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

Ulla Christensen, Dario Vazquez Albacete, Karina Marie Søgaard, Tonja Hobel, Morten Thrane Nielsen, Scott James Harrison, Anders Holmgaard Hansen, Birger Lindberg Møller, Susanna Seppala, Morten Nørholm

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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.
Original languageEnglish
JournalApplied Microbiology and Biotechnology
Volume101
Issue number10
Pages (from-to)4103-4113
Number of pages11
ISSN0175-7598
DOIs
Publication statusPublished - 2017

Keywords

  • Cell factories
  • Cytochromes P450
  • Medicinal compounds
  • Membrane protein
  • Protein production

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