Characterizing and Engineering Rhamnose-Inducible Regulatory Systems for Dynamic Control of Metabolic Pathways in .

Fine-tuning gene expression is of great interest for synthetic biotechnological applications. This is particularly true for the genus , 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 species, and the removal of the pathway in the model organism 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 (). A synthetic biology-based strategy was then deployed to build rhamnose-inducible regulatory systems, referred to as and , by assembling the repressor/operator pair RhaR/ with the well-defined constitutive promoter. Both and exhibited a high level of induced reporter activity, with no leaky expression. has been proven successful for the programmable production of actinorhodin and violacein in . Our study expanded the toolkit of inducible regulatory systems that will be broadly applicable to many other species.