Academic Editor: Stefano Serra

Received: 3 March 2019; Accepted: 14 March 2019; Published: 18 March 2019

**Abstract:** The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (*E*/*Z*)-citral to (*R*)-citronellal—a key intermediate in the synthesis of L-menthol. The applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity. Here, the (*R*)-enantioselectivity of Old Yellow Enzyme from *Saccharomyces cerevisiae* CICC1060 (OYE2y) was enhanced through protein engineering. The single mutations of OYE2y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2y. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (*R*)-enantioselectivity in the (*E*/*Z*)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% *e.e.* (*S*) to 71.92% *e.e.* (*R*) and 37.50% *e.e.* (*R*), respectively. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity. Remarkably, the double substitutions of R330H/P76M, P76G/R330H, or P76S/R330H further improved (*R*)-enantioselectivity to >99% *e.e.* in the reduction of (*E*)-citral or (*E*/*Z*)-citral. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y.

**Keywords:** asymmetric reduction; citral; citronellal; enantioselectivity; Old Yellow Enzyme; site-saturation mutagenesis; substrate binding mode
