Abstract: Disclosed is a high-throughput transcriptional assay format in Actinomycete bacteria, and Streptomyces spp. in particular, that leverages eGFP, inserted both at a neutral site and inside the biosynthetic cluster of interest, as a read-out for secondary metabolite synthesis. Using this approach, a silent gene cluster in Streptomyces albus J1074 was induced. The cytotoxins etoposide and ivermectin were revealed as potent inducers, allowing the isolation and structural characterization of nearly 20 novel small molecule products of the chosen cluster. One of these molecules is a novel antifungal, while several others inhibit a cysteine protease implicated in cancer. Studies addressing the mechanism of induction by the two elicitors led to the identification of a pathway-specific transcriptional repressor that silences the gene cluster under normal growth conditions.

Abstract: Bacteria harbor an immense reservoir of potentially new and therapeutic small molecules in the form of “silent” biosynthetic gene clusters. These clusters can be identified bioinformatically but are at best sparingly expressed under normal laboratory growth conditions; their products are therefore not interrogated during bioactivity screening exercises. An estimated 80-90% of biosynthetic loci are silent, meaning that routine bioactivity screens miss the majority of microbial biosynthetic potential. Disclosed herein is a method that allows access to this vast hidden metabolome, thereby allowing researchers to screen the complete metabolomes of microorganisms in the search of new therapeutic leads. The disclosed approach, “Bioactivity-HiTES”, broadly activates the secondary metabolomes of bacteria and links the cryptic metabolites produced to a desired biological activity.

Abstract: Disclosed is a high-throughput transcriptional assay format in Actinomycete bacteria, and Streptomyces spp. in particular, that leverages eGFP, inserted both at a neutral site and inside the biosynthetic cluster of interest, as a read-out for secondary metabolite synthesis. Using this approach, a silent gene cluster in Streptomyces albus J1074 was induced. The cytotoxins etoposide and ivermectin were revealed as potent inducers, allowing the isolation and structural characterization of nearly 20 novel small molecule products of the chosen cluster. One of these molecules is a novel antifungal, while several others inhibit a cysteine protease implicated in cancer. Studies addressing the mechanism of induction by the two elicitors led to the identification of a pathway-specific transcriptional repressor that silences the gene cluster under normal growth conditions.