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. 2007 Jun 19:8:179.
doi: 10.1186/1471-2164-8-179.

Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions

Brigitte Gasser  1 , Michael Maurer, Jari Rautio, Michael Sauer, Anamitra Bhattacharyya, Markku Saloheimo, Merja Penttilä, Diethard Mattanovich
Affiliations

Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions

Brigitte Gasser et al. BMC Genomics. .

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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Figures

Figure 1
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.
Figure 2
Figure 2
UPR regulation in P. pastoris. A: Log2 ratio of Hac1p-overproducing strains compared to the wild type, Hac1p-dependend up- or downregulatated genes are highlighted; B: Log2 ratio of cultures producing 2F5 Fab under control of the GAP promoter compared to the wild type, in the same order as A. Red bars: change in transcript up > 2-fold; yellow bars: up > 1.5 fold; white bars: unchanged; blue bars: down > 1.5 fold.
Figure 3
Figure 3
Effect of engineered folding factors on the transcriptional level. A: Constitutively UPR induced 2F5 Fab producing SMD1168 (Fab+Hac1) compared to its control strain (containing only the Fab expression cassette). B: 2F5 Fab producing SMD1168 co-expressing S. cerevisiae PDI1 compared to the control strain. Both diagrams are ordered from the lowest to the highest log2 ratio. Colour legend as in figure 2.
Figure 4
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
Figure 5
Figure 5
Intracellular product retention and BiP development analysed by immunofluorescent staining and flow cytometer analysis during cultivation at two different temperatures. Orange bars: relative fluorescence intensities per cell size obtained at 25 °C steady state; Blue bars: 20 °C steady state. BiP: intracellular signals for the UPR marker BiP/Kar2p (detected with anti-Grp78/BiP specific IgG). HC: intracellular signals for Fab heavy chain (obtained with anti-h-Fab specific IgG); LC: intracellular signals for light chain (analyzed with anti-kappa light chain IgG).
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