Project Summary/Abstract Natural products (NPs) form a validated and preeminent source of new drug leads, but the low rate of new discoveries and the limited access to bioactive compounds are challenging current NPs-based drug discovery and development. Over the past decades, genome mining has become an important way for NP discovery. This transformative research strategy combines functional genomics and bioinformatics to associate biosynthetic gene clusters (BGCs) with potential chemical entities. The success of this strategy is supported by increasing and rapid access to (meta)genomic data, from which diverse rule-based and -free bioinformatics tools enable depicting the BGCs of different compound families and even assessing their novelty and potential bioactivity. However, a fundamental challenge faced by genome mining research is how to produce sufficient amounts of novel, bioactive NPs from mined BGCs. The overall goal of the PI's research program is to access and expand the therapeutically relevant chemical diversity of microbes directly from their genomes. The central hypothesis of this work is that microbial genomes can be exploited to produce bioactive NPs and analogs through synthetic biology (SynBio) and enzymology research. SynBio has achieved many notable successes in the production of bioactive compounds over the past two decades. In the NP discovery, SynBio studies can allow the expression of natural and designed BGCs in capable chasses. In parallel, the biosynthesis of microbial NPs is enriched with functionally diverse enzymes that lend enabling strategies to produce key intermediates and analogs of bioactive NPs. To achieve the goal and test the hypothesis, this renewal will pursue two interlinked research directions. Direction 1 will focus on the discovery and production of bioactive NPs from less-explored resources, particularly in marine entrainment. To maximize the success, our SynBio research will develop and use chasses of multiple bacterial phyla. Research direction 2 of this proposal will discover and characterize synthetically significant enzymes using a diverse set of approaches, aiding the access and expansion of therapeutically relevant chemical diversity of NPs. New enzymes found in Direction 1 will be characterized in Direction 2 and can then support the mining of new BGCs in Direction 1, leading to tight integration and mutual support of the two directions. Together, these two directions can afford innovative strategies that enable the effective exploitation of bioactive NPs from the genomes of less-studied microbes. These studies can also boost the transformation of NP research from small-scale pursuits to a genome-based high-throughput endeavor, therefore supporting the paradigm shift in NP-based drug research.