Details
| Original language | English |
|---|---|
| Pages (from-to) | 144-156 |
| Number of pages | 13 |
| Journal | Biotechnology and bioengineering |
| Volume | 76 |
| Issue number | 2 |
| Publication status | Published - 8 Aug 2001 |
| Externally published | Yes |
Abstract
Fluxes in central carbon metabolism of a genetically engineered, riboflavin-producing Bacillus subtilis strain were investigated in glucose-limited chemostat cultures at low (0.11 h-1) and high (0.44 h-1) dilution rates. Using a mixture of 10% [U-13C] and 90% glucose labeled at natural abundance, 13C-labeling experiments were carried out to provide additional information for metabolic flux balancing. The resulting labeling pattern in the proteinogenic amino acids were analyzed by two-dimensional [13C, 1H] nuclear magnetic resonance (NMR) spectroscopy. To account rigorously for all available data from these experiments, we developed a comprehensive isotopomer model of B. subtilis central metabolism. Using this model, intracellular carbon net and exchange fluxes were estimated on the basis of validated physiological data and biomass composition in combination with 2D NMR data from 45 individual carbon atom spectra in the amino acids. Glucose catabolism proceeded primarily via glycolysis but pentose phosphate pathway fluxes increased with increasing growth rate. Moreover, significant back fluxes from the TCA cycle to the lower part of glycolysis via the gluconeogenic PEP carboxykinase were detected. The malic enzyme reaction, in contrast, was found to be inactive. A thorough statistical analysis was performed to prove the reliability of the isotopomer balance model and the obtained results. Specifically, a X2 test was applied to validate the model and the chi-square criterion was used to explore the sensitivity of model predictions to the experimental data.
Keywords
- Bacillus subtilis, Central metabolism, Isotopomer model, Metabolic flux analysis, NMR spectroscopy
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Chemical Engineering(all)
- Bioengineering
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
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In: Biotechnology and bioengineering, Vol. 76, No. 2, 08.08.2001, p. 144-156.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis
AU - Wiebe, Marilyn G.
AU - Karandikar, Atul
AU - Robson, Geoff D.
AU - Trinci, Anthony P.J.
AU - Candia, Juana L.Flores
AU - Trappe, Susanne
AU - Wallis, Gregg
AU - Rinas, Ursula
AU - Derkx, Patrick M.F.
AU - Madrid, Susan M.
AU - Sisniega, Heidi
AU - Faus, Ignacio
AU - Montijn, Roy
AU - Van Den Hondel, Cees A.M.J.J.
AU - Punt, Peter J.
PY - 2001/8/8
Y1 - 2001/8/8
N2 - Fluxes in central carbon metabolism of a genetically engineered, riboflavin-producing Bacillus subtilis strain were investigated in glucose-limited chemostat cultures at low (0.11 h-1) and high (0.44 h-1) dilution rates. Using a mixture of 10% [U-13C] and 90% glucose labeled at natural abundance, 13C-labeling experiments were carried out to provide additional information for metabolic flux balancing. The resulting labeling pattern in the proteinogenic amino acids were analyzed by two-dimensional [13C, 1H] nuclear magnetic resonance (NMR) spectroscopy. To account rigorously for all available data from these experiments, we developed a comprehensive isotopomer model of B. subtilis central metabolism. Using this model, intracellular carbon net and exchange fluxes were estimated on the basis of validated physiological data and biomass composition in combination with 2D NMR data from 45 individual carbon atom spectra in the amino acids. Glucose catabolism proceeded primarily via glycolysis but pentose phosphate pathway fluxes increased with increasing growth rate. Moreover, significant back fluxes from the TCA cycle to the lower part of glycolysis via the gluconeogenic PEP carboxykinase were detected. The malic enzyme reaction, in contrast, was found to be inactive. A thorough statistical analysis was performed to prove the reliability of the isotopomer balance model and the obtained results. Specifically, a X2 test was applied to validate the model and the chi-square criterion was used to explore the sensitivity of model predictions to the experimental data.
AB - Fluxes in central carbon metabolism of a genetically engineered, riboflavin-producing Bacillus subtilis strain were investigated in glucose-limited chemostat cultures at low (0.11 h-1) and high (0.44 h-1) dilution rates. Using a mixture of 10% [U-13C] and 90% glucose labeled at natural abundance, 13C-labeling experiments were carried out to provide additional information for metabolic flux balancing. The resulting labeling pattern in the proteinogenic amino acids were analyzed by two-dimensional [13C, 1H] nuclear magnetic resonance (NMR) spectroscopy. To account rigorously for all available data from these experiments, we developed a comprehensive isotopomer model of B. subtilis central metabolism. Using this model, intracellular carbon net and exchange fluxes were estimated on the basis of validated physiological data and biomass composition in combination with 2D NMR data from 45 individual carbon atom spectra in the amino acids. Glucose catabolism proceeded primarily via glycolysis but pentose phosphate pathway fluxes increased with increasing growth rate. Moreover, significant back fluxes from the TCA cycle to the lower part of glycolysis via the gluconeogenic PEP carboxykinase were detected. The malic enzyme reaction, in contrast, was found to be inactive. A thorough statistical analysis was performed to prove the reliability of the isotopomer balance model and the obtained results. Specifically, a X2 test was applied to validate the model and the chi-square criterion was used to explore the sensitivity of model predictions to the experimental data.
KW - Bacillus subtilis
KW - Central metabolism
KW - Isotopomer model
KW - Metabolic flux analysis
KW - NMR spectroscopy
UR - http://www.scopus.com/inward/record.url?scp=0034847930&partnerID=8YFLogxK
U2 - 10.1002/bit.1154
DO - 10.1002/bit.1154
M3 - Article
C2 - 11505384
AN - SCOPUS:0034847930
VL - 76
SP - 144
EP - 156
JO - Biotechnology and bioengineering
JF - Biotechnology and bioengineering
SN - 0006-3592
IS - 2
ER -