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Desensitizing Plant EPSP Synthase to Glyphosate: Optimized Global Sequence Context Accommodates a Glycine-To-Alanine Change in the Active Site

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Desensitizing Plant EPSP Synthase to Glyphosate: Optimized Global Sequence Context Accommodates a Glycine-To-Alanine Change in the Active Site JBC Papers in Press. Published on November 13, 2018 as Manuscript RA118.006134 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.006134 Optimized, glyphosate-desensitized plant EPSPS Desensitizing plant EPSP synthase to glyphosate: Optimized global sequence context accommodates a glycine-to-alanine change in the active site Yuxia Dong1, Emily Ng1, Jian Lu, Tamara Fenwick, Yumin Tao, Sean Bertain, Marian Sandoval, Ericka Bermudez, Zhenglin Hou, Phil Patten2, Michael Lassner3, Daniel Siehl* From Corteva AgriscienceTM, Agriculture Division of DowDuPont, 4010 Point Eden Way, Hayward, CA 94545 Running title: Optimized, glyphosate-desensitized plant EPSPS 1Equal contributors 2Present address; DiCE Molecules, 220 Penobscot Dr., Redwood City, CA 94063 3Present address; Amfora, Inc., San Francisco, CA 94104 TM *To whom correspondence should be addressed: Daniel L. Siehl, Corteva Agriscience , Agriculture Downloaded from Division of DowDuPont, 4010 Point Eden Way, Hayward, CA, 510 780-7156, [email protected] Keywords: molecular evolution, enzyme inhibitor, biotechnology, plant biochemistry, protein engineering, glyphosate, glyphosate resistance, plant EPSP synthase, herbicide resistance, 5- http://www.jbc.org/ enolpyruvylshikimate-3-phosphate synthase Abstract Å of glyphosate, which together reduced Km for 5-Enolpyruvylshikimate-3-phosphate synthase PEP to 43 M. Though the original optimization by guest on December 7, 2018 (EPSPS) catalyzes the transfer of a carboxyvinyl was conducted with maize EPSPS, contextually group from phosphoenolpyruvate (PEP) to homologous substitutions conferred similar shikimate-3-phosphate and in plants is the target properties to the EPSPSs of other crops. We also of the herbicide glyphosate. EPSPSs with high discovered a variant having the known catalytic efficiency and insensitivity to glyphosate-desensitizing substitution P106L plus glyphosate are of microbial origin, including the three additional ones that reduced the Km for PEP enzyme from Agrobacterium strain CP4, in from 47 M, observed with P106L alone, to 10.3 which insensitivity is conferred by an active site M. The improvements obtained with both Ala- alanine. In the sequence context of plant EPSPSs, 101 and Leu-106 have implications regarding alanine in place of glycine at the equivalent glyphosate-tolerant crops and weeds. position interferes with the binding of both glyphosate and PEP. We show here that iterative optimization of maize EPSPS containing the 5-Enolpyruvylshikimate-3-phosphate G101A substitution yielded variants on par with synthase (EPSPS; EC 2.5.1.19) catalyzes the CP4 in terms of catalytic activity in the presence transfer of a carboxyvinyl group from of glyphosate. The improvement relative to phosphoenolpyruvate (PEP) to shikimate-3- G101A alone was entirely due to reduction in Km phosphate (S3P, Figure 1). The enzyme is for PEP from 333 μM to 18 μM, versus 9.5 μM inhibited by glyphosate, competitively with PEP for native maize EPSPS. A large portion of the (1,2). The herbicidal efficacy of glyphosate reduction in Km was conferred by two down- against all plant species, its low cost, low sizing substitutions (L97C and V332A) within 8 mammalian toxicity and benign environmental 1 Optimized, glyphosate-desensitized plant EPSPS impact favor its use in crops that are endowed Depending on the substitution, the degree of with a tolerance mechanism (3-7). Glyphosate desensitization (increase in Ki) and whole plant tolerant crops have been created by expressing resistance are proportional and are in the range of glyphosate-insensitive bacterial EPSPS encoded two- to nine-fold (14,15,17). on a transgene (8,9). A second glyphosate-desensitized EPSPS With gene editing facilitated by variant is a double-mutant maize enzyme in Cas9/CRISPR technology (10), it is theoretically which threonine at position 102 is changed to possible to create glyphosate tolerance without a isoleucine in concert with the P106S mutation. foreign transgene by introducing a set of The enzyme, termed TIPS, is highly desensitized mutations into the EPSPS gene that effectively to glyphosate, but while its Km for desensitizes the enzyme to glyphosate while phosphoenolpyruvate is nearly normal, it has maintaining sufficient catalytic capacity. USDA, only 5% of the kcat of the native enzyme, as shown the American agency regulating genetically here and in earlier studies (18,19). Like the Pro106 modified crops, has given “unregulated” status to mutations, the TIPS mutations exert their effect 101 many traits created with gene editing, without the by shifting Gly closer to the glyphosate binding Downloaded from usual lengthy and costly (~$35 million) process site (18). While catalytic capacity for TIPS required for a transgenic trait EPSPS is insufficient if the mutations are created (https://www.aphis.usda.gov/aphis/ourfocus/biot by natural mutagenesis and gene editing (see echnology/am-i-regulated). Thus, gene editing Discussion), sufficiently high transgenic http://www.jbc.org/ could make glyphosate tolerance economically expression can result in competitive performance, accessible for low volume crops or for staple as in GA21 maize (20). Very recently, a mutation crops in developing countries. In the latter case, resulting in the same threonine (position 102) the hand weeding prevalent in sub-Saharan being changed to serine was shown to account for by guest on December 7, 2018 Africa is typically far from optimal for preserving glyphosate resistance in a tropical weed (21). We yield, is enormously time-consuming and often here provide the first kinetic characterization of results in spinal deformation (11). However, the variant, which shows it to be no fitter than enabling glyphosate tolerance by editing the P106S and thus unsuitable for gene editing. native EPSPS gene will require a set of mutations The other known de-sensitizing that result in a much fitter enzyme than the plant substitution is alanine for glycine at the variants currently known. equivalent of our maize position 101, first The known mutations in plant EPSPS and reported with the enzyme from a glyphosate- close homologs (termed Class I EPSPS) all resistant strain of Klebsiella (22). Alanine occurs involve modulations of active site Gly101 naturally at this position in EPSPS from (numbering according to mature maize EPSPS Agrobacterium strain CP4, the enzyme present in [CAA44974.1, Figure 2]; 96 in E. coli) that can RoundupReadyTM crops (8,9). The CP4 enzyme create interference with the binding of glyphosate is highly insensitive to inhibition by glyphosate through one of its phosphonate oxygens (2). and has 11-15% of the catalytic efficiency 106 Changing Pro to glycine, serine, threonine, (kcat/Km) of the plant enzyme (23,24). Plant leucine or alanine has the effect of moving the EPSPS with the G101A mutation has similar alpha carbon of Gly101 closer to glyphosate, insensitivity to CP4 but only 1.4% of the catalytic causing moderately reduced affinity (12). These efficiency of the native plant enzyme, largely due mutations were discovered by mutagenesis of to the 40-fold increase in Km for PEP imposed by bacterial genes (13) and are present in seven the additional methyl group (23,25). Though the species of glyphosate-resistant weeds (14-16). structures of the CP4 and E. coli enzymes show 2 Optimized, glyphosate-desensitized plant EPSPS that they share the same structural fold and mutations; in all, 43 substitutions at 29 positions. topology (26), they share only 23% identity. In The library was synthesized entirely from contrast, the E. coli enzyme shares >50% identity oligonucleotides, using the technique of synthetic with plant EPSPS. This divergence in sequence shuffling (27). The vector DNA of the library was homology was the basis for designating CP4 and transformed into our BL21(DE3) Tuner-AroA homologous microbial EPSPSs as Class II. The knockout strain and the cells were plated onto divergent amino acid sequence of CP4 has been minimal medium with glyphosate (see Figure S1 thought to provide the structural context for an for details on library design, host cells, screening optimal spatial location of the alanine methyl conditions, advancement criteria). The variant group that results in interference with the longer forms of EPSPS from the 184 colonies that grew glyphosate but not with PEP. were evaluated by enzyme assay and the best of The G101A mutation has not been found those were subjected to substrate saturation in a glyphosate resistant weed, likely due to its kinetic analysis. poor affinity for PEP. We here show that with 17 Three variants each included one of the or more additional mutations, the enzyme from previously known mutations or pair of mutations Downloaded from maize, a Class I EPSPS, can be adapted to that reduce sensitivity of EPSPS to inhibition by accommodate the G101A mutation, resulting in glyphosate. Variant P106L+3 has leucine kinetic parameters equal to or better than those of substituted for proline at position 106 and three CP4. The variants are no closer in homology to additional mutations (A100S, E301S and http://www.jbc.org/ CP4 than is the native enzyme. A337S). Alone, the P106L mutation raised Ki for glyphosate 60-fold, but also raised Km for PEP RESULTS five-fold (Table 1). The three additional Mutagenesis, maize native EPSPS – mutations served to lower Km for PEP from 47 by guest on December 7, 2018 Because the use of degenerate oligonucleotides M, seen with P106L alone, to 10.3 M with for saturation mutagenesis theoretically can 60% retention of Ki. The overall result was 30- access all 19 changes at each position, we began fold improved fitness [(kcat/Km)*Ki] compared to by searching for single desensitizing mutations in native maize EPSPS and seven-fold improved native maize EPSPS that may have been missed relative to P106S. by earlier methodology. Saturation mutagenesis Along with two other mutations (T277G, was performed and positional libraries screened A337S), variant TIPS+2 has the T102I and P106S as described in Experimental Procedures. No new (TIPS) mutations present in the GA21 maize desensitizing mutations were found.
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