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

ABSTRACT Plants have evolved to produce diverse molecules that inhibit protein translation. A lead example is homoharringtonine (HHT), both a key tool for ribosomal profiling and an FDA-approved treatment for chronic myeloid leukemia. HHT is commercially produced through semi-synthesis by esterifying the alkaloid core cephalotaxine (CET) extracted from endangered Cephalotaxus species. Despite its medicinal significance, a biosynthetic pathway to CET and HHT has not been described. Here, we use paired untargeted metabolomics (stable-isotope labeled precursor feeding) and transcriptomics to elucidate a near-complete biosynthesis to CET without prior knowledge of intermediates and biosynthetic genes. We show that while CET alkaloid core is actively biosynthesized only in growing root tips, both CET and HHT accumulate throughout the plant. We discovered and characterized seven CET pathway intermediates and six novel biosynthetic enzymes that, together, can be used to produce cephalotaxinone, the likely direct precursor of CET. Included are non-canonical cytochrome P450s, an atypical short-chain dehydrogenase, and a 2-oxogluatrate-dependent dioxygenase that together result in carbon excision and the formation of the characteristic pentacyclic backbone of HHT alkaloids. Our data support a model where cephalotaxinone is the last pathway intermediate produced specifically in the root tips, and its distribution throughout the plant is likely the starting point for subsequent elaboration to HHT. This study not only establishes a metabolic route to the core scaffold of HHT—enabling future sustainable, large- scale production of this valuable drug—but also suggests how Cephalotaxus species employ a whole plant coordination to regulate the biosynthesis of eukaryotic ribosomal toxins.