RUI: Development of New Green-Chemical Catalysts Based on Rieske Dioxygenase Scaffolds Through Directed Evolution

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Enzymes could reduce the environmental impact of processes. A class of enzymes found in soil bacteria enables them to thrive in petroleum-contaminated environments. Previously uncharacterized dioxygenases will be the focus of the project. Evolving these enzymes to expand the breadth of molecules on which they operate is a key objective, and they will be screened for the ability to degrade pollutants and synthesize valuable chemical intermediates. This project will also provide contemporary education and training experiences for undergraduate and master’s level students.Rieske dioxygenases have frequently been utilized in organic synthesis and bioremediation. Applications are restricted by limited substrate ranges and strict product selectivity. Directed molecular evolution will generate new Rieske dioxygenases exhibiting: 1) expanded reactivity for aromatic compounds and 2) expanded substrate range and increased reactivity in the dihydroxylation of alkyl olefins. Enzyme engineering will follow a “design-test-build-learn” cycle, wherein large Rieske dioxygenase variant libraries will be produced using both rational engineering and random mutagenesis strategies and will be screened for enhanced activity using a “substrate multiplexing” approach. Potential contributions of this research include A) new, sustainable catalysts with valuable applications in organic synthesis and bioremediation; B) the first reliable, enzymatic alternative to chemical methods for performing the asymmetric dihydroxylation of alkyl olefins; and C) development of a blueprint for the successful engineering of Rieske dioxygenases.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.