Cancer causes 1 of every 4 deaths in the US, and it is a critical research goal to optimize available anticancer drugs and discover and develop fundamentally new, clinically useful ones. Natural products have an excellent track record as sources of anticancer drugs but traditional natural product drug discovery approaches face many problems and challenges. As the combinatorial biosynthesis component of the UW NCDDG program, we propose to produce small libraries of a carefully selected set of promising natural product leads of micribial origin by genetic manipulations of their biosynthetic pathways. Our hypotheses are: (1) application of combinatorial biosynthetic methods to the biosynthetic machinery of proven anticancer drugs will result in the production of novel analogs with an improved therapeutic index; (2) application of combinatorial biosynthetic methods to biosynthetic machinery of natural products with promising antitumor activities but unresolved modes of action will generate libraries of the given molecular scaffold, facilitating the identification of novel targets and leading to the discovery of new classes of anticancer drugs; (3) engineering of natural product carrier proteins with cancer targeting peptides (CTPs) and/or tumor-specific activation sites for prodrug design will enable the delivery of the natural product drugs to specific cancer cells. The long-term goal of this project is to apply combinatorial biosynthetic methods to the various biosynthetic pathways that have already been characterized in our laboratories for anticancer drug discovery. The specific aims for this proposal are: (1) engineering and production of novel analogs of natural product anticancer drugs with known modes of action such as the enediynes C-1027, neocarzinostatin, maduropetin, calicheamicin, dynamicin, leinamycin, hedamycin, and rebeccamycin; (2) engineering and production of novel analogs of natural products with promising antitumor activities but unresolved modes of action such as migrastatin, dorrigocin, lactimidomycin, tautomycin, tautomycetin, and fredericamycin; and (3) engineering and production of CTP-containing chromoproteins such as C-1027 and neicarzinostatin and of cathepsin D and matrix-metalloprotease-2 specific prodrugs such as bleomycin and mitomycin C. The outcomes of these studies will lead to the identification of novel targets for anticancer drug discovery and to the development of fundamentally new, clinically useful anticancer drugs