Structural Elucidation and Engineering of the ( S )‐scoulerine 2‐ O ‐Methyltransferase Enabling Regioselective Epiberberine Biosynthesis in Coptis chinensis

Protoberberine alkaloids are a characteristic group of natural products in Coptis plants known for their notable pharmacological activities. However, the structural similarity and the substrate promiscuity of their biosynthetic enzymes have left the precise synthetic pathways remain unclarified, posing challenges to regulate product formation. In this study, we identified CcOMT8, a key enzyme responsible for C2-methoxylation in the biosynthesis of epiberberine in C. chinensis, through methyl jasmonate elicitation analysis and comparative genomics-based microsynteny analysis. Functional characterisation demonstrated that CcOMT8 specifically catalyses 2-O-methylation of (S)-scoulerine, as verified by heterologous expression in both microbial and plant systems. Its lack of activity toward (S)-cheilanthifoline further confirmed the specific route for epiberberine biosynthesis. Structural investigations of CcOMT8 and its complexes revealed key aspects of substrate recognition and a catalytic mechanism mediated by the His253-Asp254-Glu312 triad. Comparative structural analysis with 9-O-methyltransferases indicated that hydrophilic residues and reduced steric hindrance in the substrate binding pocket govern the regioselectivity of CcOMT8. Using focused rational iterative site-specific mutagenesis (FRISM), we developed an optimised mutant, S109L/C250A/L300A, with 4.88-fold enhanced catalytic efficiency. This study elucidates the biosynthetic pathway of epiberberine in Coptis, clarifies the molecular basis of enzyme-directed metabolic flux, and provides efficient biocatalysts for the synthetic biosynthesis of protoberberine alkaloids.