Engineering of flavonoid 3′-O-methyltransferase for improved biosynthesis of chrysoeriol in Escherichia coli

Abstract: O-Methylation catalyzed by O-methyltransferases (OMTs) is an important modification of flavonoids for improving the transport efficiency across membranes and metabolic stability in mammalian cells. Chrysoeriol, also known as 3′-O-methylated luteolin, is a methylated flavonoid compound with health-promoting activities. The generation of chrysoeriol from luteolin can be catalyzed by a rice-derived 3′-OMT named ROMT-9, which has a high regiospecificity and activity toward flavonoids in vitro. Herein, we explored the potential of ROMT-9 for in vivo biosynthesis of chrysoeriol in Escherichia coli and adopted semi-rational enzyme engineering guided by homology modeling and molecular docking to improve the bio-production. Two positive variants including L34Q and W284A were obtained which promoted chrysoeriol formation to more than 85 mg/L from 200 mg/L of luteolin in 24 h compared with a titer of 55 mg/L for the strain expressing the native enzyme. Further biochemical analysis confirmed that such improvement in production stemmed from a higher enzyme expression level for the L34Q variant and higher efficiency in substrate binding and catalysis for the W284A variant. This study provides some insights into the engineering of other flavonoid OMTs and will facilitate high-level biosynthesis of methylated flavonoids in engineered microorganisms. Key points: • Biosynthesis of chrysoeriol from luteolin in E. coli using ROMT-9 • Engineering of ROMT-9 for better bio-production • ROMT-9 variants promote production via better expression or better catalysis.y.