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.
