Biocatalytic Epoxidation By Evolved Cytochrome P450

Epoxides are synthetically valuable intermediates, since their facile ring openingallows the introduction of various functionalities in a stereo-and region-selectivemanner with nucleophiles. Many optically pure compounds can be synthesized owingto their chemical versatility. They have widespread application in the medicine，pharmacy and other fields. However, conventional chemical synthesis of epoxides isoften energy-intensive and time-consuming and may entail the need for severalprotection and de-protection reactions. Unexpected byproducts formed during theprocess can reduce the overall productivity and increase the costs of the synthesis. Asefficient and environmentally friendly alternatives to chemical methods, enzyme-based biocatalytic methods carry out a wide range of regio-, stereo-, andchemo-specific reactions under mild conditions. In this paper, in order to obtain theepoxidation capability at C16,17site in progesterone and at double-bound incyclohexene, styrene, the cytochrome P450BM-3139-3was subjected to investigatethe catalytic characterization and epoxidation capability by directed evolution.Firstly, the P450BM-3139-3monooxygenase, Squalene epoxidase (SE) and3-Ketosteroid-9α-Hydroxylase (KshAB) from Bacillus Megaterium, Saccharomycescerevisiae Y01, Kluyveromyces lactis NRRL Y-1140, Pichia Pastoris GS115andRhodococcus rhodochrous4.1480were cloned respectively. Only the P450BM-3139-3and KshAB were expressed in E.coli. They were as the biocatalyst candidatesfor the production of target compounds.Secondly, a colorimetric high-throughput screening method (Picric acid assay)based on the Picric acid to detect16,17-epoxysterol and alkenes oxide had been set up.The PA assay reported in this study was a technically simple, sensitive andcost-effectiveness assay to detect16,17-epoxysterol compounds that could be adaptedto other common epoxides in a96-well format for high-throughput screening assays.The total detection time of the Picric acid assay was within2h.Thirdly, two random mutation methods of library construction, error-prone PCRand DNA shuffling, were designed to engineer P450BM-3139-3in directedevolution researehes. The mutant libraries were iteratively sereened by the7-ethoxycoumarin deethylation assay for general enzyme activity screening and the modified picric acid assay for epoxidation activity detection. Four P450BM-3139-3variants with higher epoxydation activities were screened and aligned with parentsequence. The relationship between structure and function of P450BM-3139-3variants were discussed.Finally, the following substrates, progesterone, cholesterol, cPPA, cyclohexene,styrene were selected to investigate the biocatalytic epoxidation by evolved P450BM-3and new-cloned enzymes in whole-cell and in vitro (cell-free enzymes) reaction.The double-bound in cyclohexene, styrene and C16,17site in progesterone could bepremilinarily epoxidized in biocatalysis process.