Biocatalytic Reduction Of Citral To(R)-Citronellal

Citronellal,i.e.,3,7-dimethyl-6-octenal,contains a chiral carbon atom and thus has both(R)-and(S)-configuration.Citronellal has a strong aroma of green citrus and a mild smell of woody,which is widely used as a fragrant agent in foodstuff preparation.Among the two widely used isomers,(R)-citronellal is a key intermedia for the synthesis of L-menthol,one of the world's most important flavoring chemicals.At present,chemocatalysis was the main method for the production of(R)-citronellal,such as chiral BINAP-Rh complex-catalyzed enantioselective isomerization of geranylamine to(R)-citronellal presented by Takasago International Corporation,and enantioselective reduction of(Z)-citral presented by BASF.For the former method,the catalyst is quite harsh to prepare and difficult to recover.Rectification of(E/Z)-citral to obtain high purity(Z)-citral and special synthetic metal catalyst are needed for the latter method.Compared to asymmetric chemocatalysis,enoate reductase-mediated reduction of citral to citronellal has many inherent merits,such as environmental friendliness,mild reaction conditions,and high stereoselectivity.However,few ene-reductases capable of reduction of(E/Z)-citral to(R)-citronellal with high enantioselectivity and high activity have been reported.Most ene-reductases preferably produce(S)-citronellal from(E/Z)-citral.In order to establish an efficient biosynthesis of(R)-citronellal,screening and enzymatic properties of ene-reductases with high enantioselectivity toward(E/Z)-citral were described in this study.And then,appropriate whole-cell biocatalyst and catalytic reaction system were developed to minimize by-products.The study consisted of the following parts:Firstly,screening and characterization of the(R)-selective ene-reductases.A NAD(P)H dependent ene-reductase(OYE2p)from Saccharomyces cerevisiae YJM1341 was selected from a library of 23 ene-reductases constructed in our previous study,which could reduce(E/Z)-citral(an approximately 9:10 mixture of geometric E-isomer and Z-isomer)to(R)-citronellal with high enantioselectivity(84.5%ee).OYE2p showed the highest activity at 40 ? and pH 7.5,while this enzyme was relatively stable under 30 ? and at pH 7-9.The substrate spectrum analysis showed that OYE2p had high activities toward unsaturated carbonyl compounds with two electron withdrawing groups near the carbon carbon double bond,such as maleimide,N-phenyl maleimide,ketoisophorone and dimethyl maleate.Secondly,Influence factors of the enantioselectivity of OYE2p towards citral and OYE2p-catalyzed synthesis of(R)-citronellal from(E/Z)-citral.For(E)-citral(geranial)and(Z)-citral(neral),OYE2p exhibited different selectivity patterns.OYE2p showed strict enantioselectivity toward(E)-citral,producing(R)-citronellal with ee>99%,and the enantioselectivity was not affected by pH.OYE2p produced both enantiomers of citronellal from(Z)-citral,but showed(R)-citronellal formation tendency.With regard to the(Z)-citral by OYE2p,the ee value of(R)-citronellal was increased with the increase of pH in the reaction system.With in situ NAD(P)H-regeneration system involving a glucose dehydrogenase(GDH)and glucose,200 mM geranial could be completely converted to optically pure(R)-citronellal employing purified OYE2p as catalyst,resulting in ee value of 98%(R),conversion of>99%and a yield of 87.03%.Under similar conditions,200 mM E/Z mixtures of citral(an approximately 9:10 mixture of geometric E-isomer and Z-isomer),ee value of 89%(R),the conversion of 98.4%and the yield of 87.2%were obtained.Thirdly,fusion expression of OYE2p and formate dehydrogenase(FDH)and whole cells-catalyzed synthesis of(R)-citronellal from citral.Considering the complexity of coenzyme recycling system,the stability of enzyme and the cost of preparation,recombinant E.coli co-expressed OYE2p and FDH was constructed and the whole cells were employed as biocatalysts to catalyze reduction of citral to produce(R)-citronellal.During the whole cells-mediated bioreduction of citral to(R)-citronellal,the competing reaction of the conversion of the activating carbonyl group of citral and produced citronellal to the equivalent alcohols due to the presence of NAD(P)H-dependent carbonyl reductases in E.coli.Besides,the produced citronellal would be further biotransformed or metabolized.Two different linker peptides were used for the fusion expression of OYE2p and FDH in E.coli.The best recombinant strain E.coli(pET21d-fdh-(ggggs)3-oye2p)showed the higher protein expression and the higher enzyme activity.It is interesting that the competing reaction of the conversion of the activating carbonyl group of citral and produced citronellal disappeared when employing E.coli(pET21d-fdh-(ggggs)3-oye2p)whole cells to catalyze the citral to citronellal.Main reason is that strengthening of the enzyme's spatial proximity effect by the fusion expression enabled NADH to recycle in the fusion enzymes,hence,the NAD(P)H-dependent carbonyl reductases could not catalyze the reduction of carbonyl group without coenzyme NAD(P)H.And then,a two-phase reaction system composed of toluene(20%,v/v)and PBS buffer was developed to reduce the further degradation of citronellal.