Activating Silent Biosynthetic Gene Clusters in Streptomyces Spp. with Crispri

Antibiotic-resistant bacteria are a looming threat for healthcare, and illustrate the need for the discovery of novel antimicrobial agents. The bacterial genus Streptomyces represents a promising resource, as it is the most prominent producer of antimicrobial natural products. This ability lies in the expression of enzymatic pathways encoded by genomic regions called Biosynthetic Gene Clusters (BGCs). BGCs are rarely expressed under laboratory culturing conditions (i.e. are silent), often due to the repressive action of gene regulatory networks. Therefore, many BGCs are uncharacterized and a potential source of new compounds. Here we describe a novel approach to activate silent BGCs by using CRISPR interference (CRISPRi) transcriptional repressors to relieve repressive regulation and activate natural product synthesis. To do this, we develop a CRISPRi system for Streptomyces and optimize its repression capability by tuning the expression levels of its components. We then harness CRISPRi to inhibit the activity of known BGC repressors and successfully induce production of natural products. Together, this work provides a new approach to activate BGC expression in Streptomyces. This strategy circumvents the need for time-consuming genome engineering efforts and only requires straightforward transformation of easily designable plasmids, thus providing a simple method for BGC activation.