JP2007000098A - Recombinant bacterium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a microorganism for improving productivity of protein or polypeptide. <P>SOLUTION: The microorganism has a gene encoding a target protein or a target polypeptide and has an enhanced transcription initiation control region for controlling a gene encoding Fus protein and TufA protein or TuFA protein or an enhanced transcription initiation control region and ribosome binding site or the microorganism to which a gene encoding Fus protein and TufA protein or TufA protein is transferred is obtained. <P>COPYRIGHT: (C)2007,JPO&INPIT

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 and tufA gene of Bacillus subtilis encode translation elongation factors called EF-Tu and EF-G, respectively. EF-Tu is known to contribute to the progression of the correct amino acid ligation reaction by binding to transfer RNA (aminoacyl-tRNA) that transports amino acids to ribosomes during translation and through temporary pairing. ing. As a more detailed mechanism, when aminoacyl-tRNA bound to EF-Tu binds to the A (aminoacyl) site on the ribosome, binding between the growing polypeptide chain and the amino acid carried by the transfer RNA (tRNA) EF-Tu is temporarily inhibited, and it is involved in ensuring the necessary time for whether or not the anticodon of tRNA is appropriate for the messenger RNA (mRNA) sequence. That is, if the anticodon of tRNA is inappropriate, aminoacyl tRNA is released from the A site before the amino acid ligation reaction occurs. On the other hand, when the anticodon is appropriate, EF-Tu dissociates from the aminoacyl tRNA at the A site, and a peptide bond is formed between the carboxy terminus of the polypeptide chain during elongation and the amino terminus of the aminoacyl tRNA amino acid. . At this time, a polypeptide chain extended by one amino acid is bound to the tRNA at the A site. In this state, EF-G moves the peptidyl tRNA at the A site to the P (peptidyl) site on the ribosome. It is known to have a role of allowing the next amino acid elongation reaction to be moved (Non-patent Document 3). 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.

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 Biochemistry 3rd Edition, Edited by L. Stryer, WH FREEMAN AND COMPANY, pp733, (1988)

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

  The present inventors modify and enhance the transcription initiation regulatory region or the transcription initiation regulatory region and the ribosome binding site of the gene encoding the TufA protein, which is one of the translation elongation factors, or the Fus protein and the TufA protein. Alternatively, it has been found that the productivity of the target protein or target polypeptide of the host microorganism is improved by introducing the gene and increasing the expression level of these TufA protein, or Fus protein and TufA protein.

  That is, the present invention is a microorganism having a gene encoding a target protein or polypeptide, wherein the transcription start control region of the gene encoding the TufA protein or the transcription start control region and the ribosome binding site are enhanced, or TufA A microorganism obtained by introducing a gene encoding a protein is provided.

  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 realizes high productivity of the target protein or target 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 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).

  The microorganism of the present invention is a microorganism having a gene encoding a target protein or target polypeptide, and a transcription initiation control region or transcription initiation control region of a gene encoding a TufA protein, or a gene encoding a Fus protein and a TufA protein The ribosome binding site is strengthened or the gene is introduced.

  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 producing 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 Fus protein and a TufA 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.

Fus protein and TufA protein are translation elongation factors called EF-Tu and EF-G, respectively. EF-Tu binds to a transfer RNA (aminoacyl tRNA) that transports amino acids to ribosomes during translation, and forms a temporary pairing. As a result, EF-Tu is a factor that contributes to the synthesis of a protein having the correct amino acid sequence by participating in the process of correctly incorporating amino acids carried by tRNA having an anticodon specified in mRNA into the amino acid ligation reaction. EF-G is a factor that plays a role in allowing the next amino acid elongation reaction by moving the peptidyl tRNA at the A site on the ribosome to the P (peptidyl) site on the ribosome. In addition to Fus protein and TufA protein, EFP proteins involved in the first peptide bond formation in the translation process are known as translation elongation factors. The EFP protein (translation elongation factor EF-P) is encoded by the efp gene.

Examples of the Fus protein, TufA 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 in SEQ ID NOs: 8, 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 BG11939, BG11056, and BG11460, respectively.

  Transcription initiation control region or transcription initiation control region and ribosome binding site controlling the gene encoding TufA protein, or transcription initiation control region or transcription initiation control region and ribosome binding site of genes encoding Fus protein and TufA protein The transcription initiation regulatory region or the transcription initiation regulatory region that controls the gene encoding the TufA protein or the gene encoding the Fus protein and the TufA protein and all or part of the ribosome binding site are transferred to other transcription initiation regulatory regions or transcriptions. It can be performed by substituting an initiation control region and a ribosome binding site, or by inserting another transcription initiation control region or transcription initiation control region and a ribosome binding site. Since the genes encoding Fus protein and TufA protein are adjacent to each other in the genome of Bacillus subtilis, Escherichia coli, etc., the insertion of the other transcription initiation control region or the transcription initiation control region and the ribosome binding site is performed for each gene. It may be performed upstream or only upstream of the gene encoding the Fus 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. Similarly to the genes encoding the TufA protein, the Fus protein and the TufA protein, the transcription initiation control region or the transcription initiation control region and the ribosome binding site can be strengthened for the gene encoding the EFP protein.

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. The transcriptional initiation regulatory region of the spoVG gene, more specifically, the region comprising the nucleotide sequence of nucleotide numbers 38-210 of SEQ ID NO: 13, or a function as a transcriptional initiation regulatory region consists these sequences homologous 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 nucleotide sequence comprising a DNA that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence of 38 to 230, (B) a nucleotide sequence of nucleotide numbers 38 to 210 or nucleotide numbers 38 to 230 represented by SEQ ID NO: 13 and 90 % Or more, preferably 95% or more, 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 1 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, a DNA fragment containing an upstream region of the transcription initiation control region of the fus gene and / or tufA gene upstream of the transcription initiation control region or a DNA fragment comprising the transcription initiation control region and a ribosome binding site, and drug resistance. the gene fragment, whereas the downstream, tufA gene or fus gene and tufA gene translational initiation regulatory region and part or all of the structural gene region or a part or all of the structural gene region of tufA gene or fus gene and tufA gene The contained DNA fragment is bound by a known method such as SOE (splicing by overlap extension) -PCR method (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 a TufA protein, or the introduction of a gene encoding a Fus protein and a gene encoding a TufA protein is a nucleic acid fragment comprising a TufA protein or a nucleotide sequence encoding a Fus protein and a TufA protein, What is necessary is just to introduce | transduce the nucleic acid fragment couple | bonded with the DNA fragment containing a transcription start control region or a transcription start control region, and a ribosome binding site in the upstream upstream. 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 TufA protein to be introduced, or the base sequence encoding the Fus protein and TufA protein is the base sequence in which the nucleic acid fragment to be introduced encodes the TufA protein, or the base sequence encoding the Fus protein and TufA protein. As long as it is, the base sequence encoding the TufA protein inherent in the microorganism or the Fus protein and the TufA protein may not be used. 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 the TufA protein, or the gene encoding the Fus protein and the TufA protein, and the ribosome binding site may be any one that has a function in the host microorganism. Well, for example, the gene encoding the TufA protein, or the original transcription initiation control region of the Fus protein and the gene encoding the TufA protein, or the transcription initiation control region and the ribosome binding site, or other known transcription initiation control region or transcription initiation. Examples include regulatory regions and ribosome binding sites. 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 transcription initiation control region and a DNA fragment containing a ribosome binding site and a nucleic acid fragment containing a gene base sequence can be bound by, for example, the SOE-PCR method. That is, a structural gene encoding a Fus protein and a TufA protein and an original translation initiation control region or a nucleic acid fragment containing the base sequence of the structural gene encoding a Fus protein and a TufA protein, and the transcription initiation control region or transcription initiation control region Amplifying a DNA fragment containing a ribosome binding site.In this case, for example, the structural gene encoding Fus protein and TufA protein and the original translation initiation control region or the base sequence of the structural gene encoding Fus protein and TufA protein are used. The transcription start control region or the transcription start control region and the downstream 10-30 base pair sequence of the DNA fragment containing the ribosome binding site at the 5 ′ end of the upstream primer used for amplification of the contained nucleic acid fragment Amplification of DNA fragment containing regulatory region or transcription initiation regulatory region and ribosome binding site Use a primer designed to add 10 to 30 base pairs upstream of the nucleic acid fragment containing the nucleotide sequences of the genes encoding Fus protein and TufA protein at the 5 'end of the downstream primer used for Thus, amplification is performed so that a sequence common to both of them 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 control region or the 5 ′ side of the DNA fragment containing the transcription initiation control region and the ribosome binding site, the structural gene encoding the Fus protein and TufA protein, and the original translation initiation control By performing PCR using a primer that hybridizes to the 3 ′ side of the nucleic acid fragment containing the nucleotide sequence of the structural gene encoding the region or the Fus protein and the TufA protein, the above-described binding can be performed.

  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 in SEQ ID NO: 1, the base sequence of base numbers 1 to 696 of the base sequence shown in SEQ ID NO: 3, 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.

  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.

Example 1 Construction of Fus and tufA gene-enhanced strains A mutant strain with enhanced fus and tufA gene expression was constructed as follows (see FIG. 1). 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 fus / PVG-F and tufA / Cm-R shown in Table 1 A 0.2 kb fragment (A) containing the region and the ribosome binding site and a 3.4 kb fragment (B) containing the fus and tufA genes 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), the start codon of fus gene at the position of initiation codon of the spoVG transcriptional initiation regulatory region and the ribosome binding site is linked upstream of fus and tufA gene (spoVG gene And a 4.4 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, the 5 'region of the amyE gene was cloned using the amyEfw2 and amyE / PVG2-R and amyE / Cm2-F and amyErv2 primer sets shown in Table 1. 1.0kb fragment containing (E), and 1.0kb fragment containing 3 'side region of the amyE gene (F) was 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) and (F) were combined in the order, fus and tufA genes were linked downstream of the transcription initiation control region of the spoVG gene and the ribosome binding site, and the chloramphenicol resistance gene was further linked downstream thereof. A DNA fragment (G) having a total base length of 6.4 kb in which a 4.4 kb DNA fragment was inserted in the center of the amyE gene was obtained. The obtained 6.4 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. Using the genomic DNA extracted from the resulting transformant as a template, PCR was performed using the amyEfw2 and tufA / Cm-R and fus / PVG-F and amyErv2 primer sets shown in Table 1 to perform 4.6 kb and 5.2 kb. to confirm amplification of the kb of DNA fragment, that DNA fragments fus and tufA 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 is inserted confirmed. The strain thus obtained was named 168PVG (fus-tufA) strain.

Example 2 Evaluation of Alkaline Amylase Secretion Production of Bacillus subtilis Mutant-1
The target protein productivity evaluation of the 168PVG (fus-tufA) strain obtained in Example 1 was carried out using the productivity of alkaline amylase derived from Bacillus bacteria consisting of the amino acid sequence represented by amino acid numbers 1 to 480 of SEQ ID NO: 6. The indicators were as follows.
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, alkaline amylase 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. 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 control region, translation initiation control region, and secretory signal sequence coding region of the alkaline cellulase gene 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 alkaline amylase productivity was introduced into the 168PVG (fus-tufA) strain obtained in Example 1 and the 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. As a result, as shown in Table 2, when the 168PVG (fus-tufA) strain was used as the host, higher secretion of alkaline amylase was observed compared to the control 168 strain (wild type). This is because the expression of fus and tufA genes in 168PVG (fus-tufA) strain is higher than that in the wild strain, and the amount of translation elongation factors EF-G and EF-Tu is increased. It was presumed to be due to the results.
Example 3 fus gene, like the building 168PVG of (fus-tufA) strain shown in tufA gene construct examples of strains were respectively enhanced expression alone 1, fus gene, 168PVG expressed enhanced tufA gene alone (fus ) Strain and 168PVG (tufA) 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 168PVG (fus-tufA) strain is shown in Table 3.

Example 4 Evaluation of Alkaline Amylase Secretion Production of Bacillus subtilis Mutant-2
The alkaline amylase secretion productivity of the strain constructed in Example 3 was evaluated in the same manner as in Example 2. B. subtilis strain 168 was used as a control, and the 168PVG (fus-tufA) strain constructed in Example 1 was also evaluated. As shown in Table 4, higher secretion of alkaline amylase was observed in the 168PVG (tufA) strain than in the 168 strain. The productivity of the 168PVG (fus-tufA) strain was higher than that of the 168PVG (tufA) strain. That is, it was shown that the secretion productivity of alkaline amylase is improved also by enhancement of the fus gene.

Similar to the construction of 168PVG (fus-tufA) strains shown in Comparative Example efp alkaline amylase release produced Evaluation Example 1 gene expression-enhanced strain, similarly fus genes and tufA gene, the expression of efp gene encoding translation elongation factor A strengthened 168PVG (efp) strain was constructed. The primers shown in Table 1 were used for the construction of the strain, and the correspondence between each primer and the primers used for the construction of the 168PVG (fus-tufA) strain is shown in Table 3. The alkaline amylase secretion production evaluation of the constructed 168PVG (efp) strain was carried out in the same manner as in Example 2. As shown in Table 4, the productivity of the 168PVG (efp) strain was equivalent to that of the wild strain 168 strain. Yes, it was thought that the protein translation efficiency would not improve even if the intracellular level of the translation elongation factor EF-P increased due to enhanced expression of the efp gene.

It is a figure which shows the outline of the method of introduce | transducing the gene which codes Fus protein and TufA protein.

Claims (14)

  A microorganism having a gene encoding a target protein or polypeptide and having a transcription initiation control region controlling the gene encoding the TufA protein or a transcription start control region and a ribosome binding site enhanced, or encoding a TufA protein Microorganisms introduced with a gene.   A microorganism having a gene encoding a target protein or polypeptide and controlling a transcription initiation control region or transcription initiation control region that controls the gene encoding the Fus protein and a ribosome binding site and a gene encoding the TufA protein A transcription initiation control region, a microorganism in which a transcription initiation control region and a ribosome binding site are reinforced, or a microorganism into which a gene encoding a Fus protein and a gene encoding a TufA protein are introduced.   Transcription initiation control region or transcription initiation control region and ribosome binding site enhancement encodes the original transcription initiation control region or transcription initiation control region and ribosome binding site that controls the gene encoding TufA protein, or Fus protein and TufA protein The original transcription initiation control region or the transcription initiation control region and the ribosome binding site that control the gene to be replaced are replaced with another transcription initiation control region or the transcription initiation control region and the ribosome binding site, or other transcription initiation The microorganism according to claim 1 or 2, wherein a control region or a transcription initiation control region and a ribosome binding site are inserted.   The introduction of the gene is a nucleic acid fragment containing a base sequence encoding a TufA protein, or a base sequence encoding a Fus protein and a TufA protein, and a transcription initiation control region or a transcription initiation control region and a ribosome binding site upstream thereof The microorganism according to claim 1 or 2, wherein the bound nucleic acid fragment is introduced into the microorganism. 4. The microorganism according to claim 3, wherein the other transcription initiation control region or the transcription initiation control region and the ribosome binding site are the transcription initiation control region of the Bacillus subtilis spoVG gene or the transcription initiation control region and the ribosome binding site. The microorganism according to claim 4, wherein the transcription initiation control region or the transcription initiation control region and the ribosome binding site are the transcription initiation control region of the Bacillus subtilis spoVG gene or the transcription initiation control region and the ribosome binding site.   The recombinant microorganism according to any one of claims 1 to 6, wherein at least one region selected from a transcription initiation control region, a translation initiation control region and a secretion signal region is linked upstream of a gene encoding a target protein or polypeptide. .   The recombinant microorganism according to claim 7, wherein three regions comprising a transcription initiation control region, a translation initiation control region, and a secretion signal region are combined.   The recombinant microorganism according to claim 7 or 8, wherein the secretory signal region is derived from a cellulase gene of a Bacillus bacterium, and the transcription initiation control region and the translation initiation control region are derived from a 0.6-1 kb region upstream of the cellulase gene. .   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 comprising a base sequence of base numbers 1 to 696 of the cellulase gene or a base sequence having 70% or more identity with any of the base sequences, or a base in which a part of the base sequence is deleted, substituted or added The recombinant microorganism according to claim 8, which is a DNA fragment consisting of a sequence.   The microorganism according to any one of claims 1 to 10, wherein the microorganism is a Bacillus bacterium.   The microorganism according to claim 11, wherein the Bacillus bacterium is Bacillus subtilis. The microorganism according to any one of claims 1 to 12, 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 1-13.