An Engineered B. subtilis Inducible Promoter System with over 10 000-Fold Dynamic Range.

Bacillus subtilis is the leading model Gram-positive bacterium, and a widely used chassis for industrial protein production. However, B. subtilis research is limited by a lack of inducible promoter systems with low leakiness and high dynamic range. Here, we engineer an inducible promoter system based on the T7 RNA Polymerase, the lactose repressor LacI, and a chimeric T7lac promoter PT7lac, integrated as a single copy in the B. subtilis genome. In the absence of IPTG, LacI strongly represses T7RNAP and PT7lac and minimizes leakiness. Addition of IPTG de-represses PT7lac and simultaneously induces expression of T7RNAP, resulting in very high output expression. Using green fluorescent and β-galactosidase reporter proteins, we estimate that LacI-T7 can regulate expression with a dynamic range of over 10,000, by far the largest reported for an inducible B. subtilis promoter system. Furthermore, LacI-T7 responds to similar IPTG concentrations and with similar kinetics as the widely used Phy-spank IPTG-inducible system, which we show has a dynamic range of at most 300 in a similar genetic context. Due to its superior performance, our LacI-T7 system should have broad applications in fundamental B. subtilis biology studies and biotechnological applications.