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The impact of oxygen on the transcriptome of recombinant S. cerevisiae and P. pastoris - a comparative analysis.

Baumann K, Dato L, Graf AB, Frascotti G, Dragosits M, Porro D, Mattanovich D, Ferrer P, Branduardi P - BMC Genomics (2011)

Bottom Line: Further important differences were related to Fab production, which was not significantly affected by oxygen availability in S. cerevisiae, while a clear productivity increase had been previously reported for hypoxically grown P. pastoris.The effect of three different levels of oxygen availability on the physiology of P. pastoris and S. cerevisiae revealed a very distinct remodelling of the transcriptional program, leading to novel insights into the different adaptive responses of Crabtree negative and positive yeasts to oxygen availability.Moreover, the application of such comparative genomic studies to recombinant hosts grown in different environments might lead to the identification of key factors for efficient protein production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering, Autonomous University of Barcelona, Spain.

ABSTRACT

Background: Saccharomyces cerevisiae and Pichia pastoris are two of the most relevant microbial eukaryotic platforms for the production of recombinant proteins. Their known genome sequences enabled several transcriptomic profiling studies under many different environmental conditions, thus mimicking not only perturbations and adaptations which occur in their natural surroundings, but also in industrial processes. Notably, the majority of such transcriptome analyses were performed using non-engineered strains.In this comparative study, the gene expression profiles of S. cerevisiae and P. pastoris, a Crabtree positive and Crabtree negative yeast, respectively, were analyzed for three different oxygenation conditions (normoxic, oxygen-limited and hypoxic) under recombinant protein producing conditions in chemostat cultivations.

Results: The major differences in the transcriptomes of S. cerevisiae and P. pastoris were observed between hypoxic and normoxic conditions, where the availability of oxygen strongly affected ergosterol biosynthesis, central carbon metabolism and stress responses, particularly the unfolded protein response. Steady state conditions under low oxygen set-points seemed to perturb the transcriptome of S. cerevisiae to a much lesser extent than the one of P. pastoris, reflecting the major tolerance of the baker's yeast towards oxygen limitation, and a higher fermentative capacity. Further important differences were related to Fab production, which was not significantly affected by oxygen availability in S. cerevisiae, while a clear productivity increase had been previously reported for hypoxically grown P. pastoris.

Conclusions: The effect of three different levels of oxygen availability on the physiology of P. pastoris and S. cerevisiae revealed a very distinct remodelling of the transcriptional program, leading to novel insights into the different adaptive responses of Crabtree negative and positive yeasts to oxygen availability. Moreover, the application of such comparative genomic studies to recombinant hosts grown in different environments might lead to the identification of key factors for efficient protein production.

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Global comparison of differential gene regulation. The correlation matrix of 2191 common genes (absolute normalized expressions levels) for (A) S. cerevisiae and (B) P. pastoris strains grown under normoxic (N), oxygen-limited (L) and hypoxic (H) conditions are visualized with the principle component analysis (PCA) biplot. Lines pointing in the same direction correspond to strains and oxygen conditions which are correlated. In both strains, the first and second components explain 97 % of the total variation. Overlapping sets of regulated genes in (C) S. cerevisiae, (D) P. pastoris and (E) between both species are displayed by Venn diagrams. Fab = Fab producing strain, Cont = control strain. HvsL = hypoxic vs. oxygen-limited, HvsN = hypoxic vs. normoxic, and LvsN = oxygen-limited vs. normoxic.
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Figure 1: Global comparison of differential gene regulation. The correlation matrix of 2191 common genes (absolute normalized expressions levels) for (A) S. cerevisiae and (B) P. pastoris strains grown under normoxic (N), oxygen-limited (L) and hypoxic (H) conditions are visualized with the principle component analysis (PCA) biplot. Lines pointing in the same direction correspond to strains and oxygen conditions which are correlated. In both strains, the first and second components explain 97 % of the total variation. Overlapping sets of regulated genes in (C) S. cerevisiae, (D) P. pastoris and (E) between both species are displayed by Venn diagrams. Fab = Fab producing strain, Cont = control strain. HvsL = hypoxic vs. oxygen-limited, HvsN = hypoxic vs. normoxic, and LvsN = oxygen-limited vs. normoxic.

Mentions: In a first approach, the absolute (normalized) expression data of the two yeast species were separately subjected to principal component analysis (PCA). The first and second component, accounting for 97 % of the total variability in the expression data, were plotted against each other and revealed oxygen as the major discriminating factor in both data sets (Figure 1A and 1B). Interestingly, the first principal component did not reveal any key parameter for the internal structure in our data. It was the second component that determined a correlation between oxygen availability and data distribution. We assume that this result is quite common in highly dimensional data like it is the case for microarray data since the first component represents a weighted average and distinguishes genes by their average overall expression [20]. PCA projection in the case of S. cerevisiae (Figure 1A) revealed a clear division between normoxic and hypoxic gene expression, with no difference regarding the strain genetic background (control or expressing strain). The clusters for oxygen-limited conditions were distributed along the PC2 axis, slightly separating the producing from the reference strain. The PCA for P. pastoris (Figure 1B) showed a similar behavior, since the second component was defined predominantly by oxygen, tightly grouping together hypoxic and oxygen-limited conditions. Only the producing strain in hypoxic conditions diverged from this classification and seemed to be the most influential variable. While the low oxygen 'environment' (hypoxia and oxygen limitation) in P. pastoris showed a positive correlation with the PC2, this interaction was negative in S. cerevisiae (and vice versa for the normoxic groups). Detailed analysis of the overlaps of regulated genes indicated that only few genes were differentially expressed between oxygen-limited and normoxic conditions in S. cerevisiae, while most regulation was apparent between hypoxic and normoxic conditions, almost fully overlapping with the set of genes that was differentially expressed between hypoxic and oxygen-limited conditions (Figure 1C). Differently, in P. pastoris the largest number of regulated genes appeared to be in the hypoxic vs. oxygen-limited comparison, overlapping with approximately 75 % of regulated genes between hypoxic and normoxic conditions (Figure 1D). While the transcriptomes of the P. pastoris control strain grown at limited and normal oxygen supply did not differ, there were 48 regulated genes in the Fab producing strain. The majority of differential regulation in P. pastoris emerged between hypoxic and oxygen-limited growth, while most regulation in S. cerevisiae was observed between hypoxic and normoxic growth (Figure 1E). These first results of the comparative analysis already pointed to major differences between the transcriptome of P. pastoris and S. cerevisiae in response to oxygen availability.


The impact of oxygen on the transcriptome of recombinant S. cerevisiae and P. pastoris - a comparative analysis.

Baumann K, Dato L, Graf AB, Frascotti G, Dragosits M, Porro D, Mattanovich D, Ferrer P, Branduardi P - BMC Genomics (2011)

Global comparison of differential gene regulation. The correlation matrix of 2191 common genes (absolute normalized expressions levels) for (A) S. cerevisiae and (B) P. pastoris strains grown under normoxic (N), oxygen-limited (L) and hypoxic (H) conditions are visualized with the principle component analysis (PCA) biplot. Lines pointing in the same direction correspond to strains and oxygen conditions which are correlated. In both strains, the first and second components explain 97 % of the total variation. Overlapping sets of regulated genes in (C) S. cerevisiae, (D) P. pastoris and (E) between both species are displayed by Venn diagrams. Fab = Fab producing strain, Cont = control strain. HvsL = hypoxic vs. oxygen-limited, HvsN = hypoxic vs. normoxic, and LvsN = oxygen-limited vs. normoxic.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3116504&req=5

Figure 1: Global comparison of differential gene regulation. The correlation matrix of 2191 common genes (absolute normalized expressions levels) for (A) S. cerevisiae and (B) P. pastoris strains grown under normoxic (N), oxygen-limited (L) and hypoxic (H) conditions are visualized with the principle component analysis (PCA) biplot. Lines pointing in the same direction correspond to strains and oxygen conditions which are correlated. In both strains, the first and second components explain 97 % of the total variation. Overlapping sets of regulated genes in (C) S. cerevisiae, (D) P. pastoris and (E) between both species are displayed by Venn diagrams. Fab = Fab producing strain, Cont = control strain. HvsL = hypoxic vs. oxygen-limited, HvsN = hypoxic vs. normoxic, and LvsN = oxygen-limited vs. normoxic.
Mentions: In a first approach, the absolute (normalized) expression data of the two yeast species were separately subjected to principal component analysis (PCA). The first and second component, accounting for 97 % of the total variability in the expression data, were plotted against each other and revealed oxygen as the major discriminating factor in both data sets (Figure 1A and 1B). Interestingly, the first principal component did not reveal any key parameter for the internal structure in our data. It was the second component that determined a correlation between oxygen availability and data distribution. We assume that this result is quite common in highly dimensional data like it is the case for microarray data since the first component represents a weighted average and distinguishes genes by their average overall expression [20]. PCA projection in the case of S. cerevisiae (Figure 1A) revealed a clear division between normoxic and hypoxic gene expression, with no difference regarding the strain genetic background (control or expressing strain). The clusters for oxygen-limited conditions were distributed along the PC2 axis, slightly separating the producing from the reference strain. The PCA for P. pastoris (Figure 1B) showed a similar behavior, since the second component was defined predominantly by oxygen, tightly grouping together hypoxic and oxygen-limited conditions. Only the producing strain in hypoxic conditions diverged from this classification and seemed to be the most influential variable. While the low oxygen 'environment' (hypoxia and oxygen limitation) in P. pastoris showed a positive correlation with the PC2, this interaction was negative in S. cerevisiae (and vice versa for the normoxic groups). Detailed analysis of the overlaps of regulated genes indicated that only few genes were differentially expressed between oxygen-limited and normoxic conditions in S. cerevisiae, while most regulation was apparent between hypoxic and normoxic conditions, almost fully overlapping with the set of genes that was differentially expressed between hypoxic and oxygen-limited conditions (Figure 1C). Differently, in P. pastoris the largest number of regulated genes appeared to be in the hypoxic vs. oxygen-limited comparison, overlapping with approximately 75 % of regulated genes between hypoxic and normoxic conditions (Figure 1D). While the transcriptomes of the P. pastoris control strain grown at limited and normal oxygen supply did not differ, there were 48 regulated genes in the Fab producing strain. The majority of differential regulation in P. pastoris emerged between hypoxic and oxygen-limited growth, while most regulation in S. cerevisiae was observed between hypoxic and normoxic growth (Figure 1E). These first results of the comparative analysis already pointed to major differences between the transcriptome of P. pastoris and S. cerevisiae in response to oxygen availability.

Bottom Line: Further important differences were related to Fab production, which was not significantly affected by oxygen availability in S. cerevisiae, while a clear productivity increase had been previously reported for hypoxically grown P. pastoris.The effect of three different levels of oxygen availability on the physiology of P. pastoris and S. cerevisiae revealed a very distinct remodelling of the transcriptional program, leading to novel insights into the different adaptive responses of Crabtree negative and positive yeasts to oxygen availability.Moreover, the application of such comparative genomic studies to recombinant hosts grown in different environments might lead to the identification of key factors for efficient protein production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical Engineering, Autonomous University of Barcelona, Spain.

ABSTRACT

Background: Saccharomyces cerevisiae and Pichia pastoris are two of the most relevant microbial eukaryotic platforms for the production of recombinant proteins. Their known genome sequences enabled several transcriptomic profiling studies under many different environmental conditions, thus mimicking not only perturbations and adaptations which occur in their natural surroundings, but also in industrial processes. Notably, the majority of such transcriptome analyses were performed using non-engineered strains.In this comparative study, the gene expression profiles of S. cerevisiae and P. pastoris, a Crabtree positive and Crabtree negative yeast, respectively, were analyzed for three different oxygenation conditions (normoxic, oxygen-limited and hypoxic) under recombinant protein producing conditions in chemostat cultivations.

Results: The major differences in the transcriptomes of S. cerevisiae and P. pastoris were observed between hypoxic and normoxic conditions, where the availability of oxygen strongly affected ergosterol biosynthesis, central carbon metabolism and stress responses, particularly the unfolded protein response. Steady state conditions under low oxygen set-points seemed to perturb the transcriptome of S. cerevisiae to a much lesser extent than the one of P. pastoris, reflecting the major tolerance of the baker's yeast towards oxygen limitation, and a higher fermentative capacity. Further important differences were related to Fab production, which was not significantly affected by oxygen availability in S. cerevisiae, while a clear productivity increase had been previously reported for hypoxically grown P. pastoris.

Conclusions: The effect of three different levels of oxygen availability on the physiology of P. pastoris and S. cerevisiae revealed a very distinct remodelling of the transcriptional program, leading to novel insights into the different adaptive responses of Crabtree negative and positive yeasts to oxygen availability. Moreover, the application of such comparative genomic studies to recombinant hosts grown in different environments might lead to the identification of key factors for efficient protein production.

Show MeSH
Related in: MedlinePlus