Study On Molecular Modification Of P450DA And Its Application In Asymmetric Synthesis Of Chiral β-Halohydrins

Objective:The objectives of this study was:(1)to obtain a self-sufficient P450monooxygenase that could stereoselectively catalyze asymmetric hydroxylation of the1-chloro-2-phenylethane to chiral 2-chloro-1-phenylethanol by enzyme screening;(2)to improve the catalytic performance of the target P450 monooxygenase by directed evolution;(3)to analyze the P450’s substrate spectrum and develop a biocatalytical route to chiralβ-halohydrins via P450-catalyzed asymmetric hydroxylation of halohydrocarbons.Methods:1.1-Chloro-2-phenylethane was used as a template substrate to screen the bacterial strains preserved in our laboratory for a P450 monooxygenase displaying a high level of catalytic activity and stereoselectivity for asymmetric hydroxylation of the benzylic C-H bond.2.Halohydrin dehalogenase was used to contruct a cascade reaction system with P450-catalyzed hydroxylation of 1-chloro-2-phenylethane,to establish a high-throughput screening method for colorimetrically evaluating the P450-catalyzed hydroxylation activity through detection of the released chloride ion(Cl~-).3.The target P450 gene was used as parent DNA template to conduct random mutation by error-prone PCR and MEGAWHOP PCR methods.The mutant library was studied to screen out P450 variants of interest based on the high-throughput screening method developed in step 2.The mutant displaying excellent catalytic performances was further applied to iterative saturation mutagenesis to obtain P450 mutant with better properties.4.The reaction p H and temperature of the whole-cell biotransformation were investigated to establish the optimal reaction conditions.The substrate spectrum of the P450DA mutant was investigated using a variety of substrates bearing different substituents on the benzyl ring of the 1-halo-2-phenylethane.Results:1.By strain screening,a self-sufficient P450DA from Deinococcus apachensis that exhibitted good activity and S stereoselectivity for asymmetric hydroxylation of1-chloro-2-phenylethane was selected.Using 10 g cdw/L of recombinant P450DA cells,2m M of 1-chloro-2-phenylethane was converted to(S)-2-chloro-1-phenylethanol with 16%yield and 83.4%ee in 24 h.2.A high-throughput screening method was developed for activity evaluation of the selected P450DA enzyme.In this method,halohydrin dehalogenase Hhe D6 with high dehalogenation activity was selected to construct a cascade system with P450.The released Cl~-in reaction were detected by colorimetric analysis at 460 nm.The detected absorbance showed a good linear relationship(R~2=0.9997)with the concentration of2-chloro-1-phenylethanol.3.(1)A P450DA mutant,H6(N190D),with increased activity,was successfully obtained in the first round of random mutation.Using 10 g cdw/L of recombinant H6 cells,2 m M of1-chloro-2-phenylethane was converted to(S)-2-chloro-1-phenylethanol with 51%yield and 83.8%ee.(2)Another P450DA mutant G4(N190F/V356L/A486E)was further screened out by iterative saturation mutagenesis and the second round of random mutation.Using 20 g cdw/L of recombinant G4 cells,10 m M of 1-chloro-2-phenylethane was converted to(S)-2-chloro-1-phenylethanol with 91%yield and 80.3%ee.(3)The total turnover number(TTN)of the mutant G4 was 13 times higher than that of the wild-type P450DA.4.Condition optimization indicated that P450DA was an NADH-dependent enzyme,with the optimal reaction p H at 8.5 and temperature at30℃.Under the optimal reaction conditions,P450DA-G4 catalyzed asymmetric hydroxylation of 10 m M of1-halo-2-phenylethane derivatives 1b-1q to the corresponding(S)-2-halo-1-phenethyl alcohols 2b-2q with yields ranging from 7%-68%.Using recombinant P450DA-G4 cells as biocatalyst,1-halo-2-phenylethane with methyl group substituted at meta-position on the phenyl ring of substrate was converted to the corresponding(S)-β-halohydrin with 46%yield and 97.5%ee,and 1-halo-2-phenylethane with bromo group substituted at meta-position was converted to corresponding(S)-β-halohydrin with 68%yield and 97.9%ee.Conclusion:1.A self-sufficient P450DA was obtained by strain screening,which could catalyze asymmetric hydroxylation of 1-chloro-2-phenylethane to(S)-2-chloro-1-phenylethanol.2.A colorimetric high-throughput screening method was developed for evalusting the P450DA’s hydroxylation activity toward 1-chloro-2-phenylethane.Combing directed revolution with the colorimetric method developed,a mutant P450DA-G4 showing 13-fold higher TTN than that of the wild type P450DA was successfully screened out.3.Collective ananlysis of the condition optimization and substrate spectrum,it could be concluded that the P450DA-G4 was a NADH-dependent P450 enzyme.The optimum reaction temperature and p H for the asymmetric hydroxylation of halohydrocarbons were determined at 30℃ and 8.5,respectively.P450DA-G4 displayed good catalytic activity for the substrate bearing electron-withdrawing substituent at the benzyl ring,and excellent stereoselectivity toward the substrates substituents at the meta-position of the substrate benzyl ring.