(759d) Organelles By Design: Custom Nanobioreactors in Bacteria

Despite the vast array of enzymatic activities available to metabolic engineers, many biosyntheses are impractical due to the production of toxic intermediates and off-target products, poor kinetic performance, and cofactor imbalances. We aim to address these roadblocks by organizing biosynthetic pathways inside custom organelles within cells. Bacterial microcompartments, porous proteinaceous organelles found in Salmonella and other bacteria, provide an ideal starting point for the engineering of subcellular bacterial organelles. To this end, we develop tools for the control of microcompartment formation, enzyme encapsulation, and small molecule transport across the compartment shell, as well as computational tools to study native compartment function and identify promising target pathways for encapsulation.

In order to tune the catalytic activity of the microcompartments, we orchestrate microcompartment formation and cargo expression to control when the organelles are formed and how much heterologous cargo is encapsulated. We also create new localization signals that allow fine control over the loading of multiple enzymes to the compartments simultaneously. We are investigating the role of pore residues in small molecule transport across the shells of virus-like particles and the microcompartments themselves, as well as using protein engineering approaches to examine virus-like particle assembly as a model system. Lastly, we use computational tools to model microcompartment function in order to facilitate the selection of appropriate pathways for encapsulation. Together, these tools allow the creation of custom biosynthetic organelles with user-defined enzyme content and transport properties, enhancing flux and decreasing the toxicity of heterologous biosynthetic pathways.