Combinatorial Biosynthesis of New Alkaloids in Periwinkle

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Intellectual Merit:The overall goal of this project is to initiate metabolic engineering efforts in Catharanthus roseus madagascar periwinkle) to produce non-natural alkaloids. Combinatorial biosynthesis, which entails the swapping of wild type enzymes with enzymes having altered substrate specificity, has been successfully applied to a variety of prokaryotic biosynthetic pathways to yield nonnatural versions of natural products. This research describes how combinatorial biosynthesis can be accomplished in C. roseus to yield complex terpene indole alkaloid analogues. The substrate flexibility of the terpene indole alkaloid biosynthetic machinery in C. roseus has been shown to be tolerant to non-natural substrates. Additionally, the first committed enzyme of the pathway (strictosidine synthase) has been reengineered to display altered substrate specificity. These reengineered enzymes will be transformed into C. roseus cell culture and the production levels of terpene indole alkaloid analogues will be monitored in the transgenic C. roseus strains.Broader Impact:Graduate, undergraduate and post-doctoral researchers are exposed to chemical synthesis, protein expression and mutagenesis and molecular biology protocols. Since this research uses a multidisciplinary approach to solve problems in the area of natural product biosynthesis, students have numerous opportunities to interact with other labs with complementary areas of expertise. Furthermore, a graduate level course in natural product biosynthesis has been developed and is being refined. This course material is made available to the general public through MIT's open courseware site URL. Strategies to enhance the teaching of an introductory chemistry course are also described.