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.
Anchorage of recombinant proteins onto the outer membrane of gram-negative bacteria is an attractive solution for protein library screening and whole cell biocatalysis if a membrane environment is required or mass transfer into the cell is limiting. Autotransporters have been successfully applied for surface display of various heterologous proteins. Still, many underlying parameters for achieving active enzymes are not known. Here, we systematically tested different linkers between passenger and the membrane embedded β-barrel of the autotransporter. The linker can have influence on aspects such as steric orientation of the passenger, distance to the outer membrane and accessibility of active sites. Six linker variants for display of the cytochrome P450 reductase were tested. Cytochrome c reduction by the cytochrome P450 reductase varied fivefold and was highest by introduction of a flexible glycine-serine region. When these variants were co-expressed with surface displayed CYP1A2, product concentration for paracetamol differed between 0.22 μM and 2.5 μM and for resorufin between 0.23 μM to 1 μM. The best glycine/serine containing sequence, that turned out to be best for CPR display, was then introduced into the linker for displaying CYP1A2. In comparison, up to 7.9 μM paracetamol and up to 1.69 μM resorufin were obtained with this new variant. The differences were not caused by changes in the number of displayed enzymes. To our knowledge, this is the first systematic study on engineering the linker for surface display of recombinant enzymes.
A: Mechanism of passenger translocation on the surface with an autotransporter. 1: Transport over inner membrane through Sec-Translocon; 2: Signal peptide is cleaved off. Protein is kept in an unfolded state by chaperones; 3: Insertion of the β-barrel into the outer membrane; 4: Passenger is translocated onto the surface. Schematic view of the autotransporter fusion precursor protein.
B:Co-expression of cytochrome P450 monooxygenase and cytrochrome P450 reductase toward drug metabolites studies using autodisplay. With both enzymes displayed on the surface substrate accessbility and electron supply are given.
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