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Background: Industrial processes for recombinant protein production challenge production hosts, such as the yeast Pichia pastoris, on multiple levels. During a common P. pastoris fed-batch process, cells experience strong adaptations to different metabolic states or suffer from environmental stresses due to high cell density cultivation. Additionally, recombinant protein production and nutrient limitations are challenging in these processes. Results: Pichia pastoris producing porcine carboxypeptidase B (CpB) was cultivated in glucose or methanol-limited fed-batch mode, and the cellular response was analyzed using microarrays. Thereby, strong transcriptional regulations in transport-, regulatory- and metabolic processes connected to sulfur, phosphorus and nitrogen metabolism became obvious. The induction of these genes was observed in both glucose- and methanol- limited fed batch cultivations, but were stronger in the latter condition. As the transcriptional pattern was indicative for nutrient limitations, we performed fed-batch cultivations where we added the respective nutrients and compared them to non-supplemented cultures regarding cell growth, productivity and expression levels of selected biomarker genes. In the non-supplemented reference cultures we observed a strong increase in transcript levels of up to 89-fold for phosphorus limitation marker genes in the late fed-batch phase. Transcript levels of sulfur limitation marker genes were up to 35-fold increased. By addition of (NH4)2SO4 or (NH4)2HPO4, respectively, we were able to suppress the transcriptional response of the marker genes to levels initially observed at the start of the fed batch. Additionally, supplementation had also a positive impact on biomass generation and recombinant protein production. Supplementation with (NH4)2SO4 led to 5% increase in biomass and 52% higher CpB activity in the supernatant, compared to the non-supplemented reference cultivations. In (NH4)2HPO4 supplemented cultures 9% higher biomass concentrations and 60% more CpB activity were reached. Conclusions: Transcriptional analysis of P. pastoris fed-batch cultivations led to the identification of nutrient limitations in the later phases, and respective biomarker genes for indication of limitations. Supplementation of the cultivation media with those nutrients eliminated the limitations on the transcriptional level, and was also shown to enhance productivity of a recombinant protein. The biomarker genes are versatily applicable to media and process optimization approaches, where tailor-made solutions are envisioned. [ABSTRACT FROM AUTHOR]

Background: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date. Results: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose- 1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation. Conclusions: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology. [ABSTRACT FROM AUTHOR]

Background: Yeasts are attractive expression platforms for many recombinant proteins, and there is evidence for an important interrelation between the protein secretion machinery and environmental stresses. While adaptive responses to such stresses are extensively studied in Saccharomyces cerevisiae, little is known about their impact on the physiology of Pichia pastoris. We have recently reported a beneficial effect of hypoxia on recombinant Fab secretion in P. pastoris chemostat cultivations. As a consequence, a systems biology approach was used to comprehensively identify cellular adaptations to low oxygen availability and the additional burden of protein production. Gene expression profiling was combined with proteomic analyses and the 13C isotope labelling based experimental determination of metabolic fluxes in the central carbon metabolism. Results: The physiological adaptation of P. pastoris to hypoxia showed distinct traits in relation to the model yeast S. cerevisiae. There was a positive correlation between the transcriptomic, proteomic and metabolic fluxes adaptation of P. pastoris core metabolism to hypoxia, yielding clear evidence of a strong transcriptional regulation component of key pathways such as glycolysis, pentose phosphate pathway and TCA cycle. In addition, the adaptation to reduced oxygen revealed important changes in lipid metabolism, stress responses, as well as protein folding and trafficking. Conclusions: This systems level study helped to understand the physiological adaptations of cellular mechanisms to low oxygen availability in a recombinant P. pastoris strain. Remarkably, the integration of data from three different levels allowed for the identification of differences in the regulation of the core metabolism between P. pastoris and S. cerevisiae. Detailed comparative analysis of the transcriptomic data also led to new insights into the gene expression profiles of several cellular processes that are not only susceptible to low oxygen concentrations, but might also contribute to enhanced protein secretion. [ABSTRACT FROM AUTHOR]

The compartmentalization of metabolic and regulatory pathways is a common pattern of living organisms. Eukaryotic cells are subdivided into several organelles enclosed by lipid membranes. Organelle proteomes define their functions. Yeasts, as simple eukaryotic single cell organisms, are valuable models for higher eukaryotes and frequently used for biotechnological applications. While the subcellular distribution of proteins is well studied in Saccharomyces cerevisiae , this is not the case for other yeasts like Komagataella phaffii (syn. Pichia pastoris). Different to most well-studied yeasts, K. phaffii can grow on methanol, which provides specific features for production of heterologous proteins and as a model for peroxisome biology. We isolated microsomes, very early Golgi, early Golgi, plasma membrane, vacuole, cytosol, peroxisomes and mitochondria of K. phaffii from glucose- and methanol-grown cultures, quantified their proteomes by liquid chromatography-electrospray ionization-mass spectrometry of either unlabeled or tandem mass tag-labeled samples. Classification of the proteins by their relative enrichment, allowed the separation of enriched proteins from potential contaminants in all cellular compartments except the peroxisomes. We discuss differences to S. cerevisiae , outline organelle specific findings and the major metabolic pathways and provide an interactive map of the subcellular localization of proteins in K. phaffii. [ABSTRACT FROM AUTHOR]