Abstract
BackgroundHuman cytochrome P450 (CYP) enzymes mediate the first step in the breakdown of most drugs and are strongly involved in drug–drug interactions, drug clearance and activation of prodrugs. Their biocatalytic behavior is a key parameter during drug development which requires preparative synthesis of CYP related drug metabolites. However, recombinant expression of CYP enzymes is a challenging bottleneck for drug metabolite biosynthesis. Therefore, we developed a novel approach by displaying human cytochrome P450 1A2 (CYP1A2) and cytochrome P450 reductase (CPR) on the surface of Escherichia coli.ResultsTo present human CYP1A2 and CPR on the surface, we employed autodisplay. Both enzymes were displayed on the surface which was demonstrated by protease and antibody accessibility tests. CPR activity was first confirmed with the protein substrate cytochrome c. Cells co-expressing CYP1A2 and CPR were capable of catalyzing the conversion of the known CYP1A2 substrates 7-ethoxyresorufin, phenacetin and the artificial substrate luciferin-MultiCYP, which would not have been possible without interaction of both enzymes. Biocatalytic activity was strongly influenced by the composition of the growth medium. Addition of 5-aminolevulinic acid was necessary to obtain a fully active whole cell biocatalyst and was superior to the addition of heme.ConclusionWe demonstrated that CYP1A2 and CPR can be co-expressed catalytically active on the cell surface of E. coli. It is a promising step towards pharmaceutical applications such as the synthesis of drug metabolites.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0427-5) contains supplementary material, which is available to authorized users.
Highlights
Human cytochrome P450 (CYP) enzymes mediate the first step in the breakdown of most drugs and are strongly involved in drug–drug interactions, drug clearance and activation of prodrugs
We report on the first successful coexpression of cytochrome P450 1A2 (CYP1A2) and cytochrome P450 reductase (CPR) on the surface of E. coli
The obtained fusion protein consists of an N-terminal cholera toxin B signal peptide (CtxB) signal peptide for translocation through the Sec pathway, the CPR or CYP1A2 passenger without the transmembrane domains and the C-terminal autotransporter unit for outer membrane anchorage (Fig. 2)
Summary
Human cytochrome P450 (CYP) enzymes mediate the first step in the breakdown of most drugs and are strongly involved in drug–drug interactions, drug clearance and activation of prodrugs. Their biocatalytic behavior is a key parameter during drug development which requires preparative synthesis of CYP related drug metabolites. As part of the phase-I-metabolism these heme-containing proteins catalyze a huge variety of oxidation reactions accepting a broad range of endogenous and xenobiotic substrates [2] They hereby convert lipophilic into more reactive and hydrophilic metabolites as first step for their elimination from the body. The usage of purified CYPs suffers from low catalytic activities, purification costs and poor enzyme stability
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