Periodic Reporting for period 3 - MedPlant (Harnessing the Molecules of Medicinal Plants) Reporting period: 2020-10-01 to 2022-03-31 Summary of the context and overall objectives of the project Plants, as sessile organisms, synthesize complex molecules for defense and signaling. Humans have long exploited the potent medicinal activities of these plant natural products: artemisinin from sweet wormwood is used to cure malaria, vincristine from Madagascar periwinkle is used to treat cancer, and morphine from poppy alleviates pain. Synthetic biology approaches are being used with increasing success to overproduce these expensive molecules, which are often present at low levels in the plant. However, to pursue such approaches effectively, we must fully understand the biosynthetic pathways that generate these molecules. This pathway discovery process has been a major bottleneck in harnessing the chemical power of plants. This grant focuses on elucidation of the biosynthetic enzymes for monoterpene indole alkaloids. The goal is to produce molecules that have medicinal applications, and also to understand how chemical diversity evolved in these plant producers. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In this project reporting period we have achieved several major objectives.1) We have found the biosynthetic genes for many of the molecules described in the proposal: ibogaine, quinine, strychnine, mitragynine as well as strychnine. Efforts for ipecac are underway.2) We have successfully reengineered reductases and determined the mechanistic basis for aldehyde, 1-2 and 1-4 reduction.3) We have successfully engineered cyclases to show the mechanistic basis of selective cyclization. Our efforts to engineer cyclases to make alternative scaffolds have not succeeded. Instead, we have shown that we can use alternative enzymatic strategies to generate isomers of the cyclase substrate, and this instead leads to two of the alternative scaffolds that we proposed to engineer in the proposal.4) We have solved the structures of 9 biosynthetic enzymes through crystallography and are using these structures to understand the interactions among biosynthetic enzymes.5) We have successfully developed a much higher throughput method for cloning in Nicotiana.6) Strictosidine analogues (15) have been prepared for production of analogues in Nicotiana. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We have combined chemistry, structural biology, enzymology and plant engineering to produce new complex alkaloids. We have also used these approaches to understand the evolutionary basis for the emergence of this chemical diversity.