Moody, Gawain; (2022) Biochemical characterisation of the Glycyl Radical Enzyme Containing Microcompartment (GRM2) from Proteus mirabilis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Bacterial microcompartments (BMCs) are organelle-like structures which consist of a large, polyhedral protein-based shell, encapsulating an enzymatic core. The most studied and engineered systems to date are the 1,2-propanediol utilising (Pdu) and ethanolamine utilising (Eut) BMCs. The self-assembly properties and modularity of BMCs, and their native capacity to encapsulate cargos, have made them appealing for biotechnologists who want to create novel bio-architectures. For example, they can be used to package or display enzymes for increased efficiency and reduction of intermediate toxicity, for the construction of recombinant metabolic pathways. Bottlenecks impeding these efforts have been inefficient assembly and encapsulation of cargo enzymes. More recently, the family of the glycyl radical-enzyme containing microcompartments (GRM) was identified. A sub-class within this family, the choline-metabolising GRM2, is the focus of this work because of its interesting features. In particular, it possesses fewer shell proteins than current well characterised microcompartments, and may have novel and different assembly mechanisms. As such, studying the GRM2 may reveal new assembly principles, and potential tools for future biotechnology applications. In this work, strains have been produced using an extended hexameric shell protein (Eh), which have been found to readily produce large, evenly spaced sheet structures with various properties (stiffness, length, thickness) when recombinantly expressed in Escherichia coli. When truncated, Eh is still capable of forming higher order structures, though they are more flexible, thinner, and disorganised. Two predicted targeting peptides from the GRM2 were also investigated for their ability to interact with themselves, recombinant shell proteins (from this and other compartments), and with a GRM2-associated core enzyme. Finally, this work built useful constructs that may be engineered in future to create dynamic and modular bio-architectures, such as enzyme scaffolding, or perhaps form the basis of a diagnostic display system, or even provide the foundation for an engineered living material.

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