This proposal describes an approach of using hybrid reconstitution of enzyme subunits from native, cognate biosynthetic systems to generate variants of nonribosomal/polyketide natural products. The natural products involved are the epothilones, yersiniabactin, and pyochelin, all of which have known therapeutic value in cancer therapy and antibiotic treatment. The ability of the hybrid, noncognate enzyme assembly lines to successfully produce the desired variants will be assessed by evaluating the effectiveness of the acyl-transfer step across the hybrid interface. The functional properties of these hybrid systems will be correlated to the protein/protein recognition characteristics between the noncognate enzyme subunits. Genetic protein engineering such as linker/domain swapping or methyltransferase domain insertion will also be employed to either improve the efficiency of acyl-transfer or increase the level of hybridization for generation of more diverse variants of the natural products. The """"""""unnatural"""""""" variants of therapeutically important natural products will be used as probes to help discover unrealized medicinal benefits. The information gained on the relationship between the functional and structural aspects of the engineered systems will further expand the capabilities of combinatorial biosynthesis as a novel strategy to overcome cancer and antibiotic resistance.
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