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Abstract
Carbohydrates, or glycans, represent a significant class of cell surface modifications in both eukaryotic and prokaryotic cells. In bacteria, strategic regulation of the display of distinct glycan structures endows cellular fitness and competitive advantages in otherwise uninhabitable or inhospitable environments. These structures have long been known to promote antimicrobial resistance, evasion of host defenses, and potent activation of host immune responses. Moreover, bacteria possess a massive repertoire of unique glycan monomers compared to mammalian cells and, consequently, immensely varied carbohydrate structures between and within species. Together, glycan biosynthesis pathways and unique structures represent advantageous targets for new antimicrobials, attenuation of antimicrobial resistance, and significantly underutilized sources of diverse carbohydrate structures. While potential applications of polysaccharides continue to develop, new tools and techniques to isolate and probe glycan structures and biosynthetic pathways have trailed behind other macromolecules, such as proteins and nucleic acids. This research narrows in on foundational, conserved aspects of bacterial glycan biosynthesis to probe initial stages of lipid-dependent glycan biosynthesis. Specifically, fluorescent analogues of critical lipid precursors were used to probe mechanisms of antimicrobial resistance in Escherichia coli and early stages of glycan biosynthesis in Caulobacter crescentus, while addressing potential setbacks to current methods of in vitro reconstitution of glycan biosynthesis. Finally, a recombinant system for overproduction of capsular polysaccharide A from Bacteroides fragilis was engineered in E. coli, effectively modeling a framework for identifying critical bottlenecks that can be broadly applied to other recombinant glycan systems.