Site-directed mutagenesis, expression and biological activity of E. coli 5- enolpyruvylshikimate 3-phosphate synthase gene

Site-directed mutagenesis (SDM) as a powerful technique was used to change two important and conserved amino acids in 5-enolpyruvylshikimate 3- phosphate synthase (EPSPS) gene of E. coli. The mutations changed glycine 96 to alanine and alanine 183 to threonine. These two amino acids are very important for intraction of the wide spectrum herbicide, glyphosate, to EPSP synthase enzymes. By designing mutagen primers and overlapping extension method, three kinds of altered bacterial EPSPS enzymes with first, second and both mutations were produced. These modified enzymes are expected to show decreased affinity for herbicide, with least alteration in their enzymatic activity. These altered genes were cloned under the control of chemically inducible T7 promoter and over expressed in E. coli. Biological activity analyses in the presence of glyphosate show that the bacteria containing the mutated enzymes, especially the enzyme with two mutations, were more tolerant to glyphosate.