Bioreductive production of enantiopure (S)-duloxetine intermediates catalyzed with ketoreductase ChKRED15

• Ketoreductase ChKRED15 catalyzed the bioreduction of five duloxetine intermediates.
• (S)-Alcohols were achieved with >99% ee for all five intermediates.
• The S12G mutant was constructed to recover the G-rich motif.
• The S12G mutant displayed improved activity and stability.
• Up to 50 g substrate/l could be completely reduced within 24 h.

The blockbuster antidepressant drug duloxetine contains one stereo-center derived from chiral alcohol intermediates. The stereoselective bioreductive production of five of such intermediates could be achieved using the recombinant ketoreductase ChKRED15, yielding the enantiopure (S)-alcohols with >99% ee. Sequence alignment indicated that ChKRED15 lacks the conserved G-rich motif, which was then amended by a single mutation of S12G. The resulting S12G mutant displayed significantly improved catalytic activity and protein stability. When coupled with a cofactor recycling system, the S12G mutant was able to catalyze the complete conversion of ethyl 3-oxo-3-(thiophen-2-yl)propanoate within 6 h and N-methyl-3-oxo-3-(thiophen-2-yl)propanamide within 24 h at substrate concentrations of 10 and 50 g/l, respectively, without the compromise of enantioselectivity.

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