title: Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris.
abstract: Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins.
authors.affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Gumpendorfer Strasse 1a, 1060 Vienna, Austria. simona.capone@tuwien.ac.at.
authors.forename: Simona
authors.initials: S
authors.lastname: Capone
authors.affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Gumpendorfer Strasse 1a, 1060 Vienna, Austria. lejla.corajevic91@gmail.com.
authors.forename: Lejla
authors.initials: L
authors.lastname: Ćorajević
authors.affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Gumpendorfer Strasse 1a, 1060 Vienna, Austria. guenther.bonifert@chello.at.
authors.forename: Günther
authors.initials: G
authors.lastname: Bonifert
authors.affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Gumpendorfer Strasse 1a, 1060 Vienna, Austria. patrick.murth@gmx.at.
authors.forename: Patrick
authors.initials: P
authors.lastname: Murth
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, 1180 Vienna, Austria. maresch@gmx.at.
authors.forename: Daniel
authors.initials: D
authors.lastname: Maresch
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, 1180 Vienna, Austria. friedrich.altmann@boku.ac.at.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Gumpendorfer Strasse 1a, 1060 Vienna, Austria. christoph.herwig@tuwien.ac.at.
authors.forename: Christoph
authors.initials: C
authors.lastname: Herwig
authors.affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Gumpendorfer Strasse 1a, 1060 Vienna, Austria. oliver.spadiut@tuwien.ac.at.
authors.forename: Oliver
authors.initials: O
authors.lastname: Spadiut
mesh_terms: D019149:Bioreactors; D060846:Cell Engineering; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D006735:Horseradish Peroxidase
keywords: OCH1; bioreactor cultivation; glyco-engineering; horseradish peroxidase; site-directed mutagenesis; strain engineering