Dissection of specialized metabolism in plants using multiplexed perturbation - PROJECT SUMMARY: Most plants have evolved the capacity to synthetize large, complex metabolites that potently target animal physiology. While we have adopted some of these molecules as useful medicines (Taxol, colchicine, morphine, Velban), we understand a miniscule fraction of the chemistry that most plants are capable of. Most of the 3k-15k enzymes per genome involved in specialized metabolism have unknown roles, including in the genomes of plants we regularly consume as food. Learning the roles of these genes would not only illuminate what molecules a plant can synthesize, but also would allow us to (1) produce these molecules exogenously, (2) to directly assess their bioactivity, (3) make informed dietary decisions, and (4) genetically delete molecules from dietary plants. However, this is a challenging task because plant genetics is limited, plant genomes are large, and biosynthetic genes are often silent except under specific conditions. The aim of this proposal is to develop and apply tools that will systematically activate and identify different biosynthetic gene sets, efficiently defining a plants’ arsenal of biochemical pathways. In Aim 1, I will achieve this by the targeted activation of hundreds of transcription factors that regulate diverse biosynthetic pathways. During my K99 phase, I will develop this approach in tomato, a plant with relatively good genetic tools, optimizing a combination of multiplexed gene activation and single-cell measurement. I will also train with Dr. Jennifer Brophy’s lab in in vitro plant cell assays, so that during the R00 phase I can expand this approach to studying the biosynthetic dark matter of wheat and sorghum genomes. In Aim 2, I develop a variant of this methodology compatible with genetically intractable plants. I will use panels of chemical perturbations to activate biosynthesis, and a newly developed microfluidic technology to isolate and characterize cells with specialized biosynthetic roles. This will enable an accelerated dissection of the biochemistry of medicinal plants like Taxus, our sole source of the blockbuster drug Taxol. To achieve my long-term goal of becoming an independent researcher and pioneering a new scale of plant science, I have assembled a mentorship team that will complement my training in systems and synthetic biology: Dr. Elizabeth Sattely (mentor, plant biochemistry), Dr. Polly Fordyce (co-mentor, microfluidics and high throughput biology), Dr. Jonathan Weissman (advisor, single-cell biology and screening), Dr. Jennifer Brophy (advisor, plant synthetic biology), Dr. Bo Wang (advisor, single-cell technologies in noncanonical organisms). My mentors and I have designed a plan with specific goals for scientific training, career development coaching, scientific presentation, and coursework. The resources and thriving scientific environment at Stanford will help me achieve these goals. With the training and mentorship provided by this K99 opportunity, I anticipate a smooth transition to managing my own research group. There, I will establish a unique research program to develop systematic approaches to study the biosynthesis and health impacts of specialized metabolites.
