Old Yellow Enzyme OYE2-catalyzed Synthesis Of Citronellal Through Asymmetric Reduction Of Citral

As a kind of very important flavour and fragrance,citral has been widely used in food,household chemicals,pharmaceuticals and other industries.The structure of citral has three double bonds(two C=C and a C=O)and the hydrogenation of double bonds can form various hydrogenated products.The?,?-unsaturated double bond of citral can be hydrogenated to form(R)-citronellal,a key intermediate in the synthesis of L-menthol.Takasago Inc.has achieved the industrial production of(R)-citronellal using myrcene as the starting materials,which is limited by expensive metal catalyst and low yield.Thus,it is valuable to develop a new synthesis routine for(R)-citronellal.Here,(R)-citronellal was synthesized through the asymmetric reduction of citral using recombinant old yellow enzyme OYE2 as biocatalyst.The asymmetric reduction of citral was coupled with formate dehydrogenase FDHCB-catalyzed coenzyme regeneration to improve the substrate concentration and the product yield.Under the optimized conditions,the asymmetric reduction of citral was scaled up and coupled with amino acid-catalyzed isomerization of(E/Z)-citral to improve the e.e.value of(R)-citronellal.In addition,the engineering of OYE2 was conducted to further improve the e.e.value of(R)-citronellal and the enzyme activity.The results are listed as follows:1.The gene encoding old yellow enzyme OYE2 was cloned from the genomic DNA of Saccharomyces cerevisiae,and the recombinant strain E.coli BL21(DE3)/pEASY-E1-oye2 was successfully constructed.Asymmectric reduction of citral and coenzyme regeneration were catalyzed by OYE2 and FDHCB,respectively.The optimized reaction mixture is listed as follows:0.42 mg/mL OYE2,0.2 U/mL FDHCB,100 mmol/L sodium formate,0.5 mmol/L NAD~+,25 mmol/L citral and 50 mM PIPES buffer(pH 7.0).After the redution of 25 and 30 mmol/L citral was carried out at pH 7.0 and 30?for 10 h,the yields of citronellal were 92.0%and88.5%,respectively.2.To achieve the asymmetric reduction of citral with high substrate load,various factors(e.g.,controlled pH,volume of the reaction mixture,the substrate concentration,types of solvents)were investigated in a 50 mL bioreactor.The results indicated that the yield of citronellal in the case of controlled pH was 10.9%higher than the control.As solvent,2-octanol was slightly superior to isopropanol.When 50mmol/L and 100mmol/L citral was reduced in 10 mL reaction mixture for 8 h,the final citronellal yields were 94%and 97.8%,respectively.As the concentration of citral was increased up to 150 mmol/L and 200mmol/L,the yields were 80.6%and51.4%,respectively.The hydrogenated product was proved to citronellal by the analyses of FTIR and NMR.Furthermore,the asymmetric reduction of citral was coupled with amino acid-catalyzed the isomerization of(E/Z)-citral in order to improve the e.e.value of(R)-citronellal.Among 12examed amino acids,glycine-catalyzed isomerization after 4 h led to the best catalytic performance with the yield of 100%and the e.e.value of>65%,the latter value of which was 40%greater than that of asymmetric reduction without isomerization.3.In order to improve the activity and stereoselectivity,OYE2 was engineered through the strategy of rational design.Ten mutation sites were selected,and 7 mutants were successfully constructed.All the mutants were overexpressed and purified,and their catalytic performances were investigated as biocatalyst.The activity of each mutant was decreased to some extend,and only K151H showed increased e.e.value by 15%.In addition,the mutant W116F showed no enzyme activity after successful over-expression.