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

• DOI: 10.1021/acs.jafc.5c00610
• Publication Date: 2025-04-19T13:04:31Z
• Authors: Yunhao He; Yu Hua; Xinyi Chen; Xin Zhang; Lei Lei; Yongjun Lin; Gaobing Wu
• Keywords: Halomonas

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 from Halomonas sp. through directed evolution. After two screening rounds, best variant (HoEPSPS-4M) with four mutations (G112A, M155 V, I285F, D326E) exhibited a 22-fold increase in glyphosate resistance (kcat/Km × Ki) and 2.3-fold improvement catalytic efficiency (kcat/Km). Isothermal titration calorimetry (ITC) showed 1.7-fold reduction binding affinity. Molecular docking dynamics simulations revealed that G112A mutation at active site decreased binding, while distal modulated enzymatic activity altering protein functional networks. Overexpression of HoEPSPS-4 M Nicotiana benthamiana conferred high demonstrating its potential developing herbicide-resistant crops. This work expands genetic resources provides insights optimizing engineering.