Bacterial microcompartments (MCPs) are protein-based organelles that consist of metabolic enzymes encapsulated within a protein shell. The function of MCPs is to optimize metabolic pathways by increasing reaction rates and sequestering toxic pathway intermediates. A substantial amount of effort has been directed toward engineering synthetic MCPs as intracellular nanoreactors for the improved production of renewable chemicals. A key challenge in this area is engineering protein shells that allow the entry of desired substrates. In this study, we used site-directed mutagenesis of the PduT shell protein to remove its central iron-sulfur cluster and create openings (pores) in the shell of the Pdu MCP that have vari... More
Bacterial microcompartments (MCPs) are protein-based organelles that consist of metabolic enzymes encapsulated within a protein shell. The function of MCPs is to optimize metabolic pathways by increasing reaction rates and sequestering toxic pathway intermediates. A substantial amount of effort has been directed toward engineering synthetic MCPs as intracellular nanoreactors for the improved production of renewable chemicals. A key challenge in this area is engineering protein shells that allow the entry of desired substrates. In this study, we used site-directed mutagenesis of the PduT shell protein to remove its central iron-sulfur cluster and create openings (pores) in the shell of the Pdu MCP that have varied chemical properties. Subsequently, in vivo and in vitro studies were used to show that PduT-C38S and PduT-C38A variants increased the diffusion of 1,2-propanediol, propionaldehyde, NAD+ and NADH across the shell of the MCP. In contrast, PduT-C38I and PduT-C38W eliminated the iron-sulfur cluster without altering the permeability of the Pdu MCP, suggesting that the side-chains of C38I and C38W occluded the opening formed by removal of the iron-sulfur cluster. Thus, genetic modification offers an approach to engineering the movement of larger molecules (such as NAD/H) across MCP shells, as well as a method for blocking transport through trimeric bacterial microcompartment (BMC) domain shell proteins.
