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高锰素的完全生物合成


贯叶金丝桃素是导致圣约翰草 （） 作为抗抑郁药有效性的化合物，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高孔素生物合成中的缺失步骤。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，我们确定了一种细胞，“超细胞”，其中贯叶素的生物合成发生在叶子和花中。通过在酵母和烟草中构建途径，我们鉴定并表征了四种位于质体包膜的跨膜异戊二烯基转移酶 （HpPT1-4） 并完成了贯穿金丝桃素生物合成途径。高锰菊素多环支架是由涉及不规则异戊二烯偶联和串联环化的反应级联形成的。我们的研究结果揭示了贯叶金丝桃素的生物合成方式和位置，从而能够合成生物学重建完整途径。因此，本研究不仅加深了我们对细胞水平特殊代谢的理解，而且为阐明植物中其他特殊代谢物的生物合成途径提供了战略指导。