RUI: A platform technology for elucidating design rules of the Epoxomicin synthase, enabling biosynthesis of complex anti-tumor molecules

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Bio-active natural products (pharmaceuticals, nutraceuticals) are generally difficult to replicate chemically, because they result from a sequence of reactions that add, piece by piece, different subunits to the growing molecule. As a result, they are harvested primarily from plant material, usually with low yield of product. This project will investigate a strategy that manufactures subunits of the larger drug molecule in the absence of cells, then brings the subunits together to create the final product. If successful, this could dramatically improve the efficiency of large molecule drug production, and could also increase the speed of identification of more effective drug molecules. Research efforts will be driven primarily by undergraduate students who will participate in all proposed experiments and broad dissemination of results. Undergraduate students will also lead inquiry-based learning modules for K-12 outreach through an established "Learn by Doing Lab" and through expansion of the SciTrek program into local schools. Current approaches to the biomanufacturing of previously intractable natural products are limited in their capacity to biosynthesize new natural products of interest on-demand. The primary objective of this project is to enable and facilitate on-demand biosynthesis of natural products and their derivatives. Specifically, a novel approach to elucidating the mechanistic underpinnings of epoxomicin natural product biosynthesis machinery will be developed by 1) "dissecting" the non-ribosomal peptide - polyketide synthase (NRP-PKS) mega-enzyme that synthesizes epoxomicin, 2) identifying the molecular determinants of substrate specificity, and 3) engineering the epoxomicin biosynthesis machinery for the generation of derivatives. These metabolic engineering efforts will be pursued in a cell-free environment; the open nature of this platform will provide direct access to metabolic reaction conditions for design-build-test cycles that are more rapid than traditional platforms for metabolic engineering. Potential contributions include A) knowledge of biochemical principles by which NRP-PK mega-enzymes biosynthesize complex natural products; and B) a platform technology that enables engineering and bio-manufacturing of complex natural products on-demand. Advances made through this work will apply directly to characterization and engineering of a myriad of other NRP-PKS mega-enzymes. In addition, this work will provide a foundation for training undergraduate students in conducting biotechnological research and communicating science to their community.