Molecular Modification Of Yeast Old Yellow Enzyme OYE3 And Its Application In Asymmetric Synthesis Of(R)-Citronellal

As an acyclic monoterpene aldehyde,citronellal has an asymmetric carbon atom and often exists in the form of(R/S)-citronellal mixture.Among them,(R)-citronellal can be used for the synthesis of natural vitamin E and pharmaceuticals.It is also a key intermediate in the synthesis of L-menthol,which is one of the three.most popular favors in the world.At present,the synthesis of(R)-citronellal includes chemical method and biological method.The industrial synthesis of(R)-citronellal mainly relies on the chemical synthesis method invented by Takasago Company in Japan and BASF in Germany.However,the chemical method suffers from catalyst recovery and utilization.In contrast,the biological method is gradually applied to the production of(R)-citronellal because of its environmental friendliness.The enoate reductase is the most widely used and most studied biocatalyst in the biological method.The enoate reductase can catalyze the trans-hydrogenation of the carbon-carbon double bond of citral in the presence of coenzyme NAD(P)H to produce citronellal.The enoate reductase-mediated synthesis of(R)-citronellal is hampered by its low stereoselectivity and insufficient activity.Here,the old yellow enzyme OYE3 from Saccharomyces.cerevisiae S288C was chosed as biocatalyst.In order to improve the catalytic performance of OYE3 in the synthesis of(R)-citronellal,the studies were conducted as following:1.Based on the structure of OYE3 and researches of the known catalytic mechanism,OYE3 was engineered through semi-rational design and a mutant enzyme with high enantioselectivity was finally obtained.2.The catalytic conditions of the mutant enzyme were optimized to improve its(R)-citronellal yield.3.The fusion of OYE3 and GDH was fulfilled to promote coenzyme cycle and thus the fusion enzyme can catalyze the asymmetric reduction of high concentration(E)-citral.Firstly,OYE3 was modified by semi-rational design to achieve highly selective synthesis of(R)-citronellal.The amino acids in the loop region around the OYE3catalytic pocket were selected for site-directed mutagenesis.Among all mutants,S296F and W116A showed high stereoselectivity(>99%)when they catalyzed(E)-citral,and the product configuration was reversed from(S)-To(R)-citronellal when(Z)-citral was used as substrate.The homology modeling and molecular docking suggested that the binding mode of S296F and W116A with the substrate(Z)-citral had"flipped",which was consistent with the experimental results.Mutants with high stereoselectivity,such as S296F and W116G,were obtained through the optimization of amino acid substitution.Furthermore,the combination mutation led to a mutant enzyme S296F/W116G that selectively catalyzed(E)-citral but not(Z)-citral.Secondly,the catalytic efficiency in(R)-citronellal synthesis was improved by optimizing the catalytic conditions since the mutant enzyme S296F/W116G has high stereoselectivity and low catalytic activity.The effects of catalytic conditions such as temperature,p H,coenzyme NADP~+concentration,co-substrate D-glucose concentration,the ratio of GDH and S296F/W116G enzyme activity on the product yield of(E)-citral were investigated by a single variable method.Then we add 20 kinds of amino acids to couple the citral isomerization reaction to improve the(Z)-citral utilization efficiency,and explore the effect of amino acid concentration on the catalytic results.The optimized conditions were listed as following:the temperature 30?,p H7.0,NADP~+0.4 m M,D-glucose 60 m M,GDH:S296F/W116G=1:1,adding 0.5 M serine.Under the optimized conditions,S296F/W116G had a higher catalytic efficiency.For 10 m M(E)-citral,the yield of(R)-citronellal was increased from 41.31%to 58.64%.For 10 m M(Z)-citral,the yield of(R)-citronellal was increased from 0 to 55.85%.And for(E/Z)-citral,the yield of(R)-citronellal was increased from 23.53%to 48.74%.Finally,the fusion of OYE3 and GDH to the same vector through a linker peptide was conducted and a dual enzyme co-expression system was constructed to promote coenzyme cycle and increase substrate conversion rate.When 100 m M(E)-citral was catalyzed,the yields of fusion enzyme-expressing cells E.coli BL21(DE3)/p ET28b-GDH-(GSG)-OYE3 and E.coli BL21(DE3)/p ET28b-GDH-(GSG)-S296F reached above 99%and 93.98%respectively.However,the e.e.value of the product decreased significantly.The E.coli BL21(DE3)/p ET28b-GDH-(GSG)-S296F/W116G fusion enzyme expressing bacteria catalyzed 100 m M(E)-citral,the e.e.value and the product yield were 83.53%and 36.96%,respectively.It is inferred that the expression of enzyme fusion would change the binding mode between single enzyme and substrate and affect the product configuration.In addition,compared with non-fusion bacteria,the fusion-expressing bacteria had almost no by-products such as citronellol.