Characterizing and Engineering Rhamnose-Inducible Regulatory Systems for Dynamic Control of Metabolic Pathways in <i>Streptomyces</i>

Fine-tuning gene expression is of great interest for synthetic biotechnological applications. This is particularly true for the genus <i>Streptomyces</i>, which is well-known as a prolific producer of diverse natural products. Currently, there is an increasing demand to develop effective gene induction systems. In this study, bioinformatic analysis revealed a putative rhamnose catabolic pathway in multiple <i>Streptomyces</i> species, and the removal of the pathway in the model organism <i>Streptomyces coelicolor</i> impaired its growth on minimal media with rhamnose as the sole carbon source. To unravel the regulatory mechanism of RhaR, a LacI family transcriptional regulator of the catabolic pathway, electrophoretic mobility shift assays (EMSAs) were performed to identify potential target promoters. Multiple sequence alignments retrieved a consensus sequence of the RhaR operator (<i>rhaO</i>). A synthetic biology-based strategy was then deployed to build rhamnose-inducible regulatory systems, referred to as <i>rhaRS1</i> and <i>rhaRS2</i>, by assembling the repressor/operator pair RhaR/<i>rhaO</i> with the well-defined constitutive <i>kasO*</i> promoter. Both <i>rhaRS1</i> and <i>rhaRS2</i> exhibited a high level of induced reporter activity, with no leaky expression. <i>rhaRS2</i> has been proven successful for the programmable production of actinorhodin and violacein in <i>Streptomyces</i>. Our study expanded the toolkit of inducible regulatory systems that will be broadly applicable to many other <i>Streptomyces</i> species.