A minimal four-enzyme suite enables biosynthesis of the C₂₀ diterpenoid alkaloid atisinium

Diterpenoid alkaloids (DAs) are pharmaceutically important metabolites and mainly occur in the Ranunculaceae. Despite their structural complexity, all DAs originate from C₂₀ diterpenoid scaffolds, making the elucidation of C₂₀-DA biosynthesis pivotal for understanding and engineering this entire alkaloid class. However, the molecular basis for DA core assembly has remained unresolved despite decades of research. Here, we define a minimal four-enzyme suite comprising three cytochrome P450 monooxygenases (CYP701A216, CYP71FH3, and CYP729G3) and one neo-functionalized short-chain dehydrogenase/reductase (AgDAS) that converts ent-atiserene into the C₂₀ atisine-type alkaloid atisinium in Aconitum gymnandrum via an enzymatic-spontaneous-enzymatic process. Specifically, the multifunctional CYP701A216 and CYP71FH3 act in relay to generate the key intermediate ent-atiseren-19,20-dial, which undergoes spontaneous condensation with ethanolamine to form a hemiaminal. AgDAS then catalyzes atypical imine reduction to generate 15-deoxyatisinium bearing the characteristic atisine skeleton, and CYP729G3 mediates C15 hydroxylation to finalize atisinium biosynthesis. The physiological relevance of these enzymes was further investigated using virus-induced gene silencing in A. gymnandrum, demonstrating the critical role of AgDAS in this biosynthetic pathway. This compact enzyme module is highly conserved across Aconitum and Delphinium within the Ranunculaceae, pointing to an evolutionarily preserved core biosynthetic pathway. Collectively, our work establishes the complete molecular logic for DA scaffold assembly and its heterologous reconstitution in tobacco and yeast, opening routes to synthetic biology of medicinally relevant DAs.