Development of a bifunctional dynamic control system for effective product synthesis in Bacillus licheniformis DW2

Bacillus licheniformis DW2 has emerged as a promising microbial chassis for biosynthesis of industrial enzymes, lipopeptides and biochemicals. However, the lack of dynamic control tools to date has limited its expansion in basic research and industrial applications. Here, a well- performing bifunctional dynamic control system was developed for the dynamic up- and down- regulation of genes, as well as the effective biosynthesis of poly-γ-glutamic acid (γ-PGA). Firstly, a dynamic turn-on module was developed in B. licheniformis DW2, including an artificially designed broad-range regulated xylose-inducible expression system with a regulation range of 1.03 ∼ 352.37-fold, and an artificially designed highly sensitive IPTG-inducible expression system that can respond to 0.001 mM IPTG. Subsequently, a dynamic turn-off module based on the ClpXP protease was developed, which could regulate the degradation efficiency of target protein by selecting different ssrA tags. Then, a bifunctional dynamic control system based on the coupling of LacI and ClpXP was established in B. licheniformis for the first time, through combining the dynamic turn- on and off modules, which realized the dynamic up- and down- regulation of the expression of rfp and gfp genes simultaneously. Finally, it was successfully applied to dynamically optimize the metabolic flux of γ-PGA, increasing the yields of γ-PGA by 33.51 %. This work not only developed a well-performance bifunctional dynamic control tool for gene expression regulation, but also offered a strong support for the efficient production of diversified products by B. licheniformis.