Engineering of Flavonoid 3′-O-Methyltransferase for Improved Biomodification of Fisetin in <i>Escherichia coli</i>

Fisetin is mainly extracted from <i>Rhus succedanea</i> L., but it is also found in a variety of foods, vegetables, and herbs, and is commonly used in dietary supplements for its health benefits. However, its limited stability, low oral bioavailability, and poor absorption hinder its developmental applications. Methylation modification has emerged as an effective strategy to enhance the solubility, stability, and lipid solubility of fisetin. In this study, we identified a novel 3'-O-methyltransferase, PeCCoAOMT, characterized its enzymatic properties <i>in vitro</i>, and investigated its potential for producing 3'-O-methylated fisetin in <i>Escherichia coli</i>. Through strain screening, selection of protein tags and plasmid vectors, and optimization of culture conditions, the strain BTP was fermented in Lysogeny broth medium containing 5 g/L glycerol for 48 h at 37 °C. Finally, the strain BTP produced 530.44 mg/L of 3'-O-methylated fisetin, with a molar conversion rate of 63.02%, representing a 6.63-fold increase in titer compared to the initial strain, which is the highest level reported to date. This study provides valuable insights into the engineering of flavonoid O-methyltransferases and lays the foundation for the high-level biosynthesis of engineered microbial methylated flavonoids.