Metabolomics: quantification of intracellular metabolite dynamics.
The rational improvement of microbial strains for the production of primary and secondary metabolites ('metabolic engineering') requires a quantitative understanding of microbial metabolism. A process by which this information can be derived from dynamic fermentation experiments is presented. By applying a substrate pulse to a substrate-limited, steady state culture, cellular metabolism is shifted away from its metabolic steady state. With the aid of a rapid sampling and quenching routine it is possible to take 4-5 samples per second during this process, thus capturing the metabolic response to this stimulus. Over 30 metabolites, nucleotides and cofactors from Escherichia coli metabolism can be extracted and analysed using a range of different techniques, for example enzymatic assays, HPLC and LC-MS methods. Using different substrates as limiting and pulse-substrates (glucose, glycerol), different metabolic pathways and substrate uptake systems are investigated. The resulting plots of intracellular metabolite concentrations against time serve as a data basis for modelling microbial metabolic networks.
Buchholz
Arne
A
Institute of Biotechnology 2, Forschungszentrum Jülich, Germany.
Hurlebaus
Jochen
J
Wandrey
Christian
C
Takors
Ralf
R
D019149:Bioreactors; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003198:Computer Simulation; D004734:Energy Metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D016680:Genome, Bacterial; D005947:Glucose / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism*; D016014:Linear Models; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D008956:Models, Chemical; D013379:Substrate Specificity
D005947:Glucose; D005990:Glycerol