A thermostable 5-enolpyruvylshikimate-3-phosphate synthase from Thermotoga maritima enhances glyphosate tolerance in Escherichia coli and transgenic Arabidopsis

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) overexpression, attempting to provide excess EPSPS to combine with glyphosate, is one way to improve glyphosate resistance of plants. The EPSPS in extremophiles which is selected by nature to withstand the evolutionary pressure may possess some potential-specific biological functions. In this study, we reported the cloning, expression and enzymatic characterization of a novel Class II EPSPS AroAT. maritima from Thermotoga maritima MSB8. The enzyme showed low sequence identities with other EPSPSs, and was one of the most thermostable EPSPSs so far, which showed the optimum enzyme activity at 80 °C. The enzyme maintains the activity below 50 °C and in a wide range of pH 4.0–10, which indicated its stability under rough environment, especially in tropical regions and alkaline soil. Excellent Ki/Km value of AroAT. maritima suggested that the enzyme showed powerful competitive binding capacity of PEP over glyphosate and high glyphosate tolerance. Furthermore, aroAT. maritima gene was transformed into Arabidopsis thaliana. The transgenic lines were resistant to 15 mM glyphosate, which proved the application value in the cultivation of glyphosate-tolerant plants.