Development of MEMS directed evolution strategy for multiplied throughput and convergent evolution of cytochrome P450 enzymes.

Autor: Ma, Li, Li, Fengwei, Zhang, Xingwang, Chen, Hui, Huang, Qian, Su, Jing, Liu, Xiaohui, Sun, Tianjian, Fang, Bo, Liu, Kun, Tang, Dandan, Wu, Dalei, Zhang, Wei, Du, Lei, Li, Shengying
Zdroj: SCIENCE CHINA Life Sciences; Mar2022, Vol. 65 Issue 3, p550-560, 11p
Abstrakt: 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. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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