Effects of EGTA on cell surface structures of Corynebacterium glutamicum.

The mycolic acid layer and S-layer of Corynebacterium glutamicum have been considered as permeability barriers against lytic agents. EGTA, a calcium chelator, inhibited C. glutamicum growth at relatively lower concentrations compared with other Gram-positive bacteria. We investigated the effect of EGTA on C. glutamicum cell surface structures. Simultaneous addition of EGTA and lysozyme resulted in cell lysis, whereas addition of these reagents separately had no such effect. Analysis of cell surface proteins showed that CspB, an S-layer protein, was released into the culture media and degraded to several sizes upon EGTA treatment. These findings suggest that EGTA treatment causes release and proteolysis of the CspB protein, resulting in increased cell surface permeability. FE-SEM visualization further confirmed alteration of cell surface structures in EGTA-treated cells. This is the first report suggesting the importance of calcium ions in cell surface integrity of C. glutamicum.

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum.

BACKGROUND: Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. RESULTS: The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. CONCLUSION: There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system.