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Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions.

Gasser B, Maurer M, Rautio J, Sauer M, Bhattacharyya A, Saloheimo M, Penttilä M, Mattanovich D - BMC Genomics (2007)

Bottom Line: Reduction of cultivation temperature from 25 degrees C to 20 degrees 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Natural Resources and Applied Life Sciences Vienna, Department of Biotechnology, Institute of Applied Microbiology, Vienna, Austria. brigitte.gasser@boku.ac.at <brigitte.gasser@boku.ac.at>

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 degrees C to 20 degrees 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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Comparison of the UPR response in P. pastoris and S. cerevisiae. Abbreviations for P. pastoris strains are explained in Table 3, all data are derived from comparison to the wild type. Data from S. cerevisiae were taken from [21], where UPR was induced with DTT or tunicamycin. ScD60 (treatment with DTT after 60 min); ScD120 (treatment with DTT after 120 min); ScT60 (treatment with tunicamycin after 60 min), all compared to a non-treated culture. Cluster analysis was made using EPClust [47], Euklidian distance with complete linkage. Subclusters are shown for the following: A: genes induced in both yeasts; B: upregulated in P. pastoris, down-regulated in S. cerevisiae; C: down-regulated to unchanged in P. pastoris, upregulated in S. cerevisiae; D: reduced in both yeasts. Subclusters of genes that are unchanged in both organisms are not displayed. The brightest colouring corresponds to the log2 regulation ≥ ± 2.
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Figure 1: Comparison of the UPR response in P. pastoris and S. cerevisiae. Abbreviations for P. pastoris strains are explained in Table 3, all data are derived from comparison to the wild type. Data from S. cerevisiae were taken from [21], where UPR was induced with DTT or tunicamycin. ScD60 (treatment with DTT after 60 min); ScD120 (treatment with DTT after 120 min); ScT60 (treatment with tunicamycin after 60 min), all compared to a non-treated culture. Cluster analysis was made using EPClust [47], Euklidian distance with complete linkage. Subclusters are shown for the following: A: genes induced in both yeasts; B: upregulated in P. pastoris, down-regulated in S. cerevisiae; C: down-regulated to unchanged in P. pastoris, upregulated in S. cerevisiae; D: reduced in both yeasts. Subclusters of genes that are unchanged in both organisms are not displayed. The brightest colouring corresponds to the log2 regulation ≥ ± 2.

Mentions: More than a 2-fold induction of genes like KAR2 (BiP, 5-fold), PDI1 (protein disulfide isomerase, 3-fold), ERO1 (Pdi oxidase, 2-fold) and SEC61 (part of translocon complex into the ER, 2-fold) was observed in P. pastoris (Figure 1A), analogous to the UPR response described for S. cerevisiae[21]. Strong enhancement in the transcription of genes involved in the ER quality control and glycosylation such as CNE1 (calnexin), ROT2 (glucosidase II) and SEC53 (phosphomannomutase) was predominant in P. pastoris, while components of the ER-associated protein degradation (ERAD) represented by HRD1 and UBC1 showed a more consistent upregulation, as compared to S. cerevisiae. Also the down-regulation of core metabolism genes such as PFK1 (glycolysis), CIT1 (TCA cycle), GLT1 and ARO4 (amino acid biosynthesis) and ribosomal genes (RPS23B, RPL3) during UPR induction seems to be a common feature for both of the yeasts (Figure 1D), while the transcription of other genes is differentially regulated between the two organisms.


Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions.

Gasser B, Maurer M, Rautio J, Sauer M, Bhattacharyya A, Saloheimo M, Penttilä M, Mattanovich D - BMC Genomics (2007)

Comparison of the UPR response in P. pastoris and S. cerevisiae. Abbreviations for P. pastoris strains are explained in Table 3, all data are derived from comparison to the wild type. Data from S. cerevisiae were taken from [21], where UPR was induced with DTT or tunicamycin. ScD60 (treatment with DTT after 60 min); ScD120 (treatment with DTT after 120 min); ScT60 (treatment with tunicamycin after 60 min), all compared to a non-treated culture. Cluster analysis was made using EPClust [47], Euklidian distance with complete linkage. Subclusters are shown for the following: A: genes induced in both yeasts; B: upregulated in P. pastoris, down-regulated in S. cerevisiae; C: down-regulated to unchanged in P. pastoris, upregulated in S. cerevisiae; D: reduced in both yeasts. Subclusters of genes that are unchanged in both organisms are not displayed. The brightest colouring corresponds to the log2 regulation ≥ ± 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919374&req=5

Figure 1: Comparison of the UPR response in P. pastoris and S. cerevisiae. Abbreviations for P. pastoris strains are explained in Table 3, all data are derived from comparison to the wild type. Data from S. cerevisiae were taken from [21], where UPR was induced with DTT or tunicamycin. ScD60 (treatment with DTT after 60 min); ScD120 (treatment with DTT after 120 min); ScT60 (treatment with tunicamycin after 60 min), all compared to a non-treated culture. Cluster analysis was made using EPClust [47], Euklidian distance with complete linkage. Subclusters are shown for the following: A: genes induced in both yeasts; B: upregulated in P. pastoris, down-regulated in S. cerevisiae; C: down-regulated to unchanged in P. pastoris, upregulated in S. cerevisiae; D: reduced in both yeasts. Subclusters of genes that are unchanged in both organisms are not displayed. The brightest colouring corresponds to the log2 regulation ≥ ± 2.
Mentions: More than a 2-fold induction of genes like KAR2 (BiP, 5-fold), PDI1 (protein disulfide isomerase, 3-fold), ERO1 (Pdi oxidase, 2-fold) and SEC61 (part of translocon complex into the ER, 2-fold) was observed in P. pastoris (Figure 1A), analogous to the UPR response described for S. cerevisiae[21]. Strong enhancement in the transcription of genes involved in the ER quality control and glycosylation such as CNE1 (calnexin), ROT2 (glucosidase II) and SEC53 (phosphomannomutase) was predominant in P. pastoris, while components of the ER-associated protein degradation (ERAD) represented by HRD1 and UBC1 showed a more consistent upregulation, as compared to S. cerevisiae. Also the down-regulation of core metabolism genes such as PFK1 (glycolysis), CIT1 (TCA cycle), GLT1 and ARO4 (amino acid biosynthesis) and ribosomal genes (RPS23B, RPL3) during UPR induction seems to be a common feature for both of the yeasts (Figure 1D), while the transcription of other genes is differentially regulated between the two organisms.

Bottom Line: Reduction of cultivation temperature from 25 degrees C to 20 degrees 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Natural Resources and Applied Life Sciences Vienna, Department of Biotechnology, Institute of Applied Microbiology, Vienna, Austria. brigitte.gasser@boku.ac.at <brigitte.gasser@boku.ac.at>

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 degrees C to 20 degrees 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.

Show MeSH
Related in: MedlinePlus