Monitoring and Modeling of the Reaction Dynamics in the Valine/Leucine Synthesis Pathway in Corynebacterium glutamicum

Abstract

The intracellular concentrations of the valine and leucine pathway intermediates in a Corynebacterium glutamicum strain were measured during a transient state. The data were obtained by performing a glucose stimulus-response experiment with the use of a rapid sampling device and advanced mass spectrometry. The glucose stimulus resulted in a 3-fold increase in the intracellular pyruvate concentration within less than a second, demonstrating the very fast interactions in metabolic networks. The samples were taken at subsecond intervals for a time period of 25 s. The time courses of the metabolite concentrations formed the experimental basis of a mathematical model simulating the fluxes and concentrations in the valine/leucine pathway. The implementation of a model selection criterion based on the second law of thermodynamics is demonstrated to be essential for the identification of realistic and unique models. Large differences between the enzyme properties determined in vitro and those determined in vivo by the model were observed with the in vivo maximal rates being almost an order of magnitude larger than the in vitro maximal rates. The transamination of ketoisovalerate (KIV) to valine is carried out mainly by the transaminase B enzyme, with the transaminase C enzyme playing a minor role. The availability of the cofactors NADP and NADPH only has modest influence on the flux through the valine pathway, while the influence of NAD and NADH on the flux through the leucine pathway is negligible.

Impact of Thermodynamic Principles in Systems Biology

“…Using an extensive steady state dataset for lower glycolysis [38] showed that elasticity values are easily obtained using linear regression. Also lin-log kinetics was used to set-up a kinetic model for leucine/valine synthesis [40], glycolysis in Lactococcus lactis [30] and a batch fermentation [41]. In the last two studies it was shown that lin-log kinetics should not be applied to datasets where metabolite concentrations become zero (which is obvious).…”

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

“…We are still far from that. For C. glutamicum detailed dynamic models, based on mechanistic equations for the participating enzymes and concentration measurements of the pathway intermediates involved, have been developed at least for the biosynthesis of lysine [78] and valine [79]. Through metabolic control analysis, such models allow the prediction of bottlenecks and of optimal factors, e.g., enzyme concentrations to achieve increased flux.…”

“…Admittedly, for most pathways of C. glutamicum such kinetic information is still not available. This can be partly overcome by the application of power law or lin-log kinetics in the underlying equations [79,80]. Dynamic models are also needed for dynamic labeling experiments for flux analysis as has been shown for E. coli [81].…”

“…For many years, dynamic macroscopic models have been used to describe dynamic phenomena of growth and production and derive control or feeding strategies for optimized bioprocesses. A recent example deals with fed-batch production of valine [79]. Such overall phenomenological models are surely useful but do not provide any detailed description of network activities [86].…”

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

“…Stimulus response experiments are widely applied to study the dynamic behavior of metabolic pathways. [1][2][3][4] Commonly, a pulse of carbon source is added to a continuous culture and intracellular metabolite concentrations are observed during several minutes. It may be assumed that enzyme levels do not change over this short time period and the responses can be attributed to the kinetic interactions at the metabolome level alone.…”