Efficient expression of recombinant proteins in Bacillus subtilis using a rewired gene circuit of quorum sensing

Bacillus subtilis is a favored chassis for high productivity of several high value-added product in synthetic biology. Efficient production of recombinant proteins is critical but challenging using this chassis because these expression systems in use, such as constitutive and inducible expression systems, demand for coordination of cell growth with production and addition of chemical inducers. These systems compete for intracellular resources with the host, eventually resulting in dysfunction of cell survival. To overcome the problem, in this study, LuxRI quorum sensing (QS) system from Aliivibrio fischeri was functionally reconstituted in B. subtilis for achieving coordinated protein overproduction with cell growth in a cell-density-dependent manner. Furthermore, the output-controlling promoter, P_luxI, was engineered through two rounds of evolution, by which we identified four mutants, P22, P47, P56, and P58 that exhibited elevated activity compared to the original P_luxI. By incorporating a strong terminator (TB5) downstream of the target gene further enhanced expression level. The expression level of this system surpasses commonly used promoter-based systems in B. subtilis like P43 and P_ylbP. The LuxRI QS system proves to be a potent platform for recombinant protein overproduction in B. subtilis.