Hyperforin is the compound responsible for the effectiveness of St. John’s wort () 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 . genome assembled into 32 chromosomes with the scaffold N50 value of 42.44 Mb. By single-cell RNA sequencing, we identified a type of cell, “Hyper cells”, wherein hyperforin biosynthesis 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 hyperforin 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.

中文翻译：

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单细胞 RNA 测序有助于阐明圣约翰草中抗抑郁药金丝桃素的完整生物合成


金丝桃素是圣约翰草抗抑郁药功效的化合物，但其完整的生物合成途径仍然未知。基于大量 RNA 测序数据的共表达分析或基因组挖掘的基因发现未能发现金丝桃素生物合成中缺失的步骤。在这项研究中，我们对 1.54-Gb 四倍体进行了测序。基因组组装成32条染色体，支架N50值为42.44 Mb。通过单细胞RNA测序，我们鉴定出了一种细胞“Hyper细胞”，其中金丝桃素的生物合成发生在叶子和花中。通过酵母和烟草中的途径重建，我们鉴定并表征了四种跨膜异戊烯基转移酶（HpPT1-4），它们位于质体包膜并完成金丝桃素生物合成途径。金丝桃素多环支架是通过涉及不规则类异戊二烯偶联和串联环化的级联反应产生的。我们的研究结果揭示了金丝桃素的生物合成方式和地点，从而实现了完整途径的合成生物学重建。因此，这项研究不仅加深了我们对细胞水平上专门代谢的理解，而且为阐明植物中其他专门代谢物的生物合成途径提供了战略指导。