JP6059416B2 - Microorganism in which lysis is inhibited, method for producing the microorganism, method for producing protein using the microorganism, and method for inhibiting lysis of microorganism - Google Patents

Description

  The present invention relates to a microorganism in which lysis is suppressed, a method for producing the microorganism, a method for producing a protein or polypeptide using the microorganism, and a method for inhibiting lysis of the 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 application has been extended to a wide range of fields from foods, medicines, daily necessaries such as detergents and cosmetics to various chemical raw materials.

In industrial production of useful substances by such microorganisms, improvement of productivity is one of the important issues, and breeding of produced bacteria by genetic techniques such as mutation has been performed as a technique. In recent years, the development of microbial genetics and biotechnology has led to more efficient breeding of production microorganisms using genetic recombination techniques, and the development of host microorganisms for genetic recombination has been promoted. It has been. For example, a strain obtained by further improving a microbial strain recognized as safe and excellent as a host microorganism, such as Bacillus subtilis Marburg No.168 strain, has been developed.

  In the case of producing a substance using such a microorganism, it is desirable to be able to control the lysis phenomenon of the microorganism to be used, and various methods have been proposed so far as methods for inhibiting lysis of microorganisms. For example, a method for suppressing lysis of microorganisms by deleting a gene encoding a cell wall lytic enzyme (see Patent Document 1 and Non-Patent Document 1), and inactivating cell wall lytic enzyme by high expression of cell wall lytic enzyme inhibitory protein And a method for improving resistance to cell wall lytic enzymes (see Patent Document 3) have been proposed.

JP 2005-295830 A JP 2008-118985 A JP 2009-55858 A

Journal of Bioscience and Bioengineering, Vol.103, No.1, p.13-21 (2007)

  An object of the present invention is to provide a method for suppressing lysis in a microorganism that is a host for substance production and a microorganism in which lysis is suppressed. Another object of the present invention is to provide a method for producing a target substance such as a protein using a microorganism in which lysis is suppressed. Furthermore, an object of the present invention is to provide a method for producing a microorganism in which lysis is suppressed.

  In view of the above problems, the inventors have performed artificial manipulation such as deletion on a group of cell wall lytic enzymes (hereinafter also referred to as cell wall degrading enzymes or lytic enzymes) that are important factors for cell division and cell proliferation. We searched for a method to effectively suppress the lysis of microorganisms and improve the productivity of target substances such as proteins. Focusing on the fact that cell wall lytic enzymes bind to anionic polymers present in bacterial cell walls, and reducing the amount of anionic polymer in the cell walls, the binding force of cell wall lytic enzymes to the cell walls can be reduced. We thought that it might become a technique for suppressing lysis Based on this idea, we intensively studied a method for reducing the amount of anionic polymer in the cell wall, and by suppressing the expression of cell wall teichoic acid synthase-related gene derived from Bacillus subtilis, the amount of teichoic acid in the cell wall decreases, As a result, it was found that lysis was suppressed in microorganisms with a reduced amount of teichoic acid in the cell wall. Furthermore, the present inventors have found that this microorganism can be suitably used for producing target substances such as proteins, and have completed the present invention.

The present invention relates to a microorganism in which expression of at least one gene of a cell wall teichoic acid synthase-related gene of Bacillus subtilis or a gene corresponding to the gene is suppressed and lysis is suppressed.
The present invention also relates to a method for producing a protein or polypeptide using the microorganism.
The present invention also relates to a cell wall teichoic acid synthase-related gene possessed by Bacillus subtilis or a microorganism corresponding to the gene, wherein at least one of the cell wall teichoic acid synthase-related gene possessed by Bacillus subtilis or the gene corresponding to the gene. The present invention relates to a method for suppressing lysis of microorganisms, which comprises a step of suppressing expression of one gene.
Furthermore, the present invention relates to a method for producing a microorganism with suppressed lysis characterized by suppressing the expression of at least one gene of a cell wall teichoic acid synthase-related gene or a gene corresponding to the gene possessed by Bacillus subtilis. About.

According to the present invention, it is possible to provide a method for suppressing lysis and a microorganism in which lysis is suppressed in a microorganism that is a host for substance production. Further, according to the present invention, a method for efficiently producing a target substance such as a protein or polypeptide can be provided by using a microorganism in which lysis is suppressed. Furthermore, according to the present invention, a method for producing a microorganism in which lysis is suppressed can be provided.
The microorganism according to the present invention can significantly suppress the lysis in a mutant microbial strain having a faster lysis rate than, for example, a wild strain, and the microorganism strain is cultured for a long time in the course of production of the target substance. That is, the target substance can be efficiently produced for a long time. Furthermore, there is no leakage of intracellular proteins or nucleic acids due to cell wall lysis, and the target protein and the like can be easily recovered from the culture solution. Therefore, by using the microorganism according to the present invention, it is possible to construct a production system in which the productivity of the target substance is improved.

As one of the preferred embodiments for constructing the microorganism of the present invention, a method for constructing a microorganism strain (Dpr9 / PtagF strain) in which the expression of the tagF gene can be controlled by replacing the promoter upstream of the tagF gene with a spac promoter. It is the schematic diagram which showed. It is the figure which showed typically the method of preparing the DNA fragment for gene deletion introduction by SOE-PCR, and deleting the target gene using the said DNA fragment (substitution with a drug resistance gene). It is the graph which showed the time-dependent change of the cell growth curve when Dpr9 / PtagF strain | stump | stock was culture | cultivated on the culture conditions which do not contain IPTG so that expression of a tagF gene may be suppressed. As a control, the results using the Dpr9 strain are also shown. It is a figure which shows the result of having analyzed the lytic enzyme activity of Dpr9 / PtagF strain | stump | stock and Dpr9 strain | stump | stock as a control | contrast by a zymogram. A white band corresponds to lytic enzyme activity. The Dpr9 / PtagF (pHLApm) strain and, as a control, the Dpr9 (pHLApm) strain were cultured, and the lipase productivity in the culture supernatant was electrophoresed at each time point after 25 hours, 50 hours and 75 hours from the start of the culture ( It is a figure which shows the result analyzed by SDS-PAGE.

  The microorganism according to the present invention is a cell wall teichoic acid synthase-related gene possessed by Bacillus subtilis or a gene in which expression of at least one of genes corresponding to the gene is suppressed. More specifically, the microorganism according to the present invention, for example, suppresses the expression of at least one cell wall teichoic acid synthase-related gene or a gene corresponding to the gene in a mutant strain having a faster lysis rate than the wild type. Thus, the lysis is significantly suppressed, and it can be suitably used in a production system for a target substance such as a protein using a microorganism.

Generally, bacteria have various lytic enzymes, and these enzymes generally have a high isoelectric point, have a binding domain for the cell wall, and ionic bond with an anionic polymer that is a constituent of the cell wall. Bacterial cell walls contain anionic polymers such as cell wall teichoic acid (major teichoic acid and minor teichoic acid), teicuronic acid, and lipoteichoic acid. Among them, it is known that the abundance ratio of cell wall teichoic acid formed by combining major teichoic acid and peptidoglycan is high. However, there are multiple factors involved in cell wall lysis by lytic enzymes, and the knowledge of whether or not the lysis phenomenon in bacteria can be suppressed by reducing the amount of anionic polymers in the cell wall including major teichoic acid Not obtained. The present invention reduces the cell wall teichoic acid formed by binding major teichoic acid and peptidoglycan as an anionic polymer, thereby reducing the electrostatic interaction between the cell wall and the lytic enzyme, and a new lysis It was made based on the new knowledge of establishing a suppression technology.
That is, the microorganism according to the present invention suppresses the expression of at least one of a cell wall teichoic acid synthase-related gene or a gene corresponding to the gene possessed by Bacillus subtilis, so that the amount of anionic polymer that is a constituent component of the cell wall is reduced. Reduced ionic binding force between cell wall and lytic enzyme, enabling to significantly suppress lysis even for mutant microorganisms that were originally easier to lyse than wild type Is. A microorganism capable of suppressing such lysis is suitable as a host for producing a target substance such as a protein or a polypeptide because it is suitable for long-term culture and thus contributes to an improvement in the production rate of the target substance.
Hereinafter, the present invention will be described in detail.

In the microorganism according to the present invention, the expression of at least one of the cell wall teichoic acid synthase-related gene or the gene corresponding to the gene of Bacillus subtilis is suppressed as compared with the wild type. Bacillus subtilis cell wall teichoic acid synthase-related genes are a series of enzymes involved in the biosynthesis of cell wall teichoic acid formed by binding major teichoic acid and peptidoglycan specifically present on the cell surface of Bacillus subtilis. A group of genes encoding a group, specifically, tagA gene derived from Bacillus subtilis, tagB gene, tagD gene, tagF gene, tagG gene, tagH gene and tagO gene (for example, A. Formstone et al., Journal of Bacteriology, Vol. 190, p.1812-1821 (2008)). If at least one function of these gene groups can be suppressed, the entire cell wall teichoic acid (major teichoic acid) biosynthesis system can be inhibited. Therefore, in the microorganism of the present invention, at least one gene of the above gene group is used. As long as the function can be suppressed. Among them, from the viewpoint of reducing the amount of cell wall teichoic acid, it is preferable that the function of the tagF gene encoding an enzyme that catalyzes the addition reaction of major teichoic acid (polyglycerol phosphate chain) is suppressed.

In the present invention, the genes whose expression is to be suppressed are, as described above, the cell wall teichoic acid synthase-related gene group of Bacillus subtilis that is a gene group encoding a series of enzymes involved in the biosynthesis of cell wall teichoic acid in Bacillus subtilis. And a gene corresponding to the gene, specifically, the tagA gene, tagB gene, tagD gene, tagF gene, tagG gene, tagH gene and tagO gene derived from Bacillus subtilis shown in Table 1 below, and the gene It is selected from the corresponding gene group. The names, numbers, functions, etc. of each gene in the table were reported in Nature, 390, 249-256, (1997) and published on the Internet at JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) (http: //Bacillus.genome.ad.jp/, updated on June 17, 2003), based on genome data of Bacillus subtilis. Further, the base sequence of the tagA gene derived from Bacillus subtilis and the amino acid sequence of the protein encoding the gene are shown in SEQ ID NOs: 1 and 8, respectively. The base sequence of the tagB gene derived from Bacillus subtilis and the amino acid sequence of the protein encoding the gene are shown in SEQ ID NOs: 2 and 9, respectively. The base sequence of the tagD gene derived from Bacillus subtilis and the amino acid sequence of the protein encoding the gene are shown in SEQ ID NOs: 3 and 10, respectively. The base sequence of the tagF gene derived from Bacillus subtilis and the amino acid sequence of the protein encoding the gene are shown in SEQ ID NOs: 4 and 11, respectively. The base sequence of the tagG gene derived from Bacillus subtilis and the amino acid sequence of the protein encoding the gene are shown in SEQ ID NOs: 5 and 12, respectively. The base sequence of the tagH gene derived from Bacillus subtilis and the amino acid sequence of the protein encoding the gene are shown in SEQ ID NOs: 6 and 13, respectively. The nucleotide sequence of the tagO gene derived from Bacillus subtilis and the amino acid sequence of the protein encoding the gene are shown in SEQ ID NOs: 7 and 14, respectively.

As will be described later, the host of the microorganism of the present invention is not limited to Bacillus subtilis, and may be prepared from a microorganism other than Bacillus subtilis. When a microorganism other than Bacillus subtilis is used as a host, a gene corresponding to a cell wall teichoic acid synthase-related gene group possessed by Bacillus subtilis is identified according to the host to be used, and the expression of the gene is suppressed. Microorganisms can be obtained.
In the present invention, "a gene corresponding to a cell wall teichoic acid synthase-related gene of Bacillus subtilis" refers to the aforementioned Bacillus subtilis tagA gene, tagB gene, tagD gene, tagF gene, tagG gene, tagH gene or tagO gene. It is defined as a functionally identical or equivalent gene. Here, the functionally equivalent or equivalent gene is functionally equivalent to the tagA gene, tagB gene, tagD gene, tagF gene, tagG gene, tagH gene or tagO gene of Bacillus subtilis and is encoded by the gene. Is a protein involved in the biosynthesis system of cell wall teichoic acid. Specifically, the following genes (1) or (2) can be mentioned. These are included in genes that should be suppressed in the present invention as “genes corresponding to cell wall teichoic acid synthase-related genes of Bacillus subtilis”.

(1) In the base sequence of the tagA gene, tagB gene, tagD gene, tagF gene, tagG gene, tagH gene or tagO gene derived from Bacillus subtilis represented by any one of SEQ ID NOs: 1 to 7, the base sequence and 70% or more A gene consisting of a base sequence having an identity of preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, and functionally equivalent to or equivalent to the gene. .
(2) One to several nucleotide sequences in the base sequence of the tagA gene, tagB gene, tagD gene, tagF gene, tagG gene, tagH gene or tagO gene derived from Bacillus subtilis represented by any one of SEQ ID NOs: 1 to 7 Is a gene whose nucleotide sequence is deleted, substituted, inserted and / or added, and which is functionally identical or equivalent to the gene.
As these “genes corresponding to cell wall teichoic acid synthase-related genes of Bacillus subtilis”, genes derived from fungi belonging to the genus Bacillus are preferable.
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 analysis with a unit size to compare (ktup) of 2 as a parameter using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

Specific examples of "a gene corresponding to a cell wall teichoic acid synthase-related gene of Bacillus subtilis " include the tarO gene, tarA gene, tarB gene, tarI gene, tarJ gene of Bacillus subtilis W23 strain , tarK gene, tarL gene, tarD gene, tarF gene, (Microbiology, 148,815-824, (2002 )), tarO gene, tarA gene of Staphylococcus aureus (Staphylococcus aureus), tarB gene, tarI gene, tarJ Gene, tarK gene, tarL gene, tarD gene, tarF gene (J. Bacteriol., 190, 3046-3056, (2008)). In the above microorganisms, it is known that cell wall teichoic acid is composed of ribitol phosphate instead of glycerol phosphate. There is also a report that the functions of the tagA gene of Bacillus subtilis 168 can be complemented by the tarA gene of Bacillus subtilis W23 (J. Bacteriol., 186, 7865-7873, (2004)).
Hereinafter, cell wall teichoic acid synthase-related genes possessed by Bacillus subtilis and genes corresponding to the genes are collectively referred to as simply teichoic acid synthase-related genes.

In other words, the microorganism according to the present invention can be prepared from a microorganism having a teichoic acid synthase-related gene as described above. However, the microorganism according to the present invention is not limited to those prepared from microorganisms known to have a teichoic acid synthase-related gene. That is, the microorganism according to the present invention is based on the above-described identity from a database storing gene sequences and protein amino acid sequences based on the above-described base sequence of the teichoic acid synthase-related gene or the amino acid sequence of the protein. A microorganism identified as having a teichoic acid synthase-related gene by searching for a gene corresponding to the acid synthase-related gene may be used.
In view of the presence of teichoic acid in the cell wall, the microorganism according to the present invention is preferably prepared from a Gram-positive bacterium, such as Bacillus, Bacillus subtilis, Streptococcus, Listeria, Staphylo. Preferably, the fungus belongs to the genus Bacillus, preferably from the genus Staphylococcus, Enterococcus, Lactobacillus, Corynebacterium, Clostridium. It is more preferable to prepare from. Furthermore, the microorganism according to the present invention is a Bacillus subtilis from the point that whole genome information has been clarified, genetic engineering and genome engineering techniques have been established, and the ability to secrete and produce proteins or polypeptides outside the cells. It is particularly preferable to prepare from The microorganism of the present invention is constructed with these host microorganisms as parent microorganisms. These parent microorganisms are not limited to wild-type microorganisms, and for example, mutant strains having various mutations can be targeted.

Furthermore, in view of the gist of the present invention to obtain a microorganism in which lysis is significantly suppressed, it is a microorganism having a teichoic acid synthase-related gene as described above, and its lysis rate is faster than that of a wild strain. It is also a preferred embodiment to produce a microorganism according to the present invention using a microorganism strain. A microorganism strain having a higher lysis rate than such a wild strain can be identified by measuring the lysis rate. In general, when bacteria are continuously cultured, the turbidity of the culture solution decreases as bacterial lysis progresses. Therefore, the lysis rate can be calculated as the rate of decrease in medium turbidity with respect to the culture time. That is, the slope of the graph obtained with the horizontal axis as the culture time and the vertical axis as the medium turbidity can be the lysis rate. The medium turbidity can be measured with a spectrophotometer as the absorbance at 600 nm, for example. For measurement and comparison of the actual lysis rate, Example 2 described later can be referred to.
Similarly, in the microorganism of the present invention, it can also be confirmed as described above whether lysis of the microorganism is suppressed as a result of the suppression of the expression of the teichoic acid synthase-related gene. Specifically, culturing is suppressed by cultivating mutant microorganisms that suppress the expression of teichoic acid synthase-related genes and wild-type microorganisms that do not suppress the expression, and compare the medium turbidity and lysis rate. Can be confirmed.
Specifically, as a microbial strain whose lysis rate is faster than that of a wild strain, it is a bacterium belonging to the genus Bacillus having a teichoic acid synthase-related gene as described above, and the lysis rate compared to a wild strain. However, for example, a microbial strain that is 2 to 3 times, preferably 3 to 10 times faster than the wild type strain.

Among them, the microorganism according to the present invention is preferably a microorganism strain (hereinafter also referred to as a protease-deficient strain) in which the activity of at least one protease is reduced or not active as compared to a wild strain.
In general, for industrial production of target substances such as proteins using microorganisms, mutant strains with various improvements added to improve the production efficiency are used. For example, when a protein is produced as a target substance, wild-type microorganisms have a wide variety of endogenous proteolytic enzymes (proteases, peptidases), which degrade the target protein and produce a foreign protein. May be an obstacle. Therefore, attempts have been made to prevent degradation of the target protein produced by using mutant microorganisms with modifications such as deletion of these proteolytic enzyme genes. Since the mutant strain modified in this manner is deficient in protease, it is suitable as a host for production of substances such as useful proteins. However, in such cells with extremely low protease activity, cell wall lytic enzymes are not decomposed by proteases and accumulate on the cell surface layer, and there is a report that lysis is observed in the stationary phase (J. Biosci. Bioeng). ., 103, 13-21, (2007)).
Therefore, using such a protease mutant strain as a microorganism according to the present invention, lysis can be suppressed by suppressing the expression of the teichoic acid synthase-related gene of the mutant strain, and in a system for producing a target substance such as a protein, The microorganism can be cultured for a relatively long time. As a result, the production efficiency of the target substance can be improved, and the production cost of the substance can be reduced. The protease-deficient strain preferably used in the present invention includes microbial strains that have reduced or no peptidase activity.

Specific examples of protease-deficient strains preferably used in the present invention include mutant strains in which one or more protease genes are deleted among the plurality of protease genes originally possessed by the wild strain, for example, Mutant microorganisms (J. Bacteriol., 158, 411, (1984), J, deficient in genes encoding AprE , the major extracellular alkaline protease in Bacillus subtilis, or NprE , a neutral protease, or both genes) , Bacteriol., 160, 15, (1984), J. Bacteriol., 160, 442, (1984)), 8 types derived from Bacillus subtilis ( aprE gene, nprB gene, nprE gene, bpr gene, vpr gene, mpr genes, epr gene, wprA gene and aprX gene) protease, peptidase gene gene-deficient microorganism for the further these eight protease, peptidase gene All missing B. subtilis strain (Appl.Environ.Microbiol., 68,3261, (2002 ) see), in addition to the eight protease gene, further microorganisms deficient in aprX gene from Bacillus subtilis (JP 2006-174707 No., J. Biosci. Bioeng., 104, 135-143, (2007)), and these can be preferably used in the present invention.

Since the present invention suppresses lysis by reducing the electrostatic interaction between the cell wall and the lytic enzyme, the protease-deficient strain that can be used in the present invention includes a protease capable of degrading the lytic enzyme. It is preferable that the strain has a reduced activity or no activity. Examples of the lytic enzyme include lysozyme, glucosaminidase, amidase, L, D-endopeptidase, D, L-endopeptidase and the like. Among them, lytic enzymes such as LytC (amidase), LytE, and LytF (D, L-endopeptidase) are particularly strongly affected by a decrease in the amount of cell wall teichoic acid. Can be preferably suppressed.
Examples of the gene encoding a protease capable of degrading the lytic enzyme described above include aprX gene, aprE gene, nprB gene, nprE gene, bpr gene, vpr gene, mpr gene, epr gene, and wprA gene derived from Bacillus subtilis. A protease deficient strain in which these genes or a gene corresponding to the gene is deleted or inactivated can be preferably used. The gene corresponding to the gene encoding the protease possessed by Bacillus subtilis is defined in the same manner as the gene corresponding to the cell wall teichoic acid synthase-related gene possessed by Bacillus subtilis described above, and the gene encoding the protease possessed by Bacillus subtilis. It can be defined as a gene functionally consistent with The range of identity and the calculation method are also the same range and method as described above for the gene corresponding to the cell wall teichoic acid synthase-related gene of Bacillus subtilis.
Specifically, one or more genes selected from aprE , nprB , nprE , bpr , vpr , mpr , epr , wprA and aprX genes or nine genes corresponding to the genes are deleted or inactivated. Among them, the aprX gene, the aprE gene encoding the major extracellular alkaline protease and the nprE gene encoding the neutral protease, or the three genes corresponding to the gene are deleted or inactivated. More preferably, all of the aprE , nprB , nprE , bpr , vpr , mpr , epr , wprA and aprX genes, or all nine genes corresponding to the genes have been deleted or inactivated. Those are particularly preferred. Table 2 below shows the gene numbers and gene functions of these protease genes. The names, numbers, functions, etc. of each gene in the table were reported in Nature, 390, 249-256, (1997), and published on the Internet on JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) (http: //Bacillus.genome.ad.jp/, updated June 17, 2003), based on genome data of Bacillus subtilis. Furthermore, details of these protease genes and protease-deficient strains lacking the genes can be referred to the description in JP-A No. 2006-174707. As a method for deleting or inactivating a gene encoding a protease capable of degrading a lytic enzyme, it can be carried out according to the method shown as a method for suppressing the expression of a teichoic acid synthase-related gene, which will be described later. A method of destroying a gene by inactivation by loss or introduction of mutation can be preferably used. Reference can also be made to the description of JP-A-2006-174707.

Furthermore, examples of the protease-deficient strain preferably used in the present invention include strains that suppress the expression of a transcription factor that positively controls transcription of the protease to be deleted. For example, in Bacillus subtilis, examples of the transcription factor include a transcription factor encoded by the spo0A gene. Bacillus subtilis mutants lacking the spo0A gene are produced in a specific amount of protease (specifically, the proteases encoded by the aforementioned aprE , bpr , epr , mpr , nprB , nprE , vpr and wprA genes). (See J. Biosci. Bioeng. 103, 13 (2007)). Of course, in bacteria other than Bacillus subtilis, a protease-deficient strain derived from the bacteria can be prepared by deleting the homologous gene of spo0A gene. The spo0A gene homologous gene is defined in the same manner as the gene corresponding to the cell wall teichoic acid synthase-related gene of Bacillus subtilis described above, and can be defined as a gene functionally identical to the spo0A gene of Bacillus subtilis. The range of identity and the calculation method are also the same range and method as described above for the gene corresponding to the cell wall teichoic acid synthase-related gene of Bacillus subtilis. The Spo0A protein encoded by the spo0A gene from Bacillus subtilis is a major transcriptional regulator of spore formation. Examples of the homologous gene of the spoOA gene include the spo0A gene encoding a major transcriptional control factor for spore formation in spore-forming bacteria.
Table 2 below shows the gene number and gene function of the spo0A gene derived from Bacillus subtilis. Furthermore, details of the spo0A gene and a protease-deficient strain lacking the gene can be referred to the description in JP-A No. 2005-295830. As a method for reducing the expression of the spo0A gene of a bacterial strain or a homologous gene thereof compared to a wild strain, it can be performed according to the method shown as a method for suppressing the expression of a teichoic acid synthase-related gene described below, and in particular, the gene A method of destroying a gene by inactivation by introducing a deletion or mutation can be preferably used. The description in JP-A-2005-295830 can also be referred to.

  Furthermore, the construction of the microorganism of the present invention can be combined with a defect in a gene group other than the protease described above.

  The microorganism of the present invention is characterized in that the expression of teichoic acid synthase-related gene is suppressed as compared to the wild type. The method for suppressing gene expression is not particularly limited as long as it can significantly lower or inactivate the function of the gene or the protein encoded by it than the original function in a wild-type microorganism. Engineering techniques can be used. Examples thereof include a method for destroying a teichoic acid synthase-related gene, a method for suppressing transcription of a teichoic acid synthase-related gene, and a method for suppressing translation of a teichoic acid synthase-related gene. In addition, teichoic acid synthase-related genes or transcription / translation regulatory regions of the genes may be directly modified, and other factors that control expression (eg, transcription factors, antisense RNA, etc.) are controlled or modified. It may be. In addition, the microorganism according to the present invention is not only a recombinant strain in which expression of a teichoic acid synthase-related gene is artificially suppressed from a wild-type microorganism by the genetic engineering technique as described above, but also such an artificial technique. Regardless of this, naturally occurring mutants that suppress the expression of the teichoic acid synthase-related gene or a gene corresponding to the gene are also included.

Hereinafter, a method for suppressing gene expression will be described in detail.
In the present invention, suppression of expression of a teichoic acid synthase-related gene is preferably a method for suppressing transcription or translation of the gene, and more preferably a method for suppressing transcription. Since it is possible to regulate the level of gene expression and reports that teichoic acid synthase-related genes are essential genes for growth (for example, Journal of General Microbiology, 131, p.929-941 (1991), Journal of bacteriology) , 183, p.6688-6693 (2001)).

As a method for suppressing gene transcription, a method of modifying a host microorganism so that transcription of a teichoic acid synthase-related gene is suppressed is preferable. For example, the transcription promoter region of the teichoic acid synthase-related gene of the target microorganism by homologous recombination etc. is transformed with a transcriptional repressor or transcription-inducible promoter and then transformed, and the transformant is subjected to transcriptional repression or transcriptional induction conditions. And a method of culturing in the above.
In particular, it is preferable to use the aforementioned protease-deficient strain as a host and modify the host so that the teichoic acid synthase-related gene of the microorganism is expressed under the control of a transcription-inducible promoter. Microorganisms constructed in this way can prevent degradation of useful proteins and the like to be produced, and thus can achieve better production efficiency.

Specifically, using a vector or expression cassette having a transcription-inducible promoter sequence, a host microorganism is transformed according to a conventional method, and the promoter sequence is incorporated upstream of a teichoic acid synthase-related gene such as the tagF gene. preferable. 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. For example, expression of a downstream gene in the presence of IPTG (isopropyl-β-D-thiogalactopyranoside) controlled by lacI, a repressor of the lac operon. Examples include an inducible promoter (Pspac) and a promoter (Pxyl) that induces expression of a downstream gene in the presence of xylose (Gene, 181, 71-76, (1996)).

The transformation method is not particularly limited as long as it is a method capable of introducing a target DNA 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. The introduction of the target DNA fragment can also be confirmed by PCR method using a genome as a template.

Hereinafter, as an example of a method for suppressing expression of a teichoic acid synthase-related gene using a transcription-inducible promoter, a method for introducing a Pspac transcription initiation control region upstream of a tagF gene in a genome will be described with reference to FIG. The present invention is not limited to this method.
First, the 5'-side partial sequence of the tagF gene containing the SD sequence is amplified by PCR using specific primers, and the resulting DNA fragment is treated with a restriction enzyme. This DNA fragment is ligated with a plasmid having a Pspac transcription initiation control region treated with the same restriction enzyme to obtain a recombinant plasmid DNA in which the DNA fragment is inserted downstream of the Pspac transcription initiation control region. The insertion of the target DNA fragment into the obtained recombinant plasmid can be confirmed by a usual confirmation method such as sequencing. The Pspac transcription initiation control region was developed as a promoter that can be controlled by an inducer in Bacillus subtilis. When the lac repressor gene is present, the expression of the Pspac promoter is suppressed by the repressor, but the repressor is removed by adding IPTG (isopropyl-beta-D-thiogalactopyranoside), and the promoter is activated. (Yansura, D.G. and D.J. Henner, Genetics and Biotechnology of Bacilli (1984) Development of an inducible promoter for controlled gene expression in Bacillus subtilis).
Next, using the obtained recombinant plasmid, a protease-deficient strain is transformed by a transformation method such as competent cell method, and colonies grown on a medium containing erythromycin and IPTG are isolated as transformants. . The genome of the obtained transformant is extracted, and it can be confirmed by PCR that the Pspac transcription initiation control region has been introduced upstream of the tagF gene in the genome.
The transformant believed to be the homologous recombination of such single crossing shown in FIG. 1 was obtained as a result of occurring are expressed controlled as tagDEF operon tagF native promoter (TAGD upstream of the promoter ) Is replaced with the spac promoter, and the expression of the tagF gene can be artificially controlled by appropriately changing the IPTG concentration to be added. The expression of teichoic acid synthase-related gene can be controlled to a desired level by adding the concentration of IPTG, which is an expression inducer. Therefore, taking into consideration factors such as microbial growth and protein production, teichoic acid It is possible to control the strength of suppressing the expression of a synthase-related gene and to establish more optimal conditions for protein production.

In the method for disrupting a gene, at least one of a plurality of teichoic acid synthase-related genes is disrupted. The gene can be disrupted, for example, by deleting the gene or introducing a mutation into the gene.
For example, a method by homologous recombination can be used for gene disruption. 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 is also possible to inactivate by disrupting the target gene on the genome.
In particular, when Bacillus subtilis is used as a parent microorganism for constructing the microorganism of the present invention, there have already been several reports on methods for deleting or inactivating a target gene by homologous recombination (Mol. Gen. Genet., 223, 268 (1990) etc.) By repeating such a method, the host microorganism of the present invention can be obtained.

  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 using site-directed mutagenesis (for example, Mutant-K (TAKARA) or Mutant-G (TAKARA)), or TAKARA LA PCR in vitro Mutagenesis Mutation may be introduced using a series kit.

  An example of a method for deleting a gene by homologous recombination will be described with reference to FIG. First, in the first PCR, a host gene DNA (for example, bacterial genomic DNA) is adjacent to a gene to be deleted (for example, a teichoic acid synthase-related gene, a gene encoding a protease that degrades a lytic enzyme, etc.) 5 A DNA fragment containing a 'side region (A fragment: eg 0.5-3 kbp), a DNA fragment containing a 3' side region adjacent to the deletion target gene (C fragment: eg 0.5-3 kbp), and a drug A DNA fragment (B fragment) containing a 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 to add a homologous sequence (for example, 10 to 30 bases) at the 5 'end of the B fragment to the 3' end of the A fragment is used. Similarly, in PCR for C fragment amplification, primers 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 are used.

  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. Can be obtained.

  Furthermore, by introducing the obtained PCR product D into a host (for example, a bacterium), homologous recombination occurs between the host genomic DNA and the PCR product D, and the PCR product is present at the target gene site of the host genomic DNA. At the same time that the drug resistance gene in D is incorporated, the deletion target gene in the host genomic DNA can be deleted. At this time, whether or not transformation has been performed can be confirmed using the resistance of the drug corresponding to the drug resistance gene incorporated in the host genomic DNA as an index.

  As a method for deleting a predetermined gene, a two-step single crossover method using a deletion-introducing plasmid into which a deletion-introducing DNA fragment prepared by the SOE-PCR method is inserted may be used. About this method, it is possible to implement with reference to Unexamined-Japanese-Patent No. 2006-174707.

  Moreover, as a method for suppressing the translation of teichoic acid synthase-related gene, a method using so-called antisense RNA can be mentioned. Specifically, a gene that transcribes an antisense RNA against the mRNA of a teichoic acid synthase-related gene is incorporated into bacterial genomic DNA, and the antisense RNA is overexpressed to translate the teichoic acid synthase-related gene mRNA. Is suppressed.

  Furthermore, in addition to the method described above, the target microorganism strain can also be obtained by giving a random gene mutation and then performing protein productivity evaluation and gene analysis by an appropriate method. For example, a method of causing homologous recombination similar to the above-described method using a randomly cloned DNA fragment, a method of irradiating a parent microorganism with γ rays, and the like can be mentioned.

  The microorganism of the present invention constructed by the above method can be suitably used for producing a target substance such as protein. The target substance that can be produced by the microorganism is not particularly limited and can be appropriately selected according to the type of the host microorganism to be used. For example, when the microorganism according to the present invention is the above protease-deficient strain, It can be suitably used for protein or polypeptide production. Specifically, by introducing a gene encoding a target protein or polypeptide into the microorganism according to the present invention, a recombinant microorganism that highly produces the protein or polypeptide can be obtained.

Examples of the protein or polypeptide that can be produced using the microorganism of the present invention include proteins and polypeptides such as enzymes and physiologically active factors that are useful for various industrial uses such as detergents, foods, fibers, feeds, chemicals, medicine, and diagnosis. Peptides are mentioned. 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). However, it is preferable to select an optimal enzyme appropriately depending on the type of host to be used and the type of protease that is deficient. For example, when a protease-deficient strain that degrades the lytic enzyme described above is used, genes for hydrolase such as cellulase, α-amylase, protease, and lipase are preferable.
Specifically, cellulases belonging to Family 5 are listed in the classification of polysaccharide hydrolases (Biochem. J., 280, 309 (1991)), and among them, cellulases derived from microorganisms, particularly from Bacillus bacteria. As more specific examples, alkaline cellulase derived from a Bacillus bacterium comprising the amino acid sequence represented by SEQ ID NO: 2 or 4 described in JP-A-2006-174707, or one or several amino acids of the amino acid sequence And alkaline cellulase having an amino acid sequence deleted, substituted or added, and 70%, preferably 80%, more preferably 90% or more, still more preferably 95% or more, particularly preferably the amino acid sequence. Include cellulases composed of amino acid sequences having 98% or more identity.
Specific examples of α-amylase include α-amylase derived from microorganisms, and liquefied amylase derived from bacteria belonging to the genus Bacillus is particularly preferable. As more specific examples, alkaline amylase derived from Bacillus bacteria consisting of the amino acid sequence represented by SEQ ID NO: 61 described in JP-A No. 2006-174707, the amino acid sequence and 70%, preferably 80%, An amylase consisting of an amino acid sequence having preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more is mentioned. The amino acid sequence identity is calculated by the above-mentioned Lipman-Pearson method (Science, 227, 1435, (1985)).
Specific examples of proteases include serine proteases, metal proteases, and the like derived from microorganisms, particularly from Bacillus bacteria.
Specific examples of lipases include lipases classified as true lipases, lipases classified as GDSL families, and lipases classified as HSL families according to the classification of Jaeger et al. (Biochem. J., 343, 177-183 (1999)). In addition, lipases classified into families III to VIII can be mentioned. Among these, the LipA protein encoded by the LipA gene of Bacillus subtilis (the amino acid sequence of the protein is shown in SEQ ID NO: 20) and the LipA protein derived from Serratia marcescens (Journal) known to be localized in the membrane. of bioscience and bioengineering, 2001 (4), 409-415) is more preferable.
Furthermore, examples of the protein produced by the present invention include physiologically active proteins and enzymes derived from higher organisms such as humans. Preferable examples include interferon α, interferon β, growth hormone, salivary gland amylase and the like. Moreover, a part of domains constituting a physiologically active protein can be expressed, and examples thereof include the antigen recognition domain PreS2 of human hepatitis C virus antibody.

In addition, a gene encoding the target protein or polypeptide has a control region related to transcription, translation, and secretion of the gene upstream, that is, a transcription initiation control region including a promoter and a transcription initiation site, a ribosome binding site and an initiation codon. It is desirable that at least one region selected from the translation initiation region and secretory signal peptide region to be contained is bound in an appropriate form. In particular, it is preferable that three regions comprising a transcription initiation control region, a translation initiation control region, and a secretory signal region are bound.
The transcription initiation control region, translation initiation control region, and secretion signal region are not limited as long as they have a desired function, but a region derived from a bacterium belonging to the genus Bacillus is particularly preferable. Furthermore, the secretion signal peptide region is derived from a lipase gene of the genus Bacillus, and the transcription initiation region and the translation initiation region are 0.6 to 1 kb upstream of the cellulase gene, which is appropriate for the target protein or polypeptide gene. It is desirable that they are connected in a form. For example, the bacterium belonging to the genus Bacillus described in JP 2000-210081, JP 4-190793, etc., that is, KSM-S237 strain (FERM BP-7875), KSM-64 strain (FERM BP-2886) It is desirable that the transcription initiation control region and the translation initiation region of the cellulase gene are appropriately combined with the structural gene of the target protein or polypeptide together with the secretory signal peptide region. More specific transcription initiation control regions and translation initiation control regions include the nucleotide sequences of nucleotide numbers 1 to 578 of the nucleotide sequence represented by SEQ ID NO: 1 described in JP-A-2006-174707, and JP-A-2006-174707. The base sequence of base numbers 1 to 615 of the cellulase gene consisting of the base sequence represented by SEQ ID NO: 3 described in the gazette and the homology (identical to the base sequence of 70% or more, 80% or more or 90% or more) More preferably 95% or more, still more preferably 96% or more, particularly preferably 97% or more, more particularly preferably 98% or more, and most preferably 99% or more. Or a DNA fragment consisting of a base sequence from which a part of any of the above base sequences is deleted is a target protein or It is desirable that are properly coupled with the structural gene of Ripepuchido.
As a more specific secretion signal region, the base sequence of base numbers 1 to 93 of SEQ ID NO: 19 which is a lipase gene of Bacillus bacteria, or 70% or more, 80% or more or 90% or more homology to the base sequence More preferably 95% or more, still more preferably 96% or more, particularly preferably 97% or more, more particularly preferably 98% or more, and most preferably 99% or more from a nucleotide sequence having identity. It is desirable that the DNA fragment or the DNA fragment consisting of the base sequence from which any of the above base sequences is deleted is appropriately bound to the structural gene of the target protein or polypeptide.
Here, a DNA fragment consisting of a base sequence from which a part of the base sequence has been deleted is a part of the base sequence that has been deleted, but retains functions related to gene transcription, translation, and secretion. Means a DNA fragment.

By incorporating a recombinant plasmid in which a DNA fragment containing the above target protein or polypeptide gene and an appropriate plasmid vector are combined into a microorganism according to the present invention by a general transformation method, the target protein or polypeptide is obtained. Recombinant microorganisms to produce can be obtained. The recombinant microorganism can also be obtained by using a DNA fragment obtained by binding an appropriate homologous region with the genome of the microorganism according to the present invention to the DNA fragment and directly integrating it into the genome.
For production of the target protein or polypeptide using recombinant microorganisms, the strain is inoculated into a medium containing an assimilable carbon source, nitrogen source, and other essential components, cultured by a conventional microorganism culture method, and the culture is completed. Thereafter, the protein or polypeptide may be collected and purified. The components and composition of the medium are not particularly limited, but preferably better results can be obtained by using a medium containing maltose or malto-oligosaccharide as a carbon source.
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, and organic solvents such as ethanol Precipitation by adding, concentration using a ultrafiltration membrane or the like, desalting, ion exchange, purification using various chromatographies such as gel filtration, etc. can be used.

  As described above, according to the method for inhibiting lysis according to the present invention, by suppressing the expression of at least one teichoic acid synthase-related gene, a microorganism in which the lysis is significantly inhibited can be obtained. The method for inhibiting lysis according to the present invention can be suitably used for, for example, a microbial strain that is more easily lysed than the wild type, and allows the culture of the microbial strain to be continued for a long time. When such a microorganism is used for production of a target substance such as protein, the culture time of each microbial cell can be lengthened, so that the productivity of the substance can be improved. Therefore, the microorganism according to the present invention can be suitably used for production of a target substance such as protein.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

Example 1 Construction of Dpr9 / PtagF strain In this example, a protease gene 9-deficient strain (Dpr9 strain) was used as a host, and a tagF gene expression control strain (Dpr9), which is a cell wall teichoic acid synthase-related gene derived from Bacillus subtilis. / PtagF) is shown.
Using primers tagF-21F and tagF + 444R shown in Table 3, 5'-end DNA fragment (465 bp) containing the SD sequence of tagF gene was amplified by PCR using genomic DNA extracted from Bacillus subtilis 168 strain as a template. Then, it was treated with restriction enzymes Eco RI and Bam HI (DNA fragment A). On the other hand, plasmid pMutin4 with spac promoter sequence (Microbiology, 144,3097-3104, (1998) ) was treated with restriction enzymes Eco RI and Bam HI, and as shown in FIG. 1, DNA fragments of tagF gene in this plasmid A was inserted. This plasmid DNA was transformed into Escherichia coli JM109 strain, and it was confirmed by sequence that the desired DNA fragment A was inserted into the obtained recombinant plasmid DNA, and the plasmid confirmed to be inserted was designated as pM4PtagF ( Figure 1: pM4PtagF). pM4PtagF was further transformed into Escherichia coli C600 strain to obtain polymerized plasmid DNA. Using the polymerized pM4PtagF, transformation of Bacillus subtilis Dpr9 strain (Kao9 strain prepared in Examples 1 to 5 of JP 2006-174707 A) was performed by a competent method. Bacillus subtilis Dpr9 strain (Kao9 strain) is a 9-deficient strain of protease gene, and aprE gene, nprB gene, nprE gene, bpr gene, vpr gene, mpr gene, epr gene, wprA gene and aprX of Bacillus subtilis It is a Bacillus subtilis mutant lacking each gene of the gene. After transformation, colonies grown on LB agar medium containing erythromycin (0.3 μg / mL) and 1 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) were isolated as transformants. Extract the genome of the resulting transformant, and the target mutant strain in which the upstream region of tagF gene is replaced by the spac promoter from the original tagF promoter (expression of tagDEF operon is controlled by the tagD upstream promoter) by PCR. It was confirmed that (Dpr9 / PtagF strain) could be constructed. A tagF gene expression control strain (Dpr9 / PtagF strain) was constructed as described above.

Example 2 Inhibition of Bacterial Lysis of Dpr9 / PtagF Strain The Dpr9 / PtagF strain prepared in Example 1 was examined for its lysis inhibiting action.
The Dpr9 / PtagF strain obtained in Example 1 and the Bacillus subtilis strain Dpr9 as a control were inoculated into an LB agar medium (containing 0.3 ppm erythromycin and 1 mM IPTG when culturing the Dpr9 / PtagF strain), 37 The culture was performed at 0 ° C. for 8 hours. Thereafter, shake culture is performed overnight at 30 ° C. in 5 mL of LB medium (when Dpr9 / PtagF strain is cultured, containing 0.3 ppm erythromycin and 0.4 mM IPTG) so that OD600 nm = 0.01. 0.6 mL of the solution was inoculated into 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), 30 Shaking culture was performed at 2 ° C. for 2 days. After culturing, the turbidity of the culture solution was measured at OD 600 nm, and the lysis rate in each strain was compared. The results are shown in FIG.

As is clear from FIG. 3, in the Dpr9 strain, the culture turbidity decreased after 35 hours from the culturing, and lysis occurred. In contrast, the culture turbidity of the Dpr9 / PtagF strain in the absence of IPTG did not decrease, and lysis was suppressed. This indicates that the Dpr9 / PtagF strain can suppress lysis in the absence of IPTG, that is, under the conditions where the tagF gene is not expressed.

Example 3 Cell wall lytic enzyme (lytic enzyme) activity of the Dpr9 / PtagF strain Using the Dpr9 / PtagF strain obtained in Example 1 and the Dpr9 strain as a control, the cell wall lytic enzyme (lytic enzyme) activity in each strain was zymogram. Analysis (see J. Bacteriol., 174, 464, (1992); J. Bacteriol., 175, 6260, (1993)). According to the culture method shown in Example 2, these strains were cultured, and a lytic enzyme on the cell surface was prepared (FEMS Microbiol. Lett., 112, 135-140 (1993)) and used for zymogram analysis. The results are shown in FIG.

  As is clear from FIG. 4, in the Dpr9 strain, a plurality of cell wall lytic enzyme activities were observed 35 hours after culturing in which the lysis phenomenon was confirmed (see FIG. 3). On the other hand, in the Dpr9 / PtagF strain in the absence of IPTG, it was confirmed that the cell wall lytic enzyme activity significantly decreased in the same time zone.

Example 4 Measurement of teichoic acid content in cell wall of Dpr9 / PtagF strain Preparation of Teichoic Acid of Bacillus subtilis Dpr9 / PtagF strain obtained in Example 1 and Bacillus subtilis Dpr9 strain as a control include LB agar medium (when Dpr9 / PtagF strain is cultured, 0.3 ppm erythromycin and 1 mM IPTG are included) ) And cultured at 37 ° C. for 8 hours. Thereafter, shake culture is performed overnight at 30 ° C. in 5 mL of LB medium (when Dpr9 / PtagF strain is cultured, containing 0.3 ppm erythromycin and 0.4 mM IPTG) so that OD600 nm = 0.01. 0.6 mL of the solution was inoculated into 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), 30 Shaking culture was performed at 2 ° C. for 2 days. The cells were collected by centrifugation (10000 rpm, 10 min, 20 ° C.) so that the total culture turbidity was about OD3,000, and the cells were suspended in 20 ml of 3M LiCl. After the bacterial suspension was boiled for 10 minutes, 0.1 mm-sized glass beads were added and homogenized (using NISSEI Ace Homogenizer AM-8), and disruption of the bacterial cells was confirmed by microscopic observation. Then, about 1 L of ion exchange water was added, the supernatant was centrifuged (KUBOTA KR-2000c rotor: using RA-6, 3000 rpm, 20 min, 4 ° C.), and the resulting supernatant was further centrifuged (KUBOTA KR -2000c rotor: using 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, further centrifugation (KUBOTA KR-2000c rotor: using 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: using RA-3). 12,000 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 replaced with 10 ml of ion exchange water about 5 times, and then suspended in 2 ml of ion exchange water. This cell wall suspension was freeze-dried to obtain a powdered cell wall.
30 mg of the obtained cell wall was suspended in 600 μL of 10% TCA (trichloroacetic acid) and incubated at 65 ° C. for 10 hours or more, followed by centrifugation (KUBOTA KR-2000c rotor: using RA-3, 12,000 rpm, 20 min). ), And the supernatant fraction was collected as a cell wall teichoic acid sample. Further, the precipitate was suspended in 1400 μL of ion-exchanged water, and after centrifugation, this supernatant fraction was also collected as a cell wall teichoic acid sample.

2. Determination of phosphate in cell walls
The cell wall teichoic acid amount of the Dpr9 / PtagF strain and the Dpr9 strain as a control were quantified using the phosphoric acid amount as an index. The cell wall teichoic acid sample obtained above was quantified for phosphoric acid according to the molybdenum blue method (see Microbiology, 143, 947-956, (1997)). The results are shown in Table 4.

As is clear from Table 4, the amounts of phosphate per 1 mg of cell wall in the Dpr9 strain and Dpr9 / PtagF strain were 1.21 nmol and 0.81 nmol, respectively. That is, it was confirmed that the amount of teichoic acid in the cell wall of the Dpr9 / PtagF strain was reduced by about 30% compared to the Dpr9 strain. From the amino acid sequence information, TagF protein is considered to catalyze the polymerization reaction of glycerol phosphate, which is a constituent of cell wall teichoic acid. In the Dpr9 / PtagF strain, since the tagF gene was not expressed in the absence of IPTG, the amount of glycerol phosphate contained in the cell wall was lower than that in the Dpr9 strain expressing the tagF gene.

Example 5 Production of Lipase Using the Dpr9 / PtagF strain obtained in Example 1 and the Bacillus subtilis Dpr9 strain as a control, an alkaline cellulase gene derived from the Bacillus sp. KSM-S237 strain ( A transcription initiation control region and a translation initiation control promoter region (regions of base numbers 13 to 578 of SEQ ID NO: 17) of SEQ ID NO: 17 (the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 18), Bacillus subtilis A DNA fragment encoding the signal peptide region of LipA, the mature region of LipA and the terminator site of lipA (base region 1 to 725 of SEQ ID NO: 19) is inserted into the Bam HI and Xba I restriction enzyme cleavage points of shuttle vector pHY300PLK. The recombinant plasmid pHLApm (see JP-A-2009-038985) was introduced by a competent transformation method. These strains were designated as Dpr9 / PtagF (pHLApm) strain and Dpr9 (pHLApm) strain, respectively. The Dpr9 / PtagF (pHLApm) and Dpr9 (pHLApm) strains are inoculated in an LB agar medium containing 15 ppm tetracycline (when Dpr9 / PtagF (pHLApm) strains are cultured, 0.3 ppm erythromycin and 1 mM IPTG are further included). And cultured at 37 ° C. for 8 hours. Then, overnight at 30 ° C. in LB medium containing 5 mL of 15 ppm tetracycline (additional 0.3 ppm erythromycin and 0.4 mM IPTG when culturing Dpr9 / PtagF (pHLApm) strain) so that OD600 nm = 0.01. Then, 0.6 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 shaking culture at 30 ° C. for 3 days. After culture, the cells were removed by centrifugation, and the lipase in the culture supernatant was confirmed by electrophoresis (SDS-PAGE). The results are shown in FIG.

As is apparent from FIG. 5, the Dpr9 / PtagF strain showed the same lipase productivity as compared to the control Dpr9 strain. In other words, even when compared with the Dpr9 strain, suppression of the expression of the tagF gene does not negatively affect the secretory production of the protein, and the Dpr9 / PtagF strain in which the expression of the tagF gene is suppressed is also a protease-deficient strain. It is thought that it can be used suitably for protein production similarly to.

Claims (10)

A Gram-positive bacterium with reduced or no activity for at least one protease, a gene corresponding to a cell wall teichoic acid synthase-related gene of Bacillus subtilis, or a tagA gene, tagB gene, tagD derived from Bacillus subtilis At least one gene of genes, tagF genes, tagG genes, tagH genes or tagO genes having a nucleotide sequence having 90% or more identity and functionally matching or equivalent to that gene Gram-positive bacteria whose expression can be controlled and lysis can be suppressed under the control of an exogenous transcription-inhibitory or transcription-inducible promoter. The tagF gene derived from Bacillus subtilis or the nucleotide sequence of the gene and 90% of the at least one gene whose expression is controlled under the control of an exogenous transcription repressor or transcription-inducible promoter according to claim 1 The gram-positive bacterium according to claim 1, wherein the gram-positive bacterium comprises the nucleotide sequences having the above identity and is functionally identical or equivalent to the gene.   The gram-positive bacterium according to claim 1 or 2, wherein the protease decomposes a cell wall lytic enzyme. The gene encoding the protease is aprX gene derived from Bacillus subtilis, aprE gene, nprB gene, nprE gene, bpr gene, vpr gene, mpr gene, epr gene and wprA gene, and the nucleotide sequence of the gene of 90% or more. The base sequence according to any one of claims 1 to 3, wherein the base sequence has at least one selected from the group consisting of genes that are functionally identical to or equivalent to the gene. Gram-positive bacteria. The gram-positive bacterium according to any one of claims 1 to 4, which is a bacterium belonging to the genus Bacillus . 6. The Gram-positive bacterium according to claim 5, which is Bacillus subtilis .   The gram-positive bacterium according to any one of claims 1 to 6, wherein a gene encoding a target protein or polypeptide is introduced.   The manufacturing method of the protein or polypeptide which uses the Gram positive microbe of any one of Claims 1-7. A Gram-positive bacterium with reduced or no activity for at least one protease, a gene corresponding to a cell wall teichoic acid synthase-related gene of Bacillus subtilis, or a tagA gene, tagB gene, tagD derived from Bacillus subtilis In Gram-positive bacteria consisting of a base sequence having 90% or more identity with the base sequence of the gene, tagF gene, tagG gene, tagH gene or tagO gene, and having a gene functionally identical to or equivalent to the gene, Cell wall teichoic acid synthase-related gene of Bacillus subtilis or base sequence having 90% or more identity with the base sequence of tagA gene, tagB gene, tagD gene, tagF gene, tagG gene, tagH gene or tagO gene derived from Bacillus subtilis At least one of the genes that are functionally identical to or equivalent to the gene. Lysis method of controlling Gram-positive bacteria comprising the step of controlling the expression under the control of the child exogenous transcriptional inhibitory or transcription inducible promoter. The tagF gene derived from Bacillus subtilis or the nucleotide sequence of the gene and 90% or more of the at least one gene that controls expression under the control of an exogenous transcription repressor or transcription-inducible promoter according to claim 9 The lysis control method according to claim 9, which comprises a nucleotide sequence having the same identity and is functionally identical or equivalent to the gene.