Periodic Reporting for period 4 - MedPlant (Harnessing the Molecules of Medicinal Plants)
Reporting period: 2022-04-01 to 2023-12-31
The monoterpene indole alkaloids are a group of ca. 3000 molecules produced by plants. The monoterpene indole alkaloids have a wide range biological and pharmacological activities, and are used in medicine and biology for a variety of applications. For example, vinblastine is a chemotherapy drug used to treat a variety of cancers, ibogaine is a molecule under study for the treatment of addiction, quinine is an important anti-malarial drug, mitragynine is being investigated as a new anti-pain medication and strychnine is a well-known neurotoxin. Unfortunately, plants produce these molecules in small quantities, making application of these compounds challenging. If we discover the genes that encode the biosynthesis of these compounds, then we can use engineering approaches to overproduce these compounds in heterologous hosts.
The objectives of this project were to 1) discover and characterize the genes that are responsible for the biosynthesis of some of the most medically important monoterpene indole alkaloids, 2) understand the mechanism by which these enzymes catalyze the steps of biosynthesis, and use this information to engineer these enzymes to be more useful for metabolic engineering efforts, and 3) explore next generation metabolic engineering approaches by understanding the roles that inter-cellular localization plays in controlling and regulating these pathways.
The objectives of this project were to 1) discover and characterize the genes that are responsible for the biosynthesis of some of the most medically important monoterpene indole alkaloids, 2) understand the mechanism by which these enzymes catalyze the steps of biosynthesis, and use this information to engineer these enzymes to be more useful for metabolic engineering efforts, and 3) explore next generation metabolic engineering approaches by understanding the roles that inter-cellular localization plays in controlling and regulating these pathways.
Overall, the aims of the proposal were achieved. In Objective 1, we identified the vast majority of the undiscovered genes of the pharmacologically important monoterpene indole alkaloids that we set out to study. We also identified genes of related monoterpene indole alkaloids not explicitly described in the proposal. We could in these cases reconstitute the biosynthesis of these compounds in a tobacco plant. Highlights include pathway discovery and reconstitution of ibogaine, mitragynine and strychnine. We also discovered a key dimerization enzyme involved in the biosynthesis of vinblastine. In Objective 2, we showed how we can rationally predict and modulate the chemical reactivity of a range of reductases involved in alkaloid biosynthesis. Moreover, we showed how a crucial cytochrome P450 can be tuned to generate 4 structurally different scaffolds from a single starting substrate. Finally, we demonstrated how an esterase enzyme could be converted into an enzyme that catalyzes an unprecedented cyclization reaction. We also showed how redox enzymes can interact with these cyclases to tune the structure of the final cyclized product. In addition to providing enzyme tools for biocatalysis, these studies provide important insights into how evolution of natural product enzymes may occur. In Objective 3, we demonstrated how we could use plants as a heterologous host for reconstitution of medically important natural products. Moreover, we developed beyond state of the art single cell analytical techniques to probe the cell-specific localization of biosynthetic genes. Therefore, we can now probe in an unprecedented manner how modulating cell type specific localization impacts the titer of medically important natural products.
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. The results of the project make pharmacologically active compounds produced by plants more accessible for use in modern medicine.