Functional Identification,Semi-rational Design Modification And Application Of OYE From Corynebacterium Glutamicum

Old Yellow Enzyme（OYE,EC 1.6.99.1）is a family of oxidoreductases containing flavin mononucleotides（FMN）,the natural reaction of which involved the reduction of Cr（VI）,and C.glutamicum was shown to be effective in the remediation of contaminated soil with heavy metals.However,the OYE of the genus has never been explored.In addition,OYE has the ene-reductase activity that can catalyze citronellal production from citral in aqueous-based media and under mild conditions,while the class of enzymes was generally characterized by low catalytic activity and poor stability,and its applications are limited.In this study,an Old Yellow Enzyme（Cg OYE）with hexavalent chromium reductase activity as well as ene-reductase activity was identified for the first time in Corynebacterium glutamicum ATCC 13032.In this paper,we successfully improved the reductase activity of the enzyme for the substrate Cr O₃ and the ene-reductase activity for the substrate citral as well as the stereoselectivity by semi-rational design by semi-rational design.Cg OYE and its variants were applied to the reduction of Cr O₃ and the production of（R）-citronellal or（S）-citronellal.The specific studies are as follows:（1）The coding sequence（CDS）of Old Yellow Enzyme from C.glutamicum was cloned and expressed on E.coli BL21/p XMJ-19 with Cr O₃ as the substrate,the specific enzyme activity of 6.5 U·mg^-1,optimum temperature of 40°C and optimum p H of 7.0,and good stability at 40-50°C and p H in the range of 7.0-7.5.Thus,Cg OYE shows good stability in the range of40-50°C and p H 7.5-8.0;（2）To address the problems of low activity and poor stability of Cg OYE hexavalent chromium reductase.We used Cr O₃ as the substrate and predicted the relevant sites affecting Cg OYE hexavalent chromium reductase activity by molecular dynamics（MD）simulations.A variant I81G was successfully screened by alanine scanning mutagenesis and saturation mutagenesis.The specific enzyme activity of variant I81G was 18.0 U·mg^-1,which is 2.77-fold higher than the wild-type specific enzyme activity,with improved thermal stability and p H tolerance,and was expressed in E.coli BL21 and applied to the reduction of hexavalent chromium.The Cr（VI）reduction rate of E.coli BL21/p XMJ-19-Cg OYE was 38.1%after 48 h,while E.coli BL21/p XMJ-19_I81G reduced all Cr（VI）to Cr（III）at the same time,and the Cr（VI）reduction rate was 2.62 times higher than that of the control,and the OD₆₀₀ of the bacterium was 1.52 times higher than that of the control;（3）Cg OYE shows the ability to reduce the C=C bonds and was applied to catalyze（E/Z）-citral to produce（R）-/（S）-citronellal,as the（E/Z）-citral is a mixed isomer significantly affecting the catalytic efficiency and stereoselectivity of Cg OYE.To address the low activity of Cg OYE catalyzed（E/Z）-citral to（R）-citronellal,the C=C bonds asymmetric reduction activity of the enzyme was improved by truncating the peptide chain（Met1-Arg31）in the N-terminal region of Cg OYE.Among variants N8,N20,and N31,N31 showed the highest specific enzyme activity with an increase of about 4.86-fold and a significant increase in thermal stability and p H tolerance.However,N31-Cg OYE showed a low（R）-stereoselectivity with ee 47.4%;（4）To address the stereoselectivity problem of N31-Cg OYE,the Focused Physicochemical Iterative Site-specific Mutagenesis（FRISM）was used as a theoretical guide for the Cg OYE mutation,and the variant N31 was semi-rationalized.The two variants N31-Cg OYE_{I81L/W128A/H190A} and N31-Cg OYE_H198A/H295L were obtained according to the product conformation,where the mutation results showed that the conserved Cg OYE site H190 can act as a"tuneable gating"for the biocatalytic citral to citronellal.The site affects the substrate’s conformation binding to the enzyme’s active pocket,thus changing the（R）-type or（S）-type of the product citronellal.Finally,the reaction system was optimized by coupling the Cg OYE variants with glucose dehydrogenase（Bs GDH）from Bacillus subtilis,and the results showed that the optimal temperature of the dual enzyme coupling system was 40°C and the optimal p H was 8.0.The amount of（R）-citronellal catalyzed by coupling the two mutants with Bs GDH using 220 m M（E/Z）-citronellal as the substrate,respectively.The variant N31-Cg OYE_{I81L/W128A/H190A} provided（R）-citronellal in an absolute yield of 2452.8 mg with conversion and ee value of 99.4%and 99.0%,respectively,and the other variant N31-Cg OYE_H198A/H295L gave（S）-citronellal in an absolute yield of 2781.2 mg with conversion and ee value of were 99.6%and 98.7%,respectively.