Protein secretion production method

Abstract

The present invention addresses the problem of providing a method for secreting and producing a heterologous protein by developing a novel technique for improving the secreting and producing of a heterologous protein by coryneform bacteria. The present invention relates to a method for secreting and producing a heterologous protein, comprising culturing a coryneform bacterium which has the ability to secrete and produce a heterologous protein and which has been modified such that the activity of an Mdh protein is reduced, and secreting and producing the heterologous protein.

Description

Technical Field

[0001] The present invention relates to a method for secretory production of heterologous proteins. Background Art

[0002] Regarding the secretory production of heterologous proteins by microorganisms, there have been reports on secretory production of heterologous proteins by Bacillus bacteria (Non-Patent Document 1), methanol-assimilating yeast Pichia pastoris (Non-Patent Document 2), filamentous fungi of the genus Aspergillus (Non-Patent Documents 3 and 4), etc.

[0003] In addition, attempts have been made to produce heterologous proteins by secretion from coryneform bacteria. Regarding the secretion production of heterologous proteins by coryneform bacteria, reports have included the secretion of nucleases and lipases by Corynebacterium glutamicum (hereinafter also abbreviated as C. glutamicum) (Patent Document 1, Non-Patent Document 5), the secretion of proteases such as subtilisin (Non-Patent Document 6), the secretion of proteins using the signal peptides of the cell surface proteins PS1 and PS2 (also known as CspB) of coryneform bacteria (Patent Document 2), the secretion of fibronectin-binding protein using the signal peptide of PS2 (CspB) (Non-Patent Document 7), the secretion of protransglutaminase using the signal peptide of PS2 (CspB) or SlpA (also known as CspA) (Patent Document 3), the secretion of proteins using a mutant secretion system (Patent Document 4), and the secretion of protransglutaminase by mutant strains (Patent Document 5). In addition, known technologies for increasing the secretory production of heterologous proteins by coryneform bacteria include: reducing the activity of cell surface proteins (Patent Documents 6 and 7), reducing the activity of penicillin-binding proteins (Patent Document 6), enhancing the expression of genes encoding metallopeptidase (Patent Document 7), introducing mutations into the ribosomal protein S1 gene (Patent Document 8), and expressing heterologous proteins by inserting an amino acid sequence containing Gln-Glu-Thr between the signal peptide and the heterologous protein (Patent Document 9).

[0004] The general protein secretion pathway is a pathway that is widely present from prokaryotes to eukaryotes and is known as the "Sec system". However, a protein secretion pathway that is completely different from the Sec system has recently been discovered in the thylakoid membrane of plant cell chloroplasts (non-patent document 8). With regard to this new secretion pathway, since arginine-arginine sequences are commonly present in the signal sequences of proteins secreted by the above-mentioned pathway (non-patent document 8), this new secretion pathway has been named the "Tat system" (Twin-Arginine Translocation system). It is known that in the Sec system, proteins are secreted in a state before forming a higher structure, while in the Tat system, proteins are secreted after forming a higher structure in the cell through the cell membrane (non-patent document 9). For rod-shaped bacteria, secretory production of proteins using Tat-dependent signal peptides has also been reported (patent documents 8 and 10).

[0005] Malate dehydrogenase (malate dehydrogenase) is the oxidoreductase that catalyzes the reaction that malic acid is converted into oxaloacetic acid and / or its reverse reaction.Similar to many other bacteria, C.glutamicum has two kinds of malate dehydrogenases, i.e. the malate dehydrogenase (Mdh) of the cytoplasmic type encoded by the mdh gene and the malate:quinone-oxidoreductase (apple tree:quinone oxidoreductase, Mqo) of the transmembrane type encoded by the mqo gene, and these two kinds of malate dehydrogenases act synergistically.Under common culture conditions, Mqo bears the major part of the reaction that malic acid is converted into oxaloacetic acid, and Mdh only plays a function when the concentration of oxaloacetic acid is low and the concentration of malic acid is high.About Corynebacterium glutamicum, it is believed that the mqo gene deletion strain cannot grow in basal medium, but the mdh gene deletion strain grows similarly to wild-type strain, so Mqo can compensate for the function of Mdh (non-patent literature 10).

[0006] The following are several findings regarding the relationship between the mdh gene and substance production in Corynebacterium glutamicum. Specifically, it is known that mdh gene deletion improves isobutanol production (Non-Patent Document 11), that enhanced Mdh activity improves L-amino acid production (Patent Document 11), and that enhanced Mdh activity improves succinate production (Patent Document 12).

[0007] However, the relationship between Mdh and the secretory production of heterologous proteins in coryneform bacteria remains unclear.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: U.S. Patent 4965197

[0011] Patent Document 2: Japanese Patent No. 6-502548

[0012] Patent Document 3: Japanese Patent No. 4320769

[0013] Patent Document 4: Japanese Patent Application Laid-Open No. 11-169182

[0014] Patent Document 5: Japanese Patent No. 4362651

[0015] Patent Document 6: WO2013 / 065869

[0016] Patent Document 7: WO2013 / 065772

[0017] Patent Document 8: WO2013 / 118544

[0018] Patent Document 9: WO2013 / 062029

[0019] Patent Document 10: Japanese Patent No. 4730302

[0020] Patent Document 11: Japanese Patent Application Laid-Open No. 2003-144161

[0021] Patent Document 12: Japanese Patent No. 4760121

[0022] Non-patent literature

[0023] Non-patent document 1: Microbiol. rev., 57, 109-137 (1993)

[0024] Non-patent document 2: Biotechnol., 11, 905-910 (1993)

[0025] Non-patent document 3: Biotechnol., 6, 1419-1422 (1988)

[0026] Non-patent document 4: Biotechnol., 9, 976-981 (1991)

[0027] Non-patent document 5: J. Bacteriol., 174, 1854-1861 (1992)

[0028] Non-patent document 6: Appl. Environ. Microbiol., 61, 1610-1613 (1995)

[0029] Non-patent document 7: Appl. Environ. Microbiol., 63, 4392-4400 (1997)

[0030] Non-patent document 8: EMBO J., 14, 2715-2722 (1995)

[0031] Non-patent literature 9: J. Biol. Chem., 25;273(52), 34868-74(1998)

[0032] Non-patent document 10: J. Bacteriol., 182, 6884-6891 (2000)

[0033] Non-patent document 11: Appl. Environ. Microb., 77, 3300-3310 (2011) Summary of the Invention

[0034] Technical problem to be solved by the invention

[0035] The object of the present invention is to develop a new technology for improving secretory production of heterologous proteins using coryneform bacteria, thereby providing a method for secretory production of heterologous proteins using coryneform bacteria.

[0036] Technical means to solve the problem

[0037] The present inventors conducted intensive studies to solve the above-mentioned technical problems and, as a result, found that the secretory production ability of coryneform bacteria is improved by modifying coryneform bacteria so as to reduce the activity of the Mdh protein, thereby completing the present invention.

[0038] That is, the present invention can be described as follows.

[0039] [1] A method for producing a heterologous protein, comprising:

[0040] Cultivating a coryneform bacterium having a gene construct for secretory expression of a heterologous protein; and

[0041] Recover the heterologous protein produced by secretion,

[0042] The coryneform bacterium is modified so that the activity of the Mdh protein is reduced compared to an unmodified strain,

[0043] The gene construct comprises, in the 5' to 3' direction: a promoter sequence that functions in coryneform bacteria, a nucleic acid sequence encoding a signal peptide that functions in coryneform bacteria, and a nucleic acid sequence encoding a heterologous protein.

[0044] The heterologous protein is expressed as a fusion protein with the signal peptide.

[0045] [2] The method according to the above (specifically, the method described in [1]), wherein

[0046] The Mdh protein is a protein described in the following (a), (b) or (c): (a) a protein comprising the amino acid sequence shown in SEQ ID NO. 40; (b) a protein comprising an amino acid sequence containing substitutions, deletions, insertions and / or additions of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO. 40, and having malate dehydrogenase activity; (c) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO. 40, and having malate dehydrogenase activity.

[0047] [3] The method according to the above (specifically, the method described in [1] or [2]), wherein

[0048] By reducing the expression of the mdh gene or disrupting the mdh gene, the activity of the Mdh protein is reduced.

[0049] [4] The method according to the above (specifically, any one of [1] to [3]), wherein

[0050] The activity of the Mdh protein is reduced by partially or completely deleting the amino acid sequence of the Mdh protein.

[0051] [5] The method according to the above (specifically, the method according to [4]), wherein:

[0052] At least the portion of the amino acid sequence of the Mdh protein corresponding to positions 313 to 328 of SEQ ID NO. 40 is deleted.

[0053] [6] According to the above method (specifically, the method described in [4]), wherein

[0054] At least the C-terminal 16 residues of the amino acid sequence of the Mdh protein are deleted.

[0055] [7] The method according to the above (specifically, any one of [4] to [6]), wherein

[0056] The deletion is generated by:

[0057] Deletion of part or all of the coding region of the mdh gene, introduction of a stop codon into the coding region of the mdh gene, frameshift in the coding region of the mdh gene, or a combination thereof.

[0058] [8] The method according to the above (specifically, any one of [1] to [7]), wherein

[0059] The coryneform bacterium is further modified so as to retain the phoS gene encoding the mutant PhoS protein.

[0060] [9] According to the above method (specifically, the method described in [8]), wherein

[0061] The mutation is a mutation in which the amino acid residue corresponding to the tryptophan residue at position 302 of SEQ ID NO. 2 in the wild-type PhoS protein is replaced with an amino acid residue other than aromatic amino acid and histidine.

[0062]

[10] According to the above method (specifically, the method described in [9]), wherein

[0063] The amino acid residues other than the aromatic amino acids and histidine are: lysine residues, alanine residues, valine residues, serine residues, cysteine ​​residues, methionine residues, aspartic acid residues or asparagine residues.

[0064]

[11] The method according to the above (specifically, the method described in [9] or

[10] ), wherein

[0065] The wild-type PhoS protein is the protein described in (a), (b) or (c) below:

[0066] (a) a protein comprising the amino acid sequence shown in any one of SEQ ID NOs. 2 to 7;

[0067] (b) a protein comprising an amino acid sequence having a substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence represented by any one of SEQ ID NOs. 2 to 7, and having a function as a sensor kinase of the PhoRS system;

[0068] (c) A protein comprising an amino acid sequence having 90% or greater identity with the amino acid sequence represented by any one of SEQ ID NOs. 2 to 7 and functioning as a sensor kinase of the PhoRS system.

[0069]

[12] The method according to any one of the above-mentioned steps [1] to

[11] , wherein:

[0070] The signal peptide is a Tat-dependent signal peptide.

[0071]

[13] According to the above method (specifically, the method described in

[12] ), wherein

[0072] The Tat-dependent signal peptide is any one 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.

[0073]

[14] The method according to the above (specifically, the method described in

[12] or

[13] ), wherein

[0074] The coryneform bacterium is further modified so that the expression of one or more genes selected from genes encoding the Tat secretion system is increased compared to an unmodified strain.

[0075]

[15] According to the above method (specifically, the method described in

[14] ), wherein

[0076] The genes encoding the Tat secretion system include tatA gene, tatB gene, tatC gene and tatE gene.

[0077]

[16] The method according to any one of the above-mentioned steps [1] to

[11] , wherein:

[0078] The signal peptide is a Sec-dependent signal peptide.

[0079]

[17] According to the above method (specifically, the method described in

[16] ), wherein

[0080] The Sec-dependent signal peptide is any one signal peptide selected from the group consisting of a PS1 signal peptide, a PS2 signal peptide, and a SlpA signal peptide.

[0081]

[18] The method according to any one of the above-mentioned steps [1] to

[17] , wherein:

[0082] The gene construct further comprises a nucleic acid sequence encoding an amino acid sequence containing Gln-Glu-Thr between a nucleic acid sequence encoding a signal peptide that functions in coryneform bacteria and a nucleic acid sequence encoding a heterologous protein.

[0083]

[19] According to the above method (specifically, the method described in

[18] ), wherein

[0084] The gene construct further comprises a nucleic acid sequence encoding an amino acid sequence for enzyme cleavage between the nucleic acid sequence encoding the amino acid sequence containing Gln-Glu-Thr and the nucleic acid sequence encoding the heterologous protein.

[0085]

[20] The method according to any one of the above-mentioned steps [1] to

[19] , wherein:

[0086] The coryneform bacteria are bacteria of the genus Corynebacterium.

[0087]

[21] According to the above method (specifically, the method described in

[20] ), wherein

[0088] The coryneform bacteria is Corynebacterium glutamicum.

[0089]

[22] According to the above method (specifically, the method described in

[21] ), wherein

[0090] The coryneform bacteria are a modified strain derived from Corynebacterium glutamicum AJ12036 (FERM BP-734) or a modified strain derived from Corynebacterium glutamicum ATCC 13869.

[0091]

[23] The method according to any one of the above-mentioned steps [1] to

[22] , wherein:

[0092] The coryneform bacteria are coryneform bacteria in which the number of molecules per cell of cell surface protein is reduced compared to an unmodified strain.

[0093] Effects of the Invention

[0094] According to the present invention, heterologous proteins can be secreted and produced efficiently. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 These are photographs showing the results of SDS-PAGE when protein L (Protein L, the antibody-binding domain of Protein L fused to the signal peptide of CspA) was expressed in the Corynebacterium glutamicum YDK010::phoS(W302C) strain and its mdh gene-deficient strain.

[0096] Figure 2 The photographs show the results of SDS-PAGE when protein L (Protein L, the antibody-binding domain of Protein L fused to the signal peptide of CspA) was expressed in the Corynebacterium glutamicum YDK010::phoS(W302C) strain and a strain in which the start and end codons of the mdh gene were inserted.

[0097] Figure 3 The photographs show the SDS-PAGE results when CspB6Xa-LFABP (LFABP fused with the signal peptide of CspB, the N-terminal sequence of mature CspB, and the Factor Xa protease recognition sequence) was expressed in the Corynebacterium glutamicum YDK010::phoS(W302C) strain and its mdh gene start and end codon insertion strain.

[0098] Figure 4These are photographs showing the results of SDS-PAGE when PTG (transglutaminase proenzyme fused with the signal peptide of CspA) was expressed in the Corynebacterium glutamicum YDK010::phoS(W302C) strain and its mdh gene start and end codon inserted strain.

[0099] Figure 5 These are photographs showing the results of SDS-PAGE when PTG (transglutaminase proenzyme fused with a TorA signal peptide) was expressed in the Corynebacterium glutamicum YDK010::phoS(W302C) strain and its mdh gene start and end codon insertion strain. DETAILED DESCRIPTION

[0100] Hereinafter, the present invention will be described in detail.

[0101] The method of the present invention is a method for producing a heterologous protein, comprising: culturing a coryneform bacterium having a gene construct for secretory expression of the heterologous protein; and recovering the heterologous protein produced by secretion, wherein the coryneform bacterium is modified in such a way that the activity of the Mdh protein is reduced.

[0102] <1> Coryneform bacteria used in the method of the present invention

[0103] The coryneform bacteria used in the methods of the present invention are coryneform bacteria that harbor a gene construct for secretory expression of a heterologous protein and have been modified to reduce the activity of the Mdh protein. It should be noted that the coryneform bacteria used in the methods of the present invention are referred to as "the bacteria of the present invention" or "the coryneform bacteria of the present invention." Furthermore, the gene construct for secretory expression of a heterologous protein harbored by the bacteria of the present invention is also referred to as "the gene construct used in the present invention." Furthermore, the bacteria of the present invention or the strain used to construct them are also referred to as "hosts."

[0104] <1-1> Coryneform bacteria capable of secreting and producing heterologous proteins

[0105] The coryneform bacteria of the present invention have the ability to secrete and produce heterologous proteins. The coryneform bacteria of the present invention possess at least a gene construct for secretory expression of a heterologous protein (the gene construct used in the present invention), and thus possess the ability to secrete and produce heterologous proteins. Specifically, the coryneform bacteria of the present invention can possess the ability to secrete and produce heterologous proteins by possessing a gene construct for secretory expression of a heterologous protein, or by combining a gene construct for secretory expression of a heterologous protein with other properties. Examples of other properties include modifications that reduce the activity of the Mdh protein and the other properties described below.

[0106] In the present invention, the so-called "secreted" protein refers to the protein being transferred to the outside of the bacterial cell (extracellularly). Examples of the outside of the bacterial cell (extracellularly) include the culture medium and the cell surface. That is, secreted protein molecules may, for example, be present in the culture medium, on the cell surface, or both in the culture medium and on the cell surface. In other words, when a protein is "secreted", it is not limited to the case where all molecules of the protein are completely free in the culture medium. For example, it also includes the case where all molecules of the protein are present on the cell surface, or a portion of the protein molecules are present in the culture medium and the remaining molecules are present on the cell surface.

[0107] That is, in the present invention, the "ability to secrete and produce heterologous proteins" refers to the ability of the bacteria of the present invention, when cultured in a culture medium, to secrete heterologous proteins into the culture medium and / or on the bacterial surface, accumulating them to a level that allows them to be recovered from the culture medium and / or on the bacterial surface. The amount of accumulation, for example, in the culture medium is preferably 10 μg / L or greater, more preferably 1 mg / L or greater, particularly preferably 100 mg / L or greater, and even more preferably 1 g / L or greater. Furthermore, the amount of accumulation, for example, on the bacterial surface, can be such that, when the heterologous protein is recovered from the bacterial surface and suspended in a liquid equal in volume to the culture medium, the concentration of the heterologous protein in the suspension is preferably 10 μg / L or greater, more preferably 1 mg / L or greater, and particularly preferably 100 mg / L or greater. It should be noted that, in the present invention, the term "protein" secreted and produced encompasses peptides such as oligopeptides and polypeptides.

[0108] In the present invention, a "heterologous protein" refers to a protein that is exogenous to the rod-shaped bacteria that express and secrete it. For example, a heterologous protein can be a protein of microbial origin, a plant-derived protein, an animal-derived protein, a viral-derived protein, or even a protein whose amino acid sequence is artificially designed. In particular, a heterologous protein can be a human-derived protein. A heterologous protein can be a monomeric protein or a multimeric protein. A multimeric protein refers to a protein that can exist as a multimer comprising two or more subunits. In a multimer, the subunits can be linked by covalent bonds such as disulfide bonds; by non-covalent bonds such as hydrogen bonds and hydrophobic interactions; or by a combination of covalent and non-covalent bonds. In a multimer, one or more intermolecular disulfide bonds are preferably present. A multimer can be a homomultimer comprising a single type of subunit or a heteromultimer comprising two or more subunits. It should be noted that when a multimeric protein is a heteromultimer, at least one of the subunits comprising the multimer may be a heterologous protein. That is, all subunits may be heterologous, or only some may be heterologous. The heterologous protein may be a naturally secreted protein or a naturally non-secreted protein, preferably a naturally secreted protein. Furthermore, the heterologous protein may be a naturally secreted Tat-dependent protein or a naturally secreted Sec-dependent protein. Specific examples of "heterologous proteins" are described below.

[0109] The heterologous protein to be produced may be a single species, or two or more species. Furthermore, 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. That is, "secretory production of a heterologous protein" encompasses both the secretory production of all subunits that comprise the target heterologous protein and the secretory production of only a portion of the subunits that comprise the target heterologous protein.

[0110] Rod-shaped bacteria are aerobic, Gram-positive bacteria. Examples of rod-shaped bacteria include bacteria of the genera Corynebacterium, Brevibacterium, and Microbacterium. Advantages of using rod-shaped bacteria include: compared to filamentous fungi, yeast, and Bacillus bacteria, which have been used for secretory production of heterologous proteins, the amount of protein secreted externally by the bacteria is extremely small, allowing for simplified or omitted purification processes when secreting heterologous proteins; and they grow well in simple culture media containing sugars, ammonia, and inorganic salts, resulting in superior culture media costs, culture methods, and culture productivity.

[0111] Specific examples of coryneform bacteria include the following species.

[0112] Corynebacterium acetoacidophilum

[0113] Corynebacterium acetoglutamicum

[0114] Corynebacterium alkanolyticum

[0115] Corynebacterium callunae

[0116] Corynebacterium crenatum

[0117] Corynebacterium glutamicum

[0118] Corynebacterium lilium

[0119] Corynebacterium melassecola

[0120] Corynebacterium thermoaminogenes (Corynebacterium efficiens)

[0121] Corynebacterium herculis

[0122] Brevibacterium divaricatum (Corynebacterium glutamicum)

[0123] Brevibacterium flavum (Corynebacterium glutamicum)

[0124] Brevibacterium immariophilum

[0125] Brevibacterium lactofermentum (Corynebacterium glutamicum)

[0126] Brevibacterium roseum

[0127] Brevibacterium saccharolyticum

[0128] Brevibacterium thiogenitalis

[0129] Corynebacterium ammoniagenes (Corynebacteriumstationis)

[0130] Brevibacterium album

[0131] Brevibacterium cerinum

[0132] Microbacterium ammoniaphilum

[0133] Specific examples of coryneform bacteria include the following strains.

[0134] Corynebacterium acetoaceticum ATCC 13870

[0135] Corynebacterium acetoglutamicum ATCC 15806

[0136] Corynebacterium ATCC 21511

[0137] Corynebacterium sclerotiorum ATCC 15991

[0138] Corynebacterium scutellariae AS1.542

[0139] Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERMBP-734

[0140] Corynebacterium lilii ATCC 15990

[0141] Corynebacterium molasses ATCC 17965

[0142] Effective Corynebacterium (Thermoammoniagenic Corynebacterium) AJ12340 (FERM BP-1539)

[0143] Corynebacterium ligusticum ATCC 13868

[0144] Brevibacterium difidum (Corynebacterium glutamicum) ATCC 14020

[0145] Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205)

[0146] Immature Brevibacterium ATCC 14068

[0147] Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869

[0148] Brevibacterium roseum ATCC 13825

[0149] Brevibacterium saccharolyticum ATCC 14066

[0150] Brevibacterium thiogenes ATCC 19240

[0151] Corynebacterium ammoniagenes (Corynebacterium stagnantum) ATCC 6871, ATCC 6872

[0152] Brevibacterium albus ATCC 15111

[0153] Brevibacterium cereus ATCC 15112

[0154] Microbacterium ammoniaphilum ATCC 15354

[0155] It should be noted that, with regard to the genus Corynebacterium, also include the bacterium that was previously classified as Brevibacterium and is now classified into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). In addition, with regard to Corynebacterium stagnate, also include the bacterium that was previously classified as Corynebacterium ammoniagenes and was reclassified into Corynebacterium stagnate by base sequence analysis of 16S rRNA etc. (Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)).

[0156] These strains can be obtained and distributed, for example, from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852, PO Box 1549, Manassas, VA 20108, United States of America). Each strain is assigned an accession number corresponding to the strain, and the accession number can be used to obtain and distribute the strain (see http: / / www.atcc.org / ). The accession number corresponding to each strain is recorded in the catalog of the American Type Culture Collection. In addition, these strains can be obtained, for example, from the depository where the strain is stored.

[0157] Especially from wild-type strain Corynebacterium glutamicum ATCC 13869 as streptomycin (Sm) resistance mutant strain Corynebacterium glutamicum AJ12036 (FERM BP-734), compared with its parent strain (wild-type strain), it is predicted that there is a mutation in the gene of the function related to the secretion of protein, and the secretion production capacity of protein is roughly 2 to 3 times as much as the accumulation under the most suitable culture conditions, which is very high and suitable as a host bacterium (WO2002 / 081694). AJ12036 was originally deposited as an international deposit by the Institute of Microbial Technology, Institute of Industrial Technology (now: Patent Organism Depository Center (NITE IPOD), independent administrative agency, product evaluation technology base organization, zip code: 292-0818, address: Room 120, 2-5-8, Kamashira, Kazusa, Kisarazu-shi, Chiba Prefecture, Japan) on March 26, 1984, and was given the deposit number FERM BP-734.

[0158] In addition, Thermoammoniagenics AJ12340 (FERM BP-1539) was originally deposited as an international deposit on March 13, 1987 by the Institute of Microbial Technology, Industrial Technology Agency (now: Patent Organism Depository Center, Independent Administrative Institution, Product Evaluation Technology Foundation, Postal Code: 292-0818, Address: Room 120, 2-5-8 Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan) and was assigned the deposit number FERM BP-1539. In addition, Brevibacterium flavum AJ12418 (FERM BP-2205) was originally deposited as an international deposit on December 24, 1988, at the Institute of Microbial Technology, Industrial Technology Agency (now the Patent Organism Depository, Japan, 2-5-8 Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan) and assigned the accession number FERM BP-2205.

[0159] Alternatively, the coryneform bacteria described above can be used as parent strains, and strains with enhanced secretory protein production capabilities can be selected by mutation or genetic recombination methods, and used as hosts. For example, strains with enhanced secretory protein production capabilities can be selected by treatment with ultraviolet light or a chemical mutagen such as N-methyl-N'-nitrosoguanidine.

[0160] Furthermore, if a strain modified in such a manner that the cell surface protein is not produced by such a strain is used as a host, the purification of the heterologous protein secreted in the culture medium or on the bacterial cell surface becomes easy, and is therefore particularly preferred. Such modification can be carried out by introducing mutations into the coding region of the cell surface protein on the chromosome or its expression regulatory region by a mutation method or a gene recombination method. As a coryneform bacterium modified in such a manner that the cell surface protein is not produced, a deletion type strain of the cell surface protein PS2 of Corynebacterium glutamicum AJ12036 (FERM BP-734), i.e., Corynebacterium glutamicum YDK010 strain (WO2004 / 029254), can be enumerated.

[0161] The gene construct used in the present invention is introduced into the above-mentioned coryneform bacteria and retained in the coryneform bacteria, thereby obtaining coryneform bacteria with the ability to secrete and produce heterologous proteins. That is, the bacteria of the present invention, for example, can be a modified strain derived from the coryneform bacteria as described above. Specifically, the bacteria of the present invention, for example, can also be a modified strain derived from Corynebacterium glutamicum AJ12036 (FERM BP-734) or a modified strain derived from Corynebacterium glutamicum ATCC 13869. It should be noted that the modified strain derived from Corynebacterium glutamicum AJ12036 (FERM BP-734) also conforms to the modified strain derived from Corynebacterium glutamicum ATCC 13869. The gene construct used in the present invention and its introduction method will be described below.

[0162] <1-2> Decreased activity of Mdh protein

[0163] The bacteria of the present invention are modified to reduce the activity of the Mdh protein. Specifically, the bacteria of the present invention are modified to reduce the activity of the Mdh protein compared to an unmodified strain. The activity of the Mdh protein can be reduced, for example, compared to Corynebacterium glutamicum AJ12036 (FERM BP-734) or Corynebacterium glutamicum ATCC 13869. More specifically, the bacteria of the present invention can be modified to reduce the expression of the mdh gene or to disrupt the mdh gene. By modifying the coryneform bacteria to reduce the activity of the Mdh protein, the ability of the bacteria to secrete and produce heterologous proteins can be improved, that is, the secretory production of heterologous proteins by the bacteria can be enhanced.

[0164] The bacteria of the present invention can be obtained by modifying a coryneform bacterium that has the ability to secrete and produce a heterologous protein to reduce the activity of the Mdh protein. Furthermore, the bacteria of the present invention can be obtained by imparting the ability to secrete and produce a heterologous protein after modifying the coryneform bacterium to reduce the activity of the Mdh protein. In the present invention, the modifications used to construct the bacteria of the present invention can be performed in any order. It should be noted that, before modification to reduce the activity of the Mdh protein, the strain used to construct the bacteria of the present invention may or may not secrete and produce the heterologous protein, assuming it harbors a gene construct for secretory expression of the heterologous protein. That is, the bacteria of the present invention may, for example, be modified to reduce the activity of the Mdh protein to acquire the ability to secrete and produce a heterologous protein. Specifically, for example, the bacteria of the present invention may be derived from a strain that, before modification to reduce the activity of the Mdh protein, was unable to secrete and produce the heterologous protein even if it harbored a gene construct for secretory expression of the heterologous protein, and then modified to reduce the activity of the Mdh protein to enable secretory production of the heterologous protein.

[0165] Hereinafter, the Mdh protein and the mdh gene encoding it will be described. The Mdh protein is a cytoplasmic malate dehydrogenase (EC 1.1.1.37). "Malate dehydrogenase" refers to a protein (enzyme) that has the activity of catalyzing the reaction of converting malate into oxaloacetate in the presence of an electron acceptor and / or the reaction of converting oxaloacetate into malate in the presence of a potential donor. This activity is also called "malate dehydrogenase activity". As electron acceptors, NAD can be cited. + As the potential donor, NADH can be mentioned.

[0166] The base sequence of the mdh gene possessed by Corynebacterium and the amino acid sequence of the Mdh protein encoded by the sequence can be obtained, for example, from public databases such as NCBI (National Center for Biotechnology Information). The base sequence of the mdh gene of Corynebacterium glutamicum ATCC 13869 and the amino acid sequence of the Mdh protein encoded by the gene are shown in SEQ ID NO.39 and 40, respectively. That is, the mdh gene can be, for example, a gene having the base sequence shown in SEQ ID NO.39. In addition, the Mdh protein can be, for example, a protein having the amino acid sequence shown in SEQ ID NO.40. It should be noted that the expression "having an (amino acid or base) sequence" includes, unless otherwise specified, the situations of "comprising an (amino acid or base) sequence" and "being composed of an (amino acid or base) sequence".

[0167] As long as its original function is maintained, the mdh gene can be a variant of the mdh gene listed above (for example, a gene having the base sequence shown in SEQ.ID.NO.39). Similarly, as long as its original function is maintained, the Mdh protein can be a variant of the Mdh protein listed above (for example, a protein having the amino acid sequence shown in SEQ.ID.NO.40). Sometimes such variants that maintain their original function are also referred to as "conservative variants." In the present invention, the term "mdh gene" is not limited to the mdh gene listed above, but includes conservative variants thereof. Similarly, the term "Mdh protein" is not limited to the Mdh protein listed above, but includes conservative variants thereof. As conservative variants, for example, homologs and artificially modified bodies of the mdh gene and Mdh protein listed above can be cited.

[0168] "Maintaining the original function" means that the variant of a gene or protein has a function (such as activity or property) corresponding to the function (such as activity or property) of the original gene or protein. That is, "maintaining the original function" can mean that, in the mdh gene, the gene variant can encode a protein (i.e., the Mdh protein) that maintains the original function. In addition, "maintaining the original function" can mean that, in the Mdh protein, the variant of the protein has the function of being an Mdh protein (for example, the function of a protein comprising the amino acid sequence shown in SEQ.ID.NO.40). In addition, "maintaining the original function" can mean that, in the Mdh protein, the variant of the protein has malate dehydrogenase activity. That is, "functioning as an Mdh protein" can specifically mean malate dehydrogenase activity.

[0169] Malate dehydrogenase activity (oxalate acetate production direction) can be detected by + The enzyme is incubated with a substrate (malate) in the presence of NADH and the production of NADH is measured. Malate dehydrogenase activity (malate production direction) can be measured by incubating the enzyme with a substrate (oxaloacetate) in the presence of NADH and measuring the decrease in NADH.

[0170] Conservative variants are exemplified below.

[0171] Homologs of the mdh gene or Mdh protein can be easily obtained from public databases, for example, by performing a BLAST search or a FASTA search using the base sequences of the mdh gene or the amino acid sequences of the Mdh protein exemplified above as queries. Furthermore, homologs of the mdh gene can be obtained, for example, by PCR using the chromosome of a coryneform bacterium as a template and oligonucleotides prepared based on the known base sequences of the mdh gene as primers.

[0172] The mdh gene, as long as its original function is maintained, may encode a protein having an amino acid sequence resulting from substitution, deletion, insertion, and / or addition of one or more amino acids at one or more positions in the amino acid sequence of the Mdh protein exemplified above (e.g., the amino acid sequence set forth in SEQ.ID.NO.40). It should be noted that the term "one or more" varies depending on the position of the amino acid residues in the protein's three-dimensional structure and the type of amino acid residues. Specifically, it may be, for example, 1 to 50, 1 to 40, or 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0173] The substitution, deletion, insertion, and / or addition of one or more amino acids is a conservative mutation that maintains the normal function of the protein. A representative example of a conservative mutation is a conservative substitution. Conservative substitutions refer to substitutions between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid; substitutions between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid; substitutions between Gln and Asn when the substitution site is a polar amino acid; substitutions between Lys, Arg, and His when the substitution site is a basic amino acid; substitutions between Asp and Glu when the substitution site is an acidic amino acid; and substitutions between Ser and Thr when the substitution site is a hydroxyl-containing amino acid. Specific examples of substitutions considered to be conservative substitutions include substitutions of Ala with Ser or Thr, Arg with Gln, His or Lys, Asn with Glu, Gln, Lys, His or Asp, Asp with Asn, Glu or Gln, Cys with Ser or Ala, Gln with Asn, Glu, Lys, His, Asp or Arg, Glu with Gly, Asn, Gln, Lys or Asp, Gly with Pro, and His with Asn, Lys, Gln, Arg or Ty. r, Ile to Leu, Met, Val or Phe, Leu to Ile, Met, Val or Phe, Lys to Asn, Glu, Gln, His or Arg, Met to Ile, Leu, Val or Phe, Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala, Thr to Ser or Ala, Trp to Phe or Tyr, Tyr to His, Phe or Trp, and Val to Met, Ile or Leu. In addition, such amino acid substitutions, deletions, insertions or additions include those caused by naturally occurring mutations (mutants or variants) such as individual differences in bacteria from which the gene is derived and differences in species.

[0174] In addition, as long as the original function is maintained, the mdh gene can be a gene encoding a protein having the following amino acid sequence: an amino acid sequence that has an overall identity of 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more to the amino acid sequence of the above-mentioned Mdh protein (for example, the amino acid sequence shown in SEQ.ID.NO.40).

[0175] Alternatively, as long as its original function is maintained, the mdh gene may be a DNA that hybridizes under stringent conditions with a complementary sequence to the base sequence of the mdh gene exemplified above (e.g., the base sequence shown in SEQ ID NO. 39), or with a probe that can be prepared from the complementary sequence. "Stringent conditions" refers to conditions that form specific hybrids and prevent nonspecific hybridization. For example, conditions in which DNAs with high identity, for example, 80% or greater, preferably 90% or greater, more preferably 95% or greater, even more preferably 97% or greater, and particularly preferably 99% or greater, hybridize, while DNAs with lower identity do not hybridize. Alternatively, conditions in which washing is performed once, and preferably two to three times, at a salt concentration and temperature comparable to those used for conventional Southern blot hybridization washes, i.e., 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, and more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0176] The probe can be, for example, a portion of a complementary sequence to a gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on the base sequence of a known gene as primers and a DNA fragment containing this base sequence as a template. As a probe, for example, a DNA fragment of approximately 300 bp in length can be used. In such cases, hybridization washing conditions include: 50°C, 2×SSC, 0.1% SDS.

[0177] Furthermore, the mdh gene may be a gene having a base sequence obtained by replacing any codon in the base sequence of the mdh gene exemplified above or a conservative variant thereof with a codon equivalent thereto.

[0178] It should be noted that the so-called "identity" between amino acid sequences refers to the identity between amino acid sequences calculated by blastp using the default settings of Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). In addition, the so-called "identity" between base sequences refers to the identity between base sequences calculated by blastn using the default settings of Scoring Parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0179] It should be noted that the above description of variants of the genes and proteins is applicable to any protein such as PhoRS protein, cell surface protein, Tat secretion system, and the secretory heterologous protein produced in the present invention, as well as the genes encoding them.

[0180] <1-3>Other properties

[0181] As long as the bacterium of the present invention can secrete and produce heterologous proteins, it can have the desired properties. For example, the activity of cell surface proteins in the bacterium of the present invention can be reduced (WO2013 / 065869, WO2013 / 065772, WO2013 / 118544, WO2013 / 062029). In addition, the bacterium of the present invention can be modified in a manner that the activity of penicillin-binding proteins is reduced (WO2013 / 065869). In addition, the bacterium of the present invention can be modified in a manner that the expression of the gene encoding the metallopeptidase is improved (WO2013 / 065772). In addition, the bacterium of the present invention can be modified in a manner that has a mutant ribosomal protein S1 gene (mutant rpsA gene) (WO2013 / 118544). In addition, the bacterium of the present invention can be modified in a manner that has a mutant phoS gene (WO2016 / 171224). Furthermore, the bacteria of the present invention can be modified to reduce the activity of the RegX3 protein (WO2018 / 074578). Furthermore, the bacteria of the present invention can be modified to reduce the activity of the HrrSA system (WO2018 / 074579). Furthermore, the bacteria of the present invention can be modified to increase the activity of the Tat secretion system. These properties or modifications can be utilized individually or in appropriate combinations.

[0182] <1-3-1> Introduction of mutant phoS gene

[0183] The bacteria of the present invention may be modified so as to retain a mutant phoS gene. "Maintaining a mutant phoS gene" may also be referred to as "having a mutant phoS gene" or "having a mutation in the phoS gene." Furthermore, "maintaining a mutant phoS gene" may also be referred to as "having a mutant PhoS protein" or "having a mutation in the PhoS protein."

[0184] The following describes the phoS gene and PhoS protein. The phoS gene encodes the PhoS protein, which serves as the sensor kinase of the PhoRS system. The PhoRS system is a two-component control system that responds to phosphate deficiency in the environment. The PhoRS system comprises the sensor kinase PhoS, encoded by the phoS gene, and the response regulator PhoR, encoded by the phoR gene.

[0185] In the present invention, a PhoS protein with a "specific mutation" is also referred to as a "mutant PhoS protein," and the gene encoding it is also referred to as a "mutant phoS gene." In other words, a "mutant phoS gene" is a phoS gene with a "specific mutation." Furthermore, in the present invention, a PhoS protein without a "specific mutation" is also referred to as a "wild-type PhoS protein," and the gene encoding it is also referred to as a "wild-type phoS gene." In other words, a "wild-type phoS gene" is a phoS gene without a "specific mutation." It should be noted that the term "wild-type" is used for convenience in distinguishing it from "mutant type," and is not limited to naturally occurring proteins as long as they do not have a "specific mutation." "Specific mutations" are described below.

[0186] Examples of wild-type phoS genes include phoS genes from coryneform bacteria. Specific examples of phoS genes from coryneform bacteria include phoS genes from Corynebacterium glutamicum strain YDK010, Corynebacterium glutamicum strain ATCC13032, Corynebacterium glutamicum strain ATCC14067, Corynebacterium sclerotium, Corynebacterium crenulate, and Corynebacterium effusum. The base sequence of the phoS gene from Corynebacterium glutamicum strain YDK010 is shown in SEQ ID NO. 1. Furthermore, the amino acid sequences of the wild-type PhoS proteins encoded by these phoS genes are shown in SEQ ID NOs. 2 to 7, respectively.

[0187] A wild-type phoS gene can be a variant of the wild-type phoS gene as described above, as long as it does not have a "specific mutation" and maintains its original function. Similarly, a wild-type PhoS protein can be a variant of the wild-type PhoS protein as described above, as long as it does not have a "specific mutation" and maintains its original function. That is, the term "wild-type phoS gene" is not limited to the wild-type phoS gene as described above, but includes conservative variants thereof that do not have a "specific mutation." Similarly, the term "wild-type PhoS protein" is not limited to the wild-type PhoS protein as described above, but includes conservative variants thereof that do not have a "specific mutation." The description of conservative variants of the Mdh protein and mdh gene described above applies to variants of the wild-type PhoS protein and wild-type phoS gene. For example, a wild-type phoS gene can be a gene encoding a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added at one or more positions in the amino acid sequence as long as it does not have a "specific mutation" and maintains its original function. Furthermore, for example, the wild-type phoS gene may be a gene encoding a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more identical to the entire amino acid sequence, as long as it does not have a "specific mutation" and maintains its original function.

[0188] It should be noted that the phrase "maintaining the original function" may mean that the protein variant functions as a PhoS protein (for example, the function of a protein consisting of the amino acid sequences set forth in SEQ ID NOs. 2 to 7) in the wild-type PhoS protein. Furthermore, the phrase "maintaining the original function" may mean that the protein variant functions as a sensor kinase in the PhoRS system in the wild-type PhoS protein. Specifically, the phrase "functioning as a PhoS protein" may mean that the protein variant functions as a sensor kinase in the PhoRS system. Specifically, the phrase "functioning as a sensor kinase in the PhoRS system" may mean that the protein variant conjugates with the response regulator, the PhoR protein, and initiates a response to phosphate deficiency in the environment. More specifically, the phrase "functioning as a sensor kinase in the PhoRS system" may mean that the protein variant senses phosphate deficiency in the environment, undergoes autophosphorylation, and activates the PhoR protein through phosphate transfer.

[0189] Whether a variant of the PhoS protein functions as a sensor kinase of the PhoRS system can be determined, for example, by introducing a gene encoding the variant into a phoS gene-deficient strain of coryneform bacteria and confirming whether the variant complements the responsiveness to phosphate deficiency. Complementation of the responsiveness to phosphate deficiency can be detected, for example, by increasing growth and proliferation under phosphate-deficient conditions or by inducing expression of a gene known to be induced under phosphate-deficient conditions (J. Bacteriol., 188, 724-732 (2006)). PhoS gene-deficient strains of coryneform bacteria, for example, phoS gene-deficient strains of Corynebacterium glutamicum YDK010 and Corynebacterium glutamicum ATCC13032 can be used.

[0190] In the wild-type PhoS protein, the autophosphorylated histidine residue is preferably conserved. That is, the conservative mutation is preferably generated in an amino acid residue other than the autophosphorylated histidine residue. The "autophosphorylated histidine residue" refers to the histidine residue at position 276 of the wild-type PhoS protein. Furthermore, the wild-type PhoS protein preferably has, for example, the conserved sequence of the wild-type PhoS protein as described above. That is, the conservative mutation is preferably generated in an amino acid residue that is not conserved in the wild-type PhoS protein as described above.

[0191] The mutant PhoS protein has a "specific mutation" in the amino acid sequence of the wild-type PhoS protein as described above.

[0192] In other words, a mutant PhoS protein may be identical to the wild-type PhoS protein or a conservative variant thereof as described above, except for the "specific mutation." Specifically, for example, a mutant PhoS protein may have the amino acid sequence set forth in SEQ ID NOs. 2 to 7, except for the "specific mutation." Specifically, for example, a mutant PhoS protein may have an amino acid sequence containing one or more amino acid substitutions, deletions, insertions, and / or additions within the amino acid sequence set forth in SEQ ID NOs. 2 to 7, except for the "specific mutation." Specifically, for example, a mutant PhoS protein may have an amino acid sequence that is 80% or greater, preferably 90% or greater, more preferably 95% or greater, even more preferably 97% or greater, and particularly preferably 99% or greater, relative to the amino acid sequence set forth in SEQ ID NOs. 2 to 7, except for the "specific mutation."

[0193] Alternatively, a mutant PhoS protein may be a variant having a "specific mutation" in the wild-type PhoS protein as described above, and further comprising a conservative mutation at a position other than the "specific mutation." Specifically, for example, a mutant PhoS protein may be a protein having an amino acid sequence having a "specific mutation" in the amino acid sequence set forth in SEQ ID NOs. 2 to 7, and further comprising one or more amino acid substitutions, deletions, insertions, and / or additions at a position other than the "specific mutation."

[0194] The mutant phoS gene is not particularly limited as long as it encodes the mutant PhoS protein as described above.

[0195] Hereinafter, the "specific mutation" possessed by the mutant PhoS protein will be described.

[0196] The "specific mutation" is not particularly limited as long as it modifies the amino acid sequence of the wild-type PhoS protein as described above and is effective for secretory production of heterologous proteins.

[0197] "Specific mutations" are preferably mutations that increase the secretion production of heterologous proteins. The so-called "increasing the secretion production of heterologous proteins" refers to a rod-shaped bacterium (modified strain) modified in a manner having a mutant phoS gene, which can secrete and produce a larger amount of heterologous protein than an unmodified strain. The so-called "unmodified strain" refers to a control strain that does not have a mutation in the phoS gene, that is, a control strain that does not have a mutant phoS gene, for example, it can be a wild-type strain or a parent strain. The so-called "secretion and production of a larger amount of heterologous protein than an unmodified strain" means that as long as the secretion production of heterologous protein is increased compared to the unmodified strain, there is no particular limitation. For example, as the accumulation amount in the culture medium and / or on the surface of the bacteria, the heterologous protein can be secreted and produced in an amount preferably 1.1 times or more of that of the unmodified strain, more preferably 1.2 times or more, further preferably 1.3 times or more, further preferably 2 times or more, and particularly preferably 5 times or more. Furthermore, the phrase "secretory production of a heterologous protein in a greater amount than that of an unmodified strain" means that the heterologous protein cannot be detected when the culture supernatant of the unmodified strain is subjected to SDS-PAGE and stained with CBB, whereas the heterologous protein can be detected when the culture supernatant of the modified strain is subjected to SDS-PAGE and stained with CBB. It should be noted that the phrase "increasing the secretory production of a heterologous protein" does not necessarily mean increasing the secretory production of all heterologous proteins; rather, it suffices to increase the secretory production of a specific heterologous protein as set as the target for secretory production. Specifically, the phrase "increasing the secretory production of a heterologous protein" can refer to, for example, increasing the secretory production of a heterologous protein as described in the Examples.

[0198] Whether a certain mutation increases the secretory production of a heterologous protein can be confirmed, for example, by preparing a modified strain based on a coryneform bacterium strain, wherein the strain is modified to have a gene encoding a PhoS protein having the mutation, quantifying the amount of the heterologous protein secreted and produced when the modified strain is cultured in a medium, and comparing the amount of the heterologous protein secreted and produced when the strain before modification (unmodified strain) is cultured in a medium.

[0199] As a change in the amino acid sequence, the substitution of amino acid residues is preferred. That is, the "specific mutation" is preferably the substitution of any amino acid residue in the wild-type PhoS protein with another amino acid residue. The amino acid residue substituted according to the "specific mutation" can be one residue, two residues, or a combination of two or more residues. The amino acid residue substituted according to the "specific mutation" is preferably an amino acid residue other than the autophosphorylated histidine residue. The amino acid residue substituted according to the "specific mutation" is more preferably an amino acid residue in the HisKA domain other than the autophosphorylated histidine residue. The so-called "autophosphorylated histidine residue" refers to the histidine residue at position 276 of the wild-type PhoS protein. The so-called "HisKA domain" refers to the region comprising amino acid residues at positions 266 to 330 of the wild-type PhoS protein. The amino acid residue substituted according to the "specific mutation" is particularly preferably the tryptophan residue (W302) at position 302 of the wild-type PhoS protein.

[0200] In the above mutations, examples of the substituted amino acid residues include K (Lys), R (Arg), H (His), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), T (Thr), P (Pro), F (Phe), W (Trp), Y (Tyr), C (Cys), M (Met), D (Asp), E (Glu), N (Asn), and Q (Gln), other than the original amino acid residues. As the substituted amino acid residue, for example, an amino acid residue that increases the secretion production of the heterologous protein can be selected.

[0201] When W302 is substituted, the amino acid residue after the substitution includes amino acid residues other than aromatic amino acids and histidine. Specific examples of "amino acid residues other than aromatic amino acids and histidine" include K (Lys), R (Arg), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), T (Thr), P (Pro), C (Cys), M (Met), D (Asp), E (Glu), N (Asn), and Q (Gln). More specific examples of "amino acid residues other than aromatic amino acids and histidine" include K (Lys), A (Ala), V (Val), S (Ser), C (Cys), M (Met), D (Asp), and N (Asn).

[0202] It should be noted that the so-called "specific mutation" in the phoS gene refers to a mutation in the base sequence that causes the "specific mutation" described above to occur in the encoded PhoS protein.

[0203] In the present invention, the term "amino acid residue at position X of the wild-type PhoS protein" refers to the amino acid residue equivalent to the amino acid residue at position X of SEQ ID NO. 2. For example, "W302" refers to the amino acid residue equivalent to the tryptophan residue at position 302 of SEQ ID NO. 2. The positions of the amino acid residues are relative, and their absolute positions may vary due to deletions, insertions, or additions of amino acids. For example, if an amino acid residue is deleted or inserted at a position closer to the N-terminus than position X in the wild-type PhoS protein composed of the amino acid sequence of SEQ ID NO. 2, then the amino acid residue at position X-1 or X+1, respectively, counting from the N-terminus, is considered the "amino acid residue at position X of the wild-type PhoS protein." Specifically, for example, in the amino acid sequences of the wild-type PhoS proteins set forth in SEQ ID NOs. 2 to 7, "W302" refers to the tryptophan residues at positions 302, 302, 302, 321, 275, and 286, respectively. Furthermore, in the amino acid sequences of the wild-type PhoS proteins set forth in SEQ ID NOs. 2 to 7, "the histidine residue at position 276 of the wild-type PhoS protein (autophosphorylated histidine residue)" refers to the histidine residues at positions 276, 276, 276, 295, 249, and 260, respectively. Furthermore, in the amino acid sequences of the wild-type PhoS proteins shown in SEQ ID NOs. 2 to 7, the term "region comprising amino acid residues 266 to 330 of the wild-type PhoS protein (HisKA domain)" refers to regions comprising amino acid residues 266 to 330, 266 to 330, 266 to 330, 285 to 349, 239 to 303, and 250 to 314, respectively.

[0204] It should be noted that "W302" as referred to herein generally refers to a tryptophan residue, but does not necessarily have to be a tryptophan residue. That is, when the wild-type PhoS protein has an amino acid sequence other than the amino acid sequence set forth in SEQ ID NOs. 2 to 7, "W302" may not be a tryptophan residue. Therefore, for example, "a mutation in which W302 is substituted with a cysteine ​​residue" is not limited to mutations in which "W302" is a tryptophan residue and the tryptophan residue is substituted with a cysteine ​​residue. It also includes mutations in which "W302" is K (Lys), R (Arg), H (His), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), T (Thr), P (Pro), F (Phe), Y (Tyr), M (Met), D (Asp), E (Glu), N (Asn), or Q (Gln), in which the amino acid residue is substituted with a cysteine ​​residue. The same applies to other mutations.

[0205] In the amino acid sequence of any PhoS protein, which amino acid residue is "the amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO. 2" can be determined by aligning the amino acid sequence of the arbitrary PhoS protein with the amino acid sequence of SEQ ID NO. 2. Alignment can be performed, for example, using known gene analysis software. Specific examples of such software include DNASIS manufactured by HITACHI SOLUTIONS and GENETYX manufactured by GENETYX (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24(1), 72-96, 1991; Barton GJ et al., Journal of Molecular Biology, 198(2), 327-37, 1987).

[0206] Mutant phoS genes can be obtained, for example, by modifying a wild-type phoS gene so that the encoded PhoS protein has the aforementioned "specific mutation." The wild-type phoS gene used as the source of modification can be obtained, for example, by cloning from an organism harboring the wild-type phoS gene or by chemical synthesis. Furthermore, mutant phoS genes can be obtained independently of wild-type phoS genes. For example, mutant phoS genes can be directly obtained by chemical synthesis. The resulting mutant phoS gene can be further modified and utilized.

[0207] Gene modification can be carried out by known methods. For example, by site-specific mutagenesis, a target mutation is introduced into a target site of DNA. As site-specific mutagenesis, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)), a method using phage (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)) can be cited.

[0208] Hereinafter, a method for modifying coryneform bacteria so as to have a mutant phoS gene will be described.

[0209] Modification of coryneform bacteria to harbor a mutant phoS gene can be achieved by introducing the mutant phoS gene into the coryneform bacteria. Furthermore, modification of coryneform bacteria to harbor a mutant phoS gene can be achieved by introducing the aforementioned "specific mutation" into the phoS gene on the chromosome of the coryneform bacteria. Introduction of mutations into chromosomal genes can be achieved through natural mutation, mutagen treatment, or genetic engineering.

[0210] The method for introducing the mutant phoS gene into coryneform bacteria is not particularly limited. In the bacteria of the present invention, the mutant phoS gene is maintained and can be expressed under the control of a promoter that functions in coryneform bacteria. The promoter can be a promoter derived from the host or a promoter derived from a heterologous source. The promoter can also be a promoter inherent to the phoS gene or a promoter of another gene. In the bacteria of the present invention, the mutant phoS gene can be present on a vector that autonomously propagates outside the chromosome, such as a plasmid, or can be integrated into the chromosome. The bacteria of the present invention can also have only one copy of the mutant phoS gene, or can have two or more copies. The bacteria of the present invention can also have only one mutant phoS gene, or can have two or more mutant phoS genes. The introduction of the mutant phoS gene can be carried out in the same manner as the introduction of the gene in the method for increasing gene expression and the introduction of the gene construct used in the present invention, for example, as described below.

[0211] The bacterium of the present invention may or may not have the wild-type phoS gene, but preferably does not have it.

[0212] Coryneform bacteria that do not have a wild-type phoS gene can be obtained by disrupting the wild-type phoS gene on the chromosome. Disruption of the wild-type phoS gene can be performed using known methods. Specifically, for example, the wild-type phoS gene can be disrupted by partially or completely deleting the promoter region and / or coding region of the wild-type phoS gene.

[0213] Furthermore, by replacing the wild-type phoS gene on the chromosome with a mutant-type phoS gene, a coryneform bacterium modified to have a mutant-type phoS gene instead of a wild-type phoS gene can be obtained. Examples of methods for performing such gene replacement include the method called "Red-driven integration" (Datsenko, K.A., and Wanner, BL Proc. Natl. Acad. Sci. USA 97: 6640-6645 (2000)), a method combining Red-mediated gene recombination with the excision mechanism derived from lambda phage (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)) (see WO2005 / 010175), methods using linear DNA, methods using a plasmid containing a temperature-sensitive replication origin, methods using a plasmid capable of conjugative transfer, and methods using a suicide vector that does not retain a replication origin that functions in the host (U.S. Patent No. 6,303,383, Japanese Patent Application Laid-Open No. 05-007491).

[0214] The PhoS protein conjugates with the PhoR protein, which acts as a response regulator, and functions, that is, it causes a response to a lack of phosphate in the environment. Therefore, the bacteria of the present invention possess a phoR gene in a manner that allows the mutant PhoS protein to function. The phoR gene is a gene that encodes the PhoR protein, which acts as a response regulator of the PhoRS system. "Having a phoR gene" is also referred to as "having a PhoR protein." Generally, the PhoR protein originally possessed by the bacteria of the present invention conjugates with the mutant PhoS protein and functions. On the other hand, an appropriate phoR gene can be introduced into the bacteria of the present invention in addition to or instead of the phoR gene originally possessed by the bacteria of the present invention. The introduced phoR gene is not particularly limited as long as it encodes a PhoR protein that conjugates with the mutant PhoS protein and functions.

[0215] Examples of phoR genes include those from coryneform bacteria. Specifically, examples of phoR genes from coryneform bacteria include those from Corynebacterium glutamicum YDK010, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium styracifolium, Corynebacterium crenulate, and Corynebacterium effusum. The base sequence of the phoR gene and the amino acid sequence of the PhoR protein from Corynebacterium glutamicum ATCC13032 are represented by SEQ ID NOs. 8 and 9, respectively.

[0216] The phoR gene may be a variant of the phoR gene as described above, as long as it maintains its original function. Similarly, the PhoR protein may be a variant of the PhoR protein as described above, as long as it maintains its original function. That is, the term "phoR gene" encompasses the phoR gene as described above and its conservative variants. Similarly, the term "PhoR protein" encompasses the PhoR protein as described above and its conservative variants. The description regarding the conservative variants of the Mdh protein and mdh gene applies to variants of the PhoR protein and phoR gene. For example, the phoR gene may encode a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added at one or more positions in the amino acid sequence, as long as it maintains its original function. Furthermore, for example, the phoR gene may encode a protein having an amino acid sequence that is 80% or greater, preferably 90% or greater, more preferably 95% or greater, even more preferably 97% or greater, and particularly preferably 99% or greater identical to the entire amino acid sequence, as long as it maintains its original function. It should be noted that the phrase "maintaining the original function" may mean that the protein variant has the function of a PhoR protein (for example, the function of a protein comprising the amino acid sequence shown in SEQ ID NO.9) in the PhoR protein. Furthermore, the phrase "maintaining the original function" may mean that the protein variant has the function of a response regulator of the PhoRS system in the PhoR protein. Specifically, the phrase "functioning as a PhoR protein" may mean that the protein variant has the function of a response regulator of the PhoRS system. Specifically, the phrase "functioning as a response regulator of the PhoRS system" may mean that the protein variant conjugates with the PhoS protein, which is a sensor kinase, and causes a response to phosphate deficiency in the environment. More specifically, the phrase "functioning as a response regulator of the PhoRS system" may mean that the protein variant is activated by sensing phosphate deficiency in the environment and transferring a phosphate group from the autophosphorylated PhoS protein, thereby controlling the expression of genes that respond to phosphate deficiency in the environment.

[0217] Whether a variant of the PhoR protein functions as a response regulator of the PhoRS system can be confirmed, for example, by introducing a gene encoding the variant into a phoR gene-deficient strain of a coryneform bacterium and confirming whether it complements the responsiveness to phosphate deficiency. Complementation of responsiveness to phosphate deficiency can be detected, for example, by increasing growth and proliferation under phosphate deficiency conditions or by inducing expression of a gene known to be induced under phosphate deficiency conditions (J. Bacteriol., 188, 724-732 (2006)). As a phoR gene-deficient strain of a coryneform bacterium, for example, a phoR gene-deficient strain of Corynebacterium glutamicum YDK010 strain or a phoR gene-deficient strain of Corynebacterium glutamicum ATCC13032 strain can be used.

[0218] <1-3-2> Decreased activity of cell surface proteins

[0219] The bacteria of the present invention may have reduced activity of cell surface proteins. Specifically, the bacteria of the present invention may have reduced activity of cell surface proteins compared to unmodified strains. "Reduced activity of cell surface proteins" specifically refers to a reduction in the number of molecules per cell of the cell surface protein. Cell surface proteins and genes encoding them are described below.

[0220] Cell surface proteins are proteins that constitute the cell surface (S layer) of bacteria and archaea. Examples of cell surface proteins of coryneform bacteria include PS1 and PS2 (CspB) of Corynebacterium glutamicum (Japanese Patent Publication No. 6-502548) and SlpA (CspA) of Corynebacterium stagnantum (Japanese Patent Application Laid-Open No. 10-108675). Of these, those that reduce the activity of the PS2 protein are preferred.

[0221] The base sequence of the cspB gene of Corynebacterium glutamicum ATCC13869 and the amino acid sequence of the PS2 protein (CspB protein) encoded by the gene are represented by SEQ ID NOs. 10 and 11, respectively.

[0222] In addition, for example, the amino acid sequences of CspB homologs of 28 strains of Corynebacterium glutamicum have been reported (J Biotechnol., 112, 177-193 (2004)). The GenBank accession numbers of these 28 strains of Corynebacterium glutamicum and the cspB gene homologs in the NCBI database are shown below as examples (the GenBank accession numbers are shown in parentheses).

[0223] Corynebacterium glutamicum ATCC13058 (AY524990)

[0224] Corynebacterium glutamicum ATCC13744 (AY524991)

[0225] Corynebacterium glutamicum ATCC13745 (AY524992)

[0226] Corynebacterium glutamicum ATCC14017 (AY524993)

[0227] Corynebacterium glutamicum ATCC14020 (AY525009)

[0228] Corynebacterium glutamicum ATCC14067 (AY524994)

[0229] Corynebacterium glutamicum ATCC14068 (AY525010)

[0230] Corynebacterium glutamicum ATCC14747 (AY525011)

[0231] Corynebacterium glutamicum ATCC14751 (AY524995)

[0232] Corynebacterium glutamicum ATCC14752 (AY524996)

[0233] Corynebacterium glutamicum ATCC14915 (AY524997)

[0234] Corynebacterium glutamicum ATCC15243 (AY524998)

[0235] Corynebacterium glutamicum ATCC15354 (AY524999)

[0236] Corynebacterium glutamicum ATCC17965 (AY525000)

[0237] Corynebacterium glutamicum ATCC17966 (AY525001)

[0238] Corynebacterium glutamicum ATCC19223 (AY525002)

[0239] Corynebacterium glutamicum ATCC19240 (AY525012)

[0240] Corynebacterium glutamicum ATCC21341 (AY525003)

[0241] Corynebacterium glutamicum ATCC21645 (AY525004)

[0242] Corynebacterium glutamicum ATCC31808 (AY525013)

[0243] Corynebacterium glutamicum ATCC31830 (AY525007)

[0244] Corynebacterium glutamicum ATCC31832 (AY525008)

[0245] Corynebacterium glutamicum LP-6 (AY525014)

[0246] Corynebacterium glutamicum DSM20137 (AY525015)

[0247] Corynebacterium glutamicum DSM20598 (AY525016)

[0248] Corynebacterium glutamicum DSM46307 (AY525017)

[0249] Corynebacterium glutamicum 22220 (AY525005)

[0250] Corynebacterium glutamicum 22243 (AY525006)

[0251] The base sequences of genes encoding cell surface proteins vary depending on the species or strain of coryneform bacteria. Therefore, as long as the gene encoding a cell surface protein maintains its original function, it can be a variant of the gene encoding a cell surface protein as described above. Similarly, as long as the cell surface protein maintains its original function, it can be a variant of the cell surface protein as described above. For example, the term "cspB gene" encompasses the cspB gene as described above and its conservative variants. Similarly, the term "CspB protein" encompasses the CspB protein as described above and its conservative variants. Regarding variants of cell surface proteins and genes encoding them, the descriptions regarding conservative variants of the Mdh protein and mdh gene can be applied. For example, as long as the gene encoding a cell surface protein maintains its original function, it can encode a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added at one or more positions in the amino acid sequence. Furthermore, for example, a gene encoding a cell surface protein may be a gene encoding a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the entire amino acid sequence, as long as the original function is maintained. It should be noted that "maintaining the original function" may mean, for example, that the cell surface protein has the property of increasing the secretory production of a heterologous protein compared to an unmodified strain when its activity is reduced in coryneform bacteria.

[0252] The term "property of increasing the secretion and production of a heterologous protein compared to an unmodified strain when activity is reduced in a coryneform bacterium" refers to a property that confers upon the coryneform bacterium the ability to secrete and produce a greater amount of a heterologous protein than an unmodified strain when activity is reduced. An "unmodified strain" refers to a control strain in which the activity of a cell surface protein is not reduced, and may be, for example, a wild-type strain or a parent strain. The term "secretion and production of a heterologous protein compared to an unmodified strain" is not particularly limited as long as the secretion and production of the heterologous protein is increased compared to an unmodified strain. For example, the amount of heterologous protein accumulated in the culture medium and / or on the bacterial surface may be secreted and produced, preferably at least 1.1 times, more preferably at least 1.2 times, even more preferably at least 1.3 times, and particularly preferably at least 2 times that of an unmodified strain. In addition, the term "secretion and production of a larger amount of heterologous protein than that of an unmodified strain" means that the heterologous protein cannot be detected when the culture supernatant of an unmodified strain without concentration is subjected to SDS-PAGE and stained with CBB, while the heterologous protein can be detected when the culture supernatant of a modified strain without concentration is subjected to SDS-PAGE and stained with CBB.

[0253] Whether or not certain proteins have the property of increasing the secretory production of heterologous proteins compared to unmodified strains when their activity is reduced in coryneform bacteria can be confirmed by preparing a strain that has been modified based on a coryneform bacterium so as to reduce the activity of its protein, quantifying the amount of heterologous protein secreted and produced when the modified strain is cultured in a culture medium, and comparing the amount of heterologous protein secreted and produced when the strain before modification (unmodified strain) is cultured in a culture medium.

[0254] In the present invention, the phrase "reduced cell surface protein activity" encompasses cases where the coryneform bacterium has been modified to reduce the activity of the cell surface protein, as well as cases where the activity of the cell surface protein is already reduced in the coryneform bacterium. The phrase "cases where the activity of the cell surface protein is already reduced in the coryneform bacterium" also encompasses cases where the coryneform bacterium does not originally possess the cell surface protein. In other words, coryneform bacteria with reduced cell surface protein activity include, for example, coryneform bacteria that do not originally possess the cell surface protein. Examples of "cases where the coryneform bacterium does not originally possess the cell surface protein" include cases where the coryneform bacterium does not originally possess the gene encoding the cell surface protein. It should be noted that "cases where the coryneform bacterium does not originally possess the cell surface protein" may mean that the coryneform bacterium does not originally possess one or more cell surface proteins found in other strains of the species to which the coryneform bacterium belongs. For example, "Corynebacterium glutamicum does not originally possess a cell surface protein" means that the Corynebacterium glutamicum strain does not originally possess one or more proteins selected from cell surface proteins found in other Corynebacterium glutamicum strains, for example, PS1 and / or PS2 (CspB). Examples of coryneform bacteria that do not originally possess a cell surface protein include Corynebacterium glutamicum ATCC 13032, which does not originally possess the cspB gene.

[0255] <1-3-3> Protein secretion system

[0256] The bacterium of the present invention has a protein secretion system. The protein secretion system is not particularly limited as long as it can secrete the target protein. As the protein secretion system, the Sec system (Sec secretion system) and the Tat system (Tat secretion system) can be mentioned. The protein secretion system of the bacterium of the present invention can be enhanced. For example, the bacterium of the present invention can be modified to enhance the expression of one or more genes selected from the genes encoding the Tat secretion system. In the present invention, such modification is also referred to as "enhancement of the Tat secretion system". The enhancement of the Tat secretion system is particularly preferably used in cases where a Tat-dependent signal peptide is used to secrete and produce heterologous proteins. The method for increasing the expression of genes encoding the Tat secretion system is described in Japanese Patent No. 4730302.

[0257] Examples of genes encoding the Tat secretion system include the tatA gene, the tatB gene, the tatC gene, and the tatE gene.

[0258] Specific examples of genes encoding the Tat secretion system include the tatA gene, tatB gene, and tatC gene of Corynebacterium glutamicum. The tatA gene, tatB gene, and tatC gene of Corynebacterium glutamicum ATCC 13032 correspond to the complementary sequences of the sequence at positions 1571065 to 1571382, the complementary sequences of the sequence at positions 1167110 to 1167580, and the complementary sequences of the sequence at positions 1569929 to 1570873, respectively, in the genome sequence registered in the NCBI database as GenBank accession NC_003450 (VERSION NC_003450.3 GI:58036263). In addition, the TatA protein, TatB protein, and TatC protein of Corynebacterium glutamicum ATCC 13032 are registered as 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"), respectively. The base sequences of the tatA gene, tatB gene, and tatC gene of Corynebacterium glutamicum ATCC 13032, and the amino acid sequences of the TatA protein, TatB protein, and TatC protein are represented by SEQ ID NOs. 12 to 17.

[0259] Specific examples of genes encoding the Tat secretion system include the tatA, tatB, tatC, and tatE genes of Escherichia coli. The tatA, tatB, tatC, and tatE genes of Escherichia coli K-12 MG1655 correspond to sequences at positions 4019968 to 4020237, 4020241 to 4020756, 4020759 to 4021535, and 658170 to 658373, respectively, in the genome sequence registered in the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI:49175990). In addition, the TatA protein, TatB protein, TatC protein, and TatE protein of Escherichia coli K-12 MG1655 were accessioned as 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”) logged in.

[0260] The gene encoding the Tat secretion system can be a variant of the gene encoding the Tat secretion system as described above, as long as it maintains its original function. Similarly, the Tat secretion system can be a variant of the Tat secretion system as described above, as long as it maintains its original function. For example, the terms "tatA gene," "tatB gene," "tatC gene," and "tatE gene" encompass not only the tatA gene, tatB gene, tatC gene, and tatE gene as described above, respectively, but also conservative variants thereof. Similarly, the terms "TatA protein," "TatB protein," "TatC protein," and "TatE protein" encompass not only the TatA protein, TatB protein, TatC protein, and TatE protein as described above, respectively, but also conservative variants thereof. Regarding variants of the Tat secretion system and the gene encoding it, the description regarding conservative variants of the Mdh protein and mdh gene can be applied. For example, a gene encoding a Tat secretion system may encode a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added at one or more positions in the amino acid sequence, as long as the original function is maintained. Furthermore, for example, a gene encoding a Tat secretion system may encode a protein having an amino acid sequence that is 80% or greater, preferably 90% or greater, more preferably 95% or greater, even more preferably 97% or greater, and particularly preferably 99% or greater identical to the entire amino acid sequence, as long as the original function is maintained. The phrase "maintaining the original function" may mean that the Tat secretion system has the function of secreting a protein to which a Tat-dependent signal peptide is added at the N-terminus.

[0261] <1-4> Methods for reducing protein activity

[0262] Hereinafter, a method for reducing the activity of a protein such as the Mdh protein will be described. It should be noted that the method for reducing the activity of a protein described below can also be used to disrupt the wild-type PhoS protein.

[0263] So-called "reduction in the activity of a protein" means that the activity of the protein is reduced compared to an unmodified strain. So-called "reduction in the activity of a protein" specifically means that the activity per unit cell of the protein is reduced compared to an unmodified strain. So-called "unmodified strain" herein means a control strain that has not been modified to reduce the activity of the target protein. As unmodified strains, wild-type strains and parent strains can be mentioned. In addition, as unmodified strains, specifically, the standard strains (type strains) of each bacterial species can be mentioned. In addition, as unmodified strains, specifically, the strains exemplified in the description of rod-shaped bacteria can be mentioned. That is, in one embodiment, the activity of a protein can be reduced compared to a standard strain (i.e., the standard strain of the species to which the bacterium of the present invention belongs). In addition, in another embodiment, the activity of a protein can be reduced compared to Corynebacterium glutamicum ATCC 13032. In addition, in another embodiment, the activity of a protein can be reduced compared to Corynebacterium glutamicum ATCC 13869. In addition, in another embodiment, the activity of the protein can be reduced compared to Corynebacterium glutamicum AJ12036 (FERM BP-734). In addition, in another embodiment, the activity of the protein can be reduced compared to Corynebacterium glutamicum YDK010 strain. It should be noted that, in "the activity of the protein is reduced", the situation where the activity of the protein disappears completely is also included. The so-called "the activity of the protein is reduced", more specifically, means that the number of molecules per unit cell of the protein is reduced and / or the function of the unit molecule of the protein is reduced compared to the unmodified strain. That is, the "activity" in the case of the so-called "reduction in the activity of the protein" is not limited to the catalytic activity of the protein, but can also mean the transcription amount (mRNA amount) or translation amount (amount of protein) of the gene encoding the protein. The so-called "number of molecules per unit cell of the protein" can mean the average value of the number of molecules per unit cell of the protein. It should be noted that, with respect to "the number of molecules per unit cell of the protein is reduced", the situation where the protein does not exist at all is also included. In addition, with respect to "the function of the unit molecule of the protein is reduced", the situation where the function of the unit molecule of the protein disappears completely is also included. The extent of reduction in protein activity is not particularly limited as long as the activity of the protein is reduced compared to that of the unmodified strain. For example, the activity of the protein can be reduced to less than 50%, less than 20%, less than 10%, less than 5%, or 0% of that of the unmodified strain.

[0264] Modification that reduces the activity of a protein can be achieved, for example, by reducing the expression of the gene encoding the protein. The so-called "reduced expression of a gene" means that the expression of the gene is reduced compared to the unmodified strain. The so-called "reduced expression of a gene" specifically means that the expression level of the gene per unit cell is reduced compared to the unmodified strain. The so-called "expression level of a gene per unit cell" can mean the average value of the expression level of the gene per unit cell. The so-called "reduced expression of a gene" more specifically means that the transcription level (mRNA level) of the gene is reduced and / or the translation level (protein level) of the gene is reduced. As far as "reduced expression of a gene" is concerned, it also includes the situation where the gene is not expressed at all. It should be noted that "reduced expression of a gene" is also referred to as "weakened expression of a gene". The expression of a gene, for example, can be reduced to less than 50%, less than 20%, less than 10%, less than 5% or 0% of that of the unmodified strain.

[0265] The reduction in gene expression can be due to, for example, a reduction in transcription efficiency, a reduction in translation efficiency, or a combination thereof. The reduction in gene expression can be achieved, for example, by modifying the expression regulatory sequence of the gene. The so-called "expression regulatory sequence" is a general term for sites that affect gene expression, such as promoters, Shine-Dalgarno (SD) sequences (also known as ribosome binding sites (RBS)), and the spacer region between the RBS and the start codon. The expression regulatory sequence can be determined, for example, using gene analysis software such as promoter search vectors and GENETYX. When modifying the expression regulatory sequence, the expression regulatory sequence is preferably modified by more than one base, more preferably more than two bases, and particularly preferably more than three bases. The reduction in gene transcription efficiency can be achieved, for example, by replacing the promoter of the gene on the chromosome with a weaker promoter. The so-called "weaker promoter" means a promoter whose transcription of the gene is weakened compared to the wild-type promoter that originally existed. As a weaker promoter, for example, an inducible promoter can be mentioned. That is, the inducible promoter can function as a weaker promoter under non-inducing conditions (for example, in the absence of an inducing substance). In addition, a part or all of the regional deletions (defects) of the expression regulatory sequence can be made. In addition, the reduction in gene expression, for example, can also be achieved by operating the factors relevant to expression control. As the factors relevant to expression control, low molecules (inducing substances, inhibitory substances, etc.), proteins (transcription factors, etc.), nucleic acids (siRNA, etc.) relevant to transcription and translation control can be enumerated. In addition, the reduction in gene expression, for example, can also be achieved by introducing mutations into the coding region of the gene in a manner such that the expression of the gene is reduced. For example, the codons in the coding region of the gene can be replaced by synonymous codons that are utilized with lower frequency in the host, so that the expression of the gene is reduced. In addition, for example, the expression of the gene itself can be reduced by the destruction of the gene as described below.

[0266] Furthermore, modifications that reduce protein activity can be achieved, for example, by disrupting the gene encoding the protein. "Gene disruption" means modifying the gene so that it does not produce a normally functioning protein. "Not producing a normally functioning protein" includes situations where the gene is completely unable to produce protein, and situations where the gene can produce a protein whose function (e.g., activity, properties) is reduced or absent on average.

[0267] The destruction of a gene, for example, can be achieved by deleting (deleting) a gene on a chromosome. The so-called "deletion of a gene" refers to the deletion of a portion or all of the coding region of a gene. Furthermore, the sequences before and after the coding region of a gene on a chromosome can be included, and the entire gene can be deleted. The sequences before and after the coding region of a gene, for example, can include gene expression regulatory sequences. As long as the reduction in protein activity can be achieved, the deleted region can be any region such as the N-terminal region (the region encoding the N-terminal side of the protein), the internal region, or the C-terminal region (the region encoding the C-terminal side of the protein). Generally, when the deleted region is longer, the gene can be reliably inactivated. The deleted region, for example, can be a region with a length of more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or more than 95% of the total length of the coding region of the gene. In addition, the sequences before and after the deleted region are preferably inconsistent in reading frame. By inconsistent reading frames, frameshifts can be generated downstream of the deleted region.

[0268] In addition, gene disruption can be achieved, for example, by introducing an amino acid substitution (missense mutation), a stop codon (nonsense mutation), or an addition or deletion of one to two bases (frameshift mutation) into the coding region of a gene on a chromosome (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)).

[0269] In addition, gene disruption can be achieved, for example, by inserting other base sequences into the coding region of the gene on the chromosome. The insertion site can be any region of the gene, and the gene can be reliably inactivated when the inserted base sequence is longer. In addition, the sequences before and after the insertion site are preferably inconsistent in reading frame. Due to the inconsistency of the reading frame, a frameshift can be generated downstream of the insertion site. As other base sequences, there are no particular limitations if they reduce or eliminate the activity of the encoded protein. For example, marker genes such as antibiotic resistance genes and genes useful for the production of target products can be mentioned.

[0270] In particular, gene disruption can be implemented in a manner that causes a deletion (deletion) of the amino acid sequence of the encoded protein. In other words, modifications that reduce protein activity can be achieved, for example, by deleting the protein's amino acid sequence (partial or complete region of the amino acid sequence), specifically, by modifying the gene in a manner that encodes a protein in which the amino acid sequence (partial or complete region of the amino acid sequence) is deleted. It should be noted that the so-called "deletion of the amino acid sequence of a protein" refers to the deletion of part or all of the region of the protein's amino acid sequence. Furthermore, the so-called "deletion of the amino acid sequence of a protein" refers to the absence of the original amino acid sequence in the protein, and also includes the case where the original amino acid sequence is replaced by another amino acid sequence. For example, a region that is replaced by another amino acid sequence through a frameshift can be considered a deleted region. Typically, the deletion of the protein's amino acid sequence shortens its overall length, but there are also cases where the protein's overall length remains unchanged or is extended. For example, the deletion of part or all of the coding region of a gene can cause the region encoded by the deleted region to be deleted in the amino acid sequence of the encoded protein. In addition, for example, can be by importing terminator codon to the coding region of gene, in the aminoacid sequence of coded protein, make the regional deletion coded in the region near the downstream of this introduction position. In addition, for example, can make the regional deletion coded in this frameshift position by the frameshift in the coding region of gene. About the position and length of the region deleted in the deletion of aminoacid sequence, the explanation of the position and length of the region deleted in the deletion of gene can be used.

[0271] In the case of the Mdh protein, for example, at least one residue, three residues, five residues, seven residues, ten residues, thirteen residues, fourteen residues, fifteen residues, sixteen residues, seventeen residues, eighteen residues, or nineteen residues at the C-terminus of its amino acid sequence can be deleted. In the case of the Mdh protein, in particular, at least the sixteen residues at the C-terminus of its amino acid sequence can be deleted. For example, in the case of the Mdh protein represented by SEQ ID NO. 40, the amino acid sequence from positions 313 to 328 of SEQ ID NO. 40 is referred to as the "sixteen residues at the C-terminus." Furthermore, in the case of the Mdh protein, for example, at least a portion of the amino acid sequence of the Mdh protein corresponding to the C-terminus of SEQ ID NO. 40 (e.g., the sixteen residues at the C-terminus) can be deleted. The description regarding the position of the "amino acid residue at position X of the wild-type PhoS protein" can be applied to the position of the "portion corresponding to the C-terminus of SEQ ID NO. 40" in any Mdh protein.

[0272] Modifying a chromosomal gene as described above can be achieved, for example, by preparing a disrupted gene modified so that it does not produce a normally functioning protein, transforming a host with a recombinant DNA containing the disrupted gene, and allowing homologous recombination between the disrupted gene and the wild-type gene on the chromosome to occur, thereby replacing the wild-type gene on the chromosome with the disrupted gene. In this case, depending on the host's nutritional requirements and other characteristics, it is easier to manipulate the recombinant DNA if it contains a marker gene in advance. Examples of disrupted genes include genes with a portion or all of the coding region of a gene deleted, genes with introduced missense mutations, genes with introduced nonsense mutations, genes with introduced frameshift mutations, and genes with insertion sequences such as transposons and marker genes. The structure of the recombinant DNA used in homologous recombination is not particularly limited as long as homologous recombination occurs in the desired manner. For example, a linear DNA containing the disrupted gene and having upstream and downstream sequences of the wild-type gene on the chromosome at either end can be used to transform the host, allowing homologous recombination to occur upstream and downstream of the wild-type gene, thereby replacing the wild-type gene with the disrupted gene in a single step. Even if the protein encoded by the disrupted 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 based on gene replacement using homologous recombination has been established, and there are the following methods: a method called "Red-driven integration" (Datsenko, K.A. and Wanner, BL Proc. Natl. Acad. Sci. USA 97: 6640-6645 (2000)), a method combining Red-mediated integration with the excision mechanism derived from λ phage (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)) (see WO2005 / 010175), methods using linear DNA, methods using plasmids containing temperature-sensitive replication origins, methods using conjugatively transferable plasmids, methods using suicide vectors that do not retain replication origins that function in the host, and the like (U.S. Patent No. 6303383, Japanese Patent Application Laid-Open No. 05-007491). It should be noted that such a method of modifying chromosomes using homologous recombination is not limited to disruption of a target gene, but can be used for any modification of chromosomes, such as modification of expression regulatory sequences.

[0273] Furthermore, modifications that reduce protein activity can be performed, for example, by mutagenesis. Examples of mutagenesis include X-ray irradiation, ultraviolet irradiation, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0274] The above-mentioned methods for reducing protein activity may be used alone or in any combination.

[0275] A decrease in protein activity can be confirmed by measuring the activity of the protein.

[0276] A decrease in protein activity can also be confirmed by confirming a decrease in the expression of a gene encoding the protein. A decrease in gene expression can be confirmed by confirming a decrease in the transcription level of the gene or a decrease in the amount of protein expressed from the gene.

[0277] The confirmation of the reduction in the transcription amount of a gene can be carried out by comparing the amount of mRNA transcribed by the gene with that of an unmodified strain. As a method for evaluating the amount of mRNA, Northern hybridization, RT-PCR, microarray, RNA-Seq, etc. (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001) can be mentioned. The amount of mRNA, for example, can be reduced to less than 50%, less than 20%, less than 10%, less than 5% or 0% of that of the unmodified strain.

[0278] Confirmation of the reduction in the amount of protein can be carried out by performing SDS-PAGE and confirming the intensity of the separated protein bands. In addition, confirmation of the reduction in the amount of protein can be carried out by Western blotting using antibodies (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of protein (e.g., the number of molecules per unit cell) can be reduced to, for example, less than 50%, less than 20%, less than 10%, less than 5% or 0% of the unmodified strain.

[0279] Whether a gene is disrupted can be confirmed by determining the base sequence, restriction enzyme map, or full length of a portion or all of the gene, depending on the means used for the disruption.

[0280] The method for reducing protein activity can be used to reduce the activity of any protein or reduce the expression of any gene.

[0281] <1-5> Methods for increasing gene expression

[0282] Hereinafter, methods for increasing the expression of genes such as a gene encoding a Tat secretion system will be described.

[0283] "The expression of a gene is improved" means that the expression of the gene is higher than that of an unmodified strain. "The expression of a gene is improved" specifically means that the expression level per unit cell of the gene has been improved compared to an unmodified strain. Here, so-called "unmodified strain" refers to a control strain that has not been modified in a manner such that the expression of the target gene is improved. Unmodified strains include wild-type strains and parent strains. As unmodified strains, specifically, the standard strains (type strains) of each bacterial species can be enumerated. In addition, specifically, as unmodified strains, the strains exemplified in the description of rod-shaped bacteria can be enumerated. That is, in one embodiment, the expression of a gene can be improved compared to a standard strain (i.e., the standard strain of the species to which the bacterium of the present invention belongs). In addition, in another embodiment, the expression of a gene can be improved compared to Corynebacterium glutamicum ATCC 13032. In addition, in another embodiment, the expression of a gene can be improved compared to Corynebacterium glutamicum ATCC 13869. In addition, in another embodiment, the expression of a gene can be improved compared to Corynebacterium glutamicum AJ12036 (FERM BP-734). In addition, in another embodiment, the expression of a gene can be improved compared with the Corynebacterium glutamicum YDK010 strain. The so-called "expression level per unit cell of a gene" can refer to the average value per unit cell of the expression level of the gene. The so-called "increase in gene expression" can more specifically refer to an increase in the transcription level (mRNA level) of a gene and / or an increase in the translation level (protein level) of a gene. It should be noted that "increase in gene expression" is also referred to as "enhanced gene expression". The degree of increase in gene expression is not particularly limited as long as the expression of the gene is higher than that of an unmodified strain. The expression of a gene can be increased to preferably more than 1.5 times that of an unmodified strain, more preferably more than 2 times, and further preferably more than 3 times. In addition, "increase in gene expression" is not only included in increasing the expression level of the gene in the strain that originally expressed the target gene, but also included in expressing the gene in the strain that originally did not express the target gene. That is, "increase in gene expression" includes, for example, introducing the gene into a strain that does not have the target gene and expressing the gene.

[0284] Increased gene expression can be achieved, for example, by increasing the copy number of the gene.

[0285] The increase of the copy number of gene can be reached by importing this gene to the chromosome of the host.For example, homologous recombination can be utilized to carry out (MillerI, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory) to the importing of the gene of chromosome. As the gene import method utilizing homologous recombination, for example, the gene recombination (Red-driven integration) method (Datsenko, K.A, and Wanner, BL Proc.Natl.Acad.Sci.USA.97:6640-6645 (2000)) etc. that Red mediates can be enumerated: the method using linear DNA, the method using the plasmid comprising temperature-sensitive replication origin, the method using the plasmid that can be joined to transfer, the method using the suicide vector of the replication origin that does not remain on and play a function in the host, the transduction (transduction) method using phage. Specifically, the recombinant DNA that will comprise the target gene can be transformed into the host, homologous recombination occurs with the target site on the chromosome of the host, thus the gene is imported on the chromosome of the host. The structure of the recombinant DNA used in homologous recombination is not particularly limited if homologous recombination occurs in a desired manner. For example, a linear DNA comprising a target gene, i.e., a linear DNA having sequences upstream and downstream of the target site on the chromosome at both ends of the gene, can be used to transform the host, and homologous recombination can occur upstream and downstream of the target site, thereby replacing the target site with the gene. The recombinant DNA used for homologous recombination can have a marker gene for selecting transformants. The gene can be imported into only one copy or two or more copies. For example, homologous recombination can be carried out by targeting a base sequence having multiple copies on the chromosome, and multiple copies of the gene can be introduced into the chromosome. As base sequences having multiple copies on the chromosome, repetitive DNA sequences and inverted repeats present at both ends of a transposon can be cited. In addition, appropriate base sequences on chromosomes such as genes not required for the production of the target product can be used as targets for homologous recombination. In addition, genes can be randomly introduced into chromosomes using transposons or Mini-Mu (Japanese Patent Application Publication No. 2-109985, US5,882,888, EP0805867B1). As transposons, artificial transposons (Japanese Patent Application Publication No. 9-70291) can be used. It should be noted that the chromosome modification method utilizing such homologous recombination is not limited to the introduction of the target gene, and can be used for any modification of chromosomes such as the modification of expression regulatory sequences.

[0286] Confirmation of the introduction of the target gene into the chromosome can be performed by Southern blot hybridization using a probe having a sequence complementary to all or part of the gene, or PCR using primers prepared based on the sequence of the gene.

[0287] In addition, the increase in the copy number of a gene can also be achieved by introducing a vector containing the gene into the host. For example, an expression vector of the gene can be constructed by connecting a DNA fragment containing the target gene to a vector that functions in the host, and the host is transformed by the expression vector, thereby increasing the copy number of the gene. The DNA fragment containing the target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism with the target gene as a template. As a vector, a vector that can replicate autonomously in the host's cells can be used. The vector is preferably a multicopy vector. In addition, in order to select transformants, the vector preferably has markers such as antibiotic resistance genes. In addition, the vector can be equipped with a promoter and a terminator for expressing the inserted gene. The vector, for example, can be a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a phage, a clay 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 modified from these plasmids to confer drug resistance genes; pCRY30 (Japanese Patent Application Laid-Open No. 3-210184); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (Japanese Patent Application Laid-Open No. 2-72876, U.S. Patent No. 5,185,262); pCRY2 and pCRY3 (Japanese Patent Application Laid-Open No. 1-191686); pAJ655, pAJ611 , pAJ1844 (Japanese Patent Application Laid-Open No. 58-192900); pCG1 (Japanese Patent Application Laid-Open No. 57-134500); pCG2 (Japanese Patent Application Laid-Open No. 58-35197); pCG4 and pCG11 (Japanese Patent Application Laid-Open No. 57-183799); pVK7 (Japanese Patent Application Laid-Open No. 10-215883); pVK9 (US2006-0141588); pVC7 (Japanese Patent Application Laid-Open No. 9-070291); and pVS7 (WO2013 / 069634).

[0288] When introducing a gene, it is sufficient to maintain the gene in the host and enable it to be expressed. Specifically, the gene is maintained in a manner such that it is expressed under the control of a promoter that functions in the host. The promoter is not particularly limited as long as it functions in the host. The so-called "promoter that functions in the host" refers to a promoter that has promoter activity in the host. The promoter may be a promoter derived from the host or a promoter from a heterologous source. The promoter may be a promoter inherent to the gene to be introduced or a promoter from another gene. As the promoter, the promoter that functions in coryneform bacteria described later may be used.

[0289] Downstream of the gene, a terminator for terminating transcription may be configured. The terminator is not particularly limited as long as it functions in the host. The terminator may be derived from the host or from a heterologous source. The terminator may be intrinsic to the gene to be introduced or from another gene.

[0290] Vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, "Basic Course in Microbiology 8: Genetic Engineering, Kyoritsu Publishing, 1987," and these can be used.

[0291] In addition, when two or more genes are imported, each gene can be maintained in the host and can be expressed. For example, each gene can be maintained in a single expression vector or in a chromosome. In addition, each gene can be maintained in multiple expression vectors or in a single or multiple expression vectors and in a chromosome. In addition, an operon consisting of two or more genes can also be imported.

[0292] The gene to be introduced is not particularly limited as long as it encodes a protein that functions in the host. The gene to be introduced may be a gene of host origin or a gene of heterologous origin. The gene to be introduced, for example, can be obtained by PCR using primers designed based on the base sequence of the gene, using the genomic DNA of an organism having the gene, a plasmid carrying the gene, etc. as a template. In addition, the gene to be introduced, for example, can be fully synthesized based on the base sequence of the gene (Gene, 60(1), 115-127 (1987)). The obtained gene can be used as is or with appropriate modifications. That is, a variant thereof can be obtained by modifying the gene. The modification of the gene can be carried out by known methods. For example, a target mutation can be introduced into a target site of DNA by a site-specific mutagenesis method. That is, for example, a site-specific mutagenesis method can be used to modify the coding region of the gene so that the encoded protein contains a substitution, deletion, insertion and / or addition of amino acid residues in a specific site. Examples of site-specific mutagenesis methods include methods 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)). Alternatively, fully synthesized gene variants may be used.

[0293] In addition, the improvement of gene expression can be achieved by improving the transcription efficiency of the gene. In addition, the improvement of gene expression can be achieved by improving the translation efficiency of the gene. With regard to the improvement of the transcription efficiency and translation efficiency of the gene, for example, it can be achieved by modifying the expression regulatory sequence. The so-called "expression regulatory sequence" is a general term for the position that affects the expression of the gene. As an expression regulatory sequence, for example, a promoter, a Shine-Dalgarno (SD) sequence (also referred to as a ribosome binding site (RBS)) and the spacer region between the RBS and the start codon can be enumerated. The expression regulatory sequence can be determined using gene analysis software such as promoter search vectors and GENETYX. The modification of these expression regulatory sequences can be carried out, for example, by homologous recombination. As a modification method utilizing homologous recombination, a method using a temperature-sensitive vector and Red-mediated gene recombination (WO2005 / 010175) can be enumerated.

[0294] Improving gene transcription efficiency can be achieved, for example, by replacing the gene promoter on the chromosome with a stronger promoter. A "stronger promoter" refers to a promoter that increases gene transcription compared to the native wild-type promoter. Examples of stronger promoters that can be used in coryneform bacteria include: the artificially modified P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679 (2000)); the pta, aceA, aceB, adh, and amyE promoters that can be induced by acetic acid, ethanol, pyruvic acid, and the like in coryneform bacteria; the cspB, SOD, and tuf (EF-Tu) promoters, which are strong promoters with high expression levels in coryneform bacteria (Journal of Biotechnology, 104 (2003) 311-323; Appl. Environ. Microbiol., 2005 Dec; 71 (12): 8587-96); the lac promoter; the tac promoter; and the trc promoter. Furthermore, as stronger promoters, various reporter genes can be used to obtain highly active versions of existing promoters. For example, the activity of the promoter can be improved by making the -35 and -10 regions in the promoter region approach the consensus sequence (International Publication No. 00 / 18935). As highly active promoters, various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574) can be enumerated. The evaluation method of the intensity of the promoter and the example of strong promoters are recorded in the paper (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)) of Goldstein et al.

[0295] The improvement of the translation efficiency of a gene can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also known as the ribosome binding site (RBS)) of the gene on the chromosome with a more powerful SD sequence. The so-called "more powerful SD sequence" means an SD sequence whose translation of mRNA is improved compared to the wild-type SD sequence that originally existed. As a more powerful SD sequence, for example, the RBS of gene 10 derived from bacteriophage T7 can be cited (Olins PO et al, Gene, 1988, 73, 227-235). Furthermore, it is known that the replacement, insertion or deletion of multiple nucleotides in the spacer region between the RBS and the start codon, especially the sequence immediately upstream of the start codon (5'-UTR), has a great influence on the stability and translation efficiency of mRNA, and the translation efficiency of the gene can also be improved by modifying them.

[0296] The improvement of gene translation efficiency can also be achieved by, for example, modifying codons. For example, the translation efficiency of a gene can be improved by replacing rare codons present in the gene with synonymous codons that are used at a higher frequency. That is, the gene to be introduced can be modified in a manner having the most suitable codons according to the codon usage frequency of the host used. The replacement of codons can be carried out, for example, by site-specific mutagenesis. In addition, gene fragments after codon replacement can be fully synthesized. The usage frequency of codons in various organisms is disclosed in the "Codon Usage Database" (http: / / www.kazusa.or.jp / codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)).

[0297] Furthermore, the increase in gene expression can also be achieved by amplifying a regulatory gene that increases gene expression, or by deleting or weakening a regulatory gene that decreases gene expression.

[0298] The above-mentioned methods for increasing gene expression may be used alone or in any combination.

[0299] The transformation method is not particularly limited, and conventionally known methods can be used. For example, a method reported for Escherichia coli K-12, in which recipient cells are treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162); or a method reported for Bacillus subtilis, in which competent cells are prepared from cells in the proliferation stage and DNA is introduced (Duncan, CH, Wilson, GA and Young, FE., 1977. Gene 1: 153-167). Alternatively, a method known for Bacillus subtilis, actinomycetes, and yeast can be applied, in which the cells of a DNA-receiving bacterium are converted into a protoplast or spheroplast state that easily takes up recombinant DNA, and the recombinant DNA is introduced into the DNA-receiving bacterium (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). Specifically, transformation of coryneform bacteria can be performed by, for example, the protoplast method (Gene, 39, 281-286 (1985)), the electroporation method (Bio / Technology, 7, 1067-1070 (1989)), the electric pulse method (Japanese Patent Application Laid-Open No. 2-207791), and the like.

[0300] Increased gene expression can be confirmed, for example, by confirming an increase in the activity of a protein expressed by the gene. Increased protein activity can be confirmed by measuring the activity of the protein. For example, increased activity of the Tat secretion system can be confirmed, for example, by confirming an increase in the secretory production of a protein to which a Tat-dependent signal peptide has been added at its N-terminus. In this case, the secretory production of the protein to which a Tat-dependent signal peptide has been added at its N-terminus is preferably increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more compared to an unmodified strain.

[0301] Furthermore, increased gene expression can be confirmed, for example, by confirming an increase in the transcription level of the gene or an increase in the amount of protein expressed by the gene.

[0302] The confirmation that the transcription amount of a gene is improved can be carried out by comparing the amount of the mRNA transcribed from the gene with an unmodified strain such as a wild-type strain or a parent strain. As a method for evaluating the amount of mRNA, Northern hybridization, RT-PCR, microarray, RNA-Seq, etc. (Sambrook, J., et al., Molecular Cloning A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001) can be enumerated. The amount of mRNA (for example, the number of molecules per unit cell) can, for example, be increased to more than 1.5 times, more than 2 times, or more than 3 times that of the unmodified strain.

[0303] The increase in protein amount can be confirmed by performing SDS-PAGE and confirming the intensity of the separated protein bands. In addition, the increase in protein amount can be confirmed by Western blotting using antibodies (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of protein (e.g., the number of molecules per unit cell) can be increased to, for example, 1.5 times or more, 2 times or more, or 3 times or more of that of the unmodified strain.

[0304] The method for increasing gene expression can be used to enhance the expression of any gene.

[0305] <1-6> Gene constructs for secretory expression of heterologous proteins and their introduction

[0306] It is known that secretory proteins are typically translated as protein precursors (also known as prepeptides) or preproproteins (also known as prepropeptides), and then processed into mature proteins. Specifically, secretory proteins are typically translated as protein precursors or preproproteins. A signal peptide, which serves as a leader moiety, is cleaved by a protease (commonly known as a signal peptidase) to convert them into mature proteins or proproteins. The proprotein is then further cleaved by a protease to become a mature protein. Therefore, in the methods of the present invention, a signal peptide is used for secretory production of heterologous proteins. It should be noted that, in the present invention, protein precursors or preproproteins of secretory proteins may sometimes be collectively referred to as "secretory protein precursors." In the present invention, a "signal peptide" (also known as a "signal sequence") refers to an amino acid sequence present at the N-terminus of a secretory protein precursor and that is generally not present in the native mature protein.

[0307] The gene construct used in the present invention comprises, in the 5' to 3' direction, a promoter sequence that functions in coryneform bacteria, a nucleic acid sequence encoding a signal peptide that functions in coryneform bacteria, and a nucleic acid sequence encoding a heterologous protein. The nucleic acid sequence encoding the signal peptide may be linked downstream of the promoter sequence in such a manner 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 in such a manner that the heterologous protein is expressed as a fusion protein with the signal peptide. This fusion protein is also referred to as the "fusion protein of the present invention." It should be noted that in the fusion protein of the present invention, the signal peptide and the heterologous protein may be adjacent to each other or not. That is, "the heterologous protein is expressed as a fusion protein with the signal peptide" is not limited to the case where the heterologous protein is expressed as a fusion protein with the signal peptide adjacent to the signal peptide, but also includes the case where the heterologous protein is expressed as a fusion protein with the signal peptide via other amino acid sequences. For example, as described below, in the fusion protein of the present invention, an amino acid sequence containing Gln-Glu-Thr, an amino acid sequence for enzyme cleavage, etc. may be included between the signal peptide and the heterologous protein. In addition, as described below, the heterologous protein finally obtained may not have a signal peptide. That is, with respect to "expression of the heterologous protein in the form of a fusion protein with a signal peptide", as long as the heterologous protein constitutes a fusion protein with a signal peptide when expressed, the heterologous protein finally obtained does not need to constitute a fusion protein with a signal peptide. The promoter sequence is also referred to as a "promoter". Nucleic acid sequence can also be referred to as a "gene" in other words. For example, the nucleic acid sequence encoding the heterologous protein is also referred to as "gene encoding the heterologous protein" or "heterologous protein gene". As a nucleic acid sequence, DNA can be cited. In addition, the gene construct used in the present invention can have an effective control sequence (operator, SD sequence, terminator, etc.) for expressing the fusion protein of the present invention in rod-shaped bacteria at an appropriate position, and enable them to function.

[0308] The promoter used in the present invention is not particularly limited as long as it functions in coryneform bacteria. The promoter may be derived from coryneform bacteria (e.g., the host) or from a heterologous source. The promoter may be a promoter inherent to the heterologous protein gene or a promoter of another gene. A "promoter that functions in coryneform bacteria" refers to a promoter that has promoter activity in coryneform bacteria.

[0309] Examples of heterologous promoters include Escherichia coli promoters such as the tac promoter, lac promoter, trp promoter, and araBAD promoter. Among them, strong promoters such as the tac promoter and inducible promoters such as the araBAD promoter are preferred.

[0310] Examples of promoters derived from coryneform bacteria include promoters of cell surface proteins PS1 and PS2 (also called CspB), SlpA (also called CspA) genes, and promoters of various amino acid biosynthesis system genes. Specific examples of promoters of genes in various amino acid biosynthesis systems include promoters of the following genes: the glutamate dehydrogenase gene of the glutamate biosynthesis system, the glutamine synthetase gene of the glutamine biosynthesis system, the aspartate kinase gene of the lysine biosynthesis system, the homoserine dehydrogenase gene of the threonine biosynthesis system, the acetohydroxyacid synthetase gene of the isoleucine and valine biosynthesis system, the 2-isopropylmalate synthetase gene of the leucine biosynthesis system, the glutamate kinase gene of the proline and arginine biosynthesis system, the phosphoribosyl-ATP pyrophosphorylase gene of the histidine biosynthesis system, the 3-deoxy-D-arabinoheptulosonic acid-7-phosphate (DAHP) synthase gene of the biosynthesis system of aromatic amino acids such as tryptophan, tyrosine, and phenylalanine, the phosphoribosylpyrophosphate (PRPP) amidotransferase gene of nucleic acid biosynthesis systems such as inosinic acid and guanylic acid, the inosinic acid dehydrogenase gene, and the guanylate synthetase gene.

[0311] In addition, as the promoter that plays a role in coryneform bacteria, can enumerate: the stronger promoter that can be used for coryneform bacteria as described above. In addition, as promoter, the highly active type of existing promoter can be obtained and used by using various reporter genes. For example, the activity of promoter can be improved by making the -35, -10 region in the promoter region approach the consensus sequence (International Publication No. 00 / 18935). The evaluation method of the intensity of promoter and the example of strong promoter are recorded in the paper (Prokaryotic promoters in biotechnology.Biotechnol.Annu.Rev., 1,105-128 (1995)) of Goldstein etc. Further, the spacer region between the known ribosome bind site (RBS) and the start codon, particularly the displacement or insertion or deletion of multiple nucleotides in the upstream sequence (5 '-UTR) of the immediate vicinity of the start codon brings great impact for the stability and translation efficiency of mRNA, and they can also be modified.

[0312] The signal peptide used in the present invention is not particularly limited as long as it is a signal peptide that functions in coryneform bacteria. The signal peptide may be a signal peptide derived from coryneform bacteria (e.g., host-derived) or a signal peptide of heterologous origin. The signal peptide may be a signal peptide inherent to a heterologous protein or a signal peptide of another protein. The so-called "signal peptide that functions in coryneform bacteria" refers to a peptide that, when linked to the N-terminus of a target protein, can cause coryneform bacteria to secrete the protein. Whether some signal peptides function in coryneform bacteria can be confirmed, for example, by expressing a target protein by fusing it with the signal peptide and confirming whether the protein is secreted.

[0313] Examples of the signal peptide include Tat-dependent signal peptides and Sec-dependent signal peptides.

[0314] The term "Tat-dependent signal peptide" refers to a signal peptide recognized by the Tat system. Specifically, the term "Tat-dependent signal peptide" refers to a peptide that, when linked to the N-terminus of a target protein, causes the protein to be secreted by the Tat secretion system.

[0315] Examples of Tat-dependent signal peptides include the signal peptide of the TorA protein (trimethylamine N-oxidoreductase) of Escherichia coli, the signal peptide of the SufI protein (inhibitor of ftsI) of Escherichia coli, the signal peptide of the PhoD protein (phosphodiesterase) of Bacillus subtilis, the signal peptide of the LipA protein (lipoic acid synthase) of Bacillus subtilis, and the signal peptide of the IMD protein (isomaltodextranase) of Arthrobacter globiformis. The amino acid sequences of these signal peptides are shown below.

[0316] TorA signal peptide: MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO. 18)

[0317] SufI signal peptide: MSLSRRQFIQASGIALCAGAVPLKASA (SEQ ID NO. 19)

[0318] PhoD signal peptide: MAYDSRFDEWVQKLKEESFQNNTFDRRKFIQGAGKIAGLSLGLTIAQS (SEQ IDNO.20)

[0319] LipA ​​signal peptide: MKFVKRRTTALVTTLMLSVTSLFALQPSAKAAEH (SEQ ID NO.21)

[0320] IMD signal peptide: MMNLSRRTLLTTGSAATLAYALGMAGSAQA (SEQ ID NO. 22)

[0321] The Tat-dependent signal peptide has a twin-arginine motif. Examples of the twin-arginine motif include S / TRRXFLK (SEQ ID NO. 23) and RRX-#-# (X: naturally occurring amino acid residue; #: hydrophobic amino acid residue).

[0322] The term "Sec-dependent signal peptide" refers to a signal peptide recognized by the Sec system. Specifically, the term "Sec-dependent signal peptide" may be a peptide that, when linked to the N-terminus of a target protein, causes the protein to be secreted via the Sec secretion system.

[0323] As the Sec-dependent signal peptide, for example, the signal peptide of the cell surface protein of coryneform bacteria can be enumerated. For the cell surface protein of coryneform bacteria, as described above. As the cell surface protein of coryneform bacteria, PS1 and PS2 (CspB) (Japanese Patent Publication No. 6-502548) derived from Corynebacterium glutamicum and SlpA (CspA) (Japanese Patent Publication No. 10-108675) derived from Corynebacterium stagnation can be enumerated. The amino acid sequence of the signal peptide (PS1 signal peptide) of PS1 of Corynebacterium glutamicum is shown in SEQ ID NO.25, the amino acid sequence of the signal peptide (PS2 signal peptide) of PS2 (CspB) of Corynebacterium glutamicum is shown in SEQ ID NO.26, and the amino acid sequence of the signal peptide (SlpA signal peptide) of SlpA (CspA) of Corynebacterium stagnation is represented by SEQ ID NO.27.

[0324] As long as the Tat-dependent signal peptide has a twin-arginine motif and maintains its original function, it can also be a variant of the Tat-dependent signal peptide described above. In addition, as long as the Sec-dependent signal peptide maintains its original function, it can also be a variant of the Sec-dependent signal peptide described above. Regarding variants of signal peptides and the genes encoding them, the descriptions of conservative variants of the Mdh protein and mdh gene can be applied. For example, the signal peptide can be a peptide having the following amino acid sequence, wherein the amino acids have one or more amino acid substitutions, deletions, insertions and / or additions at one or more positions in the amino acid sequence of the signal peptide described above. It should be noted that the "one or more" 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. It should be noted that in the present invention, the terms "TorA signal peptide", "SufI signal peptide", "PhoD signal peptide", "LipA signal peptide", "IMD signal peptide", "PS1 signal peptide", "PS2 signal peptide" and "SlpA signal peptide" refer to the peptides described in SEQ ID NOs. 18 to 22 and 25 to 27 and their conservative variants, respectively.

[0325] The phrase "retaining the original function" of a Tat-dependent signal peptide means that it is recognized by the Tat system. Specifically, when linked to the N-terminus of a target protein, it can function to secrete the protein via the Tat secretion system. Whether a peptide functions as a Tat-dependent signal peptide can be confirmed, for example, by confirming that the secretory production of a protein to which the peptide is attached at the N-terminus increases with enhancement of the Tat secretion system, or by confirming that the secretory production of a protein to which the peptide is attached at the N-terminus decreases with the loss of the Tat secretion system.

[0326] For a Sec-dependent signal peptide to "retain its original function" means that it is recognized by the Sec system. Specifically, when linked to the N-terminus of a target protein, it can function to secrete that protein via the Sec secretion system. Whether a peptide functions as a Sec-dependent signal peptide can be confirmed by, for example, confirming that the secretory production of a protein with the peptide added to its N-terminus increases with enhancement of the Sec secretion system, or confirming that the secretory production of a protein with the peptide added to its N-terminus decreases with deficiency of the Sec secretion system.

[0327] As for the signal peptide, it is usually cut by a signal peptidase when the translation product is secreted outside the bacterium. That is, the heterologous protein finally obtained may not have a signal peptide. It should be noted that the gene encoding the signal peptide can be used as a natural gene or modified in a manner that has the most appropriate codons according to the codon usage frequency of the host used.

[0328] In the gene construct used in the present invention, a nucleic acid sequence encoding an amino acid sequence containing Gln-Glu-Thr can be inserted between the nucleic acid sequence encoding the signal peptide and the nucleic acid sequence encoding the heterologous protein (WO2013 / 062029). It should be noted that the "amino acid sequence containing Gln-Glu-Thr" is also referred to as the "insertion sequence used in the present invention". As the insertion sequence used in the present invention, the amino acid sequence containing Gln-Glu-Thr as described in WO2013 / 062029 can be mentioned. The insertion sequence used in the present invention can be suitably used in combination with a Sec-dependent signal peptide, in particular.

[0329] The insertion sequence used in the present invention is preferably a sequence comprising three or more amino acid residues from the N-terminus of the mature protein of the cell surface protein CspB of coryneform bacteria (hereinafter also referred to as "mature CspB" or "CspB mature protein"). The term "sequence comprising three or more amino acid residues from the N-terminus" refers to the amino acid sequence starting from the amino acid residue at position 1 of the N-terminus to the amino acid residue at position 3 or higher.

[0330] The cell surface protein CspB of coryneform bacteria is as described above. Specific examples of CspB include CspB from Corynebacterium glutamicum ATCC13869, or the CspB from the 28 strains of Corynebacterium glutamicum listed above, and variants thereof. In the amino acid sequence of CspB from Corynebacterium glutamicum ATCC13869 shown in SEQ.ID.NO.11, amino acid residues 1 to 30 correspond to the signal peptide, and amino acid residues 31 to 499 correspond to the mature CspB protein. The amino acid sequence of the mature CspB protein from Corynebacterium glutamicum ATCC13869, excluding the 30 amino acid residues in the signal peptide portion, is shown in SEQ.ID.NO.28. Note that in the mature CspB from Corynebacterium glutamicum ATCC13869, amino acid residues 1 to 3 at the N-terminus correspond to Gln-Glu-Thr.

[0331] The insertion sequence used in the present invention is preferably an amino acid sequence starting from the amino acid residue at position 1 of mature CspB to any one of amino acid residues 3 to 50. The insertion sequence used in the present invention is more preferably an amino acid sequence starting from the amino acid residue at position 1 of mature CspB to any one of amino acid residues 3 to 8, 17, and 50. The insertion sequence used in the present invention is particularly preferably an amino acid sequence starting from the amino acid residue at position 1 to any one of amino acid residues 4, 6, 17, and 50 of mature CspB.

[0332] The insertion sequence used in the present invention is preferably an amino acid sequence selected from the following amino acid sequences (A) to (H), for example.

[0333] (A)Gln-Glu-Thr

[0334] (B)Gln-Glu-Thr-Xaa1

[0335] (C)Gln-Glu-Thr-Xaa1-Xaa2

[0336] (D)Gln-Glu-Thr-Xaa1-Xaa2-Xaa3

[0337] (E) Amino acid sequence with amino acid residues 4 to 7 of mature CspB added to Gln-Glu-Thr

[0338] (F) Amino acid sequence with amino acid residues 4 to 8 of mature CspB added to Gln-Glu-Thr

[0339] (G) Amino acid sequence with amino acid residues 4 to 17 of mature CspB added to Gln-Glu-Thr

[0340] (H) Amino acid sequence with amino acid residues 4 to 50 of mature CspB added to Gln-Glu-Thr

[0341] In the amino acid sequences of (A) to (H), Xaa1 is Asn, Gly, Thr, Pro, or Ala, Xaa2 is Pro, Thr, or Val, and Xaa3 is Thr or Tyr. In addition, in the amino acid sequences of (A) to (H), the phrase "amino acid residues at positions 4 to X of mature CspB are added to Gln-Glu-Thr" means that amino acid residues from positions 4 to X of the N-terminus of mature CspB are added to Thr of Gln-Glu-Thr. It should be noted that, generally, the amino acid residues at positions 1 to 3 of the N-terminus of mature CspB are Gln-Glu-Thr. In this case, the phrase "amino acid sequence having amino acid residues at positions 4 to X of mature CspB added to Gln-Glu-Thr" has the same meaning as an amino acid sequence comprising amino acid residues at positions 1 to X of mature CspB.

[0342] Furthermore, specifically, the insertion sequence used in the present invention is preferably an amino acid sequence selected from the group consisting of Gln-Glu-Thr-Asn-Pro-Thr (SEQ ID NO. 32), Gln-Glu-Thr-Gly-Thr-Tyr (SEQ ID NO. 33), Gln-Glu-Thr-Thr-Val-Thr (SEQ ID NO. 34), Gln-Glu-Thr-Pro-Val-Thr (SEQ ID NO. 35), and Gln-Glu-Thr-Ala-Val-Thr (SEQ ID NO. 36).

[0343] In the present invention, the "amino acid residue at position X of mature CspB" refers to the amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO. 28. Whether an amino acid residue in any mature CspB amino acid sequence is an "amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO. 28" can be determined by aligning the amino acid sequence of any mature CspB with the amino acid sequence of SEQ ID NO. 28.

[0344] Examples of the heterologous protein secreted and produced by the method of the present invention include physiologically active proteins, receptor proteins, antigenic proteins used as vaccines, enzymes, and other arbitrary proteins.

[0345] Examples of the enzyme include transglutaminase, protein glutaminase, isomaltodextranase, protease, endopeptidase, exopeptidase, aminopeptidase, carboxypeptidase, collagenase, and chitinase. Examples of transglutaminases include secretory transglutaminases from actinomycetes such as Streptoverticillium mobaraense IFO 13819 (WO 01 / 23591), Streptoverticillium cinnamoneum IFO 12852, Streptoverticillium griseocarneum IFO 12776, and Streptomyces lydicus (WO 9606931), and filamentous fungi such as Oomycotes (WO 9622366). Examples of protein glutaminases include Chryseobacterium proteolyticum protein glutaminase (WO 2005 / 103278). As isomaltoglucanase, isomaltoglucanase of Arthrobacter sphaericus (WO2005 / 103278) is mentioned, for example.

[0346] Examples of physiologically active proteins include growth factors (proliferation factors), hormones, cytokines, and antibody-related molecules.

[0347] Specific examples of growth factors (growth factors) include epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), 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 (EPO), thrombopoietin (TPO), acidic fibroblast growth factor (AFGF), and leukocyte-derived growth factor (LGF). fibroblast growth factor (aFGF or FGF1), basic fibroblast growth factor (bFGF or FGF2), keratinocyte growth factor (KGF-1 or FGF7, KGF-2 or FGF10), hepatocyte growth factor (HGF).

[0348] Specific examples of hormones include insulin, glucagon, somatostatin, human growth hormone (hGH), parathyroid hormone (PTH), calcitonin, and exenatide.

[0349] Specific examples of cytokines include interleukins, interferons, and tumor necrosis factors (TNF).

[0350] It should be noted that growth factors (proliferation factors), hormones, and cytokines may not be strictly distinguished from each other. For example, a physiologically active protein may belong to one or more selected from growth factors (proliferation factors), hormones, and cytokines.

[0351] Furthermore, the physiologically active protein may be the entire protein or a portion of the protein. Examples of the portion of the protein include a portion having physiological activity. Specifically, examples of the portion having physiological activity include teriparatide, a physiologically active peptide comprising the N-terminal 34 amino acid residues of the mature form of parathyroid hormone (PTH).

[0352] "Antibody-related molecules" refer to proteins comprising the following molecular species, which include a single domain selected from the domains constituting a complete antibody, or a combination of two or more domains. As domains constituting a complete antibody, VH, CH1, CH2, and CH3 as heavy chain domains, and VL and CL as light chain domains can be cited. As long as the antibody-related molecules include the above-mentioned molecular species, they can be monomeric proteins or multimeric proteins. It should be noted that when the antibody-related molecules are multimeric proteins, they can be homomultimers consisting of subunits of a single type, or heteromultimers consisting of two or more subunits. Specific examples of antibody-related molecules include complete antibodies, Fab, F(ab'), F(ab')2, Fc, dimers comprising a heavy chain (H chain) and a light chain (L chain), Fc fusion proteins, heavy chain (H chain), light chain (L chain), single-chain Fv (scFv), sc(Fv)2, disulfide-bonded Fv (sdFv), bifunctional antibodies (diabodies), and VHH fragments (Nanobody (registered trademark)). More specific examples of antibody-related molecules include trastuzumab, adalimumab, and nivolumab.

[0353] 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. Furthermore, the receptor protein may be an orphan receptor whose corresponding ligand is unknown.

[0354] The antigen protein used as a vaccine is not particularly limited as long as it can induce an immune response, and may be appropriately selected depending on the intended target of the immune response.

[0355] Other proteins include liver-type fatty acid-binding protein (LFABP), fluorescent proteins, immunoglobulin-binding proteins, albumin, and extracellular proteins. Fluorescent proteins include green fluorescent protein (GFP). Immunoglobulin-binding proteins include protein A, protein G, and protein L. Albumin includes human serum albumin.

[0356] Examples of extracellular proteins include fibronectin, vitronectin, collagen, osteopontin, laminin, and partial sequences thereof. Laminin is a protein having a heterotrimeric structure composed of α, β, and γ chains. Examples of laminin include mammalian laminin. Examples of mammals include humans, monkeys, chimpanzees, and other primates, mice, rats, hamsters, guinea pigs, and other rodents, rabbits, horses, cattle, sheep, goats, pigs, dogs, cats, and other mammals. In particular, humans are included as mammals. Examples of laminin subunit chains (i.e., α, β, and γ chains) include five α chains (α1 to α5), three β chains (β1 to β3), and three γ chains (γ1 to γ3). Laminin is composed of various subtypes based on the combination of these subunit chains. Specific examples of laminin include laminin 111, laminin 121, laminin 211, laminin 213, laminin 221, laminin 311, laminin 321, laminin 332, laminin 411, laminin 421, laminin 423, laminin 511, laminin 521, and laminin 523. Partial sequences of laminin include the laminin E8 fragment, i.e., laminin E8. Specifically, laminin E8 is a protein having a heterotrimeric structure composed of an α-chain E8 fragment (α-chain E8), a β-chain E8 fragment (β-chain E8), and a γ-chain E8 fragment (γ-chain E8). The subunit chains of laminin E8 (i.e., α chain E8, β chain E8, and γ chain E8) are collectively referred to as "E8 subunit chains". Examples of the E8 subunit chains include the E8 fragments of the laminin subunit chains described above. Laminin E8 constitutes various subtypes depending on the combination of these E8 subunit chains. Specifically, examples of laminin E8 include laminin 111E8, laminin 121E8, laminin 211E8, laminin 221E8, laminin 332E8, laminin 421E8, laminin 411E8, laminin 511E8, and laminin 521E8.

[0357] Genes encoding heterologous proteins, such as these, can be used directly or after appropriate modification. For example, genes encoding heterologous proteins can be modified depending on the host used and / or the desired activity. For example, genes encoding heterologous proteins can be modified so that the amino acid sequence of the encoded heterologous protein includes substitutions, deletions, insertions, and / or additions of one or more amino acids. The above description regarding variants of the Mdh protein and the mdh gene also applies to heterologous proteins secreted and produced by the methods of the present invention and the genes encoding them. It should be noted that proteins specified by the biological species of origin are not limited to proteins found in that biological species, but include proteins having the amino acid sequence of proteins found in that biological species and their variants. Such variants may or may not be found in that biological species. For example, the term "human protein" is not limited to proteins found in humans, but includes proteins having the amino acid sequence of proteins found in humans and their variants. Furthermore, genes encoding heterologous proteins can be genes in which any codon is replaced with an equivalent codon. For example, a gene encoding a heterologous protein can be modified in such a way that it has the most appropriate codons according to the codon usage frequency of the host used.

[0358] The gene construct of the present invention may further include a nucleic acid sequence encoding an amino acid sequence for enzymatic cleavage between the nucleic acid sequence encoding the amino acid sequence containing Gln-Glu-Thr and the nucleic acid sequence encoding the heterologous protein. If the amino acid sequence for enzymatic cleavage is inserted into the fusion protein of the present invention, the expressed fusion protein can be enzymatically cleaved to obtain the target heterologous protein.

[0359] The amino acid sequence for enzyme cleavage is not particularly limited, as long as it is a sequence that is recognized and sheared by an enzyme that hydrolyzes peptide bonds, and the sequence that can be used can be appropriately selected based on the amino acid sequence of the target heterologous protein. The nucleic acid sequence encoding the amino acid sequence for enzyme cleavage can be suitably designed based on the amino acid sequence. For example, the nucleic acid sequence encoding the amino acid sequence for enzyme cleavage can be designed in a manner that has the most appropriate codons based on the codon usage frequency of the host used.

[0360] The amino acid sequence used for enzymatic cleavage is preferably a recognition sequence for a protease with high substrate specificity. Specifically, examples of such amino acid sequences include the recognition sequence for Factor Xa protease and the recognition sequence for proTEV protease. Factor Xa protease recognizes the amino acid sequence Ile-Glu-Gly-Arg (=IEGR) (SEQ ID NO. 37) in proteins, and proTEV protease recognizes the amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln (=ENLYFQ) (SEQ ID NO. 38) in proteins, specifically cleaving the C-terminal side of each sequence.

[0361] The N-terminal region of the heterologous protein ultimately obtained by the method of the present invention may be identical to or different from the native protein. For example, the N-terminal region of the heterologous protein ultimately obtained may have one or more amino acids added or deleted compared to the native protein. It should be noted that the "one or more" may vary depending on the full length or structure of the target heterologous protein. Specifically, it is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0362] Furthermore, the secretory heterologous protein can be a protein with a pro-moiety added (proprotein). When the secretory heterologous protein is a proprotein, the resulting heterologous protein may or may not be a proprotein. Specifically, the proprotein can be converted to a mature protein by cleaving the pro-moiety. Cleavage can be performed, for example, by a protease. When a protease is used, from the perspective of the activity of the resulting protein, the proprotein is generally preferably cleaved at a position substantially identical to the native protein, and more preferably at a position identical to the native protein, yielding a mature protein identical to the native protein. Therefore, a specific protease that cleaves the proprotein at a position identical to the naturally occurring mature protein is generally most preferred. However, as described above, the N-terminal region of the resulting heterologous protein may differ from that of the native protein. For example, depending on the type and intended use of the heterologous protein being produced, a protein with an N-terminal region that is one to several amino acid residues longer or shorter than the native protein may exhibit more suitable activity. Among the proteases that can be used in the present invention, in addition to commercially available proteases such as Dispase (manufactured by BOEHRINGER MANNHEIM), proteases obtained from culture fluids of microorganisms, for example, culture fluids of actinomycetes, etc. are also included. Such proteases can be used in an unpurified state or can be used after being purified to an appropriate purity as needed. It should be noted that, when the original structure is cut to obtain a mature protein, the inserted amino acid sequence containing Gln-Glu-Thr is removed along with the original structure. Therefore, even if an amino acid sequence for enzymatic cleavage is not configured after the amino acid sequence containing Gln-Glu-Thr, the target protein can be obtained.

[0363] The method for introducing the gene construct used in the present invention into rod-shaped bacteria is not particularly limited. "Introduction of the gene construct used in the present invention" refers to the process of maintaining the gene construct in the host. "Introduction of the gene construct used in the present invention" not only includes the process of introducing the pre-constructed gene construct into the host, but also includes the process of at least introducing a heterologous protein gene into the host and constructing the gene construct in the host. In the bacterium of the present invention, the gene construct used in the present invention may also be present on a plasmid, a vector that propagates autonomously outside the chromosome, or may be integrated into the chromosome. For example, the gene construct used in the present invention may be introduced in the same manner as the gene introduction in the above-mentioned method for improving gene expression. It should be noted that, when constructing the bacterium of the present invention, the following steps may be performed in any order: introduction of the gene construct used in the present invention, reduction of the activity of the Mdh protein, and other modifications.

[0364] The gene construct used in the present invention can be introduced into a host using, for example, a vector containing the gene construct. For example, the gene construct used in the present invention can be linked to a vector to construct an expression vector for the gene construct, and the host can be transformed with the expression vector to introduce the gene construct into the host. In addition, when the vector has a promoter that functions in coryneform bacteria, a base sequence encoding the fusion protein of the present invention can be linked downstream of the promoter to construct an expression vector for the gene construct used in the present invention. The vector is not particularly limited as long as it can autonomously replicate in coryneform bacteria. Vectors that can be used in coryneform bacteria are as described above.

[0365] In addition, the gene construct used in the present invention can use transposons such as artificial transposons to import on the host's chromosome. When using a transposon, by homologous recombination or the transfer ability of the transposon itself, the gene construct used in the present invention is imported on the chromosome. In addition, the gene construct used in the present invention can also be imported on the host's chromosome by other introduction methods that have utilized homologous recombination. As the introduction method utilizing homologous recombination, for example, methods such as suicide vectors using linear DNA, plasmids comprising a temperature-sensitive replication origin, plasmids that can be conjugated and transferred, or replication origins that do not function in the host can be enumerated. In addition, at least a heterologous protein gene can be imported on the chromosome so that the gene construct used in the present invention is constructed on the chromosome. In this case, part or all of the constituent elements of the gene construct used in the present invention, other than the heterologous protein gene, can originally be present on the host's chromosome. Specifically, for example, by directly utilizing a promoter sequence originally present on the host chromosome and a nucleic acid sequence encoding a signal peptide linked downstream of the promoter, and replacing only the gene linked downstream of the nucleic acid sequence encoding the signal peptide with the desired heterologous protein gene, the gene construct used in the present invention can be constructed on the chromosome, and the bacterium of the present invention can be constructed. The introduction of a portion of the gene construct used in the present invention, such as a heterologous protein gene, into the chromosome can be carried out in the same manner as the introduction of the gene construct used in the present invention into the chromosome.

[0366] The gene construct used in the present invention and its constituent elements (promoter sequence, nucleic acid sequence encoding signal peptide, nucleic acid sequence encoding heterologous protein, etc.) can be obtained, for example, by cloning. Specifically, for example, a heterologous protein gene can be obtained by cloning an organism having a target heterologous protein, and modifications such as the introduction of a base sequence encoding a signal peptide and the introduction of a promoter sequence can be performed to obtain the gene construct used in the present invention. In addition, the gene construct used in the present invention or its constituent elements can be obtained by chemical synthesis (Gene, 60 (1), 115-127 (1987)). The obtained gene construct and its constituent elements can be used as is or with appropriate modifications.

[0367] It should be noted that when expressing two or more types of proteins, the gene constructs for secretory expression of each protein can be maintained in the bacteria of the present invention in a manner that achieves secretory expression of the target heterologous protein. Specifically, for example, the gene constructs for secretory expression of each protein can all be maintained on a single expression vector, or all can be maintained on the chromosome. In addition, the gene constructs for secretory expression of each protein can be maintained separately on multiple expression vectors, or can be maintained separately on a single or multiple expression vectors and on the chromosome. "Expressing two or more types of proteins" refers, for example, to the secretory production of two or more types of heterologous proteins or the secretory production of heteromultimeric proteins.

[0368] The method for introducing the gene construct used in the present invention into coryneform bacteria is not particularly limited, and commonly used methods such as the protoplast method (Gene, 39, 281-286 (1985)), the electroporation method (Bio / Technology, 7, 1067-1070 (1989)), and the electric pulse method (Japanese Patent Application Laid-Open No. 2-207791) can be used.

[0369] <2> Method for producing heterologous protein

[0370] The bacterium of the present invention obtained above is cultured to express a heterologous protein, thereby obtaining a large amount of the heterologous protein secreted outside the bacterium.

[0371] The bacterium of the present invention can be cultured according to commonly used methods and conditions. For example, the bacterium of the present invention can be cultured in a conventional culture medium containing a carbon source, a nitrogen source, and inorganic ions. In order to achieve higher proliferation, organic micronutrients such as vitamins and amino acids can also be added as needed.

[0372] As a carbon source, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols, etc. can be used. As a nitrogen source, ammonia gas, ammonia water, ammonium salts, etc. can be used. As inorganic ions, calcium ions, magnesium ions, phosphate ions, potassium ions, iron ions, etc. can be appropriately used as needed. Culture for about 1 to 7 days under a suitable range of pH 5.0 to 8.5 and 15 to 37°C and aerobic conditions. In addition, the culture conditions in the production of L-amino acids in rod-shaped bacteria and the conditions described in the secretory production method of proteins using other Sec-dependent or Tat-dependent signal peptides can be used (see WO01 / 23591, WO2005 / 103278). In addition, when an inducible promoter for the expression of heterologous proteins is used, a promoter inducer can be added to the culture medium for cultivation. By culturing the bacterium of the present invention under such conditions, a large amount of target protein can be produced in the bacterium and efficiently secreted outside the bacterium. It should be noted that, according to the method of the present invention, since the produced heterologous protein is secreted outside the microorganism, even proteins such as transglutaminase, which are usually lethal when accumulated in large quantities in the microorganism, can be produced continuously without being affected by lethality.

[0373] The heterologous protein secreted in the culture medium by the method of the present invention can be separated and purified from the culture medium after cultivation according to methods well known to those skilled in the art. For example, after the thalline centrifugation etc. is removed, separation and purification can be carried out by known appropriate methods such as salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium and high pressure liquid chromatography, reversed phase chromatography, hydrophobic chromatography, or by combining these methods. In addition, in some cases, culture or culture supernatant can be directly used. With regard to the protein secreted on the thalline surface according to the method of the present invention, after solubilization by methods well known to those skilled in the art, such as the raising of salt concentration, the use of surfactant, etc., separation and purification can be carried out in the same manner as secretion in the culture medium. In addition, in some cases, the protein secreted on the thalline surface can be solubilized without being solubilized, and can be used as an immobilized enzyme, for example.

[0374] The secretory production of the target heterologous protein can be determined by subjecting the fraction containing the culture supernatant and / or the cell surface as a sample to SDS-PAGE and determining the molecular weight of the separated protein band. Alternatively, the secretory production of the target heterologous protein can be determined by using the fraction containing the culture supernatant and / or the cell surface as a sample and confirming it by Western blotting using an antibody (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). Furthermore, the secretory production of the target heterologous protein can be determined by detecting the N-terminal amino acid sequence of the target protein using a protein sequencer. Furthermore, the secretory production of the target heterologous protein can be confirmed by determining the mass of the target protein using a mass spectrometer. Furthermore, when the target heterologous protein is an enzyme or a protein having a measurable physiological activity, the secretory production of the target heterologous protein can be determined by using the fraction containing the culture supernatant and / or the cell surface as a sample and measuring the enzymatic activity or physiological activity of the target heterologous protein.

[0375] Example

[0376] Hereinafter, the present invention will be further specifically described with reference to non-limiting examples.

[0377] Example 1: Construction of Corynebacterium glutamicum with a deleted malate dehydrogenase gene mdh

[0378] (1) Construction of the mdh gene deletion vector pBS5TΔ2278

[0379] The genome sequence of Corynebacterium glutamicum ATCC13869 strain and the base sequence of the mdh gene encoding malate dehydrogenase (hereinafter also referred to as Mdh) have been determined (Genbank Accession No. AP017557, NCBI locus_tag CGBL_0122780). The base sequence of the mdh gene is shown in<SEQ ID NO.39> The amino acid sequence of Mdh is shown in<SEQID NO.40> .

[0380] To use PurElute TMThe chromosomal DNA of Corynebacterium glutamicum ATCC13869 strain prepared by genomic DNA kit (EdgeBio) was used as template.<SEQ.ID.NO.41> and<SEQ.ID.NO.42> The region about 1 kbp upstream of the 5' side of the mdh gene was amplified by PCR using primers<SEQ.ID.NO.43> and<SEQ.ID.NO.44> The primers were used to amplify the region of about 1 kbp downstream of the 3' side of the mdh gene by PCR. Pyrobest (R) DNA polymerase (TakaraBio) was used for PCR, and the reaction conditions were set to the solution recommended by the manufacturer. The DNA fragments of about 1 kbp amplified by each amplification were subjected to agarose gel electrophoresis, and then the target band was cut out and recovered from the gel using Wizard (R) SV Gel and PCR Clean-Up System (Promega). The two DNA fragments recovered were inserted into the SmaI site of pBS5T described in WO2006 / 057450 by seamless cloning (Infusion) reaction to obtain the mdh gene deletion vector pBS5TΔ2278. In-Fusion (R) HD Cloning Kit (Takara Bio) was used in the seamless cloning (Infusion) reaction, and the reaction conditions were set to the solution recommended by the manufacturer.

[0381] (2) Construction of the mdh gene deletion strain of YDK010::phoS(W302C)

[0382] The YDK010::phoS(W302C) strain described in WO2016 / 171224 was transformed with pBS5TΔ2278 constructed in Example 1(1). From the resulting transformants, strains were selected according to the method described in WO2006 / 057450 to obtain the YDK010::phoS(W302C)Δ2278 strain with a deletion in the mdh gene.

[0383] Example 2: Construction of Corynebacterium glutamicum with a start and stop codon inserted into the malate dehydrogenase gene mdh

[0384] (1) Construction of vector pBS5Ts2278 for insertion into the start and end codons of the mdh gene

[0385] This strain was obtained by inserting a start and end codon into the mdh gene by the following method, thereby modifying the gene to encode a C-terminal deletion-type Mdh in which the amino acid sequence after position 313 of the 328-residue Mdh is deleted.

[0386] To use PurElute TMThe chromosomal DNA of Corynebacterium glutamicum ATCC13869 strain prepared by genomic DNA kit (EdgeBio) was used as template.<SEQ.ID.NO.45> and<SEQ.ID.NO.46> The region about 1 kbp upstream of the GCG at position 312 of Ala encoding Mdh was amplified by PCR using primers<SEQ ID NO.47> and<SEQID NO.48> A region of approximately 1 kbp 3' downstream of the AAT encoding the Asn at position 313 of Mdh was amplified by PCR using primers.<SEQ ID NO.46> The primers were designed to insert two stop codons (TAGTAG) between the GCG encoding Ala at position 312 and the AAT encoding Asn at position 313. PCR was performed using Pyrobest (R) DNA polymerase (Takara Bio), and the reaction conditions were set to the manufacturer's recommended protocol. The DNA fragments of approximately 1 kbp each amplified were subjected to agarose gel electrophoresis, the target band was then cut out, and the Wizard (R) SV Gel and PCR Clean-Up System (Promega) was used to recover the fragments from the gel. The two recovered DNA fragments were inserted into the SmaI site of the pBS5T described in WO2006 / 057450 by seamless cloning (Infusion) reaction, and the vector pBS5Ts2278 for inserting the start and end codons of the mdh gene was obtained. In the seamless cloning (Infusion) reaction, In-Fusion (R) HD Cloning Kit (Takara Bio) was used, and the reaction conditions were set to the manufacturer's recommended protocol.

[0387] (2) Construction of a strain in which the start and end codons of the mdh gene of the YDK010::phoS(W302C) strain were inserted

[0388] The YDK010::phoS(W302C) strain described in WO2016 / 171224 was transformed with pBS5Ts2278 constructed in Example 2(1). From the resulting transformants, strains were selected according to the method described in WO2006 / 057450 to obtain the YDK010::phoS(W302C)::s2278 strain in which a stop codon was inserted into the mdh gene.

[0389] Example 3: Secretory expression of protein L using Corynebacterium glutamicum with a deleted mdh gene

[0390] The protein L secretion expression plasmid pPK4_CspAss_ProteinL described in Japanese Patent Application No. 2016-206702 was used to transform the YDK010::phoS(W302C) strain and the YDK010::phoS(W302C)Δ2278 strain obtained from Example 1(2), thereby obtaining the YDK010::phoS(W302C) / pPK4_CspAss_ProteinL strain and the YDK010::phoS(W302C)Δ2278 / pPK4_CspAss_ProteinL strain.

[0391] Each of the transformants obtained was cultured in MMTG liquid medium (120 g of glucose, 3 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate, 1.5 g of potassium dihydrogen phosphate, 0.03 g of iron sulfate heptahydrate, 0.03 g of manganese sulfate pentahydrate, 0.45 mg of thiamine hydrochloride, 0.45 mg of biotin, 0.15 g of DL-methionine, soybean hydrochloride (total nitrogen content 0.2 g), 50 g of calcium carbonate, made up to 1 L with water and adjusted to pH 7.0) containing 25 mg / L of kanamycin at 30°C for 72 hours.

[0392] After the culture was completed, 3.0 μl of the culture supernatant obtained by centrifugation of each culture solution was subjected to reducing SDS-PAGE and stained with SYPRO Ruby (Life technologies). The results showed that the protein L secretion level of the YDK010::phoS(W302C)Δ2278 strain was significantly increased compared to the YDK010::phoS(W302C) strain ( Figure 1 ).

[0393] After staining, the protein L band intensity was quantified using the image analysis software Multi Gauge (FUJIFILM). The average band intensity when protein L was expressed in the YDK010::phoS(W302C)Δ2278 strain was calculated as a relative value, with the average band intensity when protein L was expressed in the YDK010::phoS(W302C) strain being set to 1.00. The results confirmed that protein L secretion in the YDK010::phoS(W302C)Δ2278 strain was approximately 2.30-fold higher than that in the YDK010::phoS(W302C) strain (Table 1).

[0394] This indicates that the Δmdh mutation (deletion of the mdh gene) is an effective mutation for increasing the secretion amount in the secretory production of protein L using the CspA secretion signal of the Sec system.

[0395] [Table 1]

[0396]

[0397] Example 4: Secretory Expression of Protein L Using Corynebacterium glutamicum with Insertion of Start and Stop Codons in the mdh Gene

[0398] The protein L secretory expression plasmid pPK4_CspAss_ProteinL was used to transform the YDK010::phoS(W302C) strain and the YDK010::phoS(W302C)::s2278 strain obtained from Example 2(2), thereby obtaining the YDK010::phoS(W302C) / pPK4_CspAss_ProteinL strain and the YDK010::phoS(W302C)::s2278 / pPK4_CspAss_ProteinL strain.

[0399] Each of the transformants obtained was cultured in MMTG liquid medium (120 g of glucose, 3 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate, 1.5 g of potassium dihydrogen phosphate, 0.03 g of iron sulfate heptahydrate, 0.03 g of manganese sulfate pentahydrate, 0.45 mg of thiamine hydrochloride, 0.45 mg of biotin, 0.15 g of DL-methionine, soybean hydrochloride (total nitrogen content 0.2 g), 50 g of calcium carbonate, made up to 1 L with water and adjusted to pH 7.0) containing 25 mg / L of kanamycin at 30°C for 72 hours.

[0400] After the culture was completed, 3.0 μl of the culture supernatant obtained by centrifugation of each culture solution was subjected to reducing SDS-PAGE and stained with SYPRO Ruby (Life technologies). The results showed that the protein L secretion level of the YDK010::phoS(W302C)::s2278 strain was significantly increased compared to the YDK010::phoS(W302C) strain ( Figure 2 ).

[0401] After staining, the protein L band intensity was quantified using the image analysis software Multi Gauge (FUJIFILM). The average band intensity when protein L was expressed in the YDK010::phoS(W302C)::s2278 strain was calculated as a relative value, with the average band intensity when protein L was expressed in the YDK010::phoS(W302C) strain being set to 1.00. The results confirmed that protein L secretion in the YDK010::phoS(W302C)::s2278 strain was approximately 2.78-fold higher than that in the YDK010::phoS(W302C) strain (Table 2).

[0402] This indicates that in the secretory production of protein L using the CspA secretion signal of the Sec system, not only the Δ2278 mutation (deletion of the mdh gene) but also the s2278 mutation (insertion of the start and end codons of the mdh gene) are effective mutations for increasing secretion. It is believed that disruption of the mdh gene by insertion of the stop codon of the mdh gene results in the expression of properties similar to those of the mdh gene-deficient strain.

[0403] [Table 2]

[0404]

[0405] Example 5: Secretory expression of liver-type fatty acid-binding protein (LFABP) using Corynebacterium glutamicum with an mdh gene inserted with a start and end codon

[0406] The YDK010::phoS(W302C) strain and the YDK010::phoS(W302C)::s2278 strain obtained in Example 2(2) were transformed using pPK4_CspB6Xa-LFABP described in WO2016 / 171224, respectively, to obtain the YDK010::phoS(W302C) / pPK4_CspB6Xa-LFABP strain and the YDK010::phoS(W302C)::s2278 / pPK4_CspB6Xa-LFABP strain. pPK4_CspB6Xa-LFABP is a secretory expression plasmid for human liver-type fatty acid binding protein (hereinafter referred to as LFABP). pPK4_CspB6Xa-LFABP has: a promoter of the cspB gene (PS2 gene) derived from Corynebacterium glutamicum ATCC13869 strain; and a gene encoding a fusion protein of 30 amino acid residues of the signal peptide of CspB derived from Corynebacterium glutamicum ATCC13869 strain, the N-terminal 6 amino acid residues of the mature protein of CspB derived from the strain, the recognition sequence IEGR of the Xa factor protease, and LFABP (hereinafter referred to as CspB6Xa-LFABP) connected in an expressible manner downstream of the promoter.

[0407] Each of the transformants obtained was cultured in MMTG liquid medium (120 g of glucose, 3 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate, 1.5 g of potassium dihydrogen phosphate, 0.03 g of iron sulfate heptahydrate, 0.03 g of manganese sulfate pentahydrate, 0.45 mg of thiamine hydrochloride, 0.45 mg of biotin, 0.15 g of DL-methionine, soybean hydrochloride (total nitrogen content 0.2 g), 50 g of calcium carbonate, made up to 1 L with water and adjusted to pH 7.0) containing 25 mg / L of kanamycin at 30°C for 72 hours.

[0408] After the culture was completed, 2.0 μl of the culture supernatant obtained by centrifugation of each culture solution was subjected to reducing SDS-PAGE and stained with SYPRO Ruby (Life technologies). The results showed that the secretion of CspB6Xa-LFABP in the YDK010::phoS(W302C)::s2278 strain was increased compared to that in the YDK010::phoS(W302C) strain ( Figure 3 ).

[0409] After staining, the band intensity of CspB6Xa-LFABP was quantified using the image analysis software Multi Gauge (FUJIFILM). The average band intensity when CspB6Xa-LFABP was expressed in the YDK010::phoS(W302C)::s2278 strain was calculated as a relative value, with the average band intensity when CspB6Xa-LFABP was expressed in the YDK010::phoS(W302C) strain being set to 1.00. The results confirmed that the secretion of CspB6Xa-LFABP in the YDK010::phoS(W302C)::s2278 strain was approximately 1.53 times higher than that in the YDK010::phoS(W302C) strain (Table 3).

[0410] This indicates that, in the secretory production of CspB6Xa-LFABP by the YDK010::phoS(W302C) strain, disruption of the mdh gene is also an effective mutation for increasing the secretion amount.

[0411] [Table 3]

[0412]

[0413] Example 6: Secretory Expression of Protransglutaminase from the Sec Secretory Pathway of Corynebacterium glutamicum Using an Insertion of a Stop Codon in the mdh Gene

[0414] The YDK010::phoS(W302C) strain and the YDK010::phoS(W302C)::s2278 strain obtained in Example 2(2) were transformed using pPKSPTG1 described in WO2001 / 23591 to obtain the YDK010::phoS(W302C) / pPKSPTG1 strain and the YDK010::phoS(W302C)::s2278 / pPKSPTG1 strain, respectively. pPKSPTG1 is a secretory expression plasmid for the protransglutaminase (transglutaminase with a pro-structural portion; hereinafter also referred to as PTG) derived from Streptoverticillum mobara. pPKSPTG1 contains: a promoter of the cspB gene (PS2 gene) derived from Corynebacterium glutamicum strain ATCC13869, and a gene encoding a fusion protein of the 25 amino acid residues of the signal peptide of CspA (SlpA; Genbank Accession No. BAB62413) derived from Corynebacterium ammoniagenes strain ATCC6872 and PTG, linked downstream of the promoter in an expressible manner.

[0415] Each of the transformants obtained was cultured in MMTG liquid medium (120 g of glucose, 3 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate, 1.5 g of potassium dihydrogen phosphate, 0.03 g of iron sulfate heptahydrate, 0.03 g of manganese sulfate pentahydrate, 0.45 mg of thiamine hydrochloride, 0.45 mg of biotin, 0.15 g of DL-methionine, soybean hydrochloride (total nitrogen content 0.2 g), 50 g of calcium carbonate, made up to 1 L with water and adjusted to pH 7.0) containing 25 mg / L of kanamycin at 30°C for 72 hours.

[0416] After the culture was completed, 2.5 μl of the culture supernatant obtained by centrifugation of each culture solution was subjected to reducing SDS-PAGE and stained with SYPRO Ruby (Life technologies). The results showed that the PTG secretion amount of the YDK010::phoS(W302C)::s2278 strain was increased compared with the YDK010::phoS(W302C) strain ( Figure 4 ).

[0417] After staining, the PTG band intensity was quantified using the image analysis software Multi Gauge (FUJIFILM). The average band intensity when PTG was expressed in the YDK010::phoS(W302C)::s2278 strain was calculated as a relative value, with the average band intensity when PTG was expressed in the YDK010::phoS(W302C) strain being set to 1.00. The results confirmed that PTG secretion in the YDK010::phoS(W302C)::s2278 strain was approximately 1.86-fold higher than that in the YDK010::phoS(W302C) strain (Table 4).

[0418] These results indicate that, in the secretory production of PTG by the YDK010::phoS(W302C) strain, disruption of the mdh gene is an effective mutation that leads to an increase in secretion.

[0419] [Table 4]

[0420]

[0421] Example 7: Secretory Expression of Protransglutaminase from the Tat Secretory Pathway of Corynebacterium glutamicum Using an Insertion of a Start and Stop Codon in the mdh Gene

[0422] The YDK010::phoS(W302C) strain and the YDK010::phoS(W302C)::s2278 strain obtained in Example 2(2) were transformed using pPK6_T_PTG described in WO2016 / 171224, respectively, to obtain the YDK010::phoS(W302C) / pPK6_T_PTG strain and the YDK010::phoS(W302C)::s2278 / pPK6_T_PTG strain. pPK6_T_PTG is a co-expression plasmid for the TatABC secretion system and the pro-transglutaminase zymogen from Streptovillaria mobara (transglutaminase with a pro-structural portion; hereinafter also referred to as PTG). pPK6_T_PTG has a promoter of the cspB gene (PS2 gene) derived from Corynebacterium glutamicum ATCC13869 strain, and a gene encoding a fusion protein of the TorA signal peptide derived from Escherichia coli and PTG linked to the downstream of the promoter in an expressible manner.

[0423] Each of the transformants obtained was cultured in MMTG liquid medium (120 g of glucose, 3 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate, 1.5 g of potassium dihydrogen phosphate, 0.03 g of iron sulfate heptahydrate, 0.03 g of manganese sulfate pentahydrate, 0.45 mg of thiamine hydrochloride, 0.45 mg of biotin, 0.15 g of DL-methionine, soybean hydrochloride (total nitrogen content 0.2 g), 50 g of calcium carbonate, made up to 1 L with water and adjusted to pH 7.0) containing 25 mg / L of kanamycin at 30°C for 72 hours.

[0424] After the culture was completed, 1.0 μl of the culture supernatant obtained by centrifugation of each culture solution was subjected to reducing SDS-PAGE and stained with SYPRO Ruby (Life technologies). The results showed that the PTG secretion amount of the YDK010::phoS(W302C)::s2278 strain was significantly increased compared with the YDK010::phoS(W302C) strain ( Figure 5 ).

[0425] After staining, the PTG band intensity was quantified using the image analysis software Multi Gauge (FUJIFILM). The average band intensity when PTG was expressed in the YDK010::phoS(W302C)::s2278 strain was calculated as a relative value, with the average band intensity when PTG was expressed in the YDK010::phoS(W302C) strain being set to 1.00. The results confirmed that PTG secretion in the YDK010::phoS(W302C)::s2278 strain was approximately 1.83-fold higher than that in the YDK010::phoS(W302C) strain (Table 5).

[0426] This demonstrates that mdh gene disruption is also an effective mutation for increasing secretion levels in PTG secretion production using the TorA signal sequence of the YDK010::phoS(W302C) strain. Specifically, it was shown that mdh gene disruption can significantly increase secretion levels of heterologous proteins, not only in the Sec secretion pathway but also in the Tat secretion pathway.

[0427] [Table 5]

[0428]

[0429] [Explanation of Sequence Listing]

[0430] SEQ ID NO.1: Base sequence of the phoS gene of Corynebacterium glutamicum YDK010

[0431] SEQ ID NO.2: Amino acid sequence of PhoS protein of Corynebacterium glutamicum YDK010

[0432] SEQ ID NO.3: Amino acid sequence of PhoS protein of Corynebacterium glutamicum ATCC 13032

[0433] SEQ ID NO.4: Amino acid sequence of PhoS protein of Corynebacterium glutamicum ATCC 14067

[0434] SEQ ID NO.5: Amino acid sequence of PhoS protein of Corynebacterium sclerotiorum

[0435] SEQ ID NO.6: Amino acid sequence of PhoS protein of Corynebacterium crenata

[0436] SEQ ID NO.7: Amino acid sequence of PhoS protein of effective Corynebacterium

[0437] SEQ ID NO.8: Base sequence of the phoR gene of Corynebacterium glutamicum ATCC 13032

[0438] SEQ ID NO.9: Amino acid sequence of PhoR protein of Corynebacterium glutamicum ATCC 13032

[0439] SEQ ID NO.10: Base sequence of cspB gene of Corynebacterium glutamicum ATCC 13869

[0440] SEQ ID NO.11: Amino acid sequence of CspB protein of Corynebacterium glutamicum ATCC 13869

[0441] SEQ ID NO.12: Base sequence of the tatA gene of Corynebacterium glutamicum ATCC 13032

[0442] SEQ ID NO.13: Amino acid sequence of TatA protein of Corynebacterium glutamicum ATCC 13032

[0443] SEQ ID NO.14: Base sequence of tatB gene of Corynebacterium glutamicum ATCC 13032

[0444] SEQ ID NO.15: Amino acid sequence of TatB protein of Corynebacterium glutamicum ATCC 13032

[0445] SEQ ID NO.16: Base sequence of the tatC gene of Corynebacterium glutamicum ATCC 13032

[0446] SEQ ID NO.17: Amino acid sequence of TatC protein of Corynebacterium glutamicum ATCC 13032

[0447] SEQ ID NO.18: Amino acid sequence of TorA signal peptide

[0448] SEQ ID NO.19: Amino acid sequence of SufI signal peptide

[0449] SEQ ID NO.20: Amino acid sequence of PhoD signal peptide

[0450] SEQ ID NO.21: Amino acid sequence of LipA signal peptide

[0451] SEQ ID NO.22: Amino acid sequence of IMD signal peptide

[0452] SEQ ID NO.23: Amino acid sequence of twin-arginine motif

[0453] SEQ ID NO.24: skipped sequence

[0454] SEQ ID NO.25: Amino acid sequence of PS1 signal peptide

[0455] SEQ ID NO.26: Amino acid sequence of PS2 signal peptide

[0456] SEQ ID NO.27: Amino acid sequence of SlpA signal peptide

[0457] SEQ ID NO.28: Amino acid sequence of CspB mature protein of Corynebacterium glutamicum ATCC 13869

[0458] SEQ ID NO. 29-31: skipped sequence

[0459] SEQ ID NO. 32-36: Amino acid sequences of one embodiment of the insertion sequence used in the present invention

[0460] SEQ ID NO.37: Recognition sequence of factor Xa protease

[0461] SEQ ID NO.38: Recognition sequence of ProTEV protease

[0462] SEQ ID NO.39: Base sequence of the mdh gene of Corynebacterium glutamicum ATCC 13869

[0463] SEQ ID NO.40: Amino acid sequence of the Mdh protein of Corynebacterium glutamicum ATCC 13869

[0464] SEQ ID NO. 41~48: Primers

Claims

1. A method for producing a heterologous protein, comprising: Cultivating a coryneform bacterium having a gene construct for secretory expression of a heterologous protein; and Recover the heterologous protein produced by secretion, The coryneform bacterium is modified so that the activity of the Mdh protein is reduced compared to an unmodified strain, The gene construct comprises, in the 5' to 3' direction: a promoter sequence that functions in coryneform bacteria, a nucleic acid sequence encoding a signal peptide that functions in coryneform bacteria, and a nucleic acid sequence encoding a heterologous protein. The heterologous protein is expressed as a fusion protein with the signal peptide.

2. The method according to claim 1, wherein The Mdh protein is the protein described in (a), (b) or (c) below: (a) a protein comprising the amino acid sequence shown in SEQ ID NO.40; (b) a protein comprising an amino acid sequence having substitutions, deletions, insertions and / or additions of 1 to 10 amino acid residues in the amino acid sequence of SEQ ID NO. 40, and having malate dehydrogenase activity; (c) A protein comprising an amino acid sequence having 90% or greater identity with the amino acid sequence of SEQ ID NO: 40 and having malate dehydrogenase activity.

3. The method according to claim 1 or 2, wherein By reducing the expression of the mdh gene or disrupting the mdh gene, the activity of the Mdh protein is reduced.

4. The method according to claim 1 or 2, wherein: The activity of the Mdh protein is reduced by partially or completely deleting the amino acid sequence of the Mdh protein.

5. The method according to claim 4, wherein At least the portion of the amino acid sequence of the Mdh protein corresponding to positions 313 to 328 of SEQ ID NO. 40 is deleted.

6. The method according to claim 4, wherein: At least the C-terminal 16 residues of the amino acid sequence of the Mdh protein are deleted.

7. The method according to claim 4, wherein: The deletion is generated by: Deletion of part or all of the coding region of the mdh gene, introduction of a stop codon into the coding region of the mdh gene, frameshift in the coding region of the mdh gene, or a combination thereof.

8. The method according to claim 1 or 2, wherein: The coryneform bacterium is further modified so as to retain the phoS gene encoding the mutant PhoS protein.

9. The method according to claim 8, wherein The mutation is a mutation in which the amino acid residue corresponding to the tryptophan residue at position 302 of SEQ ID NO. 2 in the wild-type PhoS protein is replaced with an amino acid residue other than aromatic amino acid and histidine.

10. The method according to claim 9, wherein: The amino acid residues other than the aromatic amino acids and histidine are: lysine residues, alanine residues, valine residues, serine residues, cysteine ​​residues, methionine residues, aspartic acid residues or asparagine residues.

11. The method according to claim 9, wherein The wild-type PhoS protein is the protein described in (a), (b) or (c) below: (a) a protein comprising the amino acid sequence shown in any one of SEQ ID NOs. 2 to 7; (b) a protein comprising an amino acid sequence having a substitution, deletion, insertion and / or addition of 1 to 10 amino acid residues in the amino acid sequence represented by any one of SEQ ID NOs. 2 to 7, and having a function as a sensor kinase of the PhoRS system; (c) A protein comprising an amino acid sequence having 90% or greater identity with the amino acid sequence represented by any one of SEQ ID NOs. 2 to 7 and functioning as a sensor kinase of the PhoRS system.

12. The method according to claim 1 or 2, wherein: The signal peptide is a Tat-dependent signal peptide.

13. The method according to claim 12, wherein: The Tat-dependent signal peptide is any one 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.

14. The method according to claim 12, wherein: The coryneform bacterium is further modified so that the expression of one or more genes selected from genes encoding the Tat secretion system is increased compared to an unmodified strain.

15. The method according to claim 14, wherein The genes encoding the Tat secretion system include tatA gene, tatB gene, tatC gene and tatE gene.

16. The method according to claim 1 or 2, wherein: The signal peptide is a Sec-dependent signal peptide.

17. The method according to claim 16, wherein The Sec-dependent signal peptide is any one signal peptide selected from the group consisting of a PS1 signal peptide, a PS2 signal peptide, and a SlpA signal peptide.

18. The method according to claim 1 or 2, wherein: The gene construct further comprises a nucleic acid sequence encoding an amino acid sequence containing Gln-Glu-Thr between a nucleic acid sequence encoding a signal peptide that functions in coryneform bacteria and a nucleic acid sequence encoding a heterologous protein.

19. The method according to claim 18, wherein The gene construct further comprises a nucleic acid sequence encoding an amino acid sequence for enzyme cleavage between the nucleic acid sequence encoding the amino acid sequence containing Gln-Glu-Thr and the nucleic acid sequence encoding the heterologous protein.

20. The method according to claim 1 or 2, wherein: The coryneform bacteria are bacteria of the genus Corynebacterium.

21. The method according to claim 20, wherein The coryneform bacteria is Corynebacterium glutamicum.

22. The method according to claim 21, wherein The coryneform bacteria are a modified strain derived from Corynebacterium glutamicum AJ12036 (FERM BP-734) or a modified strain derived from Corynebacterium glutamicum ATCC 13869.

23. The method according to claim 1 or 2, wherein The coryneform bacteria are coryneform bacteria in which the number of molecules per cell of cell surface protein is reduced compared to an unmodified strain.

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