Co-expression of Functional Human Cytochrome P450 1A2 and its Reductase on the Surface of Escherichia coli

Quehl P, Riemer J, Jose J

Abstract

Cytochrome P450 monooxygenases (CYPs) take part in the breakdown of almost every drug catalyzing a variety of oxidations of a broad range of substrates1. The wider usage of purified human cytochrome P450 enzymes as biocatalyst is impeded by factors such as their difficult recombinant expression and subsequent purification, low catalytic activities, requirement for membrane surroundings, and poor stability of the proteins. Many of these challenges can be circumvented by the application of a bacterial whole cell catalyst. Major advantages are the inexpensive and easy cultivation, feasibility of large-scale applications and reusability of the biocatalyst. Additionally, common expression hosts like Escherichia coli have no own cytochrome P450 background. In our work, we tested the co-expression of human cytochrome P450 reductase (CPR)2 together with cytochrome P450 1A2 monooxygenases (CYP1A2) on the cell surface of E. coli. Surface display can be advantageous in terms of offering a membrane environment, overcoming mass transfer limitations, and improved protein stability. Previously, it had been shown that CYP3A4 can be displayed in an active form on the surface of E. coli with externally added CPR 3. Surface display is facilitated by usage of the autotransporter secretion pathway for which the protein of interest is combined with an N-terminal signal peptide, a C-terminal linker and beta-barrel domain4. The fusion protein is transported through the inner membrane and inserted into the outer membrane. In our study, we confirmed surface exposure of both enzymes by protease accessibility tests and flow cytometry. The surface displayed CPR is able to reduce cytochrome C5. We could confirm through a HPLC based assay that the whole cell biocatalyst co-expressing CPR and CYP1A2 shows high enzymatic activity towards 7-ethoxyresorufin. This is a promising first step towards a whole cell biocatalyst for pharmaceutical applications such as the synthesis of drug metabolites.