Reconstruction of a self-sustaining system of oscillating glycolytic reactions in artificial microreactors

Synthetic cell design is one of the daunting challenges of modern biology. While the genome synthesis problem has already been solved technologically, the understanding of the molecular composition of the intracellular environment required for initiating artificial life still remains elusive. One of the mechanisms of self-organization in prebiotic systems is the oscillatory processes (oscillations) arising from feedback regulation. Such dynamics are inherent to glycolysis as the metabolic core of the cell. The objective of this study was to reconstruct the oscillatory glycolytic chain and investigate its behavior upon confinement to microcompartments and upon interaction with nucleic acids. We used the Saccharomyces cerevisiae extract as a model and demonstrated the presence of fluctuations in the concentrations of glycolytic metabolites: ATP, NADH, hexose-6-phosphates, glyceraldehyde-3-phosphate, 3-phosphoglycerate, and phosphoenolpyruvate (PEP). Moreover, the PEP and pyruvate concentrations had a dramatic effect on the stability of oscillations. We also demonstrated that the reduction of the system volume accompanying the encapsulation into a microchip leads to a more rapid onset of oscillations. Furthermore, synthetic mRNA and DNA were found to reduce the amplitude and period of oscillations in a translation-independent manner. Hence, this study demonstrates the feasibility of reconstructing metabolic oscillations in microvolumes and reveals the effect of nucleic acids on the glycolytic dynamics, which is important for the development of protocellular systems.