Remodeling Bacillus amyloliquefaciens Cell Wall Rigidity to Reduce Cell Lysis and Increase the Yield of Heterologous Proteins.

Bacillus amyloliquefaciens has great potential as a host for heterologous protein production, but its severe autolytic behavior has precluded its industrial application to date. Because d,l-endopeptidase activity-guided cell wall rigidity is considered essential for autolysis resistance, we investigated the effects of d,l-endopeptidase genes lytE , lytF , cwlO , and cwlS play on the growth, lysis, and morphology remodeling of B. amyloliquefaciens strain TCCC11018. Individual and combinatorial deletion of lytE , lytF , and cwlS enhanced the cell growth and delayed cell lysis. For the best mutant with the lytF and cwlS double deletion, the viable cell number at 24 h increased by 11.90% and the cell wall thickness at 6 h increased by 25.87%. Transcriptomic and proteomic analyses indicated that the improvement was caused by enhanced peptidoglycan synthesis. With the lytF and cwlS double deletion, the extracellular green fluorescent protein and phospholipase D expression levels increased by 113 and 55.89%, respectively. This work broadens our understanding of the relationship between d,l-endopeptidases and B. amyloliquefaciens cell characteristics, which provides an effective strategy to improve the heterologous protein expression in B. amyloliquefaciens -based cell factories.