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A subcellular proteome atlas of the yeast Komagataella phaffii.

ABSTRACT: 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.

SUBMITTER: Valli M

PROVIDER: S-EPMC6981350 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Correction: Transcriptomic profiling of the yeast Komagataella phaffii in response to environmental alkalinization.

Project description: Not available

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What makes Komagataella phaffii non-conventional?

Project description:The important industrial protein production host Komagataella phaffii (syn Pichia pastoris) is classified as a non-conventional yeast. But what exactly makes K. phaffii non-conventional? In this review, we set out to address the main differences to the 'conventional' yeast Saccharomyces cerevisiae, but also pinpoint differences to other non-conventional yeasts used in biotechnology. Apart from its methylotrophic lifestyle, K. phaffii is a Crabtree-negative yeast species. But even within the methylotrophs, K. phaffii possesses distinct regulatory features such as glycerol-repression of the methanol-utilization pathway or the lack of nitrate assimilation. Rewiring of the transcriptional networks regulating carbon (and nitrogen) source utilization clearly contributes to our understanding of genetic events occurring during evolution of yeast species. The mechanisms of mating-type switching and the triggers of morphogenic phenotypes represent further examples for how K. phaffii is distinguished from the model yeast S. cerevisiae. With respect to heterologous protein production, K. phaffii features high secretory capacity but secretes only low amounts of endogenous proteins. Different to S. cerevisiae, the Golgi apparatus of K. phaffii is stacked like in mammals. While it is tempting to speculate that Golgi architecture is correlated to the high secretion levels or the different N-glycan structures observed in K. phaffii, there is recent evidence against this. We conclude that K. phaffii is a yeast with unique features that has a lot of potential to explore both fundamental research questions and industrial applications.

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A native phosphoglycolate salvage pathway of the synthetic autotrophic yeast <i>Komagataella phaffii</i>.

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Project description:Transcription preinitiation complex assembly on the promoters of protein encoding genes is nucleated in vivo by TFIID composed of the TATA-box Binding Protein (TBP) and 13 TBP-associate factors (Tafs) providing regulatory and chromatin binding functions. We used CXMS to study the organization and structure of the purified TFIID complex from Komagataella phaffii. Together with Cryo-EM, We confirm the unique subunit stoichiometry prevailing in TFIID and uncover a hexameric arrangement of Tafs containing a HF domain. Interaction with promoter DNA highlights two non-selective binding sites consistent with a DNA scanning mode.

2018-10-08 | PXD011092 | Pride

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Project description:The current trend in industrial biotechnology is to move from batch or fed-batch fermentations to continuous operations. The success of this transition will require the development of genetically stable production strains, the use of strong constitutive promoters, and the development of new medium formulations that allow an appropriate balance between cell growth and product formation. We identified genes that showed high expression in Komagataella phaffii during different steady-state conditions and explored the utility of promoters of these genes (Chr1-4_0586 and FragB_0052) in optimizing the expression of two different r-proteins, human lysozyme (HuLy), and the anti-idiotypic antibody fragment, Fab-3H6, in comparison with the widely used glyceraldehyde-3-phosphate dehydrogenase promoter. Our results showed that the promoter strength was highly dependent on the cultivation conditions and thus constructs should be tested under a range of conditions to determine both the best performing clone and the ideal promoter for the expression of the protein of interest. An important benefit of continuous production is that it facilitates the use of the genome-scale metabolic models in the design of strains and cultivation media. In silico flux distributions showed that production of either protein increased the flux through aromatic amino acid biosynthesis. Tyrosine supplementation increased the productivity for both proteins, whereas tryptophan addition did not cause any significant change and, phenylalanine addition increased the expression of HuLy but decreased that of Fab-3H6. These results showed that a genome-scale metabolic model can be used to assess the metabolic burden imposed by the synthesis of a specific r-protein and then this information can be used to tailor a cultivation medium to increase production.

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Project description:Komagataella phaffii yeast is one of the most important biocompounds producing microorganisms in modern biotechnology. Optimization of media recipes and cultivation strategies is key to successful synthesis of recombinant proteins. The complex effects of proline on gene expression in the yeast K. phaffii was analyzed on the transcriptome level in this work. Our analysis revealed drastic changes in gene expression when K. phaffii was grown in proline-containing media in comparison to ammonium sulphate-containing media. Around 18.9% of all protein-encoding genes were differentially expressed in the experimental conditions. Proline is catabolized by K. phaffii even in the presence of other nitrogen, carbon and energy sources. This results in the repression of genes involved in the utilization of other element sources, namely methanol. We also found that the repression of AOX1 gene promoter with proline can be partially reversed by the deletion of the KpPUT4.2 gene.

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Project description:BACKGROUND:Komagataella phaffii is a yeast widely used in the pharmaceutical and biotechnology industries, and is one of the two species that were previously called Pichia pastoris. However, almost all laboratory work on K. phaffii has utilized strains derived from a single natural isolate, CBS7435. There is little information about the sequence diversity of K. phaffii or the genetic properties of this species. RESULTS:We sequenced the genomes of all the known isolates of K. phaffii. We made a genetic cross between derivatives of two isolates that differ at 44,000 single nucleotide polymorphism sites, and used this cross to analyze the rate and landscape of meiotic recombination. We conducted tetrad analysis by making use of the property that K. phaffii haploids do not mate in rich media, which enabled us to isolate and sequence the four types of haploid cell that are present in the colony that forms when a tetra-type ascus germinates. CONCLUSIONS:We found that only four distinct natural isolates of K. phaffii exist in public yeast culture collections. The meiotic recombination rate in K. phaffii is approximately 3.5 times lower than in Saccharomyces cerevisiae, with an average of 25 crossovers per meiosis. Recombination is suppressed, and genetic diversity among natural isolates is low, in a region around centromeres that is much larger than the centromeres themselves. Our work lays a foundation for future quantitative trait locus analysis in K. phaffii.

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Transcriptomic profiling of the yeast Komagataella phaffii in response to environmental alkalinization

Project description:Adaptation to alkalinization of the medium in fungi involves an extensive remodeling of gene expression. Komagataella phaffii is an ascomycetous yeast that has become an organism widely used for heterologous protein expression. We explore here the transcriptional impact of moderate alkalinization in this yeast. In spite of a minor effect on growth, shifting the cultures from pH 5.5 to 8.0 or 8.2 provokes significant changes in the mRNA levels of over 700 genes. Functional categories such as arginine and methionine biosynthesis, non-reductive iron uptake and phosphate metabolism are enriched in induced genes, whereas many genes encoding iron-sulfur proteins or members of the respirasome were repressed. We also show that alkalinization is accompanied by oxidative stress and we propose this circumstance as a common trigger of a subset of the observed changes. PHO89, encoding a Na+/Pi cotransporter, appears among the most potently induced genes by high pH. We demonstrate that this response is mainly based on two calcineurin-dependent response elements located in its promoter, thus indicating that alkalinization triggers a calcium-mediated signal in K. phaffii. Further characterization of alkaline pH responsive promoters may lead to developing novel pH-controlled systems for heterologous protein expression in this fungus.

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