Co-expression of catalytic active human cytochrome P450 reductase and cytochrome P450 1A2 on the surface of Escherichia Coli

Quehl P, Riemer J, Jose J

Abstract

The wider usage of purified human cytochrome P450 enzymes as biocatalyst is impeded by several 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) 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 alone can be displayed in an active form on the surface of E. coli with externally added CPR (Schumacher und Jose, 2012). 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 a beta-barrel domain (Schüürmann et al., 2014). The resulting 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 cytometric analysis. The surface displayed CPR is able to reduce cytochrome C and ferricyanid. Fluorescence spectra analysis indicates that the CPR and CYP1A2 fusion proteins bind their respective co-factors. Furthermore, we could confirm through a HPLC based assay that the whole cell biocatalyst co-expressing CPR and CYP1A2 shows enzymatic activity towards the test substrate 7-ethoxyresorufin. These results are a promising first step of creating a whole cell biocatalyst for pharmaceutical relevant applications such as the synthesis of drug metabolites.