MEMS directed evolution of two cytochrome P450 enzymes revealing distinct active-sites for convergent function

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-enzyme-for-multi-substrate (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 (2Px7S) in one pot. With the significantly improved throughput and hit-rate, we witness a series of convergent evolution events of the two archetypal cytochrome P450 enzymes (P450 BM3 and P450cam) in laboratory. Further structural analysis of the two functionally convergent P450 variants provide important insights into how distinct active-sites can reach a common catalytic goal.