Preparative Production of Functionalized (N- and O-Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a Bioreactor

Biomolecules, vol. 12, pp. 153

Abstract

Polycyclic aromatic hydrocarbons (PAHs) and their N- and O-containing derivatives (N-/O-PAHs) are environmental pollutants and synthetically attractive building blocks in pharmaceuticals. Functionalization of PAHs can be achieved via C-H activation by cytochrome P450 enzymes (e.g., P450 CYP3A4) in an environmentally friendly manner. Despite its broad substrate scope, the contribution of CYP3A4 to metabolize common PAHs in humans was found to be small. We recently showcased the potential of CYP3A4 in whole-cell biocatalysis with recombinant yeast Komagataella phaffii (Pichia pastoris) catalysts for the preparative-scale synthesis of naturally occurring metabolites in humans. In this study, we aimed at exploring the substrate scope of CYP3A4 towards (N-/O)-PAHs and conducted a bioconversion experiment at 10 L scale to validate the synthetic potential of CYP3A4 for the preparative-scale production of functionalized PAH metabolites. Hydroxylated products were purified and characterized using HPLC and NMR analysis. In total, 237 mg of fluorenol and 48 mg of fluorenone were produced from 498 mg of fluorene, with peak productivities of 27.7 μmol/L/h for fluorenol and 5.9 μmol/L/h for fluorenone; the latter confirmed that CYP3A4 is an excellent whole-cell biocatalyst for producing authentic human metabolites.

Authors 6

1. Matic Srdič Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology, RWTH Aachen University, 52074 Aachen, Germany

   SeSaM-Biotech GmbH, 52074 Aachen, Germany

2. Nico D. Fessner

   Graz University of Technology · Nawi Graz

   Affiliation as printed

   Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, 8010 Graz, Austria

3. Deniz Yıldız Aachen DWI - Leibniz Institute for Interactive Materials Institute for Technical and Macromolecular Chemistry

   RWTH Aachen University · DWI – Leibniz Institute for Interactive Materials

   Affiliation as printed

   DWI—Leibniz Institute for Interactive Materials, 52074 Aachen, Germany

   Institute for Technical and Macromolecular Chemistry, RWTH Aachen University, 52074 Aachen, Germany

4. Anton Glieder
   Affiliation as printed

   Bisy GmbH, 8200 Hofstaetten an der Raab, Austria

5. Markus Spiertz corresponding
   Affiliation as printed

   SeSaM-Biotech GmbH, 52074 Aachen, Germany

6. Ulrich Schwaneberg corresponding Aachen DWI - Leibniz Institute for Interactive Materials Institute of Biotechnology

   RWTH Aachen University · DWI – Leibniz Institute for Interactive Materials

   Affiliation as printed

   DWI—Leibniz Institute for Interactive Materials, 52074 Aachen, Germany

   Institute of Biotechnology, RWTH Aachen University, 52074 Aachen, Germany