Kinetic Resolution of Citronellol by an Engineered Alcohol Oxidase for the Production of (R)-Citronellal and (S)-Citronellol.

(R)-citronellal is an important intermediate for aroma compounds and pharmaceuticals. Herein, we engineered the choline oxidase, AcCO6, from Arthrobacter chlorophenolicus, to enhance enantioselective oxidation of citronellol. Through site-directed and combinatorial mutagenesis, a quadruple mutant, AcCO6_M4a (W332V/A89S/W331F/N463E) was obtained, exhibiting 336% of the wild-type activity and a 10.2-fold improvement in enantioselectivity (E = 24.2). Structural analysis and MD simulations revealed that the mutations expanded the binding pocket, promoting favorable interactions with (R)-citronellol and enhancing hydride transfer, while disfavoring the (S)-enantiomer. Consequently, the catalytic efficiency (kcat/KM) for (R)-citronellol was 12.5-fold higher than that for (S)-citronellol. Kinetic resolution of racemic citronellol using AcCO6_M4a achieved approximately 50% conversion, yielding (R)-citronellal (86.7% ee) and (S)-citronellol (97.9% ee) with 39.1% and 42.0% yield, respectively. This work demonstrates the potential of engineered AcCO6 for enantioselective biocatalysis, offering an efficient route to synthesize chiral alcohols and aldehydes as intermediates for fragrances and pharmaceuticals.