# A Highly Glyphosate-Resistant EPSPS Mutant from Laboratory Evolution
## 4 Discussion  
In recent years, various methods have been used to obtain EPSPS mutants with increased glyphosate resistance. ALE can provide a large number of adaptive mutations and accumulate adaptive changes, which may be a good choice for acquiring beneficial mutations [29]. [PERSON] [35] et al. obtained a mesophilic subtilisin-like protease mutant adapted to low temperature using a strategy of laboratory evolution. [PERSON] [36] et al. used laboratory evolution to optimize the specificity of a modified amyloid-\(\beta\) protein and generated a sortase variant, SrtA\(\beta\), resulting in a >1400-fold change in substrate preference. In this study, the GR79(Y40I)-EPSPS mutant was obtained using the ALE strategy. The results showed that the mutant enzyme can endow the _E. coli_ strain to grow in minimal medium containing 300 mM glyphosate, and the glyphosate resistance was nearly 1.5-fold higher than that of the wild-type enzyme. Therefore, we discussed the relationship between glyphosate resistance and the mutation site of mutant GR79(Y40I)-EPSPS. Studies have shown that when EPSPS binds to ligands, the two domains are closed, resulting in the formation of an active site in the cracks between domains [37]. Modification of amino acid residues in or around the active region will significantly change the activity and glyphosate resistance of EPSPS. For example, the alterations of Gly-96-Ala in _E. coli_ and Pro-101-Ser in _S. typhimurium_ have been shown to confer glyphosate resistance [38; 39]. The Pro-106 mutant in _Zea mays_ is not directly involved in catalysis or glyphosate binding, but changes the binding of glyphosate by changing the spatial force of adjacent amino acids [10]. In our study, the 40 th amino acid Y of GR79- EPSPS was immediately adjacent to the conserved motif region 1 (GDKX) of Class II EPSPS. Studies have shown that although EPSPSs are divided into several categories, the active sites are highly conserved and rarely change in some organisms [40]. Class I and II EPSPSs active sites have some similarities [41]. Therefore, the substitution of this site will indirectly affect the resistance of EPSPS glyphosate. Figure 3: Ribbon diagram of the crystal structure of EPSPS and the active site in glyphosate-liganded EPSPS. (**A**) Crystal structure of the open conformation of GR79-EPSPS. Tyr-40 is shown in purple. (**B**) Crystal structure of the open conformation of GR79(Y40I)-EPSPS. Ile-40 is shown in purple. (**C**) The structure of the GR79-EPSPS-GLP complex. (**D**) The structure of the GR79(Y40I)-EPSPS-GLP complex. The yellow dotted line represents the hydrogen bond. The multiple sequence alignment shows that the 40 th amino acids are all nonpolar, and the Y of GR79-EPSPS is specific and contains a carbon ring. However, the amino acids of the remaining Class I EPSPSs are L, I, or V, and all have no ring structure. This result is very similar to the phenomenon that mutation of the Pro-106 site leads to an increase in glyphosate resistance. It has been reported that glyphosate resistance is improved after Pro-106 is mutated to S, A, T and L in _barnyard grass_, _Eleusine India_ and _Lolium rigidum Gaud_[5; 17; 18]. Because the Pro (P) has a ring structure, the ring prevents the \(\Phi\) peptide bond (C\(\alpha\)-N). After being mutated, the spatial direction of Arg-105 and Asp-99 may change, and Alycyl-101 can form hydrogen bonds with glyphosate [42]. This series of structural changes reduced the binding cavity of glyphosate on EPSPS and increased glyphosate resistance [42]. Our docking and molecular dynamics analyses indicate that Tyr-40 of GR79 is immediately adjacent to Lys-38. Lys-38 has a variety of interactions with glyphosate. When Y40I substitution occurs, the local space of the 38 th amino acid is released, which makes Lys-38 undergo more spatial changes.