Defined medium: 5 g/L of 1-13C glucose, 10 g/L NaNO3, 3 g/L KH2PO4, 2 g/L MgSO4.7H2O, 2 g/L NaCl, 0.2 g/L CaCl2.2H2O, 0.5 ml/L antifoam and 1 ml/L trace element solution 2. The trace element solution 2 contained 14.3 g/L ZnSO4.7H2O, 2.5 g/L CuSO4.5H2O, 0.5 g/L NiCl2.6H2O, 7 g/L MnCl2.2H2O and 13.8 g/L FeSO4.7H2O.
General protocol information
Flux analysis method:
13C constrained MFA
Platform:
GC-MS
Methods description - Notes
GC–MS analysis - GC–MS analysis was performed on an Agilent (PaloAlto,CA, USA) HP 6890 gas chromatograph coupled to a HP5973 quadrupole mass selective detector in positive electron impact ionization (EI+) operated at an electron energy of 70 eV. The GC was equipped with a 4.
... 0mm i.d. Siltek gooseneck splitless deactivated liner (Restek, Bellefonte, PA, USA), and a Supelco (Bellefonte,PA,USA) SLB-5 MS column, 15m, 0.25mm i.d., 0.25 µm film. Helium of a purity of 99.999% was used as carrier gas at a constant linear gas velocity of 35 cm/s. Transfer line temperature was 280 ºC, quadrupole temperature 150 ºC and MS source 200 ºC. The GC–MS system was controlled from Agilent MSD Chemstation v. D.01.02.16. For both methods (ECF and DMF/DMA) 1 mL sample was injected in the splitless (30s, split 1:20) mode at 200 ºC using a hot needle.The MS scanrange was m/z 40–400 with 2.88 s/scan. To avoid sample carry-over the syringe was cleaned using five times 5mL acetone, then five times5 mL dichloromethane and finally 3 times rinsing with 3 µL sample.
Metabolic network analysis - The fluxes were determined using an in-house program code in Matlab V7.0.4 , which implements the metabolic network analysis framework from [1].
Defined medium: 5g/L of 1-13C glucose, 10g/L NaNO3, 3g/L KH2PO4, 2g/L MgSO4.7H2O, 2g/L NaCl, 0.2g/L CaCl2.2H2O, 0.5ml/L antifoam and 1ml/L trace element solution 2. The trace element solution 2 contained 14.3g/L ZnSO4.7H2O, 2.5g/L CuSO4.5H2O, 0.5g/L NiCl2.6H2O,7g/L MnCl2.2H2O and 13.8g/L FeSO4.7H2O.
General protocol information
Flux analysis method: 13C constrained MFA
Platform: GC-MS
Methods description - Notes
GC–MS analysis - GC–MS analysis was performed on an Agilent (PaloAlto,CA, USA) HP 6890 gas chromatograph coupled to a HP5973 quadrupole mass selective detector in positive electron impact ionization (EI+) operated at an electron energy of 70 eV. The GC was equipped with a 4.0mm i.d. Siltek gooseneck splitless deactivated liner (Restek, Bellefonte, PA, USA), and a Supelco (Bellefonte,PA,USA) SLB-5 MS column, 15m, 0.25mm i.d., 0.25 µm film. Helium of a purity of 99.999% was used as carrier gas at a constant linear gas velocity of 35 cm/s. Transfer line temperature was 280 ºC, quadrupole temperature 150 ºC and MS source 200 ºC. The GC–MS system was controlled from Agilent MSD Chemstation v. D.01.02.16. For both methods (ECF and DMF/DMA) 1 mL sample was injected in the splitless (30s, split 1:20) mode at 200 ºC using a hot needle.The MS scanrange was m/z 40–400 with 2.88 s/scan. To avoid sample carry-over the syringe was cleaned using five times 5mL acetone, then five times5 mL dichloromethane and finally 3 times rinsing with 3 µL sample.
Metabolic network analysis - The fluxes were determined using an in-house program code in Matlab V7.0.4 , which implements the metabolic network analysis framework from [1].
--------------References------------------
[1] Wiechert, W.,2001. C13 metabolic flux analysis. Metab. Eng. 3, 195–206.
KiMoSys (https://kimosys.org). Data EntryID 73 (Aspergillus niger). [online], [Accessed 12 October 2024]. Available from: https://doi.org/10.34619/9pzn-h470