The State of Flux: Stability As a Criterion for Model Building and Prediction

Stability in a metabolic system may be unattainable if incorrect amounts of enzymes are used.  Upon loss of stability, some metabolites may accumulate or deplete leading to the irreversible loss of the desired operating point.  Changes in enzyme amount which cause loss of stability in continuous systems can lead to lower production even in batch quasi-steady systems by the inability to maintain productivity. Here we use Ensemble Modelling for Robustness Analysis (EMRA) to analyze stability to enzyme changes in three cell-free enzymatic systems.  In these systems, variations in the level of certain enzymes or substrate feed rate have been predicted to cause instability in steady state simulations. Instability can be caused by a kinetic trap in which two reactions both require the same metabolite.  The predictions of instability by EMRA are supported by the lower productivity in batch experimental tests reported in the literature.  The EMRA method uses no a priori knowledge of the kinetic parameter values of the enzymes, and is generalizable to in vivo systems.