Ongoing studies of the biosynthesis of macrolide, polyether, and terpenoid antibiotics will be continued and extended. Among the microbial metabolites to be studied are the macrolides erythromycin (1), methymycin (2), oleandomycin (3) and nargenicin (4); the polyethers monensin (5), lenoremycin (6), and portmicin (7); and the sesquiterpene pentalenolactone (8). Each is 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 broadly applicable to an understanding of polyketide and terpenoid biosynthesis as well. 1) Based on the use of N-acetylcysteamine (NAC) thioesters as advanced precursors, a systematic study is planned of the polyketide chain-building reactions in the biosynthesis of three closely related macrolide antibiotics erythromycin (1), methymycin (2), and oleandomycin (3) by incorporation specifically labeled, synthetic two-and three-subunit substrates. The biosynthesis of a structurally distinct, but biosynthetically closely related metabolite, nargenicin (4) will also be studied using the same or structurally analogous precursors. These studies are intended to establish the detailed mechanism of chain- elongation in the biosynthesis of reduced polyketides. Following these investigations, studies of the cell-free formation of erythromycin and the 16-membered macrolide, tylosin (9) will be initiated. 2) To test a general stereochemical model of polyether biosynthesis, experiments have been designed to elucidate the details of polyketide chain elongation in the biosynthesis of the polyether ionophoric antibiotics monensin (5), lenoremycin (6), and portmicin (7). Specifically labeled two-, three-, and four-subunit precursors will be fed as the NAC-thioesters in order to test their role in the biosynthesis of polyethers and to demonstrate the intermediacy of olefinic acids in the formation of the reduced polyketide chain. 3) The terpenoid cyclase pentalenene synthetase, an enzyme isolated from Streptomyces UC5319, catalyzes the conversion of farnesyl pyrophosphate to the sesquiterpene pentalenene (10), the parent hydrocarbon of the pentalenolactone family of antibiotics. Using a combination of oligonucleotide and antibody screening, the pentalenene synthetase gene will be cloned and expressed. The availability of cloned DNA will allow sequencing of the synthetase while the increased quantities of cyclase will be used for continued studies of the mechanism and stereochemistry of the cyclization reaction itself.
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