Summary Bioproduction of optical pure ( R )‐citronellal from ( E /Z)‐citral at high substrate loading remains challenging. Low catalytic efficiency of ( R )‐stereoselective ene reductases towards crude citral mixture is one of the major bottlenecks. Herein, a structure‐based engineering strategy was adopted to enhance the catalytic efficiency and stereoselectivity of an ene reductase (OYE2p) from Saccharomyces cerevisiae YJM1341 towards ( E / Z )‐citral. On basis of homologous modelling, molecular docking analysis and alanine scanning at the binding pocket of OYE2p, a mutant Y84A was obtained with simultaneous increase in catalytic efficiency and stereoselectivity. Furthermore, site‐saturation mutagenesis of Y84 yielded seven mutants with improved activity and stereoselectivity in the ( E / Z )‐citral reduction. Among them, the variant Y84V exhibited an 18.3% and 71.3% rise in catalytic efficiency ( k cat / K m ) for ( Z )‐citral and ( E )‐citral respectively. Meanwhile, the stereoselectivity of Y84V was improved from 89.2% to 98.0% in the reduction in ( E / Z )‐citral. The docking analysis and molecular dynamics simulation of OYE2p and its variants revealed that the substitution Y84V enabled ( E )‐citral and ( Z )‐citral to bind with a smaller distance to the key hydrogen donors at a modified ( R )‐selective binding mode. The variant Y84V was then co‐expressed with glucose dehydrogenase from Bacillus megaterium in E . coli D4, in which competing prim ‐alcohol dehydrogenase genes were deleted to prevent the undesired reduction in the aldehyde moiety of citral and citronellal. Employing this biocatalyst, 106 g l −1 ( E / Z )‐citral was completely converted into ( R )‐citronellal with 95.4% ee value and a high space‐time yield of 121.6 g l −1 day −1 . The work highlights the synthetic potential of Y84V, which enabled the highest productivity of ( R )‐citronellal from ( E / Z )‐citral in high enantiopurity so far.

Asymmetric reduction of electronically activated alkenes by ene reductases (ERs) is an attractive approach for the production of enantiopure chiral products.