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De-bugging and maximizing plant cytochrome P450 production in Escherichia coli with C-terminal GFP fusions

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Abstract

Cytochromes P450 (CYP) are attractive enzyme targets in biotechnology as they catalyze stereospecific C-hydroxylations of complex core skeletons at positions that typically are difficult to access by chemical synthesis. Membrane bound CYPs are involved in nearly all plant pathways leading to the formation of high-value compounds. In the present study, we systematically maximize the heterologous expression of six different plant-derived CYP genes in Escherichia coli, using a workflow based on C-terminal fusions to the green fluorescent protein. The six genes can be over-expressed in both K- and B-type E. coli strains using standard growth media. Furthermore, sequences encoding a small synthetic peptide and a small bacterial membrane anchor markedly enhance the expression of all six genes. For one of the CYPs, the length of the linker region between the predicted N-terminal transmembrane segment and the soluble domain is modified, in order to verify the importance of this region for enzymatic activity. The work describes how membrane bound CYPs are optimally produced in E. coli and thus adds this plant multi-membered key enzyme family to the toolbox for bacterial cell factory design.

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Acknowledgements

We thank Victor de Lorenzo and the members of his laboratory for generously providing the pSEVA collection. We thank Tomas Laursen, Peter Naur, Søren Bak, Björn Hamberger, Johan Andersen-Ranberg, and Britta Hamberger for advice on CYPs and reductases. We thank David Drew, Daniel Daley, and Jan. Willem de Gier for discussions on E. coli gene expression and the GFP-based expression platform. SS is the recipient of VILLUM Foundation’s Young Investigator Programme grant VKR023128. This work was supported by the Novo Nordisk Foundation, from the VILLUM research center of excellence “Plant Plasticity,” from the UCPH Excellence Program for Interdisciplinary Research to Center of Synthetic Biology “bioSYNergy,” and by a European Research Council Advanced Grant Project No. 323034: LightdrivenP450s.

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    1. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, Lyngby, Denmark

      Ulla Christensen, Dario Vazquez-Albacete, Karina M. Søgaard, Tonja Hobel, Morten T. Nielsen, Scott James Harrison, Anders Holmgaard Hansen, Susanna Seppälä & Morten H. H. Nørholm

    2. Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Copenhagen, Denmark

      Birger Lindberg Møller

    3. Center for Synthetic Biology: bioSYNergy, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Copenhagen, Denmark

      Birger Lindberg Møller

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    1. Ulla Christensen
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    Correspondence to Susanna Seppälä or Morten H. H. Nørholm.

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    Ulla Christensen and Dario Vazquez-Albacete contributed equally to this work.

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    Christensen, U., Vazquez-Albacete, D., Søgaard, K.M. et al. De-bugging and maximizing plant cytochrome P450 production in Escherichia coli with C-terminal GFP fusions. Appl Microbiol Biotechnol 101, 4103–4113 (2017). https://doi.org/10.1007/s00253-016-8076-5

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