Entry Steps in the Biosynthetic Pathway to DiterpenoidAlkaloids

Both the terpenoid and alkaloid classes of specialized metabolites have received considerable attention for their wide range of practical applications, however, few examples have been identified of these classes intersecting. The diterpenoid alkaloids are one such example of nitrogen-containing terpenoids which are found throughout many independent plant lineages, but primarily within the Aconitum (Wolfs-Bane) and Delphinium (Larkspur) genera. While there is considerable interest in these compounds for their wide range of bioactivities, their structural complexity often precludes their production through chemical synthesis, and little progress has been made towards elucidation of their biosynthetic pathways. Here, we employ a comparative transcriptomics approach to identify six enzymatic steps in the biosynthesis of atisinium, conserved across both Delphinium grandiflorum and Aconitum plicatum. Key to this pathway is a reductase which selectively incorporates ethanolamine over ethylamine into the diterpenoid scaffold. While the majority of diterpenoid alkaloids contain an ethylamine moiety, we demonstrate through isotope labeling in Aconitum callus cultures and a computational metabolomics approach that ethanolamine is, unintuitively, the preferred source of nitrogen for these metabolites. Identification of these enzymes and production of a key intermediate in a heterologous host paves the way for biosynthetic production of this group of metabolites with promise for medicinal applications.