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.

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