Abstract
The main requirement for metabolic flux analysis (MFA) is that the cells are in a pseudo-steady state, that there is no accumulation or depletion of intracellular metabolites. In the past, the applications of MFA were limited to the analysis of continuous cultures. This contribution introduces the concept of dynamic MFA and extends MFA so that it is applicable to transient cultures. Time series of concentration measurements are transformed into flux values. This transformation involves differentiation, which typically increases the noisiness of the data. Therefore, a noise-reducing step is needed. In this work, polynomial smoothing was used. As a test case, dynamic MFA is applied on Escherichia coli cultivations shifting from carbon limitation to nitrogen limitation and vice versa. After switching the limiting substrate from N to C, a lag phase was observed accompanied with an increase in maintenance energy requirement. This lag phase did not occur in the C- to N-limitation case.
Highlights
Material balances of reactors describe how certain compounds go in the reactor and are transformed into other compounds
Metabolic models consider the cell as the reactor and, to the exchange rates, include information on the different steps on how the input compounds are transformed to the output compounds
Biomass concentrations were calculated from optical density (OD) values, after generating a calibration curve between cell dry weight (CDW) and OD values
Summary
Material balances of reactors describe how certain compounds go in the reactor and are transformed into other compounds. Metabolic models consider the cell as the reactor and, to the exchange rates, include information on the different steps (reactions) on how the input compounds are transformed to the output compounds. This should allow to gain insight in the internal cellular fluxes. The prime example of a data-driven technique is metabolic flux analysis (MFA) where intracellular fluxes are calculated based on measured exchange rates. Exchange rates are not always sufficient to solve a stoichiometric model In such cases, the option exists to measure intracellular fluxes via 13C enrichment analysis [5]
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