**1. Introduction**

(*R*)-citronellal is a valuable intermediate for the synthesis of L-menthol through an acidic ene-cyclization and subsequent hydrogenation [1–3]. The potential of (*R*)-citronellal was also explored for the synthesis of natural vitamin E—a kind of fat-soluble vitamin with relatively high antioxidant ability [4,5]. The commercial Takasago process of (*R*)-citronellal began with myrcene to form an allylic amine, which underwent asymmetric isomerization in the presence of a 2,2 -bis(diphenylphosphino)-1,1 -binaphthyl(BINAP)-Rh complex and subsequent hydrolysis with acid to give enantiomerically pure (*R*)-citronellal [6]. In contrast to the three-step asymmetric synthesis from myrcene, the one-step enantioselective reduction of natural citral (the crude mixture of 60% geranial

and 40% neral) was a simplified process for the synthesis of (*R*)-citronellal [7]. The enantioselective hydrogenation of (*E*/*Z*)-citral to afford an identical enantiomer remained challenging since the reduction of the geometric isomers geranial and neral by the same catalyst usually yielded the enantiocomplementary products. In organocatalysis, the enantioselective hydrogenation of (*E*/*Z*)-citral to yield (*R*)-citronellal required the use of a dual catalyst system comprising of Pd/BaSO4 and chiral 2-diarylmethylpyrrolidine [8]. However, the obtained (*R*)-citronellal with 89% *e.e.* was insufficient for broad industrial applications.

To develop a greener and cost-effective alternative to organocatalysis, Old Yellow Enzymes (OYEs; EC 1.6.99.1) as biocatalysts have been widely investigated, which are capable of catalyzing the C=C bond reduction of α,β-unsaturated compounds such as (*E*/*Z*)-citral [9–13]. Past efforts have been made on the discovery of new, improved biocatalysts for suitable enantioselectivity and activity. Bacterial OYEs commonly produced (*S*)-citronellal from (*E*/*Z*)-citral reduction, while the counterparts from yeasts mainly afforded to (*R*)-enantiomer [10]. Representative yeast OYEs have been well characterized, including OYE2.6 from *Pichia stipites* [9], OYE1 from *Saccharomyces pastorianus*, and OYE2 and OYE3 from *Saccharomyces cerevisiae* [14,15]. So far, the application of OYE-mediated citral reduction still suffers from insufficient enantioselectivity and activity. Protein engineering has emerged as an attractive and powerful strategy for improving enzyme activity and selectivity [16–19]. The circular permutation of OYE1 from *S. pastorianus* yielded the variants exhibiting over an order of magnitude improved catalytic activity [20]. The activity improvement in the protein engineering of yeast OYEs commonly varied by substrate. The variant P295A of OYE1 from *S. pastorianus* showed three- and seven-fold activity for (*R*)- and (*S*)-carvone higher than those of wild type enzyme, respectively; however, it had no significant improvement for geranial and neral [21].

With regard to the alteration of OYE enantioselectivity, one of representative examples was the variant W116F of OYE1 from *S. pastorianus* partly reversed the enantioselectivity in the neral reduction from 19% *e.e.* (*S*) to 65% *e.e.* (*R*) as compared to the wild type [22]. In contrast to the substrate binding mode of the wild type enzyme, the W116F mutation enabled the substrate to bind with a flipped orientation in the active site, and thus reverse the enantioselectivity, while maintaining the same mechanism of trans-hydrogenation of C=C bond [23,24]. W116 is not the sole determinant of enantioselectivity in OYEs, and the enantioselectivity switches seemed to vary by enzyme: Y78, I113, and F247 in OYE2.6 [25]; C26, I69, and H167 in ene reductases YqjM [26]; and W66 and W100 in OYE from *Gluconobacter oxydans* (Gox0502) [27]. The study of enantioselectivity alteration in OYEs rarely use citral as substrate. The latest example was the NADH-dependent cyclohexenone ene reductase from *Zymomonas mobilis* (NCR), in which W66 was critical for controlling the orientation of (*E*/*Z*)-citral binding and, thus, the variant W66A/I231A of NCR reversed the geranial derived enantioselectivity from 99% *e.e.* (*S*) to 63% *e.e.* (*R*) [28].

The study aims to develop the asymmetric reduction of (*E*/*Z*)-citral to (*R*)-citronellal using engineered OYE coupled with formate dehydrogenase for NADH regeneration (Scheme 1). The Old Yellow Enzyme from *S. cerevisiae* CICC1060 (OYE2y) was cloned, overexpressed, and purified, which reduced geranial and neral to citronellal with 82.87% *e.e.* (*R*) and 32.66% *e.e.* (*S*), respectively. OYE2y was chosen for enantioselectivity alteration since the wild type enzyme showed higher enantioselectivity than OYE1 from *S. pastorianus* and OYE2 and OYE3 from *S. cerevisiae* in the (*E*/*Z*)-citral reduction [29]. The key residues for the enantioselectivity of OYE2y were identified through the combination of sequence alignment and single-point mutations. Relying on subsequent site-saturation mutagenesis, the OYE2y variants with double substitutions exhibited full (*R*)-enantioselectivity in the reduction of (*E*)-citral or (*E*/*Z*)-citral. In addition, the role of key residues and the substrate binding modes were examined via homology modeling and molecular docking.

**Scheme 1.** OYE-mediated asymmetric reduction of (*E*/*Z*)-citral to (*R*)-citronellal coupled with formate dehydrogenase from *Candida boidinii* (FDHCB)-catalyzed NADH regeneration. The reactions were conducted at 37 ◦C and 200 rpm for 11 h.
