"Discovery and Characterization of Medicinal Plant Alkaloid Biosynthetic Pathways"
Plants have evolved to produce diverse, complex natural products that interact with biomolecular targets in various species, making them a rich resource for medicinal purposes. Leading examples include Cephalotaxus and Lycopodium alkaloids, specifically homoharringtonine (HHT) and huperzine A (HupA) respectively. HHT is a clinically used anti-cancer drug for chronic myeloid leukemia (CML) that works by preventing initial elongation step of protein synthesis, whereas HupA is a potential drug for neurodegenerative diseases due to its acetylcholinesterase inhibitory effect. Despite their biomedical significance, biosynthetic pathways of these plant alkaloids remain underexplored. Here, we use combined metabolomics, transcriptomics, enzymatic logic, and biochemical analyses to unravel their biosynthesis. For HHT, we elucidated a near-complete biosynthesis to cephalotaxine (CET), the alkaloid core of HHT, without prior knowledge of intermediates and biosynthetic genes. We discovered and characterized seven new CET pathway intermediates and six novel biosynthetic enzymes—including noncanonical cytochrome P450s, an atypical short-chain dehydrogenase, and a 2-oxoglutarate-dependent dioxygenase (2OGD)—that together produce cephalotaxinone, the likely direct precursor of CET, revealing a whole plant model for coordinated biosynthesis of HHT. For HupA, we identified and characterized several key novel enzymes responsible for the formation of HupA. Two major enzyme classes were particularly characterized: two 2OGDs that catalyze the late oxidative tailoring steps to produce HupA from huperzine B (HupB) through oxidative ring cleavage, desaturation, and double-bond isomerization, and three neofunctionalized -carbonic anhydrases (CAHs) that catalyze a stereospecific Mannich-like condensation and subsequent bicyclic scaffold generation to generate a key precursor of the Lycopodium alkaloids. Collectively, these studies uncover the complex medicinal plant biosynthetic pathways, offer deeper insights into diverse enzyme-mediated biochemical transformations, and provide a foundation for future sustainable production of clinically relevant plant alkaloids.