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Towards engineering nanobioreactors: characterization of bacterial microcompartment stability, regulation, and protein encapsulation

  • Author(s): Kim, Edward
  • Advisor(s): Tullman-Ercek, Danielle
  • et al.
Abstract

Compartmentalization of biological processes is an optimization strategy found throughout the biological world. On the cellular level, organelles act as compartments that allow for spatial and temporal control of biochemical reactions, as well as provide controlled microenvironments for these reactions. Bacteria generally lack the traditional membrane-enclosed organelles associated with eukaryotes. However, recent studies found that many bacterial species utilize microcompartments (MCPs) to carry out metabolic processes.

Bacterial MCPs have been suggested as scaffolds for nanobioreactors, in which biochemical pathways are targeted to the lumen of MCPs. The advantages of such an encapsulated pathway are many, including enhanced kinetics, sequestration of toxic intermediates, and insulation of internalized pathways from side reactions. MCPs may also prove to be an attractive protein cage for drug delivery. However, several challenges remain to be overcome to successfully repurpose naturally occurring MCPs into biotechnological tools.

Here, we first demonstrate that the propanediol utilization (Pdu) MCP of Salmonella enterica has the structural stability required for use as a nanobioreactor or in drug delivery applications. Transmission electron microscopy shows that the Pdu MCP is remarkably stable over time and is able to retain its structural integrity for several weeks at 4°C. The Pdu MCP is also able to maintain its structural integrity at temperatures up to 60°C, although aggregation of MCPs is observed above 50°C. On the other hand, the Pdu MCP is sensitive to pH and high salt, and denatures outside the range of pH 6 to pH 10, and in 1 M NaCl and 1 M urea.

We then investigate regulation of Pdu MCP formation. We find that PocR is a transcriptional activator of the pdu operon, and heterologous expression of PocR results in MCP formation in the absence of the natural inducer 1,2-propanediol. Next, we report the first instance of heterologous expression of the Pdu shell proteins in Escherichia coli in which MCPs form without morphological aberrations when compared to MCPs from the native host S. enterica.

Finally, we investigate the rules that govern the encapsulation of proteins within the Pdu MCP--a critical component for many of the proposed applications. Due to the time consuming process of MCP purification and subsequent western blot for detecting protein encapsulation using current methods, we first develop a rapid flow cytometry assay for quantifying the relative amount of protein encapsulated within MCPs based on fluorescence. Using this assay, we then characterize various MCP-targeting signal sequence mutants for their ability to encapsulate proteins and identify mutants that encapsulate a greater amount of protein than the wild type signal sequence. We demonstrate that this assay is a powerful tool for reporting protein encapsulation.

The studies presented here show that the Pdu MCP is a promising scaffold for nanobioreactors and drug delivery systems.

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