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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 marker genes expression of 2F5 producing P. pastoris during steady state conditions. Log2 ratios of the average gene expression between 20 °C and 25 °C in chemostat cultivation (D = 0.043 h-1). Genes with ratios exceeding ± one standard deviation (SD) are marked in light blue, ± two SD in yellow and ± three SD in green. The p-value (χ2-test) is given for each individual marker gene. (a) *** significance level p ≤ 0.01; ** significance level p ≤ 0.05; * significance level p ≤ 0.1
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Figure 4: Comparison of marker genes expression of 2F5 producing P. pastoris during steady state conditions. Log2 ratios of the average gene expression between 20 °C and 25 °C in chemostat cultivation (D = 0.043 h-1). Genes with ratios exceeding ± one standard deviation (SD) are marked in light blue, ± two SD in yellow and ± three SD in green. The p-value (χ2-test) is given for each individual marker gene. (a) *** significance level p ≤ 0.01; ** significance level p ≤ 0.05; * significance level p ≤ 0.1

Mentions: Although the transcriptional levels of the product genes (2F5 Fab light and heavy chain) were reduced at 20°C compared to cultivation on 25°C (Figure 4), specific productivity of the 2F5 Fab protein was significantly increased during the chemostat process at lower temperature (1.4-fold on average). Several genes related to protein targeting to the ER and folding (SSA4, SEC53, KAR2, ERO1) and core metabolism genes can be found among the genes down-regulated at 20°C. Transcription of genes involved in the regulation of vesicular transport, exocytosis, ER-associated protein degradation as well as markers for response to oxidative and hyperosmotic stress was enhanced in comparison to 25°C steady state (see Figure 4).


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 marker genes expression of 2F5 producing P. pastoris during steady state conditions. Log2 ratios of the average gene expression between 20 °C and 25 °C in chemostat cultivation (D = 0.043 h-1). Genes with ratios exceeding ± one standard deviation (SD) are marked in light blue, ± two SD in yellow and ± three SD in green. The p-value (χ2-test) is given for each individual marker gene. (a) *** significance level p ≤ 0.01; ** significance level p ≤ 0.05; * significance level p ≤ 0.1
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Comparison of marker genes expression of 2F5 producing P. pastoris during steady state conditions. Log2 ratios of the average gene expression between 20 °C and 25 °C in chemostat cultivation (D = 0.043 h-1). Genes with ratios exceeding ± one standard deviation (SD) are marked in light blue, ± two SD in yellow and ± three SD in green. The p-value (χ2-test) is given for each individual marker gene. (a) *** significance level p ≤ 0.01; ** significance level p ≤ 0.05; * significance level p ≤ 0.1
Mentions: Although the transcriptional levels of the product genes (2F5 Fab light and heavy chain) were reduced at 20°C compared to cultivation on 25°C (Figure 4), specific productivity of the 2F5 Fab protein was significantly increased during the chemostat process at lower temperature (1.4-fold on average). Several genes related to protein targeting to the ER and folding (SSA4, SEC53, KAR2, ERO1) and core metabolism genes can be found among the genes down-regulated at 20°C. Transcription of genes involved in the regulation of vesicular transport, exocytosis, ER-associated protein degradation as well as markers for response to oxidative and hyperosmotic stress was enhanced in comparison to 25°C steady state (see Figure 4).

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