Engineering Bacterial Microcompartments

Compartmentalization of biological processes is a strategy that nature uses for optimization.  On the cellular level, organelles serve as compartments that allow for metabolic and synthesis processes to occur in controlled chemical environments.  In addition, these compartments couple enzymes and their substrates in close proximity, promoting enhanced kinetics and reducing undesired side reactions.  Furthermore, compartments can sequester potentially toxic intermediates formed in an enzymatic pathway, shielding the components of the cytosol. 

It was recently discovered that some bacteria produce organelle-like compartments, contrary to the long-held belief that bacteria lack any such organization.  These protein-based bacterial microcompartments (MCPs) encapsulate related enzymes involved in a metabolic process.  One such MCP system, found in Salmonella enterica, allows the utilization of 1,2-propanediol as a carbon source. Using this system as a template, our goal is to gain the ability to engineer MCPs to carry out user-specified synthetic reactions, improving yields and expanding the array of products capable of being produced in bacteria. Toward this goal, we have been engineering an operon for the inducible expression of MCP shell proteins in Escherichia coli.  We hope to utilize this MCP system as a biological part, allowing for the modular addition of MCPs to compartmentalize enzymatic pathways.

Another key step towards engineering custom MCPs is to control the localization of enzymes into the lumen of the MCP protein shell.  In the natural MCP system in S. enterica, we visualized MCP-localized enzymes by fusing them with green fluorescent protein (GFP).  When viewed by microscopy, cells expressing these fusion proteins display punctate fluorescence while those expressing GFP alone show fluorescence evenly dispersed throughout the cell.  Furthermore, it was found that a short N-terminal peptide sequence was necessary and sufficient to target heterologous proteins into MCPs.  These results are promising first steps towards creating synthetic MCPs that serve as nanobioreactors to carry out functions of biotechnological interest.