Abstract

       N-Boc-3-pyrrolidinol is an important pharmaceutical intermediate with high economic value for both R- and S-types. Traditional chemical production methods are often costly because of the difficulty in splitting the optical isomers. The use of enzymatic asymmetrical catalysis of pre-chiral ketones for asymmetric reduction to produce optically active alcohols has high industrial application. We selected an Alcohol dehydrogenase from a thermophilic microorganism Thermoethanolicus brockii (TbsADH), which has been reported in previous work to perform expertly selective asymmetric reduction, but its reaction rate is low. Directed evolution is a powerful means to modify biological macromolecules and can be designed and screened according to the needs of the target function. Among them, structure-based rational design and continuous directed evolution systems using the organism itself can greatly reduce the size of mutation libraries and save time. We constructed an EvolvR-based sequential directed evolution system with rational design based on the structure of the enzyme to obtain TbsADH catalyzing the asymmetric reduction of N-Boc-3-pyrrolidone to N-Boc-3-pyrrolidinol.