Design of a Self-Sufficient Whole-Cell Cascade for the Production of (<i>R</i>)-Citronellal from Geraniol

(<i>R</i>)-Citronellal is a key chiral precursor of high-value chemicals, such as the best-selling flavor compound (-)-menthol; however, the conventional synthesis suffers from low yield and unsatisfactory enantioselectivity. In this study, we developed a highly atom-efficient hydrogen-borrowing cascade for the synthesis of (<i>R</i>)-citronellal from geraniol using alcohol dehydrogenase from <i>Escherichia coli</i> K12 (AdhP) and ene-reductase from <i>Saccharomyces cerevisiae</i> YJM1341 (OYE2p). The key rate-limiting enzyme, AdhP, was subjected to structure-guided semirational engineering, and the triple mutant AdhP^{260T/284A/268P} (M3) was obtained that demonstrated a 1.28-fold improvement in catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) toward geraniol. After optimization of the reaction conditions, the hydrogen-borrowing cascade system achieved the conversion of 23.14 g/L geraniol into (<i>R</i>)-citronellal at a conversion rate of 98.23% with 96.7% ee. This work represents an alternative approach for the biosynthesis of (<i>R</i>)-citronellal without sacrificing a cosubstrate or additional enzymes.