Enhancement of asymmetric bioreduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine to corresponding (S)-enantiomer by fusion of carbonyl reductase and glucose dehydrogenase

Abstract

Background: (S)-(−)-N,N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine (DHTP) is a key intermediate for the preparation of (S)-duloxetine, an important antidepressant drug. However, so far, the catalytic efficiency of (S)-DHTP synthesis by asymmetric bioreduction is yet limited. The present study aims to develop an efficient system for synthesis of (S)-DHTP by bioreduction. Results: Various recombinant carbonyl reductases were evaluated for asymmetric reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP) to produce (S)-DHTP. The NADPH-dependent carbonyl reductase CR2 was identified as the suitable candidate, giving (S)-DHTP in absolute configuration. Then the fusion protein involving CR2 and glucose dehydrogenase (CR2-L-GDH) was constructed to further improve cofactor regeneration and resulted catalytic efficiency of the enzymatic reduction. By studying the effects of reaction conditions involving cofactor regeneration, suitable catalytic system was achieved for CR2-L-GDH catalyzing (S)-DHTP synthesis. Consequently, (S)-DHTP (>99.9% e.e.) with yield of 97.66% was obtained from 20 g L−1 DKTP within 8-h reaction, employing 40 g L−1 glucose and 0.1 mmol L−1 NADP+ to drive the cofactor regeneration, resulting in the space–time yield of 2.44 g L−1 h−1. Conclusion: Optically pure (S)-DHTP with improved yield was obtained by fusion enzyme CR2-L-GDH. Fusion enzyme-mediated biocatalytic system would be promising to enhance reaction efficiency of enzyme-coupled system for preparation of optically active alcohols.