JPWO2008001597A1 - Protein production method - Google Patents

Abstract

The present invention provides an efficient method for producing protein secretion by coryneform bacteria. Provided is a method for producing a target protein, the method comprising culturing a coryneform bacterium modified to enhance serine / threonine phosphatase activity, imparted with a secretory protein production ability, and recovering the secreted target protein Is done.

Description

  The present invention relates to a method for producing a secretory protein (production and secretion) in a coryneform bacterium. In particular, the present invention relates to a method for secretory production of various proteins including industrially useful enzymes and physiologically active proteins in coryneform bacteria.

  Coryneform bacteria are very useful bacteria in the fermentation industry as L-amino acids and nucleic acid-producing bacteria including L-glutamic acid and L-lysine. In addition, coryneform bacteria originally have very few proteins secreted outside the cell compared to fungi, yeast and Bacillus bacteria, which are suitable for protein secretion, simplifying the purification process when proteins are secreted and produced. It can be abbreviated and grows quickly on simple mediums such as sugar, ammonia and inorganic salts, and is excellent in medium cost, culture method and culture productivity, and is a very useful bacterium in protein production. It is considered.

As a method for efficiently secreting and producing heterologous proteins using coryneform bacteria, secretion of nuclease and lipase by Corynebacterium glutamicum (hereinafter sometimes referred to as C. glutamicum) [US Pat. No. 4,965,197] , J. Bacteriol., 174, 1854-1861 (1992)] and secretion of proteases such as subtilisin [Non-patent document 2: Appl. Environ. Microbiol., 61, 1610-1613 (1995)], coryneform bacterium Secretion of cell surface proteins [Special Table hei 6-502548], Fibronectin-binding protein secretion using coryneform bacteria Appl. Environ. Microbiol., 63, 4392-4400 (1997)], Mutant secretion apparatus was used Protein secretion method [JP-A-11-169182], transglutaminase secretion production method [Appl. Environ. Microbiol., 69, 358-366 (2003)], transglutaminase secretion production method using mutant strain [WO02 / 81694] etc. In terms of the amount of protein accumulated, the alkaline protease gene derived from Dichelobacter nodosus was expressed using the promoter, ribosome binding site and signal peptide sequence of the subtilisin gene (aprE) derived from Bacillus subtilis. . An example of expression in glutamicum and an accumulation of about 2.5 mg / ml [Appl. Environ. Microbiol., 61, 1610-1613 (1995)], and for transglutaminase secretion up to 930 mg / L Accumulation has been confirmed [WO02 / 81694].
In addition, by utilizing the secretory pathway called Tat system, proteins such as isomalt dextranase and protein glutaminase, which were difficult to produce in the secretory pathway known as Sec system, have been used in the industry. It has been reported that useful proteins are also secreted efficiently [WO2005 / 103278].

  A pstP gene, which is a gene encoding serine / threonine phosphatase, has been found in Mycobacterium spp., A closely related species of coryneform bacteria. pstP forms an operon structure together with the rodA gene, pbpA gene involved in cell growth and morphogenesis, and the genes pknA and pknB encoding serine / threonine kinases. RodA and PbpA are controlled by phosphorylation by PknA and PknB and dephosphorylation by PstP to control cell growth and morphogenesis. PstP has the ability to dephosphorylate PbpA, PknA and PknB. [WO2005 / 007880, Molecular Microbiology (2003) 49 (6), 1493-1508, Biochemical and Biophysical Reseach Communications 311 (2003) 112-120].

  Moreover, it is known that the operon structure containing pstP also exists in coryneform bacteria [Molecular Microbiology (2003) 49 (6)]. As a gene having high homology with pstP in coryneform bacteria, NCgl0044 (gene name) is registered in Genbank in the genome sequence of Corynebacterium glutamicum ATCC13032 (accession NC_003450.3 46666..44802). However, the effect of this gene on protein secretion was not clear.

An object of the present invention is to provide a method for efficiently producing a target protein in coryneform bacteria.
More specifically, the present invention efficiently produces a target protein by producing the target protein in a coryneform bacterium and efficiently secreting the produced target protein outside the cell (secretory production). It aims to provide a method.

  The present inventors have cultivated a coryneform bacterium imparted with the ability to produce a protein and enhanced serine / threonine phosphatase activity encoded by pstP, thereby producing a transglutaminase produced in the coryneform bacterium. The present inventors have found that various proteins such as protein glutaminase are efficiently secreted and completed the present invention.

That is, the present invention includes culturing a coryneform bacterium modified so as to enhance serine / threonine phosphatase activity and imparted with the ability to produce and secrete the target protein, and collecting the secreted target protein. This is a method for producing a target protein.
The present invention is also the above production method, wherein serine / threonine phosphatase is encoded by pstP.
The present invention also provides that serine / threonine phosphatase activity is enhanced by increasing the expression of pstP, enhanced by increasing the copy number of pstP, or enhanced by modifying the expression control region of pstP. It is also a method for producing a target protein, comprising culturing a coryneform bacterium that has been modified and imparted with the ability to produce and secrete a target protein, and recovering the secreted target protein.
Furthermore, the present invention is also the above production method, wherein the target protein is a protein selected from the group consisting of transglutaminase, protein glutaminase, interferon, interleukin, insulin, IGF-1 and peptide synthase.
In particular, the present invention is a gene wherein pstP encodes a protein having an amino acid sequence having at least 80% homology with the sequence described in SEQ ID NO: 2 or 4, and having serine / threonine phosphatase activity, or SEQ ID NO: It is also the production method described above, which is a gene having a sequence of a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule having the sequence of 1 or 3.

In one embodiment of the method of the present invention, a coryneform bacterium modified so that serine / threonine phosphatase activity (PstP activity) is enhanced is imparted with the ability to produce and secrete the target protein. In another embodiment of the method of the present invention, a coryneform bacterium imparted with the ability to produce and secrete a target protein is modified so that serine / threonine phosphatase activity is enhanced. The target protein may be a naturally secreted protein or a protein that is not secreted naturally. When the protein is not secreted naturally, it can be secreted by expressing it in a form to which a signal peptide that can function in coryneform bacteria is added.
In the present invention, a phosphatase is a protein that catalyzes the hydrolysis of phosphate esters and polyphosphates. In particular, a serine / threonine phosphatase is a dephosphorylation of phosphorylated Ser and Thr residues in a protein. (EC 3.1.3.16). The present inventors named the coryneform bacterium serine / threonine phosphatase PstP and the gene encoding the protein pstP.
In the present specification, “modified so that serine / threonine phosphatase activity is enhanced” means that the number of serine / threonine phosphatase (PstP) molecules per cell is increased with respect to the parent strain or the wild strain, For example, the activity per serine / threonine phosphatase (PstP) molecule is increased. The wild strains to be compared include, for example, Corynebacterium glutamicum (Brevibacterium lactofermentum) ATCC13869 and ATCC13032 in the case of coryneform bacteria.

  In one embodiment of the method of the invention, a coryneform bacterium modified to enhance serine / threonine phosphatase activity encoded by pstP is used as a host vector system. An expression construct in which a gene of a target protein to be secreted downstream of a signal peptide that can function in a coryneform bacterium is bound is introduced into the coryneform bacterium having enhanced phosphatase activity, whereby the gene is expressed and the target protein is expressed. Is produced and secreted. Either the modification for enhancing serine / threonine phosphatase activity encoded by pstP or the provision of protein production ability may be performed first.

As a gene (pstP) encoding serine / threonine phosphatase (PstP) of coryneform bacteria, for example, NCg0044 (46666..448021 of accession NC_003450.3) of Corynebacterium glutamicum (C. glutamicum) ATCC13032 registered in Genbank Is available. The nucleotide sequence of pstP of C. glutamicum ATCC13032 is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2. Further, the nucleotide sequence of pstP of C. glutamicum ATCC13869 strain is shown in SEQ ID NO: 3, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 4.
In the present invention, pstP homologues derived from other microorganisms may be used as long as the protein encoded thereby has serine / threonine phosphatase (PstP) activity in coryneform bacteria. A homologue of pstP can be searched by referring to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 using BLAST or the like (http://blast.genome.jp/).
The pstP that can be used in the present invention is a chromosome of a coryneform bacterium using a primer prepared based on a nucleotide sequence that has already been clarified, for example, SEQ ID NO: 1 or 3, such as the primers shown in SEQ ID NOs: 5 and 6. It can be obtained by PCR using DNA as a template (PCR: polymerase chain reaction; see White, TJ et al., Trends Genet. 5, 185 (1989)). Subsequently, an area including the control area of pstP can be acquired. Other microbe pstP homologues can be obtained in the same manner using these primer pairs.

  Moreover, since there may be differences in the nucleotide sequence of pstP depending on the species and strain of coryneform bacteria, pstP used in the present invention is not limited to the sequence of SEQ ID NO: 1 or 3, and the function of the encoded PstP protein, As long as it has serine / threonine phosphatase activity, it encodes a protein having a sequence including substitution, deletion, insertion or addition of one or several amino acids at one or more positions in the amino acid sequence of SEQ ID NO: 2 or 4 It may be a mutant or artificially modified. Here, “several” differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but specifically 2 to 20, preferably 2 to 10, more preferably 2 to 5 It is a piece. Such amino acid substitutions, deletions, insertions, additions, or inversions are caused by naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences of microorganisms that retain pstP. It also includes what happens.

  The substitution is preferably a conservative substitution which is a neutral mutation that does not change functionally. A conservative mutation is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In the case of Gln and Asn, when it is a basic amino acid, between Lys, Arg and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr. More specifically, conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn , Glu or Gln substitution, Cys to Ser or Ala substitution, Gln to Asn, Glu, Lys, His, Asp or Arg substitution, Glu to Gly, Asn, Gln, Lys or Asp substitution, Gly To Pro, His to Asn, Lys, Gln, Arg or Tyr, Ile to Leu, Met, Val or Phe, Leu to Ile, Met, Val or Phe, Lys to Asn , Glu, Gln, His or Arg, Met to Ile, Leu, Val or Phe, Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala, Thr Substitution from Ser to Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu.

Furthermore, as pstP, 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more, based on the entire amino acid sequence of SEQ ID NO: 2 or 4. Preferably, a nucleic acid molecule encoding a protein having 99% or more homology and having serine / threonine phosphatase (PstP) activity can be used. As used herein, “homology” refers to the ratio of identity to the entire length of the sequence, and can be calculated by BLAST, Genetyx, and the like. Furthermore, since the degeneracy of the gene differs depending on the host to be introduced, at least one codon in these nucleic acid molecules can be replaced with a codon that is easy to use in the host into which pstP is introduced.
Similarly, as long as pstP has serine / threonine phosphatase activity, it may be an N-terminal side or C-terminal side extended or shortened. For example, the length to be extended or shortened is 50 or less, preferably 20 or less, more preferably 10 or less, and particularly preferably 5 or less in amino acid residues. More specifically, the amino acid sequence of SEQ ID NO: 2 or 4 may be 5 amino acids from the N-terminal side 50 amino acids and 5 amino acids extended / shortened from the C-terminal side 50 amino acids.

  Such a gene homologous to pstP can be obtained by using, for example, site-directed mutagenesis such that the amino acid residue at a specific site of the encoded protein includes substitution, deletion, insertion or addition. It can be obtained by modifying the nucleotide sequence of 3. It can also be obtained by a conventionally known mutation treatment as follows. Mutation treatment includes in vitro treatment of the nucleotide sequence with hydroxylamine or the like, and microorganisms carrying the gene, such as coryneform bacteria, with ultraviolet light or N-methyl-N′-nitro-N-nitrosoguanidine (NTG). Alternatively, it can be artificially converted to pstP by gene recombination using methods such as ethyl methane sulfonate (EMS), which are usually used for mutagenesis, elaprone PCR, DNA shuffling, StEP-PCR. A pstP encoding a highly active PstP can be obtained by introducing a mutation (Firth AE, Patrick WM; Bioinformatics. 2005 Jun 2; Statistics of protein library construction.). Whether or not these pstP homologous genes encode serine / threonine phosphatase can be confirmed, for example, by measuring the enzyme activity by the method described above.

  In addition, pstP is a sequence that is complementary to SEQ ID NO: 1 or SEQ ID NO: 3 or a DNA that encodes a protein having a phosphotransacetylase activity that hybridizes with a probe that can be prepared from these sequences under stringent conditions. It is done. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNAs having high homology, for example, DNAs having a homology of 80, 90, 95, 97 or 99% or more hybridize, and DNAs having lower homology do not hybridize with each other, Alternatively, a salt corresponding to the usual Southern hybridization s washing conditions of 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, 0.1% SDS. The concentration and temperature may be the condition of washing once, more preferably 2-3 times.

  As a probe, partial sequences of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 3 can also be used. Such a probe can be prepared by PCR using an oligonucleotide prepared based on the nucleotide sequence as a primer and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 as a template. For example, when a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

In the present specification, “secreted” of a protein or peptide means that the protein or peptide molecule is transferred outside the bacterial cell (extracellular), and finally the protein or peptide molecule is in the medium. Of course, it includes the case where only a part is present outside the microbial cell and the case where it is present on the surface of the microbial cell.
It is known that secreted proteins are generally translated as prepeptides or prepropeptides and then become mature proteins. That is, generally, after being translated as a pre-peptide or pre-pro peptide, the signal peptide ("pre-part") is cleaved and converted to a mature peptide or pro-peptide, and the pro-peptide is further cleaved by the protease and the pro-part is further cleaved. It is known to become. In the present specification, the “signal sequence” refers to a sequence that exists at the N-terminus of a secretory protein precursor and does not exist in a natural mature protein, and the “signal peptide” refers to such a protein precursor. A peptide that is excised from the body. In general, a signal sequence is cleaved by a protease (generally called a signal peptidase) with secretion outside the cell. Such a signal peptide has certain common sequence characteristics across species, but a signal peptide that exhibits a secretory function in one species does not necessarily exhibit a secretory function in another species. .

  In the present specification, a protein having both a signal peptide and a pro moiety, that is, a primary translation product may be referred to as a “preproprotein”, and a protein that does not have a signal peptide but has a pro moiety is referred to as a “proprotein”. May be called. The pro part of a proprotein is sometimes referred to as the “pro structure part” or simply “pro structure”, and the “pro structure part / pro structure” of the protein and the “pro part” of the protein are used interchangeably herein. Is done. In the preproprotein or preprotein, the signal peptide may be derived from a different protein or may be a signal peptide naturally present in the target protein, but may be derived from a secretory protein of the host used. preferable. Or you may modify | change so that it may have an optimal codon according to the codon usage frequency of the host to be used. Furthermore, the signal peptide that can be used for the purpose of the present invention may partially contain the N-terminal amino acid sequence of the natural mature protein from which it is derived. If the signal peptide is derived from a different protein, the preproprotein may be specifically referred to as a “heterologous fusion preproprotein”. As described above, in the present invention, the target protein may be a secreted protein or a protein that is not secreted in nature. When the protein is not secreted naturally, it can be expressed in a form to which the above-mentioned signal peptide is added when the protein is expressed. Even a naturally secreted protein may be substituted for a natural signal peptide and / or prostructure with a heterologous signal peptide and / or heterologous prostructure as described above. The target protein may or may not have a prostructure.

  For example, when the target protein is protein glutaminase, they are referred to as “preproprotein glutaminase”, “proprotein glutaminase” and “heterologous fusion preproprotein glutaminase”, respectively. The “pro-part cleaved protein” refers to a protein in which at least one or more amino acids constituting the pro part are removed by cleaving the peptide bond, and the N-terminal region is a natural mature protein. Perfectly matched proteins, and those that have one or more extra amino acids derived from the pro-part at the N-terminus compared to the native protein as long as it has the activity of the protein, and the amino acid sequence than the native mature protein Is also included. Any form of protein glutaminase described above can be efficiently secreted and produced according to the present invention.

  The coryneform bacterium referred to in the present invention is an aerobic Gram-positive gonococcus and includes bacteria that have been conventionally classified into the genus Brevibacterium but are now integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol. , 41, 255 (1981)), and the Brevibacterium genus that is very closely related to the genus Corynebacterium. The advantage of using coryneform bacteria is that there are very few proteins that are inherently secreted outside the cell compared to fungi, yeasts and Bacillus bacteria that have been suitable for protein secretion so far. The production process can be simplified and abbreviated in the case of secretory production, and because it grows easily on a simple medium containing sugar, ammonia, inorganic salts, etc., it is excellent in terms of medium cost, culture method, and culture productivity. It is included. Examples of such coryneform bacteria include the following.

Corynebacterium acetoacidophilum,
Corynebacterium acetoglutamicum,
Corynebacterium alkanolyticum,
Corynebacterium carnae,
Corynebacterium glutamicum,
Corynebacterium Lilium,
Corynebacterium melacecola,
Corynebacterium thermoaminogenes,
Corynebacterium herculis,
Brevibacterium divaricatam,
Brevibacterium flavum,
Brevibacterium immariophilum,
Brevibacterium lactofermentum,
Brevibacterium roseum,
Brevibacterium saccharolyticum,
Brevibacterium thiogenitalis,
Corynebacterium ammoniagenes,
Brevibacterium album,
Brevibacterium cerinum,
Microbacterium ammonia film.

Specifically, the following strains can be exemplified.
Corynebacterium acetoacidophilum ATCC13870,
Corynebacterium acetoglutamicum ATCC15806,
Corynebacterium alkanolyticum ATCC21511,
Corynebacterium carnae ATCC15991,
Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060, ATCC13869, FERM BP-734,
Corynebacterium lilium ATCC15990,
Corynebacterium melacecola ATCC17965,
Corynebacterium efficiens AJ12340 (FERM BP-1539),
Corynebacterium herculis ATCC13868,
Brevibacterium divaricatam ATCC14020,
Brevibacterium flavum ATCC13826, ATCC14067, AJ12418 (FERM BP-2205),
Brevibacterium in mariophyllum ATCC14068,
Brevibacterium lactofermentum ATCC13869,
Brevibacterium rose ATCC13825,
Brevibacterium saccharolyticum ATCC14066,
Brevibacterium thiogenitalis ATCC19240,
Corynebacterium ammoniagenes ATCC6871, ATCC6872,
Brevibacterium album ATCC15111,
Brevibacterium cerinum ATCC15112,
Microbacterium ammonia philus ATCC15354.

You can get them from, for example, American Type Culture Collection. That is, the registration number corresponding to each strain is given, and this registration number is described in the catalog of the American Type Culture Collection, and the distribution of each strain can be received with reference to this number.
In particular, Corynebacterium glutamicum AJ12036 (FERM BP-734) isolated as a streptomycin (Sm) resistant mutant from wild-type Corynebacterium glutamicum ATCC 13869 (deposited on March 26, 1984, Hara) , National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, Higashi 1-1-1 Tsukuba, Japan, Chuo 6th, Postal Code 305-8566) is a functional gene involved in protein secretion compared to its parent strain (wild strain) The protein is secreted and has a very high ability to secrete and produce protein, which is about 2-3 times as much as the amount accumulated under optimum culture conditions, and is suitable as a host bacterium (see WO 02/081694).
Furthermore, if a strain modified so as not to produce cell surface proteins from such a strain is used as a host, purification of the target protein secreted in the medium is facilitated, which is particularly preferable. Such modification can be performed by introducing a mutation into a cell surface protein on the chromosome or its expression regulatory region by a mutation or gene recombination method. Examples of coryneform bacteria modified so as not to produce cell surface protein include Corynebacterium glutamicum (C. glutamicum) YDK010, which is a cell surface protein (PS2) disruption strain of AJ12036 (International Publication Pamphlet WO 01/23591). .

  The coryneform bacterium imparted with the ability to produce the target protein used in the present invention can be obtained, for example, by introducing a gene construct containing a nucleic acid encoding the target protein into the aforementioned coryneform bacterium. . Here, a gene construct for conferring protein production ability to coryneform bacteria generally includes a promoter, a sequence encoding an appropriate signal peptide and a nucleic acid fragment encoding the target protein, and the target protein gene in the coryneform bacterium. It has control sequences (operator, terminator, etc.) necessary for expression at appropriate positions so that they can function. The target protein may have a prostructure at the N-terminus. The vector that can be used for this construct is not particularly limited as long as it can function in coryneform bacteria. Even if it can autonomously grow outside the chromosome like a plasmid, it can be integrated into the bacterial chromosome. There may be. Examples of such include PAM330 (Japanese Patent Laid-Open No. 58-067699), pHM1519 (Japanese Patent Laid-Open No. 58-77895), and pSFK6 (Japanese Patent Laid-Open No. 2000-262288). In addition, DNA fragments capable of autonomously replicating plasmids in coryneform bacteria are extracted from these vectors and inserted into the E. coli vector, so that they can be used as so-called shuttle vectors that can replicate in both E. coli and coryneform bacteria. I can do it. Artificial transposons can also be used. When a transposon is used, the target gene is introduced into the chromosome by homologous recombination or its own ability to transfer.

  The promoter that can be used in the present invention is not particularly limited, and can be generally used as long as it is a promoter that can function in the coryneform bacterium. Further, it can be a heterologous promoter such as a tac promoter derived from E. coli. There may be. Among them, a strong promoter such as tac promoter is more preferable. Examples of promoters derived from coryneform bacteria include promoters for cell surface proteins PS1, PS2, and SlpA, various amino acid biosynthesis systems such as glutamate biosynthesis system glutamate dehydrogenase genes, glutamine synthesis system glutamine synthase Gene, aspartokinase gene of lysine biosynthesis, homoserine dehydrogenase gene of threonine biosynthesis, acetohydroxy acid synthase gene of isoleucine and valine biosynthesis, 2-isopropylmalate synthase gene of leucine biosynthesis , Proline and arginine biosynthetic glutamate kinase gene, histidine biosynthetic phosphoribosyl-ATP pyrophosphorylase gene, aromatic amino acid biosynthetic deoxyarabinohepturonic acid such as tryptophan, tyrosine and phenylalanine Examples include phosphoric acid (DAHP) synthase gene, phosphoribosyl pyrophosphate (PRPP) amide transferase gene, inosinic acid dehydrogenase gene and guanylate synthase gene promoter in nucleic acid biosynthesis systems such as inosinic acid and guanylic acid. It is done.

  The signal peptide used in the present invention is not particularly limited as long as it is a signal peptide that can function in the cells of coryneform bacteria, and any signal peptide that can function in the cells of coryneform bacteria can be used. Therefore, a signal peptide derived from a different species, for example, E. coli or Bacillus subtilis, can be used in the present invention as long as it can function in the cells of coryneform bacteria. A part of the N-terminal amino acid sequence of the secretory protein from which the signal peptide is derived may be added. The signal sequence is cleaved by signal peptidase when the translation product is secreted outside the cell. The gene encoding the signal peptide can be used in its natural form, but may be modified so as to have an optimal codon according to the codon usage frequency of the host to be used. When these signal peptides are used, the gene encoding the target protein is arranged so as to be connected to the 3′-end side of the gene encoding the signal peptide and to be controlled for expression by the promoter.

The target protein that can be produced and secreted by the present invention is not particularly limited, and includes all proteins including intracellular proteins derived from animals, plants, and microorganisms. Even if the protein is derived from a coryneform bacterium as a host, it is a heterologous protein. It may be. Examples of proteins that can be secreted and produced according to the present invention include:
Transglutaminase (EC 2.3.2.13; Genbank Accession No. etc.);
Protein glutaminase (EC 3.5.1; Genbank Accession No. BAB21508, Eur. J. Biochem. 268. 1410-1421 (2001), etc.);
Interferon (Genbank Accession No. CAA23802 etc.);
Interleukin 2 (IL2: Genbank Accession No. AAK26665, etc., mature type is amino acid sequence 21 to 153);
Insulin (JP 07-284394 A, etc.);
IGF-1 (insulin-like growth factor 1: Genbank Accession No. CAA01954 etc.);
Peptide synthases (WO 2004/011653, WO2004 / 065610);
The genes encoding these proteins can be modified depending on the host used and / or to obtain the desired activity, including one or more amino acid additions, deletions, substitutions, etc. It is. If necessary, it may be converted to an optimal codon according to the frequency of codon usage of the host. Such general molecular biology techniques, including modification techniques, gene cloning techniques, and production protein detection techniques, are well known to those skilled in the art, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Third Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, DNA cloning: A Practical Approach, Volumes I and II (DN Glover ed. 1985), FM Ausubel et al. eds), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994), PCR Technology: Principles and Application for DNA Amplification, H. Erlich, ed., Stockton Press, and the like.

  The method of introducing the gene construct that can be used in the present invention into coryneform bacteria is not particularly limited, and a commonly used method such as a protoplast method (Gene, 39, 281-286 (1985)), an electroporation method ( Bio / Technology, 7, 1067-1070) (1989)) and the like can be used. The resulting gene-transformed transformant can be cultured according to a commonly used method and conditions. For example, the transformant can be cultured in a normal medium containing a carbon source, a nitrogen source, and inorganic ions. In order to obtain higher growth, organic micronutrients such as vitamins and amino acids can be added as necessary. As the carbon source, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols, and the like can be used. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, and others can be used. As inorganic ions, calcium ions, magnesium ions, phosphate ions, potassium ions, iron ions, and the like can be appropriately used as necessary. The culture may be performed under aerobic conditions in a suitable range of pH 5.0 to 8.5 and 15 ° C. to 37 ° C., and the culture period may be about 1 to 7 days. By culturing the transformant under such conditions, the target protein is produced in a large amount in the microbial cell and efficiently secreted outside the microbial cell.

  The coryneform bacterium imparted with the ability to produce and secrete the target protein as described above is modified so that serine / threonine phosphatase activity is enhanced, or modified in advance so that serine / threonine phosphatase activity is enhanced. Coryneform bacteria used for protein production by the method of the present invention can be obtained by imparting the ability to secrete and produce the target protein to the existing coryneform bacteria.

Hereinafter, an example of a method for constructing a coryneform bacterium modified so that serine / threonine phosphatase (PstP) activity is increased will be described. These methods can be performed according to manuals such as Molecular cloning (Cold spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). Other microorganisms can be similarly modified to increase serine / threonine phosphatase activity.
Such modification that enhances serine / threonine phosphatase activity can be achieved, for example, by enhancing the expression level of pstP. Enhancement of the expression level of pstP is achieved, for example, by increasing the copy number of pstP. More specifically, a fragment containing pstP is ligated with a vector that functions in coryneform bacteria, preferably a multicopy vector, to produce a recombinant DNA, which has the above-mentioned ability to produce L-amino acids. What is necessary is just to introduce and transform into the microorganism which has. Alternatively, the recombinant DNA may be introduced into a wild-type microorganism to obtain a transformed strain, and then the protein-producing ability may be imparted to the transformed strain. The increase in copy number can also be achieved by transferring one copy or multiple copies of a fragment encoding pstP onto a chromosome. Confirmation of the transfer of pstP onto the chromosome can be confirmed by Southern hybridization using a part of pstP as a probe. Furthermore, enhancement of pstP expression can also be achieved by modifying the expression regulatory region of pstP. For example, it can be achieved by replacing the promoter sequence of pstP with a stronger promoter or bringing the promoter sequence closer to consensus (International Publication No. WO00 / 18935).

In one embodiment of the present invention, enhancement of the expression level of pstP is achieved by increasing the copy number of pstP. Increasing the copy number can be achieved, for example, by amplifying pstP with a plasmid as follows. First, pstP is cloned from the chromosome of a coryneform bacterium. Chromosomal DNA is obtained from bacteria that are DNA donors, for example, the method of Saito and Miura (H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Biotechnology Experiments, Nippon Biotechnology, Ed., Pp. 97-98, Bafukan, 1992). Oligonucleotides used for PCR can be synthesized based on the above known information. For example, pstP can be amplified using the synthetic oligonucleotides described in SEQ ID NOs: 5 and 6.
The gene fragment containing pstP amplified by the PCR method is prepared by connecting it to a vector DNA that can replicate autonomously in cells of coryneform bacteria and preparing recombinant DNA, which is introduced into Escherichia coli. It becomes easy to operate. Examples of vectors capable of autonomous replication in Escherichia coli cells include pUC19, pUC18, pHSG299, pHSG399, pHSG398, RSF1010, pBR322, pACYC184, and pMW219.

  The DNA is introduced into a vector that can function in coryneform bacteria. The vector that functions in coryneform bacteria is, for example, a plasmid that can autonomously replicate in coryneform bacteria. Specifically, as a plasmid capable of autonomous replication in coryneform bacteria, for example, plasmid pCRY30 described in JP-A-3-210184; JP-A-2-72876 and US Pat. No. 5,185,262 Plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX; plasmids pCRY2 and pCRY3 described in JP-A-1-91686; pAM330 described in JP-A-58-67679; JP-A-58-77895 PHM1519 described; pAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; Examples include pCG4 and pCG11 described in JP-A-57-183799 and pVK7 described in JP-A-10-215883.

In order to prepare a recombinant DNA by ligating a vector that functions in pstP and coryneform bacteria, the vector can be cleaved with a restriction enzyme that matches the end of pstP. This restriction enzyme site may be introduced in advance into a synthetic oligonucleotide used for amplification of pstP. Ligation can be performed using a ligase such as T4 DNA ligase.
In order to introduce the recombinant plasmid prepared as described above into a coryneform bacterium, transformation methods that have been reported so far may be used. For example, a method for increasing DNA permeability by treating recipient cells with calcium chloride as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol , 53, 159 (1970)), and a method for preparing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Duncan, CH, Wilson, GAand Young, FE, Gene, 1, 153 (1977)). Alternatively, DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, are introduced into the DNA-receptor cells in a protoplast or spheroplast state that readily incorporates recombinant DNA. (Chang, S. and Choen, SN, Mol. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JMandHopwood, OA, Nature, 274, 398 (1978); Hinnen, A., Hicks, JBand Fink, GR, Proc. Natl. Acad. Sci. USA, 75 1929 (1978)). In addition, transformation of coryneform bacteria can also be performed by the electric pulse method (Japanese Patent Laid-Open No. 2-207791) or the junction transfer method (Biotechnology (NY). 1991 Jan; 9 (1): 84-7). Can do.

  Increasing the copy number of pstP can also be achieved by having multiple copies of pstP on the chromosomal DNA of coryneform bacteria. In order to introduce a plurality of copies of pstP into the chromosomal DNA of a coryneform bacterium, homologous recombination is performed using a sequence present in the chromosomal DNA as a target. As a sequence present in multiple copies on chromosomal DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used. Alternatively, as disclosed in JP-A-2-109985, it is possible to mount pstP on a transposon, transfer it, and introduce multiple copies onto chromosomal DNA. (JP-A-2-109985, JP-A-7-107976, Mol. Gen. Genet., 245, 397-405 (1994), Plasmid. 2000 Nov; 44 (3): 285-91).

Further, a method of introducing a pstP into a replication origin that cannot replicate in the host or a plasmid having a replication origin that cannot replicate in the host and the ability to transfer conjugation to the host and amplifying it on the chromosome can also be applied. For example, vectors that can be used are pSUP301 (Simo et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schaefer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega corporation , Madison, WI, USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemisty 269: 32678-84; US-A 5487993), pCR ^(R) Blunt (Invitrogen, Groningen, Netherlands; Bernard et al , Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173: 4510-4516) or pBGS8 (spratt et al., 1986, Gene, 41: 337-342) etc. Is mentioned. A plasmid vector containing pstP is transferred into a coryneform bacterium by conjugation or transformation. The joining method is described, for example, in Schaefer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Transformation methods include, for example, Theirbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivinan (Bio / Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343- 347 (1994)).

  In addition, as a means of increasing the activity of pstP, the expression control sequence such as the promoter of pstP on chromosomal DNA or plasmid is replaced with a strong one, and the factors involved in pstP expression regulation, such as operators and repressors, are modified. This can also be achieved by connecting powerful terminators. (Hamilton et al ,; Journal of Bacterology 171: 4617-4622) For example, lac promoter, trp promoter, trc promoter, PS2 promoter and the like are known as strong promoters. Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1995, 1, 105-128). In addition, as disclosed in International Publication WO00 / 18935, it is also possible to introduce a base substitution of several bases into the promoter region of the target gene, replace it with a sequence that is closer to consensus, and modify it to be stronger. . For example, it is conceivable to replace the −35 region with TTGACA and TTGCCA sequences and the −10 region with TATAAT and TATAAC sequences. Furthermore, it is known that the substitution of several nucleotides in the spacer between the ribosome binding site (RBS) and the start codon, particularly in the sequence immediately upstream of the start codon, greatly affects the translation efficiency of mRNA, These can be modified.

Expression control sequences such as a promoter upstream of pstP can also be determined using a promoter search vector or gene analysis software such as GENETYX. These promoter substitutions or modifications enhance pstP expression. The substitution of the expression regulatory sequence can be performed using, for example, a temperature sensitive plasmid. The modification of the expression regulatory sequence may be combined with increasing the copy number of pstP.
An increase in the expression level can also be achieved by prolonging the survival time of m-RNA or preventing degradation of the enzyme protein in the cell.

  The enhanced serine / threonine phosphatase (PstP) activity is known in the art, for example, as described by Boitel et al. (Brigitte Boitel, Miguel Ortiz-Lombardia, Rosario Duran, Frederique Pompeo, Stewart T. Cole, Carlos Cervenansky and Pedro M. Alzari (2003) Molecular Microbiology. 49 (6), 1493-1508) can be confirmed by measuring the PstP enzyme activity and comparing it with a wild type or non-modified strain. In addition, confirmation that serine / threonine phosphatase (PstP) activity was enhanced can be confirmed by comparing the amount of mRNA of the gene encoding serine / threonine phosphatase (PstP) with the wild type or unmodified strain. I can confirm. Examples of the method for confirming the expression level include Northern hybridization and RT-PCR (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). In the present invention, the enhanced serine / threonine phosphatase (PstP) activity or expression level can be any activity or expression level as long as it is statistically significantly higher than that of the wild type or non-modified strain. . According to the present invention, the serine / threonine phosphatase (PstP) activity is increased by 1.5 times or more, more preferably by 2 times or more, and even more preferably by 3 times or more compared to the wild type strain or the unmodified strain.

The protein secreted into the medium according to the present invention can be separated and purified from the cultured medium according to a method well known to those skilled in the art. For example, after removing cells by centrifugation, etc., salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium / high pressure liquid chromatography, reverse phase chromatography And can be separated and purified by a known appropriate method such as hydrophobic chromatography, or a combination thereof. The protein secreted into the cell surface by the present invention is also solubilized by methods well known to those skilled in the art, for example, by increasing the salt concentration, using a surfactant, etc., and then secreting it into the medium. It can be separated and purified. In some cases, the protein secreted into the surface of the bacterial cell may be used as, for example, an immobilized enzyme without solubilizing the protein.
The invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way.

Examples Example 1 Cloning of phosphatase gene pstP from C. glutamicum ATCC13869 and amplification effect of pstP on secretory production of protein (1) Cloning of pstP from C. glutamicum ATCC13869
The genomic sequence of C. glutamicum ATCC13032 has already been determined [Eur. J. Biochem., 257, 570-576 (1998)]. With reference to this sequence, the primers shown in SEQ ID NO: 5 and SEQ ID NO: 6 were synthesized and prepared according to a conventional method (Saito, Miura method [Biochim. Biophys. Acta, 72, 619 (1963)]). A region encoding pstP was amplified from the chromosomal DNA of glutamicum ATCC13869 by PCR. Pyrobest DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) was used for the PCR reaction, and the reaction conditions followed the protocol recommended by the manufacturer. The sequence of SEQ ID NO: 5 contains the recognition sequence of the restriction enzyme PstI, and the sequence of SEQ ID NO: 6 contains the sequence of the restriction enzyme EcoRI.

(SEQ ID NO: 5) 5'-CACGGATATCCGTACTGCAGGTACAACAGT -3 '
(SEQ ID NO: 6) 5'-CACATTTCCGTGCGGCGAATTCTTAATCGT -3 '

<Sequence Listing Free Text>
Sequence number 5, 6: PCR primer

Next, the amplified DNA fragment was digested with PstI and EcoRI, and an about 1.4 kb fragment was recovered by agarose gel electrophoresis using EASYTRAP Ver.2 (Takara Shuzo Co., Ltd.). It was inserted into the PstI and EcoRI sites of a shuttle vector that can replicate in both E. coli and coryneform bacteria, and then introduced into competent cells of Escherichia coli JM109 (Takara Shuzo). A strain carrying the plasmid in which the pstP fragment was cloned was obtained, from which the plasmid was recovered and named pVpstP. The nucleotide sequence of the fragment cloned in pVpstP was determined using a dye terminator cycle sequencing kit (PE Applied Biosystems) and a DNA sequencer 377A (PE Applied Biosystems). As a result of nucleotide sequencing, it was found that the nucleotide sequence of pstP of C. glutamicum ATCC13869 obtained was partially different from the nucleotide sequence of pstP of C. glutamicum ATCC13032. The nucleotide sequence of pstP derived from C. glutamicum ATCC13869 is shown in SEQ ID NO: 3, and the entire encoded amino acid sequence is shown in SEQ ID NO: 4. The nucleotide sequence of pstP of C. efficiens YS-314 is shown in SEQ ID NO: 7, and the entire amino acid sequence encoded is shown in SEQ ID NO: 8. FIG. 3 shows the alignment of these amino acid sequences. Thus, the sequence of pstP is well conserved among coryneform bacteria, the homology between ATCC13032 and ATCC13869 is 99.8% of the total length of the amino acid sequence, and the homology between ATCC13032 and ATCC13869 and YS-314 is the amino acid sequence. The total length was 72.3%. Creation of alignment and calculation of homology were performed using Genetyx_Version7 (manufactured by Genetics).

(2) Effect on production of protein secretion by pstP amplification in C. glutamicum ATCC13869 Using the plasmid pVpstP constructed in Example 1 (1), a streptomycin (sm) resistant mutant of C. glutamicum ATCC13869 and C. glutamlcum ATCC13869 YDK010 strain (described in WO 01/23591), a cell surface protein (PS2) -disrupted strain of AJ12036, was transformed, and CM2G agar medium containing 5 mg / l chloramphenicol (yeast extract 10 g, tryptone 10 g, glucose 5 g And 5 g of NaCl, 0.2 g of DL-methionine, 20 g of agar, and 1 L with water). Next, using these transformants and pVC7 introduced into C. glutamicum ATCC13869 and YDK010 as controls, the production of proteins secreted into the culture supernatant was compared and evaluated. A strain grown overnight at 30 ° C. on the above CM2G agar medium containing 5 mg / l chloramphenicol was obtained from an MMTG liquid medium (glucose 60 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, Iron sulfate heptahydrate 0.03g, manganese sulfate tetrahydrate 0.03g, thiamine hydrochloride 0.45mg, biotin 0.45mg, DL-methionine 0.15g, calcium carbonate 50g, adjusted to pH 7.5 with 1L with water) Inoculated into large test tubes filled with 4 ml of medium supplemented with chloramphenicol 5 mg / l, and cultured at 30 ° C. for 48 hours, respectively. The amount of protein in the culture supernatant after completion of the culture was quantified using Protein Assay CBB Solution (manufactured by Nacalai Tesque). The measurement conditions followed the protocol recommended by the vendor. As a result, in both C. glutamicum ATCC13869 and YDK010, the amount of total protein secreted into the transformed YDK010 culture supernatant increased by about 1.2 times by having pVpstP compared to the control. Tables 1 and 2 show the relative ratios of secretion with pVC7 as a control.

Table 1. Effect of pstP amplification on protein secretory production of C. glutamicum ATCC13869

Table 2. Effect of pstP amplification on protein secretion of C. glutamicum YDK010

Example 2 Amplification effect of pstP on secretory production of target protein in C. glutamicum (1) Secretion production of protransglutaminase using pstP-amplified strain of C. glutamicum ATCC13869 Using plasmid pVpstP constructed in Example 1 (1) , A C1 glutamicum ATCC13869 and YDK010 having a secretory expression plasmid pPKSPTG1 with a trans-structural transglutaminase having a Sec-type secretory signal sequence CspA signal sequence described in WO01 / 23591, and 5 mg / l chloramphenicol and 25 mg Strains grown on the CM2G agar medium containing 1 / l kanamycin were selected. Next, with respect to these transformants and those obtained by introducing pVC7 and pPKSPTG1 into C. glutamicum ATCC13869 and YDK010, the amount of secretory production was compared and evaluated. A strain grown overnight at 30 ° C on the above CM2G agar medium containing 5 mg / l chloramphenicol and 25 mg / l kanamycin was added to the above MMTG liquid medium with 5 mg / l chloramphenicol and 25 mg / l kanamycin A large test tube with 4 ml of the medium was inoculated and cultured at 30 ° C. for 48 hours, respectively. As a result of subjecting 10 μl of the culture supernatant to SDS-PAGE after completion of the culture, both C. glutamicum ATCC13869 and YDK010 strains increased by about 1.5 times compared to the control using pVC7 due to having pVpstP. Was recognized. Further, even when the Sec-type secretory signal sequence was used due to the presence of pVpstP, an increase in the secretory production of protransglutaminase was observed for both C. glutamicum ATCC13869 and YDK010 strains (FIG. 1).

(2) Production of secretory protein glutaminase using a pstP-amplified strain of C. glutamicum ATCC13869 In the same manner as in Example 2 (1), the plasmid pVpstP constructed in Example 1 (1) was used, as described in WO02 / 081694 Transform C. glutamicum ATCC13869 with the secretory expression plasmid pPKT-PPG of protein glutaminase with pro structure having Tat secretion signal sequence TorA signal sequence, containing 5 mg / l chloramphenicol and 25 mg / l kanamycin Strains grown on the CM2G agar medium were selected. Next, using this transformant and pVC7 and pPKT-PPG introduced into C. glutamicum ATCC13869 as a control, the amount of secretory production was compared and evaluated. A strain grown overnight at 30 ° C on the above CM2G agar medium containing 5 mg / l chloramphenicol and 25 mg / l kanamycin is added to the above MMTG liquid medium with 5 mg / l chloramphenicol and 25 mg / l kanamycin Inoculate 4 ml of the prepared medium into large test tubes and incubate at 30 ° C. for 48 hours, respectively. After completion of the culture, 10 μl of the culture supernatant was subjected to SDS-PAGE. As a result, about 1.5 times increase in secretion amount was recognized by having pVpstP compared with the control. Furthermore, also in the secretory production using the Tat secretion signal sequence, the production of protein glutaminase with a prostructure by transformed C. glutamicum ATCC13869 was observed by having pVpstP (FIG. 2).

  According to the present invention, the efficiency of protein secretion production using coryneform bacteria is dramatically increased. That is, according to the present invention, the coryneform bacterium with enhanced secretory efficiency of the target protein by enhancing the serine / threonine phosphatase activity (PstP activity) of the coryneform bacterium imparted with the ability to produce and secrete the target protein. Is obtained. By culturing this coryneform bacterium, a method for efficiently producing a target protein is provided.

Reference 1. US Pat. No. 4,965,1972. Japanese National Publication No. 6-502548 JP-A-11-1691824. International Publication No. 02/81694 Pamphlet 5. International Publication No. 05/103278 Pamphlet 6. International Publication No. 05/007880 Pamphlet 7. Liebl W, Sinskey AJ, Schleifer KH, Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum, J. Bacteriology (1992), 174, 1854-1861
8). Billman-Jacobe H, Wang L, Kortt A, Stewart D, Radford A, Expression and secretion of heterologous proteases by Corynebacterium glutamicum, Applied Environmental Microbiology, (1995), 61, 1610-1613
9. Salim K, Haedens V, Content J, Leblon G, Huygen K, Heterologous expression of the Mycobacterium tuberculosis gene encoding antigen 85A in Corynebacterium glutamicum, Applied Enviromental Microbiology, (1997), 63, 4392-4400
10. Kikuchi Y, Date M, Yokoyama K, Umezawa Y, Matsui H, Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus, Applied Enviromental Microbiology ( ), 69, 358-366
11. Brigitte Boitel, Miguel Ortiz-Lombardia, Rosario Duran, Frederique Pompeo, Stewart T. Cole, Carlos Cervenansky and Pedro M. Alzari, PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylatin by PstP, the cognate phospho-Ser / Thr phosphatase, in Miycobacterium tubeculosis, Molecular Microbiology, (2003), 49 (6), 1493-1508
12 Puneet Chopra, Bhuminder Singh, Ramandeep Singh, Reena Vohra, Anil Koul, Laxman S. Meena, Harshavardhan Koduri, Megha Ghildiyal, Parampal Deol, Taposh K. Das, Anil K. Tyagi, and Yogendra Singh, Phosphoprotein ryl -threonine kinases PknA and PknB, Biochemical and Biophysical Research Communications, 311, (2003), 112-120.

The effect of pstP amplification on the secretory production amount of protransglutaminase by C. glutamicum ATCC13869 and YDK010 when the Sec-type secretory signal sequence is used is shown. Lanes 1, 2 (ATCC13869 / pPKSPTG1), 5 and 6 (YDK010 / pPKSPTG1) are pstP non-amplified strains, lanes 3, 4 (ATCC13869 / pPKSPTG1), 7 and 8 (YDK010 / pPKSPTG1) are protransglutaminases in pstP amplified strains Represents the production of each. The effect of pstP amplification on the secretory production of pro-structured protein glutaminase by C. glutamicum ATCC13869 when Tat secretion signal sequence is used is shown. Lanes 1 and 2 represent protein glutaminase production in the pstP non-amplified strain (ATCC13869 / pPKT-PPG) and lanes 3 and 4 represent the pstP amplified strain (ATCC13869 / pPKT-PPG), respectively. Alignment of amino acid sequences of serine / threonine phosphatase encoded by pstP of C. glutamicum ATCC13032, C. glutamicum ATCC13869, and C. efficiens YS-314. “*” Indicates the same position in the three species, and “.” Indicates the position of the amino acid that is the same in ATCC13032 and ATCC13869 but different in YS-314.

Claims (9)

  A method for producing a target protein, comprising culturing a coryneform bacterium modified so that serine / threonine phosphatase activity is enhanced and imparted with a secretory protein production ability, and recovering the secreted target protein .   2. The method of claim 1, wherein the serine / threonine phosphatase is encoded by pstP.   The method according to claim 1, wherein the serine / threonine phosphatase activity is enhanced by increased expression of pstP.   2. The method of claim 1, wherein the serine / threonine phosphatase activity is enhanced by an increase in pstP copy number.   The method according to claim 1, wherein the serine / threonine phosphatase activity is enhanced by modifying the expression control region of pstP.   The method according to any one of claims 1 to 5, wherein the target protein is a protein selected from the group consisting of transglutaminase, protein glutaminase, interferon, interleukin, insulin, IGF-1 and peptide synthase.   The method according to any one of claims 1 to 6, wherein pstP is a gene derived from a coryneform bacterium modified so that serine / threonine phosphatase activity is enhanced.   7. The gene according to claim 1, wherein pstP is a gene having an amino acid sequence having at least 70% homology with the sequence of SEQ ID NO: 2 or 4, and encoding a protein having serine / threonine phosphatase activity. The method described.   The method according to any one of claims 1 to 6, wherein pstP has a sequence of a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule having the sequence of SEQ ID NO: 1 or 3.