(792a) Engineering Synthetic Organelles: Encapsulating Heterologous Proteins Into Bacterial Microcompartments

Biological systems often use compartmentalization as a strategy for optimization.  In bacteria, naturally occurring protein microcompartments (MCPs) of approximately 125nm in diameter are used to compartmentalize metabolic pathways, thereby concentrating enzymes and their substrates, sequestering potential toxic intermediates, and insulating the reactions within from competing enzymes or metabolites.  For the propanediol utilization (Pdu) MCP of Salmonella enterica, it has been shown that an 18 amino acid N-terminal signal peptide is sufficient for localizing heterologous proteins into the Pdu MCP.  We aim to use the Pdu MCP as a template to create customizable nanobioreactors in bacteria.  

            A key step towards encapsulating biochemical pathways is to gain the ability to tune the enzyme stoichiometry within the MCP.  Towards this goal, we have constructed a library of single amino acid substitution mutants in the signal peptide and characterized its strength for encapsulation by using a flow cytometry based assay.  We also use this flow cytometry based assay to investigate whether two different naturally occurring N-terminal signal peptides compete for encapsulation, or have orthogonal mechanisms for encapsulation.  These results help us gain an understanding of the requirements for localizing enzymes to the interior of the Pdu MCP and gain tunability in enzyme stoichiometry within the MCP by manipulating the signal sequence.