Identification of a new gene encoding 5-enolpyruvylshikimate-3-phosphate synthase using genomic library construction strategy

Applying the genomic library construction strategy and colony screening, a new aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase has been identified, cloned and overexpressed in Escherichia coli, and the enzyme was purified to homogeneity. Kinetic analysis of the AroA (P.fluorescens) indicated that the full-length enzyme exhibits 10-fold increased IC50 and an approximately 38-fold increased K (i) for glyphosate compared to those of the AroA (E.coli) , while retaining high affinity for the substrate phosphoenolpyruvate. Furthermore, we have transformed the new aroA (P.fluorescens) gene into Arabidopsis thaliana via a floral dip method, and demonstrated that transgenic A. thaliana plants exhibit significant glyphosate resistance when compared with the wild type.