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ABSTRACT: RNA-seq analysis of Komagataella phaffii

PROVIDER: PRJNA1006195 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR25660906_1.fastq.gz	Fastqsanger.gz
	SRR25660906_2.fastq.gz	Fastqsanger.gz
	SRR25660907_1.fastq.gz	Fastqsanger.gz
	SRR25660907_2.fastq.gz	Fastqsanger.gz
	SRR25660908_1.fastq.gz	Fastqsanger.gz

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Komagataella phaffii

Project description:Komagataella phaffii SNP analysis

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Identifying improved sites for heterologous gene integration using ATAC-seq

Project description:We analyzed the chromatin accessibility and nucleosome positioning by ATAC-seq of both null and transgene-expressing strains of Komagataella phaffii (Pichia pastoris) under different growth conditions. These data enabled identification of the features that determine performance of various integration sites for transgene expression. Understanding chromatin accessibility and nucleosome positioning can provide further clarity into gene regulation and expression broadly in this organism.

2020-10-02 | GSE154330 | GEO

Effect and mechanism of signal peptide and maltose on recombinant type III collagen production in Pichia pastoris

Project description:In order to investigate the specific mechanism of maltose on the production of recombinant type III collagen by Komagataella phaffii GS115, Two groups were set up, control group C using BMMY medium and experimental group M using BMMY + 1% maltose.

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RNA-seq differential expression analysis of Komagataella phaffii CBS2612 under mating conditions

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Specific features of the transcriptomic response to nitrogen starvation in methylotrophic yeast Komagataella phaffi

Project description:Non-conventional methylotrophic yeast Komagataella phaffii is an important production host in biotechnology and an emerging model organism. In this work, we studied K. phaffii response to nitrogen starvation during cultivation in media with methanol as the sole carbon source. The results were compared with well-established model yeast Saccharomyces cerevisiae. Some of the observed effects of nitrogen starvation in K. phaffii were similar to those in S. cerevisiae, despite this yeast does not have metabolic pathway for methanol utilization. The effects include activation of autophagy, transport and catabolism of nitrogen-containing compounds, interconversions of amino acids, and biosynthesis of fatty acids. K. phaffii cells also demonstrated specific response to nitrogen starvation including suppression of genes involved in methanol metabolism and other peroxisomal processes and activation of purine catabolism genes.

2024-10-06 | GSE278110 | GEO

Project description:Comparative transcriptome analysis of engineered Komagataella phaffii

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Pseudohyphal differentiation in Komagataella phaffii: investigating the FLO gene family (FAIRE-seq)

Project description:The protein production host Komagataella phaffii has possess the ability to differentiate into pseudohyphal form when cultivated at slow growth rates (µ=0.05 h-1) in glucose-limited chemostats. In this study, we investigated the K. phaffii FLO gene family in the context of pseudohyphae formation. Transcriptional analysis helped us identify 3 possible responsible genes, FLO11, FLO400 and FLO5-1, all of which are under control of Flo8, a transcription factor whose disruption prevents pseudohyphae formation. Knocking out FLO11 revealed that this is not the sole protein responsible for this phenotype. Strikingly, the expression of FLO400 and FLO5-1 was negatively correlated with pseudohyphae formation, and shown to be under epigenetic control by FAIRE-Seq analysis. Knock outs of these two genes completely inhibited the appearance of pseudohyphal cells and prevented the expression of FLO11. Even though the mechanism is unclear at present, we propose that in K. phaffii Flo400 and/or Flo5-1 act as upstream signals that lead to the induction of FLO11 expression upon severe glucose limitation in chemostats at slow growth rate, and that the expression of FLO400 and FLO5-1 is controlled by epigenetic silencing, which acts independently from the general activation of FLO gene expression by the transcriptional regulator Flo8.

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The contribution of the transcription factors Crz1 and Rim101 to the response to alkalinization in Komagataella phaffii

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

CRISPR-Cas9 knockout screen informs efficient reduction of the Komagataella phaffii secretome

Project description:The yeast Komagataella phaffii is a promising alternative host for manufacturing of therapeutic proteins. Deletion of unneeded endogenous proteins could increase the secreted titer of recombinant proteins by redirecting cellular resources. Genetic engineering in non-model hosts is hampered by limited annotation of genes, especially essential genes. In this study, we identified the set of endogenous secreted proteins in K. phaffii and attempted to disrupt these genes. We designed, transformed, and sequenced a pooled CRISPR-Cas9 knockout library to determine which genes are essential. With this knowledge, we rapidly disrupted up to 9 consecutive genes in K. phaffii. Engineered strains exhibited a ~20x increase in the production of human serum albumin and a 2x increase in the production of a monoclonal antibody. The pooled CRISPR-Cas9 library and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant therapeutic proteins and enzymes.

2024-08-08 | GSE252605 | GEO

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