Astragalus membranaceus has been used in traditional Chinese medicine for over 2,000 years. Its major active triterpenoid saponins, astragalosides, have attracted great attention due to their multiple health benefits and applications in medicine. Despite this, the biosynthetic machinery for astragalosides remains enigmatic. Here a chromosome-level genome assembly of A. membranaceus was generated. The identification of two tailoring enzymes required for astragaloside biosynthesis enabled the discovery of a triterpenoid biosynthetic gene cluster, leading to elucidation of the complete astragaloside biosynthetic pathway. This pathway is characterized by a sequence of selective hydroxylation, epoxidation and glycosylation reactions, which are mediated by three cytochrome P450s, one 2-oxoglutarate-dependent dioxygenase and two glycosyltransferases. Reconstitution of this biosynthetic machinery in Nicotiana benthamiana allowed for heterologous production of astragaloside IV. These findings build a solid foundation for addressing the sourcing issues associated with astragalosides and broaden our understanding of the diversity of terpene biosynthetic gene clusters.
© 2024. The Author(s), under exclusive licence to Springer Nature Limited.