Rapid media transition: An experimental approach for steady state analysis of metabolic pathways

Abstract

Commonly steady state analysis of microbial metabolism is performed under well defined physiological conditions in continuous cultures with fixed external rates. However, most industrial bioprocesses are operated in fed-batch mode under non-stationary conditions, which cannot be realized in chemostat cultures. A novel experimental setup-rapid media transition-enables steady state perturbation of metabolism on a time scale of several minutes in parallel to operating bioprocesses. For this purpose, cells are separated from the production process and transferred into a lab-scale stirred-tank reactor with modified environmental conditions. This new approach was evaluated experimentally in four rapid media transition experiments with Escherichia coli from a fed-batch process. We tested the reaction to different carbon sources entering at various points of central metabolism. In all cases, the applied substrates (glucose, succinate, acetate, and pyruvate) were immediately utilized by the cells. Extracellular rates and metabolome data indicate a metabolic steady state during the short-term cultivation. Stoichiometric analysis revealed distribution of intracellular fluxes, which differs drastically subject to the applied carbon source. For some reactions, the variation of flux could be correlated to changes of metabolite concentrations.

Improvement of constraint‐based flux estimation during L‐phenylalanine production with Escherichia coli using targeted knock‐out mutants

“…This was demonstrated by comparing Flux Variability Analysis of the knock‐out mutants and the reference strain. The deletion of the malic enzymes thus ensured certainty in flux estimation and therefore enables further characterization of the L‐phenylalanine production by combination of theoretical (e.g., metabolic control analysis, Hatzimanikatis and Bailey, ) and experimental approaches (e.g., Rapid media transition, Link et al, ).…”

“…Another option is making use of constraint based approaches during controlled substrate limited fed‐batch processes where extracellular fluxome [e.g., biomass formation (Link et al, )] is kept constant. Link et al () demonstrated that a constant growth rate in fed‐batch mode provided metabolic steady state. Therefore, constraint based approaches are applicable for fed‐batch processes and chemostat cultures are not necessarily essential for steady state assumptions.…”

Carbon storage in recombinant Escherichia coli during growth on glycerol and lactic acid

“…Perturbation methods are divided into dynamic approaches (Chassagnole et al, ; Heijnen and Verheijen, ; Magnus et al, ; Taymaz‐Nikerel et al, ) and steady‐state approaches (Link et al, ; Nasution et al, ). Perturbation experiments often apply techniques for metabolic analyses of cells sampled from a continuing process, to avoid any process disturbance and to repeat the perturbation experiments at several process times (Link et al, ; Taymaz‐Nikerel et al, ). Perturbation experiments may be performed with culture broth samples, which are pumped through a specially designed plug‐flow reactor (De Mey et al, ).…”

“…Alternatively, samples are rapidly centrifuged and the biomass is transferred into small‐scale stirred tank reactors. These contain fresh media with varying carbon sources to allow the measurement of metabolome and fluxome data during short‐term (<20 min) batch experiments (Link et al, ). The proteome of the cells is assumed to remain unchanged in both cases.…”

“…The application of a rapid media transition for steady‐state perturbation experiments (Link et al, ) relies on the absence of any other carbon source that may have been stored inside the cells (e.g., glycogen or trehalose in case of E. coli ) during the production process and that may be consumed in the short‐term batch experiments in addition to the individual carbon source (here: glycerol) applied in the perturbation experiments. This paper deals with the characterization and avoidance of carbon storage in recombinant E. coli cells.…”