| Human breast milk is the gold standard for infant nutrition. In human milk, the oligosaccharides may protect babies by acting as decoy receptors for pathogens. Also, human milk oligosaccharides (HMOs) enhance the proliferation of probiotics to strengthen the host immune system. Among HMOs, 2'-fucosyllactose (2-FL), composed of L-fucose and D-lactose, is known to possess an anti-infection capability against many harmful organisms such as Campylobacter jejuni, enteropathogenic Escherichia coli, and even Norovirus. Consequently, great quantities of 2-FL are being demanded for food applications and thorough investigation of its biological properties. The current synthetic methods of 2-FL including chemical production and enzymatic catalysis are complicated, expensive, and thus impractical for a large-scale synthesis. In my research study, an alternative route to produce 2-FL using a microbial cell factory was devised. First, E. coli, as a host strain to produce 2-FL, was engineered to overexpress genes in the metabolic pathway of GDP-L-fucose, a donor of L-fucose, and harbor the fucosyltransferase enzyme (FucT2) which E. colicatalyzes the transfer of L-fucose onto a lactose molecule enabling 2-FL production. Second, the production conditions of 2-FL in the engineered were optimized in order to pinpoint the appropriate conditions promoting the efficient and consistent synthesis of 2-FL. Third, the key enzyme in 2-FL production, FucT2, from different sources was compared in search for the FucT2 leading to an enhanced synthesis of 2-FL. Fourth, the possibility to produce 2-FL was demonstrated in a different host, Saccharomyces cerevisiae, which is a generally recognized as safe (GRAS) organism. Through metabolic engineering, the minimum three components required to construct a 2-FL-producing S. cerevisiae consisting of GDP-L-fucose production, lactose internalization, and functional expression of FucT2 were confirmed. Additionally, the gene target perturbation, deletion of GDA1, which resulted in an improved production of GDP-L-fucose was identified. GDP-L-fucose is a beneficial sugar nucleotide which serves as a precursor to innumerable compounds aside from 2-FL. From this study, the feasibility and scalability of employing the microbial cell factory approach for 2-FL and GDP-L-fucose production were manifested. |
