Engineering 5-Enolpyruvylshikimate-3-phosphate Synthases from <i>Halomonas</i> sp. for Augmenting Glyphosate-Resistance without Activity Penalty

Glyphosate nonselectively inhibits most herbaceous plants by targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Introducing glyphosate-resistant EPSPS genes into crops imparts herbicide resistance, essential for molecular breeding. In this study, we enhanced the EPSPS from <i>Halomonas</i> sp. through directed evolution. After two screening rounds, the best variant (<i>Ho</i>EPSPS-4M) with four mutations (G112A, M155 V, I285F, D326E) exhibited a 22-fold increase in glyphosate resistance (<i>k</i>_cat/<i>K</i>_m × <i>K</i>_<i>i</i>) and a 2.3-fold improvement in catalytic efficiency (<i>k</i>_cat/<i>K</i>_m). Isothermal titration calorimetry (ITC) showed a 1.7-fold reduction in glyphosate binding affinity. Molecular docking and dynamics simulations revealed that the G112A mutation at the active site decreased glyphosate binding, while distal mutations modulated enzymatic activity by altering protein dynamics and functional networks. Overexpression of <i>Ho</i>EPSPS-4 M in <i>Nicotiana benthamiana</i> conferred high glyphosate resistance, demonstrating its potential for developing herbicide-resistant crops. This work expands genetic resources for glyphosate resistance and provides insights into optimizing resistance and enzymatic efficiency by protein engineering.