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US 10,894,810 B2Granted
Protein secretory production method
AssigneeAjinomoto Co., Inc.
InventorsYumi Ito, Yoshihiko Matsuda
Life of this patent
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Abstract
A novel technique for improving secretory production of a heterologous protein by coryneform bacteria is described, and thereby a method for secretory production of a heterologous protein is provided. A coryneform bacterium able to secrete a heterologous protein and modified so that the activity of HrrSA system is reduced is cultured to produce the heterologous protein by secretory production.
Claims17
A method for producing a heterologous protein comprising:
- culturing a coryneform bacterium having a genetic construct for secretory expression of the heterologous protein;
- and
- collecting the heterologous protein produced by secretory production,
- wherein the coryneform bacterium has been modified to reduce molecules of an HrrS protein and/or an HrrA protein per cell as compared to a non-modified bacterium,
- wherein the coryneform bacterium is Corynebacterium glutamicum,
- 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 heterologous protein is expressed as a fusion protein with the signal peptide;
- wherein the molecules of the HrrS protein and/or the HrrA protein per cell is reduced by reducing the expression of a hrrS gene and/or a hrrA gene, or by disrupting a hrrS gene and/or a hrrA,
- wherein the HrrS protein is selected from the group consisting of:
- (a) a protein comprising the amino acid sequence of SEQ ID NO:
- 63;
- (b) a protein comprising the amino acid sequence of SEQ ID NO:
- 63, said protein comprising 1 to 10 amino acid residue substitution, deletion, insertion, and/or addition and is a sensor kinase of a HrrSA system;
- and
- (c) a protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:
- 63, and is a sensor kinase of a HrrSA system, and
- wherein the HrrA protein is selected from the group consisting of:
- (a) a protein comprising the amino acid sequence of SEQ ID NO:
- 65;
- (b) a protein comprising the amino acid sequence of SEQ ID NO:
- 65, said protein comprising 1 to 10 amino acid residue substitution, deletion, insertion, and/or addition and is a response regulator of a HrrSA system;
- and
- (c) a protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:
- 65, and is a response regulator of a HrrSA system.
21 more elements
Dependencies are inferred from the claim text.