Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions

Brigitte Gasser, Michael Maurer, Jari Rautio, Michael Sauer, Anamitra Bhattacharyya, Markku Saloheimo, Merja Penttilä, Diethard Mattanovich

Research output: Contribution to journalArticleScientificpeer-review

79 Citations (Scopus)

Abstract

Background: It has become evident that host cells react to recombinant protein production with a variety of metabolic and intrinsic stresses such as the unfolded protein response (UPR) pathway. Additionally, environmental conditions such as growth temperature may have a strong impact on cell physiology and specific productivity. However, there is little information about the molecular reactions of the host cells on a genomic level, especially in context to recombinant protein secretion. For the first time, we monitored transcriptional regulation of a subset of marker genes in the common production host Pichia pastoris to gain insights into the general physiological status of the cells under protein production conditions, with the main focus on secretion stress related genes. Results: Overexpression of the UPR activating transcription factor Hac1p was employed to identify UPR target genes in P. pastoris and the responses were compared to those known for Saccharomyces cerevisiae. Most of the folding/secretion related genes showed similar regulation patterns in both yeasts, whereas genes associated with the general stress response were differentially regulated. Secretion of an antibody Fab fragment led to induction of UPR target genes in P. pastoris, however not to the same magnitude as Hac1p overproduction. Overexpression of S. cerevisiae protein disulfide isomerase (PDI1) enhances Fab secretion rates 1.9 fold, but did not relief UPR stress. Reduction of cultivation temperature from 25°C to 20°C led to a 1.4-fold increase of specific product secretion rate in chemostat cultivations, although the transcriptional levels of the product genes (Fab light and heavy chain) were significantly reduced at the lower temperature. A subset of folding related genes appeared to be down-regulated at the reduced temperature, whereas transcription of components of the ER associated degradation and the secretory transport was enhanced. Conclusion: Monitoring of genomic regulation of marker genes with the transcriptional profiling method TRAC in P. pastoris revealed similarities and discrepancies of the responses compared to S. cerevisiae. Thus our results emphasize the importance to analyse the individual hosts under real production conditions instead of drawing conclusions from model organisms. Cultivation temperature has a significant influence on specific productivity that cannot be related just to thermodynamic effects, but strongly impacts the regulation of specific genes.

Original languageEnglish
Article number179
Number of pages18
JournalBMC Genomics
Volume8
DOIs
Publication statusPublished - 19 Jun 2007
MoE publication typeA1 Journal article-refereed

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Pichia
Unfolded Protein Response
Genes
Proteins
Temperature
Recombinant Proteins
Saccharomyces cerevisiae
Activating Transcription Factors
Protein Disulfide-Isomerases
Saccharomyces cerevisiae Proteins
Cell Physiological Phenomena
Immunoglobulin Fragments
Immunoglobulin Fab Fragments
Thermodynamics
Yeasts
Light

Cite this

Gasser, Brigitte ; Maurer, Michael ; Rautio, Jari ; Sauer, Michael ; Bhattacharyya, Anamitra ; Saloheimo, Markku ; Penttilä, Merja ; Mattanovich, Diethard. / Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions. In: BMC Genomics. 2007 ; Vol. 8.
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Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions. / Gasser, Brigitte; Maurer, Michael; Rautio, Jari; Sauer, Michael; Bhattacharyya, Anamitra; Saloheimo, Markku; Penttilä, Merja; Mattanovich, Diethard.

In: BMC Genomics, Vol. 8, 179, 19.06.2007.

Research output: Contribution to journalArticleScientificpeer-review

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AU - Gasser, Brigitte

AU - Maurer, Michael

AU - Rautio, Jari

AU - Sauer, Michael

AU - Bhattacharyya, Anamitra

AU - Saloheimo, Markku

AU - Penttilä, Merja

AU - Mattanovich, Diethard

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AB - Background: It has become evident that host cells react to recombinant protein production with a variety of metabolic and intrinsic stresses such as the unfolded protein response (UPR) pathway. Additionally, environmental conditions such as growth temperature may have a strong impact on cell physiology and specific productivity. However, there is little information about the molecular reactions of the host cells on a genomic level, especially in context to recombinant protein secretion. For the first time, we monitored transcriptional regulation of a subset of marker genes in the common production host Pichia pastoris to gain insights into the general physiological status of the cells under protein production conditions, with the main focus on secretion stress related genes. Results: Overexpression of the UPR activating transcription factor Hac1p was employed to identify UPR target genes in P. pastoris and the responses were compared to those known for Saccharomyces cerevisiae. Most of the folding/secretion related genes showed similar regulation patterns in both yeasts, whereas genes associated with the general stress response were differentially regulated. Secretion of an antibody Fab fragment led to induction of UPR target genes in P. pastoris, however not to the same magnitude as Hac1p overproduction. Overexpression of S. cerevisiae protein disulfide isomerase (PDI1) enhances Fab secretion rates 1.9 fold, but did not relief UPR stress. Reduction of cultivation temperature from 25°C to 20°C led to a 1.4-fold increase of specific product secretion rate in chemostat cultivations, although the transcriptional levels of the product genes (Fab light and heavy chain) were significantly reduced at the lower temperature. A subset of folding related genes appeared to be down-regulated at the reduced temperature, whereas transcription of components of the ER associated degradation and the secretory transport was enhanced. Conclusion: Monitoring of genomic regulation of marker genes with the transcriptional profiling method TRAC in P. pastoris revealed similarities and discrepancies of the responses compared to S. cerevisiae. Thus our results emphasize the importance to analyse the individual hosts under real production conditions instead of drawing conclusions from model organisms. Cultivation temperature has a significant influence on specific productivity that cannot be related just to thermodynamic effects, but strongly impacts the regulation of specific genes.

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