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Impact of oxygen availability on the organelle-specific redox potentials and stress in recombinant protein producing Komagataella phaffii

ABSTRACT: The methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is one of the most effective and established expression hosts for heterologous protein production. The redox balance of its secretory pathway, which is multi-organelle dependent, is of high importance for recombinant protein production. Redox imbalance and oxidative stress are two main factors that can influence protein production and secretion, especially the redox potential of the ER where protein folding and disulfide bond formation occur. Glutathione is the main redox buffer of the cell and its redox conditions can be determined by the status of the glutathione redox couple (GSH-GSSG). In this study, in vivo measurements of the glutathione redox potential in different subcellular compartments were achieved by genetically encoded redox sensitive fluorescent probes (roGFPs). Using these biosensors, the impact of oxygen availability on the redox potentials of different organelles (cytosol, ER, mitochondria and peroxisomes) of non-producing and producing K. phaffii strains in glucose-limited chemostat cultures was investigated. It was found that the transition from normoxic to hypoxic conditions affected the redox potentials of all investigated organelles. Also, as reported previously, hypoxic conditions led to increased recombinant protein secretion. Finally, transcriptome and proteome datasets analysis provided novel insights into the short-term adaptation of the cells from normoxic to hypoxic conditions. In conclusion, improved production of therapeutic disulfide-bonded proteins in industrial settings can be achieved by tuning oxidative stress and redox homeostasis in the K. phaffii strains.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Komagataella Phaffii

SUBMITTER: Sandra Maass

LAB HEAD: Dörte Becher

PROVIDER: PXD055501 | Pride | 2025-01-30

REPOSITORIES: pride

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			Action	DRS
	220928_VE1_PM4_AOs_Aliki_Strap_bRP_2-1_1.raw	Raw
	220928_VE1_PM4_AOs_Aliki_Strap_bRP_2-1_2.raw	Raw
	220928_VE1_PM4_AOs_Aliki_Strap_bRP_2-1_3.raw	Raw
	220928_VE1_PM4_AOs_Aliki_Strap_bRP_2-1_4.raw	Raw
	220929_VE1_PM4_AOs_Aliki_STrap_bRP_3-1_1.raw	Raw

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Project description:The Saccharomyces cerevisiae SFP1 is required for proper regulation of ribosome biogenesis and cell size in response to nutrients. A mutant deleted for SFP1 shows specific traits among which a slow growth phenotype, which is particularly evident during growth on glucose. To assess the effects of nutrients on the activity of Sfp1 independent by growth rate related feedback we grew an sfp1Î mutant and its isogenic reference strain in chemostat cultures, at the same specific growth rate, under glucose/ethanol-limitation. Our data show that Sfp1 is involved in the modulation of cell size and RiBi gene expression and that these two functions are differently influenced by nutrients. The continuous cultures were then pulsed with a glucose excess generating a situation of batch growth similar to shake flask cultures. The dynamic analysis of the metabolic and transcriptional response following the glucose addition suggested that Sfp1 plays a role at the crossroads of ribosome biogenesis and central carbon metabolism regulation. Finally, we show that the down-regulation of RP genes, which was observed in an sfp1Î strain during shake flask growth, cannot be directly ascribed to the absence of Sfp1 but is most probably a secondary effect due to the low growth potential of the mutant strain. Experiment Overall Design: After ten volume changes, few seconds after the samples for the steady state analysis were collected, the anaerobic glucose pulse experiments were started by sparging the medium reservoir and the fermenter with pure nitrogen gas (airflow of 0.5 L min-1, Hoek-Loos, Schiedam, <5 ppm O2). NorpreneTM tubing and butyl septa were used to minimize oxygen diffusion into the anaerobic culture. Two minutes after nitrogen sparging and just before the addition of glucose, the medium and the effluent pumps were switched off. At this time point (which we will refer to as time T=0) the 200 mM glucose pulse was injected aseptically through a rubber septum. Experiment Overall Design: Sampling from chemostats, total RNA extraction, probe preparation and hybridization to Affymetrix GenechipÂ® microarrays were performed as previously described (1). Samples were collected at steady state and then at 5, 10, 30, 60 and 120 minutes after the pulse. The results relative to steady state samples were derived from three independent cultures, those relative to the time course analysis were derived from two independent cultures. Experiment Overall Design: 1) Cipollina C., van den Brink J., Daran-Lapujade P., Pronk J.T., Vai M. and de Winde J.H. (2007) Revisiting the role of yeast Sfp1 in ribosome biogenesis and cell size control: A chemostat study. Microbiology. In press.

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Impact of oxygen availability on the organelle-specific redox potentials and stress in recombinant protein producing Komagataella phaffii

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