Interfacing bacterial microcompartment shell proteins with genetically encoded condensates

Abstract Condensates formed by liquid–liquid phase separation are promising candidates for the development of synthetic cells and organelles. Here, we show that bacterial microcompartment shell proteins from Haliangium ochraceum (BMC‐H) assemble into coatings on the surfaces of protein condensates formed by tandem RGG‐RGG domains, an engineered construct derived from the intrinsically disordered region of the RNA helicase LAF‐1. WT BMC‐H proteins formed higher‐order assemblies within RGG‐RGG droplets; however, engineered BMC‐H variants fused to RGG truncations formed coatings on droplet surfaces. These intrinsically disordered tags controlled the interaction with the condensed phase based on their length and sequence, and one of the designs, BMC‐H‐T2, assembled preferentially on the surface of the droplet and prevented droplet coalescence. The formation of the coatings is dependent on the pH and protein concentration; once formed, the coatings are stable and do not exchange with the dilute phase. Coated droplets could sequester and concentrate folded proteins, including TEV protease, with selectivity similar to uncoated droplets. Addition of TEV protease to coated droplets resulted in the digestion of RGG‐RGG to RGG and a decrease in droplet diameter, but not in the dissolution of the coatings. BMC shell protein‐coated protein condensates are entirely encodable and provide a way to control the properties of liquid–liquid phase‐separated compartments in the context of synthetic biology.