Development of synthetic sRNAs tool for targeted and high-throughput gene knockdown in diverse bacteria

Abstract

The use of synthetic sRNAs for knockdown of target genes at the translational level has been restricted to a limited number of bacteria. In this work, we report the development of a broad-host-range synthetic sRNA (BHR-sRNA) platform employing the RoxS scaffold and the Hfq chaperone from Bacillus subtilis. The BHR-sRNA platform was tested in 16 bacterial species including commensal, probiotic, pathogenic, and industrial bacteria. Using this platform, more than 50% of target gene knockdown was achieved in 12 bacteria. As a medical application, virulence factors in pathogens Staphylococcus epidermidis and Klebsiella pneumoniae were knocked down to mitigate their virulence-associated phenotypes. For metabolic engineering applications, the development of high-performance Corynebacterium glutamicum strains capable of producing valerolactam and methyl anthranilate as example bulk and fine chemicals, respectively, were demonstrated by combinatorial knockdown of target genes. To facilitate high-throughput colorimetric screening of indigoidine (natural colorant) overproducers, a genome-scale sRNA library covering 2,959 C. glutamicum genes was constructed. The BHR-sRNA platform has the potential to expedite the engineering of diverse bacteria of both industrial and medical interest. This work was further supported by the Development of Next-Generation Biorefinery Platform Technologies for Leading Bio-based Chemicals Industry Project (2022M3J5A1056072) and by the Development of Platform Technologies of Microbial Cell Factories for the Next-Generation Biorefineries Project (2022M3J5A1056117) from the National Research Foundation supported by the Korean Ministry of Science and ICT.