Polyketide-derived metabolites are found in both procaryotes (mainly actinomycetes) and eucaryotes where they play an amazing variety of functional roles. Many of these compounds have important biological activities and have been developed as antibiotics and chemotherapeutic agents in both human and veterinary medicine. The importance of these compounds is increasing as the need arises to develop agents which effectively combat recalcitrant infectious diseases, and in the development of small molecules which mimic or inhibit the activity of natural hormones and immunomodulators. The primary aim of the proposed research is to understand the molecular mechanisms controlling carbon- chain construction in the biosynthesis of polyketide antibiotics. Molecular genetic, biochemical and chemical approaches will be used to obtain information on the functional role of each enzyme comprising the polyketide synthase (PKS) including; condensing enzymes, acyl carrier proteins, acyl- and terminal transferases. Molecular genetic studies have revealed a close relationship between fatty acid and polyketide biosynthesis, and a model will be developed to investigate the mechanistic similarities and differences of these important biosynthetic systems. In order to establish an effective experimental model, we will clone and sequence the fatty acid synthase (FAS) genes of Streptomyces coelicolor. Although we expect the molecular genetic details will have important comparative value, this work will be pursued with the goal of establishing the S. coelicolor FAS as a universal indicator system to study individual components of PKS and FAS gene clusters. The versatility of the approach is provided by the ease of structural determination of fatty acids compared to complex secondary metabolites. In this respect, the invariant reductive cycle that occurs in fatty acids compared to complex secondary metabolites. In this respect, the invariant reductive cycle that occurs in fatty acid biosynthesis provides an enormous strategic advantage. Of primary importance is the question of carbon chain length determination, and the effect of altering activity of PKS condensing enzyme on chain length control in polyketide antibiotic biosynthesis. Hybrid FAS/PKS gene clusters will be constructed using trans-complementation and gene replacement technology. Structural analysis of the fatty acids produced will allow us to analyze the precise functional contributions of individual PKS genes in the hybrid system. Overall, this work should provide important insight into the molecular genetic and biochemical relationships within the FAS and PKS systems in Streptomyces. Moreover, it is hoped that this work will provide an important theoretical and experimental base for the rational production of novel polyketide-derived metabolites using molecular genetic technology.
