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Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms.

Paczia N, Nilgen A, Lehmann T, Gätgens J, Wiechert W, Noack S - Microb. Cell Fact. (2012)

Bottom Line: Most surprisingly, in all cases a great diversity of central metabolic intermediates and amino acids is found in the culture medium with extracellular concentrations varying in the micromolar range.As a result, the intermediates in the culture medium during batch growth must originate from passive or active transportation due to a new phenomenon termed "extended" overflow metabolism.In turn, this finding has consequences for metabolite balancing and, particularly, for intracellular metabolite quantification and (13)C-metabolic flux analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Bio- and Geosciences, Biotechnology, Systems Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany.

ABSTRACT
Overflow metabolism is well known for yeast, bacteria and mammalian cells. It typically occurs under glucose excess conditions and is characterized by excretions of by-products such as ethanol, acetate or lactate. This phenomenon, also denoted the short-term Crabtree effect, has been extensively studied over the past few decades, however, its basic regulatory mechanism and functional role in metabolism is still unknown. Here we present a comprehensive quantitative and time-dependent analysis of the exometabolome of Escherichia coli, Corynebacterium glutamicum, Bacillus licheniformis, and Saccharomyces cerevisiae during well-controlled bioreactor cultivations. Most surprisingly, in all cases a great diversity of central metabolic intermediates and amino acids is found in the culture medium with extracellular concentrations varying in the micromolar range. Different hypotheses for these observations are formulated and experimentally tested. As a result, the intermediates in the culture medium during batch growth must originate from passive or active transportation due to a new phenomenon termed "extended" overflow metabolism. Moreover, we provide broad evidence that this could be a common feature of all microorganism species when cultivated under conditions of carbon excess and non-inhibited carbon uptake. In turn, this finding has consequences for metabolite balancing and, particularly, for intracellular metabolite quantification and (13)C-metabolic flux analysis.

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Related in: MedlinePlus

Impact of sampling procedure on the cell integrity of E. coli WT. (A) Different sample treatments for cell washing and separation. (B) Untargeted GC-TOF-MS analysis of resulting cell-free supernatants. Here the mass peaks detected in the washing, filtrate and centrifugate samples are due to derivatized compounds of the washing solution. (C) Targeted LC-ESI-MS/MS analysis of resulting cell-free supernatants and cell extracts focusing on specific sugar phosphates from glycolysis. Abbreviations: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; PEP, phosphoenolpyruvate.
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Figure 4: Impact of sampling procedure on the cell integrity of E. coli WT. (A) Different sample treatments for cell washing and separation. (B) Untargeted GC-TOF-MS analysis of resulting cell-free supernatants. Here the mass peaks detected in the washing, filtrate and centrifugate samples are due to derivatized compounds of the washing solution. (C) Targeted LC-ESI-MS/MS analysis of resulting cell-free supernatants and cell extracts focusing on specific sugar phosphates from glycolysis. Abbreviations: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; PEP, phosphoenolpyruvate.

Mentions: In order to test this hypothesis, first a culture sample was prepared by repeated washing with fresh 37°C glucose medium to guarantee that no metabolites were present in the extracellular volume (Figure 4A). Then two different cell separation methods, i.e. filtration and centrifugation, were applied to this sample. For all processing steps the resulting cell-free supernatants were analyzed for metabolites by untargeted GC-TOF-MS and targeted LC-ESI-MS/MS (Figure 4B and 4C, left). It can be seen that the extracellular concentration of all metabolites significantly decreased along the washing steps and, more importantly, also after the centrifugation and filtration step no sudden increase in the metabolite concentrations was detectable. Nevertheless, small amounts of sugar phosphates were also found in the washing supernatants. This is most likely due to an ongoing cell metabolism converting the glucose of the washing solution and, comparable to the situation in the bioreactor, excreting metabolites in the surrounding medium.


Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms.

Paczia N, Nilgen A, Lehmann T, Gätgens J, Wiechert W, Noack S - Microb. Cell Fact. (2012)

Impact of sampling procedure on the cell integrity of E. coli WT. (A) Different sample treatments for cell washing and separation. (B) Untargeted GC-TOF-MS analysis of resulting cell-free supernatants. Here the mass peaks detected in the washing, filtrate and centrifugate samples are due to derivatized compounds of the washing solution. (C) Targeted LC-ESI-MS/MS analysis of resulting cell-free supernatants and cell extracts focusing on specific sugar phosphates from glycolysis. Abbreviations: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; PEP, phosphoenolpyruvate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526501&req=5

Figure 4: Impact of sampling procedure on the cell integrity of E. coli WT. (A) Different sample treatments for cell washing and separation. (B) Untargeted GC-TOF-MS analysis of resulting cell-free supernatants. Here the mass peaks detected in the washing, filtrate and centrifugate samples are due to derivatized compounds of the washing solution. (C) Targeted LC-ESI-MS/MS analysis of resulting cell-free supernatants and cell extracts focusing on specific sugar phosphates from glycolysis. Abbreviations: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; PEP, phosphoenolpyruvate.
Mentions: In order to test this hypothesis, first a culture sample was prepared by repeated washing with fresh 37°C glucose medium to guarantee that no metabolites were present in the extracellular volume (Figure 4A). Then two different cell separation methods, i.e. filtration and centrifugation, were applied to this sample. For all processing steps the resulting cell-free supernatants were analyzed for metabolites by untargeted GC-TOF-MS and targeted LC-ESI-MS/MS (Figure 4B and 4C, left). It can be seen that the extracellular concentration of all metabolites significantly decreased along the washing steps and, more importantly, also after the centrifugation and filtration step no sudden increase in the metabolite concentrations was detectable. Nevertheless, small amounts of sugar phosphates were also found in the washing supernatants. This is most likely due to an ongoing cell metabolism converting the glucose of the washing solution and, comparable to the situation in the bioreactor, excreting metabolites in the surrounding medium.

Bottom Line: Most surprisingly, in all cases a great diversity of central metabolic intermediates and amino acids is found in the culture medium with extracellular concentrations varying in the micromolar range.As a result, the intermediates in the culture medium during batch growth must originate from passive or active transportation due to a new phenomenon termed "extended" overflow metabolism.In turn, this finding has consequences for metabolite balancing and, particularly, for intracellular metabolite quantification and (13)C-metabolic flux analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Bio- and Geosciences, Biotechnology, Systems Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany.

ABSTRACT
Overflow metabolism is well known for yeast, bacteria and mammalian cells. It typically occurs under glucose excess conditions and is characterized by excretions of by-products such as ethanol, acetate or lactate. This phenomenon, also denoted the short-term Crabtree effect, has been extensively studied over the past few decades, however, its basic regulatory mechanism and functional role in metabolism is still unknown. Here we present a comprehensive quantitative and time-dependent analysis of the exometabolome of Escherichia coli, Corynebacterium glutamicum, Bacillus licheniformis, and Saccharomyces cerevisiae during well-controlled bioreactor cultivations. Most surprisingly, in all cases a great diversity of central metabolic intermediates and amino acids is found in the culture medium with extracellular concentrations varying in the micromolar range. Different hypotheses for these observations are formulated and experimentally tested. As a result, the intermediates in the culture medium during batch growth must originate from passive or active transportation due to a new phenomenon termed "extended" overflow metabolism. Moreover, we provide broad evidence that this could be a common feature of all microorganism species when cultivated under conditions of carbon excess and non-inhibited carbon uptake. In turn, this finding has consequences for metabolite balancing and, particularly, for intracellular metabolite quantification and (13)C-metabolic flux analysis.

Show MeSH
Related in: MedlinePlus