title: Co-expression of active human cytochrome P450 1A2 and cytochrome P450 reductase on the cell surface of Escherichia coli.
abstract: BACKGROUND
  Human 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.
  
  
  RESULTS
  To 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.
  
  
  CONCLUSIONS
  We 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.
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149, Münster, Germany. paul.quehl@uni-muenster.de.
authors.forename: Paul
authors.initials: P
authors.lastname: Quehl
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149, Münster, Germany. mail.hollender@gmail.com.|Autodisplay Biotech GmbH, Merowingerplatz 1a, 40225, Düsseldorf, Germany. mail.hollender@gmail.com.
authors.forename: Joel
authors.initials: J
authors.lastname: Hollender
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149, Münster, Germany. jan.schueuermann@uni-muenster.de.
authors.forename: Jan
authors.initials: J
authors.lastname: Schüürmann
authors.affiliation: Autodisplay Biotech GmbH, Merowingerplatz 1a, 40225, Düsseldorf, Germany. tatjana.brossette@autodisplay-biotech.com.
authors.forename: Tatjana
authors.initials: T
authors.lastname: Brossette
authors.affiliation: Autodisplay Biotech GmbH, Merowingerplatz 1a, 40225, Düsseldorf, Germany. ruth.maas@autodisplay-biotech.com.
authors.forename: Ruth
authors.initials: R
authors.lastname: Maas
authors.affiliation: Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149, Münster, Germany. joachim.jose@uni-muenster.de.
authors.forename: Joachim
authors.initials: J
authors.lastname: Jose
mesh_terms: D001483:Base Sequence; D055162:Biocatalysis; D002462:Cell Membrane / Q000378:metabolism*; D003470:Culture Media / Q000494:pharmacology; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010078:Oxazines / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010615:Phenacetin / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D003470:Culture Media; D010078:Oxazines; C007649:ethoxyresorufin; D045304:Cytochromes c; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2; D010447:Peptide Hydrolases; D010615:Phenacetin
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