Abstract：AM79 aroA (WO/2009/059485), an novel 5-enolpyruvy-shikimate-3-phosphate synthase (EPSPS) gene, is isolated from glyphosate-polluted soil using metagenomics method and showed high glyphosate resistance. However, the function mechanism of the high glyphosate resistance of AM79 aroA remains unknown. Site-directed mutagenesis to uncover the function of some specific amino acids in AM79 EPSPS was performed. Phylogenetic tree analysis showed that AM79 EPSPS was a typeⅠEPSPS. Sequence alignment indicated that the 107th Alanine (Ala), the 114th Phenylalanine (Ala), the 355th Alanine and the 356th Histidine (His) were symbols to distinguish the AM79 EPSPS from other EPSPS. To explain the function of these specific amino acids in AM79 EPSPS, point mutations were carried out via overlap extension PCR. AM79 aroA and the mutant gene were transformed into aroA defective strain ER2799, then the glyphosate resistance of the transformed strains were identified. The results showed that mutants of Phe114, Ala355 and His356, respectively, led to the decrease of glyphosate resistance, indicating that these amino acids were essential for the enzyme activity and glyphosate resistance of AM79 EPSPS. Homology modeling analysis showed that the mutation of these amino acids changed the structures of the mutated AM79 EPSPS and this might be the reason for the decreased glyphosate resistance. We hypothesized that the highlighted amino acids in AM79 EPSPS might have similar function in other typeⅠEPSPS and reverse mutations on these amino acids of Pseudomonas fluorescens G2 EPSPS and Arabidopsis thaliana EPSPS (AtEPSPS) were performed. However, the mutations of these key amino acids didn't enhance the glyphosate resistance of G2 EPSPS and AtEPSPS. This study can be useful for enhancing the glyphosate resistance of AM79 EPSPS by directed evolution process.
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