Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of ()-Citral and ()-Citral.

The importance of yeast old yellow enzymes is increasingly recognized for direct asymmetric reduction of (/)-citral to ()-citronellal. As one of the most performing old yellow enzymes, the enzyme OYE3 from S288C exhibited complementary enantioselectivity for the reduction of ()-citral and ()-citral, resulting in lower value of ()-citronellal in the reduction of (/)-citral. To develop a novel approach for the direct synthesis of enantio-pure ()-citronellal from the reduction of (/)-citral, the enzyme OYE3 was firstly modified by semi-rational design to improve its ()-enantioselectivity. The OYE3 variants W116A and S296F showed strict ()-enantioselectivity in the reduction of ()-citral, and significantly reversed the ()-enantioselectivity in the reduction of ()-citral. Next, the double substitution of OYE3 led to the unique variant S296F/W116G, which exhibited strict ()-enantioselectivity in the reduction of ()-citral and (/)-citral, but was not active on ()-citral. Relying on its capability discriminating ()-citral and ()-citral, a new cascade reaction catalyzed by the OYE3 variant S296F/W116G and glucose dehydrogenase was developed, providing the enantio-pure ()-citronellal and the retained ()-citral after complete reduction of ()-citral.