Improving Glyphosate Oxidation Activity of Glycine Oxidase from Bacillus cereus by Directed Evolution

PLoS One

Zongze Shao¹  Lili Zhang²  Yongjun Lin³  Gaobing Wu³  Kai Zhang³  Pei Yao³  Ziduo Liu³  Yangyan Chen³  Tao Zhan³ 

[1] Key Laboratory of Marine Biogenetic Resources, the Third Institute of Oceanography, State Oceanic Administration, Xiamen, Fujian, P. R. China;Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production and Construction Corps, College of Life Science, Tarim University, Alar, Xinjiang, P. R. China;State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, P. R. China

关键词: Glycine;    Mutagenesis;    Polymerase chain reaction;    Enzymes;    DNA recombination;    Directed evolution;    Library screening;    Site-directed mutagenesis;   

DOI  :  10.1371/journal.pone.0079175

学科分类：医学（综合）

来源: Public Library of Science

【 摘 要 】

Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg51 mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

【 授权许可】

CC BY