*4.5. Homology Modeling and Molecular Docking*

Using the crystal structure of OYE1 from *S. pastorianus* (PDB number: 1OYB) as a template [40], the structural model of OYE2y was obtained by a homology modeling strategy [41,42]. Molecular docking simulation was performed by AutoDock Vina [43] when the binding package was set at a distance of 15 Å from FMN N5 atom. (*E*)-citral or (*Z*)-citral acted as a ligand to molecular docking with OYE2y, and the calculation of geometric parameters and ligand structure was performed by ChemBioDraw 12.0 (CambridgeSoft, Cambridge, MA, USA). To make the results more accurate, 100 consecutive runs were performed and the highest ranked score from each run was used to calculate the average score of each flexible ligand configuration. The optimal configuration and the resulting substrate–enzyme complexes were further processed using the PYMOL software [44]. The candidate complexes were acceptable when they met both criteria: (1) the substrate carbonyl oxygen should be capable of forming hydrogen bonds with the side chains of both H192 and N195 and (2) the distance between the FMN N5 atom and the β-unsaturated carbon of the substrate molecule was be in an appropriate range from 3.5 Å to 4.1 Å [22,45,46].

## *4.6. Asymmetric Reduction of Citral Mediated by OYE2y or Its Variants*

The reaction mixture (1 mL) contained 50 mM PIPES buffer (pH 7.0), 20 mM substrate, 1 mg OYE2y or its variant, 0.6 U formate dehydrogease from *Candida boidinii* (FDHCB), and 100 mM sodium formate, 0.96 mM NAD+. The substrate included (*Z*)-citral, (*E*)-citral, or (*E*/*Z*)-citral, which stock solution was 200 mM substrate in isopropanol. The overexpression and purification of FDHCB were conducted according to the procedure described previously [39]. The reaction was conducted at 30 ◦C and 200 rpm for 11 h, unless otherwise specified. The reaction mixture was centrifuged to remove the cells, and the resulting supernatant was extracted with equal volume of ethyl acetate at 30 ◦C and 200 rpm for 2 h. Finally, the solvent phase was collected, dried over anhydrous sodium sulfate and subjected to the analyses of GC and GC–MS.
