Abstract
Directed evolution (DE) inspired by natural evolution (NE) has been achieving tremendous successes in protein/enzyme engineering. However, the conventional “one-protein-for-one-task” DE cannot match the “multi-proteins-for-multi-tasks” NE in terms of screening throughput and efficiency, thus often failing to meet the fast-growing demands for biocatalysts with desired properties. In this study, we design a novel “multi-enzymes-for-multi-substrates” (MEMS) DE model and establish the proof-of-concept by running a NE-mimicking and higher-throughput screening on the basis of “two-P450s-against-seven-substrates” (2P×7S) in one pot. With the multiplied throughput and improved hit rate, we witness a series of convergent evolution events of the two archetypal cytochrome P450 enzymes (P450 BM3 and P450cam) in laboratory. It is anticipated that the new strategy of MEMS DE will find broader application for a larger repertoire of enzymes in the future. Furthermore, structural and substrate docking analysis of the two functionally convergent P450 variants provide important insights into how distinct P450 active-sites can reach a common catalytic goal.
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Acknowledgements
This work was supported by the National Key Research and Development Program of China (2019YFA0706900), the National Natural Science Foundation of China (32025001, 31872729, 31600045, 32071266, 31800664, 82022066, and 31800041), the Natural Science Foundation of Shandong Province, China (ZR2019ZD20, ZR2016CQ05, and ZR2019QC009), the Laboratory for Marine Drugs and Bioproducts of Pilot National Laboratory for Marine Science and Technology (Qingdao) (LMDBKF-2019-01), the Tianjin Synthetic Biotechnology Innovation Capability Improvement Project (TSBICIP-KJGG-001), the State Key Laboratory of Bio-organic and Natural Products Chemistry (SKLBNPC18242), the Fundamental Research Funds of Shandong University (2019GN030 and 2019GN033), and the Foundation of Qilu University of Technology of Cultivating Subject for Biology and Biochemistry (No. 202014). We would like to thank Zhifeng Li, Jing Zhu and Jingyao Qu from the State Key laboratory of Microbial Technology of Shandong University for help and guidance in GC-MS and HRMS analysis.
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Development of MEMS directed evolution strategy for multiplied throughput and convergent evolution of cytochrome P450 enzymes
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Ma, L., Li, F., Zhang, X. et al. Development of MEMS directed evolution strategy for multiplied throughput and convergent evolution of cytochrome P450 enzymes. Sci. China Life Sci. 65, 550–560 (2022). https://doi.org/10.1007/s11427-021-1994-1
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DOI: https://doi.org/10.1007/s11427-021-1994-1