Reconstruction of enzymatic activity from split genes encoding glyphosate-tolerant EPSPS protein of Psedomonas fluorescens G2 strain by intein mediated protein complementation

N-phosphonomethylglycine, commonly referred to as glyphosate, is a broad-spectrum, non-selective herbicide used for the control of weeds in glyphosate-to- lerant crops. Glyphosate inhibits the 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), a key en…

Reconstruction of enzymatic activity from split genes encoding glyphosate-tolerant EPSPS protein of Psedomonas fluorescens G2 strain by intein mediated protein complementation

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 pro- teins 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/T296 site 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-termi- nal 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 pMEPS- N295IN and pKEPSc296Ic. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296Ic, rescu- ed growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Re- consituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.