Changing Paradigms in Natural Product Discovery: A Molecule to Microbe Approach

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1 Microbial natural products represent one of our most important sources of medicines. Traditionally, these 2 compounds have been discovered from microbes cultured in the laboratory using conditions that bear little 3 resemblance to the environments from which they were derived. The failure of traditional discovery approaches 4 to keep pace with the need for new drug leads, coupled with our improved understanding of the molecular 5 genetics of natural product biosynthesis, spurred the development of new approaches for natural product 6 discovery. These efforts have largely focused on genome mining and the genetic manipulation of orphan 7 biosynthetic gene clusters with the aim of bypassing the regulatory mechanisms that control compound 8 production. While important progress has been made, these approaches have yet to yield a wealth of new 9 chemical scaffolds. Here we propose an alternative discovery paradigm that takes a compound first approach. 10 We developed a Small Molecule In situ Resin Capture (SMIRC) technique to recover microbial natural products 11 directly from environmental samples thus by-passing the need for cultivation. We have applied this approach to 12 ocean sediments, where complex microbial communities occur and interstitial water flow facilitates compound 13 capture. Coupled with mass-spectrometry based metabolomics, we demonstrate that SMIRC can capture known 14 microbial metabolites and thousands of compounds that cannot be readily identified, thus hinting at the discovery 15 potential afforded by this technique. Here we propose to test the applications of a new capture and analysis 16 pipeline to discover microbial natural products directly from marine sediments. By introducing an antibiotic screen 17 into the workflow, we target biomedically relevant compounds for isolation and structural characterization. A 18 micro-fractionation technique will be used to separate complex mixtures and guide the isolation of active, low 19 abundance compounds. We capitalize on innovations in 'nanomole-scale' natural product characterization and 20 the integration of microcryoprobe NMR and MS analyses for sub-micromole level structure determination 21 (including stereostructures). Our ability to prioritize leads based on MS de-replication and biological activity 22 provide opportunities to tease out deep chemical diversity from complex extracts. We further propose to use 23 metagenomics to link compounds to their biogenic sources and thus gain taxonomic perspective on unrealized 24 biosynthetic potential. This approach eliminates the need to establish laboratory conditions suitable for cultivation 25 and gene cluster expression (two of the major bottlenecks hampering current natural product discovery efforts) 26 and instead allows the environment to act as a natural bioreactor. Ultimately, lead compounds discovered using 27 this approach can be produced via chemical synthesis, bulk environmental extraction, synthetic biology, or 28 metagenomics informed cultivation.