Single-gene regulated non-spore-forming Bacillus subtilis: Construction, transcriptome responses, and applications...

Bacillus subtilis, a spore-forming industrial bacterium, is widely used for production of enzymes and valuable chemicals. The spore-formation, however, always results in remarkably reduced cell-density, thereby reducing product yield. Here, we constructed different non-spore-forming B. subtilis mutants via single-gene regulation. During the three spore-forming stages: signal sensing, transduction, and sporulation, we found that deleting only a single gene of sporulation, i.e. spo0A, spoIIIE, and spoIVB, can completely block the spore generation. Interestingly, the engineered non-sporulating mutants exhibited physiological heterogeneity and distinct synthetic capabilities. The spo0A-null spore-free mutant displayed remarkably high enzyme production capacity, such as 194% enhance amylase production. However, the spoIVB-null non-spore-forming mutant was especially efficient in producing secondary metabolites, such as surfactin; its flask titer increased significantly to 16.7 g/L, with the overexpression and Leu addition strategy. Our results offer a new strategy for re-modeling B. subtilis to further improve its fermentation efficiency and application.