A genomic integration platform for heterologous cargo encapsulation in 1,2-propanediol utilization bacterial microcompartments

Bacterial microcompartments (MCPs) are protein structures that encapsulate specific metabolic pathways in bacteria. The 1,2-propanediol utilization (Pdu) MCP in Salmonella enterica serovar Typhimurium LT2 encapsulates the pathway for 1,2-propanediol degradation to sequester a toxic intermediate, enable cofactor recycling, and enhance pathway flux. The Pdu MCP has potential as an enclosed scaffold for metabolic engineering applications. To successfully use Pdu MCPs for this purpose, however, methods to enable and control heterologous cargo encapsulation are critical. To this end, we here developed a genomic expression platform for cargo encapsulation in Pdu MCPs. We integrated signal sequence-tagged fluorescent reporters into the pdu operon in place of native Pdu enzymes and evaluated the resulting expression and encapsulation levels. We found that fluorescent reporters were successfully co-encapsulated, with varying relative encapsulation levels achieved when using different integration locus and signal sequence combinations. We also observed that the native Pdu signal sequences mediated different encapsulation efficiencies independent of expression levels. This work establishes the genomic integration platform as a viable method for controlling cargo encapsulation, expanding the toolkit toward engineering the Pdu MCP as a tunable nanobioreactor.