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

Microbial Cell Factories

Technical Notes

Florian W Krainer¹  Michaela Gerstmann¹  Anton Glieder¹  Thomas Vogl¹  Simona Capone²  Martin Jäger²  Christoph Herwig²  Oliver Spadiut² 

[1] Graz University of Technology, NAWI Graz, Institute of Molecular Biotechnology, Graz, Austria;Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Gumpendorfer Strasse 1a, 1060, Vienna, Austria;

关键词: Pichia pastoris;    Recombinant protein production;    Plant peroxidase;    Horseradish peroxidase;    Metabolic engineering;    Cofactor;    Heme;    Heme biosynthesis;    Apo-protein;   

DOI  :  10.1186/s12934-014-0187-z

 received in 2014-10-29, accepted in 2014-12-26,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundInsufficient 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.ResultsIn 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.ConclusionsThe 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.

【 授权许可】

Unknown   
© Krainer et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.