Synthetic Inducible Regulatory Systems Optimized for the Modulation of Secondary Metabolite Production in Streptomyces

Biosynthesis of secondary metabolites is a highly complex process that often requires tight control of their production, as overproduction of metabolites could be toxic and also may cause metabolic burden to their hosts. Tight control of metabolite production could be achieved by expressing key biosynthetic genes under control of an inducible regulatory system. In this study, we employed the modular design approach to build a high performance synthetic inducible regulatory system that displays a large dynamic range and thus is well-suited for the modulation of secondary metabolite production in Streptomyces. To this end, an inducible regulatory system was divided into three separate functional modules: (1) the induction module, (2) the target expression module, and (3) the repressor expression module. Then, these three separate modules were individually optimized in a stepwise manner and assembled to a new system. First, the cumate (CMT) induction module was chosen as the best performing induction module based on the large dynamic range and moderate inducer sensitivity. Then the CMT induction module maintained its performance when combined with diverse constitutive target expression modules, in which overall dynamic ranges varied depending on maximum promoter strengths. Lastly, the repressor expression module was optimized to achieve complete elimination of leaky expression, further increasing the dynamic range of the system. We also demonstrate that any strong constitutive regulatory system could be converted into an inducible regulatory system by simple CRISPR/Cas9-aided markerless insertion of an operator sequence whenever tight control of gene expression is required. We believe that the synthetic inducible regulatory system we report here would become a useful tool in modulating secondary metabolite production in Streptomyces.