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.

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

“…A number of studies explored fusions of ADHs with NAD(P)H‐regenerating enzymes. Three studies fused formate dehydrogenase with a specific ADH, and in one study glucose dehydrogenase was used . Even though fusing such dehydrogenases with one another can be more challenging, owing to the typical quaternary structures of these enzymes, these studies reported successful examples of active enzyme fusions with alcohol dehydrogenases.…”

Oxidoreductase fusions: engineering enzymes for coupled reactions and stability

“…Three studies fused formate dehydrogenase with a specific ADH [37] , and in one study glucose dehydrogenase was used. [38] Even though fusing such dehydrogenases with one another can be more challenging, due to the typical quaternary structures of these enzymes, these studies reported on successful examples of active enzyme fusions with alcohol dehydrogenases.…”

“…[35] The PpKT2440-BVMO could be applied in combination with an ADH from Micrococcus luteus to convert ricinoleic acid to (Z)-11-(heptanoyloxy)undec-9-enoic acid (Scheme 2a). However, this BVMO was difficult to express in E. coli, and to improve expression several strategies were explored, including: use of chaperones [36,37] , enzyme fusion [37,38] , polyionic fusion tag engineering, and use of constitutive promotor [39] . At this point, through the polyionic tag and constitutive promotor, the reaction could reach a product concentration of 21.9 g/L on a 70 L scale.…”

“…[78][79][80] In 2013, Jeon et al developed fusions of ADHs with BVMOs to convert hydroxy fatty acids into esters, in whole cells expressing the fusion enzyme (Scheme 2a). [38] The authors could demonstrate that the fused enzyme had a higher level of conversion for the cascade reaction. A similar pair of ADH with TmCHMO was fused to produce -caprolactone from cyclohexanol (C Chapter 3).…”

“…a) Conversion of hydroxylated long-chain fatty acids to produce esters. [36][37][38][39][40][41][42][43] The cascade could also start from an unsaturated precursor with a hydratase to make the hydroxyl group, [37] or with a P450 to perform hydroxylation. b) Convergent cascade analogous to the reaction displayed in scheme 1, red box.…”