Reversible generation of coacervate droplets in an enzymatic network

Abstract

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.

Directed Growth of Biomimetic Microcompartments

“…[40,[48][49][50] Moreover, enzymes, which partition into the microdroplets, maintain activity within the highly charged and crowded interior. [38,53] In addition, growth and decay of coacervate droplets can be instigated by switching the pH of the solution by bubbling carbon dioxide and ammonia through the dispersion of coacervate droplets. [38,53] In addition, growth and decay of coacervate droplets can be instigated by switching the pH of the solution by bubbling carbon dioxide and ammonia through the dispersion of coacervate droplets.…”

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

“…[3,4] Microdroplets produced in vitro by associative (coacervates) or segregative (aqueous two-phase systems) liquid-liquid phase separation have recently been used as synthetic models of dynamic protocells. [15,[29][30][31][32] However,p latforms enabling the rapid and localized actuation of polynucleotide microphase separation have not yet been developed. [14] Polynucleotides have been used as scaffold components to assemble coacervate droplets, [15][16][17][18][19] and selective sequestration of guest polynucleotides has been demonstrated in preformed coacervates.…”

“…[12,20,21] Owing to their liquid-like nature and the weak molecular interactions,s ynthetic coacervate droplets can dynamically respond to external stimuli. [15,[29][30][31][32] However,p latforms enabling the rapid and localized actuation of polynucleotide microphase separation have not yet been developed. [15,[29][30][31][32] However,p latforms enabling the rapid and localized actuation of polynucleotide microphase separation have not yet been developed.…”

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

“…[106] Inspired by these biological regulation processes, enzymemediated chemical modifications of phase-separating species are now emerging as promising alternatives for dynamic control and programmingo fLLPSi ns ynthetic systems. [118] This process was shown to be reversible throughm odulation of the enzyme activities; however,t he two opposite enzyme reactions were not mutually compatible, and addition or removal of specific enzymec ofactors was required in order to achievereversibility.I na nother example, Spruijt and co-workers demonstrated the formation and dissolution of polylysine/ATP coacervate microdroplets under the same solution conditions by using another kinase/ phosphatase pairt oc onvert ATPi nto ADP reversibly [119] (Figure 3C). [118] This process was shown to be reversible throughm odulation of the enzyme activities; however,t he two opposite enzyme reactions were not mutually compatible, and addition or removal of specific enzymec ofactors was required in order to achievereversibility.I na nother example, Spruijt and co-workers demonstrated the formation and dissolution of polylysine/ATP coacervate microdroplets under the same solution conditions by using another kinase/ phosphatase pairt oc onvert ATPi nto ADP reversibly [119] (Figure 3C).…”

“…C) Optical images showing condensationand dissolution of ATP/PLysc oacervate droplets in response to enzyme activities.Adapted from ref [119]. B) Pictures showing pH-driven reversible assembly of oligolysine/ATP coacervate droplets.Adapted, with permission, from ref [21]…”