Preparative Production of Functionalized (<i>N</i>- and <i>O</i>-Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a Bioreactor
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 a...
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MDPI AG
2022-01-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/12/2/153 |
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| author | Matic Srdič Nico D. Fessner Deniz Yildiz Anton Glieder Markus Spiertz Ulrich Schwaneberg |
| author_facet | Matic Srdič Nico D. Fessner Deniz Yildiz Anton Glieder Markus Spiertz Ulrich Schwaneberg |
| author_sort | Matic Srdič |
| collection | DOAJ |
| description | 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 <i>Komagataella phaffii</i> (<i>Pichia pastoris</i>) 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. |
| first_indexed | 2024-03-09T22:30:56Z |
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| id | doaj.art-7812f3a2f0664b0293285c4ba36251b8 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-09T22:30:56Z |
| publishDate | 2022-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-7812f3a2f0664b0293285c4ba36251b82023-11-23T18:56:54ZengMDPI AGBiomolecules2218-273X2022-01-0112215310.3390/biom12020153Preparative Production of Functionalized (<i>N</i>- and <i>O</i>-Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a BioreactorMatic Srdič0Nico D. Fessner1Deniz Yildiz2Anton Glieder3Markus Spiertz4Ulrich Schwaneberg5SeSaM-Biotech GmbH, 52074 Aachen, GermanyInstitute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, 8010 Graz, AustriaDWI—Leibniz Institute for Interactive Materials, 52074 Aachen, GermanyBisy GmbH, 8200 Hofstaetten an der Raab, AustriaSeSaM-Biotech GmbH, 52074 Aachen, GermanyInstitute of Biotechnology, RWTH Aachen University, 52074 Aachen, GermanyPolycyclic 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 <i>Komagataella phaffii</i> (<i>Pichia pastoris</i>) 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.https://www.mdpi.com/2218-273X/12/2/153cytochrome P450 3A4CYP3A4preparative-scale synthesisPAHswhole-cell biotransformationbioreactor |
| spellingShingle | Matic Srdič Nico D. Fessner Deniz Yildiz Anton Glieder Markus Spiertz Ulrich Schwaneberg Preparative Production of Functionalized (<i>N</i>- and <i>O</i>-Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a Bioreactor Biomolecules cytochrome P450 3A4 CYP3A4 preparative-scale synthesis PAHs whole-cell biotransformation bioreactor |
| title | Preparative Production of Functionalized (<i>N</i>- and <i>O</i>-Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a Bioreactor |
| title_full | Preparative Production of Functionalized (<i>N</i>- and <i>O</i>-Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a Bioreactor |
| title_fullStr | Preparative Production of Functionalized (<i>N</i>- and <i>O</i>-Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a Bioreactor |
| title_full_unstemmed | Preparative Production of Functionalized (<i>N</i>- and <i>O</i>-Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a Bioreactor |
| title_short | Preparative Production of Functionalized (<i>N</i>- and <i>O</i>-Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a Bioreactor |
| title_sort | preparative production of functionalized i n i and i o i heterocyclic polycyclic aromatic hydrocarbons by human cytochrome p450 3a4 in a bioreactor |
| topic | cytochrome P450 3A4 CYP3A4 preparative-scale synthesis PAHs whole-cell biotransformation bioreactor |
| url | https://www.mdpi.com/2218-273X/12/2/153 |
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