JP4850011B2 - Recombinant microorganism - Google Patents

Description

  The present invention relates to a recombinant microorganism used for producing a useful protein or polypeptide, and a method for producing a protein or polypeptide.

  The industrial production of useful substances by microorganisms includes a wide variety of types including foods such as alcoholic beverages, miso and soy sauce, as well as amino acids, organic acids, nucleic acid-related substances, antibiotics, carbohydrates, lipids, proteins, etc. In addition, its use has been extended to a wide range of fields from daily necessities such as foods, medicines, detergents and cosmetics to various chemical raw materials.

In industrial production of useful substances by such microorganisms, improvement of productivity is one of the important issues, and breeding of produced bacteria by genetic techniques such as mutation has been performed as a technique. Particularly recently, with the development of microbial genetics and biotechnology, more efficient breeding of production bacteria using genetic recombination techniques has been carried out. Furthermore, in response to the rapid development of genome analysis technology in recent years, attempts have been made to decode genome information of target microorganisms and actively apply them to industry. Industrially useful host microorganisms whose genome information has been disclosed include Bacillus subtilis Marburg No. 168 (Non-patent Document 1), Escherichia coli K-12 MG1655 (Non-patent Document 2), Corynebacterium Corynebacterium glutamicum ATCC132032 and the like are mentioned, and strains with improved information have been developed using these genomic information. However, despite the above efforts, production efficiency is not always satisfactory.

The prsA gene was found in Bacillus subtilis as a protein involved in the protein secretion process, and previous studies have shown that PrsA is a chaperone-like protein that facilitates protein folding after it has been transported out of the cytoplasm across the cell membrane. It is presumed to have the function of Then, a microorganism in which the productivity of the target protein has been improved by introducing a plasmid into which the prsA gene of Bacillus subtilis containing its own promoter region is introduced and having a plurality of prsA genes in the microorganism has been reported (Non-Non- patent document) Patent Document 3). However, such a microorganism is not practical because it is difficult to control the number of a plurality of plasmids introduced per cell, and the plasmids are often dropped during production culture. Furthermore, in the above report, the cloning vector pHP13 into which the prsA gene fragment has been inserted contains the replication function of the Bacillus subtilis plasmid pTA1060 (Non-patent Document 4). It is reported that the average is 5.2 (Non-Patent Document 5). That is, since the increase in the target protein in the above report is 1 to 5 times the production of the wild strain, the increase in production per introduced prsA gene is about 0.2 to 1 times the production of the wild strain. Therefore, it cannot be said that a sufficient effect is obtained.

On the other hand, in bacteria, many carbohydrates such as glucose and maltose are taken into cells via the phosphoenolpyruvate: carbohydrate phosphotransferase (PTS) system and metabolized by the glycolytic system. Regarding the carbohydrate uptake by such a PTS system, ptsGHI operon (uptake of glucose and other PTS sugars), glv operon (uptake of maltose), tre operon (uptake of trehalose), etc. are involved. Its expression is regulated by transcription factors encoded by glcT gene, glvR gene, treR gene and the like. More particularly, ptsG gene in ptsGHI operon, ptsH gene, contains ptsI gene, their expression is positively regulated by the transcription factor GlcT the glcT gene encodes (Non-patent Document 6), the glv operon glvA gene, GLVR genes include glvC genes, their expression is positively regulated by the transcription factor GlvR the GLVR gene encodes (non-Patent Document 7), also the tre operon treP gene, tREA gene, It is known that the treR gene is contained and the expression thereof is negatively controlled by the transcription factor TreR encoded by the treR gene (Non-patent Document 8). That is, by inactivating the glcT gene, ptsG gene, ptsH gene, ptsI gene, glvA gene, glvR gene, glvC gene, treP gene, or treA gene among the above genes, the uptake of carbohydrates by each PTS system Is considered to be suppressed.
However, the prsA gene overexpressed, and microorganisms subjected to inactivation of genes suppressing incorporation of sugar by such PTS system is not known so far, especially such microorganisms prsA It is not even expected to show superior productivity of useful proteins or polypeptides compared to microorganisms overexpressing genes.
Nature, 390,249,1997 Science, 277,1453,1997 Mol. Microbiology, 8,: 727 (1993) Mol. Gene. Genet., 209: 335 (1987) Plasmid, 18: 8 (1987) Mol. Microbiol., 25:65 (1997) J. Bacteriol., 183: 5110 (2001) J. Bacteriol., 178: 4576 (1996)

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

The present inventors searched for genes that affect the production of useful proteins or polypeptides in various genes encoded on the microbial genome, and overexpressed Bacillus subtilis PrsA by enhancing the Bacillus subtilis prsA gene. When a gene encoding a target protein or polypeptide is introduced into a microorganism strain in which the expression of a specific operon involved in carbohydrate uptake by the PTS system is controlled, the productivity of the target protein or polypeptide Has been found to be improved compared to before modification.

That is, the present invention is a transcription initiation control region having a function in a microorganism or a transcription initiation control region and a ribosome binding site introduced upstream in the genome of a Bacillus subtilis prsA gene or a gene corresponding to the gene, or a microorganism Introducing a transcription initiation regulatory region having a function or a gene fragment in which a transcription initiation regulatory region and a ribosome binding site are linked upstream of a Bacillus subtilis prsA gene or a gene corresponding to the gene, and glcT gene, ptsG gene, ptsH gene PtsI gene, glvR gene, glvA gene, glvC gene, treP gene, treA gene and one or more genes selected from the genes corresponding to the gene are deleted or inactivated in the microorganism strain. The present invention relates to a recombinant microorganism into which a gene encoding a peptide is introduced.

  The present invention also relates to a method for producing a target protein or polypeptide using the recombinant microorganism.

  The recombinant microorganism of the present invention has high productivity of the target protein or polypeptide. Therefore, when the target protein or target polypeptide is produced using this, the time and cost required for production of the substance can be reduced.

  In the present invention, the identity of the amino acid sequence and the base sequence is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it is calculated by performing an analysis with Unit size to compare (ktup) set to 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (software development).

  In the present specification, the transcription initiation control region is a region including a promoter and a transcription initiation point, and the ribosome binding site is a Shine-Dalgarno (SD) sequence (Proc. Natl. Acad) that forms a translation initiation control region together with the initiation codon. Sci. USA 74, 5463 (1974)).

As the parent microorganism for constructing the microorganism of the present invention, any microorganism may be used as long as it has a ptsGHI operon, a glv operon or a tre operon of Bacillus subtilis or a gene corresponding to the gene. You may have done. Specific examples include bacteria belonging to the genus Bacillus, bacteria belonging to the genus Clostridium , yeast, etc. Among them, bacteria belonging to the genus Bacillus are preferable. Furthermore, Bacillus subtilis is particularly preferred from the viewpoint that whole genome information has been clarified, genetic engineering and genome engineering techniques have been established, and that it has the ability to secrete and produce proteins outside the cells.
The names of the genes and gene regions of Bacillus subtilis described in this specification are 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 on March 10, 2004), based on genome data of Bacillus subtilis.

The Bacillus subtilis prsA gene of the present invention refers to a gene consisting of the base sequence represented by SEQ ID NO: 1. The gene corresponding to the Bacillus subtilis prsA gene refers to a gene having a Bacillus subtilis prsA gene substantially the same function, for example, Bacillus licheniformis (Bacillus licheniformis), Bacillus anthracis (Bacillus anthracis), Bacillus cereus (Bacillus cereus), In Bacillus thuringiensis , Oceanobacillus iheyensis, etc., each prsA gene identified mainly by genome analysis can be mentioned. In some cases, three types of the genes have been identified, such as Bacillus anthracis . Examples of the gene corresponding to the Bacillus subtilis prsA gene include any of the following genes (1) to (4).

  (1) A protein comprising a nucleotide sequence having 90% or more, preferably 95% or more, more preferably 99% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and comprising an amino acid sequence represented by SEQ ID NO: 2 A gene consisting of DNA that encodes a functionally equivalent protein.

  (2) A protein that is hybridized under stringent conditions with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 and functionally equivalent to a protein comprising the amino acid sequence represented by SEQ ID NO: 2 A gene consisting of DNA encoding. Here, examples of the “stringent conditions” include a method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell., Cold Spring Harbor Laboratory Press], for example, 6 × SSC. (Composition of 1 × SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhart and 100 mg / mL herring sperm DNA together with the probe at 65 ° C. for 8 The conditions are such that the temperature is kept constant for 16 hours and hybridized.

  (3) A protein comprising an amino acid sequence having 90% or more, preferably 95% or more, more preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and comprising the amino acid sequence represented by SEQ ID NO: 2 A gene consisting of DNA that encodes a functionally equivalent protein.

(4) In the amino acid sequence shown in SEQ ID NO: 2, it consists of an amino acid sequence in which one or more amino acids are deleted, substituted or added, and is functionally equivalent to a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. A gene consisting of DNA that codes for various proteins.
Here, in the amino acid sequence represented by SEQ ID NO: 2, one or several, preferably 1 to 10 amino acids are missing in the amino acid sequence in which one or more amino acids are deleted, substituted or added. A deleted, substituted or added amino acid sequence is included, and the addition includes the addition of one to several amino acids at both ends.
A protein functionally equivalent to the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 has substantially the same function as the protein encoded by the prsA gene and is transported out of the cytoplasm through the cell membrane. A protein that is thought to have a chaperone-like function that facilitates the subsequent folding of the protein.

The transcription initiation control region or transcription initiation control region and ribosome binding site that function in the microorganism of the present invention is not particularly limited as long as it is a transcription initiation control region or transcription initiation control region and ribosome binding site that functions in the host microorganism. For example, a Bacillus subtilis spoVG gene or aprE gene or a transcription initiation control region or a transcription initiation control region located upstream of a gene corresponding to these genes and a ribosome binding site are preferred. As an example of the transcription initiation control region of the Bacillus subtilis spoVG gene, a region comprising the nucleotide sequence of base numbers 38 to 210 of SEQ ID NO: 9, or a region comprising a sequence homologous to these sequences and having a function as a transcription initiation control region Is mentioned. In addition, as an example of the transcription initiation control region and the ribosome binding site of the Bacillus subtilis spoVG gene, a region comprising the nucleotide sequence of base numbers 38 to 230 of SEQ ID NO: 9, or a transcription initiation control region comprising a sequence homologous to these sequences And a region having a function as a ribosome binding site.

Introducing the transcription initiation control region or transcription initiation control region and the ribosome binding site upstream of the prsA gene or the gene corresponding to the gene into the genome, the original prsA gene of Bacillus subtilis or the gene corresponding to the gene In addition to those that replace part or all of the transcription initiation control region, or the transcription initiation control region and the ribosome binding site, the original transcription initiation control region, or those that are inserted with the transcription initiation control region and the ribosome binding site left intact included.

The transcription initiation control region, or the substitution to the transcription initiation control region and the ribosome binding site can be performed using, for example, a known method by homologous recombination. That is, first, a DNA fragment containing an upstream region of the original transcription initiation control region of the prsA gene and a drug resistance gene fragment upstream of the transcription initiation control region or a DNA fragment comprising the transcription initiation control region and the ribosome binding site, , Downstream of the prsA gene translation initiation control region and part or all of the structural gene region, or a DNA fragment containing part or all of the structural gene region of the prsA gene, SOE (splicing by overlap extension) -PCR method (Gene , 77, 61, 1989). In this way, a DNA fragment containing the upstream region of the original transcription initiation control region of the prsA gene, a drug resistance gene fragment, the transcription initiation control region, or a DNA fragment comprising the transcription initiation control region and the ribosome binding site, the prsA gene A DNA fragment is obtained in which a translation initiation control region and a part or all of the structural gene region, or a DNA fragment containing part or all of the structural gene region of the prsA gene are joined in this order.

Next, by incorporating such a DNA fragment into the parent microbial cell by a known method, the upstream region of the original transcription initiation control region of the prsA gene of the parent microbial genome, and the translation initiation control region and structural gene of the prsA gene Double crossover homologous recombination occurs in two places of the region including part or all of the region or part or all of the structural gene region of the prsA gene. As a result, the original transcription initiation control region, or a transformant in which the transcription initiation control region and the ribosome binding site are replaced with the transcription initiation control region, or the transcription initiation control region and the ribosome binding site, is used as an indicator of the drug resistance gene. Can be separated as Thereby, the transcription initiation control region introduced upstream of the prsA gene on the genome, or the transcription initiation control region and the ribosome binding site are genetically stably maintained. In addition, as a known method for specifically introducing a DNA fragment for introduction into a host microorganism, a competent cell transformation method (J. Bacterial. 93, 1925 (1967)), a protoplast transformation method (Mol. Gen. Genet 168, 111 (1979)) or electroporation method (FEMS Microbiol. Lett. 55, 135 (1990)), etc., and competent cell transformation methods are particularly preferred. Further, in the present specification, upstream and downstream of a gene is not a position from the replication start point, and upstream indicates a region continuing on the 5 ′ side of the gene or region regarded as a target, while downstream is The region following the 3 'side of the gene or region captured as the target is shown.
In particular, when Bacillus subtilis is used as the host of the microorganism of the present invention, the transcription initiation control region of the spoVG gene, or the transcription initiation control region and the ribosome from the transcription initiation control region of the prsA gene, or the transcription initiation control region and the ribosome binding site. Substitution by homologous recombination to the binding site can be performed using the method described in Mol. Gen. Genet., 223, 268, 1990, or the like.

  In addition, for the insertion of the transcription initiation control region or transcription initiation control region and the ribosome binding site, appropriately select the transcription initiation control region to be inserted or the sequence of the DNA fragment added to both ends of the transcription initiation control region and the ribosome binding site. For example, it can be carried out by the same method as the above-described substitution method. For example, a DNA fragment containing an upstream region of the original transcription initiation control region and a drug resistance gene fragment are linked upstream of the transcription initiation control region, and a DNA containing a part or all of the original transcription initiation control region downstream. Combine the fragments. In this way, the DNA fragment containing the upstream region of the original transcription initiation control region, the drug resistance gene fragment, the transcription initiation control region, and the DNA fragment containing part or all of the original transcription initiation control region were combined in this order. Obtain a DNA fragment. Next, after inserting such a DNA fragment into a host microorganism, a transformant can be isolated using a drug resistance gene as an index. On the genome of the transformant thus separated, the original transcription initiation control region and the transcription initiation control region are stably maintained in a state where they are adjacent to each other without any gap.

In the present invention, the transcription initiation control region or the upstream in the genome where the transcription initiation control region and the ribosome binding site are introduced is not particularly limited as long as it is upstream of the start codon of the prsA gene, but within 2000 base pairs adjacent to each other. This region is preferable, a region within 500 base pairs is more preferable, a region within 100 base pairs is more preferable, and a region within 50 base pairs is particularly preferable.

In addition, the transcription initiation control region in the present invention or the gene fragment in which the transcription initiation control region and the ribosome binding site are linked upstream of the prsA gene can be obtained by a known cloning method such as PCR using the genome of Bacillus subtilis or other microorganisms as a template. Based on the obtained transcription initiation control region or transcription initiation control region and ribosome binding site fragment, and prsA gene fragment, restriction enzyme method, SOE (splicing by overlap extension) -PCR method (Gene, 77, 61, 1989), etc. It can obtain by connecting by a well-known method. Such a fragment can be introduced into the cytoplasm by a vector such as a plasmid, and homologous recombination is performed between the nucleic acid fragment introduced into the cell and the chromosome by a known transformation method. Can be introduced.

The chromosomal region to be introduced in the host is preferably a non-essential gene or a non-essential gene non-gene region upstream, for example, aprE gene, sacB gene, nprE gene, amyE gene, ybxG gene internal, or while the interior of the non-gene region of the gene upstream and the like, inside the amyE gene, or the interior of the non-gene region of ybxG gene upstream preferred. Here, a non-essential gene means a gene that allows a host to survive at least under certain conditions even if it is destroyed. In addition, no problem arises even when a part or all of a non-essential gene upstream or a non-gene region upstream of a non-essential gene is involved in introduction.

In the following, the transcription initiation control region using a DNA fragment prepared by SOE (splicing by overlap extension) -PCR method (Gene, 77, 61, 1989), or the transcription initiation control region and the ribosome binding site are designated as the prsA gene. A method of introducing the gene fragment linked upstream of the parent microorganism genome by double crossing will be described.
The DNA fragment for introduction used here is about 0.1 to 3, preferably 0.4 to 3 kb fragment (hereinafter referred to as fragment (1)) adjacent to the upstream of the introduction site on the parent microorganism genome, and about 0.1 to 3 adjacent to the downstream. Preferably, the transcription initiation control region or a fragment comprising the transcription initiation control region and a ribosome binding site (hereinafter referred to as fragment (3)), between the 0.4 to 3 kb fragment (hereinafter referred to as fragment (2)), prsA gene fragment ( Hereinafter, it is a DNA fragment into which a fragment (4)) and a drug resistance marker gene fragment (hereinafter referred to as fragment (5)) are inserted. First, five fragments of fragment (1) to fragment (5) are prepared by the first PCR.
At this time, for example, the upstream 10-30 base pair sequence of fragment (3) at the downstream end of fragment (1), and the downstream 10-30 base pair sequence of fragment (3) at the upstream end of fragment (4) Designed to add the 10-30 base pair sequence upstream of fragment (5) at the downstream end of (4), and the 10-30 base pair sequence downstream of fragment (5) upstream of fragment (2). Were used (FIG. 1).

  Next, the second PCR is carried out using the five types of PCR fragments prepared in the first round as templates and using the upstream primer of fragment (1) and the downstream primer of fragment (2). This causes annealing with the fragment (3) in the sequence of the fragment (3) added to the downstream end of the fragment (1). Similarly, the sequence of the fragment (3) added to the upstream end of the fragment (4) Annealing with fragment (3) occurs in the sequence, annealing with fragment (5) occurs in the sequence of fragment (5) added to the downstream end of fragment (4), and upstream of fragment (2) Annealing with the fragment (5) occurs in the sequence of the fragment (5) added to. As a result of PCR amplification, a DNA fragment in which the five fragments (1) to (5) are bound in the order of (1), (3), (4), (5), and (2) can be obtained (FIG. 1).

  As a drug marker gene, an erythromycin resistance gene or the like can be used. The PCR reaction performed here uses the primer set shown in Table 1 below, and uses a general PCR enzyme kit such as Pyrobest DNA Polymerase (Takara Shuzo), etc. (PCR Protocols. Current Methods and Applications, Edited by BAWhite, Humana Press pp251, 1993, Gene, 77, 61, 1989) and the like.

When the DNA fragment for introduction thus obtained is introduced into a cell by a competent method or the like, genetic recombination occurs in the cell in the homologous region upstream and downstream of the introduction site on the same genome. A cell into which a transcription initiation control region or a gene fragment in which a transcription initiation control region and a ribosome binding site are linked upstream of the prsA gene can be isolated by selection with a drug resistance marker. Selection with a drug resistance marker can be performed, for example, by isolating colonies that grow on an agar medium containing erythromycin and then selecting those that are confirmed to be introduced onto the genome by PCR using the genome as a template. Good. The drug resistance marker gene is not particularly limited as long as it can be used for selection using commonly used antibiotics. In addition to the erythromycin resistance gene, the chloramphenicol resistance gene, neomycin resistance And drug resistance marker genes such as genes, spectinomycin resistance genes, tetracycline resistance genes and blasticidin S resistance genes.

In the recombinant microorganism of the present invention, in addition to the overexpression of PrsA by the prsA gene enhancement described above, glcT gene, ptsG gene, ptsH gene, ptsI gene, glvR gene, glvA gene, glvC gene, treP gene, treA gene and One or more genes selected from genes corresponding to the gene are deleted or inactivated. As shown in Example 8, the deletion or inactivation of the gene is a modification that suppresses the uptake of carbohydrates into cells.
Such gene deletion or inactivation may be deletion or inactivation of each of the above genes alone, or a combination of two or more. Further, enhancement, deletion or inactivation of genes other than the gene may be performed together. In addition, the deletion or inactivation of a gene includes the insertion or insertion of a base into the gene in addition to the substitution / deletion of all or a part of the base in the gene.

The glcT gene, ptsG gene, ptsH gene, ptsI gene, glvA gene, glvR gene, glvC gene, treP gene, or treA gene are all genes involved in carbohydrate uptake by the PTS system. Regarding PTS system carbohydrate uptake by, PtsGHI operon (glucose and other PTS sugar uptake), GLV operon (maltose uptake), are involved such tre operon (trehalose uptake), the operon, respectively glcT gene Its expression is regulated by transcription factors encoded by glvR gene, treR gene, etc. More particularly, ptsG gene in ptsGHI operon, ptsH gene, contains ptsI gene, their expression is positively regulated by the transcription factor GlcT the glcT gene encodes, GlvA gene in glv operon, GLVR gene , include glvC genes, their expression is positively regulated by the transcription factor glvR the glvR gene encodes, also the tre operon treP gene, tREA gene, contains treR gene, their expression treR gene It is known to be negatively regulated by the transcription factor TreR encoded by Table 6 summarizes the gene numbers and functions of glcT gene, ptsG gene, ptsH gene, ptsI gene, glvA gene, glvR gene, glvC gene, treP gene, and treA gene.

The gene corresponding to the glcT gene, ptsG gene, ptsH gene, ptsI gene, glvR gene, glvA gene, glvC gene, treP gene, or treA gene refers to a gene having substantially the same function as each of the genes. , 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more of the identity of each of these genes and nucleotide sequences, derived from other microorganisms, Preferably, a gene derived from a bacterium belonging to the genus Bacillus is used. Here, the identity of the base sequence is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985).

  As a procedure for the deletion or inactivation of the above-mentioned gene, in addition to a method of systematically deleting or inactivating the gene (target gene), a random gene deletion or inactivation mutation is given. And a method for evaluating protein productivity and performing gene analysis by an appropriate method.

  In order to delete or inactivate the target gene, for example, a method by homologous recombination may be used. That is, a circular recombinant plasmid obtained by cloning a DNA fragment containing a part of the target gene into an appropriate plasmid vector is introduced into the parent microbial cell, and the parent gene is homologously recombined in a part of the target gene. It is possible to disrupt and inactivate target genes on the microbial genome. Alternatively, a target gene inactivated by mutation such as base substitution or base insertion, or a linear DNA fragment containing the upstream and downstream regions of the target gene but not the target gene as shown in FIG. Constructed by the method, this is incorporated into the parent microbial cells to cause two crossover homologous recombination at two locations outside the mutation site in the target gene of the parent microbial genome, or upstream and downstream of the target gene Thus, it is possible to replace the target gene on the genome with a deleted or inactivated gene fragment.

  In particular, when Bacillus subtilis is used as a parental 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.), the host microorganism of the present invention can be obtained by repeating these methods.

  In addition, random gene deletion or inactivation can be achieved by a method of causing homologous recombination similar to the above method using a randomly cloned DNA fragment, or by irradiating a parental microorganism with γ rays, etc. Can also be implemented.

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

  The deletion DNA fragment used in this method is between about 0.1 to 3, preferably 0.4 to 3 kb fragment adjacent to the upstream of the gene to be deleted, and about 0.1 to 3, preferably 0.4 to 3 kb fragment adjacent to the downstream. A fragment into which a drug resistance marker gene fragment is inserted. First, an upstream fragment and a downstream fragment of the gene to be deleted and three fragments of a drug resistance marker gene fragment are prepared by the first PCR. At this time, for example, at the downstream end of the upstream fragment, the upstream side of the drug resistance marker gene is prepared. A primer designed to add a 10-30 base pair sequence, and conversely, a 10-30 base pair sequence downstream of the drug resistance marker gene is used at the upstream end of the downstream fragment (FIG. 3).

  Next, using the three types of PCR fragments prepared in the first round as templates, the second round PCR is performed using the upstream primer of the upstream fragment and the downstream primer of the downstream fragment. As a result, in the drug resistance marker gene sequence added to the downstream end of the upstream fragment and the upstream end of the downstream fragment, annealing with the drug resistance marker gene fragment occurs, and as a result of PCR amplification, the upstream fragment and the downstream fragment In the meantime, a DNA fragment into which a drug resistance marker gene is inserted can be obtained (FIG. 3).

  When using a chloramphenicol resistance gene as a drug resistance marker gene, for example, using the primer set shown in Table 1, using a general PCR enzyme kit such as Pyrobest DNA polymerase (Takara Shuzo), etc. Protocols. Current Methods and Applications, Edited by BAWhite, Humana Press pp251, 1993, Gene, 77, 61, 1989) etc., by performing SOE-PCR under the usual conditions, DNA fragments for deletion of each gene can be obtained. can get.

  When the DNA fragment for deletion thus obtained is introduced into the cell by a competent method or the like, gene recombination occurs in the cell in the homologous region upstream and downstream of the same gene to be deleted, and the target gene Can be isolated by selection with a drug resistance marker. That is, when a deletion DNA fragment prepared using the primer set shown in Table 1 is introduced, colonies that grow on an agar medium containing chloramphenicol are isolated, and the genome is obtained by PCR or the like using the genome as a template. It is sufficient to confirm that the above target gene is replaced with a chloramphenicol resistance gene.

  The microorganism of the present invention can be produced by introducing a gene encoding a target protein or polypeptide into the microorganism thus prepared. Here, the “target protein or polypeptide” refers to a protein or polypeptide whose production or purification is one of the purposes. In addition, in the “microorganism having a gene encoding the target protein or polypeptide”, the gene includes not only a gene originally possessed by the microorganism but also a gene that the microorganism originally does not have, that is, a foreign gene. is there.

The target protein or target polypeptide is not particularly limited, and includes, for example, various industrial enzymes such as detergents, foods, fibers, feeds, chemicals, medical treatments, and diagnostics, and bioactive peptides. Industrial enzymes are preferred. In addition, the functions of industrial enzymes are classified into oxidoreductase (Oxidoreductase), transferase (Transferase), hydrolase (Hydrolase), elimination enzyme (Lyase), isomerase (Isomerase), and synthetic enzyme (Ligase / Synthetase). ) And the like, preferably hydrolyzing enzymes such as cellulase, α-amylase and protease. Examples of α-amylase include α-amylases derived from microorganisms, preferably derived from bacteria belonging to the genus Bacillus , more preferably derived from Bacillus sp. KSM-K38. More specific examples of α-amylase derived from Bacillus sp. KSM-K38 strain include alkaline amylase derived from Bacillus genus bacteria consisting of amino acid sequences of amino acid numbers 1 to 480 of SEQ ID NO: 4, and 70% An amylase having an amino acid sequence having an identity of preferably 80%, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more. Examples of cellulases include cellulases belonging to Family 5 in the classification of polysaccharide hydrolases (Biochem. J., 280, 309, 1991). Among them, cellulases derived from microorganisms, particularly Bacillus bacteria are mentioned. Examples include alkaline cellulases derived from Bacillus sp. KSM-S237 strain (FERM BP-7875) and Bacillus sp. KSM-64 strain (FERM BP-2886), and more preferred examples. As an alkaline cellulase derived from a Bacillus bacterium comprising the amino acid sequence of amino acid numbers 1 to 795 of SEQ ID NO: 6, or an alkaline cellulase derived from a bacterium belonging to the genus Bacillus comprising an amino acid sequence of amino acid numbers 1 to 793 of SEQ ID NO: 8, or A cellulase comprising an amino acid sequence having 70%, preferably 80%, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more identity with an amino acid sequence can be mentioned. Examples of proteases include serine proteases and metalloproteases derived from microorganisms, in particular from Bacillus bacteria.

The target protein or target polypeptide gene to be introduced into the microorganism of the present invention is upstream of the control region involved in transcription, translation, and secretion of the gene, that is, the transcription initiation control region including the promoter and transcription start site, ribosome It is desirable that at least one region selected from a translation initiation control region including a binding site and an initiation codon and a secretory signal peptide region be bound in an appropriate form. In particular, it is preferable that three regions consisting of a transcription initiation control region, a translation initiation control region, and a secretion signal region are combined, and the secretion signal peptide region is derived from a cellulase gene of a Bacillus bacterium, and the transcription initiation control region In addition, it is desirable that the translation initiation control region in the 0.6-1 kb region upstream of the cellulase gene is appropriately bound to the target protein or polypeptide gene. 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 cellulase gene and the transcription initiation control region, translation initiation control region, and secretory signal peptide region of the cellulase gene are appropriately combined with the structural gene of the target protein or polypeptide. More specifically, the base sequence of base numbers 1 to 659 of the base sequence shown by SEQ ID NO: 5, the base sequence of base numbers 1 to 696 of the base sequence shown by SEQ ID NO: 7, or 70 relative to the base sequence % Or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more. It is desirable that a DNA fragment consisting of a base sequence in which a portion is deleted, substituted or added is appropriately bound to the structural gene of the target protein or polypeptide. Here, a DNA fragment consisting of a base sequence in which a part of the base sequence is deleted, substituted or added is a part of the base sequence deleted, substituted or added, but the gene transcription, This means a DNA fragment that retains functions related to translation and secretion.

  Such a gene encoding a target protein or polypeptide can be introduced by, for example, (1) introduction by a vector and (2) insertion into a genome. (1) When introduced by a vector, upstream of it, “a transcription initiation, including a control region related to transcription, translation and secretion of the gene, ie, a transcription initiation control region including a promoter and a transcription initiation site, a ribosome binding site and an initiation codon” Competent cell transformation method, protoplast transformation method, vector containing gene encoding target protein or target polypeptide to which one or more regions selected from control region and secretory signal peptide region are appropriately linked Alternatively, it may be introduced by an appropriate transformation method such as electroporation. Here, the vector is not particularly limited as long as it is an appropriate carrier nucleic acid molecule for introducing a target gene into a host, allowing it to proliferate and express, and not only a plasmid but also an artificial such as YAC or BAC. Examples include vectors using chromosome and transposon, and cosmids. Examples of plasmids include pUB110 and pHY300PLK.

  In addition, (2) for insertion into the genome, for example, a method by homologous recombination may be used. That is, a DNA fragment in which a part of a chromosomal region that causes introduction into a gene encoding a target protein or polypeptide is combined is taken into a microbial cell, and homologous recombination occurs in a part of the chromosomal region. Can be integrated into the genome. Here, the chromosomal region causing the introduction is not particularly limited, but a non-essential gene region or a non-gene region upstream of the non-essential gene region is preferable.

  Production of the target protein or polypeptide using the recombinant microorganism of the present invention is carried out by inoculating the strain in a medium containing an assimilable carbon source, nitrogen source and other essential components, and cultivating it by a normal microorganism culture method. Then, after completion of the culture, the protein or polypeptide may be collected and purified. And as shown in the below-mentioned Example, the improvement of the productivity of the target protein or polypeptide is achieved compared with the case where the microorganisms which are not modifying the said gene are used.

  Below, the construction method of the recombinant microorganism of this invention and the production method of amylase using the said recombinant microorganism are demonstrated concretely.

For the polymerase chain reaction (PCR) for DNA fragment amplification in the following examples, GeneAmp PCR System (Applied Biosystems) is used, and DNA amplification is performed using Pyrobest DNA Polymerase (Takara Bio) and attached reagents. went. The PCR reaction solution composition was 1 μL of appropriately diluted template DNA, 20 pmol of sense and antisense primers and 2.5 U of Pyrobest DNA Polymerase, respectively, and the total amount of the reaction solution was 50 μL. PCR reaction conditions were 98 ° C for 10 seconds, 55 ° C for 30 seconds and 72 ° C for 1 to 5 minutes (adjusted according to the target amplification product, 1 minute per 1 kb). After repeating, the reaction was carried out at 72 ° C. for 5 minutes.
Further, in the following examples, upstream and downstream of a gene are not positions from the replication start point, but upstream is a region continuing on the 5 ′ side of the start codon of the gene that is regarded as a target in each operation / step. On the other hand, “downstream” indicates a region continuing 3 ′ of the stop codon of the gene taken as a target in each operation / step.

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

Example 1 Construction of a prsA gene expression-enhanced strain A mutant strain with enhanced prsA gene expression was constructed as follows (see FIG. 2). Using the genomic DNA extracted from Bacillus subtilis 168 as a template, transcription initiation control of the spoVG gene using the primer sets of PVG-FW and PVG-R and prsA / PVG-F and prsA / Em2-R shown in Table 1 A 0.2 kb fragment (A) containing the region and the ribosome binding site and a 0.9 kb fragment (B) containing the prsA gene were amplified by PCR. A 1.3 kb fragment (C) containing the erythromycin (Em) resistance gene was amplified by PCR using plasmid pMUTIN4 (Microbiology. 144, 3097 (1998)) as a template and using the primer set of emf2 and emr2 shown in Table 1. . Next, the obtained (A), (B), and (C) 3 fragments were mixed to form a template, and SOE-PCR using the PVG-FW2 and emr2 primer sets shown in Table 1 was performed to obtain 3 fragments. (a) (B) bonded to as made in the order of (C), the start codon of prsA gene at the position of initiation codon of the transcription initiation regulatory region and the ribosome binding site of spoVG gene is linked upstream of prsA gene (spoVG gene And a 2.4 kb DNA fragment (D) with an Em resistance gene bound downstream thereof was obtained. Subsequently, using the genomic DNA extracted from Bacillus subtilis 168 as a template, the 5 'region of the amyE gene was cloned using the amyEfw2 and amyE / PVG2-R and amyE / Em2-F and amyErv2 primer sets shown in Table 1. The 1.0 kb fragment containing (E) and the 1.0 kb fragment containing the 3 ′ region of the amyE gene (F) were amplified by PCR. Next, the obtained (E) (F) (D) 3 fragments were mixed to form a template, and SOE-PCR using the amyEfw1 and amyErv1 primer sets shown in Table 1 was performed to obtain the 3 fragments (E ) (D) (F), a 2.4 kb DNA fragment in which the transcription start control region of the spoVG gene and the prsA gene are linked downstream of the ribosome binding site and the Em resistance gene is further linked downstream Obtained a DNA fragment (G) having a total base length of 4.3 kb inserted in the center of the amyE gene. The resulting 4.3 kb DNA fragment (G) was transformed into Bacillus subtilis 168 strain by competent cell method and grown on LB agar medium containing erythromycin (1 μg / mL) and lincomycin (25 μg / mL). Colonies were isolated as transformants. Using the genomic DNA extracted from the resulting transformant as a template, PCR was performed using the primer sets of amyEfw2 and prsA / Em2-R and prsA / PVG-F and amyErv2 shown in Table 1 to obtain 2.4 kb and 3.3 kb. to confirm amplification of the kb of DNA fragments, DNA fragments prsA gene are linked downstream of the transcription initiation regulatory region and the ribosome binding site of spoVG gene amyE gene site on the Bacillus subtilis 168 strain genome was confirmed to be inserted . The strain obtained in this way was named prsA-Ka strain.

Example 2 Evaluation of Alkaline Amylase Secretion Production-1
Evaluation of heterologous protein productivity of the prsA-Ka strain obtained in Example 1 was carried out as follows using the productivity of alkaline amylase derived from Bacillus bacteria consisting of the amino acid sequence represented by SEQ ID NO: 4 as an index.
Using genomic DNA extracted from Bacillus sp. KSM-K38 strain (FERM BP-6946) as a template, using the primer set of K38matu-F2 (ALAA) and SP64K38-R (XbaI) shown in Table 1 PCR is performed to amplify a 1.5 kb DNA fragment (H) of the base sequence represented by SEQ ID NO: 3 encoding alkaline amylase (JP 2000-184882, Eur. J. Biochem., 268, 2974, 2001) did. Using the genomic DNA extracted from Bacillus sp. KSM-S237 strain (FERM BP-7875) as a template, the S237ppp-F2 (BamHI) and S237ppp-R2 (ALAA) primer sets shown in Table 1 were used. PCR was performed to amplify a 0.6 kb DNA fragment (I) containing a transcription initiation control region, translation initiation control region, and a region encoding a secretory signal sequence of the alkaline cellulase gene (Japanese Patent Laid-Open No. 2000-210081). Next, the obtained 2 fragments of (H) (I) are mixed to form a template, and SOE-PCR using the S237ppp-F2 (BamHI) and SP64K38-R (XbaI) primer sets shown in Table 1 is performed. As a result, a 2.1 kb DNA fragment (J) in which the alkaline amylase gene was ligated downstream of the transcription initiation regulatory region, translation initiation regulatory region, and secretory signal sequence encoding the alkaline cellulase gene was obtained. The obtained 2.2 kb DNA fragment (J) was inserted into the Bam HI- Xba I restriction enzyme cleavage point of shuttle vector pHY300PLK (Yakult) to construct alkaline amylase productivity evaluation plasmid pHYK38 (S237ps).
Plasmid pHYK38 (S237ps) for evaluating alkaline amylase productivity was introduced into the prsA-Ka strain obtained in Example 1 and Bacillus subtilis 168 strain as a control by the protoplast transformation method. The resulting strain was shaken and cultured overnight at 37 ° C. in 10 mL of LB medium, and 0.05 mL of this culture solution was added to 50 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 cultured with shaking at 30 ° C. for 5 days. After culturing, the alkaline amylase activity of the culture supernatant after removing the cells by centrifugation was measured, and the amount of alkaline amylase secreted and produced outside the cells by the culture was determined. Liquitech Amy EPS (Roche Diagnostics) was used to measure the amylase activity in the culture supernatant. In other words, 50 μL of a sample solution appropriately diluted with 1% NaCl-1 / 7.5 M phosphate buffer (pH 7.4 Wako Pure Chemical Industries) was added to 100 μL of R1 and R2 mixture (R1 (coupling enzyme): R2 (amylase substrate) ) = 5: 1 (Vol.)) Was added and mixed, and the amount of p-nitrophenol released when the reaction was performed at 30 ° C. was quantified by the change in absorbance (OD405 nm) at 405 nm. The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was defined as 1 U. As a result, as shown in Table 2, when the prsA-Ka strain was used as the host, a higher secretory production of alkaline amylase was observed compared to the control 168 strain (wild type). This increases than the expression wild strain of prsA gene in prsA-Ka strain, by PrsA protein content on the cell membrane was increased, the secretion efficiency of proteins was estimated to be due to improved results.

Example 3 Replacement of glcT gene in genome with drug resistance gene A method of replacing the glcT gene in the genome with a drug resistance gene will be described with reference to FIG. The glcT gene encodes a transcriptional regulator (anti-terminator) that positively regulates the expression of the ptsGHI operon involved in the Bacillus subtilis PTS system.
Using a genomic DNA extracted from Bacillus subtilis 168 strain as a template and using the glcT-FW and glcT / Cm-R primer sets shown in Table 1, a 1.0 kb fragment adjacent to the upstream of the glcT gene in the genome (A) Was amplified by PCR. Further, using the genomic DNA as a template, a 1.0 kb fragment (B) adjacent to the downstream of the glcT gene in the genome was amplified by PCR using the glcT / Cm-F and glcT-RV primer sets.
Further, a 0.85 kb chloramphenicol (Cm) resistance gene region (C) was prepared by PCR using the plasmid pC194 DNA as a template and the catf and catr primer sets shown in Table 1.
Next, as shown in FIG. 3, the obtained 1.0 kb fragment (A), 1.0 kb fragment (B), and Cm resistance gene region (C) 3 fragments were mixed and used as a template to produce glcT shown in Table 1. -2.8kb DNA containing 3 fragments in the order of 1.0kb fragment (A), Cm resistance gene region (C), 1.0kb fragment (B) by SOE-PCR using primer set of -FW2 and glcT-RV2 Fragment (D) was obtained.
Furthermore, 168 strains were transformed using the obtained DNA fragment (D) by the competent cell transformation method. After transformation, colonies that grew on the LB agar medium containing chloramphenicol (10 μg / mL) were isolated as transformants.
The genomic DNA of the obtained transformant was extracted, and it was confirmed by PCR that the glcT gene was replaced with the Cm resistance gene. As described above, a glcT gene deletion strain (ΔglcT strain) was constructed. In addition, by using the prsA-Ka strain constructed in Example 1 in place of the 168 strain in the above transformation, a strain (prsAKΔglcT strain) in which the glcT gene in the prsA -Ka genome was replaced with a Cm resistance gene was constructed. did.

Example 4 Replacement of glvR gene and glvC gene in the genome with drug resistance gene In the same manner as the replacement of glcT gene with the drug resistance gene shown in Example 3, chloramphenicol of glvR gene and glvC gene in 168 strain genome Substitution with a resistance gene was carried out to construct a glvR gene deletion strain (ΔglvR strain) and a glvC gene deletion strain (ΔglvC strain). The primers shown in Table 1 were used for the construction of each strain, and the correspondence between each primer and the primers used for the construction of the ΔglcT strain is shown in Table 3. The glvR gene and glvC gene are both genes encoding proteins known to be involved in maltose uptake by the PTS system of Bacillus subtilis. The function of each gene is as shown in Table 6.

  Moreover, the prsAKΔglvR strain and the prsAKΔglvC strain were constructed by substituting each of the above genes on the genome of the prsA-Ka strain constructed in Example 1 with a chloramphenicol resistance gene.

Example 5 Evaluation of Alkaline Amylase Secretion Production-2
The alkaline amylase secretion productivity evaluation of the strain constructed in Examples 3 and 4 was performed in the same manner as in Example 2. As control, Bacillus subtilis 168 strain and prsA-Ka strain constructed in Example 1 were also evaluated. As shown in FIG. 4, the prsAKΔglcT strain, the prsAKΔglvR strain, and the prsAKΔglvC strain showed higher secretory production of alkaline amylase than the prsA-Ka strain. (Corresponding to the part) was more remarkable than the increase in production (corresponding to the part shown in black in FIG. 4) for the Bacillus subtilis 168 strain shown in each of the ΔglcT strain, ΔglvR strain and ΔglvC strain. That is, in the prsAKΔglcT strain, the prsAKΔglvR strain, and the prsAKΔglvC strain, it was considered that the combination of the prsA gene expression enhancement and each gene deletion acted synergistically on the increase in the production of alkaline amylase secretion.

Example 6 Replacement of treR gene with drug resistance gene The treR gene on the genome of Bacillus subtilis 168 was replaced with the chloramphenicol resistance gene in the same manner as the replacement of the glcT gene with the drug resistance gene shown in Example 3. The ΔtreR strain was constructed. The primers used are shown in Table 1, and the correspondence between each primer and the primer used for the construction of the ΔglcT strain is shown in Table 4. In addition, the prsAKΔtreR strain was constructed by replacing the treR gene on the genome of the prsA-Ka strain constructed in Example 1 with a chloramphenicol resistance gene in the same manner. The treR gene is a gene that encodes a negative transcriptional regulator that suppresses the expression of the tre operon involved in Bacillus subtilis PTS trehalose uptake. The gene number and function of the treR gene are as shown in Table 6.

Example 7 Evaluation of Alkaline Amylase Secretion Production-3
The alkaline amylase secretion productivity evaluation of the strain constructed in Example 1 was performed in the same manner as in Example 2. As control, Bacillus subtilis 168 strain and prsA-Ka strain constructed in Example 1 were also evaluated. As shown in Table 5, the secretion productivity of alkaline amylase by deleting the treR gene from the prsA-Ka strain was significantly reduced to 168 strains or less. That is, it was shown that the synergistic effect by the combination of prsA gene expression enhancement and treR gene deletion was not recognized, but rather the effect of prsA gene expression enhancement was impaired. TreR the trer gene encodes is inhibiting repressor expression treP gene and treA gene encoding trehalose uptake system, trer gene deletion causes a low productivity of bringing high expression of treP gene and treA gene It was thought that. That is, it was estimated that productivity was improved in the inactivation of the treP gene and / or treA gene.

Example 8 Measurement of change in sugar concentration in medium
If the treP gene and treA gene are highly expressed due to the deletion of the treR gene, it is presumed that if trehalose is present in the medium, its uptake is promoted. However, in the amylase secretion production evaluation of Example 7, 2xL-maltose medium containing maltose as a main carbon source was used for culturing the strain, and examination was made as to whether treR gene deletion affects maltose uptake. Went. Changes in sugar concentration in the culture supernatant when the ΔtreR strain was cultured in a 2 × L-maltose medium together with the Bacillus subtilis 168 strain, ΔglcT strain, ΔglvR strain, and ΔglvC strain were measured. F-kit starch (Roche Diagnostics) was used for the measurement of sugar concentration. That is, 10 μL of a sample solution appropriately diluted with ion-exchanged water was mixed by adding 20 μL of solution I (sugar hydrolase) and reacted at 60 ° C. for 20 minutes to decompose maltose into glucose. Next, add 100 μL of solution II / ion exchange water / solution III mixture (solution I (NADP solution): ion exchange water: solution II (glucose converting enzyme) = 100: 100: 2 (Vol.)) And mix. The amount of NADPH produced after the reaction at room temperature for 15 minutes was quantified by measuring the absorbance at 340 nm (OD 340 nm). The amount of NADPH produced when a glucose standard solution was used instead of the sample solution was also measured, and the sugar concentration in terms of glucose in the sample solution was calculated. As a result, as shown in FIG. 5, in the ΔglcT strain, ΔglvR strain, and ΔglvC strain, the decrease in the sugar concentration in the culture broth was slower than that in the 168 strain, whereas in the ΔtreR strain, the sugar concentration decreased rapidly. Was. Most of the sugars in the culture solution are considered to be maltose, that is, deletion of glcT gene, glvR gene and glvC gene in which maltose uptake is suppressed is enhanced in prsA gene in enzyme production as shown in Example 5. On the other hand, it was revealed that deletion of the treR gene that promotes maltose uptake reduces the effect of prsA gene enhancement as shown in Example 7 (FIG. 5). This result is considered to suggest that suppression of sugar uptake in the medium is effective for enzyme production, and conversely, promotion of sugar uptake is inhibitory. The rapid uptake of maltose by treR gene deletion was thought to be due to an increase in the expression level of the trehalose uptake system encoded by the treP gene and treA gene. That is, in combination with the results of Example 7, inactivation of the treP gene and / or treA gene was considered to improve enzyme productivity by suppressing sugar uptake in combination with prsA gene enhancement. .
Table 6 shows gene numbers and functions of genes related to the present invention.

  The names, numbers, functions, etc. of these genes were reported by Kunst et al. (Nature, 390, 249-256, 1997) and published online on the JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) (http: // Bacillus.genome.ad.jp/, updated March 10, 2004), based on genome data of Bacillus subtilis.

Example 9 Bacillus cereus (Bacillus cereus) from prsA gene expression enhancement and alkaline amylase secretory production evaluation in combination with glcT gene deletion Bacillus cereus (Bacillus cereus) derived prsA gene (SEQ ID NO: 53), the Bacillus subtilis prsA gene The corresponding gene. In the same manner as in Example 1, a mutant strain with enhanced expression of the Bacillus cereus- derived prsA gene was constructed. In other words, using the genomic DNA extracted from Bacillus cereus as a template and the Bce / PVG-F and Bce / emf2-R primer sets shown in Table 7, the Bacillus cereus- derived prsA gene is included. A 0.9 kb fragment (A) was amplified by PCR. In addition, using the genomic DNA extracted from the prsA-Ka strain constructed in the above comparative example as a template, using the amyEfw2 and PVG-R and emf2 and amyErv2 primer sets shown in Table 1, the 5 'region of the amyE gene A 1.2 kb fragment (B) in which the spoVG gene transcription initiation regulatory region and a region containing a ribosome binding site are linked downstream of the erythromycin resistance gene, and a 2.3 kb fragment (C) in which the 3 'region of the amyE gene is linked downstream of the erythromycin resistance gene Amplified by PCR. Subsequently, the obtained (A), (B), and (C) 3 fragments were mixed and used as a template, and SOE-PCR using the amyEfw1 and amyErv1 primer sets shown in Table 1 was performed. ) (a) binding was as made in the order of (C), linked Bacillus cereus (Bacillus cereus) from prsA gene downstream of the transcription initiation regulatory region and the ribosome binding site of spoVG gene, more erythromycin-resistant gene downstream thereof A DNA fragment (D) having a total base length of 4.3 kb in which the combined gene fragment was inserted in the center of the amyE gene was obtained. The obtained 4.3 kb DNA fragment (D) was used to transform Bacillus subtilis 168 strain to construct prsAbc-K strain.
Next, a prsAbcKΔglcT strain was constructed by replacing the glcT gene with a chloramphenicol resistance gene from the prsAbc -K strain genome in the same manner as in Example 3.
For the constructed prsAbc-K strain and prsAbcKΔglcT strain, alkaline amylase secretion production was evaluated in the same manner as in Example 2. As controls, Bacillus subtilis 168 and ΔglcT were also evaluated. As a result, as shown in FIG. 6, the prsAbc-K strain showed secretion production greatly exceeding 168 strains. Further, in the strain lacking the glcT gene from the prsAbc-K strain, a further remarkable improvement in productivity was observed, and as was seen in Example 5, the expression enhancement of the prsA gene derived from Bacillus cereus and glcT It was considered that the combination with gene deletion acted synergistically on the increase in production of alkaline amylase secretion. That is, the use of the gene corresponding to the Bacillus subtilis prsA gene, it was confirmed that the effectiveness of the same in the case of using the Bacillus subtilis prsA gene is obtained.

1 schematically shows gene transfer using a bound nucleic acid fragment prepared by SOE-PCR. 1 schematically shows a method for preparing a DNA fragment for preparing a prsA-enhanced strain by SOE-PCR. The deletion of the target gene by the double crossover method using the SOE-PCR fragment is schematically shown. It is the graph which showed the alkaline amylase secretion productivity of this invention microorganisms. It is the graph which showed the influence which it takes on the uptake | capture of maltose of each gene deletion. It is the graph which showed the alkaline amylase secretion productivity of this invention microorganisms.

Claims (13)

A transcription initiation control region or transcription initiation control region located upstream of the Bacillus subtilis spoVG gene or aprE gene and a ribosome binding site are introduced upstream in the genome of the Bacillus subtilis prsA gene or a gene corresponding to the gene, Alternatively, a gene fragment in which a transcription initiation control region or transcription initiation control region located upstream of the Bacillus subtilis spoVG gene or aprE gene and a ribosome binding site are linked upstream of the Bacillus subtilis prsA gene or a gene corresponding to the gene is introduced into the genome. and, and glcT gene, ptsG gene, ptsH gene, ptsI gene, GLVR gene, GlvA gene, GlvC gene及 beauty microbial strains comprising one or more genes selected from the gene corresponding to the gene deleted or inactivated Or a recombinant microorganism into which a gene encoding the protein or polypeptide of interest has been introduced. glcT gene or recombinant microorganism of claim 1 Symbol placing the gene corresponding to the gene is to reduction deleted or inactivated. glvC gene, GLVR gene and recombinant microorganism of claim 1 Symbol mounting one or more genes selected from the gene corresponding to the gene is to reduction deleted or inactivated. The transcription initiation control region located upstream of the spoVG gene of Bacillus subtilis is a region comprising the base sequence of base numbers 38 to 210 of SEQ ID NO: 9, and is associated with the transcription initiation control region located upstream of the Bacillus subtilis spoVG gene The recombinant microorganism according to any one of claims 1 to 3, wherein the site is a region comprising the base sequence of base numbers 38 to 230 of SEQ ID NO: 9. A gene corresponding to the glcT gene, ptsG gene, ptsH gene, ptsI gene, glvR gene, glvA gene, glvC gene has substantially the same function as that gene, and has a nucleotide sequence identity of 90% or more. The recombinant microorganism according to any one of claims 1 to 4, which is a gene derived from a bacterium belonging to the genus Bacillus which has The recombinant microorganism according to any one of claims 1 to 5, wherein the gene corresponding to the prsA gene of Bacillus subtilis is the gene according to any one of (1) to (4) below.
  (1) Coding a protein consisting of a base sequence having 90% or more identity with the base sequence represented by SEQ ID NO: 1 and having an activity functionally equivalent to the protein consisting of the amino acid sequence represented by SEQ ID NO: 2. A gene consisting of DNA.
  (2) A protein that is hybridized under stringent conditions with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 and functionally equivalent to a protein comprising the amino acid sequence represented by SEQ ID NO: 2 A gene consisting of DNA encoding.
  (3) It consists of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and a DNA encoding a protein functionally equivalent to the protein consisting of the amino acid sequence shown in SEQ ID NO: 2. gene.
  (4) In the amino acid sequence shown in SEQ ID NO: 2, it consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and is functionally equivalent to a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. A gene consisting of DNA that codes for various proteins. The recombinant microorganism according to any one of claims 1 to 6 , wherein the microorganism is a Bacillus bacterium. The recombinant microorganism according to claim 7 , wherein the bacterium belonging to the genus Bacillus is Bacillus subtilis. The group according to any one of claims 1 to 8 , wherein any one or more of a transcription initiation control region, a translation initiation control region, and a secretion signal region are linked upstream of a gene encoding a target protein or polypeptide. Replacement microorganism. The recombinant microorganism according to claim 9, wherein three regions comprising a transcription initiation control region, a translation initiation control region, and a secretion signal region are combined. Secretion signal region are those derived from a cellulase gene of Bacillus (Bacillus) bacteria, according to claim 9 or 10 transcriptional initiation regulatory region and translational initiation regulatory region is derived from upstream 0.6~1kb region of the cellulase gene The recombinant microorganism described. Three regions comprising a transcription initiation control region, a translation initiation control region, and a secretion signal region are derived from the base sequence of base numbers 1 to 659 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 5, and the base sequence represented by SEQ ID NO: 7. a DNA fragment consisting of a nucleotide sequence having any 90% identity or more nucleotide sequences or the nucleotide sequence of nucleotide numbers 1 to 696 of cellulase gene according to claim 10 or 11 recombinant microorganism, wherein composed. The process according to claim 1 to 12 any one recombinant microorganism desired protein or polypeptide using the description of.

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