Functional identification of key enzymes in calycosin biosynthesis in Astragalus membranaceus and establishment of heterologous expression system

Astragalus membranaceus , a widely used medicinal and edible herb, contains major active isoflavonoid components such as formononetin, calycosin, and their glycosides, which exhibit a broad spectrum of pharmacological effects including anti-inflammatory, immunomodulatory, and cardioprotective activities. In this study, we identified three key enzymes: isoflavone synthase (AmIFS), O -methyltransferase (AmOMT2), and isoflavone 3’-hydroxylase (AmI3’H), elucidating the critical steps in calycosin biosynthesis and revealing a novel synthetic pathway. The research showed that AmIFS efficiently catalyzed the conversion of liquiritigenin and naringenin into the isoflavone backbone. The multifunctional O -methyltransferase AmOMT2, preferentially catalyzed the 4’- O -methylation of liquiritigenin, and the resulting product was subsequently converted by AmIFS into 2,7-dihydroxy-4’-methoxyisoflavanone, thereby broadening substrate versatility of AmIFS and revealing a new biosynthetic route. Meanwhile, AmI3’H completed the biosynthesis by catalyzing the final 3’-hydroxylation of formononetin to calycosin. Furthermore, this study successfully achieved the heterologous synthesis of calycosin precursors in yeast and tobacco, confirming the functional expression of these enzymes in plant and microbial hosts. Tissue-specific expression analysis showed differential expression of AmIFS and AmI3’H in roots, stems, and leaves of A. membranaceus , which was positively correlated with formononetin accumulation. These findings not only completed the calycosin biosynthetic pathway, but also laid a theoretical foundation and provided technical support for the scalable production of bioactive isoflavones via synthetic biology. • Identified key enzymes AmIFS, AmOMT2 and AmI3’H for calycosin biosynthesis in Astragalus membranaceus , clarifying core synthetic steps. • Discovered a novel “methylation-prior, aryl ring migration-subsequent” pathway, breaking the traditional “aryl migration-first, methylation-second” approach. • Achieved heterologous synthesis of key calycosin precursors in yeast and tobacco, laying the foundation for large-scale production. • Revealed AmOMT2’s dual-specific catalytic property and AmI3’H’s substrate specificity, expanding understanding of flavonoid-modifying enzymes.