Optimization of alkaline protease production by rational deletion of sporulation related genes in Bacillus licheniformis

BackgroundOur laboratory has constructed a Bacillus licheniformis strain that secretes alkaline protease (AprE) with excellent enzymatic properties. B. licheniformis is generally regarded as safe and has a high industrial exoenzyme secretion capacity, but the host retains some undomesticated characteristic that increase its competitiveness and survival, such as spore-formation, which increases the requirements and difficulties in industrial operations (e.g. sterilization and enzyme activity control). Furthermore, the influence of sporulation on alkaline protease production in B. licheniformis has not been elucidated in detail.ResultA series of asporogenic variants of the parent strain were constructed by individually knocking out the master regulator genes (spo0A, sigF and sigE) involved in sporulation. Most of the variants formed abortively disporic cells characterized by asymmetric septa at the poles and unable to survive incubation at 75 degrees C for 10min. Two of them (Delta sigF and Delta sigE) exhibited superior characteristics in protease production, especially improving the expression of the aprE gene. Under the currently used fermentation conditions, the vegetative production phase of Delta sigF can be prolonged to 72h, and the highest protease production of Delta sigF reached 29,4941053U/mL, which was about 19.7% higher than that of the wild-type strain.Conclusion We first constructed three key sporulation-deficient strain to investigate the effect of sporulation on alkaline protease synthesis. The sigF mutant retained important industrial properties such as facilitating the sterilization process, a prolonged stable phase of enzyme production and slower decreasing trend, which will be superior in energy conservation, simpler operations and target product controlling effect. In summary, the work provides a useful industrial host with preferable characteristics and a novel strategy to enhance the production of protease.