ABSTRACT The 10-membered enediynes [exemplified by calicheamicin (CLM), esperamicin (ESP) and dynemicins (DYN)] are arguably among the most renowned natural products (NPs) discovered to date by virtue of their unprecedented complex molecular architectures, notable anticancer and anti-infective potencies and, in the case of CLM, demonstrated clinical utility. The current study builds on a longstanding collaborative effort of achievement and discovery relating to key aspects of 10-membered enediyne biosynthesis as well as parallel innovative efforts to co-opt key biosynthetic catalysts for synthetic applications. The studies put forth will take advantage of this strong foundation and a powerful combination of genetic, biochemical, chemical and protein structural tools to elucidate the remaining unusual biosynthetic transformations and to exploit select catalysts for enediyne non-native modification. Specifically, aims 1 and 2 will focus on extending our understanding of the fundamental steps of enediyne core biosynthesis common to CLM/DYN/ESP, DYN anthraquinone biosynthesis and a selected set of unique tailoring reactions (CLM/ESP thiosugar sulfur installation and aminopentose N- alkylation, ESP C6-hydroxylation and O-glycosylation). In parallel, aim 3 will focus on tactical structural studies to augment both aims 1 and 2 and the structural study of ‘unknowns’ to facilitate functional annotation. Additional studies in aim 2 with key catalysts and corresponding non-native substrates are designed to assess the potential for strategic installation of chemoselective handles to enable novel approaches for facile, mild bioconjugation of CLM to tumor-targeting mAbs (in collaboration with Pfizer).