The Lamiaceae family is renowned for its terpenoid-based medicinal components, but Leonurus, which has traditional medicinal uses, stands out for its alkaloid-rich composition. Leonurine, the principal active compound found in Leonurus, has demonstrated promising effects in reducing blood lipids and treating strokes. However, the biosynthetic pathway of leonurine remains largely unexplored. Here, we present the chromosome-level genome sequence assemblies of Leonurus japonicus, known for its high leonurine production, and Leonurus sibiricus, characterized by very limited leonurine production. By integrating genomics, RNA sequencing, metabolomics, and enzyme activity assay data, we constructed the leonurine biosynthesis pathway and identified the arginine decarboxylase (ADC), uridine diphosphate glucosyltransferase (UGT), and serine carboxypeptidase-like (SCPL) acyltransferase enzymes that catalyze key reactions in this pathway. Further analyses revealed that the UGT–SCPL gene cluster evolved by gene duplication in the ancestor of Leonurus and neofunctionalization of SCPL in L. japonicus, which contributed to the accumulation of leonurine specifically in L. japonicus. Collectively, our comprehensive study illuminates leonurine biosynthesis and its evolution in Leonurus.

唇形科以其萜类药用成分而闻名，而具有传统药用用途的益母草则因其富含生物碱的成分而脱颖而出。益母草碱是益母草中发现的主要活性化合物，在降低血脂和治疗中风方面已显示出良好的效果。然而，益母草碱的生物合成途径在很大程度上仍未被探索。在这里，我们展示了益母草 (Leonurus japonicus)和益母草 (Leonurus sibiricus)的染色体水平基因组序列组装，前者以高益母草碱产量而闻名，后者以益母草碱产量非常有限为特征。通过整合基因组学、RNA测序、代谢组学和酶活性测定数据，我们构建了益母草碱生物合成途径，并鉴定了催化关键酶的精氨酸脱羧酶（ADC）、尿苷二磷酸葡萄糖基转移酶（UGT）和丝氨酸羧肽酶（SCPL）酰基转移酶该途径中的反应。进一步的分析表明，UGT-SCPL基因簇是通过益母草祖先中的基因复制和益母草中SCPL的新功能化而进化的。japonicus，这有助于益母草碱的积累，特别是在L . 日本刺参。总的来说，我们的综合研究阐明了益母草中益母草碱的生物合成及其进化。