Laboratory evolution of an alcohol dehydrogenase towards enantioselective reduction of difficult-to-reduce ketones

Bioresources and Bioprocessing

Yao Nie¹  Huilei Yu²  Ge Qu³  Zhoutong Sun³  Yingying Jiang³  Beibei Liu³ 

[1] School of Biotechnology, Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University;State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology;Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences;

关键词: Directed evolution;    Conformational dynamics;    Alcohol dehydrogenase;    Activity;    Enantioselectivity;   

DOI  :  10.1186/s40643-019-0253-9

来源: DOAJ

【 摘 要 】

Abstract Background A thermostable alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) has been repurposed to perform asymmetric reduction of a series of prochiral ketones with the formation of enantio-pure secondary alcohols, which are crucial chiral synthons needed in the preparation of various pharmaceuticals. However, it is incapable of asymmetric reduction when applied to bulky ketones. Recently, mutations at two key residues A85 and I86 were shown to be crucial for reshaping the substrate binding pocket. Increased flexibility of the active site loop appears to be beneficial in the directed evolution of TbSADH towards difficult-to-reduce ketones. Methods Using the reported mutant A85G/I86A as template, double-code saturation mutagenesis (DCSM) was applied at selected residues lining the substrate binding pocket with a 2-membered reduced amino acid alphabet. Results and conclusions The mutant A85G/I86A was first tested for activity in the reaction of the model substrate (4-chlorophenyl)-(pyridin-2-yl)methanone, which showed a total turnover number (TTN) of 3071. In order to further improve the turnovers, a small and smart mutant library covering a set of mutations at Q101, W110, L294, and C295 was created. Eventually, a triple-mutant A85G/I86A/Q101A was identified to be a superior catalyst that gave S-selective product with 99% ee and 6555 TTN. Docking computations explain the source of enhanced activity. Some of the best variants are also excellent catalysts in the reduction of other difficult-to-reduce ketones.

【 授权许可】

Unknown