A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS proteins were identified by the ability to rescue growth of aroA-deleted mutant ER2799 on M9 minimal media.12 unique sites, which can tolerate a 5-aa insertion,were identified. In all of the 12 sites, only F295/T296site was found to split the G2-EPSPS properly by co-transformation of plasmids into E. coli ER2799.The G2-EPSPS gene was then divided into N-terminal and C-terminal from F295/T296 site which were fused to the N-terminal and C-terminal of Ssp. DnaE intein, respectively, creating two plasmids pMEPSN295IN and pKEPSc296Ic. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296Ic, rescued growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Reconsituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.