Cells can form membraneless organelles by liquid–liquid phase separation. As these organelles are highly dynamic, it is crucial to understand the kinetics of these phase transitions. Here, we use droplet‐based microfluidics to mix reagents by chaotic advection and observe nucleation, growth, and coarsening in volumes comparable to cells (pL) and on timescales of seconds. We apply this platform to analyze the dynamics of synthetic organelles formed by the DEAD‐box ATPase Dhh1 and RNA, which are associated with the formation of processing bodies in yeast. We show that the timescale of phase separation decreases linearly as the volume of the compartment increases. Moreover, the synthetic organelles coarsen into one single droplet via gravity‐induced coalescence, which can be arrested by introducing a hydrogel matrix that mimics the cytoskeleton. This approach is an attractive platform to investigate the dynamics of compartmentalization in artificial cells.

Cells can form membraneless organelles by liquid–liquid phase separation. As these organelles are highly dynamic, it is crucial to understand the kinetics of these phase transitions. Here, we use droplet‐based microfluidics to mix reagents by chaotic advection and observe nucleation, growth, and coarsening in volumes comparable to cells (pL) and on timescales of seconds. We apply this platform to analyze the dynamics of synthetic organelles formed by the DEAD‐box ATPase Dhh1 and RNA, which are associated with the formation of processing bodies in yeast. We show that the timescale of phase separation decreases linearly as the volume of the compartment increases. Moreover, the synthetic organelles coarsen into one single droplet via gravity‐induced coalescence, which can be arrested by introducing a hydrogel matrix that mimics the cytoskeleton. This approach is an attractive platform to investigate the dynamics of compartmentalization in artificial cells.

Lignocellulosic biomass remains unharnessed for the production of renewable fuels and chemicals due to challenges in deconstruction and the toxicity its hydrolysates pose to fermentation microorganisms. Here, we show in Saccharomyces cerevisiae that engineered aldehyde reduction and elevated extracellular potassium and pH are sufficient to enable near-parity production between inhibitor-laden and inhibitor-free feedstocks. By specifically targeting the universal hydrolysate inhibitors, a single strain is enhanced to tolerate a broad diversity of highly toxified genuine feedstocks and consistently achieve industrial-scale titers (cellulosic ethanol of >100 grams per liter when toxified). Furthermore, a functionally orthogonal, lightweight design enables seamless transferability to existing metabolically engineered chassis strains: We endow full, multifeedstock tolerance on a xylose-consuming strain and one producing the biodegradable plastics precursor lactic acid. The demonstration of “drop-in” hydrolysate competence enables the potential of cost-effective, at-scale biomass utilization for cellulosic fuel and nonfuel products alike.