Optimizing cofactor availability for the production of recombinant heme peroxidase in Pichia pastoris.

Florian Krainer, Simona Capone, Martin Jäger, Thomas Vogl, Michaela Agnes Gerstmann, Anton Glieder, Christoph Herwig, Oliver Spadiut*

*Korrespondierende/r Autor/-in für diese Arbeit

Publikation: Beitrag in einer FachzeitschriftArtikelBegutachtung

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.
Originalspracheenglisch
Aufsatznummer4
Seiten (von - bis)1-9
FachzeitschriftMicrobial Cell Factories
Jahrgang14
Ausgabenummer4
DOIs
PublikationsstatusVeröffentlicht - 2015

Fields of Expertise

  • Human- & Biotechnology

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