N-terminal truncation (N-) and directional proton transfer in an old yellow enzyme enables tunable efficient producing (R)- or (S)-citronellal

(R)-Citronellal is a valuable molecule as the precursor for the industrial synthesis of ()-menthol, one of the worldwide best-selling compounds in the flavors and fragrances field. However, its biocatalytic production, even from the optically pure substrate (E)-citral, is inherently limited by the activity of Old Yellow Enzyme (OYE). Herein, we rationally designed a different approach to increase the activity of OYE in biocatalytic production. The activity of OYE from Corynebacterium glutamicum (CgOYE) is increased, as well as superior thermal stability and pH tolerance via truncating the different lengths of regions at N-terminal of CgOYE. Next, we converted the truncation mutant N31-CgOYE, a protein involved in proton transfer for the asymmetric hydrogenation of C--C bonds, into highly (R)- and (S)-stereoselective enzymes using only three mutations. The mixture of racemic (E/ Z)-citral is reduced into the (R)-citronellal with ee and conversion up to 99 % by the mutant of CgOYE, over-coming the problem of the reduction for the mixtures of (E/Z)-citral in biocatalytic reaction. The present work provides a general and effective strategy for improving the activity of OYE, in which the partially conserved histidine residues provide "tunable gating" for the enantioselectivity for both the (R)- and (S)-isomerases