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Convenient Gram-Scale Metabolite Synthesis by Engineered Fission Yeast Strains Expressing Functional Human P450 Systems

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Abstract

The growing need for the characterization of cytochrome P450 (P450) metabolites often necessitates their synthesis up to Gram-scale. This task may in principle be achieved by using various techniques including chemical synthesis, the use of laboratory animals, in vitro P450 systems or microbial biotransformation. However, these approaches are in many instances unfavorable due to low yields, laborious purification, costs of cofactors, or the formation of non-physiologic metabolites. The fission yeast Schizosaccharomyces pombe has previously been shown by others and us to be very well suited for the heterologous expression of human P450s. In this study, we demonstrate whole-cell biotransformation reactions carried out with fission yeast strains that coexpress human cytochrome P450 reductase (CPR) and one of the following P450 isoforms: CYP2B6, CYP2C9, CYP2C19, CYP2D6, or CYP3A4, respectively. These strains could successfully convert their respective standard substrates but showed different responses with respect to incubation pH, the presence of glucose, and temperature, respectively. In addition, the preparative of synthesis of 2.8 g of 4′-hydroxydiclofenac was achieved by whole-cell biotransformation of diclofenac using a CPR-CYP2C9 coexpressing fission yeast strain.

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Abbreviations

P450:

Cytochrome P450

CPR:

Cytochrome P450 reductase

DAD:

Diode array detector

EMM:

Edinburgh Minimal Medium

d.wt.:

Dry weight

LC-MS/MS:

Liquid chromatography-tandem mass spectrometer

MRM:

Multiple reaction monitoring

MTP:

Microtiter plate

NaPi:

Sodium phosphate buffer

TMS:

Tetramethylsilane

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Acknowledgements

This work was supported by a grant (ChemBioTec 13220-32) from the Deutsche Bundesstiftung Umwelt (DBU).

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Correspondence to Matthias Bureik.

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Drăgan, CA., Peters, F.T., Bour, P. et al. Convenient Gram-Scale Metabolite Synthesis by Engineered Fission Yeast Strains Expressing Functional Human P450 Systems. Appl Biochem Biotechnol 163, 965–980 (2011). https://doi.org/10.1007/s12010-010-9100-3

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  • DOI: https://doi.org/10.1007/s12010-010-9100-3

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