Abstract Glyphosate can kill most herbaceous plants by inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity. Glyphosate-resistant (GR) crops can be developed by transforming the EPSPS gene encoding the glyphosate-resistant enzyme into crops. The current commercial application of GR EPSPS is mainly derived from microorganisms, and such "cross-kingdom" gene transfer is more likely to cause people's concerns about the safety of transgenic food. Therefore, improving the EPSPS of crops and then developing GR crops using crop endogenous genes is a desirable strategy to solve this problem. In this study, the Hotspots mutant library of EPSPS of Brassica napus was constructed, and the mutants resistant to high glyphosate levels were screened. 5 mutants with improved herbicide resistance were obtained, among which mBnEPSPS showed the highest glyphosate resistance. Compared with the wild type (BnEPSPS), the kcat of the mutant decreased from 515/s to 257/s, with a decrease of 50%. In the presence of glyphosate, the turnover numbers kcat (gly) of mBnEPSPS and BnEPSPS were 116/s and 13/s, respectively, meaning an 8.9-folds increase in glyphosate resistance. The mutation did not change the adaptation of enzymes to the plant cell microenvironment. After being transformed to tobacco (Nicotiana benthamiana), mBnEPSPS could confer tobacco resistance to roundup at 5 times higher than the commercial recommended dose of Roundup (41% glyphosate-isopropylammonium), suggesting that mBnEPSPS had a high breeding application value. This study provides genetic resources and a theoretical basis for developing herbicide-resistant rape seed through transgenic and gene editing.
KE Da,HE Yun-Hao,WU Fang, et al. Improving Glyphosate-resistance of EPSPS from Brassica napus Mediated by Hotspot Mutation and Its Function Validation[J]. 农业生物技术学报, 2023, 31(5): 889-900.
[1] 李杰华, 端群, 史明涛, 等. 2021. 新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定[J]. 作物学报, 47(05): 778-787.
(Li J H, Duan Q, Shi M T, et al. 2021. Development and identification of transgenic rapeseed with a novel gene for glyphosate resistance[J]. Acta Agronomica Sinica, 47(05): 778-787.)
[2] 梁晋刚, 贺晓云, 武玉花, 等. 2020. 中国农业转基因生物安全标准体系现状与展望[J]. 农业生物技术学报, 28(05): 911-917.
(Liang J G, He X Y, Wu Y H, et al. 2020. Current status and prospects of safety standard system for agricultural genetically modified organisms in China[J]. Journal of Agricultural Biotechnology, 28(05): 911-917.)
[3] 王园园, 王敏, 相世刚, 等. 2018. 全球抗除草剂转基因作物转化事件分析[J]. 农业生物技术学报, 26(01): 167-175+183-257.
(Wang Y Y, Wang M, Xiang S G, et al. 2018. Analysis on the event of global herbicide tolerant transgenic crops[J]. Journal of Agricultural Biotechnology, 26(01): 167-175, 183-257.)
[4] Castle L A, Siehl D L, Gorton R, et al. 2004. Discovery and directed evolution of a glyphosate tolerance gene[J]. Science, 304(5674): 1151-1154.
[5] Chinnadurai P, Stojšin D, Liu K, et al. 2018. Variability of CP4 EPSPS expression in genetically engineered soybean (Glycine max L. Merrill)[J]. Transgenic Research, 27(6): 511-524.
[6] Cui Y, Huang S, Liu Z, et al. 2016. Development of novel glyphosate-tolerant japonica rice lines: A step toward commercial release[J]. Frontiers in Plant Science, 7: 1218.
[7] Dong Y, Ng E, Lu J, et al. 2019. Desensitizing plant EPSP synthase to glyphosate: Optimized global sequence context accommodates a glycine-to-alanine change in the active site[J]. Journal of Biological Chemistry, 294(2): 716-725.
[8] Dreesen R, Capt A, Oberdoerfer R, et al. 2018. Characterization and safety evaluation of HPPD W336, a modified 4- hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FGØ72-2 soybean[J]. Regulatory Toxicology and Pharmacology, 97: 170-185.
[9] Engqvist M K M, Rabe K S. 2019. Applications of Protein Engineering and Directed Evolution in Plant Research[J]. Plant Physiology, 179(3): 907-917.
[10] Eschenburg S, Healy M L, Priestman M A, et al. 2002. How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli[J]. Planta, 216(1): 129-135.
[11] Fartyal D, Agarwal A, James D, et al. 2018. Co-expression of P173S mutant rice EPSPS and igrA genes results in higher glyphosate tolerance in transgenic rice[J]. Frontiers in Plant Science, 9:144.
[12] Funke T, Han H, Healy-Fried M L, et al. 2006. Molecular basis for the herbicide resistance of roundup ready crops[J]. Proceedings of the National Academy of Sciences of the USA, 103(35): 13010-13015.
[13] Funke T, Yang Y, Han H, et al. 2009. Structural basis of glyphosate resistance resulting from the double mutation Thr97-> Ile and Pro101->Ser in 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli[J].Journal of Biological Chemistry, 284(15): 9854-9860.
[14] García-García J D, Van Gelder K, Joshi J, et al. 2022. Using continuous directed evolution to improve enzymes for plant applications[J]. Plant Physiology, 188(2): 971-983.
[15] Gherekhloo J, Fernández-Moreno P T, Alcántara-de la Cruz R, et al. 2017. Pro-106-Ser mutation and EPSPS overexpression acting together simultaneously in glyphosate-resistant goosegrass (Eleusine indica)[J]. Scientific Reports, 7(1):6702.
[16] Giraldo P A, Shinozuka H, Spangenberg G C, et al. 2019. Safety assessment of genetically modified feed: Is there any difference from food?[J]. Frontiers in Plant Science, 10: 1592.
[17] Gosavi G, Ren B, Li X, et al. 2022. A new era in herbicide-tolerant crops development by targeted genome editing[J]. ACS Agricultural Science & Technology, 2(2): 184-191.
[18] Griffin S L, Chekan J R, Lira J M, et al. 2021. Characterization of a glyphosate-tolerant enzyme from Streptomyces svecius: A distinct class of 5-enolpyruvylshikimate-3-phosphate synthases[J]. Journal of Agricultural and Food Chemistry, 69(17): 5096-5104.
[19] Guo B, Guo Y, Hong H, et al. 2015. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean[J]. Frontiers in Plant Science, 6: 847.
[20] Han H, Yu Q, Widderick M J, et al. 2016. Target-site EPSPS Pro-106 mutations: Sufficient to endow glyphosate resistance in polyploid Echinochloa colona?[J]. Pest Management Science, 72(2): 264-271.
[21] Herman R A, Ekmay R D, Schafer B W, et al. 2018. Food and feed safety of DAS-444Ø6-6 herbicide-tolerant soybean[J]. Regulatory Toxicology and Pharmacology, 94: 70-74.
[22] Kaundun S S, Dale R P, Zelaya I A, et al. 2011. A novel P106L mutation in EPSPS and an unknown mechanism (s) act additively to confer resistance to glyphosate in a South African Lolium rigidum population[J]. Journal of Agricultural and Food Chemistry, 59(7): 3227-3233.
[23] Li, H, Yang Y, Hu Y, et al. 2022. Structural analysis and engineering of aldo-keto reductase from glyphosate-resistant Echinochloa colona[J]. Journal of Hazardous Materials, 436: 129191.
[24] Liang C, Sun B, Meng Z, et al. 2017. Co-expression of GR79 EPSPS and GAT yields herbicide-resistant cotton with low glyphosate residues[J]. Plant Biotechnology Journal, 15(12): 1622-1629.
[25] Light S H, Krishna S N, Minasov G, et al. 2016. An unusual cation-binding site and distinct domain-domain interactions distinguish class Ⅱ enolpyruvylshikimate-3-phosphate synthases[J]. Biochemistry, 55(8): 1239-1245.
[26] Liu D, Yu L, Wei L, et al. 2021. BnTIR: An online transcriptome platform for exploring RNA-seq libraries for oil crop Brassica napus[J]. Plant Biotechnology Journal, 19(10): 1895-1897.
[27] Mao C, Xie H, Chen S, et al. 2017. Error-prone PCR mutation of Ls-EPSPS gene from Liriope spicata conferring to its enhanced glyphosate-resistance[J]. Pesticide Biochemistry and Physiology, 141: 90-95.
[28] McElroy J S, Hall N D. 2020. Echinochloa colona with reported resistance to glyphosate conferred by Aldo-Keto reductase also contains a Pro-106-Thr EPSPS target site mutation[J]. Plant Physiology, 183(2): 447-450.
[29] Ouyang C, Liu W, Chen S, et al. 2021. The naturally evolved epsps from goosegrass confers high glyphosate resistance to rice[J]. Frontiers in Plant Science, 12: 756116.
[30] Pan C, Wu X, Markel K, et al. 2021. CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants[J]. Nature Plants, 7(7): 942-953.
[31] Qin Y, Wu G, Guo Y, et al. 2020. Engineered glyphosate oxidase coupled to spore-based chemiluminescence system for glyphosate detection[J]. Analytica Chimica Acta, 1133: 39-47.
[32] Rao A G. 2008. The outlook for protein engineering in crop improvement[J]. Plant Physiology, 147(1): 6-12.
[33] Sumbalova L, Stourac J, Martinek T, et al. 2018. HotSpot Wizard 3.0: web server for automated design of mutations and smart libraries based on sequence input information[J]. Nucleic Acids Research, 46(W1): W356-W362.
[34] Wang C, Glenn K C, Kessenich C, et al. 2016. Safety assessment of dicamba mono-oxygenases that confer dicamba tolerance to various crops[J]. Regulatory Toxicology and Pharmacology, 81: 171-182.
[35] Xiao P, Liu Y and Cao Y. 2019. Overexpression of G10-EP-SPS in soybean provides high glyphosate tolerance[J]. Journal of Integrative Agriculture, 18(8): 1851-1858.
[36] Yu H, Ma S, Li Y, et al. 2022. Hot spots-making directed evolution easier[J]. Biotechnology Advances, 56: 107926.
[37] Zhan T, Zhang K, Chen Y, et al. 2013. Improving glyphosate oxidation activity of glycine oxidase from Bacillus cere-us by directed evolution[J]. PLOS ONE, 8(11): e79175.
