No abstract

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.

The methylotrophic yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals. Like most biotechnological production hosts, P. pastoris is heterotrophic and grows on organic feedstocks that have competing uses in the production of food and animal feed. In a step toward more sustainable industrial processes, we describe the conversion of P. pastoris into an autotroph that grows on CO 2 . By addition of eight heterologous genes and deletion of three Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: