摘要Hyperforin is the compound responsible for the effectiveness of St.John's wort(Hypericum perforatum)as an antidepressant,but its complete biosynthetic pathway remains unknown.Gene discovery based on co-expression analysis of bulk RNA-sequencing data or genome mining failed to discover the missing steps in hyperforin biosynthesis.In this study,we sequenced the 1.54-Gb tetraploid H.perforatum genome assem-bled into 32 chromosomes with the scaffold N50 value of 42.44 Mb.By single-cell RNA sequencing,we iden-tified a type of cell,"Hyper cells",wherein hyperforin biosynthesis de novo takes place in both the leaves and flowers.Through pathway reconstitution in yeast and tobacco,we identified and characterized four transmembrane prenyltransferases(HpPT1-4)that are localized at the plastid envelope and complete the hyperforin biosynthetic pathway.The hyperforin polycyclic scaffold is created by a reaction cascade involving an irregular isoprenoid coupling and a tandem cyclization.Our findings reveal how and where hy-perforin is biosynthesized,enabling synthetic-biology reconstitution of the complete pathway.Thus,this study not only deepens our comprehension of specialized metabolism at the cellular level but also provides strategic guidance for elucidation of the biosynthetic pathways of other specializied metabolites in plants.