Enediyne antibiotics are the most potent, highly active antitumor agents in existence today. Neocarzinostatin (NCS) and its conjugates have shown great clinical promise in anticancer chemotherapy, and several calicheamicin-antibody and C-1027-antibody conjugates have produced outstanding results in recent clinical trials or model studies. Therefore, it is a critical research goal to develop ways to make the enediynes and their analogs for further studies. This application proposes to clone and characterize the genes for NCS production in Streptomyces carzinostaticus, complementing our on going effort for C-1027 in Streptomyces globisporus, in an attempt to synthesize novel antitumor drugs by manipulating genes governing enediyne antibiotic biosynthesis. This proposal is based on the hypothesis that the NCS chromophore is derived from three biosynthetic building blocks an enediyne core, a deoxy aminosugar, and a polyketide naphthoic acid moiety. Three corollaries of this hypothesis are (1) that the extensive knowledge of deoxysugar and polyketide biosynthesis in actinomycetes should greatly aid the effort to clone the genes for deoxy aminosugar and naphthoic acid biosynthesis from S. carzinostaticus, (2) that the structural similarity between the enediyne cores of NCS and C-1027 provides an unique opportunity to decipher the genetic and biochemical basis of enediyne core assembly, and (3) that the genetic tools and technology developed in Streptomyces species for antibiotic biosynthesis should be directly applicable to manipulating NCS and C-1027 biosynthesis genes for yield improvement and analog or novel enediyne production.
The specific aims for the five-year award period are (1) to clone and sequence the entire NCS biosynthetic gene cluster from S. carzinostaticus, (2) to develop a genetic system for S. carzinostaticus to manipulate NCS biosynthesis in viva, (3) to confirm the cloned S. carzinostaticus DNA encoding NCS biosynthesis and to localize the boundaries of the NCS gene cluster by gene disruption and replacement experiments, and (4) to identify genes essential for the enediyne core biosynthesis by comparing the NCS and C-1027 biosynthetic gene clusters and to demonstrate the synthesis of an enediyne core structure by expressing the corresponding genes in a heterologous host. The outcome of these studies will reveal new insights into the biosynthesis of the enediyne family of antibiotics, shedding light on rational engineering of enediyne biosynthesis in these as well as other enediyne-producing organisms, and could realistically lead to the making of fundamentally new, clinically useful antitumor agents
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