Facile inactivation of a Streptomyces global regulator using a versatile base editing plasmid for secondary metabolism perturbation across multiple species.

Members of the genus Streptomyces produce a wide array of bioactive and structurally diverse secondary metabolites. However, many biosynthetic gene clusters (BGCs) remain silent (cryptic) under standard laboratory cultivation conditions, posing a significant challenge to the discovery of new secondary metabolites. We considered that the introduction of transcriptional perturbations governed by global regulators may be effective at awakening such cryptic BGCs. In this study, we developed a versatile Target-AID base-editing plasmid designed to inactivate wblA, one of the widely conserved global regulator genes, by introducing a stop codon in the coding sequence. Comparative sequence analysis of 30 Streptomyces species revealed high nucleotide sequence identities among wblA homologs, enabling us to design a single guide RNA (sgRNA) applicable across multiple species. The plasmid was used to transform several Streptomyces species to find metabolic (or phenotypic) changes. Specifically, in Streptomyceslavendulae FRI-5, the wblA-inactivated mutant strain exhibited increased production of lavencidins and indigoidine, while gene complementation restored metabolite levels, confirming the metabolic changes were due to the inactivation/restoration of wblA. Furthermore, in Streptomycesvirginiae NBRC12827, wblA inactivation induced extensive secondary metabolism perturbation caused by transcriptional changes in BGCs. These findings demonstrate that our versatile plasmid enables facile inactivation of global regulators conserved in Streptomyces species, thereby accelerating the discovery of new secondary metabolites across diverse Streptomyces species.