PROJECT SUMMARY Natural products (NPs) continue to inspire novel chemistry, enzymology, biology, and medicine. New omics technologies and accompanying computational developments have fundamentally transformed the current paradigm of NP research, revealing a tremendous diversity of NP biosynthetic gene clusters (BGCs) that far exceeds the number of known NPs. Most NP BGCs, however, are silent when the microorganisms are cultured under standard laboratory conditions. In this MIRA renewal application, we propose to continue our interdisciplinary program on NP research by leveraging the Actinobacterial Strain Collection and the Genome Database at the Natural Products Discovery Center (NPDC), UF Scripps. Our hypotheses are: (i) genome mining of the Actinobacterial Strain Collection and Genome Database will allow us to identify potential producers of the targeted NP scaffolds and predict the structural novelty of the new NPs, (ii) genetic manipulation of the most promising BGCs in their native producers or expression of them in designer heterologous hosts will allow us to produce and isolate the new NPs in sufficient quantities for structural and functional characterizations, and (iii) investigation of the biosynthetic machinery of these new NPs will allow us to discover new chemistry and enzymology. The Actinobacterial Strain Collection at NPDC, consisting of 122,522 strains that were isolated over the last eight decades and from 69 different countries, encodes NP diversities that are impossible to reproduce in laboratory settings today. We have completed sequencing 13,719 selected strains (as of May 2023), of which 10,495 genomes have been assembled and annotated. These results, together with findings from the MIRA program, have cumulated into: (i) the establishment of C- 1027 and tiancimycin as model systems for enediyne NP biosynthesis, (ii) the establishment of leinamycin and guangnanmycin as model systems for biosynthesis of the leinamycin family of NPs, (iii) the identification of 1,050 and 276 distinct BGCs, encoding new enediynes and new members of the leinamycin family of NPs, respectively, and (iv) the development of a suite of enabling technologies to discover the target NPs and study their biosynthetic machinery in native producers or designer heterologous hosts, setting the stage for the proposed studies in this renewal application. The outcomes of this application include: (i) fundamental contributions to genome mining and activation of large BGCs, in native producers and heterologous hosts, for NP production and biosynthesis, (ii) discovery of new NPs, with privileged scaffolds, to inspire new chemistry, biology, and medicine, and (iii) new insights into biosynthetic machineries and novel chemistry and enzymology for the biosynthesis of enediynes and the leinamycin family of NPs. The long-term goal of our research is to understand at a molecular level how microorganisms synthesize complex NPs and to exploit this knowledge to discover no...