MEMS directed evolution of two cytochrome P450 enzymes revealing distinct active-sites for convergent functions
Year: 2020
Type: preprint
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-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’ (2P×7S) 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.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: Shandong University, National Natural Science Foundation of China, Natural Science Foundation of Shandong Province, State Key Laboratory of Bio-organic and Natural Products Chemistry, Polit National Laboratory for Marine Science and Technology, National Key Research and Development Program of China
Awards: G1265531126, G2006013661, G2369821998, G257190257, G2627400879, G2824056657, G3100123456, G3491320212, G3545758815, G3752987688, G3798588097, G4125961296, G5483601126, G5497257764, G5628523262, G5722093652, G5872809721, G6011068467, G6343841034, G6487758424, G6918640351, G7068635514, G7110715537, G7226114419, G8345084114, G8441976522, G8553075738, G8597848909, G865850907
OpenAlex ID: W3116313985