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.

A single residue mutation of 5-enoylpyruvylshikimate-3-phosphate synthase in Pseudomonas stutzeri enhances resistance to the herbicide glyphosate

“…The BamHI and SalI fragments from PCR product were cloned into pACYC184 (New England Biolabs), then transformed into the aroA-insertion mutant of E. coli AKM4188, which is an aromatic amino acid auxotroph and fails to grow on M9 minimal medium due to the inactive aroA (Jin et al 2007). Transformants were selected on M9 plates containing chloramphenicol, kanamycin and 20 mM glyphosate and incubated at 37°C for 48 h. Phylogenetic analyses were performed using MEGA version 4 (Tamura et al 2007).…”

Functional Metagenomics as a Technique for the Discovery of Novel Enzymes and Natural Products

“…Six amidase-positive recombinant clones were recovered in this way [42]. In another example of engineered E. coli auxotrophy used for selection, 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) activity in E. coli was knocked out using a kanamycin resistance cassette that was inserted into the aroA gene [62]. This gene encodes the enzyme EPSPS, a key enzyme in the shikimate pathway required for aromatic amino acid biosynthesis.…”

“…Three recombinant clones were recovered that harbored EPSPS activity that was resistant to glyphosate. One of these clones conferred growth at glyphosate concentrations up to 150 mM [62].…”

Characterization of a class II 5-enopyruvylshikimate-3-phosphate synthase with high tolerance to glyphosate from Sinorhizobium fredii

“…The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase; EC 2.5.1.19) is a key enzyme in the shikimate pathway, which converts phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P) to EPSP and inorganic phosphate (Jin et al 2007). The enzyme is the target of glyphosate, the active ingredient of Monsanto's broad-spectrum herbicide Roundup Ò (Steinrücken and Amrhein 1980).…”