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Communication

Generation of Maize 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Variants with Improved Glyphosate Tolerance

by
Stephen M. G. Duff
,
Lei Shi
,
Shirley Guo
,
Erin Hall
,
Steven Voss
,
Oscar Sparks
,
Guillermo A. Asmar-Rovira
,
Clayton T. Larue
* and
Marguerite J. Varagona
Bayer Crop Science, 700 Chesterfield PKWY W., Chesterfield, MO 63017, USA
*
Author to whom correspondence should be addressed.
Int. J. Plant Biol. 2025, 16(3), 106; https://doi.org/10.3390/ijpb16030106
Submission received: 20 June 2025 / Revised: 30 August 2025 / Accepted: 3 September 2025 / Published: 9 September 2025
(This article belongs to the Section Plant Biochemistry and Genetics)
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Abstract

Glyphosate (N-phosphonomethylglycine) is a broad-spectrum, foliar-applied herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in plants. EPSPS catalyzes a crucial step in the shikimate pathway for the biosynthesis of folates and aromatic amino acids in plants. A variety of glyphosate-tolerant EPSPS enzymes have been reported. Some of these have been introduced into crops using biotechnology to produce glyphosate-tolerant crops. Glyphosate tolerance in crops permits the use of glyphosate to control weeds while maintaining crop yield. We endeavored to optimize the maize EPSPS enzyme with improvements in both enzymatic activity and reduction in sensitivity to glyphosate to improve the potential for herbicide tolerance in crops. Here, we have improved the glyphosate tolerance of maize EPSPS with the potential of providing an herbicide tolerance trait by utilizing enzyme optimization with in vitro and in planta screening. Overexpressing some of these EPSPS variants into maize have resulted in maize plants with robust vegetative glyphosate tolerance.

1. Introduction

Sustainable agriculture, based on integrated knowledge from sciences as diverse as agronomy, soil science, molecular biology, biochemistry, toxicology, ecology, economics, and social sciences is a rapidly expanding field that aims to produce food and energy by embracing environmentally friendly solutions [1]. Agricultural production has always been plagued by weed competition for sunlight, water, nutrients, and space. In the second half of the 20th century, synthetic herbicides were developed to combat weeds worldwide. While broad-spectrum herbicides are a useful tool, their use is limited during cropping seasons due to their potential to injure or kill the desired crop plants. Therefore, genetic modification of crops imparting tolerance to herbicide applications is an important tool for managing weeds in growers’ fields [2].
With time, heritable traits conferring phenotypic resistance (i.e., mechanisms protecting plants by a reduction in herbicide damage) have developed in weed populations allowing weeds to survive despite the herbicide application [3,4]. Herbicide resistance increases the cost of weed management and limits the herbicide options to control resistant weeds [5,6]. Developing and deploying crops with tolerance to diverse herbicide sites of action as trait stacks is essential to enable farmers to effectively manage weeds. Crops with stacked herbicide tolerance traits will also help slow the pace of herbicide-resistant weed evolution [7,8,9].
In 1974, glyphosate was first introduced [10]. Since the introduction of glyphosate-tolerant soybeans in 1996 [11], glyphosate has remained a widely used herbicide [12,13], revolutionizing crop cultivation. Roundup Ready crop lines contain a gene derived from Agrobacterium sp. strain CP4, encoding a glyphosate-tolerant enzyme called CP4 EPSP synthase [14]. Since glyphosate’s introduction, different weed species have developed resistance to glyphosate. Target site resistance has become prevalent and a concern because it provides resistance by alteration of the herbicide binding site. The first reported 5-enolypyruvylshikimate-3-phosphate synthase (EPSPS) mutant resistant to glyphosate [15] was found in Salmonella typhimurium, a target site mutation at Pro106 (based on the Zea mays mature sequence). The overexpression of this enzyme in tomato resulted in weak glyphosate-tolerant plants [16,17]. After a second mutation Thr102Ile (T102I) was added to the Pro106Ser (P106S) mutation, the resulting variant was an enzyme significantly more resistant to glyphosate [18] which had a Ki = 50 μM, making it 100-fold more resistant than wild-type EPSPS [19]. Eleusine indica (goosegrass) was first characterized with a P106S variant and later goosegrass was discovered with the T102I, P106S double mutation [20,21,22]. However, Han et al. has shown that goosegrass maintaining the TIPS mutation comes with a high fitness cost [23]. We were interested in characterizing additional variants of EPSPS that could maintain glyphosate tolerance while limiting the enzymatic penalty, similar to that reported in Reed et al. [24].
Here, we report a two-step process to engineer glyphosate-tolerant maize EPSPS variants while retaining all or most of the activity of the WT maize EPSPS. We used the maize EPSPS enzyme as a model system for our work due to the economic importance of this crop worldwide. Maize is a monocot plant that is normally sensitive to glyphosate. Additionally, the maize genome includes only a single copy of the EPSPS gene [19]. Coupled with a robust transformation and plant characterization process available in our laboratories, these features made maize a suitable model system to test our engineered EPSPS variants. We hypothesized that the engineering process described here could help uncover new variants of EPSPS with useful properties, including tolerance to glyphosate. In particular, our aim was to optimize the EPSPS enzyme to improve glyphosate insensitivity while maintaining acceptable enzymatic activity, two parameters that are important considerations for biotechnology applications. This could allow for building improved herbicide tolerance crops. We screened a library of 8000 various residue 101/102/106 TIPA-like variants in an aro-minus bacterial screen. In addition, we also screened a saturation mutagenesis library (every residue). Top variants from the bacteria screen were purified; Km (PEP), Kcat, and I0.5 (glyphosate) were determined. The best variants were transformed into maize plants which were evaluated for glyphosate tolerance.

2. Materials and Methods

2.1. Production of EPSPS Variants at Positions 101, 102 and 106

Collections of Z. mays EPSPS coding sequences with amino acid variations were created and used to produce recombinant proteins. These variant proteins were used to identify mutations in the enzyme that reduce sensitivity to glyphosate. A library of selected positional variants was produced by mutagenesis of the codons at amino acid positions 101, 102, and 106 (relative to the wild-type Z. mays EPSPS) using PCR site-directed mutagenesis (Figure 1). Two complementary primers were synthesized containing a degenerate mixture of the four bases at the three positions of the three codons. These primers were added to a starting plasmid template and thermal cycled to produce mutant DNA molecules, which were subsequently cloned into plasmids for bacterial transformation and recombinant protein expression. A collection of 8000 EPSPS variants calculated to represent all possible amino acid combinations at positions 101, 102, and 106 (G101X-T102X-P106X) was produced.

2.2. Identification of Novel EPSPS Glyphosate-Resistant Variants from Site-Saturated Mutagenesis (SSM)

Our general strategy for identifying novel EPSPS variants for higher activity or better glyphosate tolerance involved using data from several methods including the following: a site-saturated mutagenesis library (see Section 2.3) screened under glyphosate pressure, homology modeling, literature mining, and rational design (Figure 1).
Four SSM libraries were created to generate site saturation mutant libraries (Figure 1) using a variation in the technique described in Jain and Varadarajan [25]. Each library was created to produce a collection of EPSPS variants representing a mutation at every amino acid position in the starting protein. The first library was generated using the wild-type Z. mays EPSPS; the second library was generated using the T102I-P106A EPSPS variant (“TIPA”); the third library was generated using the wild-type Z. mays EPSPS but excluding mutations at positions 101, 102, and 106; and the fourth library was generated using the TIPA variant EPSPS but excluding mutations at positions 101, 102, and 106. The resulting approximately 64,000 unique EPSPS variants had changes at one or more amino acid positions in the EPSPS protein.

2.3. Aro-A Bacterial Screen

The unique coding sequences for the EPSPS variants from the four SSM libraries and the G101X-T102X-P106X library were cloned into bacterial plasmids and transformed into an aroA-defective strain of E. coli. This allowed us to test these EPSPS variants in a modified complementation experiment in which we screened for activity and sensitivity to glyphosate. The transformed cells were then grown in liquid medium containing one of six different glyphosate concentrations: 0, 0.25 mM, 0.5 mM, 1 mM, 5 mM, and 10 mM. Cultures that showed bacterial growth were harvested at 0, 16, 22, and 38 h. Over 100 variants with the best glyphosate resistance compared to either WT or TIPA were identified from the two libraries using this screen.

2.4. Novel Variants Combined with Variants at Positions 101, 102, 106

Novel combinatorial EPSPS variants were created using the G101X-T102X-P106X EPSPS variants that showed improved enzyme kinetics and the EPSPS variants generated from the four SSM libraries, homology modeling, literature mining, and rational design were used to create an additional set of combinatorial EPSPS variants. These variants combined multiple previously identified mutations that had been found individually to result in improved glyphosate tolerance in new variants. The EPSPS variants were cloned into bacterial plasmids and transformed into aroA-defective strain E. coli for activity and glyphosate sensitivity analysis.

2.5. Expression and Purification of EPSPS Variants

The seven double-mutant (two amino acid positions with changes) EPSPS variants identified as conferring glyphosate tolerance as noted above were expressed in bacteria as N-His-tagged (TVMV cleavable) proteins to obtain purified recombinant protein to use in enzyme kinetics assays. Frozen bacterial cell pellets were resuspended in a volume of lysis buffer (50 mM NaP at pH 7.0, 50 mM NaCl, 10% (v/v) glycerol, 5 mM imidazole, 2 mM MgCl2, 0.25X YPER (Yeast Protein Extraction Reagent, Thermofisher), 0.75X BPER (Bacterial Protein Extraction Reagent, Thermofisher), 1 mg/mL lysozyme, 0.1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 150 U/mL benzonase, 1 mM benzamidine, and 1X dissolved Roche protease inhibitor tablet) that was four times (4X) the weight of the pellet. The solution was stirred for 30 min at room temperature. NaCl was added to the solution at 500 mM and the suspension was centrifuged for 30 min at 30,000× g. The resulting supernatant was added to 2 mL of Ni-NTA resin slurry (Qiagen, Hilden, Germany), pre-equilibrated with H2O, and followed by IMAC wash buffer containing 50 mM sodium phosphate, 250 mM NaCl, 1% glycerol, and 5 mM imidazole. The resin was used to batch bind the cell pellet solubilization supernatants for 1 h with stirring at 4 °C, which were transferred to a 20 mL Bio-Rad disposable column and washed 3 times with 20 mL of IMAC wash buffer for 10 min each with stirring. The EPSPS protein was sequentially eluted into six (6) × 1 mL fractions using IMAC wash buffer with 500 mM imidazole. Fractions containing significant protein were pooled and dialyzed overnight against 25 mM Tris (pH = 8), 250 mM NaCl, and 0.5% glycerol.
For higher throughput analysis of variants, expression, and extraction of the EPSPS variants was performed in 24-well blocks. Two replicates consisting of 50 µL overnight stocks of E. coli were inoculated into separate wells containing 5 mL of auto induction media with 25 µg/mL kanomycin and 25 ug/mL chloramphenicol. The E. coli cultures were grown at 37 °C for 2 h, followed by 15.6 °C overnight. The cells from each replicate were combined and harvested by centrifugation. The pellets were frozen until protein extraction. Frozen pellets were thawed with metal beads to loosen the pellet and then extracted at 4 °C in 2 mL of 50 mM Tris pH 8.0, Bper: Yper 3: 1, 250 mM NaCl, 10 mM imidazole, lysozyme 1 mg/100 mL, benzonase 10 µL (750 U/µL)/100 mL. The mixture was centrifuged for 15 min, and the resulting supernatants were transferred to a new 24-well block. A total of 250 µL of Ni-NTA beads (pre-equilibrated with washing buffer) were added to each supernatant. The blocks were shaken at 4 °C for 1 h and the beads were then collected into wells in a filter plate by centrifugation for 1 min. The beads were washed successively in the filter plate by adding several bed volumes of 25 mM Tris pH 8.0, 250 mM NaCl, and 20 mM imidazole, followed by 25 mM Tris pH 8.0, 250 mM NaCl, and 50 mM imidazole, and then centrifugation at 500× g for 1 min. The protein was eluted by two successive washes of 350 µL 20 mM Tris pH 8.0, 250 mM NaCl, and 200 mM imidazole. The protein samples were desalted using Zeba desalt plates. Four washes of 250 µL 15.4 mM Tris, pH 7.4, 130 mM NaCl, and 1% glycerol were performed to equilibrate the resin, and then 100 µL of variant protein from the first 200 mM imidazole was desalted by centrifugation for 2 min. The proteins were normalized to 400 ppm into 4 × 25 µL aliquots, flash frozen, and stored until kinetic analysis.

2.6. EPSPS Activity Assay

Purified recombinant protein was used to measure enzymatic activity in a variation in the assay described in Vazquez [26]. EPSPS enzymatic activity was measured in a solution consisting of 50 mM MOPS-KOH, pH 7.2, 0.5 mM MgCl2, 15% (v/v) glycerol, 1.5 mM inosine, 0.05 mM Amplex Red, 0.2 U/mL, nucleoside phosphorylase, 0.4 U/mL xanthine oxidase, and 1.0 U/mL horseradish peroxidase, and variable amounts of phosphoenolpyruvate (PEP), shikimate 3-phosphate (S3P), and glyphosate. The assay was performed in a 96-well plate with a final volume of 50 µL using the mosquito® HV liquid handler (TTP Labtech Ltd., Hertfordshire, UK) for pipetting.

2.7. EPSPS Enzyme Kinetics

Since the bacterial screen yielded a lot of variants, they were prescreened in single- or double-point glyphosate concentrations to identify the best variants for further kinetic characterization. For kinetic determinations, a master mix of all the non-variable components was created. Purified recombinant protein, glyphosate, PEP, and S3P were then added to the master mix as required. Enzyme kinetic measurements for KM and Vmax (with PEP S3P, or both) and IC0.5 (with glyphosate) were analyzed by producing a Michaelis–Menten (for Km and Vmax) or logarithmic scale (for IC0.5) plot in GraphPad Prism 7.0 (GraphPad Software, Inc., La Jolla, CA, USA) using the average values of three concentrations of each enzyme variant (normalized to a single enzyme concentration). Fluorescence change over time during the linear portion of the assay was determined on a Safire2™ (Tecan Trading AG, Mannedorf, Switzerland). The fluorescence parameters were 555 nm for excitation and 590 nm for emission (5 nm band widths in both cases) with a manual gain of 100. Michaelis–Menten constants in the presence of PEP and S3P for each EPSPS variant were determined at saturating concentration (200 µM) of the substrate not being measured. I0.5 in the presence of glyphosate was determined at S3P saturating concentration and PEP sub-saturating concentration (80 µM).

2.8. Production and Propagation of R0 and R1 Transgenic Maize Plants Expressing EPSPS Variants

Transgenic maize plants that expressed EPSPS variants which performed the best in the microbial growth assay or kinetics were generated to determine if the variants conferred glyphosate tolerance to plants, using a testing scheme as reported in Reed [24]. The full genomic DNA sequence encoding the wild-type maize EPSPS was cloned from maize genomic DNA. Mutations were then introduced into the EPSPS coding sequence to produce each double-mutant EPSPS variant to be tested. These mutated full genomic DNA sequences were then cloned as a single expression cassette into plant transformation vectors, which were used with Agrobacterium tumefaciens and standard methods for plant transformations as described previously in other reports [8]. Transformants are chosen from selection media and transferred to soil plugs. Regenerated R0 transgenic plantlets were grown in the greenhouse, single-copy plants were identified, and these were divided into control and treatment groups. Plants in the treatment group were sprayed with glyphosate-applied postemergence (POST) at 3 lb. ae/acre (3.36 kg ae/ha) at the V3–V4 stage. Treated plants were evaluated for injury 7 to 14 days after glyphosate application. Each individual plant represented a unique event, and multiple events were tested for each EPSPS variant (recorded as “n”). Scores were averaged for individual plants within events or events within a given transformation vector.
R0 plants were self and cross pollinated to produce R1 and F1 seeds for testing at the next generation. Glyphosate spray tests were repeated on plants grown from this next generation. R1 or F1 seeds were planted and grown with standard controlled environment practices for corn. Six to ten healthy plants were sprayed with glyphosate-applied postemergence (POST) at 3 lb. ae/acre (3.36 kg ae/ha) at the V3–V4 stage. Treated plants were evaluated for injury 7 to 14 days after glyphosate application. Zygosity analysis was performed using the known transgenic elements. Injury scores were based on the average of plant reps for each event or the average of all plants per transformation vector.

3. Results

3.1. Enzyme Kinetics of EPSPS Variants

Kinetic parameters were determined for the TIPA-like (mutants at the same residues as TIPA) and novel variants (Table 1), averaged across three replicates. Since glyphosate resistance, Km, and Kcat were all important parameters and the goal was to find an EPSPS gene which could support the plant through its entire life cycle and confer resistance to glyphosate, we considered the specificity constant (Kcat/Km) and designed a new unweighted parameter to combine Kcat, Km and I0.5, kcat*I0.5/Km (Table 1) to measure our overall progress towards the goal. Our goal was to engineer a glyphosate-tolerant maize EPSPS which could substitute for the glyphosate sensitive wild-type EPSPS through the lifecycle of maize, providing glyphosate tolerance and maintaining agronomic performance.
In the absence of a glyphosate challenge, it is critical to have an EPSPS with Kcat sufficiently high and Km sufficiently low so that there is no fitness pressure (maintenance of agronomic performance) due to restrained enzyme activity. If the EPSPS enzyme struggles to maintain proper enzymatic activity to meet the biosynthetic needs of a growing plant, the plant growth will suffer, even in the absence of glyphosate challenges, impacting potential crop yield. Under high glyphosate pressure, a high I0.5 is desirable. Most active variants had high I0.5 values, which was not surprising since many variants consisted of mutations stacked onto base “scaffold” variants that have previously been identified as conferring glyphosate tolerance to plants (e.g., T102G-P106S, T102G-P106W, T102I-P106A). Another variant (T102I-A103V-P106G-L107T) had the highest I0.5, slightly higher than TIPA. Most of the variants had higher Kcat values than TIPA, some of which were slightly higher than WT. Km values did not vary greatly, with some variants being higher and some lower than WT. TIPT was the best, combining the three kinetic properties evident from a kcat*I0.5/Km of 11.5, the highest of any variant. Overall, this is a promising set of variants to be tested in plants.

3.2. Glyphosate Resistance of Transgenic Maize Expressing EPSPS Variants

Many of the R0 plants expressing variable copies of the variant genes as well as CP4 showed tolerance to glyphosate. The best single molecularly clean events were advanced to the next generation R1 (occasionally F1; see Methods) and tested for glyphosate tolerance. In plant testing, all TIPA-like variants expressed with the native maize EPSPS expression elements provided vegetative tolerance to glyphosate (Figure 2). The best novel variants provided good protection to glyphosate similar to the best TIPA-like variants and CP4. The variant which imparted the best tolerance to glyphosate was N28Q-TIPA (Figure 2). In addition, another variant at position 28, N28-S TIPA, also trended toward less injury. Interestingly, I0.5 showed a correlation (R2 of 0.5) with plant efficacy.

4. Discussion

4.1. Protein Engineering Is a Useful Technique to Improve Crops

There have been several attempts at protein engineering for plant improvement using genetically modified organisms or gene editing, including Bacillus thuringiensis toxin via truncation, domain swapping, peptide addition, and amino acid mutation [27,28]. In addition, EPSPS has been engineered to be resistant to glyphosate by variants generated by DNA shuffling [29], error-prone PCR [30], and a synthetic yeast selection [24]. We successfully engineered a microbial-sourced dioxygenase to improve enzymatic parameters, including enhanced enzymatic activity of a α-ketoglutarate-dependent (R)-dichlorprop dioxygenase for use with selected herbicides and improved temperature stability [31]. These herbicide tolerance enzyme variants with enhanced enzymatic and temperature stability parameters enabled robust herbicide tolerance from two herbicide families in transgenic maize and soybeans [31]. With the advent of gene editing, it may be possible to replace wild-type genes with variants designed for plant improvement, further enabling trait development in crops [32].
Wild-type maize EPSPS is somewhat sensitive to glyphosate with an I0.5 of 0.6 mM compared to an I0.5 of 140 mM for CP4. The well-studied TIPA variant in maize EPSPS has an I0.5 of 20 mM which is intermediate between that of wild-type maize EPSPS and CP4. It is also known that by itself the TIPA variant causes a high fitness penalty [23], making it a poor candidate for gene editing since maize has a single copy of EPSPS. This fitness penalty is believed to be a result of compromised enzymatic activity of the EPSPS TIPA variant in the absence of glyphosate, which compromises the biosynthetic pathway in the plant. Therefore, it is critical to focus on improving the Kcat/Km of the final variant with respect to the TIPA variant and eliminating the fitness penalty in order to build a robust herbicide tolerance trait if the trait is designed to function as a replacement of the native EPSPS, such as in a gene editing context.

4.2. New EPSPS Variants Show Useful Parameters That Can Be Applied to Plant Biotechnology

The TIPA-like variants of maize EPSPS have promising kinetics, providing tolerance to the enzyme with I0.5 (glyphosate) values between 10 and 20-fold higher than WT EPSPS and Kcat and Km values like WT EPSPS. However, CP4 still had more than a 10-fold higher I0.5 (glyphosate) than any of the TIPA-like variants. None of the variants had a kcat*I0.5/Km value more than 15% of CP4. Some of the novel variants had Kcat/Km values as high as the TIPA-like variants, while other variants had lower Kcat/Km values. This demonstrates that while significant advancements were achieved in the study reported here, there is likely room for additional improvements in EPSPS variants. It will be important for future research to build upon what is reported here to engineer even better variants, with these variants providing a strong foundation for these future studies. Other efforts have also looked at advanced gene optimization techniques to improve enzymatic parameters and glyphosate tolerance with success, although different modifications were uncovered, suggesting that the EPSPS enzyme can be optimized by considering a number of different modifications [24]. As more tools become available to aid in enzyme optimization, it will be important to incorporate what has been described in these reports into additional optimization efforts as we look towards continual improvement of our crops.
Many of the R0 plants expressing variable copies of the variant genes as well as CP4 showed tolerance to glyphosate. The best variants were advanced to the next generation and tested for glyphosate tolerance. However, the variants with the best efficacy in plants did not strongly correlate to any kinetic parameter or combination of kinetic parameters except for the I0.5, suggesting that additional factors such as protein stability in vivo may be important in planta. While the statistical error for the plant testing of the selected variants in general was narrow for biological tests run in a greenhouse, there were two variants that had notably larger statistical errors, suggesting that the biological response varied considerably between plants and that we should be cautious in the interpretation of the data for these two variants. Additional studies will be needed to fully elucidate all the parameters that are necessary for the robust performance of the EPSPS variants as an herbicide tolerance trait. It is also important to note that maize plants expressing these variants were not yet tested in further generations where additional characterization could be completed, such as comprehensive yield or physiology analysis. Additionally, these variants were not yet tested in a gene editing context. Both of these experiments are well beyond the scope of the research presented here, but are logical follow-up studies that can be completed to further characterize these new EPSPS variants.
With 2–4 amino acid modifications, we have engineered EPSPS variants which show glyphosate resistance up to 35-fold greater than WT EPSPS, the best of which have promise for informing gene editing efforts. Many of these variants had Kcat, Km, and specificity constants comparable to WT EPSPS and when expressed in maize imparted tolerance to glyphosate. Several variants are promising candidates that could inform gene editing of crop plants to provide additional glyphosate tolerance solutions for growers. Our results support a previous report that TIPA or TIPA-like in addition to other mutations may be necessary to obtain a maize EPSPS enzyme with good kinetic properties [24]. Further engineering the maize EPSPS gene as well as testing in plants would further explore this potential. While these studies used maize and EPSPS as the model system, our hope is that this work provides a foundation for exploring modifications of enzymes that are the target of different herbicide chemistries in maize and other plant species, including other crop species. Additionally, the EPSPS variants reported here will likely provide a good starting point for additional EPSPS optimization efforts, both for maize as well as other crops, as the interest in providing weed control options continues to remain critical to supporting agriculture practices around the world.

Author Contributions

Conceptualization, S.M.G.D., L.S., S.G., E.H., S.V., O.S., C.T.L. and M.J.V., investigation, S.M.G.D., L.S., S.G., E.H., S.V., O.S., G.A.A.-R. and C.T.L.; methodology, S.M.G.D., L.S., S.G. and C.T.L., formal analysis, S.M.G.D., L.S., S.G., E.H. and C.T.L., data curation, S.M.G.D. and E.H., resources, project administration, and supervision, S.G. and M.J.V., writing, drafting, review, and editing, S.M.G.D., E.H. and C.T.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

Authors Stephen M. G. Duff, Lei Shi, Shirley Guo, Erin Hall, Steven Voss, Oscar Sparks, Guillermo Asmar-Rovira, Clayton T. Larue and Marguerite J. Varagona were employed by the company Bayer Crop Science when the research was conducted. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Scheme overview for library design and screening strategy for maize EPSPS variant selection and advancement.
Figure 1. Scheme overview for library design and screening strategy for maize EPSPS variant selection and advancement.
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Figure 2. Glyphosate spray plant injury ratings of R1 plants expressing selected maize EPSPS variants.
Figure 2. Glyphosate spray plant injury ratings of R1 plants expressing selected maize EPSPS variants.
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Table 1. Kinetics of EPSPS variants.
Table 1. Kinetics of EPSPS variants.
Kinetics
kcat (s−1)Km (PEP) μMSpecificity Constant (kcat/Km)I0.5 (mM)kcat*I0.5/Km% kcat*I0.5/Km of CP4
Control10.418.60.60.60.30.4
T102I-P106S (TIPS)5.29.40.614.27.810.3
CP48.615.90.5140.076.2100.0
T102I-P106A (TIPA)4.38.40.519.910.313.5
T102G-P106S (TGPS)11.723.00.57.84.05.2
T102V-P106S (TVPS)11.723.00.57.84.05.2
T102I-P106T (TIPT)6.911.70.619.411.515.1
T102G-P106W (TGPW)10.018.40.513.27.29.4
T102L-P106V8.914.20.610.46.58.6
T102G-P106W-L280R10.316.00.610.56.84.8
R60E-T102G-P106S6.458.50.112.01.31.7
T102I-A103V-P106G-L107T5.229.80.220.83.64.7
T102G-A103V-P106S-L107V13.620.50.712.08.010.5
T102I-P106A-L280R4.914.00.417.66.28.1
P106I-L107S5.248.40.11.40.10.1
N28T-T102G-P106S7.333.30.214.13.13.1
N28Q-T102G-P106S9.967.90.111.21.61.6
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Duff, S.M.G.; Shi, L.; Guo, S.; Hall, E.; Voss, S.; Sparks, O.; Asmar-Rovira, G.A.; Larue, C.T.; Varagona, M.J. Generation of Maize 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Variants with Improved Glyphosate Tolerance. Int. J. Plant Biol. 2025, 16, 106. https://doi.org/10.3390/ijpb16030106

AMA Style

Duff SMG, Shi L, Guo S, Hall E, Voss S, Sparks O, Asmar-Rovira GA, Larue CT, Varagona MJ. Generation of Maize 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Variants with Improved Glyphosate Tolerance. International Journal of Plant Biology. 2025; 16(3):106. https://doi.org/10.3390/ijpb16030106

Chicago/Turabian Style

Duff, Stephen M. G., Lei Shi, Shirley Guo, Erin Hall, Steven Voss, Oscar Sparks, Guillermo A. Asmar-Rovira, Clayton T. Larue, and Marguerite J. Varagona. 2025. "Generation of Maize 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Variants with Improved Glyphosate Tolerance" International Journal of Plant Biology 16, no. 3: 106. https://doi.org/10.3390/ijpb16030106

APA Style

Duff, S. M. G., Shi, L., Guo, S., Hall, E., Voss, S., Sparks, O., Asmar-Rovira, G. A., Larue, C. T., & Varagona, M. J. (2025). Generation of Maize 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Variants with Improved Glyphosate Tolerance. International Journal of Plant Biology, 16(3), 106. https://doi.org/10.3390/ijpb16030106

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