Engineering a 4-O-methyltransferase from Myxococcus xanthus for enhanced production of 4-O-methylgallic acid in E....

The biosynthesis of 4-O-methylgallic acid (4-O-MGA) from gallic acid (GA) is catalyzed by 4-O-methyltransferases (OMTs). However, existing OMTs exhibit low catalytic efficiency for this conversion. In this study, we identified MxOMT from Myxococcus xanthus as an efficient catalyst for the para-O-methylation of GA. Using a protein engineering approach, we enhanced the catalytic efficiency of MxOMT by targeting key residues in the flexible loop and 'gatekeeper' regions. High-throughput screening identified mutants with improved activity, including L38E, R39G, T40V, and S173K, which significantly increased the yield of 4-O-MGA. The best-performing quadruple mutant L38E/R39G/T40R/S173K produced 288.33 mg/L of 4-O-MGA, representing a 282.7 % increase compared to the wild-type MxOMT. Kinetic analysis revealed that the quadruple mutant exhibited a 2.28-fold higher catalytic efficiency (k_cat/K_m) than the wild-type enzyme. Computational analysis and simulations suggested that the enhanced activity resulted from improved substrate binding and active site compaction. Furthermore, we constructed a de novo biosynthetic pathway for 4-O-MGA by incorporating the optimized MxOMT enzyme into E. coli. Through systematic engineering strategies, we achieved a final titer of 143.93 mg/L of 4-O-MGA. This work demonstrates the successful engineering of MxOMT for efficient 4-O-MGA production and provides a foundation for its industrial-scale biosynthesis.