Identification of a glyphosate-resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy

Abstract

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 C(317)-->T 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 (Ki and Km) 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.

Figure 1.

Glyphosate resistance analysis of EPSPS mutants via growth of E. coli transformant. DH5α was transformed with pBREP (wild type), pBREP-P106L, and pBREP-G101A. The transformants were cultured in liquid MOPS medium containing glyphosate in increased concentration gradients. Aliquots of culture sample were measured for OD₆₀₀ before (OD_b) and after (OD_a) 12 h of incubation in 37°C by vigorous vibration. Error bars represent the sd resulting from three repetitions. A, Bar chart; OD₆₀₀ increment was calculated as (OD_a − OD_b)/OD₀, in which OD₀ is the final value of OD₆₀₀ absent glyphosate. B, Curve chart; the curve was determined by fitting data of OD_b to f(x) = C + (D − C)/(1 + (x/LD₅₀)^b) (Seefeldt et al., 1995; Igor Pro 4.00), where C = lower limit, D = upper limit, LD₅₀ = 50% lethal dose, and b = slope of the curve at LD₅₀. The first and last points on the curve represent the data of OD₀ and OD_b.

Figure 2.

Enzyme assay results. K_m (PEP) and IC₅₀ were obtained from the least-squares fitting of the data to appropriate equations (Igor Pro 4.00). Black triangles and diamonds represent the measured data points of wild-type OsEPSPS and P106L EPSPS, respectively. Error bars represent the sd resulting from three repetitions. Dashed and solid curves represent the fitting curves of OsEPSPS and P106L mutant, respectively. S3P concentration was fixed at 1 mm. A, K_m (PEP) was determined by fitting the data to V = V_max[S]/(K_m + [S]), where V is the velocity of the reaction (expressed in U/mg), V_max is the maximum velocity, and [S] is the concentration of the substrate assayed for the K_m. B, The curve was determined by fitting data to V = V_min + (V_max − V_min)/(1 + ([I]/IC₅₀)^s), where [I] is concentration of glyphosate and s is the slope of the curve at the IC₅₀. The first point on the curve represents the data obtained with no glyphosate. K_i value was determined by the method as described (Copeland, 1996). C, K_i (glyphosate) of OsEPSPS was determined by fixed PEP concentration of 0.05, 0.067, 0.1, and 0.2 mm, respectively, and glyphosate concentration was 0, 0.05, 0.1, and 0.2 μm. D, K_i (glyphosate) of P106L mutant was determined by fixed PEP concentration of 0.067, 0.1, 0.2, and 0.5 mm, respectively, and glyphosate concentration was 0, 1, 2, and 3 μm.

Figure 3.

RSO model. Model construction was based on Michaelis-Menten equation. A to D represent the RSO model of P106L mutant, P106S mutant from goosegrass (Baerson et al., 2002), G101A mutant from petunia (Padgette et al., 1991), and wild-type OsEPSPS, respectively. Calculations for A and D were carried out using the kinetic parameters shown in Table II. Calculations for B and C were carried out using the kinetic parameters reported previously (Padgette et al., 1991; Baerson et al., 2002). Sections 1 and 3 represent the surface charts generated by Excel from Equations 1 and 2, respectively. Sections 2 and 4 represent the contour charts of 1 and 3, respectively. Gly, Abbreviation of glyphosate.

Figure 4.

T-DNA cassette containing encoding region of OsEPSPS on pTi-OsEPSPS for plant transformation. Npt-II, Neomycin phosphotransferase gene; P-nos, nopaline synthase promoter; T-nos, nopaline synthase terminator; TB(L) and TB(R), left and right border of T-DNA; P-35S, cauliflower mosaic virus 35S promoter; OsEPSPS, coding sequence of premature form of OsEPSPS; CTP, coding sequence of chloroplast transit peptide.

Figure 5.

Glyphosate resistance of transgenic tobacco. A, Photograph was taken after 30 d of culture on medium containing 0 to 0.5 mm glyphosate. (Columns 1–7 contained 0, 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 mm glyphosate, respectively.) The top line shows the leaf discs of nontransformed plants, the middle line shows the leaf discs transformed with pTi-OsEPSPS, and the bottom line shows the leaf discs of plants transformed with pTi-P106L. B, Photograph was taken at 1 month after the transplant. Seedlings were germinated and selected on medium containing 100 mg/L kanamycin for 7 to 10 d to eliminate the negative plants; the green seedlings with two to four true leaves were transplanted onto medium containing 0 to 10 mm glyphosate. (Columns 1–7 contained 0, 0.05, 0.1, 1, 2, 5, and 10 mm glyphosate, respectively.) The top line shows the seedlings of nontransformed plants, the middle line shows the seedlings of transgenic plant line CEP5 expressing wild-type OsEPSPS, and the bottom line shows the seedlings of plant line P106L-A expressing P106L mutant OsEPSPS. C, Photograph was taken at 2 weeks after the spray treatment. The top line was control plants without treatment of Roundup. The bottom line was sprayed with Roundup at a dose of 0.2 mL/m² in 1% concentration. Numbers 1 and 4 represent nontransformed plants. Numbers 2 and 5 represent transgenic plant line CEP5 expressing wild-type OsEPSPS. Numbers 3 and 6 represent transgenic plant line P106L-A expressing P106L mutant OsEPSPS.

Figure 6.

Schematic diagrams of the relationship between PEP affinity and glyphosate affinity as shown in Equation 4. Two horizontal dash lines represent minimum and maximum values of . Four vertical dotted lines represent the corresponding values of ; A is obtained when the glyphosate concentration [I] is increased, and B is obtained when the expression level n is increased.