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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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Overlay of transcriptome data on the S. cerevisiae metabolic map. Fold change data of the pairwise comparison hypoxic vs. normoxic (HvsN) of the recombinant S. cerevisiae (A) and P. pastoris (B) strain are overlapped with the metabolic map of S. cerevisiae (MetaCyc, SGD database [66]). Each node in the diagram represents a single metabolite, and each line represents a single bioreaction. In the right part of the diagram the small molecule metabolism is represented (for a complete description of the map see http://pathway.yeastgenome.org). Reaction lines are colour-coded (three colour bins) according to the fold change value of the gene: red for data values that exceed a log2 fold change threshold of 0.59, yellow for data values less than the inverse of the threshold, and blue for values in between. Detailed lists of all the regulated pathways, together with their diagrams and corresponding gene lists are provided in additional file 2 (for S. cerevisiae) and 3 (for P. pastoris). Depicted pathways are indicated by numbers, according to the table shown in additional file 4. The ergosterol pathway (n.20) and glycolysis (n.69) are indicated by dashed boxes.
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Figure 3: Overlay of transcriptome data on the S. cerevisiae metabolic map. Fold change data of the pairwise comparison hypoxic vs. normoxic (HvsN) of the recombinant S. cerevisiae (A) and P. pastoris (B) strain are overlapped with the metabolic map of S. cerevisiae (MetaCyc, SGD database [66]). Each node in the diagram represents a single metabolite, and each line represents a single bioreaction. In the right part of the diagram the small molecule metabolism is represented (for a complete description of the map see http://pathway.yeastgenome.org). Reaction lines are colour-coded (three colour bins) according to the fold change value of the gene: red for data values that exceed a log2 fold change threshold of 0.59, yellow for data values less than the inverse of the threshold, and blue for values in between. Detailed lists of all the regulated pathways, together with their diagrams and corresponding gene lists are provided in additional file 2 (for S. cerevisiae) and 3 (for P. pastoris). Depicted pathways are indicated by numbers, according to the table shown in additional file 4. The ergosterol pathway (n.20) and glycolysis (n.69) are indicated by dashed boxes.

Mentions: To obtain a general view of metabolic pathways responding to oxygen availability, we overlapped the fold change values obtained from the HvsN comparison of the producing strains with the map of S. cerevisiae core metabolism. Figure 3 shows the overview of the entire schematized map, while detailed lists of all the regulated pathways, together with their diagrams and corresponding gene lists, are provided in additional file 2 (for S. cerevisiae) and additional file 3 (for P. pastoris). All depicted pathways are indicated by numbers according to the table shown in additional file 4. The most striking differences were detected for the glycolytic pathway, ergosterol and sphingolipid biosynthesis, and the oxidative branch of the pentose phosphate pathway (Figure 3). The uniform upregulation of glycolytic genes, enzymes and metabolic fluxes in P. pastoris chemostats upon a shift to hypoxic growth conditions was recently reported in our preceding study [15], indicating a transcriptional control of the central carbon metabolism in this yeast. In hypoxically grown S. cerevisiae this picture was quite different, since glycolysis was not regulated at the transcriptome level. These observations confirm previous chemostat studies, where a poor correlation between the mRNA levels and the corresponding protein abundances or in vivo fluxes demonstrated a post-transcriptional control of glycolysis in anaerobic S. cerevisiae cultures [21-23]. De Groot [22] further estimated the pool of glycolytic enzymes to account for 21 % of the total protein in anaerobic conditions, thus occupying a considerable fraction of the S. cerevisiae translation machinery. This 'occupation' could somehow hamper or limit the translation of other proteins, e.g. the recombinant Fab antibody. Estimation of the corresponding percentage in P. pastoris might provide new insights on the different production abilities of the two microorganisms.


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)

Overlay of transcriptome data on the S. cerevisiae metabolic map. Fold change data of the pairwise comparison hypoxic vs. normoxic (HvsN) of the recombinant S. cerevisiae (A) and P. pastoris (B) strain are overlapped with the metabolic map of S. cerevisiae (MetaCyc, SGD database [66]). Each node in the diagram represents a single metabolite, and each line represents a single bioreaction. In the right part of the diagram the small molecule metabolism is represented (for a complete description of the map see http://pathway.yeastgenome.org). Reaction lines are colour-coded (three colour bins) according to the fold change value of the gene: red for data values that exceed a log2 fold change threshold of 0.59, yellow for data values less than the inverse of the threshold, and blue for values in between. Detailed lists of all the regulated pathways, together with their diagrams and corresponding gene lists are provided in additional file 2 (for S. cerevisiae) and 3 (for P. pastoris). Depicted pathways are indicated by numbers, according to the table shown in additional file 4. The ergosterol pathway (n.20) and glycolysis (n.69) are indicated by dashed boxes.
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Related In: Results  -  Collection

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Figure 3: Overlay of transcriptome data on the S. cerevisiae metabolic map. Fold change data of the pairwise comparison hypoxic vs. normoxic (HvsN) of the recombinant S. cerevisiae (A) and P. pastoris (B) strain are overlapped with the metabolic map of S. cerevisiae (MetaCyc, SGD database [66]). Each node in the diagram represents a single metabolite, and each line represents a single bioreaction. In the right part of the diagram the small molecule metabolism is represented (for a complete description of the map see http://pathway.yeastgenome.org). Reaction lines are colour-coded (three colour bins) according to the fold change value of the gene: red for data values that exceed a log2 fold change threshold of 0.59, yellow for data values less than the inverse of the threshold, and blue for values in between. Detailed lists of all the regulated pathways, together with their diagrams and corresponding gene lists are provided in additional file 2 (for S. cerevisiae) and 3 (for P. pastoris). Depicted pathways are indicated by numbers, according to the table shown in additional file 4. The ergosterol pathway (n.20) and glycolysis (n.69) are indicated by dashed boxes.
Mentions: To obtain a general view of metabolic pathways responding to oxygen availability, we overlapped the fold change values obtained from the HvsN comparison of the producing strains with the map of S. cerevisiae core metabolism. Figure 3 shows the overview of the entire schematized map, while detailed lists of all the regulated pathways, together with their diagrams and corresponding gene lists, are provided in additional file 2 (for S. cerevisiae) and additional file 3 (for P. pastoris). All depicted pathways are indicated by numbers according to the table shown in additional file 4. The most striking differences were detected for the glycolytic pathway, ergosterol and sphingolipid biosynthesis, and the oxidative branch of the pentose phosphate pathway (Figure 3). The uniform upregulation of glycolytic genes, enzymes and metabolic fluxes in P. pastoris chemostats upon a shift to hypoxic growth conditions was recently reported in our preceding study [15], indicating a transcriptional control of the central carbon metabolism in this yeast. In hypoxically grown S. cerevisiae this picture was quite different, since glycolysis was not regulated at the transcriptome level. These observations confirm previous chemostat studies, where a poor correlation between the mRNA levels and the corresponding protein abundances or in vivo fluxes demonstrated a post-transcriptional control of glycolysis in anaerobic S. cerevisiae cultures [21-23]. De Groot [22] further estimated the pool of glycolytic enzymes to account for 21 % of the total protein in anaerobic conditions, thus occupying a considerable fraction of the S. cerevisiae translation machinery. This 'occupation' could somehow hamper or limit the translation of other proteins, e.g. the recombinant Fab antibody. Estimation of the corresponding percentage in P. pastoris might provide new insights on the different production abilities of the two microorganisms.

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