JPWO2013118544A1 - Protein secretory production - Google Patents

Abstract

We develop a novel technology to improve the secretory production of heterologous proteins by coryneform bacteria and provide a method for secretory production of heterologous proteins. A coryneform bacterium having the ability to secrete and produce a heterologous protein and modified to have a mutation in the ribosomal protein S1 gene is cultured to secrete and produce the heterologous protein.

Description

  The present invention relates to a method for secretory production of a heterologous protein and a coryneform bacterium having a specific mutation.

  As secretory production of heterologous proteins by microorganisms, Bacillus bacteria (Non-patent Document 1), methanol-utilizing yeast Pichia pastoris (Non-patent Document 2), and Aspergillus fungi (Non-Patent Documents 3 and 4) ) And the like have been reported for secretory production of heterologous proteins.

  Attempts have also been made to secrete and produce heterologous proteins using coryneform bacteria. Regarding the secretory production of heterologous proteins by coryneform bacteria, nuclease and lipase secretion by Corynebacterium glutamicum (hereinafter sometimes abbreviated as C. glutamicum) (Patent Document 1, Non-patent document 5), secretion of proteases such as subtilisin (non-patent document 6), protein secretion using signal peptides of cell surface proteins PS1 and PS2 (also referred to as CspB) of coryneform bacteria (patent document 2), PS2 Secretion of fibronectin binding protein using (CspB) signal peptide (Non-patent Document 7), secretion of protransglutaminase using PS2 (CspB) and SlpA (also referred to as CspA) signal peptides (Patent Document 3), mutation Secretion of protein using type secretion apparatus (Patent Document 4), Protransglutater by mutant It has been reported, such as the secretion of kinase (Patent Document 5).

  The general protein secretion pathway is the Sec system, which is widely present from prokaryotes to eukaryotes. Recently, a protein secretion pathway that is completely different from the Sec system has been found in the thylakoid membrane of plant cell chloroplasts. (Non-Patent Document 8). This novel secretory pathway is named Tat (Twin-Arginine Translocation system) because the arginine-arginine sequence is commonly present in the signal sequence of the secreted protein (Non-patent Document 8). It was. In the Sec system, proteins are secreted in a state prior to the formation of higher-order structures, whereas in the Tat system, proteins are known to be secreted through the cell membrane after forming higher-order structures in the cell. (Non-Patent Document 9). In coryneform bacteria, there is a report of protein secretory production using a Tat-dependent signal peptide (Patent Document 6).

  Ribosomal protein S1 (also referred to as RpsA protein) is the largest protein constituting the 30S subunit of the ribosome, and controls the binding of 16S rRNA in the 30S subunit to the SD sequence of mRNA (Non-patent Document 10). . In Escherichia coli, it is known that the RpsA protein is an essential protein for growth (Non-patent Document 11). Moreover, in enterobacteria, it is known that the mutation of RpsA protein is effective for the production of L-amino acids (Patent Document 7).

  In addition, it is known that mutations in ribosomal protein S12 (also called RpsL protein) are involved in streptomycin resistance, and cell-free extracts of Escherichia coli containing mutant ribosomal protein S12 are used for protein synthesis by cell-free protein synthesis systems. It is known to be useful (Patent Document 8).

  However, in coryneform bacteria, it is not known that mutation of ribosomal protein S1 is effective for secretory production of heterologous proteins, particularly for secretory production of heterologous proteins using the Tat system. In addition, it is not known that a specific mutation in ribosomal protein S1 is effective for secretory production of a heterologous protein. In addition, a coryneform bacterium having a mutation at a specific site of ribosomal protein S1 is not known.

US Patent 4,965,197 6-502548 Patent No. 4320769 JP-A-11-169182 Patent No.4362651 Patent No.4730302 International Publication 2011/096554 JP2004-173627

Microbiol.rev., 57, 109-137 (1993) Biotechnol., 11, 905-910 (1993) Biotechnol., 6, 1419-1422 (1988) Biotechnol., 9, 976-981 (1991) J. Bacteriol., 174, 1854-1861 (1992) Appl. Environ. Microbiol., 61, 1610-1613 (1995) Appl. Environ. Microbiol., 63, 4392-4400 (1997) EMBO J., 14, 2715-2722 (1995) J. Biol. Chem., 25; 273 (52), 34868-74 (1998) RNA, 8 (9), 1137-1147 (2002) J. Mol. Biol., 280 (4), 561-569 (1998)

  An object of the present invention is to develop a novel technique for improving the secretory production of a heterologous protein by a coryneform bacterium, and to provide a method for secretory production of a heterologous protein by a coryneform bacterium.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have mutated ribosomal protein S1 of coryneform bacteria in a method for producing a heterologous protein using coryneform bacteria as an expression host. As a result of the introduction, it was found that the ability to secrete and produce heterologous proteins by the Tat secretion apparatus was improved, and the present invention was completed.

That is, the present invention is as follows.
[1] A method for producing a heterologous protein comprising culturing a coryneform bacterium having a gene construct for secretory expression of a heterologous protein and recovering the secreted heterologous protein,
The coryneform bacterium is modified so as to have a mutation in the ribosomal protein S1 gene that improves the secretory production of a heterologous protein;
The method wherein the gene construct comprises, in 5 ′ to 3 ′ direction, a promoter sequence that functions in coryneform bacteria, a nucleic acid sequence that encodes a signal peptide that functions in coryneform bacteria, and a nucleic acid sequence that encodes a heterologous protein.
[2] The method described above, wherein the mutation is a mutation that improves the secretory production of a heterologous protein more than twice as compared with a control strain having no mutation in the ribosomal protein S1 gene.
[3] The method described above, wherein the mutation is a mutation that substitutes the amino acid residue at position 173 of the wild-type ribosomal protein S1 with another amino acid residue.
[4] The method described above, wherein the substitution of the amino acid residue is substitution of a glutamic acid residue with a glycine residue.
[5] The method described above, wherein the signal peptide is a Tat-dependent signal peptide.
[6] The method, wherein the Tat-dependent signal peptide is selected from the group consisting of a TorA signal peptide, a SufI signal peptide, a PhoD signal peptide, a LipA signal peptide, and an IMD signal peptide.
[7] The method, wherein the coryneform bacterium is further modified so that expression of one or more genes selected from a gene encoding a Tat secretion apparatus is increased.
[8] The method described above, wherein the gene encoding the Tat secretion apparatus is selected from the group consisting of a tatA gene, a tatB gene, a tatC gene, and a tatE gene.
[9] The method described above, wherein the coryneform bacterium is a genus Corynebacterium.
[10] The method described above, wherein the coryneform bacterium is Corynebacterium glutamicum.
[11] The method described above, wherein the coryneform bacterium is a modified strain derived from Corynebacterium glutamicum AJ12036 (FERM BP-734).
[12] The method as described above, wherein the coryneform bacterium is a coryneform bacterium in which the activity of a cell surface protein is reduced.
[13] A coryneform bacterium modified to have a mutation in the ribosomal protein S1 gene,
A coryneform bacterium, wherein the mutation is a mutation that replaces the amino acid residue at position 173 of the wild-type ribosomal protein S1 with another amino acid residue.
[14] The coryneform bacterium, wherein the substitution of the amino acid residue is a substitution of a glutamic acid residue with a glycine residue.
[15] The coryneform bacterium, which is a genus Corynebacterium.
[16] The coryneform bacterium which is Corynebacterium glutamicum.
[17] The coryneform bacterium, which is a modified strain derived from Corynebacterium glutamicum AJ12036 (FERM BP-734).
[18] The coryneform bacterium, wherein the activity of cell surface protein is reduced.

FIG. 1 is a photograph showing the results of SDS-PAGE when protransglutaminase fused with the TorA signal sequence of E. coli was expressed in the C. glutamicum YDK010 strain and its RpsA (E173G) mutant. FIG. 2 is a diagram showing alignment of amino acid sequences of RpsA proteins derived from various coryneform bacteria. The “*” at the bottom indicates a completely matched amino acid. The amino acid sequences of the RpsA proteins of C. glutamicum THM1, C. glutamicum YDK010, C. glutamicum ATCC13032, C. efficiens YS-314, and C. stationis ATCC6872 are respectively shown in SEQ ID NOs: 2, 4, 8, 10 and 12. The amino acid sequence of RpsA protein of C. glutamicum ATCC13869 strain is the same as that of YDK010 strain (SEQ ID NO: 4). FIG. 3 is a photograph showing the results of SDS-PAGE when C. glutamicum YDK010 strain and its RpsA (E173G) mutant strain were used to express protein glutaminase with a pro-structure part fused with TorA signal sequence of E. coli. is there. FIG. 4 is a photograph showing the results of SDS-PAGE when Arthrobacter globiformis isomaltodextranase (including signal sequence) is expressed in C. glutamicum YDK010 strain and its RpsA (E173G) mutant. FIG. 5 shows SDS-expressing pro-transglutaminase fused with TorA signal sequence of E. coli in C. glutamicum YDK010 strain and its RpsA (E173G) mutant strain and their Tat secretion apparatus-enhanced strains. It is a photograph which shows the result of PAGE. FIG. 6 shows the expression of protein glutaminase with a pro-structured part fused with the TorA signal sequence of E. coli in C. glutamicum YDK010 strain and its RpsA (E173G) mutant strain and their Tat secretion apparatus-enhanced strains. It is a photograph which shows the result of SDS-PAGE. FIG. 7 shows SDS when expressing Arthrobacter globiformis isomaltdextranase (including signal sequence) in C. glutamicum YDK010 strain and its RpsA (E173G) mutant strain, and their Tat secretion apparatus-enhanced strains. It is a photograph showing the result of -PAGE. FIG. 8 shows the results of SDS-PAGE when protransglutaminase fused with the signal sequence of SlpA (CspA) of C. stationis ATCC6872 was expressed in C. glutamicum YDK010 strain and its RpsA (E173G) mutant. It is a photograph.

  Hereinafter, the present invention will be described in detail.

<1> Method for producing heterologous protein of the present invention The present invention comprises culturing a coryneform bacterium having a gene construct for secretory expression of a heterologous protein, and recovering the secreted heterologous protein. A method wherein the coryneform bacterium is modified so as to have a mutation in the ribosomal protein S1 gene (hereinafter also referred to as “the method of the present invention” or “the method for producing a heterologous protein of the present invention”). provide.

<1-1> Coryneform bacterium used in the method of the present invention <1-1-1> Coryneform bacterium having an ability to secrete and produce a heterologous protein The coryneform bacterium used in the method of the present invention secretes a heterologous protein. By having a genetic construct for expression, it has the ability to secrete and produce heterologous proteins.

  The coryneform bacterium used in the method of the present invention is also referred to as “the bacterium of the present invention” or “the coryneform bacterium of the present invention”. In addition, the gene construct for secretory expression of the heterologous protein possessed by the bacterium of the present invention is also referred to as “gene construct used in the present invention”.

  In the present invention, “secreting” a protein means that the protein is transferred out of the bacterial cell (extracellular). A protein is “secreted” when all the molecules of the protein are finally completely free in the medium, as well as when all the molecules of the protein are present on the surface of the cell. This includes the case where some molecules of the protein are present in the medium and the remaining molecules are present on the surface of the cells.

  That is, in the present invention, “the ability to secrete and produce a heterologous protein” means that when the bacterium of the present invention is cultured in a medium, the heterologous protein is secreted in the medium or on the surface of the cell, and in the medium or on the surface of the cell. The ability to accumulate to the extent that can be recovered from. The accumulation amount is, for example, preferably 10 μg / L or more, more preferably 1 mg / L or more, particularly preferably 100 mg / L or more, and further preferably 1 g / L or more as the accumulation amount in the medium. It's okay. In addition, the accumulated amount is preferably the concentration of the heterologous protein in the suspension when the heterogeneous protein on the cell surface is collected and suspended in the same amount of liquid as the medium, for example, as the amount accumulated on the cell surface. The amount may be 10 μg / L or more, more preferably 1 mg / L or more, and particularly preferably 100 mg / L or more. The “protein” secreted and produced in the present invention is a concept including an embodiment called a peptide or polypeptide.

  In the present invention, “heterologous protein” refers to a protein that is exogenous to coryneform bacteria that express and secrete the protein. The heterologous protein may be, for example, a protein derived from a microorganism, a protein derived from a plant, a protein derived from an animal, a protein derived from a virus, or an artificial protein. Alternatively, the protein may be a protein whose amino acid sequence is designed. The heterologous protein may be a monomeric protein or a multimer protein. A multimeric protein refers to a protein that can exist as a multimer composed of two or more subunits. In a multimer, each subunit may be linked by a covalent bond such as a disulfide bond, or may be linked by a non-covalent bond such as a hydrogen bond or a hydrophobic interaction, or by a combination thereof. May be. Multimers preferably contain one or more intermolecular disulfide bonds. The multimer may be a homomultimer composed of a single type of subunit or a heteromultimer composed of two or more types of subunits. When the multimeric protein is a heteromultimer, at least one subunit among the subunits constituting the multimer may be a heterologous protein. That is, all the subunits may be derived from different species, or only some of the subunits may be derived from different species. The heterologous protein may be a protein that is naturally secreted or may be a protein that is non-secreted in nature, but is preferably a protein that is naturally secreted. In addition, the heterologous protein may or may not be a secreted protein that is naturally dependent on the Tat system. Specific examples of the “heterologous protein” will be described later.

  Only one type of heterologous protein may be produced, or two or more types may be produced. When the heterologous protein is a heteromultimer, only one type of subunit may be produced, or two or more types of subunits may be produced. In other words, “secreting production of a heterologous protein” means that not only the subunits constituting the target heterologous protein but all the subunits are secreted and produced, and only some subunits are secreted and produced. Is also included.

  In the present invention, coryneform bacteria are aerobic Gram-positive bacilli, and coryneform bacteria include Corynebacterium, Brevibacterium, and Microbacterium. And genus bacteria. Coryneform bacteria previously classified as genus Brevibacterium, but now also include bacteria integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). . Corynebacterium bacteria have been classified as Corynebacterium ammoniagenes, but have been reclassified as Corynebacterium stationis by 16S rRNA nucleotide sequence analysis. (Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)). The advantage of using coryneform bacteria is that, compared to filamentous fungi, yeast, Bacillus bacteria, etc., which are conventionally used for secretory production of heterologous proteins, there are very few proteins that are secreted outside the cells. Simplification and omission of the purification process can be expected, and it grows well on simple media containing sugar, ammonia, inorganic salts, etc., and excels in medium cost, culture method, and culture productivity And so on.

Specific examples of coryneform bacteria include the following species.
Corynebacterium acetoacidophilum
Corynebacterium acetoglutamicum
Corynebacterium alkanolyticum
Corynebacterium callunae
Corynebacterium glutamicum
Corynebacterium lilium
Corynebacterium melassecola
Corynebacterium thermoaminogenes (Corynebacterium efficiens)
Corynebacterium herculis
Brevibacterium divaricatum
Brevibacterium flavum
Brevibacterium immariophilum
Brevibacterium lactofermentum (Corynebacterium glutamicum)
Brevibacterium roseum
Brevibacterium saccharolyticum
Brevibacterium thiogenitalis
Corynebacterium ammoniagenes (Corynebacterium stationis)
Brevibacterium album
Brevibacterium cerinum
Microbacterium ammoniaphilum

Specific examples of coryneform bacteria include the following strains.
Corynebacterium acetoacidophilum ATCC 13870
Corynebacterium acetoglutamicum ATCC 15806
Corynebacterium alkanolyticum ATCC 21511
Corynebacterium callunae ATCC 15991
Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734
Corynebacterium lilium ATCC 15990
Corynebacterium melassecola ATCC 17965
Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC 13868
Brevibacterium divaricatum ATCC 14020
Brevibacterium flavum ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205)
Brevibacterium immariophilum ATCC 14068
Brevibacterium lactofermentum ATCC 13869
Brevibacterium roseum ATCC 13825
Brevibacterium saccharolyticum ATCC 14066
Brevibacterium thiogenitalis ATCC 19240
Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872
Brevibacterium album ATCC 15111
Brevibacterium cerinum ATCC 15112
Microbacterium ammoniaphilum ATCC 15354

  These strains can be sold, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States of America). That is, a registration number corresponding to each strain is given, and it is possible to receive a sale using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of American Type Culture Collection.

  In particular, Corynebacterium glutamicum AJ12036 (FERM BP-734) isolated as a streptomycin (Sm) resistant mutant from wild-type Corynebacterium glutamicum ATCC 13869 is more protein-prone than its parent strain (wild-type). It is predicted that there is a mutation in the functional gene involved in the secretion of the protein, and the secretory production ability of the heterologous protein is extremely high, about 2 to 3 times as the accumulated amount under the optimum culture conditions, and is suitable as a host bacterium. AJ12036 was established on March 26, 1984, by the National Institute of Microbiology, National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation Technology, Biological Depositary Center, East 1-1-1, Tsukuba, Japan, Central 6, Postcode 305-8566) was originally deposited as an international deposit and was given the deposit number FERM BP-734.

  Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539) was established on March 13, 1987, at the Institute of Microbial Industrial Technology, National Institute of Advanced Industrial Science and Technology (now the National Institute for Product Evaluation Technology Patent Biology Center, East 1 Tsukuba, Japan). -1-1 Center No. 6, postal code 305-8566) has been deposited as an international deposit and has been given the deposit number FERM BP-1539. In addition, Brevibacterium flavum AJ12418 (FERM BP-2205) was introduced on December 24, 1988, by the National Institute of Microbiology, National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation Technology Patent Biological Depositary Center, East 1 Tsukuba, Japan). -1-1 Center No. 6, postal code 305-8566), the original deposit as an international deposit, and the deposit number FERM BP-2205 is given.

  Alternatively, the above-described coryneform bacterium as a parent strain may be selected as a host by selecting a strain having enhanced protein secretory production ability using a mutation method or a gene recombination method. For example, after treatment with a chemical mutagen such as ultraviolet irradiation or N-methyl-N′-nitrosoguanidine, a strain with enhanced protein secretory production ability can be selected.

  Furthermore, if a strain modified so as not to produce cell surface protein from such a strain is used as a host, purification of a heterologous protein secreted in the medium or on the surface of the cell body is facilitated, which is particularly preferable. Such modification can be carried out by introducing mutation into the coding region of cell surface protein on the chromosome or its expression regulatory region by mutation or gene recombination. Examples of coryneform bacteria modified so as not to produce cell surface proteins include C. glutamicum YDK010 strain (WO2004 / 029254), which is a cell line protein PS2 deficient strain of C. glutamicum AJ12036 (FERM BP-734). .

  That is, the bacterium of the present invention may have a reduced cell surface protein activity. Below, cell surface layer protein and the gene which codes it are demonstrated.

  The cell surface protein is a protein constituting the cell surface layer (S layer) of bacteria and archaea. Examples of cell surface proteins of coryneform bacteria include C1 glutamicum PS1 and PS2 (also referred to as CspB), and C. stationis SlpA (also referred to as CspA). In these, it is preferable to reduce the activity of PS2 protein.

  The nucleotide sequence of the cspB gene of C. glutamicum ATCC13869 and the amino acid sequence of the PS2 protein encoded by this gene are shown in SEQ ID NOs: 26 and 27, respectively.

For example, the amino acid sequence of a CspB homolog has been reported for 28 strains of C. glutamicum (J Biotechnol., 112, 177-193 (2004)). The GenBank accession numbers in the NCBI database of these 28 strains of C. glutamicum and cspB gene homologs are exemplified below (the GenBank accession numbers are shown in parentheses).
C. glutamicum ATCC13058 (AY524990)
C. glutamicum ATCC13744 (AY524991)
C. glutamicum ATCC13745 (AY524992)
C. glutamicum ATCC14017 (AY524993)
C. glutamicum ATCC14020 (AY525009)
C. glutamicum ATCC14067 (AY524994)
C. glutamicum ATCC14068 (AY525010)
C. glutamicum ATCC14747 (AY525011)
C. glutamicum ATCC14751 (AY524995)
C. glutamicum ATCC14752 (AY524996)
C. glutamicum ATCC14915 (AY524997)
C. glutamicum ATCC15243 (AY524998)
C. glutamicum ATCC15354 (AY524999)
C. glutamicum ATCC17965 (AY525000)
C. glutamicum ATCC17966 (AY525001)
C. glutamicum ATCC19223 (AY525002)
C. glutamicum ATCC19240 (AY525012)
C. glutamicum ATCC21341 (AY525003)
C. glutamicum ATCC21645 (AY525004)
C. glutamicum ATCC31808 (AY525013)
C. glutamicum ATCC31830 (AY525007)
C. glutamicum ATCC31832 (AY525008)
C. glutamicum LP-6 (AY525014)
C. glutamicum DSM20137 (AY525015)
C. glutamicum DSM20598 (AY525016)
C. glutamicum DSM46307 (AY525017)
C. glutamicum 22220 (AY525005)
C. glutamicum 22243 (AY525006)

  Depending on the species or strain to which the coryneform bacterium belongs, there may be a difference in the base sequence of the gene encoding the cell surface protein. Therefore, the gene encoding the cell surface protein encodes a protein whose original function is maintained. As long as it is a variant of the above base sequence. “Maintaining the original function” may mean, for example, having the property of increasing the secretory production amount of a heterologous protein as compared to an unmodified strain when the activity is reduced in a coryneform bacterium. For example, a gene encoding a cell surface protein has the property of increasing the secreted production amount of a heterologous protein compared to an unmodified strain when the activity is reduced in a protein having the original function maintained, for example, a coryneform bacterium. A gene encoding a protein having an amino acid sequence in which one or several amino acids at one or several positions are substituted, deleted, inserted or added in the above amino acid sequence. May be. Regarding the cell surface protein and the variant of the gene encoding the same, the description regarding the ribosomal protein S1 and the variant of the gene encoding the same can be applied mutatis mutandis.

  “The property of increasing the secretory production of heterologous proteins when the activity is reduced in coryneform bacteria compared to non-modified strain” means that a non-modified strain such as a wild strain is reduced in activity in coryneform bacteria. Alternatively, it refers to the property of giving coryneform bacteria the ability to secrete and produce a larger amount of heterologous protein than the parent strain. The secretory production of a larger amount of a heterologous protein than that of an unmodified strain is not particularly limited as long as the amount of secretory production of the heterologous protein is increased as compared with that of the unmodified strain. The amount of accumulation is preferably 10% or more, more preferably 20% or more, particularly preferably 30% or more, and even more preferably 100% or more of the heterologous protein compared to the unmodified strain. Good. In addition, it is said that secretory production of a larger amount of a heterologous protein than that of an unmodified strain means that the heterogeneous protein cannot be detected when the culture supernatant of an unmodified strain that has not been concentrated is subjected to SDS-PAGE and stained with CBB. It may be possible to detect a heterologous protein when the culture supernatant of a modified strain that has not been subjected to SDS-PAGE and stained with CBB.

  If the activity of coryneform bacteria is reduced, the secretory production of heterologous proteins will be higher than that of non-modified strains. A modified strain is prepared, and the amount of a heterologous protein secreted and produced when the modified strain is cultured in a medium is quantified, and secreted and produced when the unmodified strain (non-modified strain) is cultured in a medium. This can be confirmed by comparing with the amount of the heterologous protein.

  In the present invention, “the activity of the cell surface protein is reduced” means that the coryneform bacterium has been modified so that the activity of the cell surface protein is reduced, and that This includes cases where the activity is reduced. “When the activity of the cell surface protein is originally reduced in the coryneform bacterium” includes the case where the coryneform bacterium originally does not have the cell surface protein. That is, examples of coryneform bacteria in which the activity of cell surface proteins is reduced include coryneform bacteria that originally have no cell surface proteins. Examples of “when the coryneform bacterium originally has no cell surface protein” include a case where the coryneform bacterium originally does not have a gene encoding the cell surface protein. “The coryneform bacterium originally has no cell surface protein” means that the coryneform bacterium is selected from one or more cell surface proteins found in other strains of the species to which the coryneform bacterium belongs. It may be that there is no protein originally. For example, “C. glutamicum originally does not have a cell surface protein” means that the C. glutamicum strain is one or more proteins selected from cell surface proteins found in other C. glutamicum strains, For example, it may be inherently free of PS1 and / or PS2 (CspB). Examples of coryneform bacteria that originally have no cell surface protein include C. glutamicum ATCC 13032 that originally does not have a cspB gene.

  Hereinafter, a method for reducing the activity of the protein will be described.

  “Protein activity decreases” means that the activity of the target protein is reduced compared to a non-modified strain such as a wild strain or a parent strain, and includes cases where the activity is completely lost. . Specifically, “the activity of the protein is decreased” means that the number of molecules per cell of the protein is decreased and / or the function per molecule of the protein compared to the unmodified strain. Means that it is decreasing. That is, “activity” in the case of “decrease in protein activity” is not limited to the catalytic activity of the protein, and may mean the transcription amount (mRNA amount) of the gene encoding the protein or the amount of the protein. Note that “the number of molecules per cell of the protein is decreased” includes a case where the protein does not exist at all. Moreover, “the function per molecule of the protein is reduced” includes the case where the function per molecule of the protein is completely lost.

  The modification that decreases the activity of the protein is achieved, for example, by decreasing the expression of a gene encoding the protein. In addition, “the expression of the gene is reduced” is also referred to as “the expression of the gene is weakened”. The decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. Reduction of gene expression can be achieved, for example, by modifying an expression regulatory sequence such as a gene promoter or Shine-Dalgarno (SD) sequence. In the case of modifying the expression control sequence, the expression control sequence is preferably modified by 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. Further, part or all of the expression regulatory sequence may be deleted. In addition, reduction of gene expression can be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (such as inducers and inhibitors) involved in transcription and translation control, proteins (such as transcription factors), nucleic acids (such as siRNA), and the like.

  Moreover, the modification that decreases the activity of the protein can be achieved, for example, by destroying a gene encoding the protein. Gene disruption can be achieved, for example, by deleting part or all of the coding region of the gene on the chromosome. Furthermore, the entire gene including the sequences before and after the gene on the chromosome may be deleted. The region to be deleted may be any region such as an N-terminal region, an internal region, or a C-terminal region as long as a decrease in protein activity can be achieved. Usually, the longer region to be deleted can surely inactivate the gene. Moreover, it is preferable that the reading frames of the sequences before and after the region to be deleted do not match.

  In addition, gene disruption is, for example, introducing an amino acid substitution (missense mutation) into a coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting 1 to 2 bases. It can also be achieved by introducing a frameshift mutation (Journal of Biological Chemistry 272: 8611-8617 (1997) Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833 -20839 (1991)).

  In addition, gene disruption can be achieved, for example, by inserting another sequence into the coding region of the gene on the chromosome. The insertion site may be any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated. Moreover, it is preferable that the reading frames of the sequences before and after the insertion site do not match. Other sequences are not particularly limited as long as they reduce or eliminate the activity of the encoded protein, and examples include marker genes such as antibiotic resistance genes and genes useful for heterologous protein production.

  Modifying a gene on a chromosome as described above includes, for example, deleting a partial sequence of the gene and preparing a deleted gene modified so as not to produce a normally functioning protein. This can be achieved by replacing the gene on the chromosome with the deleted gene by transforming the bacterium with the recombinant DNA containing, and causing homologous recombination between the deleted gene and the gene on the chromosome. At this time, the recombinant DNA can be easily manipulated by including a marker gene in accordance with a trait such as auxotrophy of the host. Even if the protein encoded by the deletion-type gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost. Gene disruption by gene replacement using such homologous recombination has already been established, and a method called “Red-driven integration” (Datsenko, KA, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640-6645 (2000)), Red-driven integration method and excision system derived from λ phage (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002) ) And the like (see WO2005 / 010175), a method using linear DNA, a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, replication functioning in the host There is a method using a suicidal vector having no origin (US Pat. No. 6,303,383, Japanese Patent Laid-Open No. 05-007491).

  Further, the modification that decreases the activity of the protein may be performed by, for example, a mutation treatment. Mutation treatment includes X-ray irradiation or ultraviolet irradiation, or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), methylmethanesulfonate (MMS), etc. A normal mutation treatment with a mutation agent can be mentioned.

  The decrease in the activity of the target protein can be confirmed by measuring the activity of the protein. Specifically, the activity of the protein is, for example, 50% or less, preferably 20% or less, more preferably 10% or less, still more preferably 5% or less, and particularly preferably 0% compared to the unmodified strain. descend.

  The decrease in the expression of the target gene can be confirmed by confirming that the transcription amount of the gene has decreased, or by confirming that the amount of the target protein expressed from the gene has decreased.

  Confirmation that the transcription amount of the target gene has decreased can be confirmed by comparing the amount of mRNA transcribed from the same gene with that of the unmodified strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR and the like (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of mRNA is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% as compared to the unmodified strain.

  Confirmation that the amount of the target protein has decreased can be confirmed by Western blotting using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of protein is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% as compared to the unmodified strain.

  Whether the target gene has been destroyed can be confirmed by determining a part or all of the base sequence, restriction enzyme map, full length, etc. of the gene according to the means used for the destruction.

  Coryneform bacteria having the ability to secrete and produce heterologous proteins can be obtained by introducing and retaining the gene construct used in the present invention in the coryneform bacteria as described above. The gene construct used in the present invention and its introduction method will be described later.

<1-1-2> Introduction of mutant ribosomal protein S1 gene The bacterium of the present invention has been modified to have a mutation in the ribosomal protein S1 gene. In the present invention, “having a mutation in the ribosomal protein S1 gene” means having a mutant ribosomal protein S1 gene. The bacterium of the present invention can be obtained by modifying a coryneform bacterium having the ability to secrete and produce a heterologous protein so as to have a mutant ribosomal protein S1 gene. The bacterium of the present invention can also be obtained by imparting the ability to secrete and produce a heterologous protein after modifying the coryneform bacterium so as to have a mutant ribosomal protein S1 gene. In the present invention, the modification for constructing the bacterium of the present invention can be performed in any order. The bacterium of the present invention may be obtained from a strain that has been capable of secreting and producing a heterologous protein before being modified so as to have a mutant ribosomal protein S1 gene. Further, the bacterium of the present invention could not secrete and produce a heterologous protein even if it had a gene construct for secretory expression of the heterologous protein before being modified to have the mutant ribosomal protein S1 gene. It may be obtained from a strain and modified to have a mutated ribosomal protein S1 gene so that a heterologous protein can be secreted and produced.

  Hereinafter, the ribosomal protein S1 and the gene encoding it will be described. In the present invention, a ribosomal protein S1 having “mutation” described later is also referred to as a mutant ribosomal protein S1, and a gene encoding it is also referred to as a mutant ribosomal protein S1 gene. In the present invention, ribosomal protein S1 having no “mutation” described later is also referred to as wild-type ribosomal protein S1, and a gene encoding it is also referred to as wild-type ribosomal protein S1 gene.

  Examples of the wild-type ribosomal protein S1 gene include the rpsA gene of C. glutamicum YDK010 strain, C. glutamicum ATCC13032 strain, C. efficiens YS-314 strain, and C. stationis ATCC6872 strain. The nucleotide sequences of these rpsA genes are shown in SEQ ID NOs: 3, 7, 9, and 11, respectively. In addition, the amino acid sequences of RpsA proteins encoded by these rpsA genes are shown in SEQ ID NOs: 4, 8, 10, and 12, respectively.

  In addition, the wild-type ribosomal protein S1 may be a variant of the RpsA protein as long as it does not have the “mutation” described later and has a function as the ribosomal protein S1. Such variants may be referred to as “conservative variants”. The conservative variant may be, for example, a homologue or artificial variant of the above RpsA protein. In the present invention, the “function as ribosomal protein S1” may be, for example, a function of controlling the binding of 16S rRNA in the 30S subunit and the SD sequence of mRNA.

  For example, the wild-type ribosomal protein S1 preferably has a conserved sequence in the RpsA protein. FIG. 2 shows the result of alignment between RpsA proteins including the above RpsA proteins.

  The wild-type ribosomal protein S1 gene may be a variant of the rpsA gene as long as it encodes the RpsA protein or a conservative variant thereof.

  The gene encoding the homologue of the RpsA protein can be easily obtained from a public database by BLAST search or FASTA search using the above-mentioned coryneform rpsA gene (SEQ ID NO: 3, 7, 9, or 11) as a query sequence. Can be acquired. The gene encoding the homologue of the RpsA protein can be obtained by PCR using, for example, a coryneform bacterium chromosome as a template and oligonucleotides prepared based on these known gene sequences as primers.

  As long as the wild-type ribosomal protein S1 does not have the “mutation” described later and has a function as the ribosomal protein S1, one or several amino acids at one or several positions in the amino acid sequence are substituted. It may be a protein having an amino acid sequence deleted, inserted or added. The “one or several” is different depending on the position of the protein in the three-dimensional structure of the amino acid residue and the type of the amino acid residue, but specifically preferably 1 to 20, more preferably 1 to 10 More preferably, it means 1 to 5.

  One or several amino acid substitutions, deletions, insertions or additions described above are conservative mutations in which the function of the protein is maintained normally. A typical conservative mutation is a conservative substitution. Conservative substitution 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 this case, between 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. Specifically, substitutions considered as 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, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Thr to Ser or 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. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences in the bacteria from which the gene is derived. Also included by

  Furthermore, as long as the wild-type ribosomal protein S1 does not have a “mutation” described later and has a function as the ribosomal protein S1, it is 80% or more, preferably 90% or more, more than the entire amino acid sequence. It may be a protein having a homology of preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more. In the present specification, “homology” may refer to “identity”.

  In addition, the wild-type ribosomal protein S1 gene does not have a “mutation” described later, and a probe that can be prepared from a known gene sequence as long as it encodes a protein having a function as the ribosomal protein S1, for example, the above base sequence It may be a DNA that hybridizes under stringent conditions with a complementary sequence for all or a part thereof. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more DNAs having homology. Are hybridized and DNAs having lower homology do not hybridize with each other, or normal Southern hybridization washing conditions of 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C., 0 .1 × SSC, 0.1% SDS, more preferably, conditions of washing once, preferably 2-3 times at a salt concentration and temperature corresponding to 68 ° C., 0.1 × SSC, 0.1% SDS Can be mentioned.

  As described above, the probe used for the hybridization may be a part of a complementary sequence of a gene. Such a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences 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.

  Further, the wild-type ribosomal protein S1 gene may be one in which any codon is substituted with an equivalent codon as long as it encodes the wild-type ribosomal protein S1 as described above. For example, the wild-type ribosomal protein S1 gene may be modified to have an optimal codon depending on the codon usage frequency of the host to be used.

  In addition, the description regarding the said gene or protein variant is applicable mutatis mutandis to Tat type | system | group secretion apparatus, cell surface layer protein, arbitrary proteins, such as the heterologous protein secreted and produced in this invention, and the gene which codes them.

  The mutant ribosomal protein S1 has a “mutation” described later in the amino acid sequence of the wild-type ribosomal protein S1 as described above.

  That is, in other words, the mutant ribosomal protein S1 may be the RpsA protein of the aforementioned coryneform bacterium or a conservative variant thereof except that it has a “mutation” described later.

  Specifically, for example, the mutant ribosomal protein S1 may be a protein having the amino acid sequence shown in SEQ ID NO: 4, 8, 10, or 12 except that it has a “mutation” described later.

  Specifically, for example, the mutant ribosomal protein S1 is substituted with one or several amino acids in the amino acid sequence shown in SEQ ID NO: 4, 8, 10, or 12 except that it has a “mutation” described later. , A protein having an amino acid sequence containing a deletion, insertion, or addition.

  Specifically, for example, the mutant ribosomal protein S1 is 80% or more, preferably 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 4, 8, 10, or 12, except that it has a “mutation” described later. It may be a protein having an amino acid sequence having a homology of 90% or more, more preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more.

  In other words, the mutant ribosomal protein S1 is a variant having the “mutation” described later in the RpsA protein of the above-mentioned coryneform bacterium, and further containing a conservative mutation at a place other than the “mutation”. It's okay.

  Specifically, for example, the mutant ribosomal protein S1 has a “mutation” to be described later in the amino acid sequence shown in SEQ ID NO: 4, 8, 10, or 12, and is further provided at a place other than the “mutation”. It may be a protein having an amino acid sequence comprising one or several amino acid substitutions, deletions, insertions or additions.

  The mutant ribosomal protein S1 gene is not particularly limited as long as it encodes the mutant ribosomal protein S1 as described above.

  Hereinafter, the “mutation” possessed by the mutant ribosomal protein S1 will be described.

  The “mutation” is not particularly limited as long as the amino acid sequence of the wild-type ribosomal protein S1 changes as described above, but is preferably a mutation that improves the amount of secreted production of a heterologous protein. “Improving the secretory production amount of a heterologous protein” means that a coryneform bacterium modified to have a mutant ribosomal protein S1 gene can secrete and produce a larger amount of a heterologous protein than an unmodified strain. The “non-modified strain” is a control strain having no mutation in the ribosomal protein S1 gene, ie, a control strain having no mutant ribosomal protein S1 gene, and may be, for example, a wild strain or a parent strain. “Secretly produce a heterologous protein in an amount larger than that of an unmodified strain” is not particularly limited as long as the amount of secretory production of the heterologous protein is increased as compared with that of the unmodified strain. The amount accumulated on the surface layer is preferably 1.1 times or more, more preferably 1.2 times or more, particularly preferably 1.3 times or more, and even more preferably 2 times or more of that of non-modified strains. It may be the production of proteins secreted. In addition, “to secrete and produce a larger amount of heterologous protein than the unmodified strain” means that the heterogeneous protein cannot be detected when the culture supernatant of the unmodified strain that has not been concentrated is subjected to SDS-PAGE and stained with CBB. However, it may be possible to detect a heterologous protein when the culture supernatant of a non-concentrated modified strain is subjected to SDS-PAGE and stained with CBB.

  Whether a mutation is a mutation that improves the production of secreted heterologous proteins, for example, based on a strain belonging to coryneform bacteria, a strain modified to have a gene encoding ribosomal protein S1 having the mutation is prepared. And quantifying the amount of the heterologous protein secreted and produced when the modified strain is cultured in the medium, and comparing it with the amount of the heterologous protein secreted and produced when the unmodified strain (non-modified strain) is cultured in the medium. This can be confirmed.

  The “mutation” is preferably one in which any amino acid residue of the wild-type ribosomal protein S1 is replaced with another amino acid residue. The “mutation” is preferably one in which the amino acid residue at position 173 of the wild-type ribosomal protein S1 is replaced with another amino acid residue. The “mutation” is preferably one in which the amino acid residue at position 173 of the wild-type ribosomal protein S1 is replaced with a glycine residue. The “mutation” is preferably one in which the glutamic acid residue at position 173 of the wild-type ribosomal protein S1 is replaced with a glycine residue.

  In the present invention, the “amino acid residue at the X position of the wild-type ribosomal protein S1” means an amino acid residue corresponding to the X position in SEQ ID NO: 4. The “X position” in the amino acid sequence means the X position from the N terminal of the amino acid sequence, and the amino acid residue at the N terminal is the first amino acid residue. That is, the position of the amino acid residue indicates a relative position, and the position may be moved back and forth by amino acid deletion, insertion, addition, or the like. For example, “amino acid residue at position 173 of wild-type ribosomal protein S1” means an amino acid residue corresponding to position 173 in SEQ ID NO: 4, and one amino acid residue on the N-terminal side from position 173 has been deleted. In this case, it is assumed that the 172nd amino acid residue from the N-terminal is “the amino acid residue at position 173 of wild-type ribosomal protein S1”. In addition, when one amino acid residue is inserted on the N-terminal side from the 173rd position, the 174th amino acid residue from the N-terminal is assumed to be “the 173rd amino acid residue of the wild-type ribosomal protein S1”. .

  Which amino acid residue in any amino acid sequence is the “amino acid residue corresponding to the X position in SEQ ID NO: 4” can be determined by aligning the arbitrary amino acid sequence with the amino acid sequence of SEQ ID NO: 4. . The alignment can be performed using, for example, known gene analysis software. Specific software includes DNA Solutions from Hitachi Solutions and GENETYX from GENETICS (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24 (1), 72-96, 1991; Barton GJ et al. al., Journal of molecular biology, 198 (2), 327-37. 1987).

  The mutant ribosomal protein S1 gene can be obtained by modifying the wild-type ribosomal protein S1 gene so that the encoded ribosomal protein S1 has the “mutation” described above. Modification of DNA can be performed by a known method. Specifically, for example, as a site-specific mutation method for introducing a target mutation into a target site of DNA, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press) (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987) Kunkel, TA et al., Meth. In Enzymol., 154, 367 (1987)). The mutant ribosomal protein S1 gene can also be obtained by chemical synthesis.

  Hereinafter, a method for modifying coryneform bacteria so as to have a mutant ribosomal protein S1 gene will be described.

  Modifying the coryneform bacterium to have the mutant ribosomal protein S1 gene can be achieved by introducing the mutant ribosomal protein S1 gene into the coryneform bacterium. Further, modifying the coryneform bacterium so as to have a mutant ribosomal protein S1 gene can also be achieved by introducing a mutation into the ribosomal protein S1 gene by natural mutation or mutagen treatment.

  The method for introducing the mutant ribosomal protein S1 gene into coryneform bacteria is not particularly limited. In the bacterium of the present invention, the mutant ribosomal protein S1 gene only needs to be retained so that it can be expressed under the control of a promoter that functions in the coryneform bacterium. In the bacterium of the present invention, the mutant ribosomal protein S1 gene may be present on a vector that autonomously proliferates outside the chromosome, such as a plasmid, or may be integrated on the chromosome. The bacterium of the present invention may have only one copy of the mutant ribosomal protein S1 gene, or may have two or more copies. The bacterium of the present invention may have only one type of mutant ribosomal protein S1 gene or may have two or more types of mutant ribosomal protein S1 genes. The introduction of the mutant ribosomal protein S1 gene can be performed, for example, in the same manner as the introduction of the gene construct used in the present invention described later.

  The bacterium of the present invention may or may not have the wild-type ribosomal protein S1 gene, but preferably does not have it.

  Coryneform bacteria having no wild-type ribosomal protein S1 gene can be obtained by disrupting the wild-type ribosomal protein S1 gene on the chromosome. The disruption of the wild-type ribosomal protein S1 gene can be performed by a known method. Specifically, for example, the wild-type ribosomal protein S1 gene can be disrupted by deleting part or all of the promoter region and / or coding region of the wild-type ribosomal protein S1 gene. When the ribosomal protein S1 gene is an essential gene, it is preferable to first introduce the mutant ribosomal protein S1 gene and then destroy the wild-type ribosomal protein S1 gene.

  In addition, by replacing the wild-type ribosomal protein S1 gene on the chromosome with a mutant ribosomal protein S1 gene, the wild-type ribosomal protein S1 gene was modified to have no wild-type ribosomal protein S1 gene and a mutant ribosomal protein S1 gene. Coryneform bacteria can be obtained. As a method for performing such gene replacement, for example, a method called “Red-driven integration” (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97 : 6640-6645 (2000)), a combination of Red driven integration method and λ phage-derived excision system (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)) Method using linear DNA, such as a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, or a suicide vector that does not have a replication origin and functions in the host. (US Pat. No. 6,303,383, Japanese Patent Laid-Open No. 05-007491).

<1-1-3> Enhancement of Tat secretion apparatus The bacterium of the present invention is modified so that expression of one or more genes selected from genes encoding the Tat secretion apparatus is increased. Also good. In the present invention, such modification is also referred to as “enhancement of the Tat secretion apparatus”. A technique for increasing the expression of a gene encoding a Tat secretion apparatus is described in Patent Document 6 (Patent No. 4730302).

  Examples of the gene encoding the Tat secretion apparatus include C. glutamicum tatA gene, tatB gene, and tatC gene. The tatA gene, tatB gene, and tatC gene of C. glutamicum ATCC 13032 are respectively sequences of positions 1571065 to 1581382 in the genome sequence registered as GenBank accession NC_003450 (VERSION NC_003450.3 GI: 58036263) in the NCBI database. It corresponds to the complementary sequence, the sequence from positions 1167110 to 1167580, and the sequence complementary to the sequence from positions 1569929 to 1570873. In addition, the TatA protein, TatB protein, and TatC protein of C. glutamicum ATCC 13032 are GenBank accession NP_600707 (version NP_600707.1 GI: 19552705, locus_tag = "NCgl1434"), GenBank accession NP_600350 (version NP_600350.1 GI: 19552348, locus_tag = "NCgl1077"), and GenBank accession NP_600706 (version NP_600706.1 GI: 19552704, locus_tag = "NCgl1433"). The nucleotide sequences of the tatA gene, tatB gene, and tatC gene of C. glutamicum ATCC 13032 and the amino acid sequences of the TatA protein, TatB protein, and TatC protein are shown in SEQ ID NOs: 13-18.

  Examples of genes encoding the Tat secretion apparatus include E. coli tatA gene, tatB gene, tatC gene, and tatE gene. The tatA gene, tatB gene, tatC gene, and tatE gene of E.coli K-12 MG1655 are respectively in the genome sequence registered as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990) in the NCBI database. This corresponds to the sequence at position 4020237, the sequence at positions 4020241 to 4020756, the sequence at positions 4020759 to 4021535, and the sequence at positions 658170 to 658373. In addition, TatA protein, TatB protein, TatC protein, and TatE protein of E. coli K-12 MG1655 are GenBank accession NP_418280 (version NP_418280.4 GI: 90111653, locus_tag = "b3836"), GenBank accession YP_026270 (version YP_026270.1 GI: 49176428, locus_tag = "b3838"), GenBank accession NP_418282 (version NP_418282.1 GI: 16131687, locus_tag = "b3839"), and GenBank accession NP_415160 (version NP_415160.1 GI: 16128610, locus_tag = "b0627 ") Is registered.

  In addition, the gene encoding the Tat secretion apparatus may be a variant of the above base sequence as long as it encodes a protein having a function as a Tat secretion apparatus. That is, for example, “tatA gene”, “tatB gene”, “tatC gene”, and “tatE gene” each include a variant thereof in addition to the above base sequence. The above description concerning the wild-type ribosomal protein S1 and the variant of the gene encoding it can be applied mutatis mutandis to the Tat secretion apparatus and the gene encoding the same. The “function as a Tat system secretion device” may be a function of secreting a protein having a Tat system-dependent signal peptide added to the N-terminus to the outside of a cell.

  Hereinafter, a method for increasing gene expression will be described.

  In the present invention, “the expression of a gene is increased” means that the expression of a target gene is increased relative to an unmodified strain such as a wild strain or a parent strain. The expression of the gene is not particularly limited as long as it is higher than that of the non-modified strain, but is preferably 1.5 times or more, more preferably 2 times or more, more preferably 3 times or more compared to the non-modified strain. To rise. In addition, “increasing gene expression” means not only increasing the expression level of a target gene in a strain that originally expresses the target gene, but also in a strain that originally does not express the target gene. Including expressing a gene. That is, “increasing gene expression” includes, for example, introducing the gene into a strain that does not hold the target gene and expressing the gene.

  An increase in gene expression can be achieved, for example, by increasing the copy number of the gene.

  Increasing the copy number of a gene can be achieved by introducing the target gene onto the chromosome into the host microorganism. Genes can be introduced into chromosomes by transposon or Mini-Mu random introduction onto chromosomes (JP-A-2-109985, US5,882,888 EP805867B1), or sequences that exist in multiple copies on chromosomal DNA. This can be achieved by utilizing the target for homologous recombination. As a sequence present in multiple copies on the chromosomal DNA, repetitive DNA and inverted repeats present at both ends of the transposon can be used. Alternatively, it is possible to introduce a gene onto a chromosome by using the Red driven integration method (WO2005 / 010175). It is also possible to introduce a gene onto a chromosome by transduction using a phage or a conjugation transfer vector. Further, as described in WO03 / 040373, it is also possible to introduce a gene by targeting a gene unnecessary for secretory production of a heterologous protein on a chromosome. In this way, one or more copies of the gene can be introduced into the target sequence.

  Confirmation of the introduction of the target gene on the chromosome was performed using Southern hybridization using a probe having a sequence complementary to all or part of the gene, or a primer prepared based on the sequence of the gene. It can be confirmed by PCR.

  An increase in the copy number of the gene can also be achieved by introducing a vector containing the target gene into the host bacterium. For example, a DNA fragment containing a target gene is linked to a vector that functions in the host bacterium to construct an expression vector for the gene, and the host bacterium is transformed with the expression vector to increase the copy number of the gene. be able to. As the vector, a vector capable of autonomous replication in a host bacterial cell can be used. The vector is preferably a multicopy vector. In order to select a transformant, the vector preferably has a marker such as an antibiotic resistance gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid. Specific examples of vectors capable of autonomous replication in coryneform bacteria include pHM1519 (Agric, Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 ( 1984)); plasmids having improved drug resistance genes; plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21 and pCRY2KE described in JP-A-2-72876 and US Pat. No. 5,185,262. PCRY2KX, pCRY31, pCRY3KE and pCRY3KX; plasmids pCRY2 and pCRY3 described in JP-A-1-91686; pAM330 described in JP-A-58-67679; pHM1519 described in JP-A-58-77895; PAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; JP-A-57-183799 Examples include pCG4 and pCG11 described in the gazette; pVK7 described in JP-A-10-215883, pVC7 described in JP-A-9-070291, and the like.

  Moreover, the increase in gene expression can be achieved by improving the transcription efficiency of the gene. Improvement of gene transcription efficiency can be achieved, for example, by replacing a promoter of a gene on a chromosome with a stronger promoter. By “stronger promoter” is meant a promoter that improves transcription of the gene over the native wild-type promoter. As a stronger promoter, for example, a known high expression promoter such as T7 promoter, trp promoter, lac promoter, tac promoter, PL promoter and the like can be used. As a more powerful promoter, a highly active promoter of a conventional promoter may be obtained by using various reporter genes. For example, the activity of the promoter can be increased by bringing the −35 and −10 regions in the promoter region closer to the consensus sequence (International Publication No. 00/18935). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

  Moreover, the increase in gene expression can be achieved by improving the translation efficiency of the gene. Improvement of gene translation efficiency can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)) of the gene on the chromosome with a stronger SD sequence. By “a stronger SD sequence” is meant an SD sequence in which the translation of mRNA is improved over the originally existing wild-type SD sequence. An example of a stronger SD sequence is RBS of gene 10 derived from phage T7 (Olins P. O. et al, Gene, 1988, 73, 227-235). Furthermore, substitution of several nucleotides in the spacer region between the RBS and the start codon, particularly the sequence immediately upstream of the start codon (5'-UTR), or insertion or deletion, contributes to mRNA stability and translation efficiency. It is known to have a great influence, and the translation efficiency of a gene can be improved by modifying them.

  In the present invention, sites that affect gene expression, such as a promoter, an SD sequence, and a spacer region between the RBS and the start codon, are collectively referred to as an “expression control region”. The expression regulatory region can be determined using a promoter search vector or gene analysis software such as GENETYX. These expression control regions can be modified by, for example, a method using a temperature sensitive vector or a Red driven integration method (WO2005 / 010175).

  The increase in gene expression can also be achieved by amplifying a regulator that increases the expression of the target gene, or by deleting or weakening a regulator that decreases the expression of the target gene.

  The methods for increasing gene expression as described above may be used alone or in any combination.

  The method for transformation is not particularly limited, and a conventionally known method can 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. 1970, 53, 159-162) and methods for introducing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE , 1997. Gene 1: 153-167). Alternatively, DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, can be made into protoplasts or spheroplasts that readily incorporate recombinant DNA into recombinant DNA. Introduction method (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM and Hopwood, OA 1978. Nature 274: 398-400; Hinnen, A Hicks, JB and Fink, GR 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933). In addition, transformation of coryneform bacteria can also be performed by an electric pulse method (Japanese Patent Laid-Open No. 2-207791).

  The increase in the expression of the target gene can be confirmed, for example, by confirming that the activity of the target protein expressed from the gene has increased. An increase in the activity of the target protein can be confirmed by measuring the activity of the protein. The increase in the activity of the Tat secretion apparatus can be confirmed, for example, by confirming that the amount of secretory production of a protein having a Tat-dependent signal peptide added to the N-terminus has increased. The amount of secretory production of a protein with a Tat-dependent signal peptide added to the N-terminus is increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more compared to the unmodified strain. preferable.

  In addition, the increase in the expression of the target gene can be confirmed, for example, by confirming that the transcription amount of the gene has increased, or by confirming that the amount of the target protein expressed from the gene has increased. it can.

  Confirmation that the transcription amount of the target gene has increased can be carried out by comparing the amount of mRNA transcribed from the same gene with an unmodified strain such as a wild strain or a parent strain. Methods for assessing the amount of mRNA include Northern hybridization, RT-PCR, etc. (Sambrook, J., et al., Molecular Cloning A Laboratory Manual / Third Edition, Cold spring Harbor Laboratory Press, Cold spring Harbor (USA ), 2001). The amount of mRNA is preferably increased 1.5 times or more, 2 times or more, or 3 times or more, compared to the unmodified strain, for example.

  Confirmation that the amount of the target protein has increased can be performed by Western blotting using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The amount of protein is preferably increased by 1.5 times or more, 2 times or more, or 3 times or more, for example, as compared to the unmodified strain.

<1-1-4> Gene construct for secretory expression of heterologous proteins Secretory proteins are generally translated as preproteins (also referred to as prepeptides) or preproproteins (also referred to as prepropeptides), and then It is known that it becomes a mature protein by processing. Specifically, secreted proteins are generally translated as preproteins or preproproteins, and then the signal peptide, which is the prepart, is cleaved by a protease (commonly called signal peptidase) to be converted into a mature protein or proprotein. Proprotein is further cleaved by protease to become mature protein. Therefore, in the method of the present invention, a signal peptide is used for secretory production of a heterologous protein. In the present invention, preproteins and preproproteins of secretory proteins are sometimes collectively referred to as “secreted protein precursors”. In the present invention, “signal peptide” (also referred to as “signal sequence”) refers to an amino acid sequence that is present at the N-terminus of a secretory protein precursor and is not normally present in a natural mature protein.

  The gene construct used in the present invention includes, in the 5 ′ to 3 ′ direction, a promoter sequence that functions in coryneform bacteria, a nucleic acid sequence that encodes a signal peptide that functions in coryneform bacteria, and a nucleic acid sequence that encodes a heterologous protein. . The nucleic acid sequence encoding the signal peptide may be linked downstream of the promoter sequence so that the signal peptide is expressed under the control of the promoter. The nucleic acid sequence encoding the heterologous protein may be linked downstream of the nucleic acid sequence encoding the signal peptide so that the heterologous protein is expressed as a fusion protein with the signal peptide. In addition, the gene construct used in the present invention has control sequences (operators, terminators, etc.) effective for expressing heterologous protein genes in coryneform bacteria at appropriate positions so that they can function. Also good.

  The promoter used in the present invention is not particularly limited as long as it is a promoter that functions in coryneform bacteria, and may be a promoter derived from coryneform bacteria or a promoter derived from a different species. The “promoter that functions in coryneform bacteria” refers to a promoter having promoter activity in coryneform bacteria. Specific examples of heterologous promoters include promoters derived from E. coli such as tac promoter, lac promoter, trp promoter, and araBAD promoter. Among them, a strong promoter such as tac promoter is preferable, and an inducible promoter such as araBAD promoter is also preferable.

  Examples of promoters derived from coryneform bacteria include promoters of cell surface proteins PS1, PS2 (also referred to as CspB), SlpA (also referred to as CspA), and promoters of various amino acid biosynthesis genes. Specific promoters for various amino acid biosynthesis genes include, for example, glutamate biosynthesis glutamate dehydrogenase gene, glutamine synthesis glutamine synthetase gene, lysine biosynthesis aspartokinase gene, threonine biosynthesis Homoserine dehydrogenase gene, isoleucine and valine biosynthesis acetohydroxy acid synthase gene, leucine biosynthesis 2-isopropylmalate synthase gene, proline and arginine biosynthesis glutamate kinase gene, histidine biosynthesis Phosphoribosyl-ATP pyrophosphorylase gene, aromatic amino acid biosynthetic deoxyarabinohepturonic acid phosphate (DAHP) synthase genes such as tryptophan, tyrosine and phenylalanine, such as inosinic acid and guanylic acid Examples include phosphoribosyl pyrophosphate (PRPP) amide transferase gene, inosinic acid dehydrogenase gene, and guanylate synthase gene promoter in nucleic acid biosynthetic systems.

  As a promoter, a highly active type of a conventional promoter may be obtained and used by using various reporter genes. For example, the activity of the promoter can be increased by bringing the −35 and −10 regions in the promoter region closer to the consensus sequence (International Publication No. 00/18935). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)). Furthermore, substitution of several nucleotides in the spacer region between the ribosome binding site (RBS) and the start codon, especially in the sequence immediately upstream of the start codon (5'-UTR), insertion or deletion makes the mRNA stable. It is known to greatly affect sex and translation efficiency, and these can be modified.

  The signal peptide used in the present invention is not particularly limited as long as it is a signal peptide that functions in a coryneform bacterium, and may be a signal peptide derived from a coryneform bacterium or a signal peptide derived from a different species. The “signal peptide that functions in coryneform bacteria” refers to a peptide that can be secreted by coryneform bacteria when linked to the N-terminus of the target protein.

Whether or not a signal peptide functions in coryneform bacteria can be confirmed by, for example, expressing the target protein by fusing it with the signal peptide and confirming whether the protein is secreted.

  The signal peptide used in the present invention is preferably a Tat-dependent signal peptide. “Tat system-dependent signal peptide” refers to a signal peptide recognized by the Tat system. Specifically, the “Tat system-dependent signal peptide” may be a peptide in which the protein is secreted by the Tat system secretion apparatus when linked to the N-terminus of the target protein.

Examples of Tat-dependent signal peptides include E. coli TorA protein (trimethylamine-N-oxide reductase) signal peptide, E. coli SufI protein (ftsI suppressor) signal peptide, Bacillus subtilis PhoD protein (phosphodiesterase) Signal peptide of LipA protein (lipoic acid synthase) of Bacillus subtilis, signal peptide of IMD protein (isomaltodextranase) of Arthrobacter globiformis. The amino acid sequences of these signal peptides are as follows.
TorA signal peptide: MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 19)
SufI signal peptide: MSLSRRQFIQASGIALCAGAVPLKASA (SEQ ID NO: 20)
PhoD signal peptide: MAYDSRFDEWVQKLKEESFQNNTFDRRKFIQGAGKIAGLSLGLTIAQS (SEQ ID NO: 21)
LipA signal peptide: MKFVKRRTTALVTTLMLSVTSLFALQPSAKAAEH (SEQ ID NO: 22)
IMD signal peptide: MMNLSRRTLLTTGSAATLAYALGMAGSAQA (SEQ ID NO: 23)

  The Tat system-dependent signal peptide has a twin arginine motif. Examples of the twin arginine motif include S / T-R-X-F-L-K (SEQ ID NO: 24) and R-R-X-#-# (#: hydrophobic residue) (SEQ ID NO: 25).

  Further, the Tat-dependent signal peptide may be a variant of the above signal peptide as long as it has a twin arginine motif and has a function as a Tat-dependent signal peptide. The above description regarding the wild-type ribosomal protein S1 and the variant of the gene encoding the same can be applied mutatis mutandis to the signal peptide and the gene encoding the same. Specifically, for example, the variant of the signal peptide is an amino acid sequence in which one or several amino acids at one or several positions are substituted, deleted, inserted or added in the amino acid sequence of the signal peptide. It may be a peptide having. The “one or several” in the signal peptide variants is specifically preferably 1 to 7, more preferably 1 to 5, further preferably 1 to 3, and particularly preferably 1 to 2. Means individual. In the present invention, “TorA signal peptide”, “SufI signal peptide”, “PhoD signal peptide”, “LipA signal peptide”, and “IMD signal peptide” have SEQ ID NOs: 19, 20, 21, 22 respectively. In addition to the peptides described in, and 23, variants thereof are included.

  “Function as a Tat-dependent signal peptide” refers to recognition by the Tat system. Specifically, the protein is secreted by the Tat secretion apparatus when linked to the N-terminus of the target protein. It may be a function.

  Whether a certain peptide has a function as a Tat-dependent signal peptide can be confirmed, for example, by confirming that the amount of secreted protein produced by adding the peptide to the N-terminus is increased by enhancement of the Tat-based secretion apparatus, This can be confirmed by confirming that the amount of secreted protein produced by adding a peptide to the N-terminus decreases due to a deficiency in the Tat secretion apparatus.

  The signal sequence is generally cleaved by a 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.

  Examples of the heterologous protein secreted and produced by the method of the present invention include bioactive proteins, receptor proteins, antigen proteins used as vaccines, and enzymes.

  Examples of the enzyme include transglutaminase, protein glutaminase, isomalt dextranase, protease, endopeptidase, exopeptidase, aminopeptidase, carboxypeptidase, collagenase, and chitinase. Examples of transglutaminase include actinomycetes such as Streptoverticillium mobaraense IFO 13819 (WO01 / 23591), Streptoverticillium cinnamoneum IFO 12852, Streptoverticillium griseocarneum IFO 12776, and Streptomyces lydicus (WO9606931) secretory fungi such as Oomycetes (WO9622366) Examples include transglutaminase. Examples of protein glutaminase include Chryseobacterium proteolyticum protein glutaminase (WO2005 / 103278). Examples of isomalt dextranase include Arthrobacter globiformis isomaltdextranase (WO2005 / 103278).

  Examples of the physiologically active protein include growth factors (growth factors), hormones, cytokines, and antibody-related molecules.

  As growth factors (growth factors), specifically, for example, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), transforming growth factor (Transforming growth factor (TGF), Nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF), Vascular endothelial growth factor (VEGF), Granulocyte Colony-stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (Erythropoietin; EPO), Thrombopoietin (TPO), acidic fibroblast growth factor (aFGF or FGF1), basic fibroblasts Examples include growth factor (basic fibroblast growth factor; bFGF or FGF2), keratinocyte growth factor (keratinocyto growth factor; KGF-1 or FGF7, KGF-2 or FGF10), and hepatocyte growth factor (HGF).

  Specific examples of hormones include insulin, glucagon, somatostatin, human growth hormone (hGH), parathyroid hormone (PTH), and calcitonin.

  Specific examples of cytokines include interleukin, interferon, and tumor necrosis factor (TNF).

  Note that growth factors (growth factors), hormones, and cytokines do not have to be strictly distinguished from each other. For example, the bioactive protein may belong to any one group selected from growth factors (growth factors), hormones, and cytokines, and belongs to a plurality of groups selected from them. Also good.

  Further, the physiologically active protein may be the whole protein or a part thereof. As a part of protein, the part which has physiological activity is mentioned, for example. Specific examples of the physiologically active moiety include a physiologically active peptide Teriparatide consisting of an N-terminal 34 amino acid residue of a mature parathyroid hormone (PTH).

The antibody-related molecule refers to a protein containing a molecular species consisting of a single domain selected from domains constituting a complete antibody or a combination of two or more domains. Domains constituting a complete antibody include VH, CH1, CH2, and CH3, which are heavy chain domains, and VL and CL, which are light chain domains. The antibody-related molecule may be a monomeric protein or a multimeric protein as long as it contains the above-described molecular species. When the antibody-related molecule is a multimeric protein, it may be a homomultimer composed of a single type of subunit or a heteromultimer composed of two or more types of subunits. Also good. Specific examples of antibody-related molecules include, for example, complete antibodies, Fab, F (ab ′), F (ab ′) ₂ , Fc, dimer consisting of heavy chain (H chain) and light chain (L chain) , Fc fusion protein, heavy chain (H chain), light chain (L chain), single chain Fv (scFv), sc (Fv) ₂ , disulfide bond Fv (sdFv), Diabody.

  The receptor protein is not particularly limited, and may be, for example, a receptor protein for a physiologically active protein or other physiologically active substance. Examples of other physiologically active substances include neurotransmitters such as dopamine. The receptor protein may also be an orphan receptor for which the corresponding ligand is not known.

  The antigenic protein used as a vaccine is not particularly limited as long as it can elicit an immune response, and may be appropriately selected according to the target of the assumed immune response.

  The genes encoding these proteins can be modified depending on the host used and to obtain the desired activity. For example, the genes encoding these proteins may be modified so that these proteins include additions, deletions, substitutions, etc. of one or several amino acids. The above description concerning the wild-type ribosomal protein S1 and the variant of the gene encoding it can be applied mutatis mutandis to the heterologous protein secreted and produced by the method of the present invention and the gene encoding the same. In addition, the genes encoding these proteins may be those obtained by replacing an arbitrary codon with an equivalent codon. For example, the genes encoding these proteins may be converted into optimal codons depending on the codon usage frequency of the host, if necessary.

  The N-terminal region of the heterologous protein finally obtained by the method of the present invention may or may not be the same as the natural protein. For example, the N-terminal region of the finally obtained heterologous protein may have one or several extra amino acids added or deleted compared to the natural protein. The “one or several” is different depending on the total length, structure, etc. of the target heterologous protein, but is preferably 1-20, more preferably 1-10, and still more preferably 1-5. Means individual.

  In addition, the heterologous protein that is secreted and produced may be a protein (proprotein) added with a prostructure. When the secreted heterologous protein is a proprotein, the finally obtained heterologous protein may or may not be a proprotein. That is, the proprotein may be cleaved from the prostructure to become a mature protein. The cleavage can be performed by, for example, a protease. In the case of using a protease, from the viewpoint of the activity of the finally obtained protein, it is generally preferable that the proprotein is cleaved at almost the same position as the natural protein, and cleaved at the same position as the natural protein. More preferably, a mature protein identical to the natural one is obtained. Thus, in general, specific proteases that cleave proproteins at positions that produce proteins identical to naturally occurring mature proteins are most preferred. However, as described above, the N-terminal region of the finally obtained heterologous protein may not be the same as the natural protein. For example, depending on the type of the heterologous protein produced, the purpose of use, and the like, a protein having a N-terminal that is one to several amino acids longer or shorter than the natural protein may have more appropriate activity. Proteases that can be used in the present invention include those obtained from a culture solution of microorganisms, such as a culture solution of actinomycetes, in addition to those commercially available such as Dispase (manufactured by Boehringer Mannheim). Such a protease can be used in an unpurified state, or may be used after being purified to an appropriate purity as required.

  The technique for introducing the gene construct used in the present invention into coryneform bacteria is not particularly limited. In the bacterium of the present invention, the gene construct used in the present invention may be present on a vector that autonomously proliferates outside the chromosome, such as a plasmid, or may be integrated on the chromosome. In constructing the bacterium of the present invention, the introduction of the genetic construct used in the present invention, the introduction of the mutant ribosomal protein S1 gene, and other modifications can be performed in any order.

  The gene construct used in the present invention can be introduced into a host using, for example, a vector containing the gene construct. The vector is not particularly limited as long as it can autonomously replicate in coryneform bacteria, and may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid. As the vector, for example, a plasmid derived from coryneform bacteria is preferable. Specific examples of vectors capable of autonomous replication in coryneform bacteria include pHM1519 (Agric, Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 ( 1984)); plasmids having improved drug resistance genes; plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21 and pCRY2KE described in JP-A-2-72876 and US Pat. No. 5,185,262. PCRY2KX, pCRY31, pCRY3KE and pCRY3KX; plasmids pCRY2 and pCRY3 described in JP-A-1-91686; pAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; JP-A-57-134500 Examples include pCG1 described; pCG2 described in JP-A-58-35197; pCG4 and pCG11 described in JP-A-57-183799, and the like.

  Artificial transposons can also be used. When a transposon is used, a heterologous protein gene is introduced onto the chromosome by homologous recombination or its own ability to transfer. In addition, as an introduction method using homologous recombination, for example, linear DNA, a plasmid containing a temperature-sensitive replication origin, a plasmid capable of conjugation transfer, or a suspension vector that does not have a replication origin that functions in the host is used. A method is mentioned. When a heterologous protein gene is introduced onto a chromosome, as long as the gene construct used in the present invention is constructed on the chromosome, any of a promoter sequence and a nucleic acid sequence encoding a signal peptide contained in the gene construct. Either or both may be present on the host chromosome. Specifically, for example, a nucleic acid sequence encoding a signal peptide connected to the downstream of the promoter sequence that is originally present on the host chromosome is used as it is, and downstream of the nucleic acid sequence encoding the signal peptide. By replacing only the connected gene with the target heterologous protein gene, the gene construct used in the present invention is constructed on the chromosome, and the bacterium of the present invention can be constructed.

  When expressing two or more kinds of proteins, the gene construct for secretory expression of each protein may be retained in the bacterium of the present invention so that the secretory expression of the target heterologous protein can be achieved. Specifically, for example, the gene construct for secretory expression of each protein may be all retained on a single expression vector, or all may be retained on a chromosome. Moreover, the gene construct for secretory expression of each protein may be separately held on a plurality of expression vectors, or may be separately held on a single or a plurality of expression vectors and on a chromosome. “When two or more types of proteins are expressed” means, for example, when two or more types of heterologous proteins are secreted and heteromultimeric proteins are secreted and produced.

  The method of introducing the gene construct used in the present invention into the coryneform bacterium 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)).

<1-2> Method for producing heterologous protein By culturing the bacterium of the present invention obtained as described above and expressing the heterologous protein, a large amount of the heterologous protein secreted outside the cells can be obtained.

  The bacterium of the present invention can be cultured according to a commonly used method and conditions. For example, the bacterium of the present invention 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 are appropriately used as necessary. The culture is carried out under aerobic conditions in a suitable range of pH 5.0 to 8.5 and 15 ° C. to 37 ° C., and cultured for about 1 to 7 days. In addition, culture conditions for L-amino acid production of coryneform bacteria and other conditions described in the protein production method using Sec and Tat signal peptides can be used (see WO01 / 23591 and WO2005 / 103278). . Further, when an inducible promoter is used for the expression of a heterologous protein, the culture can be performed by adding a promoter inducer to the medium. By culturing the bacterium of the present invention under such conditions, the target protein is produced in large quantities in the microbial cells and efficiently secreted outside the microbial cells. According to the method of the present invention, since the produced heterologous protein is secreted outside the cell body, for example, a protein that is generally lethal when accumulated in a large amount in a cell body of a microorganism such as transglutaminase has a lethal effect. Can be continuously produced without receiving

  The protein secreted into the medium by the method of 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. In some cases, the culture or culture supernatant may be used as it is. The protein secreted into the cell surface by the method of the present invention is the same as that secreted into the medium after being solubilized by methods well known to those skilled in the art, for example, by increasing the salt concentration or using a surfactant. And 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.

  Confirm that the target heterologous protein is secreted and produced by SDS-PAGE using the fraction containing the culture supernatant and / or cell surface as a sample and confirming the molecular weight of the separated protein band. Can do. In addition, the fraction containing the culture supernatant and / or the cell surface layer can be used as a sample to confirm by Western blot using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). It can also be confirmed by determining the N-terminal amino acid sequence of the target protein using a protein sequencer. It can also be confirmed by determining the mass of the target protein using a mass spectrometer. In addition, when the target heterologous protein has an enzyme or some measurable physiological activity, the fraction containing the culture supernatant and / or cell surface layer is used as a sample, and the enzyme activity or physiological activity of the target heterologous protein is measured. By measuring the activity, it can be confirmed that the target heterologous protein is secreted and produced.

<2> Coryneform bacterium having a specific mutation The present invention also relates to a coryneform bacterium modified to have a mutation in the ribosomal protein S1 gene, wherein the mutation is the amino acid at position 173 of the wild-type ribosomal protein S1. A coryneform bacterium is provided that is a mutation that replaces a residue with another amino acid residue. The coryneform bacterium may or may not have the ability to produce a heterologous protein. That is, the coryneform bacterium may or may not have a gene construct for secretory expression of a heterologous protein. With respect to the coryneform bacterium, the description about the “coryneform bacterium used in the method of the present invention” described above can be applied mutatis mutandis, except that it does not have to have a gene construct for secretory expression of a heterologous protein. The coryneform bacterium can be suitably used for secretory production of a heterologous protein, for example, by retaining a gene construct for secretory expression of the heterologous protein.

  The invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way.

Example 1: Construction of RpsA (E173G) mutant strain derived from C. glutamicum YDK010 strain (1) Acquisition of natural mutant strain having mutation in rpsA gene
Using the protransglutaminase expression plasmid pPKT-PTG1 described in WO2005 / 103278, the C. glutamicum YDK010 strain described in WO2004 / 029254 was transformed. PPKT-PTG1 is a vector for secretory expression of protransglutaminase (transglutaminase with prostructure), a promoter derived from the PS2 gene of C. glutamicum ATCC13869 strain, and an Escherichia coli operably linked downstream of the promoter. (E. coli) is a plasmid having a TorA signal peptide-derived DNA and a protransglutaminase gene derived from S. mobaraense linked to be expressed as a fusion protein with the signal peptide (WO2005 / 103278), The C. glutamicum YDK010 strain is a strain lacking the cell surface protein PS2 of C. glutamicum AJ12036 (FERM BP-734) (WO2004 / 029254). MM liquid medium containing 25 mg / l kanamycin (glucose 60 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 450 μg, biotin 450 μg, DL-methionine 0.15 g, calcium carbonate 50 g, adjusted to pH 7.0 with 1 L with water) Cultured for 48 hours.

  After culturing, a natural mutant strain in which a mutation was introduced into the rpsA gene was selected and named THM1 strain. The nucleotide sequence of the mutant rpsA gene possessed by the THM1 strain and the amino acid sequence of the mutant RpsA protein encoded by the same gene are shown in <SEQ ID NO: 01> and <SEQ ID NO: 02>. In the mutant rpsA gene of the THM1 strain, A at position 518 in the base sequence <SEQ ID NO: 03> of the rpsA gene of the YDK010 strain is mutated to G. Due to this mutation, in the mutant RpsA protein of the THM1 strain, the glutamic acid residue at position 173 in the amino acid sequence <SEQ ID NO: 04> of the RpsA protein encoded by the rpsA gene of the YDK010 strain is replaced with a glycine residue. This mutation was named RpsA (E173G) mutation.

(2) Construction of rpsA gene replacement vector encoding mutant RpsA (E173G)
C. glutamicum THM1 strain prepared using the primers described in <SEQ ID NO: 05> and <SEQ ID NO: 06> according to a conventional method (Saito, Miura method [Biochim. Biophys. Acta, 72, 619 (1963)]) Using this chromosomal DNA as a template, a region of about 2.4 kbp containing the rpsA gene (mutant rpsA gene) encoding mutant RpsA (E173G) was amplified by PCR. Pyrobest DNA polymerase (manufactured by Takara Bio Inc.) was used for the PCR reaction, and the reaction conditions followed the protocol recommended by the manufacturer.

  Next, the amplified DNA fragment of about 2.4 kbp was recovered by agarose gel electrophoresis using WizardR SV Gel and PCR Clean-Up System (Promega) and inserted into the Sma I site of pBS5T described in WO2006 / 057450. After that, it was introduced into a competent cell of E. coli JM109 (Takara Bio Inc.). A strain holding a plasmid in which a DNA fragment containing the mutant rpsA gene was cloned was obtained, and the plasmid was recovered from this to obtain a plasmid pBS5T-RpsA (E173G) in which the mutant rpsA gene was cloned. As a result of determining the nucleotide sequence of the inserted fragment, it was confirmed that the expected gene was constructed. The base sequence was determined using BigDyeR Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 3130 Genetic Analyzer (Applied Biosystems).

(3) Introduction of RpsA (E173G) mutation into YDK010 strain C. glutamicum YDK010 strain was transformed with the plasmid pBS5T-RpsA (E173G) constructed in Example 1 (2). Strains were selected from the obtained transformants according to the method described in WO2006 / 057450 to obtain a YDK010 :: RpsA (E173G) strain in which the wild-type rpsA gene on the chromosome was replaced with the mutant rpsA gene. Note that the YDK010 :: RpsA (E173G) strain can be reproducibly constructed using, for example, a mutant rpsA gene fragment obtained by genetic engineering without using the chromosomal DNA of the THM1 strain.

Example 2: Secretion expression of protransglutaminase using TorA signal sequence by RpsA (E173G) mutant
Using the protransglutaminase expression plasmid pPKT-PTG1 described in WO2005 / 103278, the YDK010 strain and the YDK010 :: RpsA (E173G) strain obtained in Example 1 (3) were transformed, and YDK010 / pPKT-PTG1 and YDK010 :: RpsA (E173G) / pPKT-PTG1 was obtained. Each transformant obtained was treated with MM liquid medium (glucose 60 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate containing 25 mg / l kanamycin. 0.03 g of Japanese product, 0.03 g of manganese sulfate pentahydrate, 450 μg of thiamine hydrochloride, 450 μg of biotin, 0.15 g of DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with water and adjusted to pH 7.0) at 30 ℃ And cultured for 48 hours. After completion of the culture, the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE and then stained with CBB R250 (Bio-Rad). As a result, the amount of protransglutaminase secretion in the YDK010 :: RpsA (E173G) strain was significantly improved as compared with the YDK010 strain (FIG. 1). From this, it became clear that the RpsA (E173G) mutation is an effective mutation that leads to an increase in secretion amount in protransglutaminase secretion using the TorA signal sequence derived from E. coli.

  As a result of comparing the amino acid sequence of the rpsA gene homologue of various corynebacterium bacteria in the vicinity of position 173, the glutamic acid residue at position 173 was found to be highly conserved among corynebacterium bacteria. Fig. 2 shows the alignment of the amino acid sequences of RpsA proteins of C. glutamicum THM1, C. glutamicum YDK010, C. glutamicum ATCC13869, C. glutamicum ATCC13032, C. efficiens YS-314, and C. stationis ATCC6872 The amino acid residue at position 173 is shown in a box. The rpsA gene is considered to be an essential gene in E. coli and the like, and it has been difficult to predict that mutations in amino acid residues having high conservation of such genes will lead to an increase in the secretion of heterologous proteins.

Example 3: Secretory expression of pro-structured protein glutaminase using TorA signal sequence by RpsA (E173G) mutant
Using the prostructure protein glutaminase expression plasmid pPKT-PPG described in WO2005 / 103278, YDK010 strain and YDK010 :: RpsA (E173G) strain obtained in Example 1 (3) were transformed respectively. pPKT-PPG and YDK010 :: RpsA (E173G) / pPKT-PPG were obtained. In addition, pPKT-PPG is a vector for secretory expression of protein glutaminase with a pro-structure, and is a promoter derived from the PS2 gene of C. glutamicum ATCC13869 strain, and E. coli ligated to express downstream of the promoter. A plasmid having a pro-structured protein glutaminase gene derived from Chryseobacterium proteolyticum linked to be expressed as a fusion protein with the DNA encoding the TorA signal peptide derived therefrom (WO2005 / 103278). Each transformant obtained was treated with MM liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate containing 25 mg / l kanamycin. 0.03 g of Japanese product, 0.03 g of manganese sulfate pentahydrate, 450 μg of thiamine hydrochloride, 450 μg of biotin, 0.15 g of DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with water and adjusted to pH 7.0) at 30 ℃ And cultured for 72 hours. After completion of the culture, the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE and then stained with CBB R250 (Bio-Rad). As a result, YDK010 :: RpsA (E173G) strain significantly improved the amount of secreted protein glutaminase secreted compared to the YDK010 strain (FIG. 3). From this, it was revealed that the RpsA (E173G) mutation is an effective mutation that leads to an increase in the secretion amount even in the secretion of pro-structured protein glutaminase using the TorA signal sequence derived from E. coli.

Example 4: Secretion expression of isomaltdextranase using IMD signal sequence by RpsA (E173G) mutant
Each of YDK010 strain and YDK010 :: RpsA (E173G) strain obtained in Example 1 (3) was transformed with the isomaltdextranase expression plasmid pPKI-IMD described in WO2005 / 103278, and YDK010 / pPKI-IMD and YDK010 :: RpsA (E173G) / pPKI-IMD were obtained. PPKI-IMD is a vector for secretory expression of isomaltdextranase, a promoter derived from the PS2 gene of C. glutamicum ATCC13869 strain, an IMD gene derived from Arthrobacter globiformis that is operably linked downstream of the promoter ( (Including the coding region of the IMD signal sequence) (WO2005 / 103278). Each transformant obtained was treated with MM liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate containing 25 mg / l kanamycin. 0.03 g of Japanese product, 0.03 g of manganese sulfate pentahydrate, 450 μg of thiamine hydrochloride, 450 μg of biotin, 0.15 g of DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with water and adjusted to pH 7.0) at 30 ℃ And cultured for 72 hours. After completion of the culture, the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE and then stained with CBB R250 (Bio-Rad). As a result, the YDK010 :: RpsA (E173G) strain had a significantly improved isomaltdextranase secretion amount compared to the YDK010 strain (FIG. 4). From this, it was revealed that the RpsA (E173G) mutation is an effective mutation that leads to an improvement in the amount of isomaltdextranase secretion using an IMD signal sequence derived from A. globiformis.

Example 5: Combination effect of RpsA (E173G) mutation and Tat secretion apparatus amplification
YDK010 / pPKT-PTG1 and YDK010 :: RpsA (E173G) / pPKT-PTG1 constructed in Example 2 were constructed in Example 3 using pVtatABC which is an amplification plasmid of the Tat secretion apparatus described in WO2005 / 103278. YDK010 / pPKT-PPG and YDK010 :: RpsA (E173G) / pPKT-PPG, YDK010 / pPKI-IMD and YDK010 :: RpsA (E173G) / pPKI-IMD constructed in Example 4 were transformed, respectively, and the Tat system Secretion device amplified strains YDK010 / pPKT-PTG1 / pVtatABC, YDK010 :: RpsA (E173G) / pPKT-PTG1 / pVtatABC, YDK010 / pPKT-PPG / pVtatABC, YDK010 :: RpsA (E173G) / pPKT-PPG / pVtatABC, YDK010 / pPKI-IMD / pVtatABC, YDK010 :: RpsA (E173G) / pPKI-IMD / pVtatABC were obtained. Each transformant obtained was treated with MM liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, phosphate, containing 25 mg / l kanamycin and 5 mg / l chloramphenicol. Potassium dihydrogen 1.5 g, iron sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 450 μg, biotin 450 μg, DL-methionine 0.15 g, calcium carbonate 50 g, 1 L with water Each of the cells was cultured at 30 ° C. for 72 hours. After completion of the culture, the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE and then stained with CBB R250 (Bio-Rad). After staining, the band intensity of the target protein was quantified using image analysis software “Multi Gauge Ver3.0” (FUJIFILM). The band intensity of each sample was calculated as a relative value to the band intensity when the target protein was expressed in the YDK010 strain introduced with pVC7. As a result, in each of the expression strains, the secretion amount of each target protein was significantly improved in the strain introduced with pVtatABC as compared with the control strain introduced with pVC7. In addition, compared to the strains in which each of the RpsA (E173G) mutation and Tat secretion apparatus amplification were made alone, the secretion production of each target protein was remarkably improved in the strain combining these modifications. (FIGS. 5, 6, 7). From this, it was possible to dramatically improve the amount of protein secreted by the Tat system by combining the RpsA (E173G) mutation and Tat system secretion apparatus amplification.

Example 6: Effect of RpsA (E173G) mutation on protein secretion by Sec system
Using the protransglutaminase expression plasmid pPKSPTG1 described in WO01 / 23591, the C. glutamicum YDK010 strain and YDK010 :: RpsA (E173G) strain are transformed to obtain YDK010 / pPKSPTG1 and YDK010 :: RpsA (E173G) / pPKSPTG1 It was. PPKSPTG1 is a vector for secretory expression of protransglutaminase (transglutaminase with prostructure), a promoter derived from the PS2 gene of C. glutamicum ATCC13869 strain, and C. ammoniagenes operably linked downstream of the promoter. This is a plasmid having a DNA encoding a signal peptide derived from SlpA of the ATCC6872 strain and a protransglutaminase gene derived from S. mobaraense ligated to be expressed as a fusion protein with the signal peptide (WO01 / 23591). Each transformant obtained was treated with MM liquid medium (glucose 60 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate containing 25 mg / l kanamycin. 0.03 g of Japanese product, 0.03 g of manganese sulfate pentahydrate, 450 μg of thiamine hydrochloride, 450 μg of biotin, 0.15 g of DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with water and adjusted to pH 7.0) at 30 ℃ And cultured for 48 hours. After completion of the culture, the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE and then stained with CBB R250 (Bio-Rad). As a result, in the YDK010 :: RpsA (E173G) strain, the amount of protransglutaminase secretion was decreased as compared to the parent strain YDK010 (FIG. 8). From this, it was found that the RpsA (E173G) mutation is a mutation having a negative effect of decreasing the secretion amount in protein secretion by the Sec system. From this, it was confirmed that the RpsA (E173G) mutation is an effective mutation that specifically improves the secretion amount in protein secretion by the Tat system.

  According to the present invention, a heterologous protein can be efficiently secreted and produced.

[Explanation of Sequence Listing]
SEQ ID NO: 1: Nucleotide sequence of mutant rpsA gene of C. glutamicum THM1 strain SEQ ID NO: 2: Amino acid sequence of mutant RpsA protein of C. glutamicum THM1 strain SEQ ID NO: 3: Nucleotide sequence of rpsA gene of C. glutamicum YDK010 strain Number 4: amino acid sequence of RpsA protein of C. glutamicum YDK010 strain SEQ ID NO: 5, 6: primer SEQ ID NO: 7: base sequence of rpsA gene of C. glutamicum ATCC13032 sequence SEQ ID NO: 8: amino acid of RpsA protein of C. glutamicum ATCC13032 strain SEQ ID NO: 9: nucleotide sequence of rpsA gene of C. efficiens YS-314 strain SEQ ID NO: 10: amino acid sequence of RpsA protein of C. efficiens YS-314 strain SEQ ID NO: 11: nucleotide sequence of rpsA gene of C. stationis ATCC6872 strain SEQ ID NO: 12: Amino acid sequence of RpsA protein of C. stationis ATCC6872 SEQ ID NO: 13: Base sequence of tatA gene of C. glutamicum ATCC13032 SEQ ID NO: 14: TatA protein of C. glutamicum ATCC13032 Mino acid sequence SEQ ID NO: 15: nucleotide sequence of tatB gene of C. glutamicum ATCC13032 sequence SEQ ID NO: 16: amino acid sequence of TatB protein of C. glutamicum ATCC13032 strain SEQ ID NO: 17: nucleotide sequence sequence number of tatC gene of C. glutamicum ATCC13032 strain 18: amino acid sequence of TatC protein of C. glutamicum ATCC13032 strain SEQ ID NO: 19: amino acid sequence of TorA signal peptide SEQ ID NO: 20: amino acid sequence of SufI signal peptide SEQ ID NO: 21: amino acid sequence of PhoD signal peptide SEQ ID NO: 22: LipA signal peptide SEQ ID NO: 23: IMD signal peptide amino acid sequence SEQ ID NO: 24, 25: Twin arginine motif amino acid sequence SEQ ID NO: 26: C. glutamicum ATCC13869 cspB gene base sequence SEQ ID NO: 27: C. glutamicum ATCC13869 cspB Amino acid sequence of the protein encoded by the gene

Claims (18)

A method for producing a heterologous protein comprising culturing a coryneform bacterium having a gene construct for secretory expression of a heterologous protein and recovering the secreted heterologous protein,
The coryneform bacterium is modified so as to have a mutation in the ribosomal protein S1 gene that improves the secretory production of a heterologous protein;
The method wherein the gene construct comprises, in 5 ′ to 3 ′ direction, a promoter sequence that functions in coryneform bacteria, a nucleic acid sequence that encodes a signal peptide that functions in coryneform bacteria, and a nucleic acid sequence that encodes a heterologous protein.   The method according to claim 1, wherein the mutation is a mutation that improves the secretory production amount of the heterologous protein more than twice as compared with a control strain having no mutation in the ribosomal protein S1 gene.   The method according to claim 1 or 2, wherein the mutation is a mutation that substitutes the amino acid residue at position 173 of the wild-type ribosomal protein S1 with another amino acid residue.   The method according to claim 3, wherein the substitution of the amino acid residue is a substitution of a glutamic acid residue with a glycine residue.   The method according to any one of claims 1 to 4, wherein the signal peptide is a Tat-dependent signal peptide.   The method according to claim 5, wherein the Tat-dependent signal peptide is selected from the group consisting of a TorA signal peptide, a SufI signal peptide, a PhoD signal peptide, a LipA signal peptide, and an IMD signal peptide.   The method according to claim 5 or 6, wherein the coryneform bacterium is further modified so that expression of one or more genes selected from genes encoding a Tat secretion apparatus is increased.   The method according to claim 7, wherein the gene encoding the Tat secretion apparatus is selected from the group consisting of a tatA gene, a tatB gene, a tatC gene, and a tatE gene.   The method according to claim 1, wherein the coryneform bacterium is a genus Corynebacterium.   The method according to claim 9, wherein the coryneform bacterium is Corynebacterium glutamicum.   The method according to claim 10, wherein the coryneform bacterium is a modified strain derived from Corynebacterium glutamicum AJ12036 (FERM BP-734).   The method according to any one of claims 1 to 11, wherein the coryneform bacterium is a coryneform bacterium in which the activity of a cell surface protein is reduced. A coryneform bacterium modified to have a mutation in the ribosomal protein S1 gene,
A coryneform bacterium, wherein the mutation is a mutation that replaces the amino acid residue at position 173 of the wild-type ribosomal protein S1 with another amino acid residue.   The coryneform bacterium according to claim 13, wherein the amino acid residue substitution is a glutamic acid residue to glycine residue substitution.   The coryneform bacterium according to claim 13 or 14, which is a genus Corynebacterium.   The coryneform bacterium according to claim 15, which is Corynebacterium glutamicum.   The coryneform bacterium according to claim 16, which is a modified strain derived from Corynebacterium glutamicum AJ12036 (FERM BP-734).   The coryneform bacterium according to any one of claims 13 to 17, wherein the activity of the cell surface protein is reduced.