title: Optimizing cofactor availability for the production of recombinant heme peroxidase in Pichia pastoris.
abstract: BACKGROUND
  Insufficient incorporation of heme is considered a central impeding cause in the recombinant production of active heme proteins. Currently, two approaches are commonly taken to overcome this bottleneck; metabolic engineering of the heme biosynthesis pathway in the host organism to enhance intracellular heme production, and supplementation of the growth medium with the desired cofactor or precursors thereof to allow saturation of recombinantly produced apo-forms of the target protein. In this study, we investigated the effect of both, pathway engineering and medium supplementation, to optimize the recombinant production of the heme protein horseradish peroxidase in the yeast Pichia pastoris.
  
  
  RESULTS
  In contrast to studies with other hosts, co-overexpression of genes of the endogenous heme biosynthesis pathway did not improve the recombinant production of active heme protein. However, medium supplementation with hemin proved to be an efficient strategy to increase the yield of active enzyme, whereas supplementation with the commonly used precursor 5-aminolevulinic acid did not affect target protein yield.
  
  
  CONCLUSIONS
  The yield of active recombinant heme peroxidase from P. pastoris can be easily enhanced by supplementation of the cultivation medium with hemin. Thereby, secreted apo-species of the target protein are effectively saturated with cofactor, maximizing the yield of target enzyme activity.
authors.affiliation: Graz University of Technology, NAWI Graz, Institute of Molecular Biotechnology, Graz, Austria. florian.krainer@tugraz.at.
authors.forename: Florian W
authors.initials: FW
authors.lastname: Krainer
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Gumpendorfer Strasse 1a, 1060, Vienna, Austria. simona.capone@tuwien.ac.at.
authors.forename: Simona
authors.initials: S
authors.lastname: Capone
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Gumpendorfer Strasse 1a, 1060, Vienna, Austria. martinjaeger@chello.at.
authors.forename: Martin
authors.initials: M
authors.lastname: Jäger
authors.affiliation: Graz University of Technology, NAWI Graz, Institute of Molecular Biotechnology, Graz, Austria. thomas.vogl@tugraz.at.
authors.forename: Thomas
authors.initials: T
authors.lastname: Vogl
authors.affiliation: Graz University of Technology, NAWI Graz, Institute of Molecular Biotechnology, Graz, Austria. michaela.gerstmann@gmx.net.
authors.forename: Michaela
authors.initials: M
authors.lastname: Gerstmann
authors.affiliation: Graz University of Technology, NAWI Graz, Institute of Molecular Biotechnology, Graz, Austria. a.glieder@tugraz.at.
authors.forename: Anton
authors.initials: A
authors.lastname: Glieder
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Gumpendorfer Strasse 1a, 1060, Vienna, Austria. christoph.herwig@tuwien.ac.at.
authors.forename: Christoph
authors.initials: C
authors.lastname: Herwig
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Gumpendorfer Strasse 1a, 1060, Vienna, Austria. oliver.spadiut@tuwien.ac.at.
authors.forename: Oliver
authors.initials: O
authors.lastname: Spadiut
mesh_terms: D042931:Aldehyde Oxidase / Q000235:genetics; D061249:Batch Cell Culture Techniques; D005296:Ferrous Compounds / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D006418:Heme / Q000096:biosynthesis*; D006735:Horseradish Peroxidase / Q000235:genetics* / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D005296:Ferrous Compounds; D005656:Fungal Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C020748:ferrous sulfate; D006418:Heme; D006735:Horseradish Peroxidase; D042931:Aldehyde Oxidase