JP6791623B2 - Recombinant microorganisms and their use - Google Patents

Description

The present invention relates to recombinant microorganisms and their use.

Industrial production of useful substances by microorganisms covers a wide variety of types, including foods such as alcoholic beverages, miso, and soy sauce, amino acids, organic acids, nucleic acid-related substances, antibiotics, sugars, lipids, and proteins. It is also used in a wide range of fields, including daily necessities such as foods, pharmaceuticals, cleaning agents, and cosmetics, and raw materials for various chemical products.

In the industrial production of useful substances by such microorganisms, improvement of their productivity is one of the important issues, and as a method thereof, breeding of producing bacteria by a genetic method such as mutation has been carried out. In particular, recently, with the development of microbial genetics and biotechnology, more efficient breeding of producing bacteria using gene recombination technology and the like has come to be carried out. Furthermore, in response to the rapid development of genome analysis technology in recent years, attempts have been made to decipher the genome information of the target microorganisms and actively apply them to industry.

As an industrially useful host microorganism whose genomic information is open to the public, Bacillus subtilis Marburg No. 168 (Non-Patent Document 1), Escherichia coli K-12 MG1655 (Non-Patent Document 2), Corynebacterium glutamicum ATCC132032, etc. have been mentioned, and improved strains have been developed using these genomic information. There is. However, despite the above efforts, the production efficiency was not always satisfactory.

On the other hand, Bacillus subtilis AbrB plays an important role in controlling the expression of various genes involved in spore formation, competence, or acquisition of nutrient sources in the transitional phase during the transition from the logarithmic growth phase to the stationary phase and in the stationary phase. (Non-Patent Documents 3 and 4), for example, a skfA operon (skfA-H) containing a skf gene encoding a killing factor as a gene under AbrB control is negatively regulated. It has been reported (Non-Patent Document 5). In addition, it has been reported that the productivity of amylase and protease is improved in the Bacillus subtilis strain lacking the abrB gene (Patent Document 1).
However, no recombinant microorganisms that constitutively express AbrB have been reported.

JP-A-2007-74934

Kunst, F. et al., Nature, 1997, 390: 249-256 Blattner, F.R. et al., Science, 1997, 277: 1453-1462 M. A. Strauch et al., Mol. Microbiol., 1993, 7: 337-342 Z. E. V. Phillips et al., Cell Mol. Life Sci., 2002, 59: 392-402 M. Fujita et al., J. Bacteriol., 2005, 187: 1357-1368

The present invention relates to a microorganism in which the productivity of a protein or polypeptide is further improved, and to provide a method for producing a target protein or polypeptide by utilizing the microorganism.

In view of the above problems, the inventors conducted a study to obtain a microorganism with improved protein or polypeptide productivity, and as a result, a transcription factor that suppresses a group of genes specifically expressed in the transition phase and the stationary phase. It has been found that by constitutively expressing a certain AbrB, a microorganism with improved productivity of a target protein or polypeptide can be obtained.

That is, the present invention relates to the following: 1) A gene encoding a target protein or polypeptide is expressed in a microorganism whose gene is constructed so that the abrB gene of Bacillus subtilis or a gene corresponding thereto is constitutively expressed. Recombinant microorganisms that have been fortified or introduced as possible.
2) A method for producing a target protein or polypeptide, which comprises a step of culturing the recombinant microorganism according to 1) and recovering the target protein or polypeptide from the culture or cells.
3) Recombinant microorganisms including a step of constructing a gene so that the abrB gene of Bacillus subtilis or a gene corresponding thereto is constitutively expressed, and a step of enhancing or introducing a gene encoding a target protein or polypeptide so that it can be expressed. Production method.
4) Using a recombinant microorganism in which the abrB gene of bacillus or a gene corresponding thereto is constitutively expressed, and the gene encoding the target protein or polypeptide is fortified or introduced so as to be expressible. A method for improving the productivity of a target protein or polypeptide for producing a protein or polypeptide.

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. By using the recombinant microorganism of the present invention, the target protein or polypeptide can be efficiently produced with high productivity, and the time and cost required for the production of the substance can be reduced.

The method for preparing a DNA fragment for introduction by the SOE-PCR method and the method for transforming a parent strain using the DNA fragment and constructing a gene so that the target gene is constitutively expressed are schematically shown.

The names of each Bacillus subtilis gene and genomic region described herein are available on the Internet at JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) ([bacillus.genome.ad.jp/], January 2006). It is described based on the Bacillus subtilis genome data (updated on the 18th).

As used herein, amino acid and nucleotide sequence identities are calculated by the Lipman-Pearson method (Lipman, DJ., Pearson. WR .: Science, 1985, 227: 1435-1441). Specifically, the genetic information processing software Genetyx-Win Ver. It is calculated by performing an analysis with Unit size to homology (ktup) as 2 using the homology analysis (Search homology) program of 11 (software development).

Unless otherwise defined herein, the term "one or several" used with respect to the deletion, substitution, addition or insertion of an amino acid or nucleotide in an amino acid or nucleotide sequence is, for example, 1 to 18 in the amino acid sequence. The number is preferably 1 to 9, more preferably 1 to 4, still more preferably 1 to 2, and the nucleotide sequence is 1 to 56, preferably 1 to 28, more preferably 1 to 14, and further. It can be preferably 1 to 7. Also herein, "addition" of an amino acid or nucleotide includes the addition of one or several amino acids or nucleotides to one or both ends of the sequence.

Unless otherwise defined herein, a "stringent condition" for hybridization is a condition that allows the identification of a gene having a nucleotide sequence of about 80% or more, preferably about 90% or more of sequence identity. Is. Examples of the "stringent condition" include Molecular Cloning-A LABORATORY MANUAL THIRD EDITION (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory, 2001). Those skilled in the art of hybridization can appropriately create stringent conditions by adjusting the salt concentration, temperature, etc. of the hybridization solution according to the nucleotide sequence, concentration, length, etc. of the probe. For example, the above-mentioned "stringent condition" means that the hybridization condition is preferably 5 × SSC, 70 ° C. or higher, more preferably 5 × SSC, 85 ° C. or higher, or 6 × SSC, 60 ° C. The above is preferable, 6 × SSC, 65 ° C. or higher is more preferable, and as cleaning conditions, 1 × SSC, 60 ° C. or higher is preferable, and 1 × SSC, 73 ° C. or higher is more preferable. The combination of the SSC and the temperature condition is an example, and a person skilled in the art can realize an appropriate stringency by appropriately combining the above or other factors that determine the stringency of hybridization.

In the present specification, the upstream and downstream of a gene refer to regions following the 5'side and the 3'side, respectively, with the gene or region captured as a target as a base point.

In the present specification, the gene control region is a region having a function of controlling the intracellular expression of a gene downstream of the region, and preferably a region having a function of constitutively expressing or highly expressing the downstream gene. is there. Specifically, it can be defined as a region that exists upstream of the coding region in the gene and has a function of interacting with RNA polymerase to control transcription of the gene. Preferably, the control region of a gene in the present specification refers to a region of about 200 to 600 nucleotides upstream of the coding region in the gene. The control region includes a transcription initiation control region and / or a translation initiation control region of a gene, or a region from the transcription initiation control region to the translation initiation control region. The transcription initiation control region is a region containing a promoter and a transcription initiation site, and the translation initiation control region is a site corresponding to the Shine-Dalgarno (SD) sequence that forms a ribosome binding site together with the start codon (Shine, J., Dalgarno). , L., Proc. Natl. Acad. Sci. USA., 1974, 71: 1342-1346).

In the present specification, "operably linking" a target gene and a control region means connecting the control region and the target gene on DNA so that the function of controlling gene expression by the control region acts on the target gene. It means to arrange. Examples of the method for operably linking the target gene and the control region include a method of linking the target gene downstream of the control region.

As used herein, the term "transcription factor"("transcriptionregulator") means a protein that binds to a transcription initiation control region or the like on DNA and promotes or suppresses the transcription process from DNA to RNA.
Further, as used herein, the term "transitional phase" means the transition from the logarithmic growth phase or exponential growth phase to the stationary phase or quiescent phase in bacterial growth.

The recombinant microorganism of the present invention is enhanced or introduced so that a gene encoding a target protein or polypeptide can be expressed in a microorganism whose gene has been constructed so that the abrB gene of Bacillus subtilis or a gene corresponding thereto is constitutively expressed. It is a gene.
That is, the gene encoding the target protein or polypeptide can be enhanced or introduced into a modified microorganism in which the parent microorganism has been genetically modified so that the abrB gene of Bacillus subtilis or a gene corresponding thereto is constitutively expressed. It was done. This recombinant microorganism is characterized in that the abrB gene or a gene corresponding thereto is constitutively expressed, and the productivity of the protein or polypeptide is improved as compared with the parent microorganism into which the target protein or polypeptide has been introduced. Has.

The parent microorganism for constructing the microorganism of the present invention may be any one having the abrB gene of Bacillus subtilis or a gene corresponding to the gene, and these may be wild-type or mutated. Specific examples thereof include bacteria of the genus Bacillus, bacteria of the genus Clostridium, yeast and the like, and among them, bacteria of the genus Bacillus are preferable. Furthermore, Bacillus subtilis is particularly preferable because the whole genome information has been clarified, genetic engineering and genomic engineering techniques have been established, and it has the ability to secrete and produce proteins outside the cells.

In Bacillus subtilis, the abrB gene is a gene encoding the transcription factor AbrB and is expressed in the logarithmic growth phase, but its expression is suppressed in the transition phase during the transition to the stationary phase and in the subsequent stationary phase. The transcription factor AbrB directly or indirectly suppresses the expression of a large number of genes related to sporulation, competence, and acquisition of nutrient sources during the logarithmic growth phase. In the transitional phase and the stationary phase, the suppression of expression by AbrB is released, and the expression of many of the above-mentioned genes is induced.
The Bacillus subtilis abrB gene is identified as the gene number: BG10204 and encodes the protein AbrB consisting of the nucleotide sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2.

Examples of the gene corresponding to the Bacillus subtilis abrB gene include the abrB gene of a microorganism belonging to the genus Bacillus other than Bacillus subtilis. Examples of microorganisms other than Bacillus subtilis include, but are not limited to, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium, Bacillus anthracis, Bacillus cereus and the like. For example, the gene corresponding to Bacillus subtilis abrB is obtained by extracting genomic DNA from the above-mentioned microorganism and performing stringent Southern hybridization using a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 or a fragment thereof as a probe. Can be identified. Further, for example, the gene corresponding to Bacillus subtilis abrB in the above-mentioned microorganism can be identified by comparing the nucleotide sequence of the genome of the above-mentioned microorganism with the nucleotide sequence represented by SEQ ID NO: 1 based on known genome sequence information. it can.

For example, the abrB gene (SEQ ID NOs: 3 and 4) of the Bacillus amyloliquefaciens DSM7 strain, the abrB gene (SEQ ID NOs: 5 and 6) of the Bacillus licheniformis ATCC14580 strain, and the abrB gene of the Bacillus megaterium DMS319 strain (SEQ ID NO: 5 and 6). The sequence identity with the abrB gene of 168 strains is 91%, 88% and 80% in the nucleotide sequence, respectively, and 98%, 94% and 85% in their encoding amino acid sequences, respectively. These are exemplified as genes corresponding to the Bacillus subtilis abrB gene.

The Bacillus subtilis abrB gene or its equivalent genes include:
(A) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(B) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the nucleotide sequence shown in SEQ ID NO: 1. A poly encoding a protein consisting of a nucleotide sequence having preferably 98% or more, more preferably 99% or more identity, and having activity as a transcription factor of a gene induced in the transition phase and the stationary phase similar to AbrB. nucleotide;
(C) As a transcription factor of a gene that hybridizes under stringent conditions to the complementary strand of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 and is induced in the transition phase and the stationary phase in the same manner as AbrB. A polynucleotide encoding an active protein;
(D) A polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(E) A transcription factor of a gene consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and induced in the transition phase and the stationary phase in the same manner as AbrB. A polynucleotide encoding a protein with activity as;
(F) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the amino acid sequence shown in SEQ ID NO: 2. A poly encoding a protein consisting of an amino acid sequence having preferably 98% or more, more preferably 99% or more identity, and having activity as a transcription factor of a gene induced in the transition phase and the stationary phase similar to AbrB. nucleotide.

In the present invention, to construct a gene so that the bacillus abrB gene or a gene corresponding thereto is constitutively expressed means that the gene is always expressed so that the bacillus abrB gene or a gene corresponding thereto is always expressed while the microorganism is alive. It means constructing, that is, constructing a gene so that the gene is expressed even in the stationary phase after the logarithmic growth phase, which is not normally expressed. Examples of gene construction such that the bacillus abrB gene or a gene corresponding thereto are constitutively expressed include, for example, a transcription initiation control region located upstream of the bacillus abrB gene or a gene corresponding thereto, or a transcription initiation control region and a ribosome. Inactivation of the binding site or the binding site of the inhibitory regulator in the region from the transcription initiation control region to the translation initiation control region can be mentioned. Here, the "inhibitory regulator" means a protein that binds to a specific base sequence involved in the regulation of transcription on a gene and suppresses transcription. As an example of the transcription initiation control region of the bacillus abrB gene, the transcription initiation control region and the ribosome binding site, or the region from the transcription initiation control region to the translation initiation control region, it consists of the nucleotide sequence shown in SEQ ID NO: 9. Examples thereof include DNA or DNA consisting of a base sequence homologous to the sequence and having a function as a transcription initiation control region or a transcription initiation control region and a ribosome binding site, or a region from the transcription initiation control region to the translation initiation control region. .. The base sequence homologous to the base sequence shown in SEQ ID NO: 9, for example, is 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: 9. , Or a base sequence consisting of a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 9 and a DNA consisting of a DNA that hybridizes under stringent conditions.

In the above control region, as the binding site of the inhibitory control factor, for example, in the base sequence shown in SEQ ID NO: 9, from the region consisting of the 20th to 49th bases or the region corresponding thereto, the 73rd to 89th bases. Area or an area corresponding thereto.
Here, in the base sequence shown in SEQ ID NO: 9, the region corresponding to the region consisting of the 20th to 49th bases or the region consisting of the 73rd to 89th bases is composed of the base sequence shown in the above-mentioned SEQ ID NO: 9. A DNA having a base sequence homologous to DNA and having a function as a transcription initiation control region or a transcription initiation control region and a ribosome binding site, or a region from the transcription initiation control region to the translation initiation control region is shown in SEQ ID NO: 9. A region corresponding to the region when compared with the base sequence, which functions as a binding site of an inhibitory regulator.

Means for inactivating the site include mutagenesis of one or more nucleotides on the nucleotide sequence of the site, substitution or insertion of another nucleotide sequence into the nucleotide sequence, or part of the sequence of the gene. Alternatively, all deletions can be mentioned. Here, a part includes a partial sequence consisting of 1 to 40 bases, preferably 5 to 30 bases, or 10 to 17 bases. Of these, preferably, in the base sequence shown in SEQ ID NO: 9, a region consisting of the 20th to 49th bases or a region corresponding thereto, or / and a region consisting of the 73rd to 89th bases or a region corresponding thereto. Is mentioned, and more preferably, the region consisting of the 73rd to 89th bases or the region corresponding thereto is completely deleted. Specific methods for introducing the above-mentioned mutation and replacing or inserting a nucleotide sequence include ultraviolet irradiation, site-specific mutation introduction, SOE-PCR method, homologous recombination method, and the like.

FIG. 1 shows an outline of the procedure for preparing a DNA fragment for deletion by the SOE-PCR method and deleting the target region by the double crossing method using the DNA fragment for deletion. First, by the SOE-PCR method, a fragment corresponding to an approximately 0.1 to 3 kb region adjacent to the upstream of the target region to be deleted (referred to as an upstream fragment) and an approximately 0.1 to 3 kb also adjacent to the downstream. A DNA fragment in which a fragment corresponding to the region (referred to as a downstream fragment) is linked is prepared. Preferably, in order to confirm the deletion of the target region, a DNA fragment in which a marker gene fragment such as a drug resistance gene is further linked between the upstream fragment and the downstream fragment is prepared.

First, by the first PCR, three fragments of the upstream fragment A and the downstream fragment B of the region to be deleted, and, if necessary, the marker gene fragment (chloramphenicol resistance gene fragment Cm in FIG. 1) are prepared. To do. For PCR amplification of the upstream and downstream fragments, primers with the sequences of the terminal 10-30 nucleotides of the later linked fragments are used. For example, when the upstream fragment A, the drug resistance marker gene fragment Cm, and the downstream fragment B are linked in this order, the upstream side of the drug resistance marker gene fragment Cm is located at the 5'end of the primer that binds to the downstream end of the upstream fragment A. A sequence corresponding to 10 to 30 nucleotides is added (Fig. 1, primer DR1), and to the 5'end of the primer that binds to the upstream end of the downstream fragment B, to 10 to 30 nucleotides downstream of the drug resistance marker gene fragment Cm. A corresponding sequence is added (FIG. 1, primer DF2). When the upstream fragment A and the downstream fragment B are amplified by PCR using the primer set designed in this way, a region corresponding to the upstream side of the drug resistance marker gene fragment Cm is located on the downstream side of the amplified upstream fragment A'. It will be added, and a region corresponding to the downstream side of the drug resistance marker gene fragment Cm will be added to the upstream side of the amplified downstream fragment B'.

Next, the upstream fragment A', the drug resistance marker gene fragment Cm, and the downstream fragment B'prepared by the first PCR are mixed and used as a template, and a primer that binds to the upstream side of the upstream fragment (FIG. 1, primer DF1). A second PCR is performed using a pair of primers consisting of a primer (FIG. 1, primer DR2) that binds to the downstream side of the downstream fragment. By this second PCR, the DNA fragment D for deletion to which the upstream fragment A, the drug resistance marker gene fragment Cm, and the downstream fragment B are bound in this order can be amplified.

The deletion DNA fragment obtained by the above method or the like is inserted into a plasmid using a normal restriction enzyme and DNA ligase to construct a deletion introduction plasmid. Alternatively, by preparing a DNA fragment for deletion in which the upstream fragment and the downstream fragment are directly linked and then inserting the DNA fragment for deletion into a plasmid containing a drug resistance marker gene, a drug can be added to the upstream fragment and the downstream fragment. A plasmid for introducing a deletion having a resistance marker gene fragment can be constructed.

The deletion-introducing plasmid constructed in the above procedure is introduced into Bacillus subtilis to which the genomic region is to be deleted by a usual method such as a competent cell transformation method. The introduction of the plasmid causes double cross-homologous recombination between the upstream and downstream fragments on the plasmid and those homologous regions of the Bacillus subtilis genome, and the region to be deleted becomes the drug resistance marker gene. A replaced transformant is obtained (Fig. 1). The transformant may be selected using the expression of a marker gene such as a drug resistance gene present in the deletion DNA fragment as an index. For example, a bacterium transformed with a chloramphenicol resistance gene fragment is cultured in a medium containing an antibiotic (chloramphenicol, etc.), and the grown colonies are collected to delete the target region. Transformants replaced with the chloramphenicol resistance gene can be obtained. Furthermore, by extracting the genomic DNA of the transformant and performing PCR using this as a template, it can be confirmed that the target region is deleted.

In addition, as another means of gene construction such that the bacillus abrB gene or a gene corresponding thereto is constitutively expressed, the expression of the bacterium abrB gene or a gene corresponding thereto on the genome is expressed in a stationary phase. Operatively link a control region that can be enhanced (strong expression control region), and introduce the bacillus abrB gene or its equivalent gene that is operably linked to the strong expression control region into the intracellular genome or plasmid. Can be mentioned. Examples of such a strong expression control region include a transcription initiation control region or a transcription initiation control region located upstream of the bacillus rplK gene, fus gene or atpI gene, or a gene corresponding to these genes, and a ribosome binding site. ..

The microorganism of the present invention can express a gene encoding a target protein or a target polypeptide in a microorganism whose gene is constructed so that the abrB gene of Bacillus subtilis or a gene corresponding thereto is constitutively expressed. It can be produced by strengthening or introducing into.

Enhancing or introducing a gene encoding a target protein or target polypeptide so that it can be expressed means that the gene encoding the target protein or target polypeptide is placed in at least an expressible state in the microorganism of the present invention. doing.
As a means for enhancing or introducing a gene encoding a target protein or a target polypeptide so that it can be expressed, for example, a control region (strong control region) capable of enhancing expression on the genome of the parent strain as compared with the wild type and the purpose. Introducing a gene of interest operably linked to a control region, preferably a strong control region, into the genome or plasmid of a parent cell, etc. Can be mentioned. For example, the control region sequence of the gene of interest on the genome of the parent strain is replaced with a strong control region, or an expression vector in which the gene of interest is integrated at an appropriate position or a strong control region is operably linked. The vector containing the gene of interest may be incorporated into the parent strain using a general transformation method.
Further, the control region can include a control region related to secretion (for example, a secretory signal peptide region) in addition to a control region related to transcription and translation, depending on the gene product to be expressed.
Various methods for introducing a gene into a cell so as to be expressible, as exemplified above, are well known to those skilled in the art. That is, when introduced by a vector, "regulatory region involved in 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 including a ribosome binding site and an initiation codon," A vector containing a gene encoding a target protein or a target polypeptide to which "one or more regions selected from a region and a secretory signal peptide region" are properly bound can be obtained by a competent cell transformation method, a promoter transformation method, or the like. Alternatively, it may be introduced by an appropriate transformation method such as an electroporation 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, proliferating and expressing it, and is not limited to a plasmid but also an artificial artificial such as YAC or BAC. Examples thereof include a chromosome, a vector using a transposon, and a cosmid, and examples of the plasmid include pUB110 and pHY300PLK.

Further, for insertion into the genome, for example, a method by homologous recombination may be used. That is, a DNA fragment to which a part of a chromosomal region to be introduced into a gene encoding a target protein or polypeptide is bound is taken up into a microbial cell to cause homologous recombination in a part of the chromosomal region. Can be integrated into the genome. Here, the chromosomal region that causes 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.

In one embodiment, the gene encoding the target protein or polypeptide introduced into the microorganism of the present invention is a transcription initiation including a regulatory region involved in transcription, translation, and secretion of the gene upstream thereof, that is, a promoter and a transcription initiation site. It is operably linked to one or more regions selected from the control region, the translation initiation region containing the ribosome binding site and the initiation codon, and the secretory signal peptide region. Preferably, the three regions consisting of the transcription initiation control region, the translation initiation control region, and the secretory signal region are more preferably derived from the cellulase gene of a Bacillus genus bacterium, and the transcription initiation control region is more preferable. In addition, a region in which the translation initiation control region is an upstream region having a length of 0.6 to 1 kb starting from the start codon of the cellulase gene is operably linked to the upstream of the target gene.

For example, the cellulase gene, which is mentioned as an example of a gene encoding a target protein or polypeptide introduced into the microorganism of the present invention, has its structural gene described in JP-A-2000-210081, JP-A-4-1090793, and the like. Transcription initiation control regions, translation initiation regions and secretory signals of cellulase genes derived from the described Bacillus strains, namely KSM-S237 strain (FERM BP-7875) or KSM-64 strain (FERM BP-2886). It is desirable that the peptide regions are operably linked. More specifically, from the base sequences of the cellulase gene base numbers 1 to 659 consisting of the base sequence represented by SEQ ID NO: 10 and the base sequences of the cellulase gene base numbers 1 to 696 consisting of the base sequence represented by SEQ ID NO: 12. DNA fragment, consisting of a base sequence having 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more identity with respect to the base sequence, and transcribing or translating the gene. A DNA fragment having a function related to secretion, or a DNA that hybridizes with a DNA consisting of any of the above base sequences under stringent conditions and has a function related to gene transcription, translation, and secretion, or any of the above base sequences. It is desirable that a DNA fragment consisting of a base sequence in which a part of the gene is deleted and having functions related to gene transcription, translation, and secretion is operably linked to the structural gene of the target gene. Here, the DNA fragment consisting of a base sequence in which a part of the base sequence is deleted is a part of the base sequence, preferably several (for example, 1 to 50, preferably 1 to 30). It means a DNA fragment that lacks 1 to 20 bases, more preferably 1 to 10 bases), but has functions related to gene transcription, translation, and secretion.

Further, the stringent conditions referred to here include, for example, the conditions described in [Molecular cloning-a Laboratory Manual 2nd edition (Sambrook et al., 1989)]. For example, 5 × SSC, 70 ° C. or higher is preferable, 5 × SSC, 85 ° C. or higher is more preferable, or 6 × SSC, 60 ° C. or higher is preferable, 6 × SSC, 65 ° C. or higher is more preferable, and the cleaning conditions are as follows. 1, × SSC, 60 ° C. or higher is preferable, and 1 × SSC, 73 ° C. or higher is more preferable.

In the present invention, the gene encoding the target protein or polypeptide is not particularly limited as long as it encodes a protein or polypeptide whose purpose is production or purification, and even an endogenous gene is used. , It may be a foreign gene (heterologous gene).
The gene encoding the protein or polypeptide of interest includes, for example, any protein or polypeptide useful in various fields such as detergents, foods, fibers, feeds, chemicals, medicine, diagnosis, and research, such as enzymes and physiologically active peptides. , Markers, signaling substances, structural proteins, genes encoding various regulators and the like. In addition, the above-mentioned enzymes are listed by function. / Synthetase) and the like, and preferred examples include hydrolases such as cellulase, α-amylase and protease.

Here, examples of the cellulase include cellulases belonging to Family 5 in the classification of polysaccharide hydrolases (Biochem. J., 280, 309, 1991), among which cellulases derived from microorganisms, preferably cells belonging to the genus Bacillus. Is preferably mentioned. As a more specific example, an alkali cellulase derived from the Bacillus bacterium KSM-S237 strain (FERM BP-7875) consisting of the amino acid sequence represented by SEQ ID NO: 11 or a Bacillus genus bacterium consisting of the amino acid sequence represented by SEQ ID NO: 13. Alkaline cellulase derived from KSM-64 strain (FERM BP-2886), or an amino acid having 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more identity with the amino acid sequence. Examples thereof include an alkaline cellulase consisting of a sequence, or an alkaline cellulase consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence represented by SEQ ID NO: 11 or SEQ ID NO: 13.

Examples of the α-amylase include α-amylase derived from a microorganism, and preferably liquefied amylase derived from a bacterium of the genus Bacillus. As a more specific example, an alkali amylase derived from Bacillus bacterium KSM-K38 strain (FERM BP-6946) consisting of the amino acid sequence shown in SEQ ID NO: 14, or 80% or more, preferably 90% or more, with the amino acid sequence. Alkaline amylase consisting of an amino acid sequence having 95% or more, more preferably 98% or more identity, or one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 14. Alkaline amylase consisting of the amino acid sequence of the above can be mentioned.

Preferable examples of the protease include serine proteases and metalloproteinases derived from microorganisms, preferably Bacillus bacteria. As a more specific example, an alkaline serine protease derived from Bacillus clausi KSM K-16 strain (FERM BP-3376) consisting of the amino acid sequence shown in SEQ ID NO: 15, or 80% or more of the amino acid sequence, preferably 80% or more. Is a protease consisting of an amino acid sequence having 90% or more, more preferably 95% or more, still more preferably 98% or more identity, or one or several amino acids are substituted or deleted in the amino acid sequence shown by SEQ ID NO: 15. , Alkaline serine protease consisting of an inserted or added amino acid sequence.

As shown in Examples described later, the recombinant microorganism of the present invention has significantly improved productivity of the target protein or polypeptide as compared with the microorganism without the above-mentioned gene modification.
By producing the target protein or polypeptide using the microorganism of the present invention, the target protein or polypeptide can be produced efficiently with high productivity, and the time and cost required for the production of the substance can be reduced. can do.

In the production of the target protein or polypeptide using the recombinant microorganism of the present invention, the strain is inoculated into a medium containing an assimilated carbon source, nitrogen source and other essential components, and cultured by a usual microbial culture method. After the culture is completed, the protein or polypeptide may be recovered and purified from the culture or cells. For example, a protein secreted and produced by culturing under the medium and conditions shown in Examples described later can be isolated and purified from a culture supernatant or the like.
The composition and composition of the medium are not particularly limited, but it is more preferable to use maltose or a medium containing soluble starch or glucose as the carbon source.
For recovery and purification of proteins and polypeptides, for example, separation of recombinant microorganisms by centrifugation or filtration, precipitation by adding a salt such as ammonium sulfate of the protein or polypeptide in the supernatant or filtrate, or an organic solvent such as ethanol. It can be carried out by using a method such as precipitation by adding, concentration or desalting using an ultrafiltration membrane or the like, purification using various chromatographies such as ion exchange or gel filtration.

As exemplary embodiments of the invention, the following are further disclosed herein. However, the present invention is not limited to these embodiments.
<1> A recombinant microorganism in which a gene encoding a target protein or polypeptide is fortified or introduced into a microorganism whose gene is constitutively expressed so that the abrB gene of Bacillus subtilis or a gene corresponding thereto is constitutively expressed.
<2> A gene construction that constitutively expresses the abrB gene of bacillus or a gene corresponding thereto is a transcription initiation control region, a transcription initiation control region and a ribosome binding site, or transcription of the abrB gene or a gene corresponding thereto. The recombinant microorganism of <1>, which is to inactivate the binding site of the inhibitory regulator in the region from the initiation control region to the translation initiation control region.
<3> The transcription initiation control region of the abrB gene or a gene corresponding thereto, the transcription initiation control region and the ribosome binding site, or the binding site of the inhibitory regulator in the region from the transcription initiation control region to the translation initiation control region. , Transcription of <2>, which is a region consisting of the 20th to 49th bases or a region corresponding thereto, or a region consisting of the 73rd to 89th bases or a region corresponding thereto in the base sequence shown in SEQ ID NO: 9. Microbial.
<4> The gene construction that constitutively expresses the abrB gene of Bacillus subtilis or the gene corresponding thereto deletes the region consisting of the 73rd to 89th bases or the region corresponding thereto in the base sequence shown in SEQ ID NO: 9. The recombinant microorganism of <3>.
<5> A recombinant microorganism according to any one of <1> to <4>, wherein the abrB gene of Bacillus subtilis or a gene corresponding thereto is selected from the group consisting of the following (a) to (f).
(A) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(B) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the nucleotide sequence shown in SEQ ID NO: 1. A poly encoding a protein consisting of a nucleotide sequence having preferably 98% or more, more preferably 99% or more identity, and having activity as a transcription factor of a gene induced in a transitional phase and a stationary phase similar to AbrB. nucleotide;
(C) As a transcription factor of a gene that hybridizes under stringent conditions to the complementary strand of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 and is induced in the transition phase and stationary phase similar to AbrB. A polynucleotide encoding an active protein;
(D) A polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(E) A transcription factor of a gene consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and induced in a transition phase and a stationary phase similar to AbrB. A polynucleotide encoding a protein with activity as;
(F) 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more than the amino acid sequence shown in SEQ ID NO: 2. A poly encoding a protein consisting of an amino acid sequence having preferably 98% or more, more preferably 99% or more identity, and having activity as a transcription factor of a gene induced in a transitional phase and a stationary phase similar to AbrB. nucleotide.
<6> A recombinant microorganism according to any one of <1> to <5>, wherein the microorganism is a bacterium belonging to the genus Bacillus.
<7> A recombinant microorganism according to any one of <1> to <5>, wherein the microorganism is Bacillus subtilis.
<8> Any one of <1> to <7> in which one or more of a transcription initiation control region, a translation initiation control region, or a secretory signal region is bound upstream of a gene encoding a target protein or polypeptide. Recombinant microorganisms.
<9> The recombinant microorganism of <8> in which three regions consisting of a transcription initiation control region, a translation initiation control region, and a secretory signal region are bound upstream of a gene encoding a target protein or polypeptide.
<10> The secretory signal region is derived from the cellulase gene of a bacterium belonging to the genus Bacillus, and the transcription initiation control region and the translation initiation control region are derived from the upstream 0.6 to 1 kb region of the cellulase gene. A <8> or <9> recombinant bacterium.
<11> The three regions consisting of the transcription initiation control region, the translation initiation control region, and the secretory signal region are represented by the nucleotide sequences 1 to 659 of the cellulase gene consisting of the nucleotide sequence shown in SEQ ID NO: 6, and SEQ ID NO: 8. 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96 with either the base sequence of the base number 1 to 696 of the cellulase gene consisting of the base sequence or the base sequence. % Or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more of a DNA fragment consisting of a base sequence, or a base sequence in which a part of the base sequence is deleted. Recombinant microorganisms of <9> and <10> that are DNA fragments.
<12> A method for producing a target protein or polypeptide, which comprises a step of culturing the recombinant microorganism according to any one of <1> to <11> and recovering the target protein or polypeptide from the culture or bacterial cells.
<13> Recombinant microorganisms including a step of constructing a gene so that the abrB gene of Bacillus subtilis or a gene corresponding thereto is constitutively expressed, and a step of enhancing or introducing a gene encoding a target protein or polypeptide so that it can be expressed. Manufacturing method.
<14> Using a recombinant microorganism in which the abrB gene of Bacillus subtilis or a gene corresponding thereto is constitutively expressed, and the gene encoding the target protein or polypeptide is fortified or introduced so as to be expressible. A method for improving the productivity of a target protein or polypeptide for producing the protein or polypeptide.

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

Example 1 Construction of recombinant microbial strain (1-1) Release of tryptophan requirement Preparation of Bacillus subtilis mutant strain 168T strain The tryptophan requirement of 168 wild Bacillus subtilis strain was canceled. Bacillus subtilis 168 strain possesses trpC2 as a genotype. That is, the 168 strain is auxotrophic for tryptophan synthesis due to a mutation in the trpC gene encoding indole-3-glycerol phosphate synthase involved in tryptophan synthesis. In addition, Bacillus subtilis ATCC6051T strain is known to be non-tryptophan-requiring (Albertini, AM & Galizzi, A. Microbiology, 1999, 145 (Pt12): 3319-3320). When the trpC genes of Bacillus subtilis 168 strain and ATCC6051T strain were compared, it was found that the 328th to 330th nucleotide ATTs were not present in Bacillus subtilis 168 strain. Therefore, PCR was performed using the ATCC6051T strain genome as a template and the primers of trpC_F1 and trpC_R1 shown in Table 1 to construct a PCR product. Using this PCR product, 168 wild strains of Bacillus subtilis were transformed by the competent method (J. Bacteriol., 1960, 81: 741-746), and tryptophan-free SMA agar medium (0.2% ammonium sulfate, 1). .4% dipotassium hydrogen phosphate, 0.6% potassium dihydrogen phosphate, 0.1% trisodium citrate-2-hydrate, 0.5% glucose, 0.035% magnesium sulfate heptahydrate, Colonies grown on 1.5% agar) were isolated as transformants. Thus, a tryptophan non-requiring 168T strain was obtained.

(1-2) Construction of Bacillus subtilis mutant strain (168T_abrB * strain) into which the abrB gene constitutive expression mutation was introduced The abrB gene constitutive expression mutation was introduced. Using the genomic DNA of the Bacillus subtilis wild strain 168 as a template, the abrB gene upstream region fragment (A) was amplified by PCR using the primers Dspo0Abox-FF and Dspo0Abox-FR shown in Table 2. In addition, the abrB gene upstream region fragment (B) was amplified by PCR using the primers Dspo0Abox-PF and Dspo0Abox-PR shown in Table 2. In addition, the primers Dspo0Abox-BF and Dspo0Abox-BR shown in Table 2 were used to amplify the abrB gene upstream / midstream region fragment (C) by PCR. Next, using the plasmid pDLT3 (Morimoto T. et al., Microbiology, 2002, 148: 3539-3552) carrying the chloramphenicol resistance gene as a template, and using the primers rPCR-CmF2 and rPCR-CmR2 shown in Table 2, PCR amplification was performed, and the fragment (D) of the chloramphenicol resistance gene was PCR amplified.

Next, the obtained fragments (A) and (D) were bound by the SOE-PCR method using the primers Dspo0Abox-FF and rPCR-CmR2 shown in Table 2, and the fragments (B) and (C) were shown in the table. It was bound by the SOE-PCR method using the primers Dspo0Abox-PF and Dspo0Abox-BR described in 2. Finally, the fragment (A + D) and the fragment (B + C) were bound by the SOE-PCR method using the primers Dspo0Abox-FF and Dspo0Abox-BR shown in Table 2 to obtain the final PCR product (A + D + B + C). The obtained final PCR product was transformed with Bacillus subtilis mutant strain 168T to obtain Bacillus subtilis mutant strain 168T_abrB * _Cm strain.

Next, the drug resistance gene replacement plasmid pCm :: Em (Lei Y. et al., J. Bacteriol., 2013, 195: 1697-1705) was used to transform with the spore-forming mutant strain 168_abrB * _Cm strain. A mutant strain of Bacillus subtilis 168T_abrB * in which the chloramphenicol resistance gene was replaced with the erythromycin resistance gene was constructed.

It was confirmed that the target mutation was introduced at a predetermined position on the genome by PCR using the genome of the obtained Bacillus subtilis mutant strain 168T_abrB * strain and subsequent sequencing by the Sanger method.

(1-3) Construction of Vector for Alkaline Cellulase Production (pHY-S237) S237 Cellulase Gene Derived from Bacillus Bacteria KSM-S237 Strain (FERM BP-7875) (Refer to JP-A-2000-210081) (SEQ ID NO: 10) Primer Egl-S237 shown in Table 3 using the DNA fragment encoding (3.1 kb; base numbers 13 to 3124 of SEQ ID NO: 10) as a template. F and primer Egl-S237. PCR was performed using the R primer set to construct a recombinant plasmid pHY-S237 inserted at the BamHI restriction enzyme cut point of the shuttle vector pHY300PLK.

(1-4) Preparation of recombinant Bacillus subtilis mutant strain into which a vector for producing alkaline cellulase (pHY-S237) was introduced. Bacillus subtilis mutant strain 168T strain and Bacillus subtilis prepared in (1-1) and (1-2) above. The fungal mutant strain 168T_abrB * strain was used as a host.
Each host was transformed by a protoplast transformation method (Mol. Gen. Genet., 1979, 168: 111-115) using the alkali cellulase production vector (pHY-S237) prepared in (1-3) above. .. DM3 protoplast regeneration medium supplemented with 50 ppm tetracycline-hydrochloride (0.5 M sodium succinate (pH 7.3), 0.5% casamino acid (manufactured by Difco), 0.5% yeast extract (manufactured by Difco), 0 .35% K ₂ HPO ₄ , 0.15% KH ₂ PO ₄ , 0.5% glucose, 20 mM MgCl ₂ , 0.01% bovine serum albumin (manufactured by Sigma), 1% bacto agar (manufactured by Diffco)) The colonies that grew above were selected as the target Bacillus subtilis transformants.

Example 2 Evaluation of Cellulase Productivity The transformant obtained in Example 1 was subjected to 5 mL of LB medium (1.0% trypton (manufactured by Diffco), 0.5% yeast extract (Difco) containing 15 ppm tetracycline). Incubate with shaking at 30 ° C. for 15 hours in 1.0% NaCl), and then add 0.6 mL of this culture solution to 30 mL of 2 × L-maltose medium (2% trypton, 1% yeast) containing 15 ppm tetracycline. The extract was inoculated with 1% sodium chloride, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate) and cultured with shaking at 30 ° C. for 3 days. After culturing, the alkali cellulase activity of the culture supernatant from which the cells were removed by centrifugation was measured, and the amount of alkali cellulase secreted and produced outside the cells was calculated.
The productivity comparison was made by a relative value with the productivity of the parent strain as 100%. As a result, a higher amount of alkaline cellulase secreted and produced was confirmed in the 168T_abrB * strain in which abrB was constitutively expressed, as compared with the case where the 168T strain was used as the parent strain.

(Measurement Example) Measurement of Alkaline Cellulase Activity For cellulase activity measurement, 0.4 mM p-nitrophenyl- was added to 50 μL of a sample solution appropriately diluted with 1 / 7.5 M phosphate buffer (pH 7.4 manufactured by Wako Pure Chemical Industries, Ltd.). 50 μL of β-D-cellulotrioside (manufactured by Biochemical Industries, Ltd.) is added and mixed, and the amount of p-nitrophenol liberated when reacted at 30 ° C. is quantified by the change in absorbance (OD420 nm) at 420 nm. Was done by. Cellulase activity was defined as the amount of enzyme that liberates 1 μmol of p-nitrophenol per minute as 1 U.

Claims (11)

A recombinant microorganism in which a gene encoding a target protein or polypeptide is fortified or introduced into Bacillus subtilis in which the abrB gene of Bacillus subtilis or a gene corresponding thereto is constitutively expressed. ,
A recombinant microorganism in which the abrB gene of Bacillus subtilis or a gene corresponding thereto is selected from the group consisting of the following (a) , (b), (d) to (f).
(A) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(B) A protein consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 and having activity as a transcription factor of a gene induced in the transition phase and the stationary phase similar to AbrB is encoded. Polynucleotide to
(D) A polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(E) A transcription factor of a gene consisting of an amino acid sequence in which 1 to 9 amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and induced in a transition phase and a stationary phase similar to AbrB. A polynucleotide encoding a protein with activity as;
(F) A protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having activity as a transcription factor of a gene induced in the transition phase and the stationary phase similar to AbrB is encoded. Polynucleotide to be. A gene construction such that the abrB gene of bacillus or a gene corresponding thereto is constitutively expressed is a transcription initiation control region, a transcription initiation control region and a ribosome binding site, or a transcription initiation control region of the abrB gene or a gene corresponding thereto. The recombinant microorganism according to claim 1, wherein the binding site of the inhibitory regulator in the region from to the translation initiation control region is inactivated. The transcription initiation control region of the abrB gene or its corresponding gene, or the transcription initiation control region and ribosome binding site, or the binding site of the inhibitory regulator in the region from the transcription initiation control region to the translation initiation control region is the SEQ ID NO: It consists of a base sequence having 90% or more identity with the base sequence shown in 9, and is a transcription initiation control region or a transcription initiation control region and a ribosome binding site, or a region from the transcription initiation control region to the translation initiation control region. 2. A region consisting of the 20th to 49th bases of SEQ ID NO: 9 or a region corresponding thereto, or / and a region consisting of the 73rd to 89th bases or a region corresponding thereto in the DNA having the function of. Recombinant microorganisms described in the section. A gene construction such that the abrB gene of Bacillus subtilis or a gene corresponding thereto is constitutively expressed deletes the region consisting of the 73rd to 89th bases or the region corresponding thereto in the base sequence shown in SEQ ID NO: 9. The recombinant microorganism according to claim 3. The set according to any one of claims 1 to 4, wherein any one or more regions of a transcription initiation control region, a translation initiation control region, and a secretory signal region are bound upstream of a gene encoding a target protein or polypeptide. Replacement microorganisms. The recombinant microorganism according to claim 5, wherein three regions consisting of a transcription initiation control region, a translation initiation control region, and a secretory signal region are bound upstream of a gene encoding a target protein or polypeptide. Claim that the secretory signal region is derived from the cellulase gene of a bacterium belonging to the genus Bacillus, and the transcription initiation control region and the translation initiation control region are derived from the upstream 0.6 to 1 kb region of the cellulase gene. 5 or 6 Recombinant Microorganism. The three regions consisting of the transcription initiation control region, the translation initiation control region, and the secretory signal region are composed of the nucleotide sequences of the nucleotide sequences 1 to 659 of the cellulase gene consisting of the nucleotide sequence represented by SEQ ID NO: 10 or the nucleotide sequence represented by SEQ ID NO: 12. From the base sequence of base numbers 1 to 696 of the cellulase gene, or from a DNA fragment consisting of a base sequence having 90% or more identity with any of these base sequences, or a base sequence in which a part of the base sequence is deleted. The recombinant microorganism according to claims 6 and 7, which is a DNA fragment. A method for producing a target protein or polypeptide, which comprises a step of culturing the recombinant microorganism according to any one of claims 1 to 8 and recovering the target protein or polypeptide from the culture or bacterial cells. Production of recombinant Bacillus subtilis including a step of constructing a gene so that the abrB gene of Bacillus subtilis or a gene corresponding thereto is constitutively expressed, and a step of enhancing or introducing a gene encoding a target protein or polypeptide so that it can be expressed. The way,
A method in which the abrB gene of Bacillus subtilis or a gene corresponding thereto is selected from the group consisting of the following (a) , (b), (d) to (f).
(A) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(B) A protein consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 and having activity as a transcription factor of a gene induced in the transition phase and the stationary phase similar to AbrB is encoded. Polynucleotide to
(D) A polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(E) A transcription factor of a gene consisting of an amino acid sequence in which 1 to 9 amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and induced in a transition phase and a stationary phase similar to AbrB. A polynucleotide encoding a protein with activity as;
(F) A protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having activity as a transcription factor of a gene induced in the transition phase and the stationary phase similar to AbrB is encoded. Polynucleotide to be. The protein is constructed using a recombinant bacillus in which the abrB gene of the bacillus or a gene corresponding thereto is constitutively expressed, and the gene encoding the target protein or polypeptide is fortified or introduced so as to be expressible. Or a method for improving the productivity of a target protein or polypeptide, which produces a polypeptide.
A method in which the abrB gene of Bacillus subtilis or a gene corresponding thereto is selected from the group consisting of the following (a) , (b), (d) to (f).
(A) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1;
(B) A protein consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 and having activity as a transcription factor of a gene induced in the transition phase and the stationary phase similar to AbrB is encoded. Polynucleotide to
(D) A polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(E) A transcription factor of a gene consisting of an amino acid sequence in which 1 to 9 amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 and induced in a transition phase and a stationary phase similar to AbrB. A polynucleotide encoding a protein with activity as;
(F) A protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having activity as a transcription factor of a gene induced in the transition phase and the stationary phase similar to AbrB is encoded. Polynucleotide to be.