Exploring the potential of bacterial microcompartments for the spatial organization of synthetic metabolic pathways

Bacteria were long thought of as bags of enzymes with little internal structural organization. This idea has been challenged in recent years, particularly with the discovery of bacterial microcompartments (MCPs), which are polyhedral protein structures within the cytoplasm that house specific cellular reactions for functions such as carbon fixation. These MCPs may be a useful tool in the development of synthetic biology applications for which spatial control over the intracellular flow of proteins and small molecules is required. Toward this goal, we have been developing an understanding of bacterial MCPs and exploring the potential to engineer various features of the structures.

Bacterial MCPs consist of protein shells that sequester native enzymatic reactions and are thought to concentrate private cofactor pools, sequester toxic intermediates, and insulate the compartmentalized pathways from other cellular processes. Two well-characterized systems from Salmonella enterica use MCPs for 1,2-propanediol utilization (Pdu) and ethanolamine utilization (Eut). In both cases it is thought that the protein shell segregates the toxic aldehyde intermediate of the pathways from the cytosol, and that pores in the shell selectively mediate the passage of substrates and products into the shell lumen. In both the Pdu and the Eut MCP systems, short N-terminal extensions from native Pdu and Eut enzymes are sufficient to compartmentalize heterologous proteins in the protein shell. In order for these MCPs to be used for bioprocess applications, we must control the copy number, enzymatic loading and stoichiometry, size, and pore selectivity of these MCPs. To this end, we have used fluorescent reporters coupled with phenotypic and biochemical assays to study the regulation, copy number, and heterologous protein loading of the Pdu and Eut MCPs. We are simultaneously investigating the potential for controlling selectivity via shell protein pores, and have developed methods to heterologously express MCPs with tunable composition in Escherichia coli. Together, these experiments provide a framework and toolkit for the encapsulation of non-native pathways. Further efforts will enable the engineering of MCPs into tailored intracellular environments for applications such as the in vivo sequestration of synthetic metabolic pathways.