Targeted Discovery of Cryptic Enediyne Natural Products via FRET-coupled High-Throughput Elicitor Screening

Enediyne antibiotics are a striking family of DNA-cleaving natural products with high degrees of cytotoxicity and structural complexity. Microbial genome sequences that have recently accumulated point to an untapped trove of ‘cryptic’ enediynes. Most of the cognate biosynthetic gene clusters (BGCs) are sparingly expressed under standard growth conditions, making it difficult to characterize their products. Herein, we report a fluorescence-based DNA cleavage assay coupled to high-throughput elicitor screening for the rapid, targeted discovery of cryptic enediyne metabolites. We applied the approach to Streptomyces clavuligerus, which harbors two such BGCs with unknown products, identified steroids as effective elicitors, and characterized ten cryptic enediyne-derived natural products, termed clavulynes A-J with unusual carbonate and terminal olefin functionalities. Our results contribute to the growing repertoire of enediynes and provide a blueprint for identifying additional ones in the futur