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 III is superior to that of furostanol-type timosaponin II, 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. • Phytosterols and timosaponins biosynthesis pathways were investigated. • SMT1 and SSR2 control the diverging steps of phytosterol and cholesterol biosynthesis. • Conversion mechanisms from furostanol-type timosaponins to spirostanol-type timosaponins are controlled by AaF26G1. • The antifungal efficacy of timosaponin III is superior to that of timosaponin BII. 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 candidate OSC genes, AaOSCR12 was identified as the gene encoding cycloartenol synthase, which is responsible for the skeleton cyclization in timosaponin biosynthesis. Six residues (257H, 369N, 448T, 507V, 558P, 616Y) were identified as the critical catalytic active sites of CAS (AaOSCR12). Sterol methyltransferase (AaSMT1) and sterol side-chain reductase (AaSSR2) were found to be the subsequent enzymes and the branching points leading to phytosterol and cholesterol biosynthesis, respectively. Two oxide reductase genes, AaCYP90B27 and AaCYP90B2 , were responsible for post-modification of cholesterol, serving as a precursor of timosaponins. A key 26-O- β -glucosidase (AaF26G1) was identified as facilitating the conversion of furostanol-type timosaponins into spirostanol-type timosaponins. Antifungal assays revealed that spirostanol-type timosaponin III exhibits superior antifungal activity compared to furostanol-type timosaponin II, potentially linked to plant defense mechanisms involving AaF26G1. This study utilized a multi-chassis cross-identification strategy, revealing key enzymes in the timosaponin biosynthetic pathway and offering novel insights into plant defense mechanisms against microbial pathogens.