One-Step Polymethylation of Flavonoids by Novel <i>Streptomyces</i> <i>O</i> -Methyltransferase

Polymethylated flavonoids exhibit significant biological activities. Methylation at multiple sites catalyzed by <i>O</i>-methyltransferases (OMTs) enables one-step synthesis of polymethylated flavonoids, thereby facilitating structural modification and druggability research of flavonoids. To identify OMTs with both substrate and regiochemical promiscuity, we performed large-scale phylogenetic and predicted structural analyses of 3873 caffeic acid OMT (COMT) sequences. Functional characterization yielded 16 active COMTs, including a microbial enzyme from <i>Streptomyces cinnamoneus</i> that exhibits broad substrate promiscuity toward flavonoids, hydroxyindoles, anthraquinones, and polyphenols. This biocatalyst methylates up to seven distinct sites on flavonoids with a unique hydrophobic active site favoring adjacent hydroxyl groups on either the A ring or B ring and the C3-hydroxyl group of flavonoids. It enables the one-step synthesis of 42 mono- and polymethylated bioactive derivatives from 22 substrates. This work provides a powerful tool for scaffold diversification in natural product synthesis and applications in food, agriculture, and drug discovery.