Glycoengineering Escherichia coli: Identifying and characterizing increased mannose availability within the cell for N-glycoprotein production

The experiments contained within this thesis are aimed at improving N-glycoprotein production potential within Escherichia coli by increasing the availability of the N-glycan precursor mannose within the cell factory using a flow cytometric approach, chemically induced mutagenesis, Next Generation Sequencing techniques, as well as other bioinformatics tools and a glycoproteomics strategy in identifying promising candidates to achieve this aim. The eukaryotic type N-glycosylation pathway requires mannose as a key precursor, and its enhanced availability in the cell has been linked with increased glycosylation efficiency. In this work, the genetic diversity of a glycan surface display E. coli parent strain W3110 was increased via a chemical mutagen and increased mannose generating mutants were identified using flow cytometry. The isolated mutant cells showed a 2.4-fold increase in cell surface mannose display compared to the wild type strain. A Next Generation Sequencing approach was then employed in analysing and identifying the genetic level changes within the mutant strains which led to the significant increase observed compared to the wild type strain. Using bioinformatics tools and techniques, several gene variants within the cell were identified and characterized to highlight potential targeted genetic engineering gene candidates for enhanced mannose production within the E. coli cell factory. Finally, glycoproteomics strategies were employed in investigating the possibility of non-targeted N-glycosylation of native proteins in E. coli containing a glycosylation machinery. Glycosylation prediction tools were used to identify potential endogenous N-glycoproteins, while also investigating possible relationships and interactions within these proteins for predicted characteristics they possess that could significantly influence the N-glycosylation potential in this bacterial system. The work in this thesis has contributed further insights into the genetic pathways and potential for enhancement within E. coli that make it a suitable and efficient N-glycoprotein production cell factory. The findings can be taken forward to identify specific gene combinations which can be targeted to achieve even higher mannose availability within the strain. Findings from other researchers in the field of N-glycosylation engineering within E. coli particularly secretion pathway research findings can be combined with the findings in this thesis to achieve more efficient protein N-glycoprotein production in E. coli. Ascertaining the possibility of native protein N-glycosylation within E. coli will determine its industrial applicability as an efficient N-glycoprotein production factory