Increasing tolerance to glyphosate herbicide in Escherichia coli by expression of recombinant glyphosate oxidoreductase (gox) gene

Genetic manipulation of crop plants to obtain resistance to herbicide glyphosate is one of the most effective approaches for weed management. Application of the genes encoding glyphosate degrading enzymes such as glyphosate oxidoreductase (GOX) in combination with a glyphosate-tolerant EPSPS is the ultimate approach to provide commercial rates of glyphosate tolerance. The gox gene was first isolated from Ochrobactrum antrophi strain LBAA that catalyses the cleavage of glyphosate into aminomethylphosphonic acid and glyoxalate. In this study, the gox gene was synthesized and cloned in pET28a expression vector and transformed in E. coli. The expression of the target protein was confirmed by SDS-PAGE. The experiment for optimization of gene expression showed that the optimal condition was 37oC for bacterial culture, 4 hours of induction time using 1 mM IPTG in OD600=0.6. In the next step, in order to perform a bioassay on transformed bacteria, the threshold of tolerance of control bacteria in minimum phosphate-free medium with different concentrations of glyphosate was investigated and compared with recombinant bacteria. The result indicated that the wild type bacteria were not able to tolerate concentration of more than 0.5 mM glyphosate but the recombinant bacteria survived to a concentration of 1 mM. These results were confirmed by colony counting on selective media with three repetitions. Hence, glyphosate oxidoreductase gene could be a suitable candidate besides the other glyphosate resistance genes for genetic manipulation of strategic plants with the aim of obtaining higher and more sustainable levels of resistance to this herbicide.