The synthesis of chiral intermediates for the traditional antidepressant duloxetine has gained significant attention as the number of depressed patients continues to grow. S - N , N -Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamide ( S -DHTP) is a critical intermediate in the synthesis of duloxetine, and the chemical synthesis process is complex and environmentally unfriendly. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a major cost driver in the biocatalytic production of S -DHTP from N , N -Dimethyl-3-keto-3-(2-thienyl)-1-propanamide (DKTP). Here, we successfully modified the coenzyme preference of an aldo-keto reductase (AKR7-2-1) to use the cheaper reduced nicotinamide adenine dinucleotide (NADH) through a coenzyme preference modification approach. We utilized protein engineering to create a superior mutant, Y53F, which increased the coenzyme specificity of AKR7-2-1 by 875-fold and improved its thermal stability , enhancing its potential for industrial applications. Molecular dynamics simulations were performed to demonstrate the effect of mutations at key sites on the protein, revealing the altered coenzyme preference and increased thermal stability from structural and energetic changes. This study validates the viability of the coenzyme preference modification strategy for aldo-keto reductase, offering valuable insights for fellow researchers and guiding future investigations.
In recent years, with the increasing number of patients with depression, the efficient synthesis of the first-line antidepressant drug duloxetine intermediate ( S-N,N- dimethyl-3-hydroxy-3-(2-thienyl)−1-propanamine, S -DHTP) has attracted great attention. The wild-type AKR3–2–9 from Bacillus megaterium YC4-R4 exhibits high application potential of catalyzing N,N -dimethyl-3-keto-3-(2-thienyl)−1-propanamine (DKTP) to prepare S -DHTP, but there is still much room for improvement. In this work, rational design was carried out to enhance the catalytic potential of AKR3–2–9. Notably, compared to the wild-type AKR3–2–9, three mutants (Ile189Val, Asn256Asp, and Ile189Val + Asn256Asp) were obtained, and their catalytic efficiencies were increased by 1.3 times, 2.3 times, and 1.31 times, respectively. Besides, the thermal stability and organic solvent resistance were improved. More importantly, when the concentration of the substrate DKTP was 0.5 g/L, the catalytic yields of Ile189Val, Asn256Asp and Ile189Val + Asn256Asp were increased by 1.45 times, 1.86 times, and 2.05 times, respectively. Besides, the corresponding optical purities of the three mutants were 92.7 %, 94.3 % and 93.8 %. The above results indicated that the rational design of the AKR of Bacillus megaterium YC4-R4 enhanced its potential for biocatalytic preparation of S -DHTP.
危险品化学品经营许可证（带存储）