A Simple and Effective Strategy for the Development of Robust Promoter-Centric Gene Expression Tools

Promoter-centric genetic tools play a crucial role in controlling gene expression for various applications, such as strain engineering and synthetic biology studies. Hence, a critical need persists for the development of robust gene expression tools. <i>Streptomyces</i> are well-known prolific producers of natural products and exceptional surrogate hosts for the production of high-value chemical compounds and enzymes. In this study, we reported a straightforward and effective strategy for the creation of potent gene expression tools. This was primarily achieved by introducing an additional -35-like motif upstream of the original -35 region of the promoter, coupled with the integration of a palindromic <i>cis</i>-element into the 5'-UTR region. This approach has generated a collection of robust constitutive and inducible gene expression tools tailored for <i>Streptomyces</i>. Of particular note, the fully activated <u>o</u>xytetracycline-inducible gene expression system containing an engineered <u><i>k</i></u><i>asO</i>p* promoter (<i>OK</i>) exhibited nearly an order of magnitude greater activity compared to the well-established high-strength promoter <i>kasO</i>p* under the tested conditions, establishing itself as a powerful gene expression system for <i>Streptomyces</i>. This strategy is expected to be applicable in modifying various other promoters to acquire robust gene expression tools, as evidenced by the enhancement observed in the other two promoters, <i>PL</i> and <i>P21</i> in this study. Moreover, the effectiveness of these tools has been demonstrated through the augmented production of transglutaminase and daptomycin. The gene expression tools established in this study, alongside those anticipated in forthcoming research, are positioned to markedly advance pathway engineering and synthetic biology investigations in <i>Streptomyces</i> and other microbial strains.