The effect of temperature on the proteome of recombinant Pichia pastoris.
The impact of environmental factors on the productivity of yeast cells is poorly investigated so far. Therefore, it is a major concern to improve the understanding of cellular physiology of microbial protein production hosts, including the methylotrophic yeast Pichia pastoris. Two-Dimensional Fluorescence Difference Gel electrophoresis and protein identification via mass spectrometry were applied to analyze the impact of cultivation temperature on the physiology of a heterologous protein secreting P. pastoris strain. Furthermore, specific productivity was monitored and fluxes through the central carbon metabolism were calculated. Chemostat culture conditions were applied to assess the adaption to different growth temperatures (20, 25, 30 degrees C) at steady-state conditions. Many important cellular processes, including the central carbon metabolism, stress response and protein folding are affected by changing the growth temperature. A 3-fold increased specific productivity at lower cultivation temperature for an antibody Fab fragment was accompanied by a reduced flux through the TCA-cycle, reduced levels of proteins involved in oxidative stress response and lower cellular levels of molecular chaperones. These data indicate that folding stress is generally decreased at lower cultivation temperatures, enabling more efficient heterologous protein secretion in P. pastoris host cells.
Dragosits
Martin
M
Department of Biotechnology, BOKU-University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Stadlmann
Johannes
J
Albiol
Joan
J
Baumann
Kristin
K
Maurer
Michael
M
Gasser
Brigitte
B
Sauer
Michael
M
Altmann
Friedrich
F
Ferrer
Pau
P
Mattanovich
Diethard
D
D002244:Carbon / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000276:immunology / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D009154:Mutation / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D013696:Temperature
D005656:Fungal Proteins; D007140:Immunoglobulin Fab Fragments; D020543:Proteome; D002244:Carbon