Engineering Of Saccharomyces Cerevisiae OYE2 And Its Application In Asymmetric Synthesis Of (R)-Citronellal

(R)-citronellal can not only be used for the treatment of breast cancer but also serves as chiral intermediate for the synthesis of L-menthol.The synthesis of(R)-citronellal includes chemical and enzymatic approache.Enoate reductase as biocatalyst is critical for enzymatic asymmetric hydrogenation of citral to(R)-citronella.The chirality of the products from the hydrogenation of the(E)-and(Z)-citral is often complementary,resulting in low e.e.value of the product.Aiming to increase the e.e.value of the product in the asymmetric hydrogenation of(E/Z)-citral,the study using the enoate reductase OYE2 from Saccharomyces cerevisiae includes as follows:(1)the construction and optimization of OYE2-catalyzed asymmetric hydrogenation coupled with amino acid catalyzed isomerisation;(2)engineering of OYE2 through rational design to improve its stereoselectivity;(3)the exploration of screening of enaote reductase based on the fluorescence method.To investigate the correlation between substrate and product,(E)-citral,(Z)-citral or(E/Z)-citral citral was subjected to thehydrogenation reaction.After 11 h reaction,16.97% and 83.03% of(Z)-citral were converted to(R)-citronellal and(S)-citronellal,respectively;94.85% and 5.15% of(E)-citral was converted to(R)-citronellal and(S)-citronellal,respectively.In the reaction system,the addition of formate dehydrogenase and sodium formate was used to fulfill the coenzyme regeneration,and the product e.e.value was increased by coupling with amino acid-catalyzed isomerization with the asymmetric hydrogenation.The optimized amino acid in isomerization was 1 M glycine at pH 7.0.In the presence of 1 M glycine,The yield and e.e.value of(R)-citronellal were 91.84% and 75.06%,which were 23.90% and27.62% higher than those of the control(without the addition of glycine).The stereoselectivity of the enoate reductase OYE2 was improved by rational design.Homologous modeling,molecular docking and structural analysis predicted that the amino acid residues around the substrate binding pocket(e.g.,Met39,Pro75,Trp116,Thr196 and Phe296 played an important role in substrate recognition.The mutant library was constructed by the site-directed mutagenesis,and the mutant P75 M was selected for its improved stereoselectivity.The P75M-catalyzed asymmetric hydrogenation(E)-and(Z)-citral indicated that the ratio of(Z)-citral to(R)-citronellal was 23.11% higher than that of wild-type OYE2.When(E/Z)-citral was used as substrate,the product e.e.values with and without addition of glycine were 12.66% and 19.38% higherthat those of wild-type OYE2,respectively.Structural analysis revealed that the mutation of P75 M made the opposite face of the ? system of(Z)-citral accessible to hydride addition,thereby increasing the formation of(R)-citronellal.The procedure screening enoate reductase was explored based on a fluorescence method,in which isonicotinylhydrazine(INH)reacts with the ?,?-unsaturated bond of citral in the methanol solution containing aluminum salt to form a fluorescent complex.The fluorescence intensity was the strongest at the excitation wavelength Ex = 400 nm and the emission wavelength Em = 510 nm.The fluorescence intensity rose as the concentration of citral increased,while citronellal did not cause fluorescence under the above conditions.Therefore,the fluorescence signal could be used for determining the concentration of citral during the enoate reductase-catalyzed hydrogenation of citral and further the catalytic activity of the enaote reductase in the reaction system.The GC analysis verified that the fluorescence signal was highly correlated with the catalytic activity of enoate reductase.