*4.2. Preparation of (Z)-Citral and (E)-Citral*

(*Z*)-citral and (*E*)-citral were prepared by a modification of the procedure described previously [38]. Activated MnO2 (1.15 g) was added into a 50-mL three-necked bottom round flask which atmosphere was replaced with N2. One-hundred milligrams of geraniol or nerol was dissolved in 16 mL dry hexane and was charged in the flask to initiate the alcohol oxidation. The reaction was maintained at 0 ◦C and 450 rpm for 6 h. Then, the reaction solution was filtered through a filter paper and hexane in the filtrate was removed by vacuum evaporation at 45 ◦C. Finally, an aliquot of the collected product (*E*)-citral or (*Z*)-citral was dissolved in ethyl acetate and subjected to the analyses of gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS).

#### *4.3. Cloning, Expression, and Purification of OYE2y*

The gene encoding OYE2y was PCR-amplified from the genomic DNA of *S. cerevisiae* CICC1060 using a set of primers: Forward, 5 -ATGCCATTTGTTAAGGACTTTAAGCCAC-3 ; Reverse, 5 -TTAATTTTTGTCCCAACCGAGTTTTAGAGC-3 . The conditions for PCR amplification of the *oye2y* gene were 94 ◦C for 2 min for initial denaturalization, 30 cycles of 94 ◦C for 30 s, 57 ◦C for 30 s, 72 ◦C for 80 s, and 72 ◦C for 10 min for the final extension.

Following the procedure of expression and purification of ReBDH [39], the PCR products were purified and then ligated with the expression vector pEASY-E1 through the AT ligation strategy. The recombinant plasmid harboring the *oye2y* gene, designated as pEASY-E1-*oye2y*, was verified by DNA sequencing (Sangon Biotech, Shanghai, China) and then transformed into *E. coli* BL21 (DE3) competent cells, resulting in the recombinant strain *E. coli* BL21(DE3)/pEASY-E1-*oye2y*. The recombinant cells containing pEASY-E1-*oye2y* were grown in the LB medium with 100 μg/mL ampicillin at 37 ◦C and 200 rpm until the OD600 was 0.6–0.8, and then 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was supplemented to initiate the induction at 23 ◦C and 160 rpm. After 12 h of growth, recombinant *E. coli* cells were harvested by centrifugation and further washed using 50 mM Tris-HCl buffer (pH 8.0). The cells were disrupted through ultrasonication for 10 min, and the cell debris and cell lysate were removed by centrifugation to result in a clear cell extract. The crude cell extracts containing OYE2y was applied to a Ni-NTA chelating affinity column equilibrated with the binding buffer (5 mM imidazole and 300 mM NaCl dissolved in 50 mM Tris-HCl, pH 8.0). Unbound proteins were washed off by the application of the binding buffer. The recombinant OYE2y was eluted with 100 mM imidazole in 50 mM Tris-HCl (pH 8.0), desalted with 50 mM Tris-HCl buffer (pH 8.0) by ultrafiltration and then stored at −20 ◦C for further study.
