Engineering the 1,2-Propanediol Utilization Bacterial Microcompartment to Develop a Tunable Nanobioreactor

Nichols, Taylor Marie

doi:https://doi.org/10.21985/n2-xpvd-3298

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Engineering the 1,2-Propanediol Utilization Bacterial Microcompartment to Develop a Tunable Nanobioreactor

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While metabolic engineering can enable the sustainable bioproduction of new materials, efforts are often impeded by pathway bottlenecks. To mitigate the effects of toxic or reactive intermediates and resource competition resulting from heterologous pathway incorporation, bacterial microcompartments (MCPs) have recently been considered for engineered compartmentalization in bacterial host organisms. MCPs are protein shells that form natively in certain bacteria to selectively encapsulate specific metabolic processes, functioning to sequester and colocalize pathway components to enhance flux. The use of MCPs as enclosed scaffolds for metabolic engineering applications requires the control of heterologous cargo encapsulation, with pathway selection designed to efficiently utilize the native benefits of MCP systems. We specifically aim to use the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica LT2 as a model scaffold for the encapsulation of heterologous cargo and pathway components. This dissertation describes our efforts to engineer the Pdu MCP for this purpose. Chapter 2 details a genomic integration platform developed to enable and control the encapsulation of heterologous cargo within Pdu MCPs. Chapters 3 and 4 discuss tag engineering, first exploring degradation tag variants for the controlled degradation of unencapsulated cargo, and then evaluating signal sequence variants to interrogate residues contributing to encapsulation efficiency. Finally, Chapter 5 details efforts to engineer the Pdu MCP to encapsulate the pathway for the bioproduction of 1,3-propanediol from glycerol. This work establishes new tools and insights to enable and control heterologous cargo encapsulation within Pdu MCPs, furthering their development as tunable nanobioreactors for the engineered compartmentalization of non-native pathways.

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