Molecular and Structural Characterization of a Chalcone di-C-Methyltransferase RdCMT from Rhododendron dauricum and Its Application in De Novo Biosynthesis of Farrerol in Pichia pastoris

Methylation plays a crucial role in drug design and optimization. While numerous methyltransferases have been characterized from plants, C-methyltransferases, particularly those targeting phenolic skeletons, are rare. In this study, we identified a novel di-C-methyltransferase RdCMT from the medicinal plant Rhododendron dauricum. RdCMT catalyzes a sequential two-step 3'-C/5'-C-methylation of naringenin chalcone, leading to the biosynthesis of farrerol. RdCMT exhibited a strict substrate specificity for chalcones. Through combinatorial catalysis, a series of C-methylated flavonoids were synthesized. Moreover, farrerol was synthesized de novo in Nicotiana benthamiana and Pichia pastoris with yields of 0.4 mg/g (dry weight) and 149.0 mg/L, respectively. The structure of RdCMT was determined using cryo-electron microscopy (cryo-EM), revealing that residues R328 and G296 significantly influence the substrate specificity of RdCMT. This work not only introduces a potent biocatalyst for the preparation of C-methylated flavonoids but also offers insights into the catalytic mechanisms of C-methyltransferases.