Asymmetric Reduction of (E/Z)-citral to (R)-Citronellal by a Cyanobacterial-Derived Old Yellow Enzyme, CtOYE

(R)-citronellal is a key intermediate in the synthesis of L-menthol, one of the world’s three most important spices. It can be obtained by asymmetric reduction of (E/Z)-citral using old yellow enzymes (OYEs), but catalysis by the wild-type enzyme suffers from poor enantioselectivity as well as low efficient conversion. A protein-directed evolution technique was used to enhance the activity and enantioselectivity of CtOYE from Chroococcidiopsis thermalis. Upon the sequence alignment and molecular docking results, two amino acid residues at positions 103 and 351 were selected as saturation mutation sites. Finally, three single substitution variants of CtOYE-W103E, CtOYE-W103M, CtOYE-W103P with improved enantioselectivity (ee > 99%) and moderate conversion (>71%), and one double substitution variant CtOYE-Y351Q/W103E with high enantioselectivity (ee > 99%) and high conversion (83.2%) for reduction of (E/Z)-citral were obtained.