Article

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 JinAffiliated withCollege of Life Sciences, Key Laboratory of Bio-resources and Eco-environment, The Ministry of Education, Sichuan UniversityBiotechnology Research Institute, Chinese Academy of Agriculture Science
  • , Wei LuAffiliated withBiotechnology Research Institute, Chinese Academy of Agriculture Science
  • , Shuzhen PingAffiliated withBiotechnology Research Institute, Chinese Academy of Agriculture Science
  • , Wei ZhangAffiliated withBiotechnology Research Institute, Chinese Academy of Agriculture Science
  • , Jian ChenAffiliated withDepartment of Pathophysiology and Institute of High Altitude Medicine, Third Military Medical University
  • , Baoqing DunAffiliated withBiotechnology Research Institute, Chinese Academy of Agriculture Science
  • , Ruiqiang MaAffiliated withBiotechnology Research Institute, Chinese Academy of Agriculture Science
  • , Zhonglin ZhaoAffiliated withBiotechnology Research Institute, Chinese Academy of Agriculture Science
  • , Jiying ShaAffiliated withBiotechnology Research Institute, Chinese Academy of Agriculture Science
    • , Liang LiAffiliated withBiotechnology Research Institute, Chinese Academy of Agriculture Science
    • , Zhirong YangAffiliated withCollege of Life Sciences, Key Laboratory of Bio-resources and Eco-environment, The Ministry of Education, Sichuan University
    • , Ming ChenAffiliated withBiotechnology Research Institute, Chinese Academy of Agriculture Science Email author 
    • , Min LinAffiliated withBiotechnology Research Institute, Chinese Academy of Agriculture Science Email author 

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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 library EPSPS Glyphosate tolerance aroA mutant