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