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GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

Identification of a Glyphosate-Resistant Mutant of Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase Using a Directed Evolution Strategy^1,[W],[OA]

State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology (M.Z., H.X., X.W., Y.W., Z.Z.), and National Laboratory of Biomacromolecules, Institute of Biophysics (W.G.), Chinese Academy of Sciences, Beijing 100101, People's Republic of China; and Center for Bioinformatics, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China (Z.Y., L.W.)

5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C³¹⁷T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (K_i and K_m) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Zhen Zhu (zhuzhen{at}cashq.ac.cn).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.068577.

^* Corresponding author; e-mail zhuzhen{at}cashq.ac.cn; fax 86–10–64852890.

Received July 20, 2005; returned for revision September 16, 2005; accepted September 25, 2005.

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