title: Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris.
abstract: Horseradish peroxidase (HRP) is a high-demand enzyme for applications in diagnostics, bioremediation, biocatalysis and medicine. Current HRP preparations are isolated from horseradish roots as mixtures of biochemically diverse isoenzymes. Thus, there is a strong need for a recombinant production process enabling a steady supply with enzyme preparations of consistent high quality. However, most current recombinant production systems are limited at titers in the low mg/L range. In this study, we used the well-known yeast Pichia pastoris as host for recombinant HRP production. To enhance recombinant enzyme titers we systematically evaluated engineering approaches on the secretion process, coproduction of helper proteins, and compared expression from the strong methanol-inducible PAOX1 promoter, the strong constitutive PGAP promoter, and a novel bidirectional promoter PHTX1. Ultimately, coproduction of HRP and active Hac1 under PHTX1 control yielded a recombinant HRP titer of 132mg/L after 56h of cultivation in a methanol-independent and easy-to-do bioreactor cultivation process. With regard to the many versatile applications for HRP, the establishment of a microbial host system suitable for efficient recombinant HRP production was highly overdue. The novel HRP production platform in P. pastoris presented in this study sets a new benchmark for this medically relevant enzyme.
authors.affiliation: Graz University of Technology, Institute of Molecular Biotechnology, NAWI Graz, Petersgasse 14, 8010, Graz, Austria. Electronic address: flokrai@gmail.com.
authors.forename: Florian W
authors.initials: FW
authors.lastname: Krainer
authors.affiliation: Graz University of Technology, Institute of Molecular Biotechnology, NAWI Graz, Petersgasse 14, 8010, Graz, Austria. Electronic address: michaela.gerstmann@gmx.net.
authors.forename: Michaela A
authors.initials: MA
authors.lastname: Gerstmann
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB), Petersgasse 14, 8010 Graz, Austria; Medical University of Graz, Institute of Pathology, Research Unit Functional Proteomics and Metabolic Pathways, Stiftingtalstrasse 24, 8010 Graz, Austria; Omics Center Graz, BioTechMed-Graz, Stiftingtalstrasse 24, 8010 Graz, Austria. Electronic address: Barbara.Darnhofer@klinikum-graz.at.
authors.forename: Barbara
authors.initials: B
authors.lastname: Darnhofer
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB), Petersgasse 14, 8010 Graz, Austria; Medical University of Graz, Institute of Pathology, Research Unit Functional Proteomics and Metabolic Pathways, Stiftingtalstrasse 24, 8010 Graz, Austria; Omics Center Graz, BioTechMed-Graz, Stiftingtalstrasse 24, 8010 Graz, Austria. Electronic address: ruth.birner-gruenberger@medunigraz.at.
authors.forename: Ruth
authors.initials: R
authors.lastname: Birner-Gruenberger
authors.affiliation: Graz University of Technology, Institute of Molecular Biotechnology, NAWI Graz, Petersgasse 14, 8010, Graz, Austria. Electronic address: a.glieder@tugraz.at.
authors.forename: Anton
authors.initials: A
authors.lastname: Glieder
mesh_terms: D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000432:Methanol; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D006735:Horseradish Peroxidase; D000432:Methanol
keywords: Bidirectional promoter; Enzyme secretion; Pichia pastoris; Plant peroxidase; Recombinant protein production; Unfolded protein response