Current Microbiology

, Volume 55, Issue 4, pp 350–355

Identification of a New Gene Encoding EPSPS with High Glyphosate Resistance from the Metagenomic Library

  • Dan Jin
  • Wei Lu
  • Shuzhen Ping
  • Wei Zhang
  • Jian Chen
  • Baoqing Dun
  • Ruiqiang Ma
  • Zhonglin Zhao
  • Jiying Sha
  • Liang Li
  • Zhirong Yang
  • Ming Chen
  • Min Lin
Article

DOI: 10.1007/s00284-007-0268-x

Cite this article as:
Jin, D., Lu, W., Ping, S. et al. Curr Microbiol (2007) 55: 350. doi:10.1007/s00284-007-0268-x

Abstract

Glyphosate, a powerful nonselective herbicide, acts as an inhibitor of the activity of the enzyme 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by the aroA gene involved in aromatic amino acid biosynthesis. An Escherichia coli mutant AKM4188 was constructed by insertion a kanamycin cassette within the aroA coding sequence. The mutant strain is an aromatic amino acids auxotroph and fails to grow on M9 minimal media due to the inactive aroA. A DNA metagenomic library was constructed with samples from a glyphosate-polluted area and was screened by using the mutant AKM4188 as recipient. Three plasmid clones, which restored growth to the aroA mutant in M9 minimal media supplemented with chloramphenicol, kanamycin, and 50 mM glyphosate, were obtained from the DNA metagenomic library. One of them, which conferred glyphosate tolerance up to 150 mM, was further characterized. The cloned fragment encoded a polypeptide, designated RD, sharing high similarity with other Class II EPSPS proteins. A His-tagged RD fusion protein was produced into E. coli to characterize the enzymatic properties of the RD EPSP protein.

Keywords

Metagenomic libraryEPSPSGlyphosate tolerancearoA mutant

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Dan Jin
    • 1
    • 2
  • Wei Lu
    • 2
  • Shuzhen Ping
    • 2
  • Wei Zhang
    • 2
  • Jian Chen
    • 3
  • Baoqing Dun
    • 2
  • Ruiqiang Ma
    • 2
  • Zhonglin Zhao
    • 2
  • Jiying Sha
    • 2
  • Liang Li
    • 2
  • Zhirong Yang
    • 1
  • Ming Chen
    • 2
  • Min Lin
    • 2
  1. 1.College of Life Sciences, Key Laboratory of Bio-resources and Eco-environmentThe Ministry of Education, Sichuan UniversityChengduChina
  2. 2.Biotechnology Research InstituteChinese Academy of Agriculture ScienceBeijingChina
  3. 3.Department of Pathophysiology and Institute of High Altitude MedicineThird Military Medical UniversityChongqingPeople’s Republic of China