DEVELOPING ESCHERICHIA COLI AS A PLATFORM TO STUDY PROTEIN GLYCOSYLATION AND SECRETION

207 pagesEscherichia coli is an exceptional model organism, tolerant to additions and overhauls of metabolic pathways, and recombinant expression of diverse proteins making it a tractable chassis for both the study of complex biological systems and production of therapeutics. However, lab strains of E. coli are unable to perform critical posttranslational modifications, like glycosylation, that are important for the study and production of protein therapeutics. In this work, we 1) endow E. coli with the catalytic potential to produce a diverse panel of human glyco-epitopes, including immunologically relevant MUC1 VNTR motifs, bearing mucin-type O-glycans through a novel recombinant pathway constituted by an N-acetyl galactosamine transferase, PglC from A. baumannii, a galactosyl transferase, WbwC from E. coli O104, and O-oligosaccharyl transferases from N. gonorrhoeae and N. meningitidis., 2) developed a facile platform for the production of N-glycoproteins through the chromosomal engineering of E. coli at the Enterobacterial common antigen (ECA) and O-polysaccharide synthesis (O-PS) loci, creating a platform for the expression of three unique glycans – modified C. jejuni heptasaccharide, mammalian tri-mannosyl core, and E. coli O56 O-antigen, and four different glycoproteins, scFv13-R4, hGH, RNAse A and glucagon, bearing the modified C. jejuni glycan. Therefore, this work facilitates the study of glycosylation pathways and glycoconjugate products using E. coli. Similarly, we created yet another platform for the high-throughput study of bacterial protein secretion, a system of relevance to pathogenesis and applications in bioprocessing. Through a novel survival-selection scheme, we screened 3,895 single gene knockouts mutants of E. coli to identify deletions of fliH, gfcC, yaiW and ydfI as leading to hypersecretory phenotype of YebF and diverse fusion proteins. While research to augment our understanding of biological processes towards improving quality of life is important, the inherent inequality of our society limits access to the fruits of such discoveries. We believe equity in higher education can empower communities through socio-economic mobility, and therefore explore the use of podcasts, an emerging medium, to amplify the voice of role models to support students from traditionally under-represented identities persist in STEM. This work lays the foundation for follow up studies, harnessing podcasts to foster inclusion and equity