Dual Silencing of Aminoglycoside Biosynthesis for the Discovery of Novel Antimicrobials in Streptomyces.

Streptomyces species are prolific sources of antimicrobials, yet their vast biosynthetic capacity remains underexploited due to the rediscovery of common antibiotics (e.g., ~1% for streptomycin and ~10% for streptothricin). To address this challenge, we developed a dual-layer biosynthetic blockade system, designated CI-ARS3, which integrates two orthogonal silencing modules: a competitive transcriptional silencer that represses transcription of the native biosynthetic gene cluster (BGC), and an antisense RNA-based silencer that facilitates the degradation of BGC-derived mRNAs and/or translational inhibition. Implemented on a single attB site-integrating plasmid (pSET152-aafssf) encoding both modules, CI-ARS3 robustly suppresses streptomycin and streptothricin biosynthesis across 54 Streptomyces strains, achieving an inhibition efficiency of over 97% with high specificity for the target compounds under the tested conditions. The removal of these dominant backgrounds led to the discovery of enissomycin, a previously unreported antibacterial molecule. Enissomycin exhibited potent activity against Acinetobacter baumannii (MIC 4-8 μg/mL) and exhibited no significant cytotoxicity, highlighting its promise for drug development. Collectively, CI-ARS3 is a robust platform for the efficient dereplication of streptomycin and streptothricin, thereby expediting the discovery of novel antimicrobial compounds from Streptomyces.