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First functional expression of cytochrome P450 3A4 in Pichia pastoris

corresponding

GEIER MARTINA1, SCHMID CHRISTIAN1, GLIEDER ANTON1,2*
*Corresponding author
1. Graz University of Technology, Institute of Molecular Biotechnology, Petersgasse 14, 8010 Graz, Austria
2. Austrian Centre of Industrial Biotechnology (ACIB), Petersgasse 14, 8010 Graz, Austria

Abstract

Getting access to drug metabolites has become an important issue within the drug discovery and development process. Besides chemical synthesis and chromatographic separation, the selective production of these metabolites employing whole-cell biotransformations with recombinant microbes has become an emerging strategy.
Here, we report for the first time the functional expression of the human cytochrome P450 3A4 (CYP3A4), a key enzyme in drug metabolism, in the methylotrophic yeast Pichia pastoris. Co-expression of the human cytochrome b5 and/or the cytochrome P450 reductase (CPR) yielded in a functional whole-cell monooxygenase catalyst.


INTRODUCTION

Cytochrome P450 monooxygenases (CYPs) represent the mayor enzymatic system involved in the phase 1 metabolism of drugs and xenobiotics in humans (1). This superfamily of heme-containing enzymes catalyzes the insertion of one atom of molecular oxygen to a non-activated carbon bond in a regio- and stereoselective way. Such metabolites produced by CYPs are often hard to obtain by classical chemical synthesis (2). However, nowadays there is an increasing need to get access to the respective drug metabolites. They are required as reference compounds for metabolite identification, structure elucidation and in safety testings to investigate their toxicity and biological activity (3). The fast and reliable production of these compounds on an analytical, but