Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal

Abstract

The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (<i>E</i>/<i>Z</i>)-citral to (<i>R</i>)-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 (<i>R</i>)-enantioselectivity of Old Yellow Enzyme from <i>Saccharomyces cerevisiae</i> 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 (<i>R</i>)-enantioselectivity in the (<i>E</i>/<i>Z</i>)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% <i>e.e.</i> (<i>S</i>) to 71.92% <i>e.e.</i> (<i>R</i>) and 37.50% <i>e.e.</i> (<i>R</i>), 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 (<i>R</i>)-enantioselectivity to >99% <i>e.e.</i> in the reduction of (<i>E</i>)-citral or (<i>E</i>/<i>Z</i>)-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.