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Highlights • Heterologous promoters could be converted to powerful promoters in S. lividans. • P tac * RBS3 showed stronger capacity for gene expression than the P tipA in S. lividans. • Engineered promoters were also useful for the metabolic engineering of S. lividans. • This study will greatly expand the gene expression toolkit of Streptomyces. Abstract 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 σ hrdB. 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 P tac in E. coli was engineered by fusing its core promoter region with the 5′-UTR R15 from a relatively powerful Streptomyces promoter P kasO * R15 and resulted in P tac* , the activity of which was 8.1-fold that of P tac and 1.7-fold that of P kasO * R15 in S. lividans TK24. Next, the 5′-UTR R15 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 P tac * RBS3 showed about 2.1, 3.6, and 17.6 times the activity of P tac *, P kasO * R15 , and P tac , respectively, demonstrating that the heterologous P tac was converted into a type of robust Streptomyces promoters. This study thus greatly expands promoter diversity for the engineering of Streptomyces. [ABSTRACT FROM AUTHOR]