Towards drug metabolite synthesis with surface displayed human cytochrome P450 1A2 and cytochrome P450 reductase.

Quehl P, Hollender J, Jose J

Abstract

Human cytochrome P450 monooxygenases (CYPs) are involved in the metabolism of most drugs [1]. They catalyze a variety ofoxidations accepting a broad range of substrates and have a major impact on bioavailability as well as drug-drug interactions.The wider usage of purified CYPS as biocatalyst is impeded by factors such as their difficult recombinant expression andsubsequent purification, low catalytic activities, requirement for membrane surroundings and poor protein stability. Many of thesechallenges can be circumvented by the application of a bacterial whole cell catalyst. In our work, we established theco-expression of human cytochrome P450 reductase (CPR) together with the CYP1A2 monooxygenase on the surface of E. coli[2]. Surface display can be advantageous by offering a membrane environment, overcoming mass transfer limitations andimproved protein stability. Previously, it had been shown that CYP3A4 can be displayed in an active form on the surface of E. coliwith externally added CPR [3]. Surface display is facilitated by usage of the autotransporter secretion pathway for which theprotein of interest is combined with an N-terminal signal peptide, a C-terminal linker and a beta-barrel domain [4]. The resultingfusion protein is inserted into the outer membrane. In our study, we confirmed surface exposure of both enzymes by proteaseaccessibility tests and flow cytometric analysis. The surface displayed CPR is enzymatic active towards cytochrome c. The wholecell biocatalyst co-expressing CPR and CYP1A2 is active towards the three structural different substrates 7-ethoxyresorufin,phenacetin and Luciferin-MultiCYP. About 1 μM resorufin from 5 μM substrate and 2.2 μM paracetamol from 250 μM phenacetinwas obtained by whole cell biocatalysis. We identified several growth medium parameters such as expression temperature, 5-aminolevulinic acid, and Na+-, K+-, Ca2+-, and inductor concentrations that are important for activity. Conclusively, we achievedthe very first fully active whole biocatalyst with surface displayed CYP1A2 and CPR. These results are a promising first steptowards a whole cell biocatalyst for pharmaceutical applications such as the synthesis of drug metabolites