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

Abstract

Background: 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. Methods: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.Results: 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. Conclusion:All these positive features demonstrate high potential of OYE2p for practical synthesis of (R)-citronellal and in asymmetric reduction of activated alkenes.

Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal

“…The time course of ( E / Z )-citral reduction clearly indicated that the ratio of geranial and neral significantly affected the e.e. value of ( R )-citronellal, which was consistent with previous observations [7,14]. In addition, the isomerization of geranial and neral occurred under some conditions [9,30].…”

“…Furthermore, the ( Z )-citral-derived e.e. value of R330H (71.92%, R ) was much higher than that of OYE2p (26.5%, R ) [7], suggesting that the role of S13, S59, and V289 could not be ignored for the ( R )-enantioselectivity. For the single substitutions, the ( R )-enantioselectivity improvement in the ( E / Z )-citral reduction was mainly attributed to the enantioselectivity inversion in the ( Z )-citral reduction, meanwhile, the same variant reduced ( E )-citral to ( R )-citronellal with similar e.e.…”

“…The commercial Takasago process of ( R )-citronellal began with myrcene to form an allylic amine, which underwent asymmetric isomerization in the presence of a 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl(BINAP)-Rh complex and subsequent hydrolysis with acid to give enantiomerically pure ( R )-citronellal [6]. In contrast to the three-step asymmetric synthesis from myrcene, the one-step enantioselective reduction of natural citral (the crude mixture of 60% geranial and 40% neral) was a simplified process for the synthesis of ( R )-citronellal [7]. 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.…”

Characterization of the Novel Ene Reductase Ppo-Er1 from Paenibacillus Polymyxa

“…Strikingly, Ppo-Er1 retained a relative specific activity of >70% at temperatures as low as 10 • C making the enzyme an interesting candidate to be used for the transformation of thermolabile substrates such as aldehydes ( Figure 3). Overall, our Ppo-Er1 data confirm that the temperature profile of class III enzymes resembles those of their mesophilic counterparts of class I, for example NemA [45] with a reported T opt of 30-50 • C and OYE2p [48] with a T opt of 25-40 • C. Finally, we employed the ThermoFAD technique to determine the melting temperature of Ppo-Er1 and found that the ene reductase unfolds at T m = 46.5 ± 1 • C ( Figure S15). The use of cosolvents is often a "must" in biocatalytic processes due to the presence of high concentrations of various organic substrates.…”

Novel Old Yellow Enzyme Subclasses

“…Thus, the optimum temperatures for the investigated class III and IV enzymes are more similar to those of their mesophilic counterparts from class I, for example, 25–40 °C for OYE2p or 30–50 °C for NemA . In contrast, thermophilic‐like class II OYEs, which have an average increased thermal stability compared to class I enzymes, partly exhibit higher optimum temperatures as observed for CrS ( T opt =65 °C) GeoER ( T opt =70 °C) and FOYE1 ( T opt =50 °C) .…”