An Alternative Pathway for Formononetin Biosynthesis in Pueraria lobata

The O-methylation is an important tailing process in Pueraria lobata isoflavone metabolism, but the molecular mechanism governing it remains not elucidated. This manuscript describes the mining of key O-methyltransferases (OMTs) involved in the process. Using our previously constructed P lobata transcriptome, the OMT candidates were searched, extensively analyzed, and their functions were investigated by expression in yeast, Escherichla coil, or GIycine max hairy roots. Here, we report the identification of the key OMT gene responsible for formononetin production in P lobata (designated as PIOMT9). PIOMT9 primarily functions as an isoflavone-specific 4'-O-methyltransferase, although it shows high sequence identities with isoflavone 7-O-methyltransferases. Moreover, unlike the previously reported OMTs that catalyze the 4'-O-methylation for formononetin biosynthesis at the isoflavanone stage, PIOMT9 performs this modifying step at the isoflavone level, using daidzein rather than 2,7,4'-trihydroxy-isoflavanone as the substrate. Gene expression analyses and metabolite profiling supported its proposed roles in P. lobata. Using the system of transgenic G. max hairy roots, the role of PIOMT9 in the biosynthesis of formononetin was further demonstrated in vivo.