Bioengineering of Escherichia coli Flagellar Type III Secretion system (FT3SS) for improved efficiency of protein export

Summary In order to produce high quality and yield of recombinant proteins one goal of the biotechnology industry is to reduce production costs as a large proportion (up to 80 %) goes into downstream processing. These processing costs could be reduced by direct export of protein product in to the medium. This in turn could be achieved by re-engineering of bacterial flagella type III secretion system (FT3SS) by converting it into a high efficiency protein export nano-machine. The flagella are comprised of motor and filament structures that provides the cells with motility but is also assembled de novo via protein exports of ~1700 subunit min-1 through the export apparatus. In terms of industrial biotechnology (IB), the FT3SS may enable a high efficiency, one-step export route from cytoplasm to the extracellular medium where the secretion rate is significantly higher than the range of IB relevant secretion systems. The main aim of this research project was to fully explore and engineer the FT3SS in order to convert Escherichia coli into a ‘Super-Secretor’ strain, that would export proteins directly in to the medium. This was achieved by using a secretion construct which harboured Cutinase cargo along with an element of native flagellin (untranslated region and late secretion signal) and purifications tags. Following the optimisation of a high throughput assay, the most efficient secretion signal and engineered strain combination was investigated with a systems biology-proteomics approach (iTRAQ) to highlight protein regulation existed during secretion. In parallel, a genomic screening approach was set-up to highlight novel genes to improve protein export. Finally, the genes encoding the highlighted proteins along with potential regulatory small non-coding RNA (snRNA) were further tested in order to investigate whether they would improve protein secretion, however, the engineered ΔclpX ΔarcZ strain was obtained which significantly improved the protein export via the modified FT3SS. These approaches comprehensively allowed secretion yields to be improved significantly, alongside the wealth of knowledge gained in this thesis is completely explored it suggest that the FT3SS platform might outcompete the current commercial protein secretion systems utilised in the IB industry.