Method for secretory production of protein

1 . A method for producing a heterologous protein, the method comprising:

culturing a coryneform bacterium having a genetic construct for secretory expression of the heterologous protein, and wherein as a result of said culturing, said coryneform bacterium secretes said heterologous protein; and

collecting the heterologous protein,

wherein the coryneform bacterium has at least two modifications so that secretory production of the heterologous protein by the coryneform bacterium is increased, wherein said modifications are:

(A) the activity of a RegX3 protein has completely disappeared as compared with a non-modified strain of the coryneform bacterium,

(B) the activity of an HrrSA system is reduced as compared with a non-modified strain of the coryneform bacterium, and

(C) the activity of an HrcA protein has completely disappeared as compared with a non-modified strain of the coryneform bacterium;

wherein modification (A) is obtained by deleting a regX3 gene;

wherein modification (C) is obtained by deleting an hrcA gene;

wherein the genetic construct comprises, in the direction from 5′ to 3′, a promoter sequence that functions in the coryneform bacterium, a nucleic acid sequence encoding a signal peptide that functions in the coryneform bacterium, and a nucleic acid sequence encoding the heterologous protein;

wherein the coryneform bacterium has been further modified so as to harbor a phoS gene encoding a mutant PhoS protein,

wherein the mutant PhoS protein comprises a mutation of replacing an amino acid residue corresponding to a tryptophan residue at position 302 in SEQ ID NO: 2 with a cysteine residue in a wild-type PhoS protein,

wherein the wild-type PhoS protein is selected from the group consisting of:

(a) a protein comprising any of the amino acid sequences of SEQ ID NOS: 2 to 7;

(b) a protein comprising any of the amino acid sequences of SEQ ID NOS: 2 to 7, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, wherein said protein has a function as a sensor kinase of a PhoRS system; and

(c) a protein comprising an amino acid sequence showing an identity of 90% or higher to any of the amino acid sequences of SEQ ID NOS: 2 to 7, wherein said protein has a function as a sensor kinase of a PhoRS system,

wherein the signal peptide is a TorA signal peptide or a Sec-dependent signal peptide;

wherein the heterologous protein is expressed as a fusion protein with the signal peptide; and

wherein the coryneform bacterium is Corynebacterium glutamicum.

2 . The method according to claim 1 , wherein a number of molecules of the RegX3 protein per cell is reduced as compared with a non-modified strain of the coryneform bacterium; the number of molecules of either or both of an HrrS protein and an HrrA protein per cell is reduced as compared with a non-modified strain of the coryneform bacterium; and/or the number of molecules of the HrcA protein per cell is reduced as compared with a non-modified strain of the coryneform bacterium.

3 . The method according to claim 1 , wherein the coryneform bacterium has a combination of modifications (A), (B), and (C).

4 . The method according to claim 1 , wherein the modification (B) is obtained by reducing the activity of one or both of an HrrS protein and an HrrA protein.

5 . The method according to claim 4 , wherein the modification (B) is obtained by reducing at least the activity of the HrrA protein.

6 . The method according to claim 1 , wherein the modification (B) is obtained by reducing the number of molecules of one or both of an HrrS protein and an HrrA protein per cell.

7 . The method according to claim 6 , wherein the modification (B) is obtained by reducing at least the number of molecules of the HrrA protein per cell.

8 . The method according to claim 1 , wherein the RegX3 protein is selected from the group consisting of:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 42;

(b) a protein comprising the amino acid sequence of SEQ ID NO: 42, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, wherein said protein has a function as a response regulator of a SenX3-RegX3 system; and

(c) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 42, wherein said protein has a function as a response regulator of a SenX3-RegX3 system.

9 . The method according to claim 2 , wherein the HrrS protein is selected from the group consisting of:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 44;

(b) a protein comprising the amino acid sequence of SEQ ID NO: 44, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, wherein said protein has a function as a sensor kinase of an HrrSA system; and

(c) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 44, wherein said protein has a function as a sensor kinase of an HrrSA system.

10 . The method according to claim 2 , wherein the HrrA protein is selected from the group consisting of:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 46;

(b) a protein comprising the amino acid sequence of SEQ ID NO: 46, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, wherein said protein has a function as a response regulator of an HrrSA system; and

(c) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 46, wherein said protein has a function as a response regulator of an HrrSA system.

11 . The method according to claim 1 , wherein the HrcA protein is selected from the group consisting of:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 48;

(b) a protein comprising the amino acid sequence of SEQ ID NO: 48, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, wherein said protein has a function as a transcription repressor of heat-shock proteins; and

(c) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 48, wherein said protein has a function as a transcription repressor of heat-shock proteins.

12 . The method according to claim 1 , wherein the coryneform bacterium has been further modified so that expression of one or more genes encoding a Tat secretion system is increased as compared with a non-modified strain of the coryneform bacterium.

13 . The method according to claim 12 , wherein the one or more genes encoding a Tat secretion system are a tatA gene, tatB gene, tatC gene, and tatE gene.

14 . The method according to claim 1 , wherein the Sec-dependent signal peptide is a signal peptide selected from the group consisting of a PS1 signal peptide, PS2 signal peptide, and SlpA signal peptide.

15 . The method according to claim 1 , wherein the genetic construct further comprises a nucleic acid sequence encoding an amino acid sequence comprising Gln-Glu-Thr between the nucleic acid sequence encoding the signal peptide that functions in the coryneform bacterium and the nucleic acid sequence encoding the heterologous protein.

16 . The method according to claim 15 , wherein the genetic construct further comprises a nucleic acid sequence encoding an amino acid sequence used for enzymatic digestion between the nucleic acid sequence encoding the amino acid sequence comprising Gln-Glu-Thr and the nucleic acid sequence encoding the heterologous protein.

17 . The method according to claim 1 , wherein the coryneform bacterium is Corynebacterium glutamicum AJ12036 or Corynebacterium glutamicum ATCC 13869.

18 . The method according to claim 1 , wherein a number of molecules of a cell surface layer protein per cell of the coryneform bacterium is reduced as compared with a non-modified strain of the coryneform bacterium.

19 . The method according to claim 1 , wherein the promoter is cspB promoter.