Development of novel bioreaction platforms for the highly efficient process of cytochrome P450

Abstract

Cytochrome P450 (CYP or P450) belong to a superfamily of multifunctional monooxygenase concerning oxidative metabolism of endogenous or exogenous molecules. P450s can catalyze various regioselective and stereospecific oxidation reactions of non-functionalized hydrocarbons. P450s containing heme molecule as a prosthetic group have been found from most living organisms. Since discovery of P450s in 1958, P450s have been interested from various industrial fields because of its various catalytic activity to large range of substrates. While there are many interests and a lot of researchers have studied about P450s, however, several characteristics have made application of P450s difficult. Briefly, major reasons for limitations of P450s application are dependence on expensive electron donor cofactor (such as NADPH or NADH), additional electron transfer domain (reductase) and heme molecule on their active site. Also, they need to be engineered for industrial uses and enzyme engineering have been difficult. To overcome these limitations, various techniques are even now being developed and some successful cases were reported. In this study, I have focused to improve P450s utilization by following experiments. First, cell surface display of P450s on the E. coli was developed to efficient whole-cell reaction. As P450s were displayed on cell membrane, they could freely use the electron donor and showed normal activity in stable. As a result, it could be free from limitation of NAD(P)H supplying. Furthermore, I changed conventional electron donor to inexpensive eosin Y (EY). In contrast with NAD(P)H, EY has good permeability to cell membrane and could easily penetrate into E. coli. Also, it can directly transfer electrons to active site, thus more efficient whole-cell reaction can be possible without conventional electron donor and reductase. Also, EY-mediated P450 reactions can directly transfer the electron in absence of heme. It means P450 reactions should not be affected by the amount of heme molecule bound to P450s and harsh handling of P450 enzyme for efficient production can be applied. Finally, general high-throughput screening method was developed to enhance the activity or the regioselectivity from wild-type of P450s. This engineering tool was initially designed to be applied regardless of kind of P450s and substrates. Therefore, following experiments should increase the value of P450s in industrial uses.