Combinatorial evolution of phosphotriesterase toward a robust malathion 
degrader by hierarchical iteration mutagenesis

Luo, Xiao-Jing; Zhao, Jian; Li, Chun-Xiu; Bai, Yun-Peng; Reetz, Manfred T.; Yu, Hui-Lei; Xu, Jian-He

doi:10.1002/bit.26012

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Combinatorial evolution of phosphotriesterase toward a robust malathion degrader by hierarchical iteration mutagenesis

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Reetz, Manfred T.
Philipps-Universität Marburg, Fachbereich Chemie;
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;

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http://onlinelibrary.wiley.com/doi/10.1002/bit.26012/epdf
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Citation

Luo, X.-J., Zhao, J., Li, C.-X., Bai, Y.-P., Reetz, M. T., Yu, H.-L., et al. (2016). Combinatorial evolution of phosphotriesterase toward a robust malathion degrader by hierarchical iteration mutagenesis. Biotechnology and Bioengineering, 113(11), 2350-2357. doi:10.1002/bit.26012.

Cite as: https://hdl.handle.net/11858/00-001M-0000-002B-98E7-E

Abstract

Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPH_M2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR-based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling. Finally, guided by in silico energy calculations (FoldX), thermostability of the variant was improved. Themutations extend from the core region to the enzyme surface during the evolutionary pathway. After screening <9,000 mutants, the best variant PoOPH_M9 showed 25-fold higher activity than wild-type PoOPH_M2, with a thermostability (T50 15) of 67.6C. Thus, PoOPH_M9 appears to be an efficient and robust candidate for malathion detoxification.