Discovery of homoharringtonine pathway enzymes reveals a whole plant model for coordinated biosynthesis

Plants produce diverse molecules that inhibit protein translation. A lead example is homoharringtonine (HHT), a key tool for ribosomal profiling and an FDA-approved treatment for chronic myeloid leukemia. HHT is commercially produced through semi-synthesis from the alkaloid core cephalotaxine (CET) extracted from endangered Cephalotaxus species. Despite its significance, the CET/HHT biosynthetic pathway remains unresolved. Here, we use paired untargeted metabolomics (stable-isotope precursor feeding) and transcriptomics to elucidate a near-complete biosynthesis to CET without prior knowledge of intermediates and biosynthetic genes. We show that while the CET core is biosynthesized only in growing root tips, CET and HHT accumulate throughout the plant. We discovered seven pathway intermediates and six novel enzymes that produce cephalotaxinone, the likely direct precursor of CET. Included are non-canonical cytochrome P450s, an atypical short-chain dehydrogenase, and a 2-oxoglutarate-dependent dioxygenase that together result in carbon excision and CET/HHT pentacyclic backbone formation. This study establishes a metabolic route to the HHT core scaffold and suggests a whole-plant coordination model in Cephalotaxus, where cephalotaxinone is produced in root tips and distributed throughout the plant for subsequent elaboration to HHT.