Metabolic adjustment upon repetitive substrate perturbations using dynamic 13C-tracing in yeast
Abstract Background Natural and industrial environments are dynamic with respect to substrate availability and other conditions like temperature and pH. Especially, metabolism is strongly affected by changes in the extracellular space. Here we study the dynamic flux of central carbon metabolism and...
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| Format: | Article |
| Language: | English |
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BMC
2017-09-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12934-017-0778-6 |
| _version_ | 1819161952572669952 |
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| author | C. A. Suarez-Mendez C. Ras S. A. Wahl |
| author_facet | C. A. Suarez-Mendez C. Ras S. A. Wahl |
| author_sort | C. A. Suarez-Mendez |
| collection | DOAJ |
| description | Abstract Background Natural and industrial environments are dynamic with respect to substrate availability and other conditions like temperature and pH. Especially, metabolism is strongly affected by changes in the extracellular space. Here we study the dynamic flux of central carbon metabolism and storage carbohydrate metabolism under dynamic feast/famine conditions in Saccharomyces cerevisiae. Results The metabolic flux reacts fast and sensitive to cyclic perturbations in substrate availability. Compared to well-documented stimulus–response experiments using substrate pulses, different metabolic responses are observed. Especially, cells experiencing cyclic perturbations do not show a drop in ATP with the addition of glucose, but an immediate increase in energy charge. Although a high glycolytic flux of up to 5.4 mmol g DW −1 h−1 is observed, no overflow metabolites are detected. From famine to feast the glucose uptake rate increased from 170 to 4788 μmol g DW −1 h−1 in 24 s. Intracellularly, even more drastic changes were observed. Especially, the T6P synthesis rate increased more than 100-fold upon glucose addition. This response indicates that the storage metabolism is very sensitive to changes in glycolytic flux and counterbalances these rapid changes by diverting flux into large pools to prevent substrate accelerated death and potentially refill the central metabolism when substrates become scarce. Using 13C-tracer we found a dilution in the labeling of extracellular glucose, G6P, T6P and other metabolites, indicating an influx of unlabeled carbon. It is shown that glycogen and trehalose degradation via different routes could explain these observations. Based on the 13C labeling in average 15% of the carbon inflow is recycled via trehalose and glycogen. This average fraction is comparable to the steady-state turnover, but changes significantly during the cycle, indicating the relevance for dynamic regulation of the metabolic flux. Conclusions Comparable to electric energy grids, metabolism seems to use storage units to buffer peaks and keep reserves to maintain a robust function. During the applied fast feast/famine conditions about 15% of the metabolized carbon were recycled in storage metabolism. Additionally, the resources were distributed different to steady-state conditions. Most remarkably is a fivefold increased flux towards PPP that generated a reversed flux of transaldolase and the F6P-producing transketolase reactions. Combined with slight changes in the biomass composition, the yield decrease of 5% can be explained. |
| first_indexed | 2024-12-22T17:20:31Z |
| format | Article |
| id | doaj.art-919228a01e4a47e4a0a1beed1b9cc721 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-22T17:20:31Z |
| publishDate | 2017-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-919228a01e4a47e4a0a1beed1b9cc7212022-12-21T18:18:50ZengBMCMicrobial Cell Factories1475-28592017-09-0116111410.1186/s12934-017-0778-6Metabolic adjustment upon repetitive substrate perturbations using dynamic 13C-tracing in yeastC. A. Suarez-Mendez0C. Ras1S. A. Wahl2Department of Biotechnology, Delft University of TechnologyDepartment of Biotechnology, Delft University of TechnologyDepartment of Biotechnology, Delft University of TechnologyAbstract Background Natural and industrial environments are dynamic with respect to substrate availability and other conditions like temperature and pH. Especially, metabolism is strongly affected by changes in the extracellular space. Here we study the dynamic flux of central carbon metabolism and storage carbohydrate metabolism under dynamic feast/famine conditions in Saccharomyces cerevisiae. Results The metabolic flux reacts fast and sensitive to cyclic perturbations in substrate availability. Compared to well-documented stimulus–response experiments using substrate pulses, different metabolic responses are observed. Especially, cells experiencing cyclic perturbations do not show a drop in ATP with the addition of glucose, but an immediate increase in energy charge. Although a high glycolytic flux of up to 5.4 mmol g DW −1 h−1 is observed, no overflow metabolites are detected. From famine to feast the glucose uptake rate increased from 170 to 4788 μmol g DW −1 h−1 in 24 s. Intracellularly, even more drastic changes were observed. Especially, the T6P synthesis rate increased more than 100-fold upon glucose addition. This response indicates that the storage metabolism is very sensitive to changes in glycolytic flux and counterbalances these rapid changes by diverting flux into large pools to prevent substrate accelerated death and potentially refill the central metabolism when substrates become scarce. Using 13C-tracer we found a dilution in the labeling of extracellular glucose, G6P, T6P and other metabolites, indicating an influx of unlabeled carbon. It is shown that glycogen and trehalose degradation via different routes could explain these observations. Based on the 13C labeling in average 15% of the carbon inflow is recycled via trehalose and glycogen. This average fraction is comparable to the steady-state turnover, but changes significantly during the cycle, indicating the relevance for dynamic regulation of the metabolic flux. Conclusions Comparable to electric energy grids, metabolism seems to use storage units to buffer peaks and keep reserves to maintain a robust function. During the applied fast feast/famine conditions about 15% of the metabolized carbon were recycled in storage metabolism. Additionally, the resources were distributed different to steady-state conditions. Most remarkably is a fivefold increased flux towards PPP that generated a reversed flux of transaldolase and the F6P-producing transketolase reactions. Combined with slight changes in the biomass composition, the yield decrease of 5% can be explained.http://link.springer.com/article/10.1186/s12934-017-0778-6Dynamic fluxes13C labelingMultiple perturbationsYeastStorage carbohydratesSystems biology |
| spellingShingle | C. A. Suarez-Mendez C. Ras S. A. Wahl Metabolic adjustment upon repetitive substrate perturbations using dynamic 13C-tracing in yeast Microbial Cell Factories Dynamic fluxes 13C labeling Multiple perturbations Yeast Storage carbohydrates Systems biology |
| title | Metabolic adjustment upon repetitive substrate perturbations using dynamic 13C-tracing in yeast |
| title_full | Metabolic adjustment upon repetitive substrate perturbations using dynamic 13C-tracing in yeast |
| title_fullStr | Metabolic adjustment upon repetitive substrate perturbations using dynamic 13C-tracing in yeast |
| title_full_unstemmed | Metabolic adjustment upon repetitive substrate perturbations using dynamic 13C-tracing in yeast |
| title_short | Metabolic adjustment upon repetitive substrate perturbations using dynamic 13C-tracing in yeast |
| title_sort | metabolic adjustment upon repetitive substrate perturbations using dynamic 13c tracing in yeast |
| topic | Dynamic fluxes 13C labeling Multiple perturbations Yeast Storage carbohydrates Systems biology |
| url | http://link.springer.com/article/10.1186/s12934-017-0778-6 |
| work_keys_str_mv | AT casuarezmendez metabolicadjustmentuponrepetitivesubstrateperturbationsusingdynamic13ctracinginyeast AT cras metabolicadjustmentuponrepetitivesubstrateperturbationsusingdynamic13ctracinginyeast AT sawahl metabolicadjustmentuponrepetitivesubstrateperturbationsusingdynamic13ctracinginyeast |