(582bq) Toward Microbial Factories: Mechanism and Kinetics of Enzyme Encapsulation in Bacterial Microcompartments

Bacteria such as Salmonella enterica lack organelles to spatially organize their metabolism, but have been found to express proteinaceous microcompartments (MCPs) which can perform this function. These compartments allow the optimization of enzymatic reactions by concentrating enzymes and reaction intermediates, sequestering toxic intermediates, and insulating the compartmentalized pathways from other cellular processes. S. enterica, in particular, employs a MCP for 1,2-propanediol utilization (Pdu). Two different 18-amino acid N-terminal peptides from native Pdu enzymes are sufficient to direct the compartmentalization of heterologous proteins in the Pdu MCP, allowing the encapsulation of heterologous metabolic pathways. The Pdu MCP can serve as a model system to elucidate the mechanism of enzyme encapsulation and the kinetic effects on encapsulated pathways.

The Pdu MCP is thought to sequester a toxic intermediate, propionaldehyde, of the Pdu pathway. The mechanism of this sequestration is unknown. We aim to elucidate the contributions from differential MCP pore permeability, enhanced enzyme kinetics due to increased intermediate concentrations, and increased concentrations of enzymatic cofactors such as coenzyme A. We investigate the Pdu MCP by an in vitro system, whereby Pdu MCPs with specified contents can be purified and the behavior of the compartmentalized enzymes measured. These results will aid the design of custom MCPs for heterologous pathways with enhanced enzyme kinetics and improved toxin sequestration.