Method for secretory production of protein

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.