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Cells can control the assembly and disassembly of membraneless organelles by enzymatic processes, but similar control has not been achieved in vitro yet. Here we develop ATP-based coacervate droplets as artificial membraneless organelles that can be fully controlled by two cooperating enzymes. Droplets can be generated within a minute following the addition of phosphoenolpyruvate as a substrate, and they can be dissolved within tens of seconds by adding glucose as the second substrate. We show how the rates of droplet generation and dissolution can be tuned by varying the enzyme and substrate concentrations, and we support our findings with a kinetic model of the underlying enzymatic reaction network. As all steps of the coacervate droplet life cycle, including nucleation, coarsening, and dissolution, occur under the same reaction conditions, the cycle can be repeated multiple times. In addition, by carefully balancing the rates of both enzymatic reactions, our system can be programmed to either form or dissolve droplets at specified times, acting as a chemical timer.

Reversible generation of coacervate droplets in an enzymatic network