Open AccessImprovement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Ochrobactrum 5-Enopyruvylshikimate-3-Phosphate Synthase
Author(s) -
YongSheng Tian,
Jing Xu,
AiSheng Xiong,
Zhao Wang,
Xin Fu,
RiHe Peng,
QuanHong Yao
Publication year - 2011
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.05271-11
Subject(s) - glyphosate , mutant , mutagenesis , dna shuffling , biology , amino acid , arabidopsis , aminomethylphosphonic acid , biochemistry , genetics , gene , directed evolution , microbiology and biotechnology
A mutant of 5-enopyruvylshikimate-3-phosphate synthase fromOchrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenicArabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops.