Integrative approaches for studying pentose metabolism in Saccharomyces cerevisiae

Laura Salusjärvi, Matej Oresic, Hannu Maaheimo, Eija Rintala, Helena Simolin, Laura Ruohonen, Merja Penttilä

Research output: Contribution to conferenceConference articleScientific

Abstract

We are interested in broadening the substrate utilisation range of the yeast and in connection to this to study nutritional responses and signalling at a global level. S.cerevisiae does not naturally utilise pentose sugars unlike most other fungi, and recombinant S.cerevisiae strains have been constructed that contain the xylose utilisation pathway from other yeasts. The xylose reductase enzyme prefers NADPH as a cofactor while the next step in the pathway xylitol dehydrogenase, NAD+. This is believed to create a redox cofactor imbalance in the cells and limit xylose fermentation. Other suggested rate-limiting steps in xylose utilisation and the use of xylose as a fermentative carbon source include xylose uptake and limitations in the pentose phospate pathway reactions. In order to understand redox and nutrient regulation in general, and the physiology of xylose utilising S.cerevisiae, we carried out extensive chemostat and batch cultures at various oxygenation levels on xylose as a carbon source, and compared those with glucose cultures. Transcriptional profiling, total proteomics and metabolite analyses were carried out, as well as metabolic flux analysis and measurement of metabolic fluxes with 13C-NMR. The results are in good agreement. In addition to the expected responses in cellular redox metabolism and pentose phosphate pathway, also new responses towards xylose as a carbon source were discovered such as the upregulation of pathways for alternative carbon source utilisation and responses for nutritional control and starvation. The physiology of the recombinant yeast appears to be neither fully repressed (fermentative) nor derepressed (gluconeogenic). Own references [1] H. Maaheimo, J. Fiaux, Z. P. Cakar, et al. Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids. Eur J Biochem. 268, 2464-2479 (2001). [2] Juho Rousu, Ari Rantanen, Hannu Maaheimo, Esa Pitkänen, Katja Saarela, Esko Ukkonen: A Method for Estimating Metabolic Fluxes from Incomplete Isotopomer Information. CMSB 2003: 88-103. [3] J. P. Pitkänen, A. Aristidou, L. Salusjärvi, L. Ruohonen, M. Penttilä, Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture. Metab Eng. 5, 16-31 (2003). [4] L. Salusjärvi, M. Poutanen, J. P. Pitkänen, et al. Proteome analysis of recombinant xylose-fermenting Saccharomyces cerevisiae. Yeast 20, 295-314 (2003). [5] Toivari, M.H., Aristidou, A., Ruohonen, L. & Penttilä, M. 2001. Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: Importance of xylulokinase (XKS1) and oxygen availability. Metab. Eng. 3, 236-249.
Original languageEnglish
Publication statusPublished - 2004
Event5th International Conference on Systems Biology – ICSB 2004 - Heidelberg, Germany
Duration: 9 Oct 200413 Oct 2004

Conference

Conference5th International Conference on Systems Biology – ICSB 2004
Abbreviated titleICSB 2004
CountryGermany
CityHeidelberg
Period9/10/0413/10/04

Fingerprint

pentoses
xylose
Saccharomyces cerevisiae
metabolism
carbon
yeasts
physiology
xylitol
NAD (coenzyme)
proteome
NADP (coenzyme)
proteomics
starvation
ethanol
fermentation
phosphates

Cite this

Salusjärvi, L., Oresic, M., Maaheimo, H., Rintala, E., Simolin, H., Ruohonen, L., & Penttilä, M. (2004). Integrative approaches for studying pentose metabolism in Saccharomyces cerevisiae. Paper presented at 5th International Conference on Systems Biology – ICSB 2004, Heidelberg, Germany.
Salusjärvi, Laura ; Oresic, Matej ; Maaheimo, Hannu ; Rintala, Eija ; Simolin, Helena ; Ruohonen, Laura ; Penttilä, Merja. / Integrative approaches for studying pentose metabolism in Saccharomyces cerevisiae. Paper presented at 5th International Conference on Systems Biology – ICSB 2004, Heidelberg, Germany.
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abstract = "We are interested in broadening the substrate utilisation range of the yeast and in connection to this to study nutritional responses and signalling at a global level. S.cerevisiae does not naturally utilise pentose sugars unlike most other fungi, and recombinant S.cerevisiae strains have been constructed that contain the xylose utilisation pathway from other yeasts. The xylose reductase enzyme prefers NADPH as a cofactor while the next step in the pathway xylitol dehydrogenase, NAD+. This is believed to create a redox cofactor imbalance in the cells and limit xylose fermentation. Other suggested rate-limiting steps in xylose utilisation and the use of xylose as a fermentative carbon source include xylose uptake and limitations in the pentose phospate pathway reactions. In order to understand redox and nutrient regulation in general, and the physiology of xylose utilising S.cerevisiae, we carried out extensive chemostat and batch cultures at various oxygenation levels on xylose as a carbon source, and compared those with glucose cultures. Transcriptional profiling, total proteomics and metabolite analyses were carried out, as well as metabolic flux analysis and measurement of metabolic fluxes with 13C-NMR. The results are in good agreement. In addition to the expected responses in cellular redox metabolism and pentose phosphate pathway, also new responses towards xylose as a carbon source were discovered such as the upregulation of pathways for alternative carbon source utilisation and responses for nutritional control and starvation. The physiology of the recombinant yeast appears to be neither fully repressed (fermentative) nor derepressed (gluconeogenic). Own references [1] H. Maaheimo, J. Fiaux, Z. P. Cakar, et al. Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids. Eur J Biochem. 268, 2464-2479 (2001). [2] Juho Rousu, Ari Rantanen, Hannu Maaheimo, Esa Pitk{\"a}nen, Katja Saarela, Esko Ukkonen: A Method for Estimating Metabolic Fluxes from Incomplete Isotopomer Information. CMSB 2003: 88-103. [3] J. P. Pitk{\"a}nen, A. Aristidou, L. Salusj{\"a}rvi, L. Ruohonen, M. Penttil{\"a}, Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture. Metab Eng. 5, 16-31 (2003). [4] L. Salusj{\"a}rvi, M. Poutanen, J. P. Pitk{\"a}nen, et al. Proteome analysis of recombinant xylose-fermenting Saccharomyces cerevisiae. Yeast 20, 295-314 (2003). [5] Toivari, M.H., Aristidou, A., Ruohonen, L. & Penttil{\"a}, M. 2001. Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: Importance of xylulokinase (XKS1) and oxygen availability. Metab. Eng. 3, 236-249.",
author = "Laura Salusj{\"a}rvi and Matej Oresic and Hannu Maaheimo and Eija Rintala and Helena Simolin and Laura Ruohonen and Merja Penttil{\"a}",
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Salusjärvi, L, Oresic, M, Maaheimo, H, Rintala, E, Simolin, H, Ruohonen, L & Penttilä, M 2004, 'Integrative approaches for studying pentose metabolism in Saccharomyces cerevisiae' Paper presented at 5th International Conference on Systems Biology – ICSB 2004, Heidelberg, Germany, 9/10/04 - 13/10/04, .

Integrative approaches for studying pentose metabolism in Saccharomyces cerevisiae. / Salusjärvi, Laura; Oresic, Matej; Maaheimo, Hannu; Rintala, Eija; Simolin, Helena; Ruohonen, Laura; Penttilä, Merja.

2004. Paper presented at 5th International Conference on Systems Biology – ICSB 2004, Heidelberg, Germany.

Research output: Contribution to conferenceConference articleScientific

TY - CONF

T1 - Integrative approaches for studying pentose metabolism in Saccharomyces cerevisiae

AU - Salusjärvi, Laura

AU - Oresic, Matej

AU - Maaheimo, Hannu

AU - Rintala, Eija

AU - Simolin, Helena

AU - Ruohonen, Laura

AU - Penttilä, Merja

N1 - CA2: BEL2 CA2: BEL1 CA: BEL

PY - 2004

Y1 - 2004

N2 - We are interested in broadening the substrate utilisation range of the yeast and in connection to this to study nutritional responses and signalling at a global level. S.cerevisiae does not naturally utilise pentose sugars unlike most other fungi, and recombinant S.cerevisiae strains have been constructed that contain the xylose utilisation pathway from other yeasts. The xylose reductase enzyme prefers NADPH as a cofactor while the next step in the pathway xylitol dehydrogenase, NAD+. This is believed to create a redox cofactor imbalance in the cells and limit xylose fermentation. Other suggested rate-limiting steps in xylose utilisation and the use of xylose as a fermentative carbon source include xylose uptake and limitations in the pentose phospate pathway reactions. In order to understand redox and nutrient regulation in general, and the physiology of xylose utilising S.cerevisiae, we carried out extensive chemostat and batch cultures at various oxygenation levels on xylose as a carbon source, and compared those with glucose cultures. Transcriptional profiling, total proteomics and metabolite analyses were carried out, as well as metabolic flux analysis and measurement of metabolic fluxes with 13C-NMR. The results are in good agreement. In addition to the expected responses in cellular redox metabolism and pentose phosphate pathway, also new responses towards xylose as a carbon source were discovered such as the upregulation of pathways for alternative carbon source utilisation and responses for nutritional control and starvation. The physiology of the recombinant yeast appears to be neither fully repressed (fermentative) nor derepressed (gluconeogenic). Own references [1] H. Maaheimo, J. Fiaux, Z. P. Cakar, et al. Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids. Eur J Biochem. 268, 2464-2479 (2001). [2] Juho Rousu, Ari Rantanen, Hannu Maaheimo, Esa Pitkänen, Katja Saarela, Esko Ukkonen: A Method for Estimating Metabolic Fluxes from Incomplete Isotopomer Information. CMSB 2003: 88-103. [3] J. P. Pitkänen, A. Aristidou, L. Salusjärvi, L. Ruohonen, M. Penttilä, Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture. Metab Eng. 5, 16-31 (2003). [4] L. Salusjärvi, M. Poutanen, J. P. Pitkänen, et al. Proteome analysis of recombinant xylose-fermenting Saccharomyces cerevisiae. Yeast 20, 295-314 (2003). [5] Toivari, M.H., Aristidou, A., Ruohonen, L. & Penttilä, M. 2001. Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: Importance of xylulokinase (XKS1) and oxygen availability. Metab. Eng. 3, 236-249.

AB - We are interested in broadening the substrate utilisation range of the yeast and in connection to this to study nutritional responses and signalling at a global level. S.cerevisiae does not naturally utilise pentose sugars unlike most other fungi, and recombinant S.cerevisiae strains have been constructed that contain the xylose utilisation pathway from other yeasts. The xylose reductase enzyme prefers NADPH as a cofactor while the next step in the pathway xylitol dehydrogenase, NAD+. This is believed to create a redox cofactor imbalance in the cells and limit xylose fermentation. Other suggested rate-limiting steps in xylose utilisation and the use of xylose as a fermentative carbon source include xylose uptake and limitations in the pentose phospate pathway reactions. In order to understand redox and nutrient regulation in general, and the physiology of xylose utilising S.cerevisiae, we carried out extensive chemostat and batch cultures at various oxygenation levels on xylose as a carbon source, and compared those with glucose cultures. Transcriptional profiling, total proteomics and metabolite analyses were carried out, as well as metabolic flux analysis and measurement of metabolic fluxes with 13C-NMR. The results are in good agreement. In addition to the expected responses in cellular redox metabolism and pentose phosphate pathway, also new responses towards xylose as a carbon source were discovered such as the upregulation of pathways for alternative carbon source utilisation and responses for nutritional control and starvation. The physiology of the recombinant yeast appears to be neither fully repressed (fermentative) nor derepressed (gluconeogenic). Own references [1] H. Maaheimo, J. Fiaux, Z. P. Cakar, et al. Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids. Eur J Biochem. 268, 2464-2479 (2001). [2] Juho Rousu, Ari Rantanen, Hannu Maaheimo, Esa Pitkänen, Katja Saarela, Esko Ukkonen: A Method for Estimating Metabolic Fluxes from Incomplete Isotopomer Information. CMSB 2003: 88-103. [3] J. P. Pitkänen, A. Aristidou, L. Salusjärvi, L. Ruohonen, M. Penttilä, Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture. Metab Eng. 5, 16-31 (2003). [4] L. Salusjärvi, M. Poutanen, J. P. Pitkänen, et al. Proteome analysis of recombinant xylose-fermenting Saccharomyces cerevisiae. Yeast 20, 295-314 (2003). [5] Toivari, M.H., Aristidou, A., Ruohonen, L. & Penttilä, M. 2001. Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: Importance of xylulokinase (XKS1) and oxygen availability. Metab. Eng. 3, 236-249.

M3 - Conference article

ER -

Salusjärvi L, Oresic M, Maaheimo H, Rintala E, Simolin H, Ruohonen L et al. Integrative approaches for studying pentose metabolism in Saccharomyces cerevisiae. 2004. Paper presented at 5th International Conference on Systems Biology – ICSB 2004, Heidelberg, Germany.