fragment (3) at the upstream end of fragment (4), and downstream of fragment (4) upstream of fragment (2) Use primers designed to add a 10-30 base pair sequence on the side (see Figure 1). ).

Next, using the four types of PCR fragments prepared in the first round as templates, the second PCR is performed using the upstream primer of fragment (1) and the downstream primer of fragment (2). This causes annealing with the fragment (3) in the sequence of the fragment (3) added to the downstream end of the fragment (1). Similarly, the sequence of the fragment (3) added to the upstream end of the fragment (4) Annealing with the fragment (3) occurs in the sequence, and an annealing with the fragment (4) occurs in the sequence of the fragment (4) added to the upstream end of the fragment (2). As a result of PCR amplification, a DNA fragment for introduction in which fragments (1) to (4) are bound in the order of (1), (3), (4), and (2) can be obtained (see FIG. 1).
The PCR reaction carried out here uses the primer set shown in Table 6 of the Examples described later, and uses a general PCR enzyme kit such as Pyrobest DNA Polymerase (Takara Shuzo) and the like (PCR Protocols. Current Methods and Applications, Edited by BAWhite, Humana Press pp251, 1993, Gene, 77, 61, 1989) and the like.

  The obtained DNA fragment for introduction is introduced into cells by a competent transformation method or the like. In the homologous region upstream and downstream of the introduction site on the genome having the same identity as the introduced DNA fragment, gene recombination occurs in the cell, and the transcription initiation control region inherently belongs to the teicronate synthase-related gene group. A transformant (recombinant microorganism) having a genome linked upstream of the transcription initiation regulatory region and the ribosome binding site can be obtained. Transformants can be isolated by selection with drug resistance markers. As a drug marker gene to be used, a kanamycin resistance gene or the like can be used. Selection with a drug resistance marker can be performed, for example, by isolating colonies that grow on an agar medium containing kanamycin and then selecting those that are confirmed to be introduced onto the genome by PCR using the genome as a template. Good. The drug resistance marker gene is not particularly limited as long as it can be used for selection using commonly used antibiotics. In addition to the kanamycin resistance gene, the chloramphenicol resistance gene, neomycin resistance And drug resistance marker genes such as genes, spectinomycin resistance genes, tetracycline resistance genes and blasticidin S resistance genes.

  The microorganism of the present invention can be prepared by introducing a gene encoding a target protein or polypeptide into a microorganism constructed so as to constitutively express teicronate synthase-related genes. it can. 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 is not particularly limited, and includes various industrial enzymes such as detergents, foods, fiber treatments, feed treatments, cosmetics, pharmaceuticals, and diagnostic agents, and bioactive peptides. In addition, 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 does not decrease 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: 56, and the amino acid sequence of the Bacillus subtilis CssS protein is shown in SEQ ID NO: 57, 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 CssSR 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, OP 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 does not decrease 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 does not decrease 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 0.9 or more. Specific examples of the protein or polypeptide whose productivity does not decrease in the Bacillus subtilis cssS gene-deficient strain include the following hydrolases such as cellulase and protease.

  Cellulases include, for example, cellulases belonging to Family 5 in the classification of polysaccharide hydrolases (Biochem. J., 280, 309, 1991). Among them, cellulases derived from microorganisms, particularly fungi belonging to the genus Bacillus are mentioned. It is done. As a more specific example, an alkaline cellulase derived from the Bacillus bacterium strain KSM-S237 (FERM BP-7875) comprising the amino acid sequence represented by SEQ ID NO: 19, or a Bacillus bacterium KSM comprising the amino acid sequence represented by SEQ ID NO: 21 -64 strain (FERM BP-2886) -derived alkaline cellulase or the same amino acid sequence as 70%, preferably 80%, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more. A cellulase consisting of an amino acid sequence having the property, or an alkaline cellulase consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 19 or 21 It is done.

Specific examples of proteases include those derived from microorganisms, particularly serine proteases belonging to high alkaline proteases (see Extremophiles 8, 229-235. (2004)). As a more specific example, an alkaline protease (M-protease) derived from Bacillus clausii strain KSM-K16 (FERM BP-3376) consisting of the amino acid sequence represented by SEQ ID NO: 23, or 70% Or a protease comprising an amino acid sequence having an identity of preferably 80%, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, or one or a number in the amino acid sequence represented by SEQ ID NO: 23 Examples include alkaline protease consisting of an amino acid sequence in which a single amino acid is deleted, substituted, inserted and / or added.

The target protein or target polypeptide gene to be 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, ribosome It is desirable that at least one region selected from a translation initiation control region including a binding 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 Japanese Patent Application Laid-Open No. 2000-210081, Japanese Patent Application Laid-Open No. 4-190793, etc., that is, KSM-S237 strain (FERM BP-7875), KSM-64 strain (FERM BP-2886) It is desirable that the cellulase gene derived from) and the transcription initiation control region, translation initiation control region, and secretory signal peptide region of the cellulase gene are appropriately combined with the structural gene of the target protein or polypeptide. 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: 18 or the base sequence of base numbers 1 to 696 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 20 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.
As shown in the examples described later, the microorganism of the present invention has significantly improved productivity of the target protein or polypeptide compared to the microorganism not subjected to the genetic modification. In particular, the microorganism of the present invention has the advantage that productivity is improved when a protein or polypeptide that does not decrease 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. 2). 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: 18) encoding (SEQ ID NO: 18) is inserted at the Bam HI restriction enzyme breakpoint of the 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 carried out using a primer set of -R (BglII) primer, and the signal sequence of M-protease, the pro-sequence and the nucleotide sequence (SEQ ID NO: 22) of the gene encoding M-protease (see Patent No. 3026111) A 1.2-kb DNA fragment containing the mature enzyme region (base numbers 163 to 1387 of SEQ ID NO: 22) 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 shown in SEQ ID NO: 18 (Japanese Patent Laid-Open No. 2000-210081) (base of SEQ ID NO: 18) 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 the primer set of SP64K38-R (XbaI), and the alkaline amylase AmyK38 represented by SEQ ID NO: 24 (JP 2000-184882, 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: 24) 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: 18 (Japanese Patent Laid-Open No. 2000-210081) (Base numbers 13 to 572 of SEQ ID NO: 18) 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 the alkaline protease KP-43 shown in SEQ ID NO: 26 (hereinafter simply referred to as “KP-43 protease”) (WO99 / 18218 pamphlet: GenBank accession no AB051423) a 2.0 kb DNA fragment (base numbers 1 to 2015 of SEQ ID NO: 26) 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: 18) containing the transcription initiation control region of the alkaline cellulase gene shown in SEQ ID NO: 18 (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. 3 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. 3, in the transformant in which the S237 cellulase or M-protease expression vector was introduced into the ΔcssS strain, the production amount in the case of the transformant introduced into the 168 strain (parent strain) was set to 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 of PrplU-tuaA-H Teicronate synthase-related genes ( tuaA , tuaB , tuaC , tuaD , tuaE , tuaF , tuaG, and tuaH genes) A constitutively expressed mutant 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 genomic DNA extracted from Bacillus subtilis 168 as a template, each of cwlB + 982F primer, lytC + 1494RKm primer, tuaA-177FrU primer and tuaA + 357RKm primer, rplU-482FKm primer and rplU-1R primer shown in Table 6 Using a primer set, a 534 bp fragment at the 3 ′ end of the lytC gene (1), a 553 bp fragment at the 5 ′ end of the tuaA gene containing the tua operon promoter (2), and an rplU gene encoding the 50S ribosomal protein L21 Promoter region: rplU promoter (4) was 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 (1), (2), (3) and (4) were mixed and used as a template, and by the SOE-PCR method using CB + 982F primer and tuaA + 357RKm primer, (1) − The DNA fragments for introduction were obtained by binding in the order of (3)-(4)-(2).
168 strains of Bacillus subtilis were transformed by the competent cell transformation method using the prepared DNA fragment for introduction. Thereafter, colonies that grew on the LB agar medium containing kanamycin (10 μg / ml) were isolated as transformants. Genomic DNA of the obtained transformant was extracted, and it was confirmed by PCR that the rplU promoter was inserted upstream of the tua operon promoter and the kanamycin resistance gene was located further upstream. The sequence confirmed that there was no mutation in the rplU promoter sequence.
As described above, a strain in which the rplU promoter was inserted upstream of the Bacillus subtilis tua operon promoter was constructed and named PrplU-tuaA-H.

2. Measurement of the amount of teicuronic acid on the cell surface The cellulase gene was introduced into the obtained PrplU-tuaA-H strain and the parent strain (168 strains of Bacillus subtilis), respectively. Specifically, a DNA fragment (3.1 kb; SEQ ID NO: 18) encoding the S237 cellulase gene (see Japanese Patent Application Laid-Open No. 2000-210081) (SEQ ID NO: 18) derived from the KSM-S237 strain (FERM BP-7875) of the genus Bacillus PCR was performed using the Egl-S237.F primer and Egl-S237.R primer set shown in Table 7 using the 18 base numbers 13 to 3124) as a template, and the Bam HI restriction enzyme cleavage point of the 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, PrplU-tuaA-H strain and Bacillus subtilis 168 strain as a control The amount of teicuronic acid of (parent strain) was quantified. The results are shown in Table 8.

As apparent from Table 8, the amounts of teicuronic acid per 1 mg of the cell wall of Bacillus subtilis 168 and PrplU-tuaA-H in the stationary phase were 0.24 μmol and 0.42 μmol, respectively.
Expression of teicronate synthase-related genes is induced in phosphate starvation. Since phosphorus is depleted in the stationary phase under the conditions of this medium, the teicronate synthase-related gene group is expressed to some extent even in 168 strains. Nevertheless, the amount of teicuronic acid in the cell wall of the PrplU-tuaA-H strain was increased about 1.7 times compared to the 168 strain. From this, it is clear that in PrplU-tuaA-H strain, teicronate synthase-related gene group (tua operon) is expressed at a higher level than in the wild strain.

Example 2 Measurement of protein productivity Production of S237 cellulase, M-protease, K38 amylase, and KP-43 protease in the same manner as in Test Example 2 for the PrplU-tuaA-H strain obtained in 1) and the parent strain Bacillus subtilis 168 strain (wild strain) Sex was 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 in FIG. 4 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 can be seen from FIG. 4, when PrplU-tuaA-H strain is used as the host, S237 cellulase production secreted outside the cells in comparison with the production amount of control 168 strain (parent strain) as 100 It can be seen that the amount or the amount of M-protease production is increased. Specifically, the production amount in the PrplU-tuaA-H strain was 1.28 times for the S237 cellulase and 1.36 times for the M-protease compared to the 168 strain (wild strain). On the other hand, in the transformants in which K38 amylase or KP-43 protease was introduced into the PrplU-tuaA-H strain, the productivity of these proteins was similar or slightly lower than that in the case of 168 strains. Little improvement was seen.
From these results, it was found that the mutant microorganism genetically modified to enhance the expression of the teicronate synthase-related gene group (tua operon) is useful as a protein production host. In particular, the microorganism of the present invention showed excellent productivity in the production of proteins such as S237 cellulase and M-protease that did not decrease in productivity in Bacillus subtilis cssS gene-deficient strains. 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 relatively easily secreted and hardly causes stress during secretion.

Claims (14)

Expression of genes related to teicronate synthase , consisting of the Bacillus subtilis tuaA , tuaB , tuaC , tuaD , tuaE , tuaF , tuaG, and tuaH genes, or genes corresponding to the genes, should be enhanced. A recombinant microorganism obtained by introducing a gene encoding a target protein or polypeptide into a microorganism that has been genetically constructed. The recombinant microorganism according to claim 1, wherein the target protein or polypeptide is a protein or polypeptide whose productivity does not decrease in a cssS gene-deficient strain of Bacillus subtilis.   A transcription initiation control region having a function in a microorganism is introduced upstream in the genome of the teicuronate synthase-related gene group or a gene corresponding to the gene group, and is introduced into the teicuronate synthase-related gene group or the gene group. The recombinant microorganism according to claim 1 or 2, wherein the corresponding gene is constitutively expressed. The recombinant microorganism according to claim 3, wherein the transcription initiation control region having a function in the microorganism is derived from the rplU gene of Bacillus subtilis or a gene corresponding to the gene.   The protein or polypeptide of interest is S237 cellulase having the amino acid sequence shown in SEQ ID NO: 19 or M-protease having the amino acid sequence shown in SEQ ID NO: 23. The recombinant microorganism according to item 1.   6. One or more regions of a transcription initiation control region, a translation initiation control region, or a secretion signal region are bound upstream of a gene encoding the target protein or polypeptide. The recombinant microorganism according to any one of the above.   The three regions comprising a transcription initiation control region, a translation initiation control region, and a secretion signal region are linked upstream of a gene encoding the target protein or polypeptide. Recombinant microorganism described in 1. 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 6 or 7, characterized in that The three regions comprising the transcription initiation control region, translation initiation control region and secretory signal region are DNA fragments comprising any one of the following base sequences (a) to (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: 18 or the base sequence of base numbers 1 to 696 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 20 Base sequence having 70% or more identity to any base sequence of (a) (c) A base in which a part of any base sequence of (a) is deleted, substituted, inserted and / or added Array The recombinant microorganism according to any one of claims 1 to 9, wherein the microorganism is a fungus belonging to the genus Bacillus . The microorganism according to any one of claims 1 to 10, wherein the microorganism is Bacillus subtilis .   The manufacturing method of the target protein or polypeptide using the recombinant microorganism of any one of Claims 1-11. Expression of genes related to teicronate synthase , consisting of the Bacillus subtilis tuaA , tuaB , tuaC , tuaD , tuaE , tuaF , tuaG, and tuaH genes, or genes corresponding to the genes, should be enhanced. A method for producing a recombinant microorganism, comprising constructing a microorganism and introducing a gene encoding a target protein or polypeptide. Tycronate synthase-related gene group into which a gene encoding the protein or polypeptide of interest has been introduced and which comprises the Bacillus subtilis tuaA , tuaB , tuaC , tuaD , tuaE , tuaF , tuaG, and tuaH genes Alternatively, by using a recombinant microorganism that has been constructed so that expression of genes corresponding to the gene group is enhanced, the protein or polypeptide is produced, and thus the productivity of the target protein or polypeptide is improved. How to make.

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