The biosynthesis of six complex antibiotics of microbial origin will be studied: the macrolides erythromycin and avermectin, the polyether monensin, the saturated polyketide nargenicin, and the sesquiterpenes pentalenolactone and quadrone. Each of these substances is itself representative of a broad class of related natural products. It is expected that the information gained from studying the biosynthesis of these individual metabolites will be applicable to an understanding of the formation of their parent structural families as well. A series of experiments using intact cells, cell-free extracts, and blocked mutants and exploiting a variety of modern 13C NMR techniques, including 13C180 isotope shifts, 13C 2H decoupling, and 13C13C multiple labeling will be used to investigate the mechanism and sterechemistry of the key chain-elongation and ring-forming steps of a variety of important polyketide antibiotics for which erythromycin, avermectin, monensin, and nargenicin may be taken as prototypes. A recently developed cell-free cyclase will be used to study the biosynthesis of the sesquiterpene pentalenolactone and to test a stereochemical model for the formation of humulene-derived sesquiterpenes. This model will be extend to a study of the biosynthesis of the novel sesquiterpene antibiotic quadrone. The goals are not only to elucidate characteristic precursor-product relationships and to deduce particular biosynthetic pathways, but to establish the details of the key bond-forming and bond-breaking reactions by which simple precursors are converted to complex natural products. In carrying out these investigations we also hope to continue to develop experimental tools which will be broadly applicable to biosynthetic studies in general.
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