title: Identification of a new gene encoding EPSPs with high glyphosate resistance from the metagenomic library.
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.
authors: College of Life Sciences, Key Laboratory of Bio-Resources and Eco-Environment, The Ministry of Education, Sichuan University, 610064, Chengdu, China.
authors: Dan
authors: D
authors: Jin
authors: Wei
authors: W
authors: Lu
authors: Shuzhen
authors: S
authors: Ping
authors: Wei
authors: W
authors: Zhang
authors: Jian
authors: J
authors: Chen
authors: Baoqing
authors: B
authors: Dun
authors: Ruiqiang
authors: R
authors: Ma
authors: Zhonglin
authors: Z
authors: Zhao
authors: Jiying
authors: J
authors: Sha
authors: Liang
authors: L
authors: Li
authors: Zhirong
authors: Z
authors: Yang
authors: Ming
authors: M
authors: Chen
authors: Min
authors: M
authors: Lin
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015698:Genomic Library; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D053209:Herbicide Resistance / Q000235:genetics; D006540:Herbicides / Q000494:pharmacology*; D007700:Kinetics; D008957:Models, Genetic; D009154:Mutation; D010802:Phylogeny; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D005998:Glycine; D006540:Herbicides; D000097797:Glyphosate