A class II 5-enolpyruvylshikimate-3-phosphate synthase from Pseudomonas P818 confers robust glyphosate tolerance in transgenic plants

Glyphosate is a widely used herbicide that targets 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), blocking the shikimate pathway and leading to plant death. The discovery of novel EPSPS genes is key to engineering glyphosate tolerance in crops. In this study, we isolated the glyphosate-tolerant bacterial strain Pseudomonas P818 from glyphosate-contaminated soil and cloned its class II EPSPS gene ( 818-EPSPS ). Sequence and phylogenetic analyses revealed typical motifs of class II EPSPS. Kinetic characterization revealed a high K i/ K m ratio (10.4), indicating that 818-EPSPS has low affinity for glyphosate (high K i value) while retaining high catalytic efficiency (low K m value). The heterologous expression of 818-EPSPS restored growth in E. coli strain ER2799 under glyphosate stress, confirming its functional resistance. Transgenic Arabidopsis thaliana and tobacco ( Nicotiana tabacum ) plants heterologously expressing 818-EPSPS exhibited strong tolerance to glyphosate, maintaining growth at concentrations that were lethal to the wild-type controls. We introduced codon-optimized 818-EPSPS into maize ( Zea mays ), generating stable transgenic plants. Transgenic maize line EP03, carrying a single-copy insertion, showed robust tolerance to up to four times the recommended dosage of glyphosate in field trials. Genomic sequencing revealed that the T-DNA in EP03 was inserted into chromosome 5 without disrupting any host genes. Our findings establish 818-EPSPS as a promising candidate for engineering glyphosate-tolerant crops and provide a new genetic resource for maize improvement and biosafety applications.