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EP-4737583-A1 - METHOD FOR PRODUCING PROTEIN OF INTEREST

EP4737583A1EP 4737583 A1EP4737583 A1EP 4737583A1EP-4737583-A1

Abstract

Provided is a method for producing a protein of interest using a gram-positive bacterium with improved productivity. The method for producing a protein of interest comprises: culturing a gram-positive bacterium comprising a gene encoding heterologous FK506-binding protein (FKBP) and a gene encoding the protein of interest; or culturing a mixture of a gram-positive bacterium comprising the gene encoding heterologous FKBP and a gram-positive bacterium comprising the gene encoding the protein of interest.

Inventors

  • NISHIGUCHI, HIROKI
  • KAWAHARA, AKIHITO

Assignees

  • Kao Corporation

Dates

Publication Date
20260506
Application Date
20240627

Claims (10)

  1. A method for producing a protein of interest, comprising: culturing a gram-positive bacterium comprising a gene encoding heterologous FK506-binding protein (FKBP) and a gene encoding the protein of interest; or culturing a mixture of a gram-positive bacterium comprising the gene encoding heterologous FKBP and a gram-positive bacterium comprising the gene encoding the protein of interest.
  2. The method according to claim 1, wherein the heterologous FKBP is a polypeptide which belongs to a BCFK clade and has an isomerization activity of a prolyl bond, a polypeptide consisting of an amino acid sequence of SEQ ID NO: 6, or a polypeptide which consists of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 6 and has an isomerization activity of a prolyl bond.
  3. The method according to claim 1, wherein the heterologous FKBP is a polypeptide consisting of any of amino acid sequences of SEQ ID NOs: 1 to 6, or a polypeptide which consists of an amino acid sequence having at least 80% identity to any of the amino acid sequences of SEQ ID NOs: 1 to 6 and has an isomerization activity of a prolyl bond.
  4. The method according to any one of claims 1 to 3, wherein the gene encoding heterologous FKBP is operably linked to a polynucleotide encoding a secretory signal peptide.
  5. The method according to any one of claims 1 to 4, wherein the gram-positive bacterium is Bacillus subtilis or a mutant strain thereof.
  6. The method according to any one of claims 1 to 4, wherein the gram-positive bacterium is Bacillus subtilis with at least one of extracellular protease gene deleted or inactivated.
  7. The method according to any one of claims 1 to 6, wherein the protein of interest has at least one prolyl bond isomerization site.
  8. The method according to any one of claims 1 to 7, wherein the protein of interest is an antibody-related molecule.
  9. The method according to any one of claims 1 to 7, wherein the protein of interest is a Fab.
  10. A gram-positive bacterium comprising a gene encoding heterologous FKBP, or the gene encoding heterologous FKBP and a gene encoding a protein of interest.

Description

Field of the Invention The present invention relates to a method for producing a protein of interest with improved productivity. Background of the Invention Various types of useful substances are produced industrially using microbes, including foods, amino acids, organic acids, nucleic acid-related substances, antibiotics, carbohydrates, lipids, proteins, and the like, and their application falls within a wide range of fields from daily goods such as foods, medicines, detergents, and cosmetics to various starting materials for chemical products. Examples of industrially useful host microbes include E. coli, Bacillus subtilis, yeasts, and filamentous fungi. An important challenge to the industrial production of useful substances using such microbes is improvement in its productivity. Peptidylprolyl isomerase (PPIase) is an enzyme capable of catalyzing the isomerization of prolyl bonds and assisting in protein folding (Non Patent Literature 1). PPIase consists of three families: cyclophilin, FK506-binding protein (FKBP), and parvulin, differing in sensitivity to inhibitors. The amino acid sequences of these three families of PPIase are known to share low sequence identity. All the three families of PPIase reside in the periplasm of E. coli. Among them, FkpA which belongs to FKBP has been studied in detail and reported to be capable of improving the productivity of a protein of interest with increase in its expression level (Non Patent Literature 2). It has also been reported that the region of FkpA which contributes to improvement in the productivity of a protein of interest in E. coli is an N-terminal chaperon region (Non Patent Literature 3). Moreover, the addition of PPIase belonging to any of the three families has been reported to be capable of improving the productivity of a protein of interest in a cell-free system (Patent Literature 1). Enhanced expression of parvulin including PrsA has often been studied on the secretory production of a protein of interest in gram-positive bacteria (Non Patent Literature 4). It has been reported that a gram-positive bacterium Bacillus subtilis has PrsA as endogenous chaperon and PPIase (Non Patent Literature 5), and the productivity of a protein of interest is improved by enhancing the expression level of PrsA (Patent Literature 2 and Non Patent Literatures 6 and 7). PrsA is anchored to the outer surface of a cell membrane and promotes the folding of a protein transported via translocase. Since incorrectly folded proteins are degraded by protease present in cell walls or cell membrane-cell membrane interfaces, correct folding is required immediately after cleavage of a signal peptide (Non Patent Literature 4). For FKBP, however, there has been no report so far regarding its influence on the production of a protein of interest in gram-positive bacteria. (Patent Literature 1) JP-A-2005-253432(Patent Literature 2) JP-B-4202985(Non Patent Literature 1) Journal of Molecular Biology, 2015, 427 (7): 1609-1631(Non Patent Literature 2) Biotechnol Prog. 2017, 33 (1): 212-220(Non Patent Literature 3) Microbial Cell Factories, 2010, volume 9, Article number: 22(Non Patent Literature 4) Microbial Cell Factories, 2019, volume 18, Article number: 158(Non Patent Literature 5) J Biol Chem. 2004, 279 (18): 19302-14(Non Patent Literature 6) Journal of Bacteriology, 1998, 180 (11): 2830-2835(Non Patent Literature 7) Journal of Bacteriology, 2001, 183 (6): 1881-1890 Summary of the Invention The present invention relates to the following 1) and 2). 1) A method for producing a protein of interest, comprising: culturing a gram-positive bacterium comprising a gene encoding heterologous FKBP and a gene encoding the protein of interest; or culturing a mixture of a gram-positive bacterium comprising the gene encoding heterologous FKBP and a gram-positive bacterium comprising the gene encoding the protein of interest.2) A gram-positive bacterium comprising a gene encoding heterologous FKBP, or the gene encoding heterologous FKBP and a gene encoding a protein of interest. Brief Description of the Drawings Fig. 1 illustrates an amount of Fab produced in a Fab-producing Bacillus subtilis strain allowed to express each PPIase.Fig. 2 illustrates an amount of Fab produced in a Fab-producing Bacillus subtilis strain allowed to express PrsA or FkpA.Fig. 3 illustrates an amount of Fab produced in a Fab-producing Bacillus subtilis strain allowed to express the whole region (FkpA), an N-terminal region (FkpA_N) or a C-terminal region (FkpA_C) of FkpA.Fig. 4 illustrates an amount of Fab produced in a Fab-producing Bacillus subtilis strain allowed to secretorily express FkpA (FkpA_p) or allowed to express FkpA anchored to a cell membrane (FkpA_m).Fig. 5 illustrates the phylogenetic tree of FKBP.Fig. 6 illustrates the HMM profile of BCFK clade. Detailed Description of the Invention As used herein, the identity between nucleotide sequences or amino acid sequences is calculated by the Lipman-Pearson method (Sci