Identification of a yeast old yellow enzyme for highly enantioselective reduction of citral isomers to (R)-citronellal

Abstract

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.