An expression tag toolbox for microbial production of membrane bound plant cytochromes P450

Dario Vazquez Albacete, Mafalda Cavaleiro, Ulla Christensen, Susanna Seppala, Birger Lindberg Møller, Morten Nørholm

Research output: Contribution to journalJournal articleResearchpeer-review

854 Downloads (Pure)

Abstract

Membrane-associated Cytochromes P450 (P450s) are one of the most important enzyme families for biosynthesis of plant-derived medicinal compounds. However, the hydrophobic nature of P450s makes their use in robust cell factories a challenge. Here we explore a small library of N-terminal expression tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Analysis of long-term cultures by flow cytometry showed homogeneous populations for some of the conditions. Three chimeric designs were chosen for a more complex combinatorial assembly of a multigene pathway consisting of two P450s and a redox partner. Cells expressing these recombinant enzymes catalysed the conversion of the substrate to highly different ratios of the intermediate and the final product of the pathway. Finally, the effect of a robustly performing expression tag was explored with a library of 49 different P450s from medicinal plants and nearly half of these were improved in expression by more than 2-fold. The developed toolbox serves as platform to tune P450 performance in microbial cells, thereby facilitating recombinant production of complex plant P450-derived biochemicals.
Original languageEnglish
JournalBiotechnology and Bioengineering
Volume114
Issue number4
Pages (from-to)751-760
ISSN0006-3592
DOIs
Publication statusPublished - 2017

Keywords

  • N-terminal tag
  • Cytochromes P450
  • Gene expression
  • Medicinal plants
  • Membrane protein
  • Terpenoids

Fingerprint

Dive into the research topics of 'An expression tag toolbox for microbial production of membrane bound plant cytochromes P450'. Together they form a unique fingerprint.
  • BacTory: Biorefinery Training Platform: Bacterial Factories for Sustainable Chemical and Drug Production

    Molin, S. (Project Manager), Nørholm, M. (Project Manager), Cavaleiro, M. (PhD Student), Machado, H. (PhD Student), Wendel, S. (PhD Student), van der Helm, E. (PhD Student), Bojanovic, K. (PhD Student), Vazquez Albacete, D. (Project Participant), D'Arrigo, I. (PhD Student), Li, S. (PhD Student), Yang, X. (PhD Student), Redl, S. M. A. (PhD Student), Pogrebnyakov, I. (PhD Student), Lehning, C. E. (PhD Student), Giubergia, S. (PhD Student), Calero Valdayo, P. M. (PhD Student) & Lohmann, R. (Project Manager)

    01/10/201231/01/2017

    Project: Research

Cite this