Cloning And Characterization Of A Novel Gene Involved In Glyphosate Degradation

Glyphosate is a good-absorption herbicide of broad grass-spectrum. It is a competitive inhibitor to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs), a key enzyme in the synthesis of aromatic amino acids of plants and some microorganism. Inhibition of the EPSPs leads to insufficient production of aromatic amino acids, which are prerequisites to maintain the necessary protein synthesis in vivo, and thus lead to plant death. Due to many advantages of glyphosate, it has become the one of the most successful herbicides in the 20th century. Glyphosate is a non-selective herbicide that kills plants of all kinds. This had limited its application until the transgenic crop era, when scientists discovered glyphosate resistant gene and transformed it genetically into crops. The commercialized glyphosate-resistant transgenic crops were based on the overproduction of bacterial EPSPs from Agrobacterium sp. strain CP4 (namely CP4 EPSPs). However, glyphosate uptaken by CP4 EPSPs-transgrenic crops can not be degraded and may accumulate in the plants, which existed a potential negative effects to the plants and human. Therefore, it is necessary to explore a new resistant mechanism with no glyphoste residues.In this work, a novel glycine oxidase gene (go), designated as goA, was identified from a metagenomic library of glyphosate-contaminated soil by functional screening. It had a nucleotide sequence of 1110 bp, encoding 369 amino acids, encoding product showed 88% identity to the reported wild GO from Bacillus subtilis strain 168. To verify its activity, goA was constructed into an expression vector pEASY-E1, overexpressed in Escherich coli BL21(DE3) pLysS, and then purified by affinity chromatography and anion exchange chromatography. The specific activity of the recombinant enzyme was 0.283 U/mg and total yield was 88.9% after purification. Enzyme assay showed that GOA can degrade glyphosate into aminomethyl phosphonic acid and glyoxylate. The optimal temperature and optimal pH of the GOA were 50℃and 8.5, respectively. The purified GOA exhibited better thermostability at 50℃and pH stability over a broader range than the reported GO. GOA exhibited an 8-fold higher apparent affinity (1/Km) for glyphosate substrate and a 5.5-fold higher specificity constant (kcat/Km for glyphosate over glycine) than that of the reported wild-type GO. Protein modeling study revealed that GOA binds to glyphosate closely with an increased H-bond number in comparison to GO. The goA founded in this work provides a potential candidate gene for the development of glyphosate-tolerant transgenic plants without glyphosate residues.