Redox controls metabolic robustness in the gas-fermenting acetogen Clostridium autoethanogenum

Living biological systems display a fascinating ability to self-organize their metabolism. This ability ultimately determines the metabolic robustness that is fundamental to controlling cellular behavior. However, fluctuations in metabolism can affect cellular homeostasis through transient oscillations. For example, yeast cul-tures exhibit rhythmic oscillatory behavior in high cell-density con-tinuous cultures. Oscillatory behavior provides a unique opportunity for quantitating the robustness of metabolism, as cells respond to changes by inherently compromising metabolic efficiency. Here, we quantify the limits of metabolic robustness in self-oscillating auto-trophic continuous cultures of the gas-fermenting acetogen Clostrid-ium autoethanogenum . Online gas analysis and high-resolution temporal metabolomics showed oscillations in gas uptake rates and extracellular byproducts synchronized with biomass levels. The data show initial growth on CO, followed by growth on CO and H-2 . Growth on CO and H-2 results in an accelerated growth phase, after which a downcycle is observed in synchrony with a loss in H-2 uptake. In-triguingly, oscillations are not linked to translational control, as no differences were observed in protein expression during oscillations. In-tracellular metabolomics analysis revealed decreasing levels of re-dox ratios in synchrony with the cycles. We then developed a thermodynamic metabolic flux analysis model to investigate whether regulation in acetogens is controlled at the thermody-namic level. We used endo-and exo-metabolomics data to show that the thermodynamic driving force of critical reactions collapsed as H-2 uptake is lost. The oscillations are coordinated with redox. The data indicate that metabolic oscillations in ace-togen gas fermentation are controlled at the thermodynamic level.