A NAD(P)H-dependent enoate reductase (OYE2p) from Saccharomyces cerevisiae YJM1341 was discovered by genome data mining for asymmetric reduction of (E/Z)-citral to (R)-citronellal with high enantioselectivity.This enzyme was heterologously expressed in E. coli and characterized for its biocatalytic properties. OYE2p was identified with reduction activities toward a diverse range of ɑ,β-unsaturated compounds bearing conjugated aldehyde, ketone, imide, carboxylic acid and ester.OYE2p showed the highest specific activity at 40 °C and a pH optimum at 7.0–8.0. The stability of OYE2p was rather pH-independent, and the half-life time values of the enzyme at pH 6.0–8.0 were more than 257 h. With regard to the reduction of (E)-citral and (Z)-citral, OYE2p exhibited different selectivity patterns. (E)-citral was exclusively reduced to (R)-citronellal by OYE2p in ≥ 99% ee, which was independent on pH. OYE2p produced both enantiomers of citronellal from (Z)-citral, but showed (R)-citronellal formation tendency, and the ee value of (R)-citronellal was affected by pH in the reaction system. Accordingly, the ee values for (R)-citronellal formation increased with the increasing levels of E-isomer in the (E/Z)-citral mixture as well as the increase of pH. Under the reaction conditions (30 °C and pH 8.6), using purified OYE2p as catalyst, 200 mM (E/Z)-citral (an approximately 10:9 mixture of geometric E-isomer and Z-isomer) was efficiently converted to (R)-citronellal with 88.8% ee and 87.2% yield.All these positive features demonstrate high potential of OYE2p for practical synthesis of (R)-citronellal and in asymmetric reduction of activated alkenes.
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.
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