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

Methylation plays a crucial role in drug design and optimization. While numerous methyltransferases have been characterized from plants, <i>C</i>-methyltransferases, particularly those targeting phenolic skeletons, are rare. In this study, we identified a novel di-<i>C</i>-methyltransferase RdCMT from the medicinal plant <i>Rhododendron dauricum</i>. RdCMT catalyzes a sequential two-step 3'-<i>C</i>/5'-<i>C</i>-methylation of naringenin chalcone, leading to the biosynthesis of farrerol. RdCMT exhibited a strict substrate specificity for chalcones. Through combinatorial catalysis, a series of <i>C</i>-methylated flavonoids were synthesized. Moreover, farrerol was synthesized de novo in <i>Nicotiana benthamiana</i> and <i>Pichia pastoris</i> 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 <i>C</i>-methylated flavonoids but also offers insights into the catalytic mechanisms of <i>C</i>-methyltransferases.