Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase

Microbial Cell Factories

Research

Daniel Maresch¹  Friedrich Altmann¹  Stanimira Krasteva²  Amirhossein Saadati²  Christoph Herwig²  Christoph Gmeiner²  Oliver Spadiut²  Manuela Frank² 

[1] Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria;Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Gumpendorfer Strasse 1a, 1060, Vienna, Austria;

关键词: Pichia pastoris;    Glycosylation;    OCH1;    Horseradish peroxidase;    Bioreactor cultivation;    Fed-batch;    Design of Experiments;   

DOI  :  10.1186/s12934-014-0183-3

 received in 2014-09-07, accepted in 2014-12-18,  发布年份 2015

来源: Springer

【 摘 要 】

Pichia pastoris is a prominent host for recombinant protein production, amongst other things due to its capability of glycosylation. However, N-linked glycans on recombinant proteins get hypermannosylated, causing problems in subsequent unit operations and medical applications. Hypermannosylation is triggered by an α-1,6-mannosyltransferase called OCH1. In a recent study, we knocked out OCH1 in a recombinant P. pastoris CBS7435 MutS strain (Δoch1) expressing the biopharmaceutically relevant enzyme horseradish peroxidase. We characterized the strain in the controlled environment of a bioreactor in dynamic batch cultivations and identified the strain to be physiologically impaired. We faced cell cluster formation, cell lysis and uncontrollable foam formation.In the present study, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on 1) cell physiology, 2) cell morphology, 3) cell lysis, 4) productivity and 5) product purity of the recombinant Δoch1 strain in a multivariate manner. Cultivation at 30°C resulted in low specific methanol uptake during adaptation and the risk of methanol accumulation during cultivation. Cell cluster formation was a function of the C-source rather than process parameters and went along with cell lysis. In terms of productivity and product purity a temperature of 20°C was highly beneficial. In summary, we determined cultivation conditions for a recombinant P. pastoris Δoch1 strain allowing high productivity and product purity.

【 授权许可】

Unknown   
© Gmeiner 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.