Site-selective dimethylation of flavonoids using fusion flavonoid O-methyltransferases
2025
Kyungha Lee | Seong Hee Bhoo | Sang-Won Lee | Man-Ho Cho
Abstract Flavonoids are often decorated with methyl groups, which are catalyzed by flavonoid O-methyltransferases (FOMTs). Most FOMTs methylate flavonoids in a regiospecific manner. Because of the regiospecific nature of FOMTs, the synthesis of polymethoxyflavonoids is accomplished by multiple O-methylation steps. The multistep synthesis of dimethoxyflavonoids can be efficiently performed by a one-pot procedure using a multienzyme biocatalyst. For the one-pot production of dimethoxyflavonoids, fusion FOMTs were generated by the combination of two different regiospecific FOMTs. RdOMT10 (flavonoid 3-OMT), OsNOMT (flavonoid 7-OMT), and ObFOMT5 (flavonoid 4'-OMT) were used in the FOMT fusion. The fusion FOMTs (OsNOMT/ObFOMT5, OsNOMT/RdOMT10, and ObFOMT5/RdOMT10) were heterologously expressed in Escherichia coli. Activity assays of the recombinant fusion FOMTs demonstrated that OsNOMT/ObFOMT5, OsNOMT/RdOMT10, and ObFOMT5/RdOMT10 catalyze 7/4'-O-methylations, 7/3-O-methylations, and 4'/3-O-methylations of flavonoids, respectively. OsNOMT/ObFOMT5 and OsNOMT/RdOMT10 showed strong dimethylation activity towards diverse flavonoids and were therefore used in the site-selective bioconversion of flavonoids into dimethoxyflavonoids. The E. coli cells bearing OsNOMT/ObFOMT5 successfully converted flavonoids into 7,4'-dimethoxyflavonoids. The engineered E. coli expressing OsNOMT/RdOMT10 converted flavonoids into 3,7-dimethoxyflavonoids. This result indicates that the fusion FOMTs are useful multienzyme biocatalysts for the site-selective production of dimethoxyflavonoids by one-pot bioconversion.
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