Abstract
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.
Highlights
Bacterial microcompartments (BMCs) are selectively permeable proteinaceous organelles which encapsulate segments of metabolic pathways across bacterial phyla
We had observed that facets assembled into three-dimensional particles independent of the presence of pentamers[20]; taken together, these findings suggested a strategy for producing shells lacking vertices that could be subsequently capped and purified
Encapsulation via covalent-linkage (EnCo) allows facile programming of cargo composition expressed in cis and in trans
Summary
Bacterial microcompartments (BMCs) are selectively permeable proteinaceous organelles which encapsulate segments of metabolic pathways across bacterial phyla. 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). Correspondence and requests for materials should be addressed to Bacterial microcompartments (BMCs) are ensembles of enzymes enclosed in a semi-permeable proteinaceous shell, compositionally distinguishing them from the more familiar lipid-membrane bound organelles of eukaryotes These compartments have been bioinformatically identified in the majority of bacterial phyla[1] where they perform distinct, spatially separate metabolic functions allowing their hosts to fix carbon, as in the anabolic carboxysomes, or metabolize substrates like ethanolamine that generate volatile or toxic intermediates, as in the catabolic metabolosomes. There is no disassembly/reassembly methods developed which would allow in vitro encapsulation of cargo
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