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Production of human milk oligosaccharides in the cell factory of E. coli
Havrdová, Jana ; Bojarová, Pavla (advisor) ; Smrček, Stanislav (referee)
Human milk oligosaccharides (HMOs) are among the most abundant components of human breast milk and are essential for the development of neonatal health. It is very challenging to isolate these oligosaccharides from animal milk, especially the less abundant ones. Therefore, different approaches are to be sought after. Chemical and enzymatic syntheses of these compounds are labor-intensive, and expensive affording low yields. A newly adopted approach to HMO synthesis is through bac- terial cell factories, in which genetically engineered bacterial strains can use cheap carbohydrate substrates and convert them into specific oligosaccharides. The aim of this thesis is to examine the feasibility of using selected bacterial strain (E. coli) for the production of HMOs. With lactose as a glycan substrate, the bacterial host has to be β-galactosidase deficient, otherwise, the substrate would be degraded. In order to generate higher lactose intake in the cell, a crp gene that encodes for the positive transcriptional regulator (Catabolite Activator Protein - CAP) can be incorporated into the host organism. The cloned plasmid - pRSFDuet- 1-crp was used for the transformation of into the selected bacterial strain. Lactose from the cells was purified by gel chromatography and the influence of CAP over expression...
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