Utilisation of bacterial microcompartment technology to enhance spatial organisation in Escherichia coli

Enhancing the catalytic efficiency of enzymes has been an aspiration for many years and protein engineering has been quite successful in this area. However, researchers are now turning their attention to the organisation of enzymes within cells to enhance whole pathway productivity. As such, bacterial microcompartments (BMCs) have attracted significant interest due to their ability to not only concentrate enzymes and metabolites but also prevent the release of toxic intermediates into the cell owing to their semi-permeable protein shell. The aim of the research described in this thesis was to develop a number of mechanisms to enhance the spatial organisation of proteins within E. coli and thereby increase metabolic productivity. The ability of BMC encapsulation peptides to target foreign proteins to a BMC was initially investigated. However, we found that the fusion of BMC encapsulation peptides to the enzymes of a 1,2-propanediol (1,2-PD) synthesis pathway resulted in the aggregation of the tagged enzymes rather than their localisation to a BMC. Nonetheless, the tagged enzymes were found to produce significantly more 1,2-PD than strains producing untagged enzymes, irrespective of the presence of BMCs, suggesting that it is the enzyme aggregate that results in enhanced substrate channelling. Furthermore, we showed that a highly conserved hydrophobic motif is conserved across a wide range of BMC targeting peptides and suggest that it is the amphipathic nature of these peptides that results in aggregation of tagged components. The ability of PduA, a component of the BMC shell, to form filamentous structures was investigated for its potential as a cytoplasmic scaffold. Targeting proteins to the cytoscaffold was explored through the use of de novo designed coiled-coil peptides. Localising the enzymes pyruvate decarboxylase and alcohol dehydrogenase on this scaffold by tagging them with the cognate coiled-coil peptide resulted in a significant enhancement in ethanol production in comparison to a strain lacking the scaffold. Additionally, we showed that it was possible to locate this intracellular scaffold to the inner membrane of the cell, further demonstrating the flexibility of this system. Finally, we utilised the same coiled-coil peptide technology to target fluorescent proteins to BMCs, overcoming the aggregation behaviour of the native targeting peptides. Through the re-design of a BMC shell protein we showed targeting occurs to both the external and luminal faces of these BMCs. Taken together, the evidence presented in this thesis provides a number of alternative strategies to not only enhance spatial organisation within a cell but also to increase productivity of engineered metabolic pathways