Engineering diverse eubacteria promoters for robust Gene expression in Streptomyces lividans.
Due to the lack of powerful gene regulation elements, the engineering development of Streptomyces is often limited. Here, we disclosed that the heterologous σ70 -dependent promoters, which have been reported as inefficient tools for gene expression in Streptomyces, could be efficiently recognized by Streptomyces housekeeping factor σhrdB. Therefore, an effective strategy was developed to engineer these promoters for robust gene expression in Streptomyces by fusing them with optimized 5'-untranslation regions (5'-UTRs). As a proof of concept, the widely used Ptac in E. coli was engineered by fusing its core promoter region with the 5'-UTRR15 from a relatively powerful Streptomyces promoter PkasO*R15 and resulted in Ptac*, the activity of which was 8.1-fold that of Ptac and 1.7-fold that of PkasO*R15 in S. lividans TK24. Next, the 5'-UTRR15 was optimized by randomizing the ribosome binding site (RBS). Based on the base biases of those RBSs with higher activity, eight artificial RBSs were rationally designed, and the optimal resulting promoter Ptac*RBS3 showed about 2.1, 3.6, and 17.6 times the activity of Ptac*, PkasO*R15, and Ptac, respectively, demonstrating that the heterologous Ptac was converted into a type of robust Streptomyces promoters. This study thus greatly expands promoter diversity for the engineering of Streptomyces.