Discovery and directed evolution of a glyphosate tolerance gene

Science. 2004 May 21;304(5674):1151-4. doi: 10.1126/science.1096770.

Abstract

The herbicide glyphosate is effectively detoxified by N-acetylation. We screened a collection of microbial isolates and discovered enzymes exhibiting glyphosate N-acetyltransferase (GAT) activity. Kinetic properties of the discovered enzymes were insufficient to confer glyphosate tolerance to transgenic organisms. Eleven iterations of DNA shuffling improved enzyme efficiency by nearly four orders of magnitude from 0.87 mM-1 min-1 to 8320 mM-1 min-1. From the fifth iteration and beyond, GAT enzymes conferred increasing glyphosate tolerance to Escherichia coli, Arabidopsis, tobacco, and maize. Glyphosate acetylation provides an alternative strategy for supporting glyphosate use on crops.

MeSH terms

  • Acetylation
  • Acetyltransferases / chemistry
  • Acetyltransferases / genetics*
  • Acetyltransferases / metabolism
  • Amino Acid Sequence
  • Bacillus / enzymology
  • Catalysis
  • DNA Shuffling*
  • Directed Molecular Evolution*
  • Drug Resistance
  • Escherichia coli / genetics
  • Gene Library
  • Genetic Variation
  • Glycine / analogs & derivatives*
  • Glycine / metabolism
  • Glycine / toxicity*
  • Herbicides / metabolism
  • Herbicides / toxicity*
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis
  • Plants, Genetically Modified* / drug effects
  • Plants, Genetically Modified* / genetics
  • Recombinant Proteins / metabolism
  • Recombination, Genetic
  • Tobacco / drug effects
  • Tobacco / genetics
  • Tobacco / growth & development
  • Transformation, Genetic
  • Zea mays / drug effects
  • Zea mays / genetics
  • Zea mays / growth & development

Substances

  • Herbicides
  • Recombinant Proteins
  • glyphosate
  • Acetyltransferases
  • glyphosate N-acetyltransferase
  • Glycine