Ferulic acid production in Escherichia coli by engineering caffeic acid O-methyltransferase

Methylation is a key step for the structure diversification of natural products, however, the low activity and substrate promiscuity of methyltransferase remain challenges for efficient biosynthesis. In this study, taking the synthesis of ferulic acid as a case, we attempted to improve the catalytic efficiency of caffeic acid O-methyltransferase (AtCOMT) by engineering its binding pocket and interactions with substrate. Through reshaping the substrate binding pocket and increasing the hydrogen bond network, L125R/I317E mutant with highly catalytic efficiency and substrate specificity was obtained. Compared with the wild-type AtCOMT, the k_cat/K_m increased by 5.79-fold. Molecular dynamics simulation verified that the increased activity and specificity of AtCOMT were derived from the tighter substrate binding pocket and stronger enzyme-substrate interactions produced by the designed hydrogen bonds and salt bridges. Eventually, 1.273 g/L ferulic acid was obtained by fed-batch fermentation, which was 21.3-fold compared with the WT. This work provided the foundation for efficient synthesis of ferulic acid by AtCOMT, and also illustrated that rational design of substrate binding pocket and hydrogen bond network would be an effective strategy for improving the activity and substrate specificity of enzyme.