The contribution of the transcription factors Crz1 and Rim101 to the response to alkalinization in Komagataella phaffii
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PROVIDER: PRJNA1262845 | ENA |
REPOSITORIES: ENA
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Project description:In the industrially relevant yeast Komagataella phaffii, as in other yeasts, alkalinization of the medium results in fast and extensive transcriptional remodeling. This response has been useful to design novel methanol-free, regulatable platforms for heterologous protein expression. However, its molecular bases are fully unknown. Crz1 and Rim101/PacC are transcription factors known to contribute to the alkaline pH transcriptional responses in many fungi. We identified here the transcription factors Crz1 and Rim101 in K. phaffii and used CRISPR/Cas9 techniques to create single and double mutant strains. Phenotypic characterization shows that crz1 cells are strongly sensitive to Ca2+ ions, mildly sensitive to alkaline pH and hypertolerant to SDS. In contrast, rim101 cells are markedly hypersensitive to Na+ and Li+ ions, to alkaline pH and to SDS. RNA-seq transcriptomic analysis revealed that mutation of RIM101 affects 55% of genes whose expression is altered at pH 8.0, whereas lack of Crz1 alters only 11% of these. Thirty-eight genes are co-regulated by both Crz1 and Rim101. The PMC1 vacuolar Ca+2-ATPase is regulated by Crz1, whereas the ENA2 Na+-ATPase, the PHO89 and PHO84 phosphate transporters, and diverse genes involved in iron homeostasis are Rim101-dependent. These effects were confirmed by proteomic analyses after 3 h of alkalinization. Therefore, while Rim101 appears as a major player in the transcriptional response to alkalinization, nearly 40% of the alkaline pH transcriptional response in K. phaffii is controlled still unknown pathways.
2025-09-02 | GSE297128 | GEO
Project description:In the industrially relevant yeast Komagataella phaffii, as in other yeasts, alkalinization of the medium results in fast and extensive transcriptional remodeling. This response has been useful to design novel methanol-free, regulatable platforms for heterologous protein expression. However, its molecular bases are fully unknown. Crz1 and Rim101/PacC are transcription factors known to contribute to the alkaline pH transcriptional responses in many fungi. We identified here the transcription factors Crz1 and Rim1 in K. phaffii and used CRISPR/Cas9 techniques to create single and double mutant strains. Phenotypic characterization shows that crz1 cells are strongly sensitive to Ca2+ ions, mildly sensitive to alkaline pH and hypertolerant to SDS. In contrast, rim101 cells are markedly hypersensitive to Na+ and Li+ ions, to alkaline pH and to SDS. RNA-seq transcriptomic analysis revealed that mutation of RIM101 affects 55% of genes whose expression is altered at pH 8.0, whereas lack of Crz1 alters only 11% of these. Thirty-eight genes are co-regulated by both Crz1 and Rim101. The PMC1 vacuolar Ca+2-ATPase is regulated by Crz1, whereas the ENA2 Na+-ATPase, the PHO89 and PHO84 phosphate transporters, and diverse genes involved in iron homeostasis are Rim101-dependent. These effects were confirmed by proteomic analyses after 3 h of alkalinization. Therefore, while Rim101 appears as a major player in the transcriptional response to alkalinization, nearly 40% of the alkaline pH transcriptional response in K. phaffii is controlled still unknown pathways.
2025-09-02 | PXD064682 | Pride
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.
2023-04-12 | GSE224427 | GEO
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