Modification of carbonyl reductase based on substrate pocket loop regions alteration: an application for synthesis of duloxetine chiral intermediate in high efficiency

Duloxetine, a prominent 5-hydroxytryptamine norepinephrine reuptake inhibitor, is deployed mainly in the management of adult depression, showcasing minimal side effects, swift therapeutic onset, and a robust safety profile. Ethyl ( S )-3-hydroxy-3-(2-thienyl)propionate (( S )-HEES) is the crucial chiral intermediate for duloxetine production. Asymmetric synthesis of ( S )-HEES using carbonyl reductase as the biocatalyst has exhibited advantages including mild reaction conditions, high catalytic efficiency and environmental friendliness. In the present study, a loop region alteration strategy was developed to screen for a carbonyl reductase for ( S )-HEES synthesis and Ea SDR6 from Exiguobacterium sp. s126 was identified with considerable catalytic performance and broad substrate adaptability. Site-directed mutagenesis was subsequently performed, Mut-R142A/N204A was identified with a 3.6-fold enhancement in activity relative to the wild-type Ea SDR6. The mutant k cat value was 52.5 s −1 , 2.9-fold compared to the wild type, and the total catalytic efficiency ( k cat / K M ) was 24.9 mM −1  s −1 , 1.9-fold higher than the wild type. The n -butyl acetate–aqueous biphasic bioreaction system was established for the asymmetric synthesis of ( S )-HEES with the conversion of ethyl 3-oxo-3-(2-thienyl)propionate (KEES) of 90.2%, the product e.e. of > 99% after 8 h reaction at a substrate concentration of 200 g/L. The spatiotemporal yield reached 22.5 g/(L·h), which was higher than the ever reports about ( S )-HEES biosynthesis. The present research provides new knowledge and technology for the construction of stereoselective carbonyl reductase and the green biosynthesis of chiral alcohol pharmaceutical intermediates. Graphical abstract