Abstract

How new enzymatic activities become integrated with existing metabolic networks to generate lineage-restricted plant chemistry remains a central question in metabolic evolution, exemplified by gingerol biosynthesis in Zingiberaceae, whose complete enzymatic basis has remained elusive. Here, we resolve this problem by elucidating the evolutionary origins and the full biosynthetic pathway of gingerol through single-cell transcriptomics, functional screening, structural biology, comparative genomics and pathway reconstruction. A coordinated metabolic program operates in ginger oil cells, thereby guiding the identification of diketide-CoA synthases, gingerol synthases (GS) and gingerol reductases that together constitute the complete pathway. GS remodels the conserved type III polyketide synthase scaffold to organize a fatty acyl-CoA and a phenylpropanoid-derived intermediate for gingerol formation. Furthermore, GS arose through duplication and limited active-site remodeling of a curcumin synthase-like enzyme, accompanied by coordinated recruitment of precursor-supply and reduction pathways. Translating these principles into yeast achieves the first de novo microbial production of 6-gingerol from glucose at 12.4 mg/L. These findings reveal the evolutionary logic by which plants recruit and coordinate novel enzymatic activities to establish lineage-restricted metabolic program, providing a framework for understanding and engineering specialized metabolism across the plant kingdom.