Expression of an evolved engineered variant of a bacterial glycine oxidase leads to glyphosate resistance in alfalfa

• A bacterial glycine oxidase (GO) variant previously engineered for efficient glyphosate degradation was targeted to the cytoplasm and chloroplast of alfalfa transgenic plants.
• The plastid-imported GO variant was more effective than cytoplasmic for glyphosate resistance.
• Two transgenic lines showed moderate resistance to the herbicide both in vitro and in vivo, which may translate into useful resistance in field conditions.
• The GO-mediated glyphosate resistance is based on herbicide degradation, and has the potential to reduce glyphosate phytotoxicity to the crops.

The main strategy for resistance to the herbicide glyphosate in plants is the overexpression of an herbicide insensitive, bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). A glyphosate resistance strategy based on the ability to degrade the herbicide can be useful to reduce glyphosate phytotoxicity to the crops. Here we present the characterization of glyphosate resistance in transgenic alfalfa (Medicago sativa L.) expressing a plant-optimized variant of glycine oxidase (GO) from Bacillus subtilis, evolved in vitro by a protein engineering approach to efficiently degrade glyphosate. Two constructs were used, one with (GOTP+) and one without (GOTP−) the pea rbcS plastid transit peptide. Molecular and biochemical analyses confirmed the stable integration of the transgene and the correct localization of the plastid-imported GO protein. Transgenic alfalfa plants were tested for glyphosate resistance both in vitro and in vivo. Two GOTP+ lines showed moderate resistance to the herbicide in both conditions. Optimization of expression of this GO variant may allow to attain sufficient field resistance to glyphosate herbicides, thus providing a resistance strategy based on herbicide degradation