Elucidation of the biosynthetic pathways of timosaponins reveals the antifungal mechanisms in Anemarrhena asphodeloides

2,3-oxidosqualene cyclase AaOSCR12 is the only identified CAS in Anemarrhena asphodeloides, which directly determines the synthesis and accumulation of subsequent timosaponins. Then the divergence step catalyzed by AaSMT1 and AaSSR2, separately metabolizing into phytosterol and cholesterol pathway. Furthermore, the antifungal efficacy of spirostanol-type timosaponin AⅢ is superior to that of furostanol-type timosaponin BⅡ, potentially associated with the plant defense mechanisms involving the steroid sapogenin 26-O-β-glucosidase AaF26G1 by unraveled the logic of the conversion from furostanol-type timosaponins to spirostanol-type timosaponins. [Display omitted] •This work elucidated the biosynthesis pathways of phytosterols and timosaponins in Anemarrhena asphodeloides. Specifically, the catalytic mechanism of the cycloartenol synthase AaOSCR12 was extensively investigated. Six amino acid residues of AaOSCR12 (257H-369N-448T-507V-558P-616Y) were identified as critical catalytic active sites.•This work elucidated the divergence step catalyzed by sterol methyltransferase and side-chain reductase from A. asphodeloides, which separately metabolize into the phytosterol and cholesterol pathways.•This work elucidated the conversion mechanism from furostanol-type timosaponins to spirostanol-type timosaponins.•This work demonstrated that the antifungal efficacy of spirostanol-type timosaponin AⅢ is superior to that of furostanol-type timosaponin BⅡ, potentially due to plant defense mechanisms involving furostanol-type steroid sapogenin 26-O-β-glucosidase from A. asphodeloides. Timosaponins, as steroidal saponins, are the primary active constituents and quality biomarkers in Anemarrhena asphodeloides Bunge. Despite their significance, the biosynthetic pathways of timosaponins have not been thoroughly investigated. This study aims to delineate the biosynthetic pathway of timosaponins in A. asphodeloides, elucidate the catalytic mechanisms of the key cycloartenol synthase (CAS), and investigate the antifungal properties of timosaponins. Genes were cloned from A. asphodeloides and heterologous expressed in yeast, tobacco or bacillus coli. Site-directed mutagenesis and molecular docking were used to elucidate the catalytic mechanism of CAS. Antifungal assays were conducted to evaluate the antifungal activities of timosaponins. In this study, we elucidated the biochemical functions of seven genes involved in timosaponins biosynthesis in A. asphodeloides. Among three cand