The self-assembly of bacterial microcompartments is the result of several genetic, biochemical, and physical stimuli orchestrating inside the bacterial cell. In this work, we use 1,2-propanediol utilization microcompartments as a paradigm to identify the factors that physically drive the self-assembly of MCP proteins in vitro using its major shell protein and major encapsulated enzyme. We find that a major shell protein PduBB′ tends to self-assemble under macromolecular crowded environment and suitable ionic strength. Microscopic visualization and biophysical studies reveal phase separation to be the principle mechanism behind the self-association of shell protein in the presence of salts and macromolecular crowding. The shell protein PduBB′ interacts with the enzyme diol-dehydratase PduCDE and co-assemble into phase separated liquid droplets. The co-assembly of PduCDE and PduBB′ results in the enhancement of catalytic activity of the enzyme. The shell proteins that make up PduBB′ (PduB and PduB′) have contrasting self-assembly behavior. While N-terminal truncated PduB′ has a high self-associating property and forms solid assemblies that separates out of solution, the longer component of the shell protein PduBM38L is more soluble and shows least tendency to undergo phase separation. A combination of spectroscopic, imaging and biochemical techniques shows the relevance of divalent cation Mg2+ in providing stability to intact PduMCP. Together our results suggest a combination of protein–protein interactions and phase separation guiding the self-assembly of Pdu shell protein and enzyme in the solution phase.

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