Engineering biosynthesis of Lycopodium alkaloids, promising natural products for the treatment of neurodegenerative disease Plant natural products make up many of the pharmaceuticals currently used in the clinic (e.g. Taxol, etoposide, and vinblastine as chemotherapeutics). Strikingly, numerous plant-derived compounds can potently modulate neurotransmission and signaling (e.g. morphine, hyoscyamine, and galantamine), and thus plant natural products serve as a promising source for neurological disease treatment. However, the biosynthetic machinery remains unknown for most of these compounds, including for many diverse, medicinal plant alkaloids that are derived from a distinctive imine-coupling reaction for which no catalytic enzyme has yet been identified. One such alkaloid that is currently in clinical trials as a treatment for neurodegenerative diseases such as Alzheimer?s and dementia is huperzine A (HupA), which belongs to a group of compounds called the Lycopodium alkaloids. The plants that produce these compounds are slow growing and difficult to cultivate, and thus a more feasible source of HupA will be necessary to supply this compound for clinical study and eventual pharmaceutical use. Discovery of HupA biosynthetic genes will allow for its metabolic engineering in other systems (e.g. yeast), thereby providing a potential for sustained production. Furthermore, discovery of novel enzymatic activity will inform upon the biosynthesis of other medicinal alkaloids, particularly those derived from analogous imine- coupling reactions. In this study, metabolomics, RNA-seq, and heterologous protein expression will be used to uncover HupA biosynthetic enzymes. In the 1st specific aim, HupA production will be profiled in live plants, and this data will be used to develop an RNA-seq experiment, wherein biosynthetic genes are predicted to be expressed in tissue where HupA is produced. Differential expression analysis and enzymatic logic will then be used to determine candidate genes likely to be involved in HupA biosynthesis. In the 2nd aim, candidate enzymes involved in the crucial imine-coupling reaction will be biochemically characterized, which will have biosynthetic implications not only for HupA, but also for many other medically-relevant alkaloids. Lastly, the 3rd aim will entail the biochemical characterization of tailoring enzymes that are necessary to produce HupA from a precursor scaffold molecule. Overall, study of plant natural products that modulate neural activity will be crucial for the continued discovery and development of drugs for treating human neurological disorders. Understanding HupA biosynthesis will not only provide a route to engineered HupA production, but will also offer insight into the biosynthetic logic of other imine-containing alkaloids used in medicine. The proposed research will be carried out under the mentorship of Professor Elizabeth Sattely (Chemical Engineering, Stanford University), who is an expert in the study of plant natural products. Professor Sattely, and Stanford University in general, will provide an excellent environment for me to accomplish the research and training outlined in this fellowship application.
Numerous plant natural products have promise as pharmaceuticals for treating neurological disorders, including neurodegenerative diseases such as Alzheimer?s and dementia. Plant compounds show striking diversity in their ability to modulate brain function in humans, and thus study of their biosynthesis and chemistry will be useful for discovering novel therapeutics for the treatment of neurological disease. Uncovering the enzymes that synthesize huperzine A (HupA), a plant alkaloid currently being explored as a treatment for several neurological diseases, will not only allow for production of this valuable compound through metabolic engineering, but will also provide insight into the biosynthetic machinery for other structurally-related plant natural products that have potential as pharmaceuticals, thereby accelerating the future research of these compounds.
