Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris

Florian W. Krainer, Michaela A. Gerstmann, Barbara Darnhofer, Ruth Birner-Gruenberger, Anton Glieder

Research output: Contribution to journalArticleResearchpeer-review

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.

Original languageEnglish
Pages (from-to)181-9
Number of pages9
JournalJournal of Biotechnology
Volume233
DOIs
Publication statusPublished - 10 Sep 2016

Fingerprint

Pichia
Horseradish Peroxidase
Peroxidase
Enzymes
Methanol
Armoracia
Biocatalysis
Benchmarking
Environmental Biodegradation
Secretory Pathway
Bioreactors
Isoenzymes
Yeasts
Medicine

Keywords

  • Journal Article

Cite this

Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris. / Krainer, Florian W.; Gerstmann, Michaela A.; Darnhofer, Barbara; Birner-Gruenberger, Ruth; Glieder, Anton.

In: Journal of Biotechnology, Vol. 233, 10.09.2016, p. 181-9.

Research output: Contribution to journalArticleResearchpeer-review

Krainer, Florian W. ; Gerstmann, Michaela A. ; Darnhofer, Barbara ; Birner-Gruenberger, Ruth ; Glieder, Anton. / Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris. In: Journal of Biotechnology. 2016 ; Vol. 233. pp. 181-9.
@article{240ef29c5aed4cd38d11a8ac08c7b2e2,
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.",
keywords = "Journal Article",
author = "Krainer, {Florian W.} and Gerstmann, {Michaela A.} and Barbara Darnhofer and Ruth Birner-Gruenberger and Anton Glieder",
note = "Copyright {\circledC} 2016 The Authors. Published by Elsevier B.V. All rights reserved.",
year = "2016",
month = "9",
day = "10",
doi = "10.1016/j.jbiotec.2016.07.012",
language = "English",
volume = "233",
pages = "181--9",
journal = "Journal of Biotechnology",
issn = "0168-1656",
publisher = "Elsevier B.V.",

}

TY - JOUR

T1 - Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris

AU - Krainer, Florian W.

AU - Gerstmann, Michaela A.

AU - Darnhofer, Barbara

AU - Birner-Gruenberger, Ruth

AU - Glieder, Anton

N1 - Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

PY - 2016/9/10

Y1 - 2016/9/10

N2 - 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.

AB - 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.

KW - Journal Article

U2 - 10.1016/j.jbiotec.2016.07.012

DO - 10.1016/j.jbiotec.2016.07.012

M3 - Article

VL - 233

SP - 181

EP - 189

JO - Journal of Biotechnology

JF - Journal of Biotechnology

SN - 0168-1656

ER -