Protometabolically Generated NADH Mediates Material Properties of Aqueous Dispersions to Coacervate Microdroplets.

Macromolecular assembly between biomolecules dictates the material state of chemically complex aqueous dispersions such as the cytoplasm. The formation of protein precipitates, fibers, or liquid droplets have been associated with metabolic regulation and disease. However, the effect of metabolic flux on the material properties of aqueous dispersions remains underexplored. Here, we use the protometabolic reduction of NAD⁺ to NADH by pyruvate to study the effect of NADH production on the phase separation properties of polyarginine. We show that reduction of NAD⁺ in the presence of polyarginine can tune the material properties of the dispersion between precipitates, homogeneous solution, and liquid droplets depending on the buffer concentration. In situ droplet formation results in 2-3 times higher reaction rate and NADH yield, compared to homogeneous solution. Our study provides a setting for coupling protometabolism to active protocell environments in the absence of enzymes and sheds light on the self-regulation of metabolic flux on mediating biomolecular phase separation.