Biotechnological advances in bacterial microcompartment technology

Bacterial microcompartments (BMCs) represent proteinaceous macromolecular nano-bioreactors that are found in a broad range of bacteria, and which are associated with either anabolic or catabolic processes. They consist of a semipermeable outer shell that packages a central metabolic enzyme or pathway, providing both enhanced flux and protection against toxic intermediates. Recombinant production of BMCs has led to their repurposing with the incorporation of altogether new pathways. Deconstruction of BMCs into their component parts has shown that some individual shell proteins self-associate into filaments that can be further modified into a cytoplasmic scaffold to which enzymes/proteins can be targeted. BMCs therefore represent a modular system that is highly suited for the engineering of biological systems for useful purposes

Effect of metabolosome encapsulation peptides on enzyme activity, co-aggregation, incorporation and bacterial microcompartment formation

Metabolosomes, catabolic bacterial microcompartments, are proteinaceous organelles that are associated with the breakdown of metabolites such as propanediol and ethanolamine. They are composed of an outer multi-component protein shell that encases a specific metabolic pathway. Protein cargo found within BMCs is directed by the presence of an encapsulation peptide that appears to trigger aggregation prior to the formation of the outer shell. We investigated the effect of three distinct encapsulation peptides on foreign cargo in a recombinant BMC system. Our data demonstrate that these peptides cause variation with respect to enzyme activity and protein aggregation. We observed that the level of protein aggregation generally correlates with the size of metabolosomes, while in the absence of cargo BMCs self-assemble into smaller compartments. The results agree with a flexible model for BMC formation based around the ability of the BMC shell to associate with an aggregate formed due to the interaction of encapsulation peptides

A study into the properties and applications of bacterial microcompartments and polyphosphate metabolism

Bacterial microcompartments were initially observed in the 1950s and since that time, research in the field has focused on the unique properties of their proteinaceous structures and their role in the bacterial cell. This thesis investigates the properties of the propanediol utilization (Pdu) and ethanolamine (Eut) utilization microcompartments and applications of their components in the cells of Lactobacillus reuteri, and Escherichia coli predominantly, with additional investigation into Yersinia spp. The experimental chapters of this thesis initially investigate the application of bacterial microcompartments as tools for compartmentalizing metabolic reactions, in particular regarding the uptake of phosphate from extracellular environment and the retention of polyphosphate intracellularly. This was observed experimentally through the engineering of recombinant constructs containing genes encoding microcompartment-directed polyphosphate kinase enzyme. Bacterial phenotypes conferred by these constructs with respect to enhanced net uptake of phosphate from the environment were defined, and potential applications as a clinical therapy addressed in an animal trial, which utilized the phosphate-uptake constructs which had been created. In the trial, recombinant bacteria expressing these constructs were successfully delivered in therapeutic quantities to the target (small intestine) of rats experiencing induced renal failure, with no adverse effects were observed. However inter-group animal variation regarding baseline blood mineral content was larger than the expected therapeutic effect and therefore no therapeutic effect could be demonstrated. Additionally, novel phenotypic differences conferred by induction of Pdu and Eut microcompartments and empty recombinant microcompartments on host cells were identified. These identified an observable phenotypic difference in buoyant density between strains expressing the Pdu and Eut microcompartments. Further investigation into the potential function and phenotypic differences of strains expressing microcompartments were supported by methodological developments including the use of imaging instruments such as the FIB-SEM, and microcompartment extraction from cells. These technical developments enabled additional observations regarding the characteristics of microcompartments and provided novel information