Exploring a New Dimension of Microbial Secondary Metabolism

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ABSTRACT Bacterial natural products have provided an immense source of therapeutic agents and driven innovation in chemistry, biology, and pharmacology. In the past decade, it has become evident that the capacity of bacteria to synthesize natural products was vastly underestimated. Advances in genome and transcriptome sequencing combined with bioinformatic methods have shown that most biosynthetic genes are not expressed or, at best, sparingly expressed during standard laboratory growth; their products are therefore not synthesized at sufficient titers for detection and structural/functional characterization. These so-called ‘silent’ or ‘cryptic’ biosynthetic gene clusters outnumber constitutively active ones by a factor of 5-10. As such, they represent a new dimension of bacterial metabolism, and unlocking it will allow access to a wealth of new natural products and to deeper insights into microbial physiology and biochemistry. We recently contributed the High-Throughput Elicitor Screening (HiTES) methodology toward the discovery of cryptic metabolites. In this approach, small molecule libraries are screened to identify inducers of selected silent gene clusters. With elicitors identified, the cryptic metabolite and the regulation of the silent cluster can be investigated. Several aspects of HiTES remain unexplored and they form the basis for the current application: HiTES has only been applied to Burkholderia spp. and a few actinomycetes. Other prolific genera have not yet been targeted. Moreover, the mechanism underlying HiTES remains to be determined. Low-dose antibiotics have repeatedly been identified as the most effective elicitors, but the regulatory pathways that underpin this response remain to be elucidated. In the current application, we seek address these topics by expanding HiTES to prolific and understudied bacterial genera for the discovery of new, cryptic natural products with desired biological activities, and by elucidating the mechanisms with which low-dose antibiotics elicit cryptic metabolite biosynthesis, focusing on the β-lactam antibiotics and their stimulatory effect on the model organism Burkholderia thailandensis. Collectively, these studies will shed light on an emerging dimension of bacterial secondary metabolism, unearth new regulatory circuits that drive expression of silent gene clusters, and provide novel natural products as possible therapeutic leads and sources of biosynthetic and pharmacological investigations.