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Improvement of P450BM-3 whole-cell biocatalysis by integrating heterologous cofactor regeneration combining glucose facilitator and dehydrogenase in E. coli

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Abstract

Escherichia coli BL21, expressing a quintuple mutant of P450BM-3, oxyfunctionalizes α-pinene in an NADPH-dependent reaction to α-pinene oxide, verbenol, and myrtenol. We optimized the whole-cell biocatalyst by integrating a recombinant intracellular NADPH regeneration system through co-expression of a glucose facilitator from Zymomonas mobilis for uptake of unphosphorylated glucose and a NADP+-dependent glucose dehydrogenase from Bacillus megaterium that oxidizes glucose to gluconolactone. The engineered strain showed a nine times higher initial α-pinene oxide formation rate corresponding to a sixfold higher yield of 20 mg g−1 cell dry weight after 1.5 h. The initial total product formation rate was 1,000 μmol h−1 μmol−1 P450 leading to a total of 32 mg oxidized products per gram cell of dry weight after 1.5 h. The physiological functioning of the heterologous cofactor regeneration system was illustrated by a sevenfold increased α-pinene oxide yield in the presence of glucose compared to glucose-free conditions.

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Acknowledgments

This research was financially supported by the German Federal Ministry of Economics and Technology via the AiF ZUTECH program (project no. 119 ZN). We thank H. Sahm, Research Centre Juelich for plasmid pET24glcdh and pZY507glf, U. Schwaneberg, Jacobs University Bremen for the supply with 12-pNCA and R.D. Schmid, Institute of Technical Biochemstry, Stuttgart for the provision of pET28bm-3qm.

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  1. Biochemical Engineering Group, DECHEMA e.V., Karl-Winnacker-Institut, Theodor-Heuss-Allee 25, 60486, Frankfurt, Germany

    Hendrik Schewe, Bjoern-Arne Kaup & Jens Schrader

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  1. Hendrik Schewe
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  2. Bjoern-Arne Kaup
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  3. Jens Schrader
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Correspondence to Jens Schrader.

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Schewe, H., Kaup, BA. & Schrader, J. Improvement of P450BM-3 whole-cell biocatalysis by integrating heterologous cofactor regeneration combining glucose facilitator and dehydrogenase in E. coli . Appl Microbiol Biotechnol 78, 55–65 (2008). https://doi.org/10.1007/s00253-007-1277-1

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