Single-cell RNA sequencing facilitates the elucidation of the complete biosynthesis of the antidepressant hyperforin in St. John’s wort

Hyperforin is the compound responsible for the effectiveness of St. John's wort (Hypericum perforatum) as an antidepressant, but its complete biosynthesis 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. Here we sequenced the 1.54 Gb tetraploid H. perforatum genome assembled into 32 chromosomes with scaffold N50 value of 42.44 Mb. By single-cell RNA-seq, we identified a type of cells, Hyper cells, wherein hyperforin biosynthesis de novo takes place in both leaves and flowers. Through pathway reconstitution in yeast and tobacco, we identify and characterize four transmembrane prenyltransferases (HpPT1-4) to resolve hyperforin biosynthesis, which localize to the plastid envelope. 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 hyperforin is biosynthesized that enables synthetic-biology reconstitution of the complete pathway. These results deepen our comprehension of specialized metabolism at the cellular level, and we anticipate acceleration of pathway elucidation in plant metabolism.