JP4839169B2 - 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 application has been extended to a wide range of fields from foods, medicines, daily necessaries such as detergents and cosmetics to various chemical raw materials.

In industrial production of useful substances by such microorganisms, improvement of productivity is one of the important issues, and breeding of produced bacteria by genetic techniques such as mutation has been performed as a technique. 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 efforts described above, it was not always possible to say that the production efficiency of existing strains was high.

By the way, it is known that the fus gene, tufA gene, tsf gene, etc. of Bacillus subtilis encode translation elongation factors called EF-Tu, EF-G, and EF-Ts, respectively.
It has been shown in E. coli and the like that EF-Ts regulates its function by binding to EF-Tu and promoting dissociation of GDT binding to EF-Tu (Non-patent Document 3). EF-Ts is widely conserved across species, and Bacillus stearothermophilus EF-Ts strongly binds to E. coli EF-Tu (Non-patent Document 4) and It is known that a high structural similarity is observed between EF-1β corresponding to EF-Ts and EF-Ts of Escherichia coli (Non-patent Document 5).
EF-Tu binds to a transfer RNA (aminoacyl tRNA) that transports amino acids to the ribosome during translation, and the tRNA anticodon is converted into the messenger RNA (mRNA) sequence through temporary pairing. On the other hand, it is involved in securing a necessary time according to whether it is appropriate or not (Non-patent Document 6). In addition, EF-P involved in the first peptide bond formation in the translation process is known as a Bacillus subtilis translation elongation factor, and EF-P is encoded by the efp gene.

In addition, protein synthesis on the ribosome is performed according to the mRNA sequence and is terminated by the appearance of a stop codon. After such a series of protein synthesis is completed, it is indispensable for the ribosome to rapidly dissociate from the mRNA in order to quickly start the next protein synthesis. Such function, termination of protein synthesis rapidly to complete the ribosome recycling factor contributing to the translational enhancer (ribosome recycling factor), i.e. frr gene Frr protein encoded is involved.
The Frr protein is widely conserved in prokaryotes such as Escherichia coli, Pseudomonas bacteria, and Bacillus bacteria such as Bacillus subtilis, and it has been shown that the Frr gene of Pseudomonas arginosa complements the frr gene mutation of Escherichia coli. (Non-patent document 7).

Since these genes are extremely important for cell growth, and their mutations have a great influence on the growth itself, it is difficult to analyze the effect on individual functions in the cell. For this reason, it has not been known whether these genes are related to the productivity of the target protein or polypeptide.
Nature, 390,249,1997 Science, 277,1453,1997 J. Biol. Chem., 256,5591, 1981 J. Bacteriol., 153, 1266, 1983 Structure, 15, 217, 1999 Biochemistry 3rd Edition, Edited by L. Stryer, WH FREEMAN AND COMPANY, pp733, (1988) J. Bacteriol., 181, 1281, 1999

  An object of the present invention is to provide a microorganism capable of improving the productivity of a target protein or polypeptide.

  Whether the Tsf protein and / or the Frr protein, which are translation elongation factors and ribosome regeneration factors, are modified by the transcription initiation control region or the transcription initiation control region and the ribosome binding site of the gene encoding them. Alternatively, it has been found that by introducing the gene and increasing the expression level of these Tsf protein and / or Frr protein, the productivity of the target protein or target polypeptide of the host microorganism is improved.

  That is, the present invention is a microorganism having a gene encoding a target protein or target polypeptide, and strengthens the transcription initiation control region or transcription initiation control region and the ribosome binding site of the gene encoding the Tsf protein and / or Frr protein. Or a microorganism obtained by introducing a gene encoding Tsf protein and / or Frr protein.

  Moreover, this invention provides the manufacturing method of the target protein or target polypeptide which uses the said microorganisms.

  The microorganism of the present invention achieves high productivity of the target protein or polypeptide. Thereby, it is possible to suppress waste of medium components such as energy loss, a carbon source, and a nitrogen source by efficiently using the medium components for protein or polypeptide production.

  In this specification, the identity of amino acid sequences and nucleotide sequences is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985). Specifically, it is calculated by performing an analysis with a unit size to compare (ktup) of 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)).

When preparing the microorganism of the present invention, the microorganism used as a parent microorganism is not particularly limited, and may be a wild type or a mutant, but a microorganism having a Tsf protein and / or a Frr protein. Is preferred. Specifically, and Bacillus (Bacillus) bacteria such as Bacillus subtilis, Clostridium (Clostridium) bacteria, or yeast, and among them B. (Bacillus) bacteria are preferred. Furthermore, Bacillus subtilis is particularly preferred from the viewpoint that whole genome information has been clarified and genetic engineering and genomic engineering techniques have been established, and that it has the ability to secrete and produce proteins outside the cells.

  The microorganism of the present invention is a microorganism having a gene encoding a target protein or polypeptide, and strengthens a transcription initiation control region or a transcription initiation control region and a ribosome binding site of a gene encoding a Tsf protein and / or a Frr protein. Or the gene is introduced.

Tsf protein is a translation elongation factor called EF-Ts. It has been shown in E. coli and the like that EF-Ts regulates its function by binding to EF-Tu and promoting dissociation of GDT binding to EF-Tu (Non-patent Document 3). EF-Ts is widely conserved across species, and Bacillus stearothermophilus EF-Ts strongly binds to E. coli EF-Tu (Non-patent Document 4) and It is known that a high structural similarity is observed between EF-1β corresponding to EF-Ts and EF-Ts of Escherichia coli (Non-patent Document 5).
The Frr protein is a factor that has the role of dissociating ribosomes from mRNA at the end of translation and recycling the ribosomes for the next cycle of translation.
Known translation elongation factors include Tsf protein, Fus protein and TufA protein, and Efp protein involved in the first peptide bond formation in the translation process. The Efp protein (translation elongation factor EF-P) is encoded by the efp gene.

Examples of the Tsf protein, Frr protein, and Efp protein include proteins encoded by genes consisting of the nucleotide sequences represented by SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11, respectively. The amino acid sequences of such proteins are the amino acid sequences shown by SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12, respectively. Proteins consisting of such sequences have their gene numbers as published on the JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) (http://bacillus.genome.ad.jp/, updated March 10, 2004). These are proteins encoded by the genes disclosed as BG19025, BG12587, and BG11460, respectively.

Transcription initiation control region that controls Tsf protein and / or Frr protein-encoding gene, or enhancement of transcription initiation control region and ribosome binding site, inherent transcription initiation control that regulates Tsf protein and / or Frr protein-encoding gene Replacing all or part of the region or transcription initiation control region and ribosome binding site with another transcription initiation control region or transcription initiation control region and ribosome binding site, or other transcription initiation control region or transcription initiation control region and ribosome This can be done by inserting a binding site upstream of the gene encoding Tsf protein or Frr protein.
Here, the upstream of the gene is not particularly limited as long as it is upstream of the start codon of the target gene, but an adjacent region within 2000 base pairs is preferable, a region within 500 base pairs is more preferable, and 100 A region within base pairs is more preferred, and a region within 50 base pairs is particularly preferred.

Here, the other transcription initiation control region or the transcription initiation control region and the ribosome binding site include the original transcription initiation control region or the transcription initiation control region and the transcription initiation control region or the transcription initiation control region and the ribosome stronger than the ribosome binding site. The transcription initiation control region or the transcription initiation control region and the ribosome binding site may be, for example, the transcription initiation control region of the spoVG gene or the transcription initiation control region and the ribosome binding site or the transcription initiation control region of the aprE gene. Alternatively, known ones such as a transcription initiation control region and a ribosome binding site can be mentioned.
More specifically, the transcription start control region of the spoVG gene has a function as a transcription start control region consisting of a region consisting of the base sequence of base numbers 38 to 210 of SEQ ID NO: 13, or a sequence homologous to these sequences. The area | region which has is mentioned.
More specifically, the transcription initiation control region and the ribosome binding site of the spoVG gene are more specifically a region comprising the nucleotide sequence of base numbers 38 to 230 of SEQ ID NO: 13, or a sequence homologous to these sequences. Examples include a region having a function as a control region and a ribosome binding site. Examples of the base sequence of base numbers 38 to 210 represented by SEQ ID NO: 13 and the base sequence homologous to the base sequence of base numbers 38 to 230 include (A) base numbers 38 to 210 represented by SEQ ID NO: 13 or base numbers A base sequence comprising a DNA hybridizing under stringent conditions with a DNA comprising a base sequence complementary to the base sequence of 38 to 230, (B) a base number 38 to 210 represented by SEQ ID NO: 13 or a base number 38 to Examples include base sequences having the identity of 230 base sequences with 90% or more, preferably 95% or more, and more preferably 99% or more.
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 (1 × SSC composition: 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. Examples include conditions of constant temperature for 8 to 16 hours for hybridization.
The identity of the base sequence is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985). Other transcription initiation control regions or transcription initiation control regions and ribosome binding sites are enhanced by introducing mutations into the original transcription initiation control region or transcription initiation control region and ribosome binding sites using known methods. But you can.
Such a transcription initiation control region or transcription initiation control region and ribosome binding site are randomly mutated by a known method such as PCR or mutagen treatment, for example, to the original transcription initiation control region or transcription initiation control region and ribosome binding site. Or after deletion, substitution or addition of one or more bases by site-specific mutation, and under the control of the transcription initiation control region or transcription initiation control region into which the mutation has been introduced and the ribosome binding site Place an appropriate gene such as β-galactosidase or green fluorescent protein, or a drug resistance gene, and examine the expression level of that gene, and the expression level is the transcription initiation control region or transcription initiation control region before mutagenesis. Increased transcription initiation control region or transcription initiation control compared to when performed at the ribosome binding site It can be obtained by selecting the frequency and ribosome binding site.
The above deletion, substitution or addition of one or more bases includes deletion, substitution or addition of one or several, preferably 1 to 10 bases. ~ Addition of several bases included.

For the substitution to the other transcription initiation control region or the transcription initiation control region and the ribosome binding site, for example, a known method by homologous recombination may be used.
That is, first, the DNA fragment and drug containing the upstream region of the original transcription initiation control region of the tsf gene and / or frr gene upstream of the other transcription initiation control region or the DNA fragment comprising the transcription initiation control region and the ribosome binding site. On the other hand, a part of or all of the translation initiation control region and the structural gene region of the gene downstream of the other transcription initiation control region or the DNA fragment containing the transcription initiation control region and the ribosome binding site A DNA fragment containing a part or all of the structural gene region is bound by a known method such as SOE (splicing by overlap extension) -PCR (Gene, 77, 61, 1989).
Next, when the DNA fragment formed by such binding is introduced into the host microorganism by a known method, homologous recombination occurs at a site homologous to the introduced DNA fragment in the region on the chromosome in the microorganism, Isolate transformants in which the original transcription initiation control region or transcription initiation control region and the ribosome binding site are replaced with the other transcription initiation control region or transcription initiation control region and the ribosome binding site, using the drug resistance gene as an index be able to. As a result, the introduced transcription initiation control region or transcription initiation control region and the ribosome binding site are genetically stably maintained.
Specific methods for introducing the DNA fragment for introduction into the host microorganism include competent cell transformation method (J. Bacterial. 93, 1925 (1967)) and protoplast transformation method (Mol. Gen. Genet. 168). , 111 (1979)) or electroporation (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 addition, the insertion of the other transcription initiation control region or transcription initiation control region and the ribosome binding site is performed by appropriately arranging the transcription initiation control region to be inserted or the DNA fragment sequence added to both ends of the transcription initiation control region and the ribosome binding site. If selected, it can be carried out by a method similar to the above-described replacement 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.
Next, after introducing a DNA fragment formed by such binding 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 other transcription initiation control region are stably held in a state where they are adjacent to each other without any gap.

The introduction of a gene encoding Tsf protein and / or Frr protein is a nucleic acid fragment containing a base sequence encoding Tsf protein and / or Frr protein, which is upstream of the transcription initiation control region or transcription initiation control region. What is necessary is just to introduce | transduce the nucleic acid fragment couple | bonded with the DNA fragment containing a ribosome binding site in an appropriate form.
Such a fragment can be either (1) introduced as a nucleic acid fragment to which a nucleic acid fragment consisting of a partial chromosome sequence of the host is added at both ends, or (2) as a nucleic acid fragment introduced into an appropriate vector. By introducing, it can be genetically stably maintained in the host microorganism.

The base sequence encoding the Tsf protein and / or Frr protein to be introduced is the Tsf protein and / or the host microorganism originally possessed as long as the nucleic acid fragment to be introduced is a base sequence encoding the Tsf protein and / or Frr protein. It may not be the same as the base sequence encoding the Frr protein.
When the fragment (1) is introduced, homologous recombination occurs at a site corresponding to the sequence of the host chromosome added to the nucleic acid fragment, and the introduced nucleic acid fragment is incorporated into the chromosome in the microorganism. Examples of a method for introducing a nucleic acid fragment into a host microorganism include a competent cell transformation method, a protoplast transformation method, an electroporation method, and the like, and a competent cell transformation method is particularly preferable.

The chromosomal region to be introduced in the host is preferably a non-essential gene, or a non-essential gene non-gene region, such as an aprE gene, a sacB gene, an nprE gene, an amyE gene, Alternatively, the inside of the non-gene region upstream of the gene can be mentioned, but the inside of the amyE gene is preferable.
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 addition, the transcription initiation control region or transcription initiation control region that binds upstream of the gene encoding Tsf protein and / or Frr protein and the ribosome binding site may have any function in the host microorganism. For example, Tsf protein And / or the original transcription initiation control region or transcription initiation control region of the gene encoding the Frr protein and the ribosome binding site, or other known transcription initiation control region or transcription initiation control region and the ribosome binding site.
The known transcription initiation control region or transcription initiation control region and ribosome binding site are preferably the transcription initiation control region or transcription initiation control region and ribosome binding site of Bacillus subtilis spoVG gene.
Here, the transcription initiation regulatory region of the Bacillus subtilis spoVG gene or the transcription initiation regulatory region and the ribosome binding site are published on the JAFAN: Japan Functional Analysis Network for Bacillus subtilis (BSORF DB) (http://bacillus.genome.ad jp /, updated March 10, 2004), a transcription initiation control region or transcription initiation control region that controls the gene spoVG disclosed as gene number BG101126 and a ribosome binding site.
More specifically, the transcription start control region of the spoVG gene has a function as a transcription start control region consisting of a region consisting of the base sequence of base numbers 38 to 210 of SEQ ID NO: 13, or a sequence homologous to these sequences. Areas.
In addition, as a transcription initiation control region and a ribosome binding site of the spoVG gene, more specifically, a region comprising the base sequence of base numbers 38 to 230 of SEQ ID NO: 13, or a sequence homologous to these sequences, transcription start control. Examples include a region and a region having a function as a ribosome binding site.
Such a transcription initiation control region or a DNA fragment containing a transcription initiation control region and a ribosome binding site can be bound to a nucleic acid fragment containing the base sequence of a structural gene encoding a protein, for example, by SOE-PCR.
That is, a structural gene encoding Tsf protein and / or Frr protein and an original translation initiation control region or a nucleic acid fragment containing the base sequence of the structural gene encoding Tsf protein and / or Frr protein, and the transcription initiation control region or transcription Amplification of an initiation control region and a DNA fragment containing a ribosome binding site is carried out by, for example, a structural gene encoding Tsf protein and / or Frr protein and an original translation initiation control region or a structural gene encoding the protein. On the 5 ′ end of the upstream primer used for amplification of the nucleic acid fragment containing the base sequence, the transcription start control region or the 10-30 base pair sequence downstream of the DNA fragment containing the transcription start control region and the ribosome binding site is reversed. Amplification of the transcription initiation regulatory region or a DNA fragment containing the transcription initiation regulatory region and a ribosome binding site A primer designed so that 10 to 30 base pairs upstream of the nucleic acid fragment containing the nucleotide sequence of the gene encoding Tsf protein and / or Frr protein is added to the 5 ′ end of the downstream primer to be used. When used, amplification is performed so that a sequence common to both is added to one end to which each fragment binds.
Next, in the reaction system containing such a fragment as a template, the transcription initiation regulatory region or 5 'of the DNA fragment containing the transcription initiation regulatory region and the ribosome binding site, the structural gene encoding Tsf protein and / or Frr protein, and the original translation By performing PCR using a primer that hybridizes to the 3 ′ side of the nucleic acid fragment containing the base sequence of the structural gene encoding the initiation regulatory region or Tsf protein and / or Frr protein, the above-described binding is performed. Can do.

  When introduced by a vector, examples of the vector include plasmids, artificial chromosomes such as YAC and BAC, vectors using transposons, and cosmids. Examples of plasmids include pUB110 and pHY300PLK. Examples of a method for introducing the nucleic acid fragment shown above into a microorganism include a protoplast method, a competent cell method, and an electroporation method.

The microorganism of the present invention can be produced by allowing the microorganism thus produced to have a gene encoding a target protein or polypeptide.
Here, “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 not originally possessed by the microorganism, that is, a foreign gene. It is.

The target protein or target polypeptide gene is not particularly limited, and examples thereof include proteins and polypeptides such as enzymes and physiologically active factors that are useful for foods, pharmaceuticals, cosmetics, detergents, fiber treatments, medical tests, etc. And various industrial enzymes such as detergents, foods, fibers, feeds, chemicals, medicines, and diagnostics, and bioactive peptides. Industrial enzymes are preferred.
In addition, industrial enzymes are classified according to their functions: oxidoreductase, transferase, hydrolase, lyase, isomerase, and synthase (Ligase / Synthetase). ) And the like, and preferred examples include genes for hydrolases such as cellulase, α-amylase, and protease. Specific examples include α-amylase, among which α-amylase derived from microorganisms, preferably derived from bacteria belonging to the genus Bacillus , and more preferably derived from Bacillus sp. KSM-K38 strain.
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: 6, 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. The amino acid sequence identity is calculated by the Lipman-Pearson method (Science, 227, 1435, 1985).
Specific 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 bacteria derived from the genus Bacillus. . More specific examples include alkaline cellulase derived from Bacillus sp. KSM-S237 strain (FERM BP-7875) or Bacillus sp. KSM-64 strain (FERM BP-2886). As a more specific example, an alkaline cellulase derived from a Bacillus bacterium comprising the amino acid sequence of amino acid numbers 1 to 795 of SEQ ID NO: 2 or a Bacillus genus bacterium comprising an amino acid sequence of amino acid numbers 1 to 793 of SEQ ID NO: 4 Alkaline cellulase derived from, 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 the amino acid sequence. It is done.
Specific examples of proteases include serine proteases, metal proteases, and the like derived from microorganisms, particularly from Bacillus bacteria.

In addition, the target protein or polypeptide gene includes a control region related to transcription, translation, and secretion of the gene upstream, that is, a transcription initiation control region including a promoter and a transcription initiation site, a translation including a ribosome binding site and an initiation codon. It is desirable that at least one region selected from the initiation control region and the secretory signal peptide region is bound in a proper 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: 1, the base sequence of base numbers 1 to 696 of the base sequence shown by SEQ ID NO: 3, or the base sequence 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more of a DNA fragment comprising a nucleotide sequence having identity or any of the above nucleotide sequences It is desirable that a DNA fragment consisting of a base sequence partially 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.

  Providing such a gene encoding a target protein or polypeptide can be performed, for example, by a method of (1) introduction with 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.

  The microorganism thus prepared has high productivity of the target protein or target polypeptide. Such an effect is presumed to be based on an improvement in translation efficiency due to activation of the translation extension reaction or the like.

  For production of the target protein or polypeptide using the recombinant microorganism of the present invention, the strain is inoculated into a medium containing an assimilable carbon source, nitrogen source and other essential components, and cultured by an ordinary microorganism culture method. Then, after completion of the culture, the protein or polypeptide may be collected and purified.

  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.

  Further, Bacillus subtilis was transformed 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. until the degree of growth (OD600) was about 1. After shaking culture, a portion of the culture broth was added to 9 times the amount of SPII medium (0.20% ammonium sulfate, 1.40% dipotassium hydrogen phosphate, 0.60% potassium dihydrogen phosphate, 0.10% trisodium citrate dihydrate, 0.50% Inoculate glucose, 0.01% casamino acid (Difco), 5mM magnesium sulfate, 0.40μM manganese chloride, 5μg / 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.

  Liquitech Amy EPS (Roche Diagnostics) was used for measuring 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.

Example 1 Construction of a tsf gene expression-enhanced strain A mutant strain with enhanced expression of the tsf gene was constructed as follows (see FIG. 1).
Using the genomic DNA extracted from Bacillus subtilis 168 as a template, transcription start control of the spoVG gene using the primer sets of PVG-FW and PVG-R and tsf / PVG-F and tsf / Cm-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 tsf gene were amplified by PCR. A 0.85 kb fragment containing the chloramphenicol resistance gene (C) using the plasmid pC194 (J. Bacteriol. 150 (2), 815 (1982)) as a template and the catf and catr primer sets shown in Table 1 Was amplified by PCR.
Next, the 3 fragments obtained by mixing the obtained (A), (B), and (C) 3 fragments as a template and performing SOE-PCR using the PVG-FW2 and catr2 primer sets shown in Table 1 were obtained. (a) (B) bonded to as made in the order of (C), initiation codon position of tsf gene at the position of initiation codon of the spoVG transcriptional initiation regulatory region and the ribosome binding site is linked upstream of tsf gene (spoVG gene And a 1.9 kb DNA fragment (D) to which a chloramphenicol resistance gene was bound downstream. Subsequently, using the genomic DNA extracted from Bacillus subtilis 168 as a template, using the amyEfw2 and amyE / PVG2-R and amyE / Cm2-F and amyErv2 primer sets shown in Table 1, the 5 'region of the amyE gene was identified. 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) 1.9 kb in which the transcription initiation control region of the spoVG gene and the tsf gene are linked downstream of the ribosome binding site, and the chloramphenicol resistance gene is further linked downstream A DNA fragment (G) having a total base length of 3.8 kb in which the DNA fragment was inserted in the center of the amyE gene was obtained. The resulting 3.8 kb DNA fragment (G) was used to transform Bacillus subtilis 168 strain by competent cell method, and colonies grown on LB agar medium containing chloramphenicol (10 μg / mL) were transformed. Separated as a body. By using the genomic DNA extracted from the obtained transformant as a template, PCR was carried out using the primer sets of amyEfw2 and tsf / Cm-R and tsf / PVG-F and amyErv2 shown in Table 1 to obtain 2.1 kb and 2.7 to confirm amplification of the kb of DNA fragments, DNA fragments tsf 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 thus obtained was named 168PVG (tsf) strain.

Example 2 Construction of a frr gene expression-enhanced strain Of the primers used in constructing the 168PVG (tsf) strain, tsf / PVG-F is represented by frr / PVG-F shown in Table 1, and tsf / Cm-R is represented by by using instead of the frr / Cm-R shown in 1, was constructed frr gene expression-enhanced strain 168PVG (frr) strain by the same method (see FIG. 2.).

Example 3 Evaluation of Alkaline Amylase Secretion Production of Bacillus subtilis Mutant-1
The target protein productivity of the 168PVG (tsf) and 168PVG (frr) strains obtained in Examples 1 and 2 was evaluated from a Bacillus bacterium comprising the amino acid sequence represented by amino acid numbers 1 to 480 of SEQ ID NO: 6. Using alkaline amylase productivity as an index, the following procedure was performed.
Using genomic DNA extracted from Bacillus sp. KSM-K38 as a template, PCR was performed using the K38matu-F2 (ALAA) and SP64K38-R (XbaI) primer sets shown in Table 1, and alkaline amylase was used. A 1.5 kb DNA fragment (H) having the base sequence shown by SEQ ID NO: 5 encoding (Japanese Patent Laid-Open No. 2000-184882, Eur. J. Biochem., 268, 2974, 2001) was amplified. In addition, using genomic DNA extracted from Bacillus sp. KSM-S237 as a template, PCR was performed using the S237ppp-F2 (BamHI) and S237ppp-R2 (ALAA) primer sets shown in Table 1, and alkaline was performed. A 0.6 kb DNA fragment (I) containing a transcription start control region, a translation start control region and a region encoding a secretory signal sequence of the cellulase gene (Japanese Patent Laid-Open No. 2000-210081) was amplified.
Subsequently, the obtained 1.5 kb DNA fragment (H) and 0.6 kb DNA fragment (I) were mixed to form a template, and S237ppp-F2 (BamHI) and SP64K38-R (XbaI) shown in Table 1 were used. By performing SOE-PCR using the primer set, a 2.1 kb DNA fragment in which the alkaline amylase gene was ligated downstream of the transcription initiation control region, translation initiation control region and secretory signal sequence of the alkaline cellulase gene ( J) was obtained. The obtained 2.2 kb DNA fragment (J) was inserted into the BamHI - XbaI restriction enzyme cleavage point of shuttle vector pHY300PLK (Yakult) to construct alkaline pH amylase productivity evaluation plasmid pHYK38 (S237ps).

A plasmid pHYK38 (S237ps) for evaluating the productivity of alkaline amylase was introduced into the 168PVG (tsf) and 168PVG (frr) strains obtained in Example 1 and, further, as a control, Bacillus subtilis 168 strain by protoplast transformation. The resulting strain was shaken and cultured overnight in 10 mL of LB medium at 37 ° C., and 0.05 mL of this culture was further 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.
As a result, as shown in Table 2, when 168PVG (tsf) and 168PVG (frr) strains were used as hosts, secretion production of alkaline amylase was higher than that of the control 168 strain (wild type). Admitted. This is because the expression of the tsf gene in the 168PVG (tsf) strain was higher than that in the wild strain and the intracellular amount of the translation elongation factor EF-Ts (Tsf protein) was increased, or the rrf gene in the 168PVG (frr) strain. As a result of the increase in the expression of and the intracellular amount of ribosome recycling factor (Frr protein) increased from the wild type, it was speculated that the translation efficiency of the protein in each strain was improved.

Comparative Example EFP gene expression shown in alkaline amylase release produced in example 1. All evaluation enhanced strain 168PVG (tsf) similarly to the construction of strains, tsf gene similar, 168PVG having enhanced expression of the EFP gene encoding translation elongation factor (EFP ) The stock was constructed. That is, among the primers used in the construction of the 168PVG (tsf) strain, tsf / PVG-F was replaced with efp / PVG-F shown in Table 1, and tsf / Cm-R was changed to efp / Cm-R shown in Table 1. Thus, an efp gene expression enhanced strain 168PVG (efp) was constructed in the same manner. The alkaline amylase secretion production evaluation of the constructed 168PVG (efp) strain was carried out in the same manner as in Example 3. As shown in Table 3, the productivity of the 168PVG (efp) strain was equivalent to that of the wild strain 168 strain. Yes, it was thought that even if the intracellular level of translation elongation factor EF-P (Efp protein) increased due to enhanced expression of the efp gene, the translation efficiency of the protein was not improved.

It is a figure which shows the outline of the method (construction of the mutant which strengthened the expression of tsf gene) which introduce | transduces the gene which codes Tsf protein. It is a figure which shows the outline of the method (construction of the mutant which strengthened the expression of frr gene) which introduce | transduces the gene which codes Frr protein.

Claims (6)

Has a gene that encodes the target protein or polypeptide, and upstream of it is a 3 region consisting of a transcription initiation control region, a translation initiation control region, and a secretion signal region, and the secretion signal region is derived from a cellulase gene of Bacillus bacteria Transcription that regulates the gene encoding Tsf protein and / or Frr protein , wherein the transcription initiation control region and translation initiation control region are derived from the 0.6-1 kb region upstream of the cellulase gene. The transcription initiation control region of Bacillus subtilis spoVG gene is replaced with all or part of the initiation control region or transcription initiation control region and ribosome binding site, or the transcription initiation control region of Bacillus subtilis spoVG gene. Alternatively, a microorganism into which a transcription initiation control region and a ribosome binding site are inserted , or Tsf A nucleic acid fragment containing a nucleotide sequence encoding a protein and / or Frr protein, which is upstream of the transcription initiation regulatory region of the Bacillus subtilis spoVG gene or a nucleic acid fragment linked to the transcription initiation regulatory region and the ribosome binding site Microorganisms. 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: 1, and the base sequence represented by SEQ ID NO: 3. 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 1 recombinant microorganism, wherein composed. The microorganism according to claim 1 or 2 , wherein the microorganism is a Bacillus bacterium. The microorganism according to claim 3 , wherein the Bacillus bacterium is Bacillus subtilis. The microorganism according to any one of claims 1 to 4 , wherein the target protein is α-amylase derived from Bacillus sp. KSM-K38 strain. The manufacturing method of the target protein or target polypeptide using the microorganisms of any one of Claims 1-5 .

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