Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

Significance To meet the increasing demands of drug discovery and biosynthetic studies, we established a precise quantitative method based on flow cytometry at single-cell (protoplast) resolution in Streptomyces for the identification of regulatory elements. A series of native or synthetic promoters and ribosomal binding sites has been characterized. Moreover, an insulator was demonstrated to eliminate element–element interference. As a proof of concept, a native silent gene cluster was activated by the synthetic modular regulatory elements in a predictable manner. The universality of these elements is of high value to the synthetic biology of Streptomyces. There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.

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