PROJECT SUMMARY Plants synthesize complex molecules for defense and signaling using specialized metabolic pathways. These plant natural products enhance their own evolutionary fitness and many of these molecules have been used with great success as pharmaceuticals to treat a wide range of human diseases as exemplified by Paclitaxel and Vinorelbine. However, our access to plant specialized metabolites can be limited, as these molecules are often produced in small amounts as part of complex mixtures and restricted to specific cell-types. While metabolic engineering and synthetic biology has the potential to improve our access to these compounds, these approaches require in-depth knowledge of the biosynthetic genes, transporters, and/or regulatory elements of the specific pathway. Next-generation omics technologies have made elucidation of plant natural product pathways more streamlined over the last decade, yet the discovery of plant natural product genes remains challenging relative to that in microbial systems. As a consequence, successful examples of metabolic engineering to improve access to the wealth of pharmacologically active molecules encoded in plant genomes are still few in number. For example, while the plant anti-cancer agent vinblastine has been reconstituted in yeast, the titers are not commercially viable. We have recently developed a single-cell omics-enabled, genome-to-pathway discovery pipeline that accelerates the discovery of natural product biosynthetic pathway genes and their associated regulatory sequences, including transcription factors and cis-regulatory elements. In Aim 1, we will prepare single cell- omics datasets from plant materials for pathway discovery of eight key plant natural products with anti-pain, anti- inflammatory, anti-malarial, and anti-cancer activity. In Aim 2, we will select and validate biosynthetic pathway genes for these compounds in N. benthamiana. New and innovative metabolic engineering strategies for improving access to plant natural products are still needed. In Aim 3, cell-type specific regulatory sequences for these eight biosynthetic pathways will be identified. In Aim 4, synthetic biology will bes used to engineer Catharanthus roseus callus-derived suspension cultures to produce anhydrovinblastine and a subset of the eight natural products from this study. Single-cell omics will be used to examine the heterogeneity of natural product production in populations of wild-type and engineered C. roseus suspension culture cells. Even if commercially appropriate cultures are not developed within the scope of this proposal, this project will provide the first rigorous omics datasets on a plant cell culture system. In summary, the state-of-the-art omics approach in this project will lower the barrier for gene discovery of plant-derived natural products. Utilization of synthetic biology approaches empowered by cell-type-specific knowledge of biosynthetic pathways will enable a renaissance in the sustainable production of clinically-relevant compounds in plant suspension culture.

PROJECT NARRATIVE Plants produce a plethora of medicinally important natural products that have significant potential to improve human health yet are often produced in low amounts, in complex mixtures, and/or cannot be produced in a sustainable or economic manner. Omics technologies enable the discovery of the biosynthetic genes of plant natural products which can then be employed in commercial production of medically relevant compounds. In this project, we will use state-of-the-art omics technologies to efficiently discover genes responsible for the biosynthesis of eight pharmacologically relevant compounds and utilize this knowledge to revitalize an old technology, plant cell culture, to enable robust, sustainable production of these products.