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 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 cellularlevel but also provides strategic guidance for elucidation of the biosynthetic pathways of other specializied metabolites in plants.

The biosynthetic pathway of the hallucinogen mescaline and its heterologous reconstruction

Mescaline,among the earliest identified natural hallucinogens,holds great potential in psychotherapy treatment.Nonetheless,despite the existence of a postulated biosynthetic pathway for more than half a century,the specific enzymes involved in this process are yet to be identified.In this study,we investigated the cactus Lophophora williamsii(Peyote),the largest known natural producer of the phenethylamine mescaline.We employed a multi-faceted approach,combining de novo whole-genome and transcriptome sequencing with comprehensive chemical profiling,enzymatic assays,molecular modeling,and pathway engineering for pathway elucidation.We identified four groups of enzymes responsible for the six catalytic steps in the mescaline biosynthetic pathway,and an N-methyltransferase enzyme that N-methylates all phenethylamine intermediates,likely modulating mescaline levels in Peyote.Finally,we reconstructed the mescaline biosynthetic pathway in both Nicotiana benthamiana plants and yeast cells,providing novel insights into several challenges hindering complete heterologous mescaline production.Taken together,our study opens up avenues for exploration of sustainable production approaches and responsible utilization of mescaline,safeguarding this valuable natural resource for future generations.