Lawrence Berkeley National Laboratory

Bacterial microcompartments (BMCs) are selectively permeable proteinaceous organelles which encapsulate segments of metabolic pathways across bacterial phyla. They consist of an enzymatic core surrounded by a protein shell composed of multiple distinct proteins. Despite great potential in varied biotechnological applications, engineering efforts have been stymied by difficulties in their isolation and characterization and a dearth of robust methods for programming cores and shell permeability. We address these challenges by functionalizing shell proteins with affinity handles, enabling facile complementation-based affinity purification (CAP) and specific cargo docking sites for efficient encapsulation via covalent-linkage (EnCo). These shell functionalizations extend our knowledge of BMC architectural principles and enable the development of minimal shell systems of precisely defined structure and composition. The generalizability of CAP and EnCo will enable their application to functionally diverse microcompartment systems to facilitate both characterization of natural functions and the development of bespoke shells for selectively compartmentalizing proteins.