Development of MEMS directed evolution strategy for multiplied throughput and convergent evolution of cytochrome P450 enzymes
Development of MEMS directed evolution strategy for multiplied throughput and convergent evolution of cytochrome P450 enzymes
复制标题
开发MEMS定向进化策略以实现细胞色素P450酶的倍增通量和收敛进化
DOI：
10.1007/s11427-021-1994-1
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发表时间：
2021-08-31
影响因子：
9.1
通讯作者：
Li，Shengying
中科院分区：
文献类型：
--
作者：
Ma，Li;Li，Fengwei;Li，Shengying
受自然进化（NE）启发的定向进化（DE）在蛋白质/酶工程中取得了巨大的成功。然而，传统的“一蛋白一任务”DE在筛选通量和效率方面无法与“多蛋白多任务”NE相匹配，因此通常无法满足对具有所需性质的生物催化剂的快速增长的需求。本研究设计了一种新型的“多酶多底物”（multi-enzymes-for-multi-substrates，MEMS）DE模型，并在“两个P450对七个底物”（2 P ×7S）的基础上，通过一锅法模拟NE和高通量筛选，建立了概念验证。随着通量的倍增和命中率的提高，我们在实验室中目睹了两种原型细胞色素P450酶（P450 BM 3和P450 cam）的一系列趋同进化事件。预计MEMS DE的新策略将在未来为更大的酶库找到更广泛的应用。此外，两个功能收敛的P450变体的结构和底物对接分析为不同的P450活性位点如何达到共同的催化目标提供了重要的见解。
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.