Many valuable drugs, including antibiotics, immunosuppressants and anticancer compounds are polyketide natural products of actinomycete bacteria. They are produced on modular polyketide synthases (PKSs), assembly-line multienzymes containing many different enzyme active sites, housed in giant multifunctional polypeptides. A number of strategies are now used to engineer PKSs and produce altered bioactive products, especially domain and module swapping between natural PKSs.
However, many hybrid PKSs have compromised catalytic abilities. We intend here to use novel knowledge-based engineering of the active sites of enoylreductase (ER) domains, which play a key role in establishing the stereochemistry of branching alkyl groups. We propose that alteration of only a few active site residues will be sufficient to switch the stereochemical outcome of reduction. This hypothesis will be tested using two complementary approaches: (a) individual recombinant ER domains (and site-directed mutants) will be purified and their activity and stereospecificity studied in vitro; and (b) a triketide synthase model PKS will be used to assay altered ER domains in vivo. Finally, the technique will be applied to creating a designed change in the clinically important macrolide antibotic erythromycin A, by switching the active site of the ER domain from module 4 of the erythromycin-producing PKS.
This project will comprise an advanced training programme in the techniques and skills to be used in studying unique "assembly-line" multienzyme complexes, in which the host laboratory is highly experienced. This will complement the Marie-Curie Fellow's strong existing training in bio-organic chemistry, and thus consolidate and broaden her career prospects, as well as maintaining excellence in European research. Hence the project is directly relevant to the Work Programme as well as the specific aims of the Marie Curie IEF action.
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