Defined minimal medium [1], with 10 g glucose l-1 as carbon source, and supplemented with 10 mg ergosterol l-1 and 420 mg Tween 80 l-1 (a source of oleic acid).
The culture conditions and biomass determination and chemical and metabolite analyses are described in more detail by Wiebe et al. (2008) [2].
Biosyntheti
... cally directed fractional (BDF) 13C-labelling:
13C-labelling experiments were performed in at least two replicate cultures under each oxygenation condition. After reaching a metabolic steady state, as determined by constant physiological parameters including biomass production, carbon dioxide evolution and oxygen uptake rates (CER and OUR), alkali utilisation, and subsequently confirmed by the observation of constant extracellular and intracellular metabolites and gene transcription, 10% of the carbon source in the medium was replaced with [U-13C]glucose (Isotec, 99 atom% 13C). During steady state growth the active pathways in the cells will determine how the yeast biomass becomes 13C-labelled. After approximately 1.5 residence times biomass samples, 50 ml of culture broth, corresponding to 0.27 to 0.05 g CDW, were harvested by centrifugation. The cell pellets were suspended into 10 ml of 6 M HCl and the biomass was hydrolysed in sealed glass tubes at 110°C for 22 h. The suspensions were dried and dissolved in H2O for filtration through 0.2 μm filters. The filtrates were vacuum-dried and dissolved in D2O for NMR experiments. The pH of the samples was below 1 due to residual HCl.
As described previously [3, 4, 5, 6, 7, 8, 9], the calculation of metabolic flux ratios when using fractional 13C-labelling of amino acids is based on the assumption that both a metabolic and an isotopomeric steady state exist. To establish a cost-effective protocol for a larger number of 13C-labelling experiments, 13C-labelled substrate was fed to a chemostat operating in a metabolic steady state for the duration of 1.5 volume changes [10, 7] before harvesting the biomass. The fraction of unlabelled biomass produced prior to the start of 13C-labelled medium supply was calculated following simple wash-out kinetics [9].
NMR spectroscopy:
13C-HSQC nuclear magnetic resonance (NMR) spectra were acquired at 40°C on a Varian Inova spectrometer operating at a 1H-resonance frequency of 600 MHz essentially as described [6]. For each sample two spectra were acquired focusing on the aliphatic and aromatic regions. For more informatio see original paper.
METAFoR analysis:
Metabolic flux ratio (METAFoR) analysis was done based on the compartmentalized metabolic model of S. cerevisiae central carbon metabolism formulated by Maaheimo and co-workers (2001) [5]. The software FCAL (R.W. Glaser; FCAL 2.3.1) [4] was used for the integration of 13C-scalar fine structures of proteinogenic amino acid carbon signals in the 13C-HSQC NMR spectra and the calculation of relative abundances of intact carbon fragments originating from a single source molecule of glucose. For more information see original paper. --------------------------------------------References---------------------------------------
[1] Verduyn C, Postma E, Scheffers WA, van Dijken JP. Yeast. 1992, 8: 501-517. http://doi.org/fgpqk3 [2] Wiebe MG, Rintala E, Tamminen A, Simolin H, Salusjärvi L, Toivari M, Kokkonen JT, Kiuru J, Ketola RA, Jouhten P, Huuskonen A, Maaheimo H, Ruohonen L, Penttilä M. FEMS Yeast Res. 2008, 8: 140-154.
[3] Fiaux J, Ìakar PZ, Sonderegger M, Wüthrich K, Szyperski T, Sauer U. Eukaryot Cell. 2003, 2: 170-180. http://doi.org/dhwxq7 [4] Szyperski T, Glaser RW, Hochuli M, Fiaux J, Sauer U, Bailey JE, Wüthrich K. Metab Eng. 1999, 1: 189-197. http://doi.org/b36nqn [5] Maaheimo H, Fiaux J, Ìakar PZ, Bailey JE, Sauer U, Szyperski T. Eur J Biochem. 2001, 268: 2464-2479. http://doi.org/fg2ghr [6] Szyperski T. Eur J Biochem. 1995, 232: 433-448. http://doi.org/dvsfrz [7] Sauer U, Hatzimanikatis V, Bailey JE, Hochuli M, Szyperski T, Wüthrich K. Nat Biotechnol. 1997, 15: 448-452. http://doi.org/fms74w [8] Sauer U, Lasko DR, Fiaux J, Hochuli M, Glaser R, Szyperski T, Wüthrich K, Bailey JE. J Bacteriol. 1999, 181: 6679-6688.
[9] Sola A, Maaheimo H, Ylonen K, Ferrer P, Szyperski T. Eur J Biochem. 2004, 271: 2462-2470. http://doi.org/bntz8s [10] Fiaux J, Ìakar PZ, Sonderegger M, Wüthrich K, Szyperski T, Sauer U. Eukaryot Cell. 2003, 2: 170-180. http://doi.org/dhwxq7
Defined minimal medium [1], with 10 g glucose l-1 as carbon source, and supplemented with 10 mg ergosterol l-1 and 420 mg Tween 80 l-1 (a source of oleic acid).
General protocol information
Flux analysis method: 13C constrained MFA, flux ratio
Platform: NMR
Methods description - Notes
The culture conditions and biomass determination and chemical and metabolite analyses are described in more detail by Wiebe et al. (2008) [2].
Biosynthetically directed fractional (BDF) 13C-labelling:
13C-labelling experiments were performed in at least two replicate cultures under each oxygenation condition. After reaching a metabolic steady state, as determined by constant physiological parameters including biomass production, carbon dioxide evolution and oxygen uptake rates (CER and OUR), alkali utilisation, and subsequently confirmed by the observation of constant extracellular and intracellular metabolites and gene transcription, 10% of the carbon source in the medium was replaced with [U-13C]glucose (Isotec, 99 atom% 13C). During steady state growth the active pathways in the cells will determine how the yeast biomass becomes 13C-labelled. After approximately 1.5 residence times biomass samples, 50 ml of culture broth, corresponding to 0.27 to 0.05 g CDW, were harvested by centrifugation. The cell pellets were suspended into 10 ml of 6 M HCl and the biomass was hydrolysed in sealed glass tubes at 110°C for 22 h. The suspensions were dried and dissolved in H2O for filtration through 0.2 μm filters. The filtrates were vacuum-dried and dissolved in D2O for NMR experiments. The pH of the samples was below 1 due to residual HCl.
As described previously [3, 4, 5, 6, 7, 8, 9], the calculation of metabolic flux ratios when using fractional 13C-labelling of amino acids is based on the assumption that both a metabolic and an isotopomeric steady state exist. To establish a cost-effective protocol for a larger number of 13C-labelling experiments, 13C-labelled substrate was fed to a chemostat operating in a metabolic steady state for the duration of 1.5 volume changes [10, 7] before harvesting the biomass. The fraction of unlabelled biomass produced prior to the start of 13C-labelled medium supply was calculated following simple wash-out kinetics [9].
NMR spectroscopy:
13C-HSQC nuclear magnetic resonance (NMR) spectra were acquired at 40°C on a Varian Inova spectrometer operating at a 1H-resonance frequency of 600 MHz essentially as described [6]. For each sample two spectra were acquired focusing on the aliphatic and aromatic regions. For more informatio see original paper. METAFoR analysis:
Metabolic flux ratio (METAFoR) analysis was done based on the compartmentalized metabolic model of S. cerevisiae central carbon metabolism formulated by Maaheimo and co-workers (2001) [5]. The software FCAL (R.W. Glaser; FCAL 2.3.1) [4] was used for the integration of 13C-scalar fine structures of proteinogenic amino acid carbon signals in the 13C-HSQC NMR spectra and the calculation of relative abundances of intact carbon fragments originating from a single source molecule of glucose. For more information see original paper. --------------------------------------------References--------------------------------------- [1] Verduyn C, Postma E, Scheffers WA, van Dijken JP. Yeast. 1992, 8: 501-517. 10.1002/yea.320080703 [2] Wiebe MG, Rintala E, Tamminen A, Simolin H, Salusjärvi L, Toivari M, Kokkonen JT, Kiuru J, Ketola RA, Jouhten P, Huuskonen A, Maaheimo H, Ruohonen L, Penttilä M. FEMS Yeast Res. 2008, 8: 140-154. [3] Fiaux J, Ìakar PZ, Sonderegger M, Wüthrich K, Szyperski T, Sauer U. Eukaryot Cell. 2003, 2: 170-180. 10.1128/EC.2.1.170-180.2003 [4] Szyperski T, Glaser RW, Hochuli M, Fiaux J, Sauer U, Bailey JE, Wüthrich K. Metab Eng. 1999, 1: 189-197. 10.1006/mben.1999.0116 [5] Maaheimo H, Fiaux J, Ìakar PZ, Bailey JE, Sauer U, Szyperski T. Eur J Biochem. 2001, 268: 2464-2479. 10.1046/j.1432-1327.2001.02126.x [6] Szyperski T. Eur J Biochem. 1995, 232: 433-448. 10.1111/j.1432-1033.1995.tb20829.x [7] Sauer U, Hatzimanikatis V, Bailey JE, Hochuli M, Szyperski T, Wüthrich K. Nat Biotechnol. 1997, 15: 448-452. 10.1038/nbt0597-448 [8] Sauer U, Lasko DR, Fiaux J, Hochuli M, Glaser R, Szyperski T, Wüthrich K, Bailey JE. J Bacteriol. 1999, 181: 6679-6688. [9] Sola A, Maaheimo H, Ylonen K, Ferrer P, Szyperski T. Eur J Biochem. 2004, 271: 2462-2470. 10.1111/j.1432-1033.2004.04176.x [10] Fiaux J, Ìakar PZ, Sonderegger M, Wüthrich K, Szyperski T, Sauer U. Eukaryot Cell. 2003, 2: 170-180. 10.1128/EC.2.1.170-180.2003
KiMoSys (https://kimosys.org). Data EntryID 117 (Saccharomyces cerevisiae). [online], [Accessed 3 December 2024]. Available from: https://doi.org/10.34619/x818-xy86
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