title: De-bugging and maximizing plant cytochrome P450 production in Escherichia coli with C-terminal GFP fusions.
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.
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, Lyngby, Denmark.
authors.forename: Ulla
authors.initials: U
authors.lastname: Christensen
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, Lyngby, Denmark.
authors.forename: Dario
authors.initials: D
authors.lastname: Vazquez-Albacete
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, Lyngby, Denmark.
authors.forename: Karina M
authors.initials: KM
authors.lastname: Søgaard
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, Lyngby, Denmark.
authors.forename: Tonja
authors.initials: T
authors.lastname: Hobel
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, Lyngby, Denmark.
authors.forename: Morten T
authors.initials: MT
authors.lastname: Nielsen
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, Lyngby, Denmark.
authors.forename: Scott James
authors.initials: SJ
authors.lastname: Harrison
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, Lyngby, Denmark.
authors.forename: Anders Holmgaard
authors.initials: AH
authors.lastname: Hansen
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Copenhagen, Denmark.|Center for Synthetic Biology: bioSYNergy, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, Copenhagen, Denmark.
authors.forename: Birger Lindberg
authors.initials: BL
authors.lastname: Møller
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, Lyngby, Denmark. susse@biosustain.dtu.dk.
authors.forename: Susanna
authors.initials: S
authors.lastname: Seppälä
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, Lyngby, Denmark. morno@biosustain.dtu.dk.
authors.forename: Morten H H
authors.initials: MHH
authors.lastname: Nørholm
mesh_terms: D000818:Animals; D055162:Biocatalysis; D003001:Cloning, Molecular / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D049452:Green Fluorescent Proteins / Q000235:genetics; D010084:Oxidation-Reduction; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003577:Cytochrome P-450 Enzyme System
keywords: Cell factories; Cytochromes P450; Medicinal compounds; Membrane protein; Protein production