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Applied Microbiology and Biotechnology

, Volume 101, Issue 10, pp 4103–4113 | Cite as

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

  • Ulla Christensen
  • Dario Vazquez-Albacete
  • Karina M. Søgaard
  • Tonja Hobel
  • Morten T. Nielsen
  • Scott James Harrison
  • Anders Holmgaard Hansen
  • Birger Lindberg Møller
  • Susanna SeppäläEmail author
  • Morten H. H. NørholmEmail author
Biotechnologically relevant enzymes and proteins

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.

Keywords

Cytochromes P450 Protein production Membrane protein Cell factories Medicinal compounds 

Notes

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.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_8076_MOESM1_ESM.pdf (2.8 mb)
ESM 1 (PDF 2837 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Ulla Christensen
    • 1
  • Dario Vazquez-Albacete
    • 1
  • Karina M. Søgaard
    • 1
  • Tonja Hobel
    • 1
  • Morten T. Nielsen
    • 1
  • Scott James Harrison
    • 1
  • Anders Holmgaard Hansen
    • 1
  • Birger Lindberg Møller
    • 2
    • 3
  • Susanna Seppälä
    • 1
    Email author
  • Morten H. H. Nørholm
    • 1
    Email author
  1. 1.Novo Nordisk Foundation Center for BiosustainabilityTechnical University of DenmarkLyngbyDenmark
  2. 2.Plant Biochemistry Laboratory, Department of Plant and Environmental SciencesUniversity of CopenhagenFrederiksberg CDenmark
  3. 3.Center for Synthetic Biology: bioSYNergyUniversity of CopenhagenFrederiksberg CDenmark

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