Engineering Of Enoate Reductase Through Rational Design And Directed Evolution

Enoate reductases from the Old Yellow Enzyme(OYE)family are capable of catalyzing the asymmetric reduction of(E/Z)-citral to(R)-citronellal.(R)-citronellal can be used for the treatment of breast cancer and the synthesis of natural vitamin E.Also,(R)-citronellal is an important intermediate for the synthesis of L-menthol.The synthesis of(R)-citronellal includes chemical and enzymatic methods.In the enzymatic method,enoate reductase is the key enzyme for asymmetric hydrogenation of(E/Z)-citral to(R)-citronellal.The enantioselective hydrogenation of(E/Z)-citral to afford an identical enantiomer remained challenging since the reduction of the geometric isomers geranial and neral by the same catalyst usually yielded the enantiocomplementary products.So,the applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity.The following works have been carried out on the enoate reductase OYE2 y derived from Saccharomyces cerevisiae CICC1060:(1)construction and optimization of the OYE2y-mediated asymmetric hydrogenation system;(2)rational design of OYE2 y to improve its enantioselectivity;(3)site-saturation mutagenesis and combinational mutagenesis.In order to investigate the time couses of asymmetric hydrogenation,(E)-citral,(Z)-citral,and(E/Z)-citral were tested as substrates,respectively.During the first 3 h,the concentration of geranial decreased significantly faster than that of neral.Meanwhile,the concentration of(R)-citronellal increased rapidly and the e.e.value was kept at a higher level of >60%(R).Then,the conversion rate of neral turned faster from 3 h to 5 h,resulting in the decreasing of e.e.value from 65.02%(R)to 40.26%(R).After 6 h,the conversion rate of neral was nearly parallel to that of geranial,meanwhile the e.e.values were kept almost constant.The 11 h reaction was completed with 89.51% yield,giving(R)-citronellal with the e.e.value of 38.13%(R).When the enzyme OYE2 y was tested with newly-prepared(E)-citral or(Z)-citral for 4 h,the enantioselectivity of OYE2 y stayed at a relatively constant level.The(E)-citral and(Z)-citral derived e.e.values after 11 h reduction were 82.87%(R)and 32.66%(S),respectively.The(R)-enantioselectivity of old yellow enzyme from Saccharomyces cerevisiae CICC1060 OYE2 y was enhanced through protein engineering.The single mutations of OYE2 y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2 y.Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76,yielding 17 and 5 variants with improved(R)-enantioselectivity in the(E/Z)-citral reduction,respectively.Among them,the variants R330 H and P76 C partly reversed the neral derived enantioselectivity from 32.66% e.e.(S)to 71.92% e.e.(R)and 37.50% e.e.(R),respectively.Remarkably,the double substitutions of R330H/P76 M,P76G/R330 H or P76S/R330 H further improved(R)-enantioselectivity to >99% e.e.in the reduction of(E)-citral or(E/Z)-citral.The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites,e.g.,R330 in OYE2 y.To explore the molecular mechanism of OYE2 y enantioselectivity and activity,(E)-citral and(Z)-citral were docked with OYE2 y and its variants,respectively.The docking analysis of OYE2 y and its positive variants revealed that the substitutions R330 H and P76 C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity.The docking analysis of inactivated variants indicated that the carbonyl oxygen of substrate could not form hydrogen bonds with Asn195 of the inactivated variantst,which might result in the loss of OYE2 y activity.