Book of Abstracts: Albany 2005
In Planta Herbicide Detoxification with an Improved Bacterial Enzyme
Plants can be genetically modified to tolerate non-selective herbicides using two general strategies. In the first strategy, crop plants express a gene encoding a target site enzyme that is insensitive to the herbicide. This strategy is used in Roundup Ready® crops that express a 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase enzyme that is insensitive to glyphosate and in Clearfield crops that tolerate imidazolinone herbicides because they have an insensitive acetohydroxyacid synthase (AHAS) target enzyme. The second strategy involves inactivation of the herbicide either through degradation or modification. Examples of this strategy include LibertyLink® phosphinothricin tolerant crops expressing the N-acetyltransferase genes, pat or bar, and bromoxynil tolerant crops expressing the bxn nitrilase gene. Detoxification of the applied herbicide not only protects the modified crop, it also removes herbicide residues from the environment.
Until recently, there were no known enzymatic activities that could detoxify glyphosate, the largest selling agricultural herbicide, in a wide spectrum of host plants. Our advantage in developing a novel glyphosate detoxification enzyme comes from eliminating the need to discover enzymes exhibiting high biological activity because we can modify poor enzymes to improve their kinetic properties. Using a highly sensitive mass spectrometry method, we screened a microbial diversity collection to discover Bacillus glyphosate N-acetyltransferase (gat) genes. The native gat genes are not capable of conferring a glyphosate tolerant phenotype to E. coli or plants. DNA shuffling was used to increase the enzyme activity on the synthetic substrate glyphosate to within the norm for any N-acetyltransferase on its native substrate. The crystal structure of an improved GAT protein was solved and used to evaluate the reaction mechanism and to map the effects of DNA shuffling on the enzyme activity. Expression of improved gat genes in model and crop plants confers glyphosate tolerance suitable for the selection of transgenic plants. Soil-grown plants tolerate high doses of glyphosate spray application. This new herbicide tolerance strategy is being evaluated in several major crops.
References and Footnotes
Linda A. Castle*