Journal of Microbial & Biochemical Technology

Direct monitoring of the unfolded protein response in Pichia pastoris

World Congress and Expo on Applied Microbiology

August 18-20, 2015 Frankfurt, Germany

Raschmanova H1, Knejzlik Z1, Kovar K2 and Melzoch K1

1University of Chemistry and Technology Prague, Czech Republic 2Zurich University of Applied Sciences, Switzerland

Posters-Accepted Abstracts: J Microb Biochem Technol

Abstract:

High productivity, safety and the ability to secrete high quality heterologous proteins make the methylotrophic yeast Pichia pastoris a popular microbial host system for the production of recombinant proteins. However, inappropriate process conditions, as well as heterologous protein production itself induce stress in the lumen of the endoplasmic reticulum. Consequently, a cascade of signalling processes called the Unfolded Protein Response (UPR) pathway is triggered. Because the pathway results in the decrease of proteosynthesis or even protein degradation, and thus is the main factor affecting protein quantity and quality (cell productivity). It would be of great significance to monitor the UPR-activity directly during a cultivation process. Nowadays, UPR is monitored by time-consuming and expensive methods (PCR, electrophoretic methods). To analyse the state of UPR in a real time, we designed a Pichia pastoris strain in which the UPR-activity was detected by flow cytometry. A green fluorescent protein gene was used as a marker of the activity of six selected upstream regions (i.e. promoters) of the UPRrelated genes (PDI, ERO, JEM, SCJ, CAL and BiP). The UPR pathway was induced by dithiothreitol addition and the response of the promoters was evaluated as a fluorescence signal by flow cytometry. BiP1 was assessed as the most suitable promoter for stress monitoring, since the five-fold increase of its activity was observed after the treatment. The BiP1 promoter is currently being characterised. We suggested a promising method straightforward monitoring of the cellular stress that could be applied to optimize a biotechnological production of recombinant proteins.

Biography :

Raschmanova H completed her Master´s degree in the Department of Biotechnology of the University of Chemistry and Technology Prague in 2014 at the age of 24 years. Since 2014, she has been the student of PhD degree at the same institute. She specializes in the research of the Unfolded Protein Response in Pichia pastoris and its relationship to the production of recombinant proteins.