Harnessing Steroidal Molecules of Plants - Project Summary/Abstract Plants synthesize a diverse array of specialized (secondary) steroidal metabolites that are of high value pharmaceutically and nutritionally. Examples include cardenolides, bufadienolides, as well as steroidal saponins and alkaloids, many of which have been used to treat variety of human diseases. Plants natively synthesize specialized steroids, but usually in small amounts and as constituents of complex mixtures. Structural complexity often renders the chemical synthesis of steroids challenging and expensive. Therefore, our access to wide range of bioactive steroids in natural and synthetic systems remains restricted. Metabolic engineering approaches potentially can provide a new way to access steroidal molecules. However, knowledge gaps in our fundamental understanding of plant derived steroidal specialized metabolic pathways, and associated enzymes have hampered application of these approaches towards the production of steroidal compounds at scale for medicinal applications. The proposed research aims to elucidate complex steroidal biosynthetic pathways from plants and to provide access of high value steroids in sustainable bioproduction platform. Our research program starts with identification and characterization of biosynthetic enzymes that convert simple sterols to specialized steroids, for example cardenolides and bufadienolides. These classes of steroidal metabolites are produced in a wide array of plants and are known for their use in treating of congenital heart conditions, cancers and other chronic diseases. Our expertise in comparative metabolomics, genomics and transcriptomics across a diverse spectrum of plants enable us to identify candidate genes involved in biosynthetic pathway of complex steroidal molecules (e.g. cardenolides). By including plant species with both overlapping and divergent steroidal metabolite profiles, we expect to rapidly filter out large number of gene candidates for further functional characterization. The prioritized candidates will be then tested in different heterologous expression systems (e.g. E. coli, yeast, tobacco plants) to confirm their role in biosynthetic pathways (e.g. cardenolides). Our strategy will support accelerated discovery of steroidal pathways and associated enzymes across multiple plants. Building on this foundation, metabolic engineering will be used to develop efficient and sustainable ‘Plant’ based expression chassis for reconstitution of complex steroidal pathways to generate both natural and new-to-nature steroids with potential biological activity. A practical goal is to unlock the biosynthesis of steroidal metabolites with pharmacologically relevant bioactivities. Long term goals include a comprehensive understanding of the molecular mechanisms in cells through which remarkable metabolic diversity is generated in chemical structure and biological activity, laying a foundation for exploiting nature-inspired diversity in development of new medicinals.
