*2.1. OYE2y-Mediated Reduction of (E/Z)-Citral*

The yeast Old Yellow Enzyme OYE2y was heterologously expressed in *Echerichia coli* BL21(DE3), and the resulting recombinant OYE2y with *N*-terminal His tag was purified using affinity chromatography. The enzyme OYE2y with 400 amino acid residues shared the sequence identities of 91.50%, 98.75%, and 81.25% to OYE1 from *S. pastorianus* and OYE2 and OYE3 from *S. cerevisiae* [14], respectively. To investigate the enantioselectivity of OYE2y in citral reduction, the purified OYE2y rather than the whole-cell biocatalyst was used to avoid side reactions. OYE2y accepted NADH or NADPH as coenzyme, and formate/formate dehydrogenase system was used for NADH regeneration in this study. The composition of (*E*/*Z*)-citral was determined to contain 58.45% geranial and 41.55% neral. During the first 3 h, the concentration of geranial decreased significantly faster than that of neral. Meanwhile, the concentration of (*R*)-citronellal increased rapidly, and the *e.e.* value was kept at a higher level of >60% (*R*) (Figure 1A). Then, the conversion rate of neral turned faster from 3 h to 5 h, resulting in the decreasing of *e.e.* value from 65.02% (*R*) to 40.26% (*R*). After 6 h, the conversion rate of neral was nearly parallel to that of geranial, meanwhile the *e.e.* values were kept almost constant. The 11 h reaction was completed with 89.51% yield, giving (*R*)-citronellal with an *e.e.* value of 38.13% (*R*). The time course of (*E*/*Z*)-citral reduction clearly indicated that the ratio of geranial and neral significantly affected the *e.e.* value of (*R*)-citronellal, which was consistent with previous observations [7,14]. In addition, the isomerization of geranial and neral occurred under some conditions [9,30]. Thus, the use of freshly prepared geranial and neral with high purity was necessary to study the enantioselectivity of OYEs.

(**A**) **Figure 1.** *Cont*.

**Figure 1.** Asymmetric reduction of (*E*/*Z*)-citral (**A**), (*E*)-citral (**B**), and (*Z*)-citral (**C**) using the purified OYE2y. -, (*R*)-citronellal; -, (*S*)-citronellal; , geranial; , neral; , the *e.e.* value of hydrogenated product. Data present mean values ± SD from three independent experiments.

(**C**)

Considering the high cost of commercial products, geranial and neral with high purity were prepared according to the previous procedure with improvements [31]. Based on the optimized conditions, the yields of (*E*)-citral and (*Z*)-citral were increased up to 99.39% and 99.35%, respectively. The obtained (*E*)-citral sample contained 98.38% geranial and 1.62% neral, and the obtained (*Z*)-citral sample contained 96.84% neral and 3.16% geranial. When the enzyme OYE2y was tested with newly prepared (*E*)-citral or (*Z*)-citral for 4 h, the enantioselectivity of OYE2y stayed at a relatively constant level (Figure 1B,C). The (*E*)-citral and (*Z*)-citral-derived *e.e.* values after 11 h reduction were 82.87% (*R*) and 32.66% (*S*), respectively.
