Gene mining and characterization of an aromatic heterocyclic ketone reductase from Modestobacter roseus

Abstract:

[Background] (R,S)-2-chloro-1-(6-fluoro-3,4-dihydro-2H-1-benzopyran-2-yl)-ethanol [NEB-8(R,S)] is an important chiral intermediate for the synthesis of nebivolol. It is synthesized by asymmetric reduction of aromatic heterocyclic ketones under the catalysis of ketoreductase. However, the low catalytic efficiency and poor enantioselectivity of natural ketoreductases and the unclear source of commercial ketoreductases result in low conversion rates. [Objective] To develop a new method for the asymmetric synthesis of NEB-8(R,S) with high enantioselectivity, good stability, and high catalytic efficiency. [Methods] We mined a ketoreductase gene mradh from Modestobacter roseus based on the existing protein sequences of ketoreductases, expressed this gene in Escherichia coli BL21(DE3), and determined the enantioselectivity of the protein. Furthermore, we characterized the enzymatic properties and optimized its reaction conditions under high concentrations of substrate. [Results] In this study, a novel ketoreductase MrADH was identified which belongs to the medium-chain alcohol dehydrogenase superfamily and exhibits both oxidative and reductive activities. The oxidation and reduction activities of MrADH were the highest at pH 9.5 and pH 7.0, respectively. MrADH had good reduction and oxidation activities for ketoesters, meta-substituted aromatic ketones, heterocyclic ketones, and secondary alcohols. In addition, it had a half-life of up to 207.0 h at 40 ℃, demonstrating good thermal stability. MrADH efficiently catalyzed the reduction of 600 mmol/L (S)-2-chloro- 1-(6-fluoro-3,4-dihydro-2H-1-benzopyran-2-yl)-ethanone to synthesize the product alcohol with the enantiomeric excess (e.e.) near 100% and a conversion rate of 99.7%. [Conclusion] The ketoreductase from M. roseus can efficiently synthesize high-optical purity intermediate of nebivolol hydrochloride, laying a foundation for the synthesis of nebivolol hydrochloride.