Method for the secretory production of proteins

Abstract

A new technique for improving the secretion production of a heterologous protein by a coryneform bacterium is developed, and thus a method for the secretion production of a heterologous protein is provided. A coryneform bacterium having a secretion production ability of a heterologous protein and modified to maintain a phoS gene having a specific mutation is cultured to produce a heterologous protein by secretion.

Description

Invention Field

[0001] This invention relates to a method for the secretory production of heterologous proteins. Background of the Invention

[0003] Regarding the production of heterologous proteins through microbial secretion, it has been reported that heterologous proteins are produced through secretion by Bacillus bacteria (non-patent document 1), methanol-assimilating yeast, Pichia pastoris (non-patent document 2), and filamentous fungi of the Aspergillus genus (non-patent documents 3 and 4).

[0004] They also explored the production of heterologous proteins through the secretion of Corynebacterium bacteria. Regarding the production of heterologous proteins through the secretion of Corynebacterium glutamicum (hereafter abbreviated as Corynebacterium glutamicum), it has been reported to be through Corynebacterium glutamicum (Hereinafter abbreviated as Corynebacterium glutamicum). The secretion of nucleases and lipases (Patent Document 1, Non-Patent Document 5), the secretion of proteases such as Bacillus subtilis protease (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 Corynebacterium (Patent Document 2), the secretion of fibronectin-binding proteins using the signal peptide of PS2 (CspB) (Non-Patent Document 7), the secretion of transglutaminase using the signal peptides of PS2 (CspB) and SlpA (also known as CspA) (Patent Document 3), the secretion of proteins using a variant secretion system (Patent Document 4), and the secretion of transglutaminase by a variant strain (Patent Document 5), etc. In addition, as a technique to improve the secretion yield of heterologous proteins by Corynebacterium bacteria, it is known to reduce the activity of cell surface proteins (Patent Documents 6 and 7), reduce the activity of penicillin-binding proteins (Patent Document 6), enhance the expression of genes encoding metallopeptidase (Patent Document 7), introduce mutations into the ribosomal protein S1 gene (Patent Document 8), express heterologous proteins by including an inserted Gln-Glu-Thr amino acid sequence between the signal peptide and the heterologous protein, etc.

[0005] The conventional protein secretion pathway, widely present in prokaryotes and eukaryotes, is known as the "Sec system." However, a completely different protein secretion pathway has recently been discovered in the thylakoid membrane of plant chloroplasts (Non-Patent Document 8). Because the arginine-arginine sequence is ubiquitous in the signal sequences of proteins secreted through this pathway, it has been named the "Tat system" (diarginine translocation system) (Non-Patent Document 8). It is known that proteins are secreted by the Sec system before the formation of higher-order structures, while in cells, proteins are secreted across the cell membrane via the Tat system after the formation of higher-order structures (Non-Patent Document 9). Secretory production of proteins using Tat-dependent signal peptides has also been reported in Corynebacterium bacteria (Patent Documents 8 and 10).

[0006] As a system by which bacteria respond to various changes in their internal and external environment, a signal transduction pathway known as the "two-component regulatory system" is employed. This system consists of two components: a sensor kinase responsible for sensing stimuli from environmental changes, and a regulator responsible for receiving signals from the sensor kinase and regulating the expression of downstream genes. When the sensor kinase senses a stimulus, its specific histidine residues are autophosphorylated, transducing the signal by transferring the phosphate group to a specific aspartic residue in the regulator, thereby activating the phosphorylated regulator to act as a transcription factor and regulate the expression of downstream genes.

[0007] For details regarding the two-component regulatory system of Corynebacterium glutamicum, see Non-Patent Document 10, etc. At least 13 types of systems are known as two-component regulatory systems for Corynebacterium glutamicum. One of these is the PhoRS system, which consists of the sensor kinase PhoS protein and the response regulator PhoR protein. Analysis of PhoRS-deficient strains revealed that the PhoRS system is a regulatory system that senses phosphate consumption in the environment and performs signal transduction (Non-Patent Document 11).

[0008] PhoS protein is a membrane protein with two transmembrane domains. The PhoS protein consists of a sensor domain for sensing stimuli, a linker domain called the HAMP domain, a HisKA domain with autophosphorylated histidine residues, and an HATPase domain that has ATP-binding capacity and catalyzes the autophosphorylation of histidine residues. PhoR protein is an intracellular protein. The PhoR protein consists of an receptor domain attached to the N-terminus for receiving signals and an effector domain attached to the C-terminus for regulating downstream gene expression (Non-Patent Document 10).

[0009] However, the relationship between the PhoRS system and the secretory production of heterologous proteins is unknown. It is also unknown whether mutations in the PhoS protein in Corynebacterium bacteria are effective for the secretory production of heterologous proteins. Furthermore, it is unknown whether specific mutations in the PhoS protein are effective for the secretory production of heterologous proteins.

[0010] Existing technical references

[0011] Patent documents

[0012] Patent document 1: US Patent No. 4,965,197

[0013] Patent Document 2: Japanese Patent Publication (Laid-open) (Kohyo) No. 6-502548

[0014] Patent document 3: Japanese Patent No. 4320769

[0015] Patent document 4: Japanese Patent Publication (Laid-open) (Kokai) No. 11-169182

[0016] Patent document 5: Japanese Patent No. 4362651

[0017] Patent document 6: WO2013 / 065869

[0018] Patent document 7: WO2013 / 065772

[0019] Patent document 8: WO2013 / 118544

[0020] Patent document 9: WO2013 / 062029

[0021] Patent document 10: Japanese Patent No. 4730302

[0022] Non-patent documents

[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 document 9: J. Biol. Chem., 25;273(52), 34868-74(1998)

[0032] Non-Patent Document 10: Appl. Microbiol. Biotechnol., 94, 1131-1150 (2012)

[0033] Non-patent document 11: J. Bacteriol., 188, 724-732 (2006) Invention Overview

[0035] The objective of this invention

[0036] The object of this invention is to develop new technologies to improve the secretion production of heterologous proteins by Corynebacterium bacteria, and thereby provide a method for producing heterologous proteins using Corynebacterium bacteria secretion.

[0037] means to achieve the stated purpose

[0038] To achieve the above objectives, the inventors of this invention conducted various studies. As a result, they discovered that the ability of Corynebacterium bacteria to produce heterologous proteins through secretory production could be improved by modifying the phoS gene, which retains a specific mutation, thus realizing the present invention.

[0039] Therefore, the present invention can be implemented as follows.

[0040] [1] A method for producing heterologous proteins, the method comprising:

[0041] Culturing Corynebacterium bacteria with genetic constructs for secreting the expression of the heterologous protein; and

[0042] Collect the heterologous proteins produced through secretion.

[0043] The *Corynebacterium* bacteria in question have been modified to retain the phoS gene encoding the mutant PhoS protein.

[0044] The mutant PhoS protein is a mutant PhoS protein that improves the secretory production of the heterologous protein.

[0045] The genetic construct contains, in a 5' to 3' orientation, a promoter sequence that functions in the Corynebacterium bacteria, a nucleic acid sequence encoding a signal peptide that functions in the Corynebacterium bacteria, and a nucleic acid sequence encoding the heterologous protein.

[0046] The heterologous protein is expressed as a fusion protein with the signal peptide.

[0047] [2] The above method, wherein the mutation is a mutation in wild-type PhoS protein in which an amino acid residue other than an autophosphorylated histidine residue is replaced by another amino acid residue.

[0048] [3] The above method, wherein the mutation is a mutation in the wild-type PhoS protein in which an amino acid residue in the HisKA domain, except for an autophosphorylated histidine residue, is replaced by another amino acid residue.

[0049] [4] The above method, wherein the mutation is a mutation in wild-type PhoS protein in which an amino acid residue corresponding to the tryptophan residue at position 302 in SEQ ID NO: 4 is replaced with an amino acid residue other than aromatic amino acids and histidine residues.

[0050] [5] A method for producing heterologous proteins, the method comprising:

[0051] Culturing Corynebacterium bacteria with genetic constructs for secreting the expression of the heterologous protein; and

[0052] Collect the heterologous proteins produced through secretion.

[0053] The *Corynebacterium* bacteria in question have been modified to retain the phoS gene encoding the mutant PhoS protein.

[0054] The mutant PhoS protein described therein is a PhoS protein with the following mutation: the mutation is a mutation in the wild-type PhoS protein in which an amino acid residue corresponding to the tryptophan residue at position 302 in SEQ ID NO: 4 is replaced with an amino acid residue other than aromatic amino acids and histidine residues.

[0055] The genetic construct contains, in a 5' to 3' orientation, a promoter sequence that functions in the Corynebacterium bacteria, a nucleic acid sequence encoding a signal peptide that functions in the Corynebacterium bacteria, and a nucleic acid sequence encoding the heterologous protein.

[0056] The heterologous protein is expressed as a fusion protein with the signal peptide.

[0057] [6] In the above method, the amino acid residues other than aromatic amino acids and histidine residues are lysine residues, alanine residues, valine residues, serine residues, cysteine ​​residues, methionine residues, aspartic acid residues, or asparagine residues.

[0058] [7] The above method, wherein the wild-type PhoS protein is a protein as defined in (a), (b), or (c) below:

[0059] (a) A protein containing the amino acid sequence of SEQ ID NO: 4, 54, 55, 56, 57, or 58;

[0060] (b) A protein containing an amino acid sequence of SEQ ID NO: 4, 54, 55, 56, 57, or 58, but which includes a substitution, deletion, insertion, or addition of 1 to 10 amino acid residues;

[0061] (c) A protein containing an amino acid sequence that shows 90% or higher identity with the amino acid sequence of SEQ ID NO: 4, 54, 55, 56, 57, or 58.

[0062] [8] The above method, wherein the signal peptide is a Tat-dependent signal peptide.

[0063] [9] In the above method, the Tat-dependent signal peptide is selected from the group consisting of TorA signal peptide, SufI signal peptide, PhoD signal peptide, LipA signal peptide, and IMD signal peptide.

[0064]

[10] The above method, wherein the Corynebacterium bacteria have been further modified to increase the expression of one or more genes selected from the Tat secretion system.

[0065]

[11] In the above method, the gene encoding the Tat secretion system is composed of the tatA gene, the tatB gene, the tatC gene, and the tatE gene.

[0066]

[12] The above method, wherein the signal peptide is a Sec-dependent signal peptide.

[0067]

[13] In the above method, the Sec-dependent signal peptide is selected from the group consisting of PS1 signal peptide, PS2 signal peptide, and SlpA signal peptide.

[0068]

[14] In the above method, the genetic construct further comprises a nucleic acid sequence encoding an amino acid sequence containing Gln-Glu-Thr between the nucleic acid sequence encoding the signal peptide that functions in the Corynebacterium bacteria and the nucleic acid sequence encoding the heterologous protein.

[0069]

[15] In the above method, the genetic construct further includes a nucleic acid sequence encoding an amino acid sequence for enzymatic digestion between the nucleic acid sequence encoding the amino acid sequence containing Gln-Glu-Thr and the nucleic acid sequence encoding the heterologous protein.

[0070]

[16] In the above method, the corynebacterium is a bacterium belonging to the genus Corynebacterium.

[0071]

[17] In the above method, the corynebacterium is Corynebacterium glutamicum.

[0072]

[18] In the above method, the Corynebacterium is a modified strain derived from Corynebacterium glutamicum AJ12036 (FERMBP-734) or Corynebacterium glutamicum ATCC13869.

[0073]

[19] In the above method, the Corynebacterium is a Corynebacterium with reduced activity of cell surface proteins.

[0074]

[20] Corynebacterium,

[0075] It has been modified to retain the phoS gene that encodes the mutant PhoS protein.

[0076] The mutant Phos protein described therein is a Phos protein with the following mutation, which is a mutation in the wild-type Phos protein in which an amino acid residue corresponding to the tryptophan residue at position 302 in SEQ ID NO: 4 is replaced with an amino acid residue other than aromatic amino acids and histidine residues.

[0077]

[21] The above-mentioned Corynebacterium bacteria, wherein the amino acid residues other than aromatic amino acids and histidine residues are lysine residues, alanine residues, valine residues, serine residues, cysteine ​​residues, methionine residues, aspartic acid residues, or asparagine residues.

[0078]

[22] The above-mentioned Corynebacterium bacteria, wherein the wild-type PhoS protein is a protein as defined in (a), (b), or (c) below:

[0079] (a) A protein containing the amino acid sequence of SEQ ID NO: 4, 54, 55, 56, 57, or 58;

[0080] (b) A protein containing an amino acid sequence of SEQ ID NO: 4, 54, 55, 56, 57, or 58, but which includes a substitution, deletion, insertion, or addition of 1 to 10 amino acid residues;

[0081] (c) A protein containing an amino acid sequence that shows 90% or higher identity with the amino acid sequence of SEQ ID NO: 4, 54, 55, 56, 57, or 58.

[0082]

[23] The above-mentioned Corynebacterium bacteria, wherein the Corynebacterium bacteria are bacteria belonging to the genus Corynebacterium.

[0083]

[24] The above-mentioned Corynebacterium, wherein the Corynebacterium is Corynebacterium glutamicum.

[0084]

[25] The Corynebacterium of claim 24, wherein the Corynebacterium is a modified strain derived from Corynebacterium glutamicum AJ12036 (FERM BP-734) or Corynebacterium glutamicum ATCC13869.

[0085]

[26] The above-mentioned Corynebacterium bacteria, wherein the Corynebacterium bacteria are Corynebacterium bacteria with reduced activity of cell surface proteins. Brief description of the attached diagram

[0087] Figure 1 The image shows SDS-PAGE results observed in Corynebacterium glutamicum strain YDK010, its PhoS(W302C) mutant, and PhoS-deleted strains after expression of CspB50TEV-Teri (teriparatide fused with the CspB signal sequence and the mature CspB N-terminal sequence).

[0088] Figure 2 The diagram shows the amino acid sequence alignment of the HisKA domain of the PhoS homologue of Corynebacterium bacteria.

[0089] Figure 3 The image shows the SDS-PAGE results observed in Corynebacterium glutamicum strain YDK010 and its PhoS(W302C) mutant strain after expression of CspB6Xa-LFABP (an LFABP fused with the CspB signal sequence and the mature CspB N-terminal sequence).

[0090] Figure 4 The image shows the SDS-PAGE results observed in Corynebacterium glutamicum strain YDK010 and its PhoS(W302C) mutant strain after expression of CspB6TEV-ExCP (exenatide fused with the CspB signal sequence and the mature CspB N-terminal sequence).

[0091] Figure 5 The diagram illustrates the construction scheme of the pPK6 vector.

[0092] Figure 6 The image shows the SDS-PAGE results observed in Corynebacterium glutamicum strain YDK010 and its PhoS(W302C) mutant strain after expression of transglutaminase fused with the TorA signal sequence of Escherichia coli.

[0093] Figure 7The image shows the SDS-PAGE results observed in Corynebacterium glutamicum strain YDK010 and its PhoS(W302C) mutant strain after expression of a protein containing a pro-structure moiety fused with the Escherichia coli TorA signal sequence.

[0094] Figure 8 The image shows the SDS-PAGE results observed in the *Corynebacterium glutamicum* strain YDK010 and its PhoS(W302C) mutant strain after expression of isomaltodextranase, including the signal sequence, from *Arthrobacter globiformis*.

[0095] Figure 9 The images show the SDS-PAGE results observed in the Phos-deleted strain of Corynebacterium glutamicum YDK010 and its Phos-complemented strain after expression of CspB50TEV-Teri.

[0096] Figure 10 The image shows the SDS-PAGE results observed in Phos-deleted strains of Corynebacterium glutamicum YDK010 after expression of CspB50TEV-Teri. These strains were infused with various mutant phoS(W302X) genes.

[0097] Figure 11 The image shows the SDS-PAGE results observed in Corynebacterium glutamicum ATCC13869 strain and its PhoS(W302C) mutant strain after expression of CspB6TEV-ExCP.

[0098] Figure 12 The image shows the SDS-PAGE results observed in Corynebacterium glutamicum ATCC13869ΔcspB strain and its Phos(W302C) mutant strain after expression of CspB6Xa-LFABP. Invention Details

[0100] The present invention will now be described in detail.

[0101] <1> The method for producing heterologous proteins of the present invention

[0102] The present invention provides a method for producing heterologous proteins, the method comprising culturing Corynebacterium bacteria having a genetic construct for secreting expression of heterologous proteins, and collecting the heterologous proteins produced by secretion, wherein the Corynebacterium bacteria have been modified to retain the phoS gene encoding a mutant PhoS protein (hereinafter also referred to as "the method of the present invention" or "the method of producing heterologous proteins of the present invention").

[0103] <1-1> Corynebacterium bacteria used in the method of the present invention

[0104] The Corynebacterium bacteria used in the method of the present invention are Corynebacterium bacteria having a genetic construct for secreting and expressing heterologous proteins, said Corynebacterium bacteria being modified to retain the mutant phoS gene. The Corynebacterium bacteria used in the method of the present invention are also referred to as "the bacteria of the present invention" or "the Corynebacterium bacteria of the present invention". Furthermore, the genetic construct for secreting and expressing heterologous proteins maintained by the bacteria of the present invention is also referred to as "the genetic construct used in the present invention".

[0105] <1-1-1> Corynebacteria with the ability to secrete and produce heterologous proteins

[0106] The Corynebacterium bacteria of the present invention have a genetic construct for secreting expression of heterologous proteins (the genetic construct for the method of the present invention), and thus have the ability to secrete and produce heterologous proteins.

[0107] In this invention, the expression "secreted" means that the protein is transported out of the bacterial cell (extracellular transport). Examples of extracellular locations include culture media and the cell surface. That is, the expression "secreted" is not limited to the case where all protein molecules are ultimately present in the culture medium in a completely free form, but also includes the case where all protein molecules are present in the cell surface, and the case where a portion of the protein molecules are present in the culture medium and the remainder of the protein molecules are present in the cell surface.

[0108] In other words, in this invention, the term "ability to produce heterologous proteins through secretion" refers to the ability of the bacteria of this invention, when cultured in a culture medium, to secrete heterologous proteins into the culture medium or cell surface and accumulate there to a degree that allows the heterologous proteins to be collected from the culture medium or cell surface. The accumulation amount can be, for example, in terms of accumulation in the culture medium, preferably 10 μg / L or higher, more preferably 1 mg / L or higher, particularly preferably 100 mg / L or higher, and even more preferably 1 g / L or higher. Furthermore, the accumulation amount can be, for example, in terms of accumulation in the cell surface, the amount of heterologous protein collected from the cell surface and suspended in a liquid of the same volume as the culture medium, wherein the concentration of the heterologous protein in the suspension is preferably 10 μg / L or higher, more preferably 1 mg / L or higher, and particularly preferably 100 mg / L or higher. Furthermore, in this invention, the term "protein produced through secretion" also includes the concept of so-called peptides, such as oligopeptides and polypeptides.

[0109] In this invention, the term "heterologous protein" refers to a foreign protein relative to the corynebacterium bacteria that express and secrete the protein. For example, a heterologous protein can be a protein derived from microorganisms, plants, animals, viruses, or even a protein with an artificially designed amino acid sequence. Heterologous proteins can be monomeric or multimeric proteins. The term "multimeric protein" refers to a protein that can exist as a multimer composed of two or more subunits. In a multimer, subunits can be linked by covalent bonds (e.g., disulfide bonds), by non-covalent bonds such as hydrogen bonds and hydrophobic interactions, or by a combination thereof. The multimer preferably contains one or more intermolecular disulfide bonds. The multimer can be a homopolymer composed of a single type of subunit or a heteropolymer composed of two or more types of subunits. In the case that the multimeric protein is a heteropolymer, it is sufficient that at least one subunit selected from the subunits constituting the heteropolymer is a heterologous protein. That is, all subunits can be heterologous, or only a portion of the subunits can be heterologous. While heterologous proteins can be secreted or non-secretory proteins by nature, they are preferably secreted proteins. Furthermore, heterologous proteins can be Tat-dependent secreted proteins or Sec-dependent secreted proteins by nature. Specific examples of "heterologous proteins" will be mentioned later.

[0110] The heteroprotein to be produced can consist of a single type of protein, or two or more types of proteins. Furthermore, when the heteroprotein is a heteropolymer, only one type of subunit can be produced, or two or more types of subunits can be produced. That is, the term "secretory production of a heteroprotein" includes both secreting the production of all subunits that constitute the target heteroprotein and secreting the production of only a portion of the subunits that constitute the target heteroprotein.

[0111] Corynebacteria are aerobic, Gram-positive bacilli. Examples of corynebacteria include bacteria of the genera *Corynebacterium*, *Bryophyllus*, and *Bacteroides*. The advantages of using corynebacteria include their inherent ability to secrete very small amounts of protein extracellularly compared to fungi, yeasts, and bacilli traditionally used for protein secretion production. This is expected to simplify or eliminate the purification process of heterologous proteins produced through secretion production. They can grow well in simple media containing sugars, ammonia, mineral salts, etc., and are therefore highly advantageous considering factors such as culture medium cost, culture methods, and culture productivity.

[0112] Specific examples of Corynebacterium bacteria include the following species:

[0113] Corynebacterium acetoacidophilum

[0114] Corynebacterium acetoglutamicum

[0115] Corynebacterium alkanolyticum

[0116] Corynebacterium callunae

[0117] Corynebacterium crenatum

[0118] Corynebacterium glutamicum

[0119] Corynebacterium lilium

[0120] Corynebacterium melassecola

[0121] Corynebacterium thermoaminogenes (Corynebacterium efficiens)

[0122] Corynebacterium herculis

[0123] Brevibacterium divaricatum (Glutamic acid bacteria)

[0124] Brevibacterium flavum (Corynebacterium glutamicum)

[0125] Brevibacterium immariophilum

[0126] Brevibacterium lactofermentum (Glutamic acid Corynebacterium)

[0127] Brevibacterium roseum

[0128] Brevibacterium saccharolyticum

[0129] Brevibacterium thiogenitalis

[0130] Corynebacterium ammoniagenes (Corynebacterium stationis)

[0131] Brevibacterium album

[0132] Brevibacterium cerinum

[0133] Microbacterium ammoniaphilum

[0134] Specific examples of Corynebacterium bacteria include the following strains:

[0135] Corynebacterium acetophilia ATCC 13870

[0136] Corynebacterium aceti glutamate ATCC 15806

[0137] Corynebacterium alkylate ATCC 21511

[0138] Corynebacterium tumefaciens ATCC 15991

[0139] Corynebacterium obliterans AS1.542

[0140] Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734

[0141] Corynebacterium lilium ATCC 15990

[0142] Corynebacterium molasses ATCC 17965

[0143] Corynebacterium thermoaminogenes (Corynebacterium efficiens) AJ12340(FERM BP-1539)

[0144] Corynebacterium tumefaciens ATCC 13868

[0145] Brevibacterium divaricatum (ATCC 14020)

[0146] Corynebacterium glutamicum (Yellow Short Bacterium) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205)

[0147] Brevibacterium immariophilum ATCC 14068

[0148] Brevibacterium lactofermentum (ATCC 13869)

[0149] Rose-colored short bacillus ATCC 13825

[0150] Brevibacterium saccharolyticum ATCC 14066

[0151] Thiobacillus thiocyanate ATCC 19240

[0152] Corynebacterium stationis (ATCC 6871, ATCC 6872)

[0153] Brevibacterium album ATCC 15111

[0154] Short bacillus cereus ATCC 15112

[0155] Ammonia-loving microbes ATCC 15354

[0156] Corynebacterium includes bacteria formerly classified as *Brevibacteria* that have now been merged into the genus *Corynebacterium* (Int. J. Syst. Bacteriol., 41, 255 (1991)). Furthermore, *Corynebacterium stationis* includes bacteria formerly classified as *Corynebacterium ammoniagenicum*, but now reclassified as *Corynebacterium stationis* based on nucleotide sequence analysis such as 16S rRNA (Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)).

[0157] These strains are available, for example, from the American Center for Type Culture Collection (address: 12301 Parklawn Drive, Rockville, Maryland 20852, PO Box 1549, Manassas, VA 20108, USA). That is, each strain is assigned a accession number, and these accession numbers can be used to order strains (see http: / / www.atcc.org / ). The accession numbers of the strains are listed in the American Center for Type Culture Collection's catalog. These strains can also be obtained, for example, from collections of preserved strains.

[0158] In particular, the Corynebacterium glutamicum strain AJ12036 (FERM BP-734), isolated from a streptomycin (Sm) resistant mutant of the wild-type strain Corynebacterium glutamicum ATCC 13869, was predicted to have mutations in genes responsible for protein secretion functions and showed up to about 2 to 3 times the secretory protein production capacity in terms of protein accumulation under optimal culture conditions compared to the parent strain (wild-type strain), and was therefore preferred as a host bacterium. Strain AJ12036 was initially deposited as an international collection on March 26, 1984, at the Fermentation Research Institute Agency of Industrial Science and Technology (currently an independent administrative body, National Advanced Institute of Industrial Science and Technology, International Patent Biological Collection Center, #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba Prefecture, 292-0818, Japan), and assigned the accession number FERM BP-734.

[0159] Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539) was initially deposited as an international deposit on March 13, 1987, at the Institute of Microbial Industrial Technology, Industrial Technology Institute, Ministry of International Trade and Industry (currently an independent administrative body, National Advanced Institute of Industrial Science and Technology, International Patent Biological Collection Center, #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba Prefecture, 292-0818, Japan), and was assigned the registration number FERM BP-1539. The yellow short bacillus AJ12418 (FERM BP-2205) was initially deposited as an international collection on December 24, 1988, at the Institute of Microbial Industrial Technology, Industrial Technology Institute, Ministry of International Trade and Industry (currently an independent administrative agency, National Advanced Institute of Industrial Science and Technology, International Patent Biological Collection Center, #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba Prefecture, 292-0818, Japan), and was assigned the accession number FERM BP-2205.

[0160] Furthermore, strains with enhanced ability to produce proteins through secretory production can be selected from those Corynebacterium strains used as parental strains, through mutagenesis or genetic recombination, and used as hosts. For example, strains with enhanced ability to produce proteins through secretory production can be selected after treating parental strains with ultraviolet light irradiation or chemical mutagen such as N-methyl-N'-nitrosoguanidine.

[0161] Furthermore, if a strain modified as described above to prevent the production of cell surface proteins is used as a host, purification of heterologous proteins secreted in the culture medium or on the cell surface becomes easier and is therefore particularly preferred. Such modifications can be performed by introducing mutations into the coding region or expression control region of the cell surface protein on the chromosome through mutagenesis or genetic recombination. Examples of Corynebacterium bacteria modified to prevent the production of cell surface proteins include Corynebacterium glutamicum YDK010 (WO2004 / 029254), a PS2-deficient strain of Corynebacterium glutamicum AJ12036 (FERMBP-734).

[0162] Corynebacterium bacteria capable of secreting heterologous proteins can be obtained by introducing the genetic construct used in the method of the present invention into Corynebacterium bacteria as described above, thereby creating bacteria that retain the genetic construct. The genetic construct used in the method of the present invention and its introduction method will be described below.

[0163] <1-1-2> Introduction of the mutant phoS gene

[0164] The bacteria of the present invention have been modified to retain the mutant phoS gene. The expression "retaining the mutant phoS gene" is also referred to as "having a mutant phoS gene" or "having a mutation in the phoS gene." Additionally, the expression "retaining the mutant phoS gene" is also referred to as "having a mutant PhoS protein" or "having a mutation in the PhoS protein." The bacteria of the present invention can be obtained by modifying a Corynebacterium capable of secreting heterologous proteins into a retaining mutant phoS gene. The bacteria of the present invention can also be obtained by modifying Corynebacterium bacteria into a retaining mutant phoS gene and then conferring upon them the ability to secrete heterologous proteins. In the present invention, the bacteria of the present invention can be constructed and modified in any order. Strains used to construct the bacteria of the present invention and previously modified to retain the mutant phoS gene may or may not be able to produce heterologous proteins, assuming that the strain has a genetic construct for heterologous protein secretion and expression. That is, the bacteria of the present invention can also be, for example, bacteria that have acquired the ability to secrete heterologous proteins by modifying to retain the mutant phoS gene. In particular, for example, the bacteria of the present invention can also be bacteria obtained from strains that, even if they have a genetic construct for secretory expression of heterologous proteins, were unable to produce heterologous proteins by secretory production before being modified to retain the mutant phoS gene, but became capable of producing heterologous proteins by secretory production due to modification to retain the mutant phoS gene.

[0165] The phoS gene and the PhoS protein will be explained below. The phoS gene is the gene that encodes the PhoS protein, which is the sensor kinase of the PhoRS system. The PhoRS system is a two-component regulatory system that induces a response to phosphate consumption. The PhoRS system consists of the sensor kinase PhoS encoded by the phoS gene and the regulator PhoR encoded by the phoR gene.

[0166] In this invention, a Phos protein with a “specific mutation” is also called a “mutant Phos protein,” and the gene encoding it is also called a “mutant Phos gene.” In other words, a mutant Phos gene is a Phos gene with a “specific mutation.” Furthermore, in this invention, a Phos protein without a “specific mutation” is also called a “wild-type Phos protein,” and the gene encoding it is also called a “wild-type Phos gene.” In other words, a wild-type Phos gene is a Phos gene without a “specific mutation.” The term “wild-type” is used here for ease of distinction between “wild-type” and “mutant,” and “wild-type” is not limited to those obtained as naturally occurring substances, as long as they do not have a “specific mutation.” The “specific mutation” will be described later.

[0167] Examples of wild-type phoS genes include phoS genes of Corynebacterium bacteria. Specific examples of phoS genes of Corynebacterium bacteria include the following: Corynebacterium glutamicum YDK010, Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 14067, Corynebacterium spp., Corynebacterium tumefaciens, and C. efficiens. The nucleotide sequence of the phoS gene of Corynebacterium glutamicum YDK010 is shown in SEQ ID NO: 3. The amino acid sequences of the wild-type PhoS proteins encoded by these phoS genes are shown in SEQ ID NO: 4, 54, 55, 56, 57, and 58, respectively. That is, a wild-type phoS gene can be, for example, a gene having the nucleotide sequence shown in SEQ ID NO: 3. Additionally, a wild-type PhoS protein can be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 4, 54, 55, 56, 57, or 58. The phrase "a gene or protein having a nucleotide or amino acid sequence" encompasses both cases where the gene or protein contains a nucleotide or amino acid sequence and cases where the gene or protein is composed of a nucleotide or amino acid sequence.

[0168] The wild-type phoS gene can be a variant of any of the wild-type phoS genes exemplified above, provided that it does not have the “specific mutation” and retains its original function. Similarly, the wild-type PhoS protein can be a variant of any protein encoded by the wild-type phoS gene exemplified above, provided that it does not have the “specific mutation” and retains its original function. Such variants are also referred to as “conserved variants.” In this invention, the term “wild-type phoS gene” includes not only the wild-type phoS gene exemplified above, but also its conserved variants that do not have the “specific mutation.” Similarly, the term “wild-type PhoS protein” includes not only the protein encoded by the wild-type phoS gene exemplified above, but also its conserved variants that do not have the “specific mutation.” Examples of conserved variants include, for example, homologs and artificially modified versions of the wild-type phoS gene and wild-type PhoS protein listed above.

[0169] The expression "retaining original function" means that a variant of a gene or protein possesses a function (e.g., activity or property) corresponding to that of the original gene or protein. That is, the expression "retaining original function" for the wild-type phoS gene can mean a gene variant encoding a protein that retains its original function. Furthermore, the expression "retaining original function" for the wild-type PhoS protein can mean a protein variant that functions as a sensor kinase in the PhoRS system. The term "functioning as a sensor kinase in the PhoRS system" can specifically refer to the function of inducing a response to phosphate depletion in the environment in combination with the response regulator PhoR protein. The term "functioning as a sensor kinase in the PhoRS system" can more specifically refer to the function of autophosphorylating upon sensing phosphate depletion in the environment and activating the PhoR protein via phosphate group transfer.

[0170] Whether a variant of the PhoS protein functions as a sensor kinase in the PhoRS system can be confirmed, for example, by introducing the gene encoding the variant into a phoS-gene-deleted strain of Corynebacterium bacteria and confirming whether the response to phosphate deficiency is replenished. For example, replenishment of the response to phosphate deficiency can be detected by improvement in growth under phosphate-depletion conditions or by inducing the expression of a gene (whose expression is known to be induced under phosphate-depletion conditions) (J. Bacteriol., 188, 724-732 (2006)). A phoS-gene-deleted strain of Corynebacterium bacteria can be used, such as a phoS-gene-deleted strain of Corynebacterium glutamicum YDK010 or a phoS-gene-deleted strain of Corynebacterium glutamicum ATCC13032.

[0171] The following will illustrate examples of conservative variants.

[0172] Homologues of the wild-type phoS gene can be readily obtained from public databases, for example, through a BLAST or FASTA search using the nucleotide sequence of any of the above-mentioned wild-type phoS genes as the query sequence. Furthermore, homologues of the wild-type phoS gene can be obtained, for example, through PCR using the chromosome of Corynebacterium bacteria as a template, and using oligonucleotides prepared based on any nucleotide sequence of these known wild-type phoS genes as primers.

[0173] Wild-type Phos proteins can be proteins having any of the amino acid sequences of wild-type Phos proteins exemplified above (SEQ ID NO: 4, 54, 55, 56, 57, or 58), but include amino acid sequences with substitutions, deletions, insertions, or additions of one or more amino acid residues at one or more positions, as long as they do not have a “specific mutation” and retain their original function. Although the number referred to by the term “one or more” as described above can vary depending on the position or type of amino acid residues in the three-dimensional structure of the protein, it is particularly preferred to be 1 to 20, more preferably 1 to 10, further preferably 1 to 5, and particularly preferably 1 to 3.

[0174] The substitution, deletion, insertion, or addition of one or more amino acid residues mentioned above are conserved mutations that maintain the normal function of a protein. A typical example of a conserved mutation is a conserved substitution. A conserved substitution is a mutation in which the substitution occurs between Phe, Trp, and Tyr if the substitution site is an aromatic amino acid; between Leu, Ile, and Val if it is a hydrophobic amino acid; between Gln and Asn if it is a polar amino acid; between Lys, Arg, and His if it is a basic amino acid; between Asp and Glu if it is an acidic amino acid; and between Ser and Thr if it is an amino acid with a hydroxyl group. Examples of substitutions considered conservative include, in particular, substitution of Ala with Ser or Thr; substitution of Arg with Gln, His, or Lys; substitution of Asn with Glu, Gln, Lys, His, or Asp; substitution of Asp with Asn, Glu, or Gln; substitution of Cys with Ser or Ala; substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg; substitution of Glu with Gly, Asn, Gln, Lys, or Asp; and substitution of Gly, Asn, Lys, Gln, Arg, or Ty with Pro. Replacing His with r, replacing Ile with Leu, Met, Val, or Phe, replacing Leu with Ile, Met, Val, or Phe, replacing Lys with Asn, Glu, Gln, His, or Arg, replacing Met with Ile, Leu, Val, or Phe, replacing Phe with Trp, Tyr, Met, Ile, or Leu, replacing Ser with Thr or Ala, replacing Thr with Ser or Ala, replacing Trp with Phe or Tyr, replacing Tyr with His, Phe, or Trp, and replacing Met with Met, Ile, or Leu. Furthermore, such amino acid residue substitutions, deletions, insertions, or additions include naturally occurring mutations (mutants or variants) due to individual differences or species differences in the bacteria from which the gene originates.

[0175] Wild-type Phos proteins can also be proteins having the following amino acid sequences, which show, for example, 80% or higher, preferably 90% or higher, more preferably 95% or higher, even more preferably 97% or higher, and particularly preferably 99% or higher homology with the overall amino acid sequence of any wild-type Phos protein (SEQ ID NO: 4, 54, 55, 56, 57, or 58) as exemplified above, provided that it does not have a “specific mutation” and maintains its original function. In this specification, “homology” can mean “identity”.

[0176] Preferably, the autophosphorylated histidine residue is conserved. That is, it is preferred that conserved mutations occur at amino acid residues other than the autophosphorylated histidine residue. The term "autophosphorylated histidine residue" refers to the histidine residue at position 276 of the wild-type PhoS protein. Furthermore, it is preferred, for example, that the wild-type PhoS protein has the conserved sequence of the wild-type PhoS protein as exemplified above. That is, it is preferred that conserved mutations occur, for example, at non-conserved amino acid sequences in the wild-type PhoS protein as exemplified above.

[0177] The wild-type phoS gene can also be DNA that hybridizes under stringent conditions to a complementary sequence of any nucleotide sequence of the wild-type phoS gene in the examples described above, or to a probe prepared from the complementary sequence, as long as it does not have a “specific mutation” and maintains its original function. The term “stringent conditions” refers to conditions under which so-called specific hybridization occurs and non-specific hybridization does not occur. Examples of stringent conditions include DNAs that are highly homologous to each other under these conditions, such as DNAs with at least 80% homology, preferably at least 90% homology, more preferably at least 95% homology, further more preferably at least 97% homology, and particularly preferably at least 99% homology, hybridizing to each other, and DNAs with less than the above homology not hybridizing to each other, or typical Southern hybridization washing conditions, i.e., washing once, preferably twice or three times, at a salt concentration and temperature corresponding to 1 x SSC, 0.1% SDS at 60°C, preferably 0.1 x SSC, 0.1% SDS at 60°C, more preferably 0.1 x SSC, 0.1% SDS at 68°C.

[0178] The probe can, for example, be a portion of a sequence complementary to the aforementioned gene. Such probes can be prepared by PCR using oligonucleotides as primers and DNA fragments containing these oligonucleotide sequences as templates, the oligonucleotides being prepared based on the nucleotide sequence of a known gene. As a probe, for example, a DNA fragment of approximately 300 bp in length can be used. In this case, the washing conditions for hybridization can be, for example, 50°C, 2 x SSC, and 0.1% SDS.

[0179] Furthermore, the wild-type phoS gene can be a gene having a nucleotide sequence corresponding to any nucleotide sequence of the wild-type phoS gene as exemplified above, or a conserved variant thereof, wherein any codon is replaced with a corresponding equivalent codon. For example, the wild-type phoS gene can be a gene modified to have optimal codons based on the codon frequencies in the host to be used.

[0180] The percentage of sequence identity between two sequences can be determined, for example, using mathematical algorithms. Non-restrictive examples of such mathematical algorithms include the algorithm of Myers and Miller (1988) CABIOS 4:11-17, the local homology algorithm of Smith et al (1981) Adv. Appl. Math. 2:482, the homology algorithm of Needleman and Wunsch (1970) J.Mol. Biol. 48:443-453, the homology search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and a modified version of the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0181] Sequence comparisons (i.e., alignments) for determining sequence identity can be performed using programs based on such mathematical algorithms. These programs can be appropriately executed by a computer. Examples of such programs include, but are not limited to, the PC / Gene program CLUSTAL (available from Intelligenetics, Mountain View, Calif.), the ALIGN program (version 2.0), and the Wisconsin genetics software package versions 8, including GAP, BESTFIT, BLAST, FASTA, and TFASTA (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed, for example, by using initial parameters. The CLUSTAL procedure is well described in Higgins et al. (1988) Gene 73:237-244 (1988), Higgins et al. (1989) CABIOS 5:151-153, Corpet et al. (1988) Nucleic Acids Res. 16:10881-90, Huang et al. (1992) CABIOS 8:155-65, and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.

[0182] To obtain nucleotide sequences homologous to the target nucleotide sequence, a BLAST nucleotide search can be performed, for example, using the BLASTN program with a score of 100 and a word length of 12. To obtain amino acid sequences homologous to the target protein, a BLAST protein search can be performed, for example, using the BLASTX program with a score of 50 and a word length of 3. See http: / / www.ncbi.nlm.nih.gov for BLAST nucleotide and BLAST protein searches. Additionally, Gapped BLAST (BLAST 2.0) can be used to obtain alignments including gaps for comparative purposes. PSI-BLAST can also be used for repeated searches of distal relationships between detection sequences. See Altschul et al. (1997) Nucleic Acids Res. 25:3389 for Gapped BLAST and PSI-BLAST. When using BLAST, gap BLAST, or PSI-BLAST, you can use the initial parameters for each program (e.g., BLASTN for nucleotide sequences and BLSATX for amino acid sequences). Manual alignment is also an option.

[0183] When aligning two sequences, sequence identity between the two sequences is calculated as the proportion of matching residues in the two sequences in order to maximize their correspondence with each other.

[0184] The above description of gene and protein variants can be modified as necessary to apply to any protein, such as PhoR proteins, cell surface proteins, the Tat secretion system, and heterologous proteins produced by secretion in this invention, as well as the genes encoding them.

[0185] The mutant PhoS protein has a “specific mutation” in the amino acid sequence of the wild-type PhoS protein as described above.

[0186] In other words, except that the mutant Phos protein has a “specific mutation,” the mutant Phos protein can be identical to any of the wild-type Phos proteins or their conserved variants as described above. Specifically, except that the mutant Phos protein has a “specific mutation,” the mutant Phos protein can be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 4, 54, 55, 56, 57, or 58. In particular, except that the mutant Phos protein has a “specific mutation,” the mutant Phos protein can also be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 4, 54, 55, 56, 57, or 58 but including one or more substituted, deleted, inserted, or added amino acid residues. In particular, in addition to having a “specific mutation”, the mutant Phos protein may also be a protein showing 80% or higher, preferably 90% or higher, more preferably 95% or higher, further more preferably 97% or higher, and particularly preferably 99% or higher homology with the amino acid sequence shown in SEQ ID NO: 4, 54, 55, 56, 57, or 58.

[0187] Furthermore, in other words, the mutant Phos protein can be a variant of any of the above examples of the wild-type Phos protein, having a “specific mutation” and further including a conserved mutation at a position other than the “specific mutation”. Specifically, the mutant Phos protein can be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 4, 54, 55, 56, 57 or 58 but having a “specific mutation”, and further including one or more substitutions, deletions, insertions or additions of amino acid residues at a position other than the “specific mutation”.

[0188] The mutant phoS gene is not particularly restricted, as long as it encodes the mutant PhoS protein as described above.

[0189] The following section describes the "specific mutations" in the mutant PhoS protein.

[0190] "Specific mutations" are not particularly limited, as long as they alter the amino acid sequence of the wild-type PhoS protein and are effective in secreting heterologous proteins.

[0191] Preferably, the "specific mutation" is a mutation that improves the secretion yield of heterologous proteins. The expression "improves the secretion yield of heterologous proteins" means that a Corynebacterium modified to possess the mutant phoS gene (the modified strain) can produce a greater amount of heterologous protein through secretion than can be obtained with an unmodified strain. The "unmodified strain" refers to a control strain that does not possess the "specific mutation" in the phoS gene, i.e., a control strain that does not possess any mutant phoS gene, and it can be, for example, a wild-type strain or a parental strain. While the degree of increase expressed in the statement "the amount of heterologous protein produced by secretion is greater than that obtainable with unmodified strains" is not particularly limited, as long as the secretion yield of heterologous protein is greater than that obtainable with unmodified strains, the statement can mean that, in terms of the accumulation in the culture medium and / or on the cell surface, the amount of heterologous protein produced by secretion is, for example, preferably 1.1 times or more, more preferably 1.2 times or more, even more preferably 1.3 times or more, even more preferably 2 times or more, and particularly preferably 5 times or more. Furthermore, the statement "the amount of heterologous protein produced by secretion is greater than that obtainable with unmodified strains" can also mean that when the supernatant of the unconcentrated culture medium of the unmodified strain is applied to SDS-PAGE and stained with CBB, the heterologous protein is not detectable, but when the supernatant of the unconcentrated culture medium of the modified strain is applied to SDS-PAGE and stained with CBB, the heterologous protein is detectable. Incidentally, the statement "to improve the secretory production of heterologous proteins" does not necessarily mean that the secretory yield of each heterologous protein is improved, and it is sufficient that the secretory production of the heterologous protein selected as the target of secretory production is improved. The statement "to improve the secretory production of heterologous proteins" can specifically mean, for example, that the secretory yield of heterologous proteins described in the Examples section, such as CspB50TEV-Teri, CspB6Xa-LFABP, CspB6TEV-ExCP fused with the E. coli TorA signal sequence, transglutaminase, protein glutaminase containing the prestructural portion and fused with the E. coli TorA signal sequence, or isomaltodextrinase containing the signal sequence, is improved.

[0192] Whether a specific mutation is a mutation that improves the secretion yield of heterologous proteins can be confirmed, for example, by preparing a strain of Corynebacterium bacteria modified to have a gene encoding the Phos protein, the Phos protein having the specific mutation, quantifying the amount of heterologous protein produced by secretion when the strain is cultured in a culture medium, and comparing it with the amount of heterologous protein produced by secretion when the unmodified strain is cultured in a culture medium.

[0193] Preferred examples of amino acid sequence alterations include the substitution of amino acid residues. That is, a preferred “specific mutation” is a mutation that replaces an amino acid residue with another amino acid residue. The amino acid residue substituted by the “specific mutation” can be one residue or a combination of two or more residues. The amino acid residue substituted by the “specific mutation” is preferably an amino acid residue other than an autophosphorylated histidine residue. The amino acid residue substituted by the “specific mutation” is more preferably an amino acid residue in the HisKA domain other than an autophosphorylated histidine residue. The term “autophosphorylated histidine residue” refers to the histidine residue at position 276 of the wild-type PhoS protein. The term “HisKA domain” refers to the region consisting of amino acid residues at positions 266-330 of the wild-type PhoS protein. The amino acid residue substituted by the “specific mutation” is particularly preferred to be the tryptophan residue at position 302 (W302) of the wild-type PhoS protein.

[0194] Examples of substituted amino acid residues in the above mutations 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), provided that the substituted amino acid residue is not the original amino acid residue. For example, substituted amino acid residues can be selected that lead to improved secretion yield of the heterologous protein.

[0195] When substitution occurs in W302, examples of substituted amino acid residues include amino acid residues other than aromatic amino acids and histidine residues. Specific examples of "amino acid residues other than aromatic amino acids and histidine residues" 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 residues" include K (Lys), A (Ala), V (Val), S (Ser), C (Cys), M (Met), D (Asp), and N (Asn).

[0196] Incidentally, the term "specific mutation" used for the phoS gene refers to a mutation that results in such a "specific mutation" entering the nucleotide sequence of the encoded PhoS protein.

[0197] In this invention, "the amino acid residue at position X of the wild-type Phos protein" refers to the amino acid residue corresponding to the amino acid residue phase at position X in SEQ ID NO: 4. For example, "W302" refers to the amino acid residue corresponding to the tryptophan residue at position 302 in SEQ ID NO: 4. The positions of these amino acid residues represent relative positions, and their absolute positions can be shifted due to the deletion, insertion, or addition of amino acid residues or residues. For example, if an amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 4 is deleted or inserted, the amino acid residue originally located at position X is repositioned to position X-1 or X+1 from the N-terminus; however, it is still considered "the X-position amino acid residue of the wild-type PhoS protein." Specifically, for example, "W302" refers to the tryptophan residue at positions 302, 302, 302, 321, 275, and 286 in the amino acid sequences of the wild-type PhoS proteins shown in SEQ ID NO: 4, 54, 55, 56, 57, and 58, respectively. Furthermore, "the histidine residue at position 276 of the wild-type PhoS protein" refers to the histidine residue at position 276 in SEQ ID NO: 4, 54, 55, and 58. The amino acid sequences of the wild-type Phos proteins shown in SEQ ID NOs 4, 54, 55, 56, 57, and 58 contain histidine residues at positions 276, 276, 276, 295, 249, and 260, respectively. Furthermore, the "region consisting of amino acid residues at positions 266-330 (HisKA domain)" refers to the region in the amino acid sequences of the wild-type Phos proteins shown in SEQ ID NOs 4, 54, 55, 56, 57, and 58 consisting of amino acid residues at positions 266-330, 266-330, 266-330, 285-349, 239-303, and 250-314, respectively.

[0198] Incidentally, while "W302" as referred to herein is typically a tryptophan residue, it can also be non-tryptophan. That is, when the wild-type Phos protein has an amino acid sequence other than that shown in SEQ ID NO: 4, 54, 55, 56, 57, and 58, "W302" may not be a tryptophan residue. Therefore, for example, "mutations that replace W302 with cysteine" includes not only mutations that replace a tryptophan residue with a cysteine ​​residue when "W302" is a tryptophan residue, but also mutations that replace a residue with a cysteine ​​residue when "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). It can also be modified as necessary in detail to be applicable to other mutations.

[0199] The amino acid residue in any Phos protein sequence that corresponds to the amino acid residue at position X in SEQ ID NO: 4 can be determined by comparing the amino acid sequence of any Phos protein with that of SEQ ID NO: 4. This comparison can be performed using known gene analysis software. Specific examples of such software include DNASIS (produced by Hitachi Solutions) and GENTYX (produced 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).

[0200] For example, a mutant phoS gene can be obtained by modifying the wild-type phoS gene to induce the aforementioned "specific mutation" in the encoded PhoS protein. This can be achieved, for example, by cloning from an organism possessing the wild-type phoS gene or through chemical synthesis. Alternatively, a mutant phoS gene can be obtained without using the wild-type phoS gene. For example, a mutant phoS gene can be directly obtained through chemical synthesis. The obtained mutant phoS gene can then be further modified before use.

[0201] Genes can be modified using known methods. For example, a target mutation can be introduced into a target site in DNA using site-specific mutagenesis. Examples of site-specific mutagenesis 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 bacteriophages (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)).

[0202] The following section will explain the method for modifying Corynebacterium bacteria to have a mutant phoS gene.

[0203] Corynebacterium bacteria can be modified to possess the mutant phoS gene by introducing the mutant phoS gene into them. Alternatively, the mutant phoS gene can be introduced into the phoS gene on the chromosome of Corynebacterium bacteria. The mutation can be introduced into the gene on the chromosome through natural mutation, mutagenesis, or genetic engineering.

[0204] There are no particular limitations on the method of introducing the mutant phoS gene into Corynebacterium bacteria. It is sufficient that the bacteria of the present invention maintain the mutant phoS gene such that it can be expressed under the control of a promoter that functions in Corynebacterium bacteria. The promoter can be a host promoter or a heterologous promoter. The promoter can be a natural promoter of the phoS gene or a promoter of another gene. In the bacteria of the present invention, the mutant phoS gene can be present in an extrachromosomal self-replicating vector, such as a plasmid, or incorporated into the chromosome. The bacteria of the present invention can have only one copy of the mutant phoS gene, or two or more copies of the mutant phoS gene. The bacteria of the present invention can have only one mutant phoS gene, or two or more mutant phoS genes. The mutant phoS gene can be introduced, for example, in the same manner as gene introduction in the methods for increasing gene expression described below, or in the same manner as introduction for the genetic constructs of the present invention described below.

[0205] The bacteria of the present invention may or may not have the wild-type phoS gene. Preferably, the bacteria of the present invention do not have the wild-type phoS gene.

[0206] Corynebacterium bacteria lacking the wild-type phoS gene can be obtained by disrupting the wild-type phoS gene on the chromosome. The wild-type phoS gene can be disrupted using known methods. Specifically, for example, the wild-type phoS gene can be disrupted by deleting part or all of the promoter region and / or coding region of the wild-type phoS gene.

[0207] Furthermore, by replacing the wild-type phoS gene on the chromosome with the mutant phoS gene, it is possible to obtain Corynebacterium bacteria that are modified to have the mutant phoS gene but not the wild-type phoS gene. Examples of methods for performing such gene replacements include, for example, methods using linear DNA such as those called “Red-driven integration” (Datsenko, KA, and Wanner, BL, Proc. Natl. Acad. Sci. USA, 97:6640-6645(2000)), methods utilizing a combination of Red-driven integration and excision systems derived from λ phage (Cho, EH, Gumport, RI, Gardner, JF, J. Bacteriol., 184:5200-5203 (2002)) (see WO2005 / 010175), methods using plasmids including temperature-sensitive replication regions, methods using plasmids capable of conjugative transfer, methods utilizing suicide plasmids that do not include replication regions and function in the host (US Patent No. 6,303,383, Japanese Patent Publication No. 05-007491), and so on.

[0208] The PhoS protein functions by binding to the response regulator PhoR protein (i.e., inducing a response to phosphate consumption in the environment). Therefore, the bacteria of the present invention possess a phoR gene that enables the mutant PhoS protein to function. The phoR gene is the gene encoding the PhoR protein, which is the response regulator of the PhoRS system. The statement "possessing a phoR gene" also means "possessing a PhoR protein." Generally, it is sufficient for the inherent PhoR protein of the bacteria of the present invention to function by binding to the mutant PhoS protein. Alternatively, in addition to or in place of the inherent phoR gene of the bacteria of the present invention, a suitable phoR gene may be introduced into the bacteria of the present invention. The introduced phoR gene is not particularly limited, as long as it encodes a PhoR protein that functions by binding to the mutant PhoS protein.

[0209] Examples of phoR genes include, for example, the phoR genes of Corynebacterium bacteria. Specific examples of phoR genes of Corynebacterium bacteria include, for example, the phoR genes of the following bacteria: Corynebacterium glutamicum YDK010, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC 14067, Corynebacterium spp., Corynebacterium tumefaciens, and C. efficiens. The nucleotide sequence of the phoR gene of Corynebacterium glutamicum ATCC 13032 and the amino acid sequence of its PhoR protein are shown in SEQ ID NO: 96 and 97, respectively.

[0210] The phoR gene can be a variant of any of the phoR genes listed above, as long as it retains its original function. Similarly, the phoR protein can be a variant of any of the phoR proteins listed above, as long as it retains its original function. That is, the term "phoR gene" includes not only the phoR genes listed above, but also their conserved variants. Similarly, the term "phoR protein" includes not only the phoR proteins listed above, but also their conserved variants. The above description of conserved variants of phoS genes and phoS proteins can be applied in analogous terms to variants of phoR genes and phoR proteins. For example, a phoR gene can be a gene encoding a protein having the above-described amino acid sequence, but including substitutions, deletions, insertions, or additions of one or more amino acid residues at one or more positions, as long as the gene encodes a protein that retains its original function. Incidentally, the expression "retains original function" for phoR proteins can indicate that the variant of the protein functions as a response regulator of the phoRS system. The term "function as a response regulator of the PhoRS system" can specifically refer to functions including, in combination with the sensor kinase PhoS protein, inducing a response to phosphate consumption in the environment. More specifically, the term "function as a response regulator of the PhoRS system" can refer to functions activated by the transfer of phosphate groups from the PhoS protein, which senses phosphate consumption in the environment and undergoes autophosphorylation, regulating the expression of genes responding to phosphate consumption in the environment.

[0211] For example, it can be determined whether a variant of the PhoR protein functions as a regulator of the PhoRS system by introducing the gene encoding the variant into a phoR-gene-deleted strain of Corynebacterium bacteria and confirming whether the response to phosphate consumption is complementary. Complementary responsiveness to phosphate consumption can be detected, for example, as an improvement in growth under phosphate-consuming conditions or by inducing the expression of genes known to be induced under phosphate-consuming conditions (J. Bacteriol., 188, 724-732 (2006)). For example, a phoR-gene-deleted strain of Corynebacterium glutamicum YDK010 or a phoR-gene-deleted strain of Corynebacterium glutamicum ATCC13032 can be used as a phoR-gene-deleted strain of Corynebacterium glutamicum.

[0212] <1-1-3> Decreased activity of cell surface proteins

[0213] The bacteria of this invention can be bacteria with reduced activity of cell surface proteins. The cell surface proteins and the genes encoding them will be described below.

[0214] Cell surface proteins are proteins that constitute the surface layer (S layer) of bacteria or archaea. Examples of cell surface proteins of Corynebacterium include PS1 and PS2 (CspB) of Corynebacterium glutamicum (Japanese Patent Publication No. 6-502548) and SlpA (CspA) of C. stationis (Japanese Patent Publication No. 10-108675). It is preferable to reduce the activity of PS2 protein in these.

[0215] The nucleotide sequence of the cspB gene of Corynebacterium glutamicum ATCC 13869 and the amino acid sequence of the PS2 protein (CspB protein) encoded by the gene are shown in SEQ ID NO: 67 and 68, respectively.

[0216] In addition, for example, the amino acid sequences of CspB homologs of 28 Corynebacterium glutamicum strains have been reported (J. Biotechnol., 112, 177-193 (2004)). The GenBank accession numbers of these 28 Corynebacterium glutamicum strains and their cspB gene homologs in the NCBI database are shown below (GenBank accession numbers are shown in parentheses).

[0217] Corynebacterium glutamicum ATCC 13058 (AY524990)

[0218] Corynebacterium glutamicum ATCC 13744 (AY524991)

[0219] Corynebacterium glutamicum ATCC 13745 (AY524992)

[0220] Corynebacterium glutamicum ATCC 14017 (AY524993)

[0221] Corynebacterium glutamicum ATCC 14020 (AY525009)

[0222] Corynebacterium glutamicum ATCC 14067 (AY524994)

[0223] Corynebacterium glutamicum ATCC 14068 (AY525010)

[0224] Corynebacterium glutamicum ATCC 14747 (AY525011)

[0225] Corynebacterium glutamicum ATCC 14751 (AY524995)

[0226] Corynebacterium glutamicum ATCC 14752 (AY524996)

[0227] Corynebacterium glutamicum ATCC 14915 (AY524997)

[0228] Corynebacterium glutamicum ATCC 15243 (AY524998)

[0229] Corynebacterium glutamicum ATCC 15354 (AY524999)

[0230] Corynebacterium glutamicum ATCC 17965 (AY525000)

[0231] Corynebacterium glutamicum ATCC 17966 (AY525001)

[0232] Corynebacterium glutamicum ATCC 19223 (AY525002)

[0233] Corynebacterium glutamicum ATCC 19240 (AY525012)

[0234] Corynebacterium glutamicum ATCC 21341 (AY525003)

[0235] Corynebacterium glutamicum ATCC 21645 (AY525004)

[0236] Corynebacterium glutamicum ATCC 31808 (AY525013)

[0237] Corynebacterium glutamicum ATCC 31830 (AY525007)

[0238] Corynebacterium glutamicum ATCC 31832 (AY525008)

[0239] Corynebacterium glutamicum LP-6 (AY525014)

[0240] Corynebacterium glutamicum DSM20137 (AY525015)

[0241] Corynebacterium glutamicum DSM20598 (AY525016)

[0242] Corynebacterium glutamicum DSM46307 (AY525017)

[0243] Corynebacterium glutamicum 22220 (AY525005)

[0244] Corynebacterium glutamicum 22243 (AY525006)

[0245] Since the nucleotide sequence of genes encoding cell surface proteins can vary depending on the species or strain of Corynebacterium, the gene encoding a cell surface protein can be a variant of any gene encoding the cell surface proteins exemplified above, as long as it retains its original function. Similarly, a cell surface protein can be a variant of any cell surface protein exemplified above, as long as it retains its original function. That is, the term "cspB gene" includes not only the cspB gene exemplified above, but also its conserved variants. Similarly, the term "CspB protein" includes not only the CspB protein exemplified above, but also its conserved variants. The above description of conserved variants of phoS genes and phoS proteins can be applied by analogy to variants of cell surface proteins and the genes encoding them. For example, a gene encoding a cell surface protein can be a gene encoding a protein having the above-described amino acid sequence, but including substitutions, deletions, insertions, or additions of one or more amino acid residues at one or more positions, as long as the gene encodes a protein that retains its original function. Incidentally, the expression "maintaining original function" used for cell surface proteins can mean that the protein has the property that, if the activity of the protein is reduced in Corynebacterium bacteria, the amount of heterologous protein secreted is increased compared to the amount that can be obtained using unmodified strains.

[0246] The phrase "the property of increasing the amount of heterologous protein secreted compared to that obtained using an unmodified strain when the activity of the protein is reduced in Corynebacterium bacteria" refers to the property that, when its activity is reduced in Corynebacterium bacteria, it endows Corynebacterium bacteria with the ability to produce a higher amount of heterologous protein through secretion than that obtained with an unmodified strain. "Unmodified strain" refers to a control strain in which the activity of the cell surface protein is not reduced, and it can be, for example, a wild-type strain or a parent strain. Although the degree of increase implied by "the amount of heterologous protein produced through secretion is higher than that obtained with an unmodified strain" is not particularly limited, as long as the secretion yield of the heterologous protein is increased compared to that obtained with an unmodified strain, this statement can mean, in terms of the accumulation in the culture medium and / or on the cell surface, that the amount of heterologous protein produced through secretion is, for example, 1.1 times or more, more preferably 1.2 times or more, further more preferably 1.3 times or more, and particularly preferably 2 times or more, than that obtained with an unmodified strain. Additionally, the term "the amount of heterologous protein produced by secretion is higher than that obtained with unmodified strains" can also mean that when the unconcentrated culture supernatant of unmodified strains is applied to SDS-PAGE and stained with CBB, heterologous proteins cannot be detected, while when the unconcentrated culture supernatant of modified strains is applied to SDS-PAGE and stained with CBB, heterologous proteins can be detected.

[0247] Whether a protein possesses the property of increasing the secretion yield of a heterologous protein compared to the amount obtained by an unmodified strain when the protein's activity is reduced in Corynebacterium bacteria can be confirmed by the following: preparing a modified strain that reduces the activity of a protein from a Corynebacterium strain, quantifying the observed secretion yield of the heterologous protein when the modified strain is cultured in a medium, and comparing this quantified amount with the amount of heterologous protein secreted when the unmodified strain is cultured in a medium.

[0248] In this invention, the statement "cell surface proteins are reduced" includes cases where Corynebacterium bacteria have been modified to reduce the activity of cell surface proteins, and cases where the activity of cell surface proteins is inherently reduced in Corynebacterium bacteria. "The activity of cell surface proteins is inherently reduced in Corynebacterium bacteria" includes cases where Corynebacterium bacteria inherently lack cell surface proteins. That is, examples of reduced activity of cell surface proteins in Corynebacterium bacteria include cases where Corynebacterium bacteria inherently lack cell surface proteins. "Corynebacterium bacteria inherently lack cell surface proteins" includes cases where Corynebacterium bacteria inherently lack genes encoding cell surface proteins. The statement "Corynebacterium bacteria inherently lack cell surface proteins" can mean that Corynebacterium bacteria inherently lack one or more proteins selected from cell surface proteins found in other strains belonging to the Corynebacterium species. For example, "inherently lacking cell surface proteins in Corynebacterium glutamicum" can mean that a Corynebacterium glutamicum strain inherently lacks one or more cell surface proteins selected from those found in other Corynebacterium glutamicum strains, i.e., for example, lacking PS1 and / or PS2 (CspB). Examples of Corynebacterium bacteria inherently lacking cell surface proteins include Corynebacterium glutamicum ATCC 13032, which inherently lacks the cspB gene.

[0249] The following sections describe methods for reducing the activity of proteins such as cell surface proteins. The methods described below for reducing protein activity can be used to disrupt the wild-type phoS gene.

[0250] The expression "reduced protein activity" means that the activity of the protein per cell is reduced compared to the unmodified strain. The term "unmodified strain" as used herein refers to a control strain that has not been modified to reduce the activity of the target protein. Examples of unmodified strains include wild-type strains and parental strains. The expression "reduced protein activity" also includes a state where protein activity has been completely lost. Specifically, the expression "reduced protein activity" means that the number of protein molecules per cell is reduced, and / or the function of each protein molecule is reduced compared to the unmodified strain. That is, the term "activity" in the expression "reduced protein activity" is not limited to the catalytic activity of the protein, but can also refer to the amount of transcription of the gene encoding the protein (i.e., the amount of mRNA) or the amount of translation of the protein (i.e., the amount of protein). The expression "the number of protein molecules per cell is reduced" also includes a state where the protein is not present at all. The expression "the function of each protein molecule is reduced" also includes a state where the function of each protein molecule has been completely lost. There is no particular limitation on the degree of reduction in protein activity, as long as the activity is reduced compared to the unmodified strain. Protein activity can be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the activity of the unmodified strain.

[0251] Modifications used to reduce protein activity can be achieved, for example, by reducing the expression of genes encoding proteins. The expression "reduced gene expression" means a decrease in gene expression per cell compared to unmodified strains, such as wild-type strains and parental strains. The expression "reduced gene expression" can specifically mean a decrease in the amount of gene transcription (i.e., the amount of mRNA) and / or a decrease in the amount of gene translation (i.e., the amount of protein expressed from the gene). The expression "reduced gene expression" also includes a state of complete gene non-expression. The expression "reduced gene expression" also refers to "attenuated gene expression." Gene expression can be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that in unmodified strains.

[0252] Decreased gene expression can be due to, for example, decreased transcription efficiency, decreased translation efficiency, or a combination thereof. Gene expression can be reduced by modifying expression control sequences such as promoters, Shine-Dalgarno (SD) sequences (also known as ribosome binding sites (RBS)), the spacer region between the RBS and the start site, and the gene's heuristic codon. When modifying expression control sequences, it is preferable to modify one or more nucleotides of the expression control sequence, more preferably two or more nucleotides, and particularly preferably three or more nucleotides. Furthermore, a portion or all of the expression control sequence can be deleted. Gene expression can also be reduced, for example, by manipulating factors responsible for expression control. Examples of factors responsible for expression control include low-molecular-weight molecules (inducers, inhibitors, etc.) responsible for transcription or translation control, proteins (transcription factors, etc.) responsible for transcription or translation control, nucleic acids (siRNA, etc.) responsible for transcription or translation control, and so on. Furthermore, gene expression can also be reduced, for example, by introducing mutations that reduce gene expression into the coding region of the gene. For example, gene expression can be reduced by replacing a codon in the coding region of the gene with a synonymous codon that is less frequently used in the host. In addition, gene expression can be reduced by disrupting genes, as described below.

[0253] Protein activity can be reduced by, for example, by disrupting the gene that encodes the protein. The expression "disrupt gene" means that the gene is modified to not produce a protein that functions normally. The expression "not producing a protein that functions normally" includes a state where no protein is produced from the gene at all, as well as a state where the function (such as activity or property) of each molecule of protein produced from the gene is reduced or eliminated.

[0254] Gene destruction can be achieved, for example, by deleting part or all of the coding region of a gene on a chromosome. Alternatively, the entire gene containing both upstream and downstream segments can be deleted. The deleted region can be any region, such as an N-terminal region, an internal region, or a C-terminal region, as long as it reduces protein activity. Deletion of longer regions generally results in more reliable gene inactivation. Furthermore, it is preferable that the upstream and downstream reading frames of the region to be deleted are different.

[0255] Gene disruption can also be achieved by introducing amino acid substitution mutations (missense mutations), stop codons (speechless mutations), frameshift mutations that add or delete one or two nucleotide residues into the coding region of a gene on a chromosome, etc. (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)).

[0256] Gene disruption can also be achieved, for example, by inserting another sequence into the coding region of a gene on a chromosome. The insertion site can be any region of the gene, and insertion into a longer region generally inactivates the gene more reliably. Preferably, the upstream and downstream reading frames of the sequence from the insertion site are different. Other sequences are not particularly limited, as long as they reduce or eliminate the activity of the encoded protein, and examples include, for example, marker genes, such as antibiotic resistance genes, and genes that contribute to the production of the target substance.

[0257] Such chromosomal gene modifications can be obtained, for example, by preparing a modified defective gene that cannot produce a normally functioning protein, and transforming the host with recombinant DNA containing the defective gene to induce homologous recombination between the defective gene and the wild-type gene on the chromosome, thereby replacing the wild-type gene on the chromosome with the defective gene. This process is facilitated if a marker gene selected according to host characteristics (such as a nutritional auxotroph) is included in the recombinant DNA. Examples of defective genes include genes with complete or partial gene deletions, genes with missense mutations, genes with transposon or marker gene insertions, genes with nonsense mutations, and genes with frameshift mutations. The structure of the recombinant DNA used for homologous recombination is not particularly limited, as long as it induces homologous recombination in the desired manner. For example, a linear DNA containing the defective gene and further containing upstream and downstream sequences of the wild-type gene on the chromosome at both ends can be used to induce homologous recombination, causing homologous recombination to occur upstream and downstream of the wild-type gene, thereby replacing the wild-type gene with the defective gene in one step. Proteins encoded by defective genes may have a different conformation than wild-type proteins after they are produced, and therefore their function may be reduced or eliminated. Gene disruption based on gene substitution utilizing homologous recombination has been established, and methods using linear DNA exist, such as the "Red-driven integration" method (Datsenko, KA, and Wanner, BL, Proc. Natl. Acad. Sci. USA, 97:6640-6645 (2000)), and methods utilizing a combination of Red-driven integration and excision systems derived from λ phage (Cho, EH, Gumport, RI, Gardner, JF, J. Bacteriol., 184:5200-5203 (2002)) (see WO2005 / 010175), methods using plasmids with temperature-sensitive origins of replication, methods using plasmids capable of conjugation transfer, and methods using suicide plasmids that do not have origins of replication that function in the host (US Patent No. 6,303,383, Japanese Patent Publication (Kokai) No. 05-007491), etc.

[0258] Protein activity can also be reduced through modifications such as mutagenesis. Examples of mutagenesis include X-ray or ultraviolet irradiation and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0259] A decrease in protein activity can be determined by measuring the protein's activity.

[0260] A decrease in protein activity can also be determined by identifying a reduction in the expression of the gene encoding the protein. A decrease in gene expression can be determined by identifying a reduction in the amount of gene transcription or a reduction in the amount of protein expressed from that gene.

[0261] A reduction in gene transcription can be confirmed by comparing the amount of mRNA transcribed from the gene to the amount transcribed in the unmodified strain. Examples of methods for evaluating mRNA levels include Northern hybridization, RT-PCR, etc. (Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). Preferably, the amount of mRNA is reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that in the unmodified strain.

[0262] The reduction in protein quantity can be determined by performing SDS-PAGE and determining the intensity of the separated protein bands. The reduction in protein quantity can also be determined by using Western blotting with an antibody (Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). Preferably, the protein quantity is reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

[0263] Depending on the methods used to disrupt the gene, disruption can be determined by measuring part or all of the gene, the restriction enzyme map of the gene, the full-length nucleotide sequence, etc.

[0264] <1-1-4> Protein Secretion System

[0265] The bacteria of the present invention possess a protein secretion system. The protein secretion system is not particularly limited, as long as it is capable of secreting the target protein. Examples of protein secretion systems include the Sec system (Sec secretion system) and the Tat system (Tat secretion system). The bacteria of the present invention may have been modified to enhance the protein secretion system. For example, the bacteria of the present invention may have been modified to enhance the expression of one or more genes selected from genes encoding the Tat secretion system. In the present invention, such modifications are also referred to as "enhancement of the Tat secretion system." Enhancement of the Tat secretion system is particularly preferred for cases where heterologous proteins are produced by using the secretory production of Tat-dependent signal peptides. A method for increasing the expression of genes encoding the Tat secretion system is described in Japanese Patent No. 4730302.

[0266] Examples of genes encoding the Tat secretion system include the tatA, tatB, and tatC genes of *Corynebacterium glutamicum*. The tatA, tatB, and tatC genes of *Corynebacterium glutamicum* ATCC 13032 correspond to complementary sequences at positions 1571065–1571382, 1167110–1167580, and 1569929–1570873, respectively, in the genome sequence registered in the NCBI database as GenBank entry NC_003450 (version NC_003450.3 GI:58036263). The TatA, TatB, and TatC proteins of *Corynebacterium glutamicum* ATCC 13032 have been registered in GenBank as NP_600707 (version NP_600707.1 GI:19552705, locus_tag="NCgl1434"), NP_600350 (version NP_600350.1 GI:19552348, locus_tag = "NCgl1077"), and NP_600706 (version NP_600706.1 GI:19552704, locus_tag="NCgl1433"), respectively. The nucleotide sequences of the tatA, tatB, and tatC genes of *Corynebacterium glutamicum* ATCC 13032, and the amino acid sequences of their TatA, TatB, and TatC proteins are shown in SEQ ID NO: 69-74.

[0267] Examples of genes encoding the Tat secretion system also include the tatA, tatB, tatC, and tatE genes in *E. coli*. The tatA, tatB, tatC, and tatE genes of *E. coli* K-12 MG1655 correspond to sequences at positions 4019968-4020237, 4020241-4020756, 4020759-4021535, and 658170-658373 in the genomic sequence registered in the NCBI database as GenBank accession NC_000913 (version NC_000913.2 GI:49175990), respectively. The TatA, TatB, TatC, and TatE proteins of *Escherichia coli* K-12 MG1655 have been registered in GenBank as follows: NP_418280 (version NP_418280.4 GI:90111653, locus_tag="b3836"), YP_026270 (version YP_026270.1 GI:49176428, locus_tag="b3838"), NP_418282 (version NP_418282.1 GI:16131687, locus_tag="b3839"), and NP_415160 (version NP_415160.1 GI:16128610, locus_tag="b0627").

[0268] Genes encoding the Tat secretion system can be variants of any of the genes encoding the Tat secretion system exemplified above, as long as they retain their original function. Similarly, the Tat secretion system can be a variant of any of the Tat secretion systems exemplified above, as long as it retains its original function. That is, the terms "tatA gene," "tatB gene," "tatC gene," and "tatE gene" include not only the tatA, tatB, tatC, and tatE genes exemplified above, but also their conserved variants. Similarly, the terms "TatA protein," "TatB protein," "TatC protein," and "TatE protein" include not only the TatA, TatB, TatC, and TatE proteins exemplified above, but also their conserved variants. The above description of conserved variants of the phoS gene and PhoS protein can be compared to the Tat secretion system and variants of the genes encoding it. For example, a gene encoding the Tat secretion system could be a gene encoding a protein having any of the aforementioned amino acid sequences, but including substitutions, deletions, insertions, or additions of one or more amino acid residues at one or more positions, as long as the gene encodes a protein that retains its original function. Incidentally, the expression "retains original function" for the Tat secretion system could mean that the system has the function of secreting a protein fused to an N-terminus with a Tat-dependent signal peptide out of the cell.

[0269] The following will describe methods for increasing the expression of genes, such as those encoding the Tat secretion system.

[0270] The expression "increased gene expression" means an increase in gene expression compared to an unmodified strain. In this document, the term "unmodified strain" refers to a control strain that has not been modified to increase the expression of the target gene. Examples of unmodified strains include wild-type strains and parental strains. The expression "increased gene expression" can specifically mean an increase in the amount of gene transcription (i.e., the amount of mRNA) and / or an increase in the amount of gene translation (i.e., the amount of protein expressed from the gene). The expression "increased gene expression" can also mean "enhanced gene expression." The degree of increase in gene expression is not particularly limited, as long as it is increased compared to an unmodified strain. Compared to the expression in an unmodified strain, gene expression is preferably increased by 1.5 times or more, more preferably 2 times or more, or more preferably 3 times or more. Furthermore, the expression "increased gene expression" not only indicates an increase in the expression level of the target gene in strains that inherently express the target gene, but also indicates the introduction of the gene into strains that do not inherently express the target gene. In other words, the phrase "increased gene expression" can also mean, for example, introducing a target gene into a strain that does not have the gene and expressing it therein.

[0271] Gene expression can be enhanced, for example, by increasing the number of gene copies.

[0272] Gene copy number can be increased by introducing genes into the host chromosome. Genes can be introduced into the chromosome, for example, using homologous recombination (Miller, JH, Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene transfer methods utilizing homologous recombination include methods using linear DNA, such as Red-driven integration (Datsenko, KA, and Wanner, BL, Proc. Natl. Acad. Sci. USA, 97:6640-6645 (2000)), methods using plasmids containing temperature-sensitive origins of replication, methods using plasmids capable of conjugation transfer, methods using suicide vectors that do not have origins of replication that function in the host, or methods using phage transduction. The structure of the recombinant DNA used for homologous recombination is not particularly limited, as long as it induces homologous recombination in the desired manner. For example, a host can be transformed with linear DNA containing a target gene and upstream and downstream sequences at both ends of a homologous recombination target region on the chromosome, such that homologous recombination occurs on each side upstream and downstream of the target region, thereby replacing the target region with an arbitrary sequence. The recombinant DNA used for homologous recombination may contain a marker gene for selecting transformants. Only one copy, or two or more copies of the gene, can be introduced. For example, multiple copies of a gene can be introduced into the chromosome by performing homologous recombination using a sequence present in multiple copies on the chromosome as the target. Such sequences present in multiple copies on the chromosome include repetitive DNA and inverted repeat sequences located at both ends of a transposon. Alternatively, homologous recombination can be performed by using a suitable sequence (such as a gene) on the chromosome that is not essential for the production of the target substance as the target. Furthermore, genes can be randomly introduced into the chromosome using transposons or Mini-Mu (Japanese Patent Publication No. 2-109985, US Patent No. 5,882,888, EP 805867 B1). Artificial transposons can also be used as transposons (Japanese Patent Publication No. 9-70291).

[0273] The target gene can be determined for introduction into the chromosome by Southern hybridization using probes with sequences complementary to the whole gene or a portion thereof, or by PCR based on gene sequence preparation.

[0274] Furthermore, the copy number of a gene can be increased by introducing a vector containing the gene into the host. For example, the copy number of the target gene can be increased by ligating a DNA fragment containing the target gene to a vector that functions in the host to construct a gene expression vector, and then transforming the host with the expression vector. For example, a DNA fragment containing the target gene can be obtained by PCR using microbial genomic DNA containing the target gene as a template. As a vector, a vector that replicates autonomously in the host cell can be used. The vector is preferably a multi-copy vector. Furthermore, the vector preferably has a marker such as an antibiotic resistance gene for selecting transformants. In addition, the vector may have a promoter and / or terminator for expressing the introduced gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a granule, a phage particle, etc. Specific examples of vectors capable of autonomous replication in Corynebacterium bacteria include pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); plasmids that improve upon these and possess drug resistance genes; plasmid pCRY30 described in Japanese Patent Publication No. 3-210184; and plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, and pCRY3KE described in Japanese Patent Publication No. 2-72876 and US Patent No. 5,185,262. And pCRY3KX; plasmids pCRY2 and pCRY3 described in Japanese Patent Publication No. 1-191686; pAJ655, pAJ611, and pAJ1844 described in Japanese Patent Publication No. 58-192900; pCG1 described in Japanese Patent Publication No. 57-134500; pCG2 described in Japanese Patent Publication No. 58-35197; pCG4 and pCG11 described in Japanese Patent Publication No. 57-183799; pVK7 described in Japanese Patent Publication No. 10-215883; and pVC7 described in Japanese Patent Publication No. 9-070291.

[0275] When a gene is introduced, it is sufficient that the gene can be expressibly held by the bacteria of the present invention. Specifically, it is sufficient that the gene is introduced such that it is expressed under the control of a promoter sequence that functions in the bacteria of the present invention. The promoter can be a host-derived promoter or a heterologous promoter. The promoter can be a natural promoter of the gene to be introduced or a promoter of another gene. Promoters that function in Corynebacterium bacteria as described below can be used as promoters.

[0276] A terminator for terminating gene transcription can be provided downstream of the gene. There are no particular limitations on the terminator, as long as it functions in the bacteria of this invention. The terminator can be a host-derived terminator or a heterologous terminator. The terminator can be a natural terminator of the gene to be introduced or a terminator of another gene.

[0277] The vectors, promoters, and terminators that can be used for various microorganisms are detailed in "Fundamental Microbiology Vol. 8, Genetic Engineering, KYORITSU SHUPPAN CO., LTD, 1987" and can be used.

[0278] Furthermore, when two or more genes are introduced, it is sufficient for each gene to be expressed by the bacterial family of the present invention. For example, all genes can be carried by a single expression vector or chromosome. Alternatively, genes can be carried separately by two or more expression vectors, or separately by one or two or more expression vectors and chromosomes. Operants consisting of two or more genes can also be introduced.

[0279] There are no particular restrictions on the gene to be introduced, as long as it encodes a protein that functions in the host. The gene to be introduced can be derived from the host or it can be a foreign gene. For example, the gene to be introduced can be obtained by PCR using primers designed based on the gene's nucleotide sequence and using genomic DNA of an organism that carries the gene, plasmids carrying the gene, etc., as templates. The gene to be introduced can be, for example, a completely synthesized gene based on its nucleotide sequence (Gene, 60(1), 115-127 (1987)). The obtained gene can be used on its own or modified as needed.

[0280] Furthermore, gene expression can be increased by improving gene transcription efficiency. Additionally, gene expression can be increased by improving gene translation efficiency. Gene transcription and translation efficiency can be improved, for example, by modifying the gene expression control sequence. The term "expression control sequence" collectively refers to sites that affect gene expression. Examples of expression control sequences include, for example, promoters, Shine-Dalgarno (SD) sequences (also known as ribosome binding sites (RBS)), and the spacer region between the RBS and the start codon. Expression control sequences can be identified using promoter search vectors or gene analysis software such as GENETYX. These expression control sequences can be modified, for example, by homologous recombination. Examples of methods using homologous recombination modification include methods using temperature-sensitive vectors or Red-driven integration methods (WO2005 / 010175).

[0281] Gene transcription efficiency can be improved, for example, by replacing the gene's promoter on the chromosome with a stronger promoter. A "stronger promoter" means a promoter that provides improved gene transcription compared to the gene's inherent wild-type promoter. Examples of strong promoters that can be used in Corynebacterium bacteria include the artificially modified P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679 (2000)), the pta, aceA, aceB, adh, and amyE promoters that are inducible in Corynebacterium bacteria using acetic acid, ethanol, pyruvate, etc., the cspB, SOD, and tuf (EF-Tu) promoters that can provide high expression levels in Corynebacterium bacteria (Journal of Biotechnology, 104 (2003) 311-323; Appl. Environ. Microbiol., 2005 Dec; 71(12):8587-96), as well as the lac, tac, and trc promoters. In addition, existing promoters with high activity can also be obtained as stronger promoters by using various reporter genes. For example, promoter activity can be enhanced by making the -35 and -10 regions of the shared sequence adjacent in the promoter region (WO00 / 18935). The paper by Goldstein et al. describes methods for evaluating promoter strength and examples of strong promoters (Prokaryotic Promoters in Biotechnology, Biotechnol. Annu. Rev., 1, 105-128 (1995)).

[0282] For example, gene transcription efficiency can be improved by replacing the Shine-Dalgarno (SD) sequence (also known as the ribosome binding site (RBS)) of a gene on the chromosome with a stronger SD sequence. A “stronger SD sequence” refers to an SD sequence that provides improved mRNA translation compared to the inherent wild-type SD sequence of the gene. Examples of stronger SD sequences include, for example, the RBS of gene 10 derived from bacteriophage T7 (Olins PO et al, Gene, 1988, 73, 227-235). Furthermore, substitutions, insertions, or deletions of several nucleotides in the spacer region between the RBS and the start codon (particularly in the sequence immediately upstream of the start codon (5'-UTR)) are known to significantly affect mRNA stability and translation efficiency, and therefore can also be modified to improve gene translation efficiency.

[0283] Gene translation efficiency can also be improved, for example, by modifying codons. For instance, rare codons present in a gene can be replaced with more frequently used synonyms. That is, the gene to be introduced can be modified to contain the optimal codons, for example, based on the frequency of codons observed in the host used. Codons can be replaced, for example, by site-specific mutagenesis methods used to introduce the desired mutation into the target site of DNA. Examples of site-specific mutagenesis methods include those using PCR (Higuchi, R., 61, in PCR Technology, Erlich, HA Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and those using bacteriophages (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)). Alternatively, the gene segment in which the target codon is replaced can be synthesized entirely. The frequencies of codons in various organisms are published in the "Codon Usage Database" (http: / / www.kazusa.or.jp / codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)).

[0284] In addition, gene expression can be increased by amplifying regulators that increase gene expression, or by deleting or weakening regulators that decrease gene expression.

[0285] The methods described above for increasing gene expression can be used independently or in any combination.

[0286] There are no particular limitations on the methods used for transformation, and conventionally known methods can be used. For example, the method of treating recipient cells with calcium chloride to increase DNA permeability has been reported for Escherichia coli K-12 strain (Mandel, M. and Higa, A., J. Mol. Biol., 1970, 53, 159-162), and the method of preparing competent cells from cells in the growth phase and then transforming them with DNA has been reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE, Gene, 1977, 1:153-167). Alternatively, DNA recipient cells can be prepared as protoplasts or protoplasts, which can readily take up recombinant DNA and then introduce it into the DNA recipient cells. This method is known to be applicable to Bacillus subtilis, actinomycetes, and yeasts (Chang, S. and Choen, SN, 1979, Mol. Gen. Genet., 168:111-115; Bibb, MJ, Ward, JMand Hopwood, OA, 1978, Nature, 274:398-400; Hinnen, A., Hicks, JB and Fink, GR, 1978, Proc. Natl. Acad. Sci. USA, 75:1929-1933). Corynebacterium bacteria can be transformed using methods such as protoplast method (Gene, 39, 281-286 (1985)), electroporation method (Bio / Technology, 7, 1067-1070 (1989)), and electropulse method (Japanese Patent Publication No. 2-207791).

[0287] An increase in gene expression can be determined, for example, by determining an increase in the activity of the protein expressed from the gene. An increase in protein activity can be determined by measuring the protein's activity. For example, an increase in the activity of the Tat secretion system can be determined by determining an increase in the secretion yield of a protein fused to a Tat-dependent signal peptide at its N-terminus. In such cases, it is preferable that the secretion yield of the protein fused to the Tat-dependent signal peptide at its N-terminus is increased to, for example, 1.5 times or more, 2 times or more, or 3 times or more than that of an unmodified strain.

[0288] An increase in gene transcription can also be determined by comparing the amount of mRNA transcribed from the gene to that of an unmodified strain, such as a wild-type strain or a parental strain. Examples of methods used to evaluate the amount of mRNA include Northern hybridization, RT-PCR, etc. (Sambrook, J., et al., Molecular Cloning A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). Preferably, the amount of mRNA is increased to, for example, 1.5 times or more, 2 times or more, or 3 times or more compared to an unmodified strain.

[0289] The amount of protein can be determined by performing SDS-PAGE and determining the intensity of the separated protein bands. An increase in protein amount can be determined by Western blotting using antibodies (Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). Preferably, the amount of protein is increased to, for example, 1.5 times or more, 2 times or more, or 3 times or more than that of the unmodified strain.

[0290] <1-1-5> Genetic constructs for heterologous protein secretion expression

[0291] Secretory proteins are known to be typically translated into preproteins (also called prepeptides) or preproproteins (also called prepropeptides) and then processed into mature proteins. Specifically, secretory proteins are typically translated into preproteins or preproproteins, which are then cleaved by a protease (commonly called a signal peptidase) as the premoiety, thus converting the secretory protein into a mature protein or proprotein. For proproteins, the premoiety is further cleaved by the protease, and the preprotein becomes a mature protein. Therefore, in the method of this invention, the signal peptide is used for the secretory production of heterologous proteins. In this invention, the preprotein and preproprotein of secretory proteins can be collectively referred to as "secretory protein precursors." In this invention, a "signal peptide" (also called a "signal sequence") refers to the N-terminus of a secretory protein precursor and typically does not have the same amino acid sequence as that found in native mature proteins.

[0292] The genetic construct used in this invention comprises, in a 5' to 3' orientation, a promoter sequence that functions in Corynebacterium bacteria, a signal peptide sequence that functions in Corynebacterium bacteria, and a nucleic acid sequence encoding a heterologous protein. The signal peptide can be linked downstream of the promoter sequence to express the signal peptide under the control of the promoter. A nucleic acid sequence encoding a heterologous protein can be linked downstream of the nucleic acid sequence encoding the signal peptide to express the heterologous protein as a fusion protein with the signal peptide. This fusion protein is also referred to as the "fusion protein of this invention." In the fusion protein of this invention, the signal peptide and the heterologous protein may be adjacent to each other or not. That is, the statement "expression of a heterologous protein as a fusion protein with a signal peptide" includes not only the case where the heterologous protein is expressed as a fusion protein with the signal peptide, where the signal peptide and the heterologous protein are adjacent to each other, but also the case where the heterologous protein is expressed as a fusion protein with the signal peptide, where the heterologous protein is fused to the signal peptide via another amino acid sequence. For example, as described below, the fusion protein of this invention may contain an insert sequence between the signal peptide and the heterologous protein, such as an amino acid sequence comprising Gln-Glu-Thr and an amino acid sequence for enzymatic digestion. The nucleic acid sequence may also be referred to as a "gene." For example, a nucleic acid sequence encoding a heterologous protein is also referred to as a "gene encoding a heterologous protein" or a "heterologous protein gene." Examples of nucleic acid sequences include DNA. The genetic constructs used in this invention may also include control sequences (operons, terminators, etc.) that are effective in expressing the fusion protein of this invention at appropriate positions in Corynebacterium.

[0293] The promoters used in this invention are not particularly limited, as long as they are promoters that function in Corynebacterium bacteria. The promoter can be a promoter derived from Corynebacterium bacteria (such as a promoter derived from the host), or it can be a heterologous promoter. The promoter can be a natural promoter of a heterologous protein, or it can be a promoter of another gene. "A promoter that functions in Corynebacterium bacteria" refers to a promoter that has promoter activity in Corynebacterium bacteria.

[0294] Specific examples of heterologous promoters include, for example, promoters derived from *E. coli*, such as the tac promoter, lac promoter, trp promoter, and araBAD promoter. Among these, strong promoters such as the tac promoter and inducible promoters such as the araBAD promoter are preferred.

[0295] Examples of promoters derived from Corynebacterium bacteria include promoters of cell surface proteins PS1, PS2 (also known as CspB), and SlpA (also known as CspA) genes, as well as promoters of genes for various amino acid biosynthesis systems. Specific examples of promoters for various amino acid biosynthesis systems include, for example, the promoters of the following genes: glutamate dehydrogenase gene for the glutamate biosynthesis system, glutamine synthase gene for the glutamine biosynthesis system, aspartate kinase gene for the lysine biosynthesis system, homoserine dehydrogenase gene for the threonine biosynthesis system, acetylhydroxy acid synthase gene for the isoleucine and valine biosynthesis systems, 2-isopropylmalate synthase gene for the leucine biosynthesis system, glutamate kinase gene for the proline and arginine biosynthesis systems, phosphoribosyl-ATP pyrophosphorylase gene for the histidine biosynthesis system, deoxyarabinosyl phosphate (DAHP) synthase gene for aromatic amino acid biosynthesis systems such as tryptophan, tyrosine, and phenylalanine, phosphoribosyl pyrophosphate (PRPP) amidotransferase gene for nucleic acid biosynthesis systems (such as those of inosinic acid and guanylic acid), inosinic acid dehydrogenase gene, and guanylic acid synthase gene.

[0296] Examples of promoters that function in Corynebacterium include strong promoters for use in Corynebacterium as described above. As promoters, highly active types of existing promoters can be obtained and used by employing various reporter genes. For example, promoter activity can be enhanced by making the -35 and -10 regions of the shared sequence adjacent to each other in the promoter region (WO00 / 18935). The paper by Goldstein et al. describes methods for assessing promoter strength and examples of strong promoters (Prokaryotic Promoters in Biotechnology, Biotechnol. Annu. Rev., 1, 105-128 (1995)). Furthermore, it is known that substitutions, insertions, or deletions of several nucleotides in the spacer region between the ribosome binding site (RBS) and the start codon, particularly in the sequence immediately upstream of the start codon (5'-UTR), significantly affect mRNA stability and translation efficiency, and these sequences can also be modified.

[0297] The signal peptide used in this invention is not particularly limited, as long as it functions in Corynebacterium bacteria. The signal peptide can be a signal peptide derived from Corynebacterium bacteria, such as a host-derived signal peptide, or it can be a heterologous signal peptide. The signal peptide can be a natural signal peptide of a heterologous protein or a signal peptide of another gene. "A signal peptide that functions in Corynebacterium bacteria" means that when it is attached to the N-terminus of a target protein, it allows the Corynebacterium bacteria to secrete the protein. The function of the signal peptide in Corynebacterium bacteria can be determined, for example, by expressing the target protein in a form fused with the signal peptide and confirming whether the protein is secreted.

[0298] Examples of signal peptides include Tat-dependent signal peptides and Sec-dependent signal peptides.

[0299] The term "Tat-dependent signal peptide" refers to a signal peptide recognized by the Tat system. Specifically, the term "Tat-dependent signal peptide" can refer to a signal peptide that, when linked to the N-terminus of a target protein, causes the secretion of that protein by the Tat secretion system.

[0300] Examples of Tat-dependent signal peptides include: the signal peptide of TorA protein (trimethylamine-N-oxidoreductase) from *Escherichia coli*, the signal peptide of SufI protein (an inhibitor of ftsI) from *E. coli*, the signal peptide of PhoD protein (phosphodiesterase) from *Bacillus subtilis*, the signal peptide of LipA protein (lipoic acid synthase) from *Bacillus subtilis*, and the signal peptide of IMD protein (isomaltodextrin) from *Arthrobacter spheroides*. The amino acid sequences of these signal peptides are as follows.

[0301] TorA signal peptide:

[0302] MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLTPRRATA (SEQ ID NO: 75)

[0303] SufI signal peptide:

[0304] MSLSRRQFIQASGIALCAGAVPLKASA (SEQ ID NO: 76)

[0305] PhoD signal peptide:

[0306] MAYDSRFDEWVQKLKEESFQNNTFDRRKFIQGAGKIAGLSLLGLTIAQS (SEQ ID NO: 77)

[0307] LipA ​​signal peptide:

[0308] MKFVKRRTTALVTTLMLSVTSLFALQPSAKAAEH (SEQ ID NO: 78)

[0309] IMD signal peptide:

[0310] MMNLSRRTLLTTGSAATLAYALGMAGSAQA (SEQ ID NO: 79)

[0311] Tat-dependent signal peptides possess a twin-arginine motif. Examples of twin-arginine motifs include S / TRRXFLK (SEQ ID NO: 80) and RRX-#-# (#: hydrophobic residue) (SEQ ID NO: 81).

[0312] The term "Sec-dependent signal peptide" refers to a signal peptide recognized by the Sec system. Specifically, the term "Sec-dependent signal peptide" can refer to a signal peptide that, when linked to the N-terminus of a target protein, causes the secretion of that protein by the Sec secretory system.

[0313] Examples of Sec-dependent signal peptides include signal peptides of cell surface proteins from Corynebacterium bacteria. Cell surface proteins from Corynebacterium bacteria are as described above. Examples of cell surface proteins from Corynebacterium bacteria include PS1 and PS2 (CspB) from Corynebacterium glutamicum (Japanese Patent Publication No. 6-502548) and SlpA (CspA) from Corynebacterium stationis (Japanese Patent Publication No. 10-108675). The amino acid sequence of the signal peptide of Corynebacterium glutamicum PS1 (PS1 signal peptide) is shown in SEQ ID NO: 82, the amino acid sequence of the signal peptide of Corynebacterium glutamicum PS2 is shown in SEQ ID NO: 83, and the amino acid sequence of SlpA (CspA) (SlpA signal peptide) from Corynebacterium stationis is shown in SEQ ID NO: 84. Furthermore, U.S. Patent No. 4,965,197 describes the presence of signal peptides for DNases derived from Corynebacterium bacteria, and such signal peptides can also be used in this invention.

[0314] Tat-dependent signal peptides can be variants of any of the Tat-dependent signal peptides exemplified above, provided they contain a diarginine motif and retain their original function. Sec-dependent signal peptides can be variants of any of the Sec-dependent signal peptides exemplified above, provided they contain a diarginine motif and retain their original function. The above description of conserved variants of the phoS gene and PhoS protein can be applied in parallel to variants of the signal peptide and the gene encoding it. For example, a signal peptide can be a peptide having any of the amino acid sequences described above, but including substitutions, deletions, insertions, or additions of one or more amino acid residues at one or more positions. The number for the term "one or more" used for signal peptide variants is particularly preferred to be 1 to 7, more preferably 1 to 5, even more preferably 1 to 3, and particularly preferably 1 to 2. In this 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" not only include the peptides of SEQ ID NO: 75, 76, 77, 78, 79, 82, 83 and 84, respectively, but also include their conserved variants.

[0315] The expression "retains original function" for Tat-dependent signal peptides means that the peptide is recognized by the Tat system, and more particularly, it can mean a peptide that, when linked to the N-terminus of a target protein, has the function of causing that protein to be secreted via the Tat secretion system. Whether a peptide functions as a Tat-dependent signal peptide can be determined, for example, by confirming an increase in the secretion of proteins linked to the N-terminus due to an enhanced Tat secretion system, or by confirming an increase in the secretion of proteins linked to the N-terminus due to the absence of the Tat secretion system.

[0316] The expression "retains original function" for a Sec-dependent signal peptide means that the peptide is recognized by the Sec system, and in particular, it can mean a peptide that, when linked to the N-terminus of a target protein, has the function of causing that protein to be secreted through the Sec secretion system. Whether a peptide functions as a Sec-dependent signal peptide can be determined, for example, by confirming an increase in the secretion of proteins linked to the N-terminus due to enhancement of the Sec secretion system, or by confirming an increase in the secretion of proteins linked to the N-terminus due to the absence of the Sec secretion system.

[0317] When the translation product is secreted extracellularly, the signal sequence is typically cleaved by a signal peptidase. While naturally occurring genes encoding signal peptides can be used directly, they can be modified to have optimal codons based on the codon frequencies in the host cell.

[0318] In the genetic constructs used in this invention, a nucleic acid encoding a Gln-Glu-Thr amino acid sequence may be inserted between a nucleic acid sequence encoding a signal peptide and a nucleic acid sequence encoding a heterologous protein (WO2013 / 062029). "An amino acid sequence containing Gln-Glu-Thr" is also referred to as "the insert sequence used in this invention." Examples of insert sequences used in this invention include the Gln-Glu-Thr amino acid sequence described in WO2013 / 062029. In particular, the insert sequence used in this invention may preferably be used in combination with a Sec-dependent signal peptide.

[0319] The insertion sequence used in this invention is preferably a sequence consisting of three or more amino acid residues from the N-terminus of a mature protein of Corynebacterium cell surface protein CspB (hereinafter also referred to as "mature CspB" or "CspB mature protein"). The term "preferably a sequence consisting of three or more amino acid residues from the N-terminus" refers to an amino acid residue starting at position 1 at the N-terminus and continuing to three or more distal amino acid residues.

[0320] The cell surface protein CspB of Corynebacterium glutamicum is as described above. Specific examples of CspB include, for example, the CspB of Corynebacterium glutamicum ATCC 13869, the CspB of the 28 Corynebacterium glutamicum strains mentioned above, and their variants. The amino acid sequence of the CspB protein of Corynebacterium glutamicum ATCC 13869 is shown in SEQ ID NO: 68, where amino acid residues at positions 1 to 30 correspond to the signal peptide, and amino acid residues at positions 31 to 499 correspond to the mature CspB protein. The amino acid sequence of the mature CspB protein of Corynebacterium glutamicum ATCC 13869 (excluding the 30 amino acid residues that constitute the signal peptide portion) is shown in SEQ ID NO: 85. In the mature CspB of Corynebacterium glutamicum ATCC 13869, amino acid residues at positions 1 to 3 at the N-terminus correspond to Gln-Glu-Thr.

[0321] The insertion sequence used in this invention is preferably an amino acid sequence starting from position 1 of mature CspB and extending to any amino acid residues at positions 3-50. More preferably, the insertion sequence used in this invention is an amino acid sequence starting from position 1 of mature CspB and extending to any amino acid residues at positions 3-8, 17, and 50. Particularly preferred is an amino acid sequence starting from amino acid residue 1 and extending to any amino acid residues at positions 4, 6, 17, and 50.

[0322] The insertion sequence used in this invention is preferably an amino acid sequence selected from the group consisting of the following amino acid sequences (A) to (H):

[0323] (A) Gln-Glu-Thr

[0324] (B) Gln-Glu-Thr-Xaa1 (SEQ ID NO: 86)

[0325] (C) Gln-Glu-Thr-Xaa1-Xaa2 (SEQ ID NO: 87)

[0326] (D) Gln-Glu-Thr-Xaa1-Xaa2-Xaa3 (SEQ ID NO: 88)

[0327] (E) An amino acid sequence consisting of Gln-Glu-Thr fused with amino acid residues at positions 4-7 of mature CspB.

[0328] (F) The amino acid sequence consisting of Gln-Glu-Thr, which is fused with amino acid residues at positions 4-8 of the mature CspB.

[0329] (G) An amino acid sequence consisting of Gln-Glu-Thr fused with amino acid residues at positions 4-17 of mature CspB.

[0330] (H) An amino acid sequence consisting of Gln-Glu-Thr fused with amino acid residues at positions 4-50 of the mature CspB.

[0331] In amino acid sequences (A) through (H), Xaa1 is Asn, Gly, Thr, Pro, or Ala; Xaa2 is Pro, Thr, or Val; and Xaa3 is Thr or Tyr. For amino acid sequences (A) through (H), "Gln-Glu-Thr fused with amino acid residues at positions 4-X of mature CspB" means that the amino acid residues at positions 4-X of the N-terminus of mature CspB are fused with Thr of Gln-Glu-Thr. The first to third amino acid residues at the N-terminus of mature CspB are usually Gln-Glu-Thr, and in this case, "amino acid sequence consisting of Gln-Glu-Thr fused with amino acid residues at positions 4-X of mature CspB" has the same meaning as amino acid sequence consisting of amino acid residues at positions 1-X of mature CspB.

[0332] Furthermore, specifically, the insertion sequence used in this invention is preferably an amino acid sequence selected from the group consisting of: Gln-Glu-Thr-Asn-Pro-Thr (SEQ ID NO: 89), Gln-Glu-Thr-Gly-Thr-Tyr (SEQ ID NO: 90), Gln-Glu-Thr-Thr-Val-Thr (SEQ ID NO: 91), Gln-Glu-Thr-Pro-Val-Thr (SEQ ID NO: 92), and Gln-Glu-Thr-Ala-Val-Thr (SEQ ID NO: 93).

[0333] In this invention, "the amino acid residue at position X of mature CspB" refers to the amino acid residue corresponding to position X in SEQ ID NO: 85. The amino acid residue in any mature CspB sequence that corresponds to "the amino acid residue at position X in SEQ ID NO: 85" can be determined by comparing the amino acid sequence of any mature CspB with the amino acid sequence of SEQ ID NO: 85.

[0334] Examples of heterologous proteins produced by secretion according to the method of the present invention include, for example, physiologically active proteins, receptor proteins, antigen proteins used as vaccines, and enzymes.

[0335] Examples of enzymes include transglutaminase, protein glutaminase, isomaltodextrinase, protease, endopeptidase, exopeptidase, aminopeptidase, carboxypeptidase, collagenase, chitinase, etc. Examples of transglutaminases include secretory transglutaminases of actinomycetes and any filamentous fungi (such as oomycetes (WO96 / 22366)), such as *Streptoverticillium mobaraense* IFO 13819 (WO01 / 23591), *Streptoverticillium cinnamoneum* IFO 12852, *Streptoverticillium griseocarneum* IFO 12776, and *Streptomyces lydicus* (WO96 / 06931). Examples of protein glutaminases include protein glutaminase of *Chryseobacterium proteolyticum* (WO2005 / 103278). Examples of protein isomaltodextrinases include isomaltodextrinase from Arthrobacter spp. (WO2005 / 103278).

[0336] Examples of physiologically active proteins include, for example, growth factors, hormones, cytokines, and antibody-related molecules.

[0337] Specific examples of 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), vesicular 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 or FGF1), basic fibroblast growth factor (bFGF or FGF2), keratinocyte growth factor (KGF-1 or FGF7, and KGF-2 or FGF10), and hepatocyte growth factor (HGF).

[0338] Specific examples of hormones include, for example, insulin, glucagon, somatostatin, human growth hormone (hGH), parathyroid hormone (PTH), calcitonin, and exenatide.

[0339] Specific examples of cytokines include, for example, interleukins, interferons, and tumor necrosis factors (TNFs).

[0340] Growth factors, hormones, and cytokines do not need to be strictly distinguished from each other. For example, physiologically active proteins may belong to a single group selected from growth factors, hormones, and cytokines, or they may belong to multiple groups selected from them.

[0341] Furthermore, physiologically active proteins can be complete proteins or a portion of a protein. Examples of a portion of a protein include, for example, a physiologically active part. Specific examples of physiologically active parts include, for example, teriparatide, a physiologically active peptide composed of the N-terminal 34 amino acid residues of parathyroid hormone (PTH).

[0342] The term "antibody-associated molecule" refers to a protein containing a group of molecules composed of a single domain or a combination of two or more domains selected from those constituting a complete antibody. Examples of domains constituting a complete antibody include heavy chain domains VH, CH1, CH2, and CH3 and light chain domains VL and CL. Antibody-associated molecules can be monomeric or multimeric proteins, as long as they contain the aforementioned molecular groups. When an antibody-associated molecule is a multimeric protein, it can be a homopolymer composed of a single subunit or a heteropolymer composed of two or more subunits. Specific examples of antibody-associated molecules include, for example, complete antibodies, Fab, F(ab'), F(ab')2, Fc, dimers composed of a heavy chain (H chain) and a light chain (L chain), Fc-fusion proteins, heavy chain (H chain), light chain (L chain), light chain Fv (scFv), sc(Fv)2, disulfide-bonded Fv (sdFv), and bifunctional antibodies.

[0343] There are no particular restrictions on receptor proteins. Receptor proteins can be, for example, any physiologically active protein or receptor for other physiologically active substances. Examples of other physiologically active substances include neurotransmitters such as dopamine. Furthermore, receptor proteins can be orphan receptors with unknown corresponding ligands.

[0344] There are no particular restrictions on the antigen proteins used in vaccines, as long as they are proteins that can induce an immune response. The antigen proteins can be appropriately selected based on the intended target of the immune response.

[0345] In addition, other examples of proteins include liver-type fatty acid-binding protein (LFABP).

[0346] The genes encoding these proteins can be modified to obtain the desired activity depending on the host to which they are intended. For example, the genes encoding these proteins can be modified individually to include the addition, deletion, substitution, etc., of one or more amino acid residues. The above description of variants of the PhoS protein and phoS gene can be applied by analogy to heterologous proteins produced by secretion using the methods of the present invention and the genes encoding them. Furthermore, in the genes encoding these proteins, any codons can be replaced with equivalent codons. For example, in the genes encoding these proteins, codons can be optimized as needed based on codon frequencies observed in the host.

[0347] The genetic construct of the present invention may further include a nucleic acid sequence encoding an amino acid sequence for enzymatic digestion between a nucleic acid sequence encoding an amino acid sequence comprising Gln-Glu-Thr and a nucleic acid sequence encoding a heterologous protein. If the amino acid sequence for enzymatic digestion is inserted into the fusion protein of the present invention, the expressed fusion protein can be enzymatically digested to obtain the target heterologous protein.

[0348] There are no particular restrictions on the amino acid sequence used for enzymatic digestion, as long as it is a sequence that can be recognized and digested by enzymes that hydrolyze peptide bonds. Furthermore, the usable sequence can be appropriately selected based on the amino acid sequence of the target heterologous protein. Nucleic acid sequences encoding the amino acid sequence used for enzymatic digestion can be designed based on the amino acid sequence used for digestion, and, for example, optimal codons can be used based on codon frequencies observed in the host.

[0349] The amino acid sequence used for enzymatic digestion is preferably a recognition sequence of a protease exhibiting high substrate specificity. Specific examples of such amino acid sequences include, for example, the recognition sequences of factor Xa protease and proTEV protease. Factor Xa protease and proTEV protease recognize the amino acid sequences Ile-Glu-Gly-Arg (= IEGR, SEQ ID NO: 94) and Glu-Asn-Leu-Tyr-Phe-Gln (= ENLYFQ, SEQ ID NO: 95) in proteins, respectively, to specifically cleave the protein at the C-terminus of each recognition sequence.

[0350] The N-terminal region of the heterologous protein finally obtained by the method of the present invention may be the same as that of the natural protein, or it may not be the same as that of the natural protein. For example, the N-terminal region of the finally obtained heterologous protein may be the N-terminal region of a natural protein including the addition or deletion of one or more amino acid residues. Although the number of "one or more" amino acid residues may vary depending on the full length or structure of the target heterologous protein, it is particularly preferred to be 1 to 20, more preferably 1 to 10, further preferably 1 to 5, and particularly preferably 1 to 3.

[0351] Furthermore, the heterologous protein to be produced through secretory production can be a protein containing the original structural moiety (the original protein). When the heterologous protein to be produced through secretory production is the original protein, the final heterologous protein may or may not be the original protein. That is, the original protein can be processed into a mature protein by cleaving the pre-structural moiety. Cleavage can be achieved, for example, with a protease. When using a protease, generally, considering the activity of the final protein, the original protein is cleaved at a position substantially the same as the natural protein, or more preferably at the exact same position as the natural protein, to obtain a mature protein identical to the naturally occurring mature protein. Therefore, generally, a specific protease that cleaves the original protein at the position that produces a protein identical to the naturally occurring mature protein is most preferred. However, the N-terminal region of the final heterologous protein may differ from the N-terminal region of the natural protein as described above. For example, depending on the type, purpose, etc., of the heterologous protein to be produced, a protein with a longer or shorter N-terminus by one to several amino acid residues may have more suitable activity compared to the natural protein. Proteases that can be used in this invention include, for example, commercially available proteases such as Dispase (produced by Boehringer Mannheim) and those obtainable from microbial cultures (such as actinomycete cultures). Such proteases can be used in their unpurified state or, if necessary, purified to an appropriate purity. When the original structural moiety is cleaved to obtain the mature protein, the inserted amino acid sequence containing Gln-Glu-Thr is removed from the original structural moiety, and thus the target protein can be obtained without providing an amino acid sequence downstream of the amino acid sequence containing Gln-Glu-Thr for enzymatic digestion.

[0352] There are no particular limitations on the method of introducing the genetic construct used in this invention into Corynebacterium bacteria. The term "introducing the genetic construct used in this invention" refers to creating a host carrying the construct. The term "introducing the genetic construct used in this invention" includes not only the case of introducing the initially constructed genetic construct into the host, but also the case of introducing at least a heterologous protein gene into the host and constructing the genetic construct in the host. In the bacteria of this invention, the genetic construct used in this invention can exist in an extrachromosomal autonomously replicating vector (e.g., plasmid), or can be incorporated into the chromosome. For example, the genetic construct used in this invention can be introduced in a manner similar to the introduction of genes for increasing the expression of the aforementioned genes. Furthermore, in order to construct the bacteria of this invention, the introduction of the genetic structure used in this invention, the introduction of the mutant phoS gene, and other modifications can be performed in any order.

[0353] The genetic constructs used in this invention can be introduced into a host, for example, by using a vector containing the genetic construct. For instance, the genetic construct can be introduced into a host by connecting the genetic construct to a vector to construct an expression vector of the genetic construct, and then transforming the host with the expression vector. Furthermore, when the vector contains a promoter that functions in Corynebacterium bacteria, an expression vector for the genetic construct used in this invention can be constructed by linking a nucleic acid sequence encoding the fusion protein of this invention downstream of the promoter. There are no particular limitations on the vector, as long as it is a vector that replicates autonomously in Corynebacterium bacteria. Vectors that can be used in Corynebacterium bacteria are as described above.

[0354] Furthermore, genetic constructs used in this invention can be introduced into a host chromosome using, for example, transposons (e.g., artificial transposons). When using transposons, the genetic constructs used in this invention are introduced into the chromosome through the transposon's own homologous recombination or translocation capabilities. Additionally, genetic constructs used in this invention can be introduced into the chromosome using other introduction methods utilizing homologous recombination. Examples of introduction methods utilizing homologous recombination include methods using linear DNA, plasmids with temperature-sensitive replication origins, plasmids capable of conjugation transfer, suicide plasmids without replication origins that function in the host, etc. Furthermore, at least a heterologous protein gene can be introduced into the chromosome so that the genetic constructs used in this invention are constructed on the chromosome. In this case, some or all of the components of the genetic construct, except for the heterologous protein gene, may be inherently present on the host chromosome. Specifically, for example, by using a promoter sequence inherently present on the host chromosome and a nucleic acid sequence encoding a signal peptide inherently present on the host chromosome and linked downstream of the promoter, and by replacing only the gene linked downstream of the nucleic acid sequence encoding the signal peptide with the target heterologous protein gene, a genetic construct for this invention can be constructed on the chromosome, and thus the bacteria of this invention can be constructed. A portion of the genetic construct used in this invention (e.g., a heterologous protein gene) can be introduced into the chromosome in the same manner as the genetic construct used in this invention for chromosome introduction.

[0355] Genetic constructs or components thereof used in this invention can be obtained, for example, by cloning, such as promoter sequences, nucleic acid sequences encoding signal peptides, or nucleic acid sequences encoding heterologous proteins. Specifically, for example, a genetic construct for use in this invention can be obtained by cloning a gene for a target heterologous protein from an organism possessing that protein, followed by modifications such as introducing a nucleic acid sequence encoding a signal peptide and introducing a promoter sequence. Alternatively, genetic constructs or components thereof used in this invention can be obtained through chemical synthesis. The resulting genetic constructs used in this invention or its components can be used on their own or modified as needed.

[0356] Furthermore, when expressing two or more proteins, it is sufficient for the bacteria of the present invention to maintain the genetic constructs for secreting the expressed proteins so that the secretion of the target heterologous protein can be achieved. Specifically, for example, all genetic constructs for secretory protein expression can be maintained on a single expression vector or on a chromosome. Alternatively, the genetic constructs for secretory protein expression can be maintained on multiple expression vectors, or on one or more expression vectors and chromosomes, respectively. "The case of expressing two or more proteins" refers, for example, to the case of producing two or more heterologous proteins through secretory production, or the case of producing heteropolymeric proteins through secretory production.

[0357] The method of introducing the genetic construct used in this invention into Corynebacterium bacteria is not particularly limited, and commonly used methods such as protoplast method (Gene, 39, 281-286 (1985)), electroporation method (Bio / Technology, 7, 1067-1070 (1989)), and electropulse method (Japanese Patent Publication No. 2-207791) can be used.

[0358] <1-2> Methods for producing heterologous proteins

[0359] By culturing the bacteria of the present invention obtained as described above to express heterologous proteins, a large amount of heterologous proteins secreted from cells are obtained.

[0360] The bacteria of the present invention can be cultured according to commonly used methods and conditions. For example, the bacteria of the present invention can be cultured in a conventional culture medium containing a carbon source, a nitrogen source, and inorganic ions. To obtain higher proliferation rates, organic micronutrients such as vitamins and amino acids can be added as needed.

[0361] As a carbon source, carbohydrates such as glucose and sucrose, and organic acids such as acetic acid and alcohols can be used. As a nitrogen source, ammonia, ammonia water, and ammonium salts can be used. As inorganic ions, calcium ions, magnesium ions, phosphate ions, potassium ions, and iron ions can be used as appropriate. Culture is carried out under aerobic conditions within a suitable range of pH 5.0-8.5 and 15-37°C for 1 to 7 days. Furthermore, culture conditions for the production of L-amino acids by Corynebacterium bacteria and other conditions described in the methods for protein preparation using Sec- or Tat-dependent signal peptides can be used (see WO01 / 23591 and WO2005 / 103278). In addition, when an inducible promoter is used for heterologous protein expression, culture can be carried out by adding an inducer to the culture medium. By culturing the bacteria of the present invention under such conditions, a large amount of the target protein is produced in the cells and efficiently secreted extracellularly. Furthermore, according to the method of the present invention, the generated heterologous proteins are secreted from the cells, and thus proteins that are usually lethal if they accumulate in large quantities in the cells of microorganisms (such as transglutaminase) can be produced without having a lethal effect.

[0362] Proteins secreted in a culture medium according to the method of the present invention can be separated and purified from the culture medium by methods well known to those skilled in the art. For example, after removing cells by centrifugation, proteins can be separated and purified by known suitable methods, such as salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium- and high-performance liquid chromatography, reversed-phase chromatography, and hydrophobic chromatography, or combinations thereof. Furthermore, in some cases, the culture or culture supernatant can be used directly. Proteins secreted in the cell surface layer according to the method of the present invention can also be separated and purified in the same manner as proteins secreted in the culture medium after dissolution by methods well known to those skilled in the art (e.g., increasing the salt concentration and using surfactants). Furthermore, in some cases, proteins secreted in the cell surface layer can be used, for example, as immobilized enzymes without dissolving them.

[0363] The secretory production of a target heterologous protein can be determined by performing SDS-PAGE on the culture supernatant and / or containing cell surface components as a sample, and determining the molecular weight of the separated protein bands. Alternatively, the secretory yield of the target heterologous protein can be determined by performing Western blotting using antibodies on the culture supernatant and / or containing cell surface components as a sample (Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). Furthermore, the secretory production of the target protein can be determined by detecting the N-terminal amino acid sequence of the target protein using a protein sequencer. Additionally, the secretory production of the target protein can be determined by determining the mass of the target protein using a mass spectrometer. Furthermore, when the target heterologous protein is an enzyme or protein with measurable physiological activity, the secretory production of the target protein can be determined by measuring the enzyme activity or physiological activity in the culture supernatant and / or containing cell surface components as a sample.

[0364] <2> Corynebacteria that retain the mutant phoS gene

[0365] The present invention also provides a Corynebacterium carrying a mutant phoS gene. This Corynebacterium may or may not have the ability to secrete heterologous proteins. Therefore, this Corynebacterium may or may not have a genetic construct for secreting heterologous proteins. The above description of "Corynebacterium for the method of the present invention" can be applied mutably to this Corynebacterium, except that the Corynebacterium may not have a genetic construct for secreting heterologous proteins. For example, the Corynebacterium can be made to retain a genetic construct for secreting heterologous proteins and can be used to secrete heterologous proteins. That is, the embodiment of this Corynebacterium is the above-described "Corynebacterium for the method of the present invention". Example

[0366] The present invention will be further described in detail with reference to the following embodiments. However, these embodiments should not be construed in any way as limiting the scope of the invention.

[0367] Example 1: Obtaining the Phos- mutant strain derived from Corynebacterium glutamicum strain YDK010

[0368] The *Corynebacterium glutamicum* strain YDK010, disclosed in WO2002 / 081694, was transformed with pPKK50TEV-Teri (a secretory expression plasmid for the physiologically active peptide teriparatide), disclosed in WO2014 / 126260. Incidentally, pPKK50TEV-Teri is a secretory expression vector for the physiologically active peptide teriparatide, and it contains the promoter region of the *Corynebacterium glutamicum* strain ATCC13869 cspB gene and the nucleotide sequence expressibly linked downstream of the promoter encoding the following fusion protein (hereinafter referred to as CspB50TEV-Teri): the CspB signal peptide of the same strain, the N-terminal 50 amino acid residues of the mature CspB of the same strain, the ProTEV protease recognition sequence ENLYFQ, and teriparatide (WO2014 / 126260). Corynebacterium glutamicum strain YDK010 is a CspB-deficient strain (FERMBP-734) of Corynebacterium glutamicum strain AJ12036. Transformants obtained were cultured under the following conditions to form colonies: CMDex agar medium containing 25 mg / L kanamycin (5 g glucose, 0.4 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 0.01 g MnSO4·5H2O, 1 g KH2PO4, 10 µg biotin, 10 g Difco). TM Select Soytone (Becton Dickinson) , 10 gBacto TM Yeast extract (Becton Dickinson), 3 g urea, 1.2 g (as total nitrogen), soybean hydrolysate obtained with HCl, and 20 g agar powder were added to 1 L of water and adjusted to pH 6.5 and incubated at 30°C.

[0369] After culturing, a naturally occurring mutant strain carrying the mutated phoS gene was selected and named strain YDK0107. The nucleotide sequence of the mutant phoS gene and the amino acid sequence of the mutant Phos protein of strain YDK0107 are shown in SEQ ID NOS: 1 and 2, respectively. In the mutant phoS gene of strain YDK0107, the "G" at position 906 of the wild-type phoS gene of strain YDK010 has been mutated to "T". Due to this mutation, in the mutant Phos protein of strain YDK0107, the tryptophan residue at position 302 of the wild-type Phos protein of strain YDK010 has been mutated to a cysteine ​​residue. This mutation is designated as the Phos(W302C) mutation. Incidentally, PurElute was used... TMGenomic DNA was prepared using the Genomic DNA Kit (EdgeBio) and nucleotide sequencing was performed using the BigDye Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems).

[0370] (2) Construct a phoS gene substitution vector encoding the mutant PhoS(W302C)

[0371] By using primers with SEQ ID NO: 5 and 6, and with PurElute TM Genomic DNA from Corynebacterium glutamicum strain YDK0107, prepared using a genomic kit (EdgeBio), was used as a template for PCR to amplify approximately 1.5 kbp of the phoS gene (also known as the mutant phoS gene or mutant phoS(W302C) gene) encoding the mutant PhoS(W302C). PCR was performed using Pyrobest® DNA polymerase (Takara Bio) under the manufacturer's recommended conditions.

[0372] The amplified DNA fragment of approximately 1.5 kbp was then subjected to agarose gel electrophoresis, the target band was excised, and the DNA fragment was collected from the gel using a Wizard® SV gel and a PCR Clean-Up System (Promega). The collected DNA fragment was inserted into the SmaI site of pBS5T disclosed in WO2006 / 057450, and the result was introduced into *E. coli* JM109 competent cells (Takara Bio). A strain holding a plasmid containing a DNA fragment containing the mutant phoS gene was obtained; this plasmid was collected from the strain to obtain pBS5T-phoS (W302C), a plasmid containing the mutant phoS gene. Nucleotide sequencing of the inserted fragment confirmed that the expected gene was cloned. Nucleotide sequencing was performed using a BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and a 3130 Genetic Analyzer (Applied Biosystems).

[0373] (3) Construction of PhoS(W302C) mutant strain

[0374] The *Corynebacterium glutamicum* strain YDK010, disclosed in WO2002 / 081694, was transformed with the plasmid pBS5T-phoS(W302C) constructed in Example 1(2). Strains were selected from the obtained transformants according to the method disclosed in WO2006 / 057450 to obtain YDK010::phoS(W302C), i.e., a strain in which the wild-type phoS gene on the chromosome is replaced by a mutant phoS gene. Incidentally, even without using the genomic DNA of strain YDK0107, strain YDK010::phoS(W302C) can be reproducibly constructed using, for example, a mutant phoS gene obtained through genetic engineering.

[0375] (4) Construct the phoS gene deletion vector pBS5TΔphoS

[0376] PCR was performed using PurElute. TM Genomic DNA from *Corynebacterium glutamicum* strain ATCC13869, prepared using a genomic DNA kit (EdgeBio), was used as a template to amplify a 1 kbp region upstream of the 5' side of the phoS gene using primers of SEQ ID NOS: 7 and 8, and a 1 kbp region downstream of the 3' side of the phoS gene using primers of SEQ ID NOS: 9 and 10. PCR was performed using Pyrobest® DNA polymerase (Takara Bio) under the manufacturer's recommended conditions. Each approximately 1 kbp amplified DNA fragment was subjected to agarose gel electrophoresis, the target band was excised, and the DNA fragments were collected from the gel using a Wizard® SV gel and a PCR Clean-Up System (Promega). The two collected DNA fragments were inserted into the SmaI site of pBS5T disclosed in WO2006 / 057450 to obtain the phoS gene deletion vector pBS5TΔphoS. The infusion reaction was performed using the In-Fusion® HD Cloning Kit (Takara Bio) under the manufacturer’s recommended conditions.

[0377] (5) Constructing a PhoS-deleted strain of strain YDK010

[0378] The plasmid pBS5TΔphoS constructed in Example 1(4) was used to transform the Corynebacterium glutamicum strain YDK010 disclosed in WO2002 / 081694. Strains were selected from the obtained transformants according to the method disclosed in WO2006 / 057450 to obtain YDK010::phoS(W302C), a phoS gene-deficient strain.

[0379] Example 2: Production of CspB50TEV-teriparatide fusion protein using CspB signal sequence in PhoS(W302C) mutant and PhoS deletion strains

[0380] The pPKK50TEV-Teri disclosed in WO2014 / 126260 (which is a secretion plasmid of the physiologically active peptide teriparatide) was used to transform each of the following strains: Corynebacterium glutamicum YDK010 disclosed in WO2002 / 081694, the YDK0107 strain obtained in Example 1 (1), the YDK010::phoS(W302C) strain obtained in Example 1 (3), and the YDK010ΔphoS strain obtained in Example 1 (5), to obtain strains YDK010 / pPKK50TEV-Teri, YDK0107 / pPKK50TEV-Teri, YDK010::phoS(W302C) / pPKK50TEV-Teri, and YDK010ΔphoS / pPKK50TEV-Teri. Each obtained transformant was cultured in MMTG liquid medium containing 25 mg / L kanamycin (120 g glucose, 3 g MgSO4·7H2O, 30 g (NH4)2SO4, 1.5 g KH2PO4, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·5H2O, 0.45 mg thiamine hydrochloride, 0.45 mg biotin, 0.15 g DL-methionine, 0.2 g (as total nitrogen) soybean hydrolysate obtained with HCl, and 50 g CaCO3, diluted with water to 1 L and adjusted to pH 7.0) at 30°C for 72 hours. After culture, 5 μL of culture supernatant obtained by centrifugation of each culture medium was subjected to reducing SDS-PAGE and then stained with SYPRO Orange (Life Technologies). As a result, compared with strain YDK010, the secretion of CspB50TEV-Teri in strains YDK0107 and YDK010::phoS(W302C) was significantly improved. Figure 1After staining, the band intensity of CspB50TEV-Teri was digitized using the image analysis software Multi Gauge (FUJIFILM), and the average band intensity observed after CspB50TEV-Teri expression in each strain was calculated as a relative value based on the average band intensity observed after CspB50TEV-Teri expression in strain YDK010 (set to 1). The results showed that, compared to strain YDK010, the secretion of CspB50TEV-Teri was confirmed to be improved by approximately 13.2-fold in YDK0107 and by approximately 12.5-fold in strain YDK010::phoS(W302C) (Table 1). Conversely, compared to strain YDK010, the secretion of CspB50TEV-Teri was reduced by approximately 0.2-fold in strain YDK010ΔphoS. This demonstrates that the PhoS(W302C) mutation is an effective mutation that significantly improves the secretion of CspB50TEV-Teri. Conversely, the deletion of the phoS gene did not show any beneficial effect on the secretion of CspB50TEV-Teri.

[0381] Table 1

[0382]

[0383] According to Appl. Environ. Microbiol., 94, 1131-1150 (2012), the region at positions 266-330 of the Phos protein in *Corynebacterium glutamicum* strain ATCC13032 is considered to be the HisKA domain, and the HisKA domain contains an autophosphorylated histidine residue. Since the tryptophan residue at position 302 is present in the HisKA domain, the amino acid sequences of the HisKA domain of Phos protein homologs from various *Corynebacterium* species were compared. The following amino acid sequence alignments are shown in... Figure 2In the image, the amino acid residue at position 302 is indicated by a box: the amino acid sequence of the HisKA domain of the PhoS protein of *Corynebacterium glutamicum* strains YDK0107, YDK010, and ATCC13869, predicted from nucleotide sequences analyzed using the BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and 3130 Genetic Analyzer (Applied Biosystems), and strains ATCC13032 (Genbank accession number NP_601807), ATCC14067 (Genbank accession number KEI24167), *Corynebacterium tumefaciens* (Genbank accession number WP_015652043), *Corynebacterium tumefaciens* (Genbank accession number WP_031512002), and C. The amino acid sequence of the HisKA domain of a Phosphate homologue of efficien (Genbank accession number WP_006769148) was obtained from a database using a BLAST search with the amino acid sequence of SEQ ID NO: 4 as the query sequence, yielding homologs with 70% or higher homology. The results showed that the tryptophan residue at position 302 is generally conserved in Corynebacterium bacteria, except for strain YDK0107. Although Phosphate is known as a sensor kinase in a two-component regulatory system, its effect on the secretion of heterologous proteins remains unknown. Furthermore, it is difficult to predict whether mutations in highly conserved amino acid residues such as W302 would improve the secretion of heterologous proteins.

[0384] Example 3: Production of liver-type fatty acid-binding protein (LFABP) fused to the N-terminal amino acid residue of mature CspB using CspB signal sequence in the PhoS(W302C) mutant strain.

[0385] (1) Construct a secretory expression plasmid of liver-type fatty acid-binding protein (LFABP) fused with the N-terminal 6 amino acids of the mature CspB protein.

[0386] The amino acid sequence of human liver-type fatty acid-binding protein (hereinafter referred to as LFABP) has been determined (Genbank accession number NP_001434). This amino acid sequence is shown in SEQ ID NO: 11. Considering the codon frequencies of Corynebacterium glutamicum, a nucleotide sequence encoding LFABP was designed. Furthermore, a fusion protein of LFABP (hereinafter referred to as CspB6Xa-LFABP) was designed, consisting of the 30 amino acid residues of the CspB signal peptide from Corynebacterium glutamicum strain ATCC13869, the N-terminal 6 amino acid residues of the mature CspB protein from the same strain, the factor Xa protease recognition sequence IEGR, and the fusion protein itself. The nucleotide sequence encoding the fusion protein is shown in SEQ ID NO: 12, and the amino acid sequence of the fusion protein is shown in SEQ ID NO: 13.

[0387] The expression cassette for CspB6Xa-LFABP was then fully synthesized, in which the promoter of the cspB gene from Corynebacterium glutamicum ATCC13869 was linked upstream to the DNA of SEQ ID NO: 12, and KpnI sites were further added to both the 5' and 3' ends. The synthesized DNA fragment was treated with the restriction enzyme KpnI and inserted at the KpnI site of pPK4 disclosed in Japanese Patent Publication No. 9-322774 to construct pPK4_CspB6Xa-LFABP, which is the secretory expression plasmid for CspB6Xa-LFABP. Nucleotide sequencing of the inserted fragment confirmed the construction of the desired gene encoding CspB6Xa-LFABP. Nucleotide sequencing was performed using the BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems).

[0388] (2) Secretory expression of liver-type fatty acid-binding protein (LFABP) fused to the N-terminal 6 amino acid residues of the mature CspB protein.

[0389] The pPK4_CspB6Xa-LFABP constructed in Example 3(1), which is a secretory expression plasmid of LFABP fused with the N-terminal 6 amino acid residues of mature CspB and the factor Xa protease recognition sequence IEGR, was used to transform each of the Corynebacterium glutamicum strain YDK010 disclosed in WO2002 / 081694 and the YDK010::phoS(W302C) strain constructed in Example 1(3) to obtain strains YDK010 / pPK4_CspB6Xa-LFABP and YDK010::phoS(W302C) / pPK4_CspB6Xa-LFABP.

[0390] Each transformed organism was cultured in MMTG liquid medium containing 25 mg / L kanamycin (120 g glucose, 3 g MgSO4·7H2O, 30 g (NH4)2SO4, 1.5 g KH2PO4, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·5H2O, 0.45 mg thiamine hydrochloride, 0.45 mg biotin, 0.15 g DL-methionine, 0.2 g (as total nitrogen) soybean hydrolysate obtained with HCl, and 50 g CaCO3, diluted with water to 1 L and adjusted to pH 7.0) at 30°C for 72 h. After culture, 6.5 μL of culture supernatant obtained by centrifugation of each culture medium was subjected to reducing SDS-PAGE and then stained with Quick-CBB (Wako Pure Chemical Industries).

[0391] As a result, compared with strain YDK010, dense bands indicating proteins with CspB6Xa-LFABP molecular weight were detected in strain YDK010::phoS(W302C), thus confirming a significant improvement in CspB6Xa-LFABP secretion in strain YDK010::phoS(W302C). Figure 3After staining, the band intensity of CspB6Xa-LFABP was digitized using the image analysis software Multi Gauge (FUJIFILM), and the average band intensity observed in the YDK010::phoS(W302C) strain after CspB6Xa-LFABP expression was calculated as a relative value based on the average band intensity observed in the YDK010 strain after CspB6Xa-LFABP expression (set to 1). The results confirmed that the secretion of CspB50TEV-Teri increased by approximately 1.9-fold in YDK010::phoS(W302C) compared to the YDK010 strain (Table 2). This reveals that the Phos(W302C) mutation is also an effective mutation leading to a significant improvement in the secretion of CspB6Xa-LFABP.

[0392] Table 2

[0393]

[0394] Example 4: Production of exenatide precursor (ExCP) fused to the N-terminal amino acid residue of mature CspB using CspB signal sequence in PhoS(W302C) mutant strain

[0395] (1) Construct a secretory expression plasmid of exenatide precursor (ExCP) fused with the N-terminal 6 amino acid residues of mature CspB.

[0396] The amino acid sequence of the physiologically active peptide exenatide has been determined (Genbank accession number P26349). Since activated exenatide is a C-terminal amidated peptide, the amino acid sequence of exenatide with Cys-Pro added to the C-terminus (hereinafter referred to as ExCP), a precursor to an amide compound, is shown in SEQ ID NO: 14. Considering the codon frequency of Corynebacterium glutamicum, a nucleotide sequence encoding ExCP was designed. Furthermore, a fusion protein of the CspB signal peptide (30 amino acid residues) from Corynebacterium glutamicum strain ATCC13869, the N-terminal 6 amino acid residues of the mature CspB protein from the same strain, the ProTEV protease recognition sequence ENLYFQ, and ExCP (hereinafter referred to as CspB6TEV-ExCP), along with the nucleotide sequence encoding the fusion protein, was designed. The designed nucleotide sequence encoding the fusion protein is shown in SEQ ID NO: 15, and the amino acid sequence of the fusion protein is shown in SEQ ID NO: 16.

[0397] Then, an expression cassette for CspB6Xa-ExCP was fully synthesized, in which the promoter of the cspB gene of Corynebacterium glutamicum ATCC13869 was linked upstream to the DNA of SEQ ID NO: 15, and KpnI sites were further added to both the 5' and 3' ends. The synthesized DNA fragment was treated with the restriction enzyme KpnI and inserted at the KpnI site of pPK4 disclosed in Japanese Patent Publication No. 9-322774 to construct pPK4_CspB6TEV-ExCP, which is the secretory expression plasmid of CspB6TEV-ExCP. Nucleotide sequencing of the inserted fragment confirmed the construction of the desired gene encoding CspB6TEV-ExCP. Nucleotide sequencing was performed using the BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems).

[0398] (2) Secretory expression of exenatide precursor (ExCP) fused to the N-terminal 6 amino acid residues of the mature CspB protein.

[0399] The pPK4_CspB6TEV-ExCP constructed in Example 4(1), which is a secretory expression plasmid of ExCP fused with the N-terminal 6 amino acid residues of mature CspB and the ProTEV protease recognition sequence, was used to transform each of the Corynebacterium glutamicum strain YDK010 disclosed in WO2002 / 081694 and the YDK010::phoS(W302C) strain constructed in Example 1(3) to obtain strains YDK010 / pPK4_CspB6TEV-ExCP and YDK010::phoS(W302C) / pPK4_CspB6TEV-ExCP.

[0400] Each transformed organism was cultured in MMTG liquid medium containing 25 mg / L kanamycin (120 g glucose, 3 g MgSO4·7H2O, 30 g (NH4)2SO4, 1.5 g KH2PO4, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·5H2O, 0.45 mg thiamine hydrochloride, 0.45 mg biotin, 0.15 g DL-methionine, 0.2 g (as total nitrogen) soybean hydrolysate obtained with HCl, and 50 g CaCO3, diluted with water to 1 L and adjusted to pH 7.0) at 30°C for 72 h. After culture, 6.5 μL of culture supernatant obtained by centrifugation of each culture medium was subjected to reducing SDS-PAGE and then stained with Quick-CBB (Wako Pure Chemical Industries).

[0401] As a result, a dense band indicating the molecular weight of protein CspB6TEV-ExCP was detected in strain YDK010::phoS(W302C), while it was difficult to detect in strain YDK010, thus confirming a significant improvement in CspB6TEV-ExCP secretion in strain YDK010::phoS(W302C). Figure 4 In addition, the average amount of CspB6TEV-ExCP secreted in each strain was calculated and presented as a score range from "±" to "+++". Figure 3 This reveals that the PhoS(W302C) mutation is also an effective mutation that leads to a significant improvement in the secretion of CspB6TEV-ExCP.

[0402] Table 3

[0403]

[0404] Examples 2-4 disclose the CspB fusion method disclosed in WO2013 / 062029.

[0405] When using the Sec secretion system to express heterologous proteins (the method of expressing heterologous proteins by fusing them with the N-terminal amino acid residues of mature CspB), the secretory expression level of the target protein can be significantly improved by using the PhoS(W302C) mutant strain, regardless of the type of protein to be expressed, the number of N-terminal amino acid residues of the mature CspB to which it is fused, and the type of protease recognition sequence.

[0406] Example 5: Production of proglutaminease secreted using TorA signal sequence in PhoS(W302C) mutant strain

[0407] (1) Construct a co-expression plasmid of the gene encoding the Tat secretion system and the gene encoding proglutaminase with the TorA signal sequence added.

[0408] (a) Construct pPK5, which corresponds to the vector of pPK4 modified with the NaeI recognition sequence.

[0409] In pPK4 disclosed in Japanese Patent Publication No. 9-322774, a recognition sequence for the restriction enzyme NaeI is present at one site. To modify this sequence, primers SEQ ID NO: 17 and 18 were synthesized, containing the sequence gcaggc modified from the NaeI recognition sequence gccggc in pPK4 and its neighboring sequence. PCR was then performed using primers SEQ ID NO: 17 and 18 and pPK4 as a template to amplify a full-length plasmid of approximately 5.6 kbp. PCR was performed using Pyrobest® DNA polymerase (Takara Bio) under the following conditions: 95°C for 5 min, and 12 cycles (95°C for 30 s, 55°C for 1 min, and 72°C for 12 min).

[0410] The obtained PCR product was then treated with the restriction enzyme DpnI to digest the methylated template DNA. The unmethylated plasmid obtained after DpnI digestion was introduced into competent E. coli JM109 cells (Takara Bio) to obtain the plasmid. Nucleotide sequencing confirmed that the NaeI recognition sequence was the modified desired plasmid. Nucleotide sequencing was performed using the BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems). The vector thus obtained, corresponding to the pPK4 vector modified with the NaeI recognition sequence, was named pPK5.

[0411] (b) Construct the vector pPK5-tatABC, which is a vector corresponding to pPK5 carrying the tatABC gene.

[0412] Then, PCR was performed using primers SEQ ID NO: 19 and 20, and pVtatABC (an amplification plasmid of the Tat secretion system) disclosed in WO2005 / 103278 as a template to amplify a DNA fragment of approximately 3.7 kbp containing the sequence encoding the tatABC gene. Primer SEQ ID NO: 20 was designed to contain recognition sequences for the restriction enzymes KpnI and ApaI. PCR was performed using Pyrobest® DNA polymerase (Takara Bio) under the manufacturer's recommended conditions. The DNA fragment was phosphorylated at the ends using the BKL Kit (Takara Bio), treated with KpnI, blunt-end treated with the BKL Kit (Takara Bio), and inserted into a pPK5 vector dephosphorylated at the ends using CIAP (Takara Bio) to construct the pPK5-tatABC vector carrying the tatABC gene. Ligation was performed using a DNA ligation kit Ver. 2.1 (Takara Bio) under the manufacturer's recommended conditions. Nucleotide sequencing of the inserted fragment confirmed the insertion of the expected gene. Nucleotide sequencing was performed using the BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems).

[0413] (c) Construct the vector pPK6, which is the vector corresponding to the pPK5-tatABC vector modified with the KpnI and XbaI recognition sequences in the tatABC gene.

[0414] In the tatABC gene region of the pPK5-tatABC plasmid constructed in (b), recognition sequences for restriction enzymes KpnI and XbaI are present on each side. To modify these sequences, primers SEQ ID NO: 21 and 22, containing the sequence ggaacc modified from the KpnI recognition sequence ggtacc in pPK5-tatABC and its neighboring sequences, and primers SEQ ID NO: 23 and 24, containing the sequence tgtaga modified from the XbaI recognition sequence tctaga in pPK5-tatABC and its neighboring sequences, were synthesized.

[0415] First, to modify the KpnI recognition sequence in the tatABC gene region, PCR was performed using primers SEQ ID NO: 21 and 22, and pPK5-tatABC as a template. To amplify the full-length plasmid of approximately 9.4 kbp, PCR was performed using Pyrobest® DNA polymerase (Takara Bio), and the reaction conditions consisted of the following: 95°C for 5 minutes, and 12 cycles (95°C for 30 seconds, 55°C for 1 minute, and 72°C for 12 minutes).

[0416] The obtained PCR product was then treated with the restriction enzyme DpnI to digest the methylated template DNA. The unmethylated plasmid obtained after DpnI digestion was introduced into competent E. coli JM109 cells (Takara Bio) to obtain the plasmid. The pPK5-tatABCΔKpnI vector, in which the KpnI recognition sequence in the tatABC gene region was modified, was then constructed.

[0417] Then, to modify the XbaI recognition sequence in the tatABC gene region, PCR was performed using primers SEQ ID NO: 23 and 24 and pPK5-tatABCΔKpnI as a template to amplify a full-length plasmid of approximately 9.4 kbp. PCR was performed using Pyrobest® DNA polymerase (Takara Bio) under the following conditions: 95°C for 5 min, and 12 cycles (95°C for 30 s, 55°C for 1 min, and 72°C for 12 min).

[0418] The obtained PCR product was then treated with the restriction enzyme DpnI to digest the methylated template DNA. The unmethylated plasmid obtained after DpnI digestion was introduced into competent *E. coli* JM109 cells (Takara Bio) to obtain the plasmid. A modified pPK5-tatABCΔKpnIΔXbaI vector was constructed, in which the XbaI recognition sequence in the tatABC gene region is modified. Nucleotide sequencing confirmed the construction of the expected gene. Nucleotide sequencing was performed using the BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems).

[0419] The pPK4-based vector carrying the tatABC gene obtained in this way is named pPK6. The procedure for constructing pPK6 from pPK4 is as follows: Figure 5As shown. To amplify the TatABC secretion system in the secretory expression of proteins using the Tat system, the method in WO2005 / 103278 requires the use of two plasmids: a secretory expression plasmid for the target protein and an amplification plasmid for the Tat secretion system, pVtatABC. Conversely, the use of the pPK6 vector allows for expression of the target protein and amplification of the TatABC secretion system within a single plasmid.

[0420] (d) Construct pPK6-TorAss, which is a vector corresponding to a pPK6 vector carrying a cspB promoter and a TorA signal sequence.

[0421] PCR was performed using primers SEQ ID NO: 25 and 26 and pPTGFP, disclosed in Appl. Environ. Microbiol., 72, 7183-7192 (2006), as a template to amplify a DNA fragment of approximately 0.7 kbp containing the promoter region of the cspB gene from Corynebacterium glutamicum strain ATCC13869 and the nucleotide sequence encoding the TorA signal sequence of Escherichia coli. Primer SEQ ID NO: 26 was designed to contain recognition sequences for the restriction enzymes ApaI and NaeI. PCR was performed using Pyrobest® DNA polymerase (Takara Bio) under the manufacturer's recommended conditions. DNA fragments were treated with KpnI and ApaI and inserted into the KpnI-ApaI site of the pPK6 vector constructed in (c) to construct pPK6-TorAss, a vector carrying the promoter region of the cspB gene from Corynebacterium glutamicum ATCC13869 and the nucleotide sequence encoding the TorA signal sequence of Escherichia coli. Ligation was performed using a DNA ligation kit Ver. 2.1 (Takara Bio) under the manufacturer's recommended conditions. Nucleotide sequencing of the inserted fragment confirmed the insertion of the expected gene. Nucleotide sequencing was performed using a BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and a 3130 Genetic Analyzer (Applied Biosystems).

[0422] (e) Constructing a transglutaminase secretory expression vector using the pPK6-TorAss vector.

[0423] PCR was performed using primers SEQ ID NO: 27 and 28 and pPKSPTG1 (a transglutaminase secretory expression vector) disclosed in WO2001 / 23591 as templates to amplify a DNA fragment encoding transglutaminase of approximately 1.1 kbp. Primer SEQ ID NO: 28 was designed to contain the recognition sequence of the restriction enzyme XbaI. PCR was performed using Pyrobest® DNA polymerase under the manufacturer's recommended reaction conditions. The DNA fragment was treated with XbaI and inserted into the NaeI-XbaI site of the pPK6-TorAss vector constructed in (d) to construct pPK6_T_PTG, a co-expression vector of the TatABC secretory system and transglutaminase with the TorA signal sequence added. Ligation was performed using a DNA ligation kit Ver. 2.1 (TakaraBio) under the manufacturer's recommended reaction conditions. Nucleotide sequencing of the inserted fragment confirmed the insertion of the intended gene. Nucleotide sequencing was performed using the BigDye® Terminator v3.1 Cyclic Sequencing Kit (AppliedBiosystems) and the 3130 Genetic Analyzer (Applied Biosystems).

[0424] (2) Using the TorA signal sequence to secrete and express proglutaminease in strains YDK010 and YDK010::phoS(W302C)

[0425] The pPK6_T_PTG obtained in Example 5(1)(e) (which is a secretory expression plasmid for transglutaminase) was used to transform the Corynebacterium glutamicum strain YDK010 disclosed in WO2002 / 081694 and the YDK010::phoS(W302C) strain obtained in Example 1(3) to obtain strains YDK010 / pPK6_T_PTG and YDK010::phoS(W302C) / pPK6_T_PTG. Each obtained transformant was cultured in MMTG liquid medium containing 25 mg / L kanamycin (120 g glucose, 3 g MgSO4·7H2O, 30 g (NH4)2SO4, 1.5 g KH2PO4, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·5H2O, 0.45 mg thiamine hydrochloride, 0.45 mg biotin, 0.15 g DL-methionine, 0.2 g (as total nitrogen) soybean hydrolysate obtained with HCl, and 50 g CaCO3, diluted with water to 1 L and adjusted to pH 7.0) at 30°C for 72 h. After culture, 5 μL of culture supernatant obtained by centrifugation of each culture medium was subjected to reducing SDS-PAGE and then stained with Quick-CBB (Wako Pure Chemical Industries). As a result, compared with strain YDK010, the secretion of transglutaminase in strain YDK010::phoS(W302C) was significantly improved. Figure 6 After staining, the band intensity of transglutaminase was digitized using the image analysis software MultiGauge (FUJIFILM), and the average band intensity observed after transglutaminase expression in each strain was calculated as a relative value based on the average band intensity observed in strain YDK010 (set as 1). The results confirmed that the secretion of transglutaminase was improved by approximately 7.2-fold in strain YDK010::phoS(W302C) compared to strain YDK010 (Table 4). This reveals that the PhoS(W302C) mutation is also an effective mutation leading to a significant improvement in transglutaminase secretion using the TorA signal sequence.

[0426] Table 4

[0427]

[0428] Example 6: Production of protein glutaminase containing a pro-structure moiety in the PhoS(W302C) mutant strain using TorA signal sequence.

[0429] (1) Construct a co-expression plasmid of the tatABC gene encoding the Tat secretion system and a gene expressing glutaminase, a protein containing the pre-structural portion of the TorA signal sequence.

[0430] PCR was performed using primers SEQ ID NO: 29 and 30 and pPKT-PPG (an expression plasmid containing the pre-structural portion of the protein glutaminase) disclosed in WO2005 / 103278 as templates to amplify the promoter region of the cspB gene from Corynebacterium glutamicum ATCC13869 strain, and the nucleotide sequence of a fusion protein that expressibly links downstream of the promoter and encodes the Escherichia coli TorA signal sequence and the protein glutaminase containing the pre-structural portion of Chryseobacterium proteolyticum. Each of primers SEQ ID NO: 29 and 30 was designed to contain the recognition sequence of the restriction enzyme XbaI. PCR was performed using Pyrobest(R) DNA polymerase under the manufacturer's recommended reaction conditions. The DNA fragment was treated with XbaI and inserted into the XbaI site of the pPK6 vector constructed in Example 5(1) to construct pPK6_T_PPG, a co-expression vector of the TatABC secretion system and a protein glutaminase containing the prestructural portion of the TorA signal sequence. This was done using a DNA ligation kit.<Mighty Mix> The ligation reaction was performed using Takara Bio under the manufacturer's recommended conditions. Nucleotide sequencing of the inserted fragment confirmed the insertion of the intended gene. Nucleotide sequencing was performed using a BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and a 3130 GeneticAnalyzer (Applied Biosystems).

[0431] (2) Using the TorA signal sequence to secrete and express a protein glutaminase containing the pre-structural part in strains YDK010 and YDK010::phoS(W302C).

[0432] The pPK6_T_PPG obtained in Example 6(1), which is a secretory expression plasmid containing the pre-structural portion of the protein glutaminase, was transformed into the Corynebacterium glutamicum strain YDK010 disclosed in WO2002 / 081694 and the YDK010::phoS(W302C) strain obtained in Example 1(3) to obtain strains YDK010 / pPK6_T_PPG and YDK010::PhoS(W302C) / pPK6_T_PPG. Each obtained transformant was cultured in MMTG liquid medium containing 25 mg / L kanamycin (120 g glucose, 3 g MgSO4·7H2O, 30 g (NH4)2SO4, 1.5 g KH2PO4, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·5H2O, 0.45 mg thiamine hydrochloride, 0.45 mg biotin, 0.15 g DL-methionine, 0.2 g (as total nitrogen) soybean hydrolysate obtained with HCl, and 50 g CaCO3, diluted with water to 1 L and adjusted to pH 7.0) at 30°C for 72 hours. After culture, 5 μL of culture supernatant obtained by centrifugation of each culture medium was subjected to reducing SDS-PAGE and then stained with Quick-CBB (Wako Pure Chemical Industries). As a result, compared with strain YDK010, the secretion of glutaminase containing the prestructural portion of protein in strain YDK010::phoS(W302C) was significantly improved. Figure 7 After staining, the band intensity of glutaminase containing the prestructural portion was digitized using the image analysis software Multi Gauge (FUJIFILM), and the average band intensity of glutaminase containing the prestructural portion observed in each strain was calculated as a relative value based on the average band intensity of glutaminase expressing the prestructural portion observed in strain YDK010 (set as 1). The results confirmed that the secretion of glutaminase containing the prestructural portion was improved by approximately 8.3-fold in strain YDK010::phoS(W302C) compared to strain YDK010 (Table 5). This reveals that the PhoS(W302C) mutation is also an effective mutation leading to a significant improvement in the secretion of glutaminase containing the prestructural portion using the TorA signal sequence.

[0433] Table 5

[0434]

[0435] Example 7: Production of isomaltodextrinase secreted using IMD signal sequence in PhoS(W302C) mutant strain

[0436] (1) Construct a co-expression plasmid of the tatABC gene encoding the Tat secretion system and the gene encoding isomaltodextrinase.

[0437] PCR was performed using primers SEQ ID NO: 29 and 31 and pPKI-IMD (an expression plasmid for isomaltodextrinase) disclosed in WO2005 / 103278 as templates to amplify the promoter region of the cspB gene of Corynebacterium glutamicum ATCC13869 strain and the IMD gene sequence (including the coding region of the IMD signal sequence) of Arthroblastus spheroidus, which is expressibly linked downstream of the promoter. Each of primers SEQ ID NO: 29 and 31 was designed to contain the recognition sequence of the restriction enzyme XbaI. PCR was performed using PrimeSTAR(R) GXL DNA polymerase (Takara Bio) and reaction conditions according to the manufacturer's recommended protocol. The DNA fragment was treated with XbaI and inserted into the XbaI site of the pPK6 vector constructed in Example 5(1) to construct pPK6_T_PPG, a co-expression vector of the TatABC secretion system and isomaltodextrinase including the IMD signal sequence. DNA ligation kit was used.<Mighty Mix> The ligation reaction was performed using Takara Bio under the manufacturer's recommended conditions. Nucleotide sequencing of the inserted fragment confirmed the insertion of the intended gene. Nucleotide sequencing was performed using a BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and a 3130 Genetic Analyzer (Applied Biosystems).

[0438] (2) IMD signal sequence was used to secrete and express isomaltodextrinase in strains YDK010 and YDK010::phoS(W302C).

[0439] The pPK6_I_IMD obtained in Example 7(1), which is a secretory expression plasmid of isomaltoid dextrinase, was used to transform the Corynebacterium glutamicum strain YDK010 disclosed in WO2002 / 081694 and the YDK010::phoS(W302C) strain obtained in Example 1(3) to obtain strains YDK010 / pPK6_I_IMD and YDK010::phoS(W302C) / pPK6_I_IMD. Each obtained transformant was cultured in MMTG liquid medium containing 25 mg / L kanamycin (120 g glucose, 3 g MgSO4·7H2O, 30 g (NH4)2SO4, 1.5 g KH2PO4, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·5H2O, 0.45 mg thiamine hydrochloride, 0.45 mg biotin, 0.15 g DL-methionine, 0.2 g (as total nitrogen) soybean hydrolysate obtained with HCl, and 50 g CaCO3, diluted with water to 1 L and adjusted to pH 7.0) at 30°C for 72 hours. After culture, 5 μL of culture supernatant obtained by centrifugation of each culture medium was subjected to reducing SDS-PAGE and then stained with Quick-CBB (Wako PureChemical Industries). As a result, compared with strain YDK010, the secretion of isomaltodextrinase in strain YDK010::phoS(W302C) was significantly improved. Figure 8 After staining, the band intensity of isomaltodextrinase was digitized using the image analysis software Multi Gauge (FUJIFILM), and the average band intensity of isomaltodextrinase expression observed in each strain was calculated as a relative value based on the average band intensity of isomaltodextrinase expression observed in strain YDK010 (set as 1). The results confirmed that the secretion of isomaltodextrinase was improved by approximately 6.6-fold in strain YDK010::phoS(W302C) compared to strain YDK010 (Table 6). This reveals that the Phos(W302C) mutation is also an effective mutation leading to a significant improvement in isomaltodextrinase secretion using the A. globiformis IMD signal sequence.

[0440] Table 6

[0441]

[0442] Examples 5-7 demonstrate that, in the case of secretory expression of heterologous proteins using the Tat secretion system disclosed in WO2005 / 103278, the secretory expression level of the target protein can be significantly improved by using the PhoS(W302C) mutant strain, regardless of the type of protein to be expressed or the type of signal sequence to be used.

[0443] Therefore, Examples 2-7 demonstrate that the secretion yield of a target protein can be significantly improved by using the PhoS(W302C) mutant strain, regardless of the type of secretion pathway used, the type of signal sequence used, or the type of protein to be expressed.

[0444] Example 8: Functional supplementation of PhoS-deleted strains by plasmid amplification of the phoS gene

[0445] (1) Constructing amplification plasmids for the wild-type phoS gene and the mutant phoS(W302C) gene.

[0446] (a) Constructing an amplification plasmid for the wild-type phoS gene

[0447] By using primers SEQ ID NO: 32 and 33, and with PurElute TM Genomic DNA from Corynebacterium glutamicum strain YDK010, prepared using an EdgeBio genomic DNA kit, was used as a template for PCR to amplify an approximately 1.5 kbp region containing the wild-type phoS gene. PCR was performed using Pyrobest® DNA polymerase (Takara Bio) under the manufacturer's recommended conditions.

[0448] The amplified DNA fragment of approximately 1.5 kbp was then subjected to agarose gel electrophoresis, the target band was excised, and the DNA fragment was collected from the gel using a Wizard® SV gel and a PCR Clean-Up System (Promega). The collected DNA fragment was inserted into the SmaI site of the pVC7 vector (a shuttle vector containing a chloramphenicol resistance gene and capable of replication in both *E. coli* and *Corynebacterium*) disclosed in Japanese Patent Publication No. 9-070291 via an infusion reaction to obtain the amplified plasmid pVphoS(WT) of the wild-type phoS gene. The infusion reaction was performed using the In-Fusion® HD Cloning Kit (Takara Bio) under the manufacturer's recommended conditions. Nucleotide sequencing of the inserted fragment confirmed the insertion of the expected gene. Nucleotide sequencing was performed using the BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems).

[0449] (b) Constructing an amplification plasmid for the mutant phoS(W302C) gene

[0450] Similarly, by using primers SEQ ID NO: 32 and 33, and with PurElute TM Genomic DNA from *Corynebacterium glutamicum* strain YDK010::phoS(W302C) prepared using an EdgeBio genomic DNA kit was used as a template for PCR to amplify an approximately 1.5 kbp region containing the mutant phoS(W302C). PCR was performed using Pyrobest® DNA polymerase (Takara Bio) under the manufacturer's recommended conditions.

[0451] The amplified DNA fragment of approximately 1.5 kbp was then subjected to agarose gel electrophoresis, the target band was excised, and the DNA fragment was collected from the gel using a Wizard® SV gel and a PCR Clean-Up System (Promega). The collected DNA fragment was inserted into the SmaI site of the pVC7 vector (a shuttle vector containing a chloramphenicol resistance gene and capable of replication in both *E. coli* and *Corynebacterium*) disclosed in Japanese Patent Publication No. 9-070291 via an infusion reaction to obtain the amplified plasmid pVphoS(W302C) of the mutant phoS(W302C) gene. The infusion reaction was performed using the In-Fusion® HD Cloning Kit (Takara Bio) under the manufacturer's recommended conditions. Nucleotide sequencing of the inserted fragment confirmed the insertion of the expected gene. Nucleotide sequencing was performed using the BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and the 3130 GeneticAnalyzer (Applied Biosystems).

[0452] (2) Amplification effect of wild-type phoS gene or mutant phoS(W302C) gene on the secretory expression of CspB50TEV-Teri in PhoS-deficient strains.

[0453] The YDK010ΔphoS strain constructed in Example 1(5) was transformed using a combination of pPKK50TEV-Teri disclosed in WO2014 / 126260 and pVphoS(WT) or pVphoS(W302C) constructed in Example 8(1). Furthermore, as a control, the YDK010ΔphoS strain constructed in Example 1(5) was transformed using a combination of the pPK4 vector disclosed in Japanese Patent Publication No. 9-322774 and pVphoS(WT) or pVphoS(W302C) constructed in Example 8(1). Each obtained transformant was cultured in MMTG liquid medium containing 25 mg / L kanamycin (120 g glucose, 3 g MgSO4·7H2O, 30 g (NH4)2SO4, 1.5 g KH2PO4, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·5H2O, 0.45 mg thiamine hydrochloride, 0.45 mg biotin, 0.15 g DL-methionine, 0.2 g (as total nitrogen) soybean hydrolysate obtained with HCl, and 50 g CaCO3, diluted with water to 1 L and adjusted to pH 7.0) at 30°C for 72 hours. After culture, 5 μL of culture supernatant obtained by centrifugation of each culture medium was subjected to reducing SDS-PAGE and then stained with SYPRO Orange (Life Technologies) to compare the secretion of CspB50TEV-Teri. Figure 9 Furthermore, the CspB50TEV-Teri secretion levels observed in some strains are shown in Table 7 using a score range from "+" to "+++". As a result, when pVphoS(WT) was introduced into the YDK010ΔphoS strain, a CspB50TEV-Teri secretion level comparable to that observed in the YDK010 strain was obtained, while when pVphoS(W302C) was introduced into the YDK010ΔphoS strain, a CspB50TEV-Teri secretion level comparable to that observed in the YDK010::phoS(W302C) strain was obtained. This demonstrates that the functional supplementation of chromosomal phoS gene deletions can be achieved by amplifying any wild-type or mutant phoS gene on a plasmid. Therefore, it was revealed that effects comparable to those achieved by using the phoS gene on a mutant chromosome can be obtained by utilizing expression plasmids of the mutant phoS gene.

[0454] Table 7

[0455]

[0456] Example 9: Using a strain expressing the mutant PhoS (W302X) to secrete and express a heterologous protein, wherein the tryptophan residue at position 302 of the wild-type PhoS protein is modified with any amino acid residue.

[0457] (1) Construct expression plasmids for mutant phoS genes encoding various mutant PhoS(W302X) proteins.

[0458] Each plasmid pVphoS(W302X) for expressing the mutant PhoS(W302X) was constructed, wherein position 302 of the wild-type PhoS protein (W302) was modified to another amino acid residue. “X” represents any amino acid residue.

[0459] For the construction of pVphoS(W302S) (which is an expression plasmid for the mutant (W302S) protein), PCR was performed using the pVphoS(WT) plasmid constructed in Example 8(1) as a template in combination with primers SEQ ID NO: 32 and 34 to amplify a region approximately 0.9 kbp from the N-terminus of the phoS gene, and in combination with primers SEQ ID NO: 33 and 35 to amplify a region approximately 0.6 kbp from the C-terminus of the phoS gene. Primers SEQ ID NOS: 34 and 35 were designed to replace the codon (tgg) encoding the tryptophan residue at position 302 of the wild-type PhoS protein with the codon (tcc) encoding the serine residue. PCR was performed using Pyrobest(R) DNA polymerase (Takara Bio) and the reaction conditions were as recommended by the manufacturer. The amplified DNA fragments were subjected to agarose gel electrophoresis, the target band was excised, and the DNA fragments were collected from the gel using a Wizard® SV gel and a PCR Clean-Up System (Promega). The two collected DNA fragments were inserted into the SmaI site of the pVC7 vector disclosed in Japanese Patent Publication No. 9-070291 via an infusion reaction to obtain the amplified plasmid pVphoS(W302C) of the mutant phoS(W302S) gene. The infusion reaction was performed using the In-Fusion® HD Cloning Kit (Takara Bio) under the manufacturer's recommended conditions. Nucleotide sequencing of the inserted fragments confirmed the cloning of the expected gene. Nucleotide sequencing was performed using the BigDye® Terminator v3.1 Cyclic Sequencing Kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems).

[0460] Similarly, plasmids pVphoS(W302A) expressing mutant PhoS(W302A), pVphoS(W302V) expressing mutant PhoS(W302V), pVphoS(W302M) expressing mutant PhoS(W302M), pVphoS(W302F) expressing mutant PhoS(W302Y), and pVphoS(W302Y) expressing mutant PhoS(W302A) and PhoS(W302F) expressing mutant PhoS(W302Y) were created. Ten plasmids expressing the mutant PhoS(W302X) protein were constructed, namely, pVphoS(W302D), pVphoS(W302N), pVphoS(W302H), and pVphoS(W302K). Figure 8 The diagram shows the primer sets used in the PCR for constructing their respective plasmids to amplify the N-terminal region of the phoS gene and the primer sets used to amplify the C-terminal region of the phoS gene.

[0461] Table 8

[0462]

[0463] (2) Amplification effect of various PhoS(W302X) proteins on the secretory expression of CspB50TEV-Teri in PhoS-deficient strains.

[0464] The YDK010ΔphoS strain constructed in Example 1(5) was transformed using each combination of pPKK50TEV-Teri disclosed in WO2014 / 126260 and various pVphoS (W302C) constructed in Example 9(1). Each obtained transformant was cultured in MMTG liquid medium (120 g glucose, 3 g MgSO4·7H2O, 30 g (NH4)2SO4, 1.5 g KH2PO4, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·5H2O, 0.45 mg thiamine hydrochloride, 0.45 mg biotin, 0.15 g DL-methionine, 0.2 g (as total nitrogen) soybean hydrolysate obtained with HCl, and 50 g CaCO3, diluted with water to 1 L and adjusted to pH 7.0) at 30°C for 72 hours. After culture, 5 μL of culture supernatant obtained by centrifugation of each culture medium was subjected to reducing SDS-PAGE and then stained with SYPRO Orange (Life Technologies) to compare the secretion of CspB50TEV-Teri. Figure 10 Furthermore, the secretion levels of CspB50TEV-Teri observed in each strain are shown in Table 9 as a score range from "+" to "+++". As a result, compared to the wild-type PhoS protein, the secretion level of CspB50TEV-Teri was significantly increased when the tryptophan residue at position 302 of the wild-type PhoS protein was replaced with amino acid residues other than aromatic amino acids and histidine residues (in one case, a cysteine ​​residue was used to replace the tryptophan residue at position 302 of the wild-type PhoS protein). Incidentally, no transformants were obtained from the strain introduced with pVphoS(W302Y), therefore the secretion level of CspB50TEV-Teri could not be evaluated.

[0465] This study revealed that mutating the tryptophan residue at position 302 of the wild-type PhoS protein to any amino acid other than aromatic amino acids and histidine residues, and mutating that residue to cysteine, led to a significant improvement in the secretion of the heterologous protein.

[0466] Table 9

[0467]

[0468] nt; Not detected

[0469] Example 10: Construction of a PhoS(W302C) mutant strain from Corynebacterium glutamicum ATCC13869 and secretion of heterologous proteins.

[0470] (1) Constructing the Corynebacterium glutamicum ATCC13869::phoS(W302C) strain

[0471] Corynebacterium glutamicum strain ATCC13869 was transformed with pBS5T-phoS(W302C) (which is a vector replacing the mutant phoS gene) constructed in Example 1(2). The transformed strains were selected from the obtained strains according to the method disclosed in WO2006 / 057450 to obtain ATCC13869::phoS(W302C), which is a strain in which the wild-type phoS gene on the chromosome is replaced by the mutant phoS gene.

[0472] (2) In the strain Corynebacterium glutamicum ATCC13869::phoS(W302C), an exenatide precursor (ExCP) fused with the N-terminal 6 amino acid residues of the mature CspB protein was secreted and expressed.

[0473] The pPK4_CspB6TEV-ExCP constructed in Example 4(1), which is a secretory expression plasmid of ExCP fused with the N-terminal 6 amino acid residues of mature CspB and the ProTEV protease recognition sequence, was used to transform Corynebacterium glutamicum strain ATCC13869 and strain ATCC13869::phoS(W302C) constructed in Example 10(1) to obtain strains ATCC13869 / pPK4_CspB6TEV-ExCP and ATCC13869::phoS(W302C) / pPK4_CspB6TEV-ExCP.

[0474] Each transformed organism was cultured in MMTG liquid medium containing 25 mg / L kanamycin (120 g glucose, 3 g MgSO4·7H2O, 30 g (NH4)2SO4, 1.5 g KH2PO4, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·5H2O, 0.45 mg thiamine hydrochloride, 0.45 mg biotin, 0.15 g DL-methionine, 0.2 g (as total nitrogen) soybean hydrolysate obtained with HCl, and 50 g CaCO3, diluted with water to 1 L and adjusted to pH 7.0) at 30°C for 72 h. After culture, 6.5 μL of culture supernatant obtained by centrifugation of each culture medium was subjected to reducing SDS-PAGE and then stained with Quick-CBB (Wako Pure Chemical Industries).

[0475] As a result, similar to the case using strain YDK010 as the genetic background, a dense band indicating the molecular weight of the protein CspB6TEV-ExCP was detected in strain ATCC13869::phoS(W302C), while it was difficult to detect in strain ATCC13869, thus confirming a significantly increased secretion of CspB6TEV-ExCP in strain ATCC13869::phoS(W302C). Figure 11 This reveals that when using ATCC13869 strain as the genetic background, the Phos(W302C) mutation is also an effective mutation that leads to a significant improvement in the secretion of CspB6TEV-ExCP.

[0476] Example 11: Constructing a CspB-deficient strain from Corynebacterium glutamicum ATCC13869::phoS(W302C) and secreting and expressing a heterologous protein.

[0477] (1) Constructing the Corynebacterium glutamicum ATCC13869::phoS(W302C)ΔcspB strain

[0478] The ATCC13869::phoS(W302C) strain was transformed with pBS5T-ΔcspB (a cspB gene deletion vector) disclosed in WO2013 / 065869. The transformed strains were then selected from the obtained transformants according to the method disclosed in WO2006 / 057450 to obtain ATCC13869::phoS(W302C)ΔcspB, a cspB gene-deficient strain.

[0479] (2) Secretory expression of liver-type fatty acid-binding protein (LFABP) fused to the N-terminal 6 amino acid residues of mature CspB protein in Corynebacterium glutamicum ATCC13869::phoS(W302C)ΔcspB strain

[0480] The pPK4_CspB6Xa-LFABP constructed in Example 3(1) (which is related to the N-terminal 6 amino acid residues of the mature CspB protein and...)

[0481] The secretory expression plasmid of LFABP fused with the factor Xa protease recognition sequence IEGR was transformed into each of the Corynebacterium glutamicum ATCC13869ΔcspB strain disclosed in WO2013 / 065869 and the ATCC13869::phoS(W302C)ΔcspB strain constructed in Example 11(1) to obtain strains ATCC13869ΔcspB / pPK4_CspB6Xa-LFABP and ATCC13869::phoS(W302C)ΔcspB / pPK4_CspB6Xa-LFABP.

[0482] Each transformed organism was cultured in MMTG liquid medium containing 25 mg / L kanamycin (120 g glucose, 3 g MgSO4·7H2O, 30 g (NH4)2SO4, 1.5 g KH2PO4, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·5H2O, 0.45 mg thiamine hydrochloride, 0.45 mg biotin, 0.15 g DL-methionine, 0.2 g (as total nitrogen) soybean hydrolysate obtained with HCl, and 50 g CaCO3, diluted with water to 1 L and adjusted to pH 7.0) at 30°C for 72 h. After culture, 6.5 μL of culture supernatant obtained by centrifugation of each culture medium was subjected to reducing SDS-PAGE and then stained with Quick-CBB (Wako Pure Chemical Industries).

[0483] As a result, similar to the case using strain YDK010 as the genetic background, bands indicating proteins with the molecular weight of CspB6Xa-LFABP were consistently and densely detected in strain ATCC13869::phoS(W302C)ΔcspB compared to strain ATCC13869ΔcspB, thus confirming a significantly increased secretion of CspB6Xa-LFABP in strain ATCC13869::phoS(W302C)ΔcspB. Figure 12 This reveals that when using ATCC13869ΔcspB strain as the genetic background, the PhoS(W302C) mutation is also an effective mutation that leads to a significant improvement in the secretion of CspB6Xa-LFABP.

[0484] Examples 10-11 demonstrate that when using ATCC13869 or ATCC13869ΔcspB strains as the genetic background, the secretory expression of the target protein can also be significantly improved by using the PhoS(W302C) mutant strain.

[0485] Therefore, Examples 2-11 demonstrate that the secretory expression of the target protein can be significantly improved by using the PhoS(W302C) mutant strain, regardless of differences in the genetic background of the host strain.

[0486] Industrial applicability

[0487] According to the present invention, heterologous proteins can be efficiently produced through secretion.

[0488] <Explanation of Sequence Lists>

[0489] SEQ ID NOS:

[0490] 1: Nucleotide sequence of the phoS gene, a mutant of Corynebacterium glutamicum YDK0107

[0491] 2: Amino acid sequence of the mutant Phos protein of Corynebacterium glutamicum YDK0107

[0492] 3: Nucleotide sequence of the wild-type phoS gene of Corynebacterium glutamicum YDK010

[0493] 4: Amino acid sequence of wild-type Phos protein from Corynebacterium glutamicum YDK010

[0494] 5 to 10: Primers

[0495] 11: Amino acid sequence of LFABP

[0496] 12: Nucleotide sequence encoding CspB6Xa-LFABP

[0497] 13: Amino acid sequence of CspB6Xa-LFABP

[0498] 14: Amino acid sequence of exenatide precursor

[0499] 15: Nucleotide sequence encoding CspB6TEV-ExCP

[0500] 16: Amino acid sequence of CspB6TEV-ExCP

[0501] 17 to 53: Primers

[0502] 54: Amino acid sequence of Phos protein from Corynebacterium glutamicum ATCC 13032

[0503] 55: Amino acid sequence of Phos protein from Corynebacterium glutamicum ATCC 14067

[0504] 56: Amino acid sequence of Phos protein from Corynebacterium tumefaciens

[0505] 57: Amino acid sequence of Phos protein from Corynebacterium tumefaciens

[0506] 58: Amino acid sequence of the Phos protein from C. efficiens

[0507] 59: Amino acid sequence of the HisAK domain of the Phos protein from Corynebacterium glutamicum YDK0107

[0508] 60: Amino acid sequence of the HisAK domain of the Phos protein from Corynebacterium glutamicum YDK010

[0509] 61: Amino acid sequence of the HisAK domain of the Phos protein from Corynebacterium glutamicum ATCC 13869

[0510] 62: Amino acid sequence of the HisAK domain of the Phos protein from Corynebacterium glutamicum ATCC 13032

[0511] 63: Amino acid sequence of the HisAK domain of the Phos protein from Corynebacterium glutamicum ATCC 14067

[0512] 64: Amino acid sequence of the HisAK domain of the Phos protein from Corynebacterium tumefaciens.

[0513] 65: Amino acid sequence of the HisAK domain of the Phos protein from Corynebacterium tumefaciens.

[0514] 66: Amino acid sequence of the HisAK domain of the Phos protein of C. efficiens

[0515] 67: Nucleotide sequence of the cspB gene in Corynebacterium glutamicum ATCC 13869

[0516] 68: Amino acid sequence of CspB protein from Corynebacterium glutamicum ATCC 13869

[0517] 69: Amino acid sequence of TatA protein from Corynebacterium glutamicum ATCC 13032

[0518] 70: Amino acid sequence of TatA protein from Corynebacterium glutamicum ATCC 13032

[0519] 71: Nucleotide sequence of the tatB gene in Corynebacterium glutamicum ATCC 13032

[0520] 72: Amino acid sequence of TatB protein from Corynebacterium glutamicum ATCC 13032

[0521] 73: Nucleotide sequence of the tatC gene in Corynebacterium glutamicum ATCC 13032

[0522] 74: Amino acid sequence of TatC protein from Corynebacterium glutamicum ATCC 13032

[0523] 75: Amino acid sequence of TorA signal peptide

[0524] 76: Amino acid sequence of the SufI signal peptide

[0525] 77: Amino acid sequence of PhoD signal peptide

[0526] 78: Amino acid sequence of LipA signal peptide

[0527] 79: Amino acid sequence of the IMD signal peptide

[0528] 80 and 81: Amino acid sequences of the diarginine motif

[0529] 82: Amino acid sequence of PS1 signal peptide

[0530] 83: Amino acid sequence of PS2 signal peptide

[0531] 84: Amino acid sequence of SlpA signal peptide

[0532] 85: Amino acid sequence of mature CspB protein from Corynebacterium glutamicum ATCC 13869

[0533] 86 to 93: In one embodiment, the amino acid sequence used for the insert sequence of the present invention

[0534] 94: Recognition sequence of factor Xa protease

[0535] 95: Recognition sequence of ProTEV protease

[0536] 96: Nucleotide sequence of the phoR gene of Corynebacterium glutamicum ATCC 13032

[0537] 97: Amino acid sequence of the Phor protein from Corynebacterium glutamicum ATCC 13032 sequence list <110> Ajinomoto Co., Ltd. <120> Protein secretion production methods <130> D757-16011 <150> JP2015-089046 <151> 2015-04-24 <160> 97 <170> PatentIn version 3.5 <210> 1 <211> 1458 <212> DNA <213> Corynebacterium glutamicum <400> 1 atggaaaacc cttatgtcgc tgcgctcgat gacgataaaa aagaagtcgg cgcaataaaa 60 gaagcagaaa aagaacctga aataggtccc atcagagctg ccggacgagc cataccgctg 120 cgcacccgca tcattttgat cgtggtgggt atcgccgggc ttggtttgct ggtcaacgcg 180 attgctgttt ccagcctcat gcgtgaagtt tcctataccc gcatggatca agagctagag 240 acctcgatgg ggacgtgggc gcataacgtt gagctgttta atttcgatgg cgtccgccaa 300 gggccaccca gcgattatta tgtggccaag gtttttcctg atggatccag cattattttc 360 aacgatgcac aatcggcacc caatctagct gaaaccacca tcggtactgg tccacacact 420 gtggatgctg ctagcggttc tgcctccaac actccgtggc gtgtgatggc ggaaaagaac 480 ggtgacatta tcaccgtggt gggtaaaagc atggggcgtg aaacaaacct gctgtaccga 540 ttggtgatgg tgcagatgat catcggcgcg ctgattctgg ttgctatttt gattacttca 600 ctcttcctag tcagacgctc gttgcggccg ttgagagaag ttgaagagac cgccaccagg 660 attgcgggcg gtgatttgga tcgacgtgtc ccgcagtggc caatgaccac agaagtcgga 720 cagctgtcga atgccctcaa tatcatgttg gagcagctcc aagcctcaat tctgaccgcc 780 cagcaaaaag aagctcagat gcgccgattc gttggcgacg cctcccacga gctccgcaca 840 ccactgacct ctgtgaaggg cttcaccgag ctgtattcat caggtgcaac agatgatgcc 900 aactgtgtca tgtccaagat cggtggcgaa gcccaacgca tgagtgtgct tgtggaagac 960 ctcctgtcac tgacgcgtgc cgaaggccag caaatggaga agcaccgcgt tgacgtgctg 1020 gaactcgcat tggcagtacg cggatccatg cgagcagcct ggccagatcg caccgtcaac 1080 gtgtccaata aagccgagtc cattccagtt gttgaaggcg acccaacccg cctccaccaa 1140 gttctcacca acctggttgc caacggactc aaccacggcg gaccggacgc ggaagtcagc 1200 attgagatca acaccgatgg gcaaaacgtg aggattctcg tggcagacaa cggtgtcgga 1260 atgtctgaag aagatgccca gcatatcttc gagcgtttct accgcgccga ttcctcccgc 1320 tcacgcgcat ccggcggatc gggcctcggc cttgcgatca cgaaatccct ggtcgaaggc 1380 cacggcggca cagtcaccgt cgacagcgtg caaggcgaag gcacggtgtt cacgatcacc 1440 ttgccggcgg tttcttaa 1458 <210> 2 <211> 485 <212> PRT <213> Corynebacterium glutamicum <400> 2 Met Glu Asn Pro Tyr Val Ala Ala Leu Asp Asp Asp Lys Lys Glu Val 1 5 10 15 Gly Ala Ile Lys Glu Ala Glu Lys Glu Pro Glu Ile Gly Pro Ile Arg 20 25 30 Ala Ala Gly Arg Ala Ile Pro Leu Arg Thr Arg Ile Ile Leu Ile Val 35 40 45 Val Gly Ile Ala Gly Leu Gly Leu Leu Val Asn Ala Ile Ala Val Ser 50 55 60 Ser Leu Met Arg Glu Val Ser Tyr Thr Arg Met Asp Gln Glu Leu Glu 65 70 75 80 Thr Ser Met Gly Thr Trp Ala His Asn Val Glu Leu Phe Asn Phe Asp 85 90 95 Gly Val Arg Gln Gly Pro Pro Ser Asp Tyr Tyr Val Ala Lys Val Phe 100 105 110 Pro Asp Gly Ser Ser Ile Ile Phe Asn Asp Ala Gln Ser Ala Pro Asn 115 120 125 Leu Ala Glu Thr Thr Ile Gly Thr Gly Pro His Thr Val Asp Ala Ala 130 135 140 Ser Gly Ser Ala Ser Asn Thr Pro Trp Arg Val Met Ala Glu Lys Asn 145 150 155 160 Gly Asp Ile Ile Thr Val Val Gly Lys Ser Met Gly Arg Glu Thr Asn 165 170 175 Leu Leu Tyr Arg Leu Val Met Val Gln Met Ile Ile Gly Ala Leu Ile 180 185 190 Leu Val Ala Ile Leu Ile Thr Ser Leu Phe Leu Val Arg Arg Ser Leu 195 200 205 Arg Pro Leu Arg Glu Val Glu Glu Thr Ala Thr Arg Ile Ala Gly Gly 210 215 220 Asp Leu Asp Arg Arg Val Pro Gln Trp Pro Met Thr Thr Glu Val Gly 225 230 235 240 Gln Leu Ser Asn Ala Leu Asn Ile Met Leu Glu Gln Leu Gln Ala Ser 245 250 255 Ile Leu Thr Ala Gln Gln Lys Glu Ala Gln Met Arg Arg Phe Val Gly 260 265 270 Asp Ala Ser His Glu Leu Arg Thr Pro Leu Thr Ser Val Lys Gly Phe 275 280 285 Thr Glu Leu Tyr Ser Ser Gly Ala Thr Asp Asp Ala Asn Cys Val Met 290 295 300 Ser Lys Ile Gly Gly Glu Ala Gln Arg Met Ser Val Leu Val Glu Asp 305 310 315 320 Leu Leu Ser Leu Thr Arg Ala Glu Gly Gln Gln Met Glu Lys His Arg 325 330 335 Val Asp Val Leu Glu Leu Ala Leu Ala Val Arg Gly Ser Met Arg Ala 340 345 350 Ala Trp Pro Asp Arg Thr Val Asn Val Ser Asn Lys Ala Glu Ser Ile 355 360 365 Pro Val Val Glu Gly Asp Pro Thr Arg Leu His Gln Val Leu Thr Asn 370 375 380 Leu Val Ala Asn Gly Leu Asn His Gly Gly Pro Asp Ala Glu Val Ser 385 390 395 400 Ile Glu Ile Asn Thr Asp Gly Gln Asn Val Arg Ile Leu Val Ala Asp 405 410 415 Asn Gly Val Gly Met Ser Glu Glu Asp Ala Gln His Ile Phe Glu Arg 420 425 430 Phe Tyr Arg Ala Asp Ser Ser Arg Ser Arg Ala Ser Gly Gly Ser Gly 435 440 445 Leu Gly Leu Ala Ile Thr Lys Ser Leu Val Glu Gly His Gly Gly Thr 450 455 460 Val Thr Val Asp Ser Val Gln Gly Glu Gly Thr Val Phe Thr Ile Thr 465 470 475 480 Leu Pro Ala Val Ser 485 <210> 3 <211> 1458 <212> DNA <213> Corynebacterium glutamicum <400> 3 atggaaaacc cttatgtcgc tgcgctcgat gacgataaaa aagaagtcgg cgcaataaaa 60 gaagcagaaa aagaacctga aataggtccc atcagagctg ccggacgagc cataccgctg 120 cgcacccgca tcattttgat cgtggtgggt atcgccgggc ttggtttgct ggtcaacgcg 180 attgctgttt ccagcctcat gcgtgaagtt tcctataccc gcatggatca agagctagag 240 acctcgatgg ggacgtgggc gcataacgtt gagctgttta atttcgatgg cgtccgccaa 300 gggccaccca gcgattatta tgtggccaag gtttttcctg atggatccag cattattttc 360 aacgatgcac aatcggcacc caatctagct gaaaccacca tcggtactgg tccacacact 420 gtggatgctg ctagcggttc tgcctccaac actccgtggc gtgtgatggc ggaaaagaac 480 ggtgacatta tcaccgtggt gggtaaaagc atggggcgtg aaacaaacct gctgtaccga 540 ttggtgatgg tgcagatgat catcggcgcg ctgattctgg ttgctatttt gattacttca 600 ctcttcctag tcagacgctc gttgcggccg ttgagagaag ttgaagagac cgccaccagg 660 attgcgggcg gtgatttgga tcgacgtgtc ccgcagtggc caatgaccac agaagtcgga 720 cagctgtcga atgccctcaa tatcatgttg gagcagctcc aagcctcaat tctgaccgcc 780 cagcaaaaag aagctcagat gcgccgattc gttggcgacg cctcccacga gctccgcaca 840 ccactgacct ctgtgaaggg cttcaccgag ctgtattcat caggtgcaac agatgatgcc 900 aactgggtca tgtccaagat cggtggcgaa gcccaacgca tgagtgtgct tgtggaagac 960 ctcctgtcac tgacgcgtgc cgaaggccag caaatggaga agcaccgcgt tgacgtgctg 1020 gaactcgcat tggcagtacg cggatccatg cgagcagcct ggccagatcg caccgtcaac 1080 gtgtccaata aagccgagtc cattccagtt gttgaaggcg acccaacccg cctccaccaa 1140 gttctcacca acctggttgc caacggactc aaccacggcg gaccggacgc ggaagtcagc 1200 attgagatca acaccgatgg gcaaaacgtg aggattctcg tggcagacaa cggtgtcgga 1260 atgtctgaag aagatgccca gcatatcttc gagcgtttct accgcgccga ttcctcccgc 1320 tcacgcgcat ccggcggatc gggcctcggc cttgcgatca cgaaatccct ggtcgaaggc 1380 cacggcggca cagtcaccgt cgacagcgtg caaggcgaag gcacggtgtt cacgatcacc 1440 ttgccggcgg tttcttaa 1458 <210> 4 <211> 485 <212> PRT <213> Corynebacterium glutamicum <400> 4 Met Glu Asn Pro Tyr Val Ala Ala Leu Asp Asp Asp Lys Lys Glu Val 1 5 10 15 Gly Ala Ile Lys Glu Ala Glu Lys Glu Pro Glu Ile Gly Pro Ile Arg 20 25 30 Ala Ala Gly Arg Ala Ile Pro Leu Arg Thr Arg Ile Ile Leu Ile Val 35 40 45 Val Gly Ile Ala Gly Leu Gly Leu Leu Val Asn Ala Ile Ala Val Ser 50 55 60 Ser Leu Met Arg Glu Val Ser Tyr Thr Arg Met Asp Gln Glu Leu Glu 65 70 75 80 Thr Ser Met Gly Thr Trp Ala His Asn Val Glu Leu Phe Asn Phe Asp 85 90 95 Gly Val Arg Gln Gly Pro Pro Ser Asp Tyr Tyr Val Ala Lys Val Phe 100 105 110 Pro Asp Gly Ser Ser Ile Ile Phe Asn Asp Ala Gln Ser Ala Pro Asn 115 120 125 Leu Ala Glu Thr Thr Ile Gly Thr Gly Pro His Thr Val Asp Ala Ala 130 135 140 Ser Gly Ser Ala Ser Asn Thr Pro Trp Arg Val Met Ala Glu Lys Asn 145 150 155 160 Gly Asp Ile Ile Thr Val Val Gly Lys Ser Met Gly Arg Glu Thr Asn 165 170 175 Leu Leu Tyr Arg Leu Val Met Val Gln Met Ile Ile Gly Ala Leu Ile 180 185 190 Leu Val Ala Ile Leu Ile Thr Ser Leu Phe Leu Val Arg Arg Ser Leu 195 200 205 Arg Pro Leu Arg Glu Val Glu Glu Thr Ala Thr Arg Ile Ala Gly Gly 210 215 220 Asp Leu Asp Arg Arg Val Pro Gln Trp Pro Met Thr Thr Glu Val Gly 225 230 235 240 Gln Leu Ser Asn Ala Leu Asn Ile Met Leu Glu Gln Leu Gln Ala Ser 245 250 255 Ile Leu Thr Ala Gln Gln Lys Glu Ala Gln Met Arg Arg Phe Val Gly 260 265 270 Asp Ala Ser His Glu Leu Arg Thr Pro Leu Thr Ser Val Lys Gly Phe 275 280 285 Thr Glu Leu Tyr Ser Ser Gly Ala Thr Asp Asp Ala Asn Trp Val Met 290 295 300 Ser Lys Ile Gly Gly Glu Ala Gln Arg Met Ser Val Leu Val Glu Asp 305 310 315 320 Leu Leu Ser Leu Thr Arg Ala Glu Gly Gln Gln Met Glu Lys His Arg 325 330 335 Val Asp Val Leu Glu Leu Ala Leu Ala Val Arg Gly Ser Met Arg Ala 340 345 350 Ala Trp Pro Asp Arg Thr Val Asn Val Ser Asn Lys Ala Glu Ser Ile 355 360 365 Pro Val Val Glu Gly Asp Pro Thr Arg Leu His Gln Val Leu Thr Asn 370 375 380 Leu Val Ala Asn Gly Leu Asn His Gly Gly Pro Asp Ala Glu Val Ser 385 390 395 400 Ile Glu Ile Asn Thr Asp Gly Gln Asn Val Arg Ile Leu Val Ala Asp 405 410 415 Asn Gly Val Gly Met Ser Glu Glu Asp Ala Gln His Ile Phe Glu Arg 420 425 430 Phe Tyr Arg Ala Asp Ser Ser Arg Ser Arg Ala Ser Gly Gly Ser Gly 435 440 445 Leu Gly Leu Ala Ile Thr Lys Ser Leu Val Glu Gly His Gly Gly Thr 450 455 460 Val Thr Val Asp Ser Val Gln Gly Glu Gly Thr Val Phe Thr Ile Thr 465 470 475 480 Leu Pro Ala Val Ser 485 <210> 5 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 5 aggcagcaaa acaccgagga ctcaa 25 <210> 6 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 6 cgggcttggtttgctggtca acgcg 25 <210> 7 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 7 tcgagctcgg tacccggcta atcctctggc ctg 33 <210> 8 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 8 taactaattt ctcctaggca tcaagggccg gaa 33 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 9 aggagaaatt agttacgtgg 20 <210> 10 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 10 ctctagagga tcccccggat gtacgtggaa gac 33 <210> 11 <211> 127 <212> PRT <213> Homo sapiens <400> 11 Met Ser Phe Ser Gly Lys Tyr Gln Leu Gln Ser Gln Glu Asn Phe Glu 1 5 10 15 Ala Phe Met Lys Ala Ile Gly Leu Pro Glu Glu Leu Ile Gln Lys Gly 20 25 30 Lys Asp Ile Lys Gly Val Ser Glu Ile Val Gln Asn Gly Lys His Phe 35 40 45 Lys Phe Thr Ile Thr Ala Gly Ser Lys Val Ile Gln Asn Glu Phe Thr 50 55 60 Val Gly Glu Glu Cys Glu Leu Glu Thr Met Thr Gly Glu Lys Val Lys 65 70 75 80 Thr Val Val Gln Leu Glu Gly Asp Asn Lys Leu Val Thr Thr Phe Lys 85 90 95 Asn Ile Lys Ser Val Thr Glu Leu Asn Gly Asp Ile Ile Thr Asn Thr 100 105 110 [[ID=2৪]]Met Thr Leu Gly Asp Ile Val Phe Lys Arg Ile Ser Lys Arg Ile 115 120 125 <210> 12 <211> 504 <212> DNA <213> Homo sapiens <400> 12 atgtttaaca accgtatccg cactgcagct ctcgctggtg caatcgcaat ctccaccgca 60 gcttccggcg tagctatccc agcattcgct caggagacca acccaaccat cgagggccgc 120 atgtccttct ccggcaagta ccagctgcag tcccaggaaa acttcgaggc attcatgaag 180 gctatcggtc tgccagaaga gctcatccag aagggcaagg atatcaaggg tgtttccgaa 240 atcgtgcaga acggcaagca cttcaagttc accatcaccg caggttccaa ggtcatccag 300 aacgagttca ccgttggcga agagtgcgaa ctcgagacca tgaccggtga aaaggttaag 360 accgtggtcc agctggaggg cgacaacaag ctcgtgacca ccttcaagaa catcaagtcc 420 gtcaccgaac tgaacggcga tatcatcacc aacaccatga ccctcggtga catcgtgttc 480 aagcgcatct ccaagcgtat ctaa 504 <210> 13 <211> 167 <212> PRT <213> Homo sapiens <400> 13 Met Phe Asn Asn Arg Ile Arg Thr Ala Ala Leu Ala Gly Ala Ile Ala 1 5 10 15 Ile Ser Thr Ala Ala Ser Gly Val Ala Ile Pro Ala Phe Ala Gln Glu 20 25 30 Thr Asn Pro Thr Ile Glu Gly Arg Met Ser Phe Ser Gly Lys Tyr Gln 35 40 45 Leu Gln Ser Gln Glu Asn Phe Glu Ala Phe Met Lys Ala Ile Gly Leu 50 55 60 Pro Glu Glu Leu Ile Gln Lys Gly Lys Asp Ile Lys Gly Val Ser Glu 65 70 75 80 Ile Val Gln Asn Gly Lys His Phe Lys Phe Thr Ile Thr Ala Gly Ser 85 90 95 Lys Val Ile Gln Asn Glu Phe Thr Val Gly Glu Glu Cys Glu Leu Glu 100 105 110 Thr Met Thr Gly Glu Lys Val Lys Thr Val Val Gln Leu Glu Gly Asp 115 120 125 Asn Lys Leu Val Thr Thr Phe Lys Asn Ile Lys Ser Val Thr Glu Leu 130 135 140 Asn Gly Asp Ile Ile Thr Asn Thr Met Thr Leu Gly Asp Ile Val Phe 145 150 155 160 Lys Arg Ile Ser Lys Arg Ile 165 <210> 14 <211> 41 <212> PRT <213> Gila monster (Heloderma suspectum) <400> 14 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Cys Pro 35 40 <210> 15 <211> 252 <212> DNA <213> Gila monster (Heloderma suspectum) <400> 15 atgtttaaca accgtatccg cactgcagct ctcgctggtg caatcgcaat ctccaccgca 60 gcttccggcg tagctatccc agcattcgct caggagacca acccaaccga aaacctgtac 120 ttccagcacg gcgagggaac cttcacgtct gatctgtcta agcagatgga ggaagaggca 180 gttcgcctgt tcattgagtg gctgaaaaat ggcggtcctt ctagcggtgc acctcccccc 240 tcctgcccat ga 252 <210> 16 <211> 83 <212> PRT <213> Gila monster (Heloderma suspectum) <400> 16 Met Phe Asn Asn Arg Ile Arg Thr Ala Ala Leu Ala Gly Ala Ile Ala 1 5 10 15 [[ID=三十九]]Ile Ser Thr Ala Ala Ser Gly Val Ala Ile Pro Ala Phe Ala Gln Glu 20 25 30 Thr Asn Pro Thr Glu Asn Leu Tyr Phe Gln His Gly Glu Gly Thr Phe 35 40 45 Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe 50 55 60 Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro 65 70 75 80 Ser Cys Pro <210> 17 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 17 cgagccacca ggcaggcggg aaaatcg 27 <210> 18 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 18 cgattttccc gcctgcctgg tggctcg 27 <210> 19 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 19 cccgcttgat cattccttta agg 23 <210> 20 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 20 aatgggccctttggtacccc taaataatat cggtcc 36 <210> twenty one <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty one cgtgctctag gggaaccgtg cgttccc 27 <210> twenty two <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty two gggacgcac ggttccccta gagcacg 27 <210> twenty three <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty three cgacgctgaa gttgtagaga tcatccg 27 <210> twenty four <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty four cggatgatct ctacaacttc agcgtcg 27 <210> 25 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 25 ggcggtaccc aaattcctgt gaagtagc 28 <210> 26 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 26 ggcgggcccg ccggcagtcg cacgtcgcgg cgttaacaat gacg 44 <210> 27 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primers <400> 27 gacaatggcg cgggggaaga gacg 24 <210> 28 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 28 caggtcgact ctagaggatc cc 22 <210> 29 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 29 atattattta ggtctagaca aattcctgtg 30 <210> 30 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 30 ctgcaggtcg actctagaat taattaaaat ccaca 35 <210> 31 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 31 ctgcaggtcg actctagatc acatgtccaa ctctatcc 38 <210> 32 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 32 ctctagagga tccccatgga aaacccttat gtcgc 35 <210> 33 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 33 tcgagctcgg tacccttaag aaaccgccgg caag 34 <210> 34 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 34 catgacggag ttggcatcat ctgttgcacc 30 <210> 35 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 35 gccaactccg tcatgtccaa gatcggtgg 29 <210> 36 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 36 catgactgcg ttggcatcat ctgttgcacc 30 <210> 37 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 37 gccaacgcag tcatgtccaa gatcggtgg 29 <210> 38 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 38 catgaccacg ttggcatcat ctgttgcacc 30 <210> 39 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 39 gccaacgtgg tcatgtccaa gatcggtgg 29 <210> 40 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 40 catgaccatg ttggcatcat ctgttgcacc 30 <210> 41 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 41 gccaacatgg tcatgtccaa gatcggtgg 29 <210> 42 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 42 catgacgaag ttggcatcat ctgttgcacc 30 <210> 43 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 43 gccaacttcg tcatgtccaa gatcggtgg 29 <210> 44 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 44 catgacgtag ttggcatcat ctgttgcacc 30 <210> 45 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 45 gccaactacg tcatgtccaa gatcggtgg 29 <210> 46 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 46 catgacatcg ttggcatcat ctgttgcacc 30 <210> 47 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 47 gccaacgatg tcatgtccaagatcggtgg 29 <210> 48 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 48 catgacgttg ttggcatcat ctgttgcacc 30 <210> 49 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 49 gccaacaacg tcatgtccaa gatcggtgg 29 <210> 50 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 50 catgacgtgg ttggcatcat ctgttgcacc 30 <210> 51 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 51 gccaaccacg tcatgtccaa gatcggtgg 29 <210> 52 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 52 catgaccttg ttggcatcat ctgttgcacc 30 <210> 53 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 53 gccaacaagg tcatgtccaa gatcggtgg 29 <210> 54 <211> 485 <212> PRT <213> Corynebacterium glutamicum <400> 54 Met Glu Asn Pro Tyr Val Ala Ala Leu Asp Asp Glu Asn Gln Glu Val 1 5 10 15 Gly Val Lys Lys Glu Ala Glu Lys Glu Pro Glu Ile Gly Pro Ile Arg 20 25 30 Ala Ala Gly Arg Ala Ile Pro Leu Arg Thr Arg Ile Ile Leu Ile Val 35 40 45 Val Gly Ile Ala Gly Leu Gly Leu Leu Val Asn Ala Ile Ala Val Ser 50 55 60 Ser Leu Met Arg Glu Val Ser Tyr Thr Arg Met Asp Gln Glu Leu Glu 65 70 75 80 Thr Ser Met Gly Thr Trp Ala His Asn Val Glu Leu Phe Asn Phe Asp 85 90 95 Gly Val Arg Gln Gly Pro Pro Ser Asp Tyr Tyr Val Ala Lys Val Phe 100 105 110 Pro Asp Gly Ser Ser Ile Ile Phe Asn Asp Ala Gln Ser Ala Pro Asp 115 120 125 Leu Ala Glu Thr Thr Ile Gly Thr Gly Pro His Thr Val Asp Ala Ala 130 135 140 Ser Gly Ser Ala Ser Asn Thr Pro Trp Arg Val Met Ala Glu Lys Asn 145 150 155 160 Gly Asp Ile Ile Thr Val Val Gly Lys Ser Met Gly Arg Glu Thr Asn 165 170 175 Leu Leu Tyr Arg Leu Val Met Val Gln Met Ile Ile Gly Ala Leu Ile 180 185 190 Leu Val Ala Ile Leu Ile Thr Ser Leu Phe Leu Val Arg Arg Ser Leu 195 200 205 Arg Pro Leu Arg Glu Val Glu Glu Thr Ala Thr Arg Ile Ala Gly Gly 210 215 220 Asp Leu Asp Arg Arg Val Pro Gln Trp Pro Met Thr Thr Glu Val Gly 225 230 235 240 Gln Leu Ser Asn Ala Leu Asn Ile Met Leu Glu Gln Leu Gln Ala Ser 245 250 255 Ile Leu Thr Ala Gln Gln Lys Glu Ala Gln Met Arg Arg Phe Val Gly 260 265 270 Asp Ala Ser His Glu Leu Arg Thr Pro Leu Thr Ser Val Lys Gly Phe 275 280 285 Thr Glu Leu Tyr Ser Ser Gly Ala Thr Asp Asp Ala Asn Trp Val Met 290 295 300 Ser Lys Ile Gly Gly Glu Ala Gln Arg Met Ser Val Leu Val Glu Asp 305 310 315 320 Leu Leu Ser Leu Thr Arg Ala Glu Gly Gln Gln Met Glu Lys His Arg 325 330 335 Val Asp Val Leu Glu Leu Ala Leu Ala Val Arg Gly Ser Met Arg Ala 340 345 350 Ala Trp Pro Asp Arg Thr Val Asn Val Ser Asn Lys Ala Glu Ser Ile 355 360 365 Pro Val Val Lys Gly Asp Pro Thr Arg Leu His Gln Val Leu Thr Asn 370 375 380 Leu Val Ala Asn Gly Leu Asn His Gly Gly Pro Asp Ala Glu Val Ser 385 390 395 400 Ile Glu Ile Asn Thr Asp Gly Gln Asn Val Arg Ile Leu Val Ala Asp 405 410 415 Asn Gly Val Gly Met Ser Glu Glu Asp Ala Gln His Ile Phe Glu Arg 420 425 430 Phe Tyr Arg Ala Asp Ser Ser Arg Ser Arg Ala Ser Gly Gly Ser Gly 435 440 445 Leu Gly Leu Ala Ile Thr Lys Ser Leu Val Glu Gly His Gly Gly Thr 450 455 460 Val Thr Val Asp Ser Val Gln Gly Glu Gly Thr Val Phe Thr Ile Thr 465 470 475 480 Leu Pro Ala Val Ser 485 <210> 55 <211> 485 <212> PRT <213> Corynebacterium glutamicum <400> 55 Met Glu Asn Pro Tyr Val Ala Ala Leu Asp Asp Glu Asn Gln Glu Val 1 5 10 15 Gly Val Lys Lys Glu Ala Glu Lys Glu Pro Glu Ile Gly Pro Ile Arg 20 25 30 Ala Ala Gly Arg Ala Ile Pro Leu Arg Thr Arg Ile Ile Leu Ile Val 35 40 45 Val Gly Ile Ala Gly Leu Gly Leu Leu Val Asn Ala Ile Ala Val Ser 50 55 60 Ser Leu Met Arg Glu Val Ser Tyr Thr Arg Met Asp Gln Glu Leu Glu 65 70 75 80 Thr Ser Met Gly Thr Trp Ala His Asn Val Glu Leu Phe Asn Phe Asp 85 90 95 Gly Val Arg Gln Gly Pro Pro Ser Asp Tyr Tyr Val Ala Lys Val Phe 100 105 110 Pro Asp Gly Ser Ser Ile Ile Phe Asn Asp Ala Gln Ser Ala Pro Asp 115 120 125 Leu Ala Glu Thr Thr Ile Gly Thr Gly Pro His Thr Val Asp Ala Ala 130 135 140 Ser Gly Ser Ala Ser Asn Thr Pro Trp Arg Val Met Ala Glu Lys Asn 145 150 155 160 Gly Asp Ile Ile Thr Val Val Gly Lys Ser Met Gly Arg Glu Thr Asn 165 170 175 Leu Leu Tyr Arg Leu Val Val Val Gln Met Ile Ile Gly Ala Leu Ile 180 185 190 Leu Val Ala Ile Leu Ile Thr Ser Leu Phe Leu Val Arg Arg Ser Leu 195 200 205 Arg Pro Leu Arg Glu Val Glu Glu Thr Ala Thr Arg Ile Ala Gly Gly 210 215 220 Asp Leu Asp Arg Arg Val Pro Gln Trp Pro Met Thr Thr Glu Val Gly 225 230 235 240 Gln Leu Ser Asn Ala Leu Asn Ile Met Leu Glu Gln Leu Gln Ala Ser 245 250 255 Ile Leu Thr Ala Gln Gln Lys Glu Ala Gln Met Arg Arg Phe Val Gly 260 265 270 Asp Ala Ser His Glu Leu Arg Thr Pro Leu Thr Ser Val Lys Gly Phe 275 280 285 Thr Glu Leu Tyr Ser Ser Gly Ala Thr Asp Asp Ala Asn Trp Val Met 290 295 300 Ser Lys Ile Gly Gly Glu Ala Gln Arg Met Ser Val Leu Val Glu Asp 305 310 315 320 Leu Leu Ser Leu Thr Arg Ala Glu Gly Gln Gln Met Glu Lys His Arg 325 330 335 Val Asp Val Leu Glu Leu Ala Leu Ala Val Arg Gly Ser Met Arg Ala 340 345 350 Ala Trp Pro Asp Arg Thr Val Asn Val Ser Asn Lys Ala Glu Ser Ile 355 360 365 Pro Val Val Glu Gly Asp Pro Thr Arg Leu His Gln Val Leu Thr Asn 370 375 380 Leu Val Ala Asn Gly Leu Asn His Gly Gly Pro Asp Ala Glu Val Ser 385 390 395 400 Ile Glu Ile Asn Thr Asp Gly Gln Asn Val Arg Ile Leu Val Ala Asp 405 410 415 Asn Gly Val Gly Met Ser Glu Glu Asp Ala Gln His Ile Phe Glu Arg 420 425 430 Phe Tyr Arg Ala Asp Ser Ser Arg Ser Arg Ala Ser Gly Gly Ser Gly 435 440 445 Leu Gly Leu Ala Ile Thr Lys Ser Leu Val Glu Gly His Gly Gly Thr 450 455 460 Val Thr Val Asp Ser Val Gln Gly Glu Gly Thr Val Phe Thr Ile Thr 465 470 475 480 Leu Pro Ala Val Ser 485 <210> 56 <211> 504 <212> PRT <213> Calluna vulgaris <400> 56 Met Glu Asn Pro Tyr Val Ala Ala Leu Asp Lys Asn Ser Asn Phe Gly 1 5 10 15 Ala Lys Asp Thr Asp Ser Ala Val Ser Asp Ser Thr Glu Val Ser Gln 20 25 30 Asn Asn Asp Gly Ile Gly Thr Pro Ala Thr Ala Glu Pro Lys Val Gly 35 40 45 Pro Ile Arg Thr Ala Gly Arg Ala Met Pro Leu Arg Thr Arg Ile Ile 50 55 60 Leu Leu Val Val Gly Ile Ala Gly Leu Gly Leu Leu Val Asn Ala Val 65 70 75 80 Ala Val Ser Ser Leu Met Arg Glu Val Ser Tyr Thr Arg Met Asp Gln 85 90 95 Asp Leu Glu Ser Ala Met Gly Thr Trp Val Arg Asn Val Glu Leu Phe 100 105 110 Asn Phe Asp Gly Val Arg Gln Gly Pro Pro Ser Asp Tyr Tyr Val Ala 115 120 125 Lys Val Phe Pro Asp Gly Ser Ser Ile Ile Phe Asn Asp Ala Glu Ser 130 135 140 Ala Pro Asp Leu Gly Gln Thr Thr Ile Gly Thr Gly Pro His Thr Val 145 150 155 160 Glu Ala Ala Glu Gly Ser Ala Ser Ser Thr His Trp Arg Val Met Ala 165 170 175 Ala Lys Asn Gly Asp Val Ile Thr Val Val Gly Lys Ser Met Gly Arg 180 185 190 Glu Ser Thr Leu Leu Tyr Arg Leu Val Val Val Gln Met Val Ile Gly 195 200 205 Val Leu Ile Leu Ile Ala Ile Leu Ile Gly Ser Phe Phe Leu Val Arg 210 215 220 Arg Ser Leu Lys Pro Leu Arg Glu Val Glu Glu Thr Ala Ser Arg Ile 225 230 235 240 Ala Gly Gly Glu Leu Asp Arg Arg Val Pro Gln Trp Pro Met Thr Thr 245 250 255 Glu Val Gly Gln Leu Ala Asn Ala Leu Asn Ile Met Leu Glu Gln Leu 260 265 270 Gln Thr Ser Ile Met Asn Ala Gln Gln Lys Glu Ala Gln Met Arg Arg 275 280 285 Phe Val Gly Asp Ala Ser His Glu Leu Arg Thr Pro Leu Thr Ser Val 290 295 300 Lys Gly Phe Thr Glu Leu Tyr Ser Ser Gly Ala Thr Gln Asp Ala Asp 305 310 315 320 Trp Val Leu Ser Lys Ile Gly Gly Glu Ala Gln Arg Met Ser Val Leu 325 330 335 Val Glu Asp Leu Leu Ser Leu Thr Arg Ala Glu Gly Gln Gln Met Glu 340 345 350 Lys His Arg Val Asp Met Leu Glu Leu Ala Leu Ala Val Arg Gly Ser 355 360 365 Leu Lys Ala Ala Trp Pro Asp Arg Thr Val Asn Val Ala Asn Arg Ser 370 375 380 Glu Asn Ile Pro Val Val Glu Gly Asp Pro Thr Arg Leu His Gln Val 385 390 395 400 Leu Thr Asn Leu Val Ala Asn Gly Leu Asn His Gly Gly Pro Glu Ala 405 410 415 Glu Val Asn Ile Gln Val Glu Thr Ala Asp Asp Lys Val Lys Ile Leu 420 425 430 Val Ile Asp Asn Gly Val Gly Met Ser Lys Glu Asp Ala Glu His Ile 435 440 445 Phe Glu Arg Phe Tyr Arg Ala Asp Thr Ser Arg Ser Arg Ala Ser Gly 450 455 460 Gly Ser Gly Leu Gly Leu Ala Ile Thr Lys Ser Leu Val Glu Gly His 465 470 475 480 Gly Gly Thr Ile Thr Val Asp Ser Glu Leu Gly Lys Gly Thr Val Phe 485 490 495 Ser Ile Ile Leu Pro Ala Ala Glu 500 <210> 57 <211> 458 <212> PRT <213> Corynebacterium crenatum <400> 57 Ile Gly Pro Ile Arg Ala Ala Gly Arg Ala Ile Pro Leu Arg Thr Arg 1 5 10 15 Ile Ile Leu Ile Val Val Gly Ile Ala Gly Leu Gly Leu Leu Val Asn 20 25 30 Ala Ile Ala Val Ser Ser Leu Met Arg Glu Val Ser Tyr Thr Arg Met 35 40 45 Asp Gln Glu Leu Glu Thr Ser Met Gly Thr Trp Ala His Asn Val Glu 50 55 60 Leu Phe Asn Phe Asp Gly Val Arg Gln Gly Pro Pro Ser Asp Tyr Tyr 65 70 75 80 Val Ala Lys Val Phe Pro Asp Gly Ser Ser Ile Ile Phe Asn Asp Ala 85 90 95 Gln Ser Ala Pro Asp Leu Ala Glu Thr Thr Ile Gly Thr Gly Pro His 100 105 110 Thr Val Asp Ala Ala Ser Gly Ser Ala Ser Asn Thr Pro Trp Arg Val 115 120 125 Met Ala Glu Lys Asn Gly Asp Ile Ile Thr Val Val Gly Lys Ser Met 130 135 140 Gly Arg Glu Thr Asn Leu Leu Tyr Arg Leu Val Met Val Gln Met Ile 145 150 155 160 Ile Gly Ala Leu Ile Leu Val Ala Ile Leu Ile Thr Ser Leu Phe Leu 165 170 175 Val Arg Arg Ser Leu Arg Pro Leu Arg Glu Val Glu Glu Thr Ala Thr 180 185 190 Arg Ile Ala Gly Gly Asp Leu Asp Arg Arg Val Pro Gln Trp Pro Met 195 200 205 Thr Thr Glu Val Gly Gln Leu Ser Asn Ala Leu Asn Ile Met Leu Glu 210 215 220 Gln Leu Gln Ala Ser Ile Leu Ser Ala Gln Gln Lys Glu Ala Gln Met 225 230 235 240 Arg Arg Phe Val Gly Asp Ala Ser His Glu Leu Arg Thr Pro Leu Thr 245 250 255 Ser Val Lys Gly Phe Thr Glu Leu Tyr Ser Ser Gly Ala Thr Asp Asp 260 265 270 Ala Asn Trp Val Met Ser Lys Ile Gly Gly Glu Ala Gln Arg Met Ser 275 280 285 Val Leu Val Glu Asp Leu Leu Ser Leu Thr Arg Ala Glu Gly Gln Gln 290 295 300 Met Glu Lys His Arg Val Asp Val Leu Glu Leu Ala Leu Ala Val Arg 305 310 315 320 Gly Ser Met Arg Ala Ala Trp Pro Asp Arg Thr Val Asn Val Ser Asn 325 330 335 Lys Ala Ala Ser Ile Pro Val Val Glu Gly Asp Pro Thr Arg Leu His 340 345 350 Gln Val Leu Thr Asn Leu Val Ala Asn Gly Leu Asn His Gly Gly Pro 355 360 365 Asp Ala Glu Val Ser Ile Glu Ile Asn Thr Asp Gly Gln Asn Val Arg 370 375 380 Ile Leu Val Ala Asp Asn Gly Val Gly Met Ser Glu Glu Asp Ala Gln 385 390 395 400 His Ile Phe Glu Arg Phe Tyr Arg Ala Asp Ser Ser Arg Ser Arg Ala 405 410 415 Ser Gly Gly Ser Gly Leu Gly Leu Ala Ile Thr Lys Ser Leu Val Glu 420 425 430 Gly His Gly Gly Thr Val Thr Val Asp Ser Val Gln Gly Glu Gly Thr 435 440 445 Val Phe Thr Ile Thr Leu Pro Ala Val Ser 450 455 <210> 58 <211> 471 <212> PRT <213> Corynebacterium efficiens <400> 58 Met Thr Ala Pro Glu Asn Pro His Ala Gln Val Thr Pro Val Gly Arg 1 5 10 15 Phe Arg Gln Ala Ala Arg Gly Val Pro Leu Arg Thr Arg Ile Ile Leu 20 25 30 Leu Val Val Gly Ile Ala Gly Leu Gly Leu Leu Val Asn Ala Ile Ala 35 40 45 Val Ser Ser Leu Met Arg Glu Val Ser Tyr Ser Arg Met Asp Gln Glu 50 55 60 Leu Glu Ser Ala Met Asn Ser Trp Ala Gln Thr Ala Glu Leu Phe Gly 65 70 75 80 Ser Ile Thr Leu Gly Pro Pro Ser Asp Tyr Tyr Val Val Arg Ile Phe 85 90 95 Pro Asp Gly Ser His Met Val Phe Asn Gln Ser Asp Ser Ala Pro Asp 100 105 110 Leu Gly Glu Thr Thr Ile Gly Ile Gly Pro His Thr Ala Ser Ala Ala 115 120 125 Pro Gly Ser Ser Ser Ser Val Pro Trp Arg Val Ile Ala Ile Ser Asp 130 135 140 Asn Gly Thr Ile Thr Val Val Gly Lys Ser Leu Ala Pro Glu Ser Met 145 150 155 160 Leu Leu Tyr Arg Leu Val Ile Val Gln Leu Val Ile Gly Met Leu Ile 165 170 175 Val Val Ala Ile Leu Leu Ser Ser Leu Tyr Leu Val Asn Arg Ser Leu 180 185 190 Arg Pro Leu Arg Glu Val Glu Lys Thr Ala Lys Ser Ile Ala Gly Gly 195 200 205 Asp Leu Asp Arg Arg Val Pro Gln Trp Pro Met Thr Thr Glu Val Gly 210 215 220 Gln Leu Ala Asn Ala Leu Asn Ile Met Leu Glu Gln Leu Gln Ala Ser 225 230 235 240 Ile Leu Ser Ala Gln Glu Lys Glu Ser Gln Met Arg Arg Phe Val Gly 245 250 255 Asp Ala Ser His Glu Leu Arg Thr Pro Leu Thr Ser Val Lys Gly Tyr 260 265 270 Ser Glu Leu Tyr His Ser Gly Ala Thr Arg Asp Ala Asp Trp Val Leu 275 280 285 Ser Lys Ile Ser Gly Glu Ala Gln Arg Met Ser Val Leu Val Glu Asp 290 295 300 Leu Leu Ser Leu Thr Arg Ala Glu Gly Gln Gln Met Glu Lys Arg Pro 305 310 315 320 Val Asp Val Leu Glu Leu Ser Leu Ser Val Ala Ser Ser Met Arg Ala 325 330 335 Ala Trp Pro Glu Arg Ser Ile Thr Val Val Asn Lys Thr Gly Ser Leu 340 345 350 Pro Val Val Glu Gly Asp Ala Thr Arg Leu His Gln Val Leu Thr Asn 355 360 365 Leu Val Asn Asn Gly Leu Asn His Gly Gly Pro Asp Ala Ser Val Glu 370 375 380 Ile Glu Ile Ser Ala Glu Gly Gly Ser Val Leu Val Arg Val Val Asp 385 390 395 400 Asp Gly Val Gly Met Thr Ala Glu Asp Ala Gln His Ile Phe Glu Arg 405 410 415 Phe Tyr Arg Thr Asp Thr Ser Arg Ser Arg Ala Ser Gly Gly Ser Gly 420 425 430 Leu Gly Leu Ala Ile Thr Lys Ser Leu Val Glu Gly His Arg Gly Thr 435 440 445 Ile Thr Val Asp Ser Glu Val Gly Glu Gly Thr Val Phe Thr Ile Thr 450 455 460 Leu Pro Ser Arg Met Glu Asp 465 470 <210> 59 <211> 65 <212> PRT <213> Corynebacterium glutamicum <400> 59 Gln Met Arg Arg Phe Val Gly Asp Ala Ser His Glu Leu Arg Thr Pro 1 5 10 15 Leu Thr Ser Val Lys Gly Phe Thr Glu Leu Tyr Ser Ser Gly Ala Thr 20 25 30 Asp Asp Ala Asn Cys Val Met Ser Lys Ile Gly Gly Glu Ala Gln Arg 35 40 45 Met Ser Val Leu Val Glu Asp Leu Leu Ser Leu Thr Arg Ala Glu Gly 50 55 60 Gln 65 <210> 60 <2​​​​​​​​ 1 5 10 15 Leu Thr Ser Val Lys Gly Phe Thr Glu Leu Tyr Ser Ser Gly Ala Thr 20 25 30 Asp Asp Ala Asn Trp Val Met Ser Lys Ile Gly Gly Glu Ala Gln Arg 35 40 45 Met Ser Val Leu Val Glu Asp Leu Leu Ser Leu Thr Arg Ala Glu Gly 50 55 60 Gln 65 <210> 61 <211> 65 <212> PRT <213> Corynebacterium glutamicum <400> 61 Gln Met Arg Arg Phe Val Gly Asp Ala Ser His Glu Leu Arg Thr Pro 1 5 10 15 Leu Thr Ser Val Lys Gly Phe Thr Glu Leu Tyr Ser Ser Gly Ala Thr 20 25 30 Asp Asp Ala Asn Trp Val Met Ser Lys Ile Gly Gly Glu Ala Gln Arg 35 40 45 Met Ser Val Leu Val Glu Asp Leu Leu Ser Leu Thr Arg Ala Glu Gly 50 55 60 Gln 65 <210> 62 <211> 65 <212> PRT <213> Corynebacterium glutamicum <400> 62 Gln Met Arg Arg Phe Val Gly Asp Ala Ser His Glu Leu Arg Thr Pro 1 5 10 15 Leu Thr Ser Val Lys Gly Phe Thr Glu Leu Tyr Ser Ser Gly Ala Thr 20 25 30 Asp Asp Ala Asn Trp Val Met Ser Lys Ile Gly Gly Glu Ala Gln Arg 35 40 45 Met Ser Val Leu Val Glu Asp Leu Leu Ser Leu Thr Arg Ala Glu Gly 50 55 60 Gln 65 <210> 63 <211> 65 <212> PRT <213> Corynebacterium glutamicum <400> 63 Gln Met Arg Arg Phe Val Gly Asp Ala Ser His Glu Leu Arg Thr Pro 1 5 10 15 Leu Thr Ser Val Lys Gly Phe Thr Glu Leu Tyr Ser Ser Gly Ala Thr 20 25 30 Asp Asp Ala Asn Trp Val Met Ser Lys Ile Gly Gly Glu Ala Gln Arg 35 40 45 Met Ser Val Leu Val Glu Asp Leu Leu Ser Leu Thr Arg Ala Glu Gly 50 55 60 Gln 65 <210> 64 <211> 65 <212> PRT <213> Corynebacterium scoparium <400> 64 Gln Met Arg Arg Phe Val Gly Asp Ala Ser His Glu Leu Arg Thr Pro 1 5 10 15 Leu Thr Ser Val Lys Gly Phe Thr Glu Leu Tyr Ser Ser Gly Ala Thr 20 25 30 Gln Asp Ala Asp Trp Val Leu Ser Lys Ile Gly Gly Glu Ala Gln Arg 35 40 45 Met Ser Val Leu Val Glu Asp Leu Leu Ser Leu Thr Arg Ala Glu Gly 50 55 60 Gln 65 <210> 65 <211> 65 <212> PRT <213> Corynebacterium crenatum <400> 65 Gln Met Arg Arg Phe Val Gly Asp Ala Ser His Glu Leu Arg Thr Pro 1 5 10 15 Leu Thr Ser Val Lys Gly Phe Thr Glu Leu Tyr Ser Ser Gly Ala Thr 20 25 30 Asp Asp Ala Asn Trp Val Met Ser Lys Ile Gly Gly Glu Ala Gln Arg 35 40 45 Met Ser Val Leu Val Glu Asp Leu Leu Ser Leu Thr Arg Ala Glu Gly 50 55 60 Gln 65 <210> 66 <211> 65 <212> PRT <213> Corynebacterium efficiens <400> 66 Gln Met Arg Arg Phe Val Gly Asp Ala Ser His Glu Leu Arg Thr Pro 1 5 10 15 Leu Thr Ser Val Lys Gly Tyr Ser Glu Leu Tyr His Ser Gly Ala Thr 20 25 30 Arg Asp Ala Asp Trp Val Leu Ser Lys Ile Ser Gly Glu Ala Gln Arg 35 40 45 Met Ser Val Leu Val Glu Asp Leu Ser Leu Thr Arg Ala Glu Gly 50 55 60 Gln 65 <210> 67 <211> 1500 <212> DNA <213> source <400> 67 atgtttaaca accgtatccg cactgcagct ctcgctggtg caatcgcaat ctccaccgca 60 gcttccggcg tagctatccc agcattcgct caggagacca acccacctt cacacaac 120 aacggcttca acgatgctga tggatccacc atccagccag tgagccagt taaccacc 180 gaggaaaccc tccggacct gactgactcc accggcgctt acctggaga gttccagtac 240 ggcaacgttg aggaaatcgt tgaagcatac ctgcaggttc aggcttccgc agacggattc 300 gatccttctg agcaggctgc ttacgaggct ttcgaggctg ctcgcgttcg tgcatcccag 360 gagctcgcgg cttccgctga gaccatcact aagacccgcg agtccgttgc ttacgcactc 420 aaggctgacc gcgaagctac cgcagctttc gaggcttacc tcagcgctct tcgtcaggtt 480 tcagtcatca acgatctgat cgctgatgct aacgccaaga acaagactga ctttgcagag 540 atcgagctct acgatgttct ttacaccgac gccgacatct ctggcgatgc tccacttctt 600 gctcctgcat acaaggagct gaaggacctt caggctgagg ttgacgcaga cttcgagtgg 660 ttgggcgagt tcgcaattga taacaatgaa gacaactacg tcattcgtac tcacatccct 720 gctgtagagg cactcaaggc agcgatcgat tcactggtcg acaccgttga gccacttcgt 780 gcagacgcta tcgctaagaa catcgaggct cagaagtctg acgttctggt tccccagctc 840 ttcctcgagc gtgcaactgc acagcgcgac accctgcgtg ttgtagaggc aatcttctct 900 acctctgctc gttacgttga actctacgag aacgtcgaga acgttaacgt tgagaacaag 960 acccttcgcc agcactactc ttccctgatc cctaacctct tcatcgcagc ggttggcaac 1020 atcaacgagc tcaacaatgc agatcaggct gcacgtgagc tcttcctcga ttgggacacc 1080 gacctcacca ccaacgatga ggacgaagct tactaccagg ctaagctcga cttcgctatc 1140 gagacctacg caaagatcct gatcaacggt gaagtttggc aggagccact cgcttacgtc 1200 cagaacctgg atgcaggcgc acgtcaggaa gcagctgacc gcgaagcaga gcgcgcagct 1260 gacgcagcat accgcgctga gcagctccgc atcgctcagg aagcagctga cgctcagaag 1320 gctctcgctg aggctcttgc taatgcaggc aacaacgaca acggtggcga caactcctcc 1380 gacgacaagg gaaccggttc ttccgacatc ggaacctggg gacctttcgc agcaattgca 1440 gctatcatcg cagcaatcgc agctatcttc ccattcctct ccggtatcgt taagttctaa 1500 <210> 68 <211> 499 <212> PRT <213> Corynebacterium glutamicum <400> 68 Met Phe Asn Asn Arg Ile Arg Thr Ala Ala Leu Ala Gly Ala Ile Ala 1 5 10 15 Ile Ser Thr Ala Ala Ser Gly Val Ala Ile Pro Ala Phe Ala Gln Glu 20 25 30 Thr Asn Pro Thr Phe Asn Ile Asn Asn Gly Phe Asn Asp Ala Asp Gly 35 40 45 Ser Thr Ile Gln Pro Val Glu Pro Val Asn His Thr Glu Glu Thr Leu 50 55 60 Arg Asp Leu Thr Asp Ser Thr Gly Ala Tyr Leu Glu Glu Phe Gln Tyr 65 70 75 80 Gly Asn Val Glu Glu Ile Val Glu Ala Tyr Leu Gln Val Gln Ala Ser 85 90 95 Ala Asp Gly Phe Asp Pro Ser Glu Gln Ala Ala Tyr Glu Ala Phe Glu 100 105 110 Ala Ala Arg Val Arg Ala Ser Gln Glu Leu Ala Ala Ser Ala Glu Thr 115 120 125 Ile Thr Lys Thr Arg Glu Ser Val Ala Tyr Ala Leu Lys Ala Asp Arg 130 135 140 Glu Ala Thr Ala Ala Phe Glu Ala Tyr Leu Ser Ala Leu Arg Gln Val 145 150 155 160 Ser Val Ile Asn Asp Leu Ile Ala Asp Ala Asn Ala Lys Asn Lys Thr 165 170 175 Asp Phe Ala Glu Ile Glu Leu Tyr Asp Val Leu Tyr Thr Asp Ala Asp 180 185 190 Ile Ser Gly Asp Ala Pro Leu Leu Ala Pro Ala Tyr Lys Glu Leu Lys 195 200 205 Asp Leu Gln Ala Glu Val Asp Ala Asp Phe Glu Trp Leu Gly Glu Phe 210 215 220 Ala Ile Asp Asn Asn Glu Asp Asn Tyr Val Ile Arg Thr His Ile Pro 225 230 235 240 Ala Val Glu Ala Leu Lys Ala Ala Ile Asp Ser Leu Val Asp Thr Val 245 250 255 Glu Pro Leu Arg Ala Asp Ala Ile Ala Lys Asn Ile Glu Ala Gln Lys 260 265 270 Ser Asp Val Leu Val Pro Gln Leu Phe Leu Glu Arg Ala Thr Ala Gln 275 280 285 Arg Asp Thr Leu Arg Val Val Glu Ala Ile Phe Ser Thr Ser Ala Arg 290 295 300 Tyr Val Glu Leu Tyr Glu Asn Val Glu Asn Val Asn Val Glu Asn Lys 305 310 315 320 Thr Leu Arg Gln His Tyr Ser Ser Leu Ile Pro Asn Leu Phe Ile Ala 325 330 335 Ala Val Gly Asn Ile Asn Glu Leu Asn Asn Ala Asp Gln Ala Ala Arg 340 345 350 Glu Leu Phe Leu Asp Trp Asp Thr Asp Leu Thr Thr Asn Asp Glu Asp 355 360 365 Glu Ala Tyr Tyr Gln Ala Lys Leu Asp Phe Ala Ile Glu Thr Tyr Ala 370 375 380 Lys Ile Leu Ile Asn Gly Glu Val Trp Gln Glu Pro Leu Ala Tyr Val 385 390 395 400 Gln Asn Leu Asp Ala Gly Ala Arg Gln Glu Ala Ala Asp Arg Glu Ala 405 410 415 Glu Arg Ala Ala Asp Ala Ala Tyr Arg Ala Glu Gln Leu Arg Ile Ala 420 425 430 Gln Glu Ala Ala Asp Ala Gln Lys Ala Leu Ala Glu Ala Leu Ala Asn 435 440 445 Ala Gly Asn Asn Asp Asn Gly Gly Asp Asn Ser Ser Asp Asp Lys Gly 450 455 460 Thr Gly Ser Ser Asp Ile Gly Thr Trp Gly Pro Phe Ala Ala Ile Ala 465 470 475 480 Ala Ile Ile Ala Ala Ile Ala Ala Ile Phe Pro Phe Leu Ser Gly Ile 485 490 495 Val Lys Phe <210> 69 <211> 318 <212> DNA <213> Pseudomonas aeruginosa <400> 69 atgtccctcg gaccatggga aattggaatc attgtcctgc tgatcatcgt gctgttcggc 60 gcgaagaagc tgcctgatgc agctcgttcc atcggccgtt ccatgcgcat cttcaagtct 120 gaagtcaaag aaatgaacaa ggacggcgat accccagaac aacagcagca gcctcagcag 180 cagattgcgc ccaaccagat cgaggctcct cagccaaact ttgagcagca ctaccaggga 240 cagcaggttc agcagcctca gaaccctcag acccctgact accgtcagaa ctacgaggat 300 ccaaaccgca cctcttaa 318 <210> 70 <211> 105 <212> PRT <213> Pseudomonas aeruginosa <400> 70 Met Ser Leu Gly Pro Trp Glu Ile Gly Ile Ile Val Leu Leu Ile Ile 1 5 10 15 Val Leu Phe Gly Ala Lys Lys Leu Pro Asp Ala Ala Arg Ser Ile Gly 20 25 30 Arg Ser Met Arg Ile Phe Lys Ser Glu Val Lys Glu Met Asn Lys Asp 35 40 45 Gly Asp Thr Pro Glu Gln Gln Gln Gln Pro Gln Gln Gln Ile Ala Pro 50 55 60 Asn Gln Ile Glu Ala Pro Gln Pro Asn Phe Glu Gln His Tyr Gln Gly 65 70 75 80 Gln Gln Val Gln Gln Pro Gln Asn Pro Gln Thr Pro Asp Tyr Arg Gln 85 90 95 Asn Tyr Glu Asp Pro Asn Arg Thr Ser 100 105 <210> 71 <211> 471 [[ID=,21]]<212> DNA <213> Corynebacterium glutamicum <400> 71 atgttttcta gcgtgggttg gggagagatc ttcctcttag tcgttgtggg ccttgttgtc 60 atcggcccgg aacggttgcc tcgtttgatc caggacgcac gcgctgcgct gctcgctgca 120 cgtaccgcta tcgacaatgc aaagcagtcg ttggacagtg attttggttc ggaatttgat 180 gaaatccgaa agccactaac ccaggttgca cagtacagcc ggatgagccc caagacggcc 240 atcactaagg cgttatttga taatgattcc tcgttcctgg atgactttga tccaaagaag 300 atcatggccg aaggaacaga aggcgaagct cagcgcaaca agcaggcagc tgacaacaat 360 gcgaatgtgg tggaacgtcc agctgatggt tccaccgcac gcccaacgca aaacgatcca 420 aaagacggcc cgaattactc aggtggcgtc tcttggaccg atattattta g 471 <210> 72 <211> 156 <212> PRT <213> Corynebacterium glutamicum <400> 72 Met Phe Ser Ser Val Gly Trp Gly Glu Ile Phe Leu Leu Val Val Val 1 5 10 15 Gly Leu Val Val Ile Gly Pro Glu Arg Leu Pro Arg Leu Ile Gln Asp 20 25 30 Ala Arg Ala Ala Leu Leu Ala Ala Arg Thr Ala Ile Asp Asn Ala Lys 35 40 45 Gln Ser Leu Asp Ser Asp Phe Gly Ser Glu Phe Asp Glu Ile Arg Lys 50 55 60 Pro Leu Thr Gln Val Ala Gln Tyr Ser Arg Met Ser Pro Lys Thr Ala 65 70 75 80 Ile Thr Lys Ala Leu Phe Asp Asn Asp Ser Ser Phe Leu Asp Asp Phe 85 90 95 Asp Pro Lys Lys Ile Met Ala Glu Gly Thr Glu Gly Glu Ala Gln Arg 100 105 110 Asn Lys Gln Ala Ala Asp Asn Asn Ala Asn Val Val Glu Arg Pro Ala 115 120 125 Asp Gly Ser Thr Ala Arg Pro Thr Gln Asn Asp Pro Lys Asp Gly Pro 130 135 140 Asn Tyr Ser Gly Gly Val Ser Trp Thr Asp Ile Ile 145 150 155 <210> 73 <211> 945 <212> DNA <213> Corynebacterium glutamicum <400> 73 atgtccattg ttgagcacat caaagagttt cgacgccgac ttcttatcgc tctggcgggc 60 atcctcgtgg gcaccattat cggctttatt tggtacgatt tctcattttg gcagatcccc 120 actttgggcg agctgctgag ggatccgtac tgttctctgc ctgctgaatc ccgctgggcc 180 atgagcgact cagaggaatg tcgactgctc gcaaccggcc cgtttgatcc attcatgctt 240 cgccttaaag tagcggcgtt ggtgggtatg gttcttggct cacccgtgtg gctgagccag 300 ctgtggggct ttatcacccc aggtttgatg aagaatgagc gccgttacac cgcaatcttc 360 gtcacgattg ctgttgtgct gtttgtcggc ggtgctgttc ttgcgtactt cgtcgttgca 420 tatggtttgg agttcctcct taccattggt ggagacaccc aggcagcggc cctgactggt 480 gataagtact tcggattctt gctcgcgttg ttggcgattt tcggcgtgag cttcgaagtt 540 ccactggtga tcggcatgct caacattgtg ggtatcttgc cttacgatgc cattaaagat 600 aagcgacgca tgatcatcat gattttgttc gtgttcgctg ctttcatgac acccggccag 660 gatcctttca ccatgttggt gttggcgctt tcactcaccg ttctggtaga gcttgccctg 720 cagttctgtc gtttcaacga caaacgccgg gacaagaagc gcccagaatg gcttgatggc 780 gatgacctct ctgcatcacc actggatact tctgctggtg gagaagatgc tccaagccca 840 gtcgaaaccc cagaggcggt ggagccttcg cggatgctga acccaagtgg ggaggcgtcg 900 ataagctata aacccgggcg cgccgacttc ggtgacgtgc tctag 945 <210> 74 <211> 314 <212> PRT <213> Corynebacterium glutamicum <400> 74 Met Ser Ile Val Glu His Ile Lys Glu Phe Arg Arg Arg Leu Leu Ile 1 5 10 15 Ala Leu Ala Gly Ile Leu Val Gly Thr Ile Ile Gly Phe Ile Trp Tyr 20 25 30 Asp Phe Ser Phe Trp Gln Ile Pro Thr Leu Gly Glu Leu Leu Arg Asp 35 40 45 Pro Tyr Cys Ser Leu Pro Ala Glu Ser Arg Trp Ala Met Ser Asp Ser 50 55 60 Glu Glu Cys Arg Leu Leu Ala Thr Gly Pro Phe Asp Pro Phe Met Leu 65 70 75 80 Arg Leu Lys Val Ala Ala Leu Val Gly Met Val Leu Gly Ser Pro Val 85 90 95 Trp Leu Ser Gln Leu Trp Gly Phe Ile Thr Pro Gly Leu Met Lys Asn 100 105 110 Glu Arg Arg Tyr Thr Ala Ile Phe Val Thr Ile Ala Val Val Leu Phe 115 120 125 Val Gly Gly Ala Val Leu Ala Tyr Phe Val Val Ala Tyr Gly Leu Glu 130 135 140 Phe Leu Leu Thr Ile Gly Gly Asp Thr Gln Ala Ala Ala Leu Thr Gly 145 150 155 160 Asp Lys Tyr Phe Gly Phe Leu Leu Ala Leu Leu Ala Ile Phe Gly Val 165 170 175 Ser Phe Glu Val Pro Leu Val Ile Gly Met Leu Asn Ile Val Gly Ile 180 185 190 Leu Pro Tyr Asp Ala Ile Lys Asp Lys Arg Arg Met Ile Ile Met Ile 195 200 205 Leu Phe Val Phe Ala Ala Phe Met Thr Pro Gly Gln Asp Pro Phe Thr 210 215 220 Met Leu Val Leu Ala Leu Ser Leu Thr Val Leu Val Glu Leu Ala Leu 225 230 235 240 Gln Phe Cys Arg Phe Asn Asp Lys Arg Arg Asp Lys Lys Arg Pro Glu 245 250 255 Trp Leu Asp Gly Asp Asp Leu Ser Ala Ser Pro Leu Asp Thr Ser Ala 260 265 270 Gly Gly Glu Asp Ala Pro Ser Pro Val Glu Thr Pro Glu Ala Val Glu 275 280 285 Pro Ser Arg Met Leu Asn Pro Ser Gly Glu Ala Ser Ile Ser Tyr Lys 290 295 300 Pro Gly Arg Ala Asp Phe Gly Asp Val Leu 305 310 <210> 75 <211> 39 <212> PRT <213> Large intestinal bacteria <400> 75 Met Asn Asn Asn Asp Leu Phe Gln Ala Ser Arg Arg Arg Phe Leu Ala 1 5 10 15 Gln Leu Gly Gly Leu Thr Val Ala Gly Met Leu Gly Pro Ser Leu Leu 20 25 30 Thr Pro Arg Arg Ala Thr Ala 35 <210> 76 <211> 27 <212> PRT <213> E. coli <400> 76 Met Ser Leu Ser Arg Arg Gln Phe Ile Gln Ala Ser Gly Ile Ala Leu 1 5 10 15 Cys Ala Gly Ala Val Pro Leu Lys Ala Ser Ala 20 25 <210> 77 <211> 48 <212> PRT <213> Bacillus subtilis <400> 77 Met Ala Tyr Asp Ser Arg Phe Asp Glu Trp Val Gln Lys Leu Lys Glu 1 5 10 15 Glu Ser Phe Gln Asn Asn Thr Phe Asp Arg Arg Lys Phe Ile Gln Gly 20 25 30 Ala Gly Lys Ile Ala Gly Leu Ser Leu Gly Leu Thr Ile Ala Gln Ser 35 40 45 <210> 78 <211> 34 <212> PRT <213> Bacillus subtilis <400> 78 Met Lys Phe Val Lys Arg Arg Thr Thr Ala Leu Val Thr Thr Leu Met 1 5 10 15 Leu Ser Val Thr Ser Leu Phe Ala Leu Gln Pro Ser Ala Lys Ala Ala 20 25 30 Glu His <210> 79 <211> 30 <212> PRT <213> Orbacterium spheroidum <400> 79 Met Met Asn Leu Ser Arg Arg Thr Leu Leu Thr Thr Gly Ser Ala Ala 1 5 10 15 Thr Leu Ala Tyr Ala Leu Gly Met Ala Gly Ser Ala Gln Ala 20 25 30 <210> 80 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Diarginine motif <220> <221> misc_feature <222> (1)..(1) <223> Xaa can be any naturally occurring amino acid. <220> <221> misc_feature <222> (4)..(4) <223> Xaa can be any naturally occurring amino acid. <400> 80 Xaa Arg Arg Xaa Phe Leu Lys 1 5 <210> 81 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Diarginine motif <220> <221> misc_feature <222> (3)..(5) <223> Xaa can be any naturally occurring amino acid. <400> 81 Arg Arg Xaa Xaa Xaa 1 5 <210> 82 <211> 43 <212> PRT <213> Corynebacterium glutamicum <400> 82 Met Arg Asp Thr Ala Phe Arg Ser Ile Lys Ala Lys Ala Gln Ala Lys 1 5 10 15 Arg Arg Ser Leu Trp Ile Ala Ala Gly Ala Val Pro Thr Ala Ile Ala 20 25 30 Leu Thr Met Ser Leu Ala Pro Met Ala Ser Ala 35 40 <210> 83 <211> 30 <212> PRT <213> Corynebacterium glutamicum <400> 83 Met Phe Asn Asn Arg Ile Arg Thr Ala Ala Leu Ala Gly Ala Ile Ala 1 5 10 15 Ile Ser Thr Ala Ala Ser Gly Val Ala Ile Pro Ala Phe Ala 20 25 30 <210> 84 <211> 25 <212> PRT <213> Corynebacterium stationis <400> 84 Met Lys Arg Met Lys Ser Leu Ala Ala Ala Leu Thr Val Ala Gly Ala 1 5 10 15 Met Leu Ala Ala Pro Val Ala Thr Ala 20 25 <210> 85 <211>​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​100 105 110 Asp Arg Glu Ala Thr Ala Ala Phe Glu Ala Tyr Leu Ser Ala Leu Arg 115 120 125 Gln Val Ser Val Ile Asn Asp Leu Ile Ala Asp Ala Asn Ala Lys Asn 130 135 140 Lys Thr Asp Phe Ala Glu Ile Glu Leu Tyr Asp Val Leu Tyr Thr Asp 145 150 155 160 Ala Asp Ile Ser Gly Asp Ala Pro Leu Leu Ala Pro Ala Tyr Lys Glu 165 170 175 Leu Lys Asp Leu Gln Ala Glu Val Asp Ala Asp Phe Glu Trp Leu Gly 180 185 190 Glu Phe Ala Ile Asp Asn Asn Glu Asp Asn Tyr Val Ile Arg Thr His 195 200 205 Ile Pro Ala Val Glu Ala Leu Lys Ala Ala Ile Asp Ser Leu Val Asp 210 215 220 Thr Val Glu Pro Leu Arg Ala Asp Ala Ile Ala Lys Asn Ile Glu Ala 225 230 235 240 Gln Lys Ser Asp Val Leu Val Pro Gln Leu Phe Leu Glu Arg Ala Thr 245 250 255 Ala Gln Arg Asp Thr Leu Arg Val Val Glu Ala Ile Phe Ser Thr Ser 260 265 270 Ala Arg Tyr Val Glu Leu Tyr Glu Asn Val Glu Asn Val Asn Val Glu 275 280 285 Asn Lys Thr Leu Arg Gln His Tyr Ser Ser Leu Ile Pro Asn Leu Phe 290 295 300 Ile Ala Ala Val Gly Asn Ile Asn Glu Leu Asn Asn Ala Asp Gln Ala 305 310 315 320 Ala Arg Glu Leu Phe Leu Asp Trp Asp Thr Asp Leu Thr Thr Asn Asp 325 330 335 Glu Asp Glu Ala Tyr Tyr Gln Ala Lys Leu Asp Phe Ala Ile Glu Thr 340 345 350 Tyr Ala Lys Ile Leu Ile Asn Gly Glu Val Trp Gln Glu Pro Leu Ala 355 360 365 Tyr Val Gln Asn Leu Asp Ala Gly Ala Arg Gln Glu Ala Ala Asp Arg 370 375 380 Glu Ala Glu Arg Ala Ala Asp Ala Ala Tyr Arg Ala Glu Gln Leu Arg 385 390 395 400 Ile Ala Gln Glu Ala Ala Asp Ala Gln Lys Ala Leu Ala Glu Ala Leu 405 410 415 Ala Asn Ala Gly Asn Asn Asp Asn Gly Gly Asp Asn Ser Ser Asp Asp 420 425 430 Lys Gly Thr Gly Ser Ser Asp Ile Gly Thr Trp Gly Pro Phe Ala Ala 435 440 445 Ile Ala Ala Ile Ile Ala Ala Ile Ala Ala Ile Phe Pro Phe Leu Ser 450 455 460 Gly Ile Val Lys Phe 465 <210> 86 <211> 4 <212> PRT <213> Corynebacterium glutamicum <220> <221> misc_feature <222> (4)..(4) <223> Xaa is Asn, Gly, Thr, Pro, or Ala <400> 86 Gln Glu Thr Xaa 1 <210> 87 <211> 5 <212> PRT <213> Corynebacterium glutamicum <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa can be Asn, Gly, Thr, Pro, or Ala <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa is Pro, Thr, or Val <400> 87 Gln Glu Thr Xaa Xaa 1 5 <210> 88 <211> 6 <212> PRT <213> Corynebacterium glutamicum <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa can be Asn, Gly, Thr, Pro, or Ala <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa represents Pro, Thr, or Val. <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa is Thr or Tyr <400> 88 Gln Glu Thr Xaa Xaa Xaa 1 5 <210> 89 <211> 6 <212> PRT <213> Corynebacterium glutamicum <400> 89 Gln Glu Thr Asn Pro Thr 1 5 <210> 90 <211> 6 <212> PRT <213> Corynebacterium glutamicum <400> 90 Gln Glu Thr Gly Thr Tyr 1 5 <210> 91 <211> 6 <212> PRT <213> Corynebacterium glutamicum <400> 91 Gln Glu Thr Thr Val Thr 1 5 <210> 92 <211> 6 <212> PRT <213> Corynebacterium glutamicum <400> 92 Gln Glu Thr Pro Val Thr 1 5 <210> 93 <211> 6 <212> PRT <213> Corynebacterium glutamicum <400> 93 Gln Glu Thr Ala Val Thr 1 5 <210> 94 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Factor Xa <400> 94 Ile Glu Gly Arg 1 <210> 95 <211> 6 <212> PRT <213> Artificial sequence <220> <223> ProTEV <400> 95 Glu Asn Leu Tyr Phe Gln 1 5 <210> 96 <211> 708 <212> DNA <213> Corynebacterium glutamicum <400> 96 atggacaacc agtctgacgg acaaatccgc gtactcgtcg ttgatgacga gccaaacatc 60 gtcgagctgc tcaccgtaag ccttaaattc caaggcttcg cagtgatgac cgccaacgat 120 ggcaatgaag ccctgaagat tgctcgtgag ttccgtccag acgcatacat cctcgatgtc 180ggcaatgaag ccctgaagat tgctcgtgag ttccgtccag acgcatacat cctcgatgtc 180 atgatgccag gaatggacgg cttcgagctg ctgaccaagc tgcgcggcga aggccttgac 240atgatgccag gaatggacgg cttcgagctg ctgaccaagc tgcgcggcga aggccttgac 240<00​​​​​​​​​​​​​​​​​​​​​​​​​1 5 10 15 Glu Pro Asn Ile Val Glu Leu Leu Thr Val Ser Leu Lys Phe Gln Gly 20 25 30 Phe Ala Val Met Thr Ala Asn Asp Gly Asn Glu Ala Leu Lys Ile Ala 35 40 45 Arg Glu Phe Arg Pro Asp Ala Tyr Ile Leu Asp Val Met Met Pro Gly 50 55 60 Met Asp Gly Phe Glu Leu Leu Thr Lys Leu Arg Gly Glu Gly Leu Asp 65 70 75 80 Ser Pro Val Leu Tyr Leu Thr Ala Lys Asp Ala Val Glu His Arg Ile 85 90 95 His Gly Leu Thr Ile Gly Ala Asp Asp Tyr Val Thr Lys Pro Phe Ser 100 105 110 Leu Glu Glu Val Ile Thr Arg Leu Arg Val Ile Leu Arg Arg Gly Gly 115 120 125 Ala Val Glu Glu Asp Thr Ser Thr Ser Leu Gln Tyr Ala Asp Leu Thr 130 135 140 Leu Asn Asp Glu Thr His Glu Val Thr Lys Ala Gly Glu Leu Ile Asp 145 150 155 160 Leu Ser Pro Thr Glu Phe Asn Leu Leu Arg Tyr Leu Met Leu Asn Ala 165 170 175 Glu Val Val Leu Ser Lys Ala Lys Ile Leu Asp Asn Val Trp His Tyr 180 185 190 Asp Phe Gly Gly Asp Gly Asn Val Val Glu Ser Tyr Ile Ser Tyr Leu 195 200 205 Arg Arg Lys Val Asp Thr Gln Asp Pro Gln Leu Ile Gln Thr Val Arg 210 215 220 Gly Val Gly Tyr Val Leu Arg Thr Pro Arg Ser 225 230 235

Claims

1. A method for producing heterologous proteins, the method comprising: Cultivate Corynebacterium bacteria with genetic constructs for secreting and expressing the heterologous protein; and Collect the heterologous proteins produced through secretion. The *Corynebacterium* bacteria in question have been modified to retain the phoS gene encoding the mutant PhoS protein. The mutant Phos protein is a Phos protein composed of the following: a mutation in the wild-type Phos protein of Corynebacterium glutamicum, in which an amino acid residue corresponding to the tryptophan residue at position 302 in SEQ ID NO: 4 is replaced by an amino acid residue selected from the group consisting of: lysine residue, alanine residue, valine residue, serine residue, cysteine ​​residue, methionine residue, aspartic acid residue, and asparagine residue, wherein the wild-type Phos protein is a protein composed of the amino acid sequence of SEQ ID NO:

4. The genetic construct contains, in a 5' to 3' orientation, a promoter sequence that functions in the Corynebacterium bacteria, a nucleic acid sequence encoding a signal peptide that functions in the Corynebacterium bacteria, and a nucleic acid sequence encoding the heterologous protein. The heterologous protein is expressed as a fusion protein with the signal peptide, and The corynebacterium mentioned is Corynebacterium glutamicum.

2. The method of claim 1, wherein the signal peptide is a Tat-dependent signal peptide.

3. The method of claim 2, wherein the Tat-dependent signal peptide is selected from the group consisting of TorA signal peptide, SufI signal peptide, PhoD signal peptide, LipA signal peptide, and IMD signal peptide.

4. The method of claim 2, wherein the Corynebacterium bacteria has been further modified to increase the expression of one or more genes selected from the Tat secretion system.

5. The method of claim 4, wherein the gene encoding the Tat secretion system is composed of the tatA gene, the tatB gene, the tatC gene, and the tatE gene.

6. The method of claim 1, wherein the signal peptide is a Sec-dependent signal peptide.

7. The method of claim 6, wherein the Sec-dependent signal peptide is selected from the group consisting of PS1 signal peptide, PS2 signal peptide, and SlpA signal peptide.

8. The method of any one of claims 1 to 7, wherein the genetic construct further comprises, between the nucleic acid sequence encoding the signal peptide that functions in the Corynebacterium bacteria and the nucleic acid sequence encoding the heterologous protein, a nucleic acid sequence encoding an amino acid sequence comprising Gln-Glu-Thr.

9. The method of claim 8, wherein the genetic construct further comprises, between the nucleic acid sequence encoding the amino acid sequence comprising Gln-Glu-Thr and the nucleic acid sequence encoding the heterologous protein, a nucleic acid sequence encoding an amino acid sequence for enzymatic digestion.

10. The method of claim 1, wherein the corynebacterium is a modified strain derived from Corynebacterium glutamicum AJ12036 or Corynebacterium glutamicum ATCC13869.

11. The method according to any one of claims 1 to 7, wherein the corynebacterium is a corynebacterium with reduced activity of cell surface proteins.

12. Corynebacterium bacteria, It has been modified to retain the phoS gene that encodes the mutant PhoS protein. The mutant Phos protein is a Phos protein composed of the following: a mutation in the wild-type Phos protein in which an amino acid residue corresponding to the tryptophan residue at position 302 in SEQ ID NO: 4 is replaced by an amino acid residue selected from the group consisting of: lysine residue, alanine residue, valine residue, serine residue, cysteine ​​residue, methionine residue, aspartic acid residue, and asparagine residue, and wherein the wild-type Phos protein is a protein composed of the amino acid sequence of SEQ ID NO:

4. The corynebacterium mentioned is Corynebacterium glutamicum.

13. The Corynebacterium of claim 12, wherein the Corynebacterium is a modified strain derived from Corynebacterium glutamicum AJ12036 or Corynebacterium glutamicum ATCC13869.

14. The Corynebacterium bacterium according to any one of claims 12 to 13, wherein the Corynebacterium bacterium is a Corynebacterium bacterium with reduced activity of cell surface proteins.

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