Improving glyphosate oxidation activity of glycine oxidase from Bacillus cereus by directed evolution

PLoS One. 2013 Nov 5;8(11):e79175. doi: 10.1371/journal.pone.0079175. eCollection 2013.

Abstract

Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg(51) mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Oxidoreductases / chemistry
  • Amino Acid Oxidoreductases / genetics
  • Amino Acid Oxidoreductases / metabolism*
  • Amino Acid Substitution
  • Arginine / chemistry
  • Arginine / genetics
  • Arginine / metabolism
  • Bacillus cereus / genetics
  • Bacillus cereus / metabolism*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites / genetics
  • Binding, Competitive
  • Directed Molecular Evolution / methods
  • Enzyme Stability / genetics
  • Glycine / analogs & derivatives*
  • Glycine / chemistry
  • Glycine / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Models, Molecular
  • Molecular Structure
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Mutation
  • Oxidation-Reduction
  • Protein Binding
  • Protein Structure, Tertiary
  • Substrate Specificity
  • Temperature

Substances

  • Bacterial Proteins
  • Mutant Proteins
  • glyphosate
  • Arginine
  • Amino Acid Oxidoreductases
  • glycine oxidase
  • Glycine

Grant support

This work was supported by grants from the National Natural Science Foundation of China (number u1170303), the Genetically Modified Organisms Breeding Major Projects of China (2011zx08001-001), and the opening project of the Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production & Construction Corps (number BRZD1101). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.