Microbial glycosyltransferases and biomedically important oligosaccharides.

摘要

The O antigen is one of the most variable cell constituents. More than 170 O serotypes have so far been identified in E. coli. E. coli O128 strains are one of the leading causes of infantile diarrhea in developing countries. E. coli 086 strains possess human blood-group B and alpha-Gal activities. In this study, the complete E. coli O128 and O86 antigens biosynthesis gene clusters were sequenced. All the genes required for the biosynthesis of these O antigens were identified by comparative sequence analysis. The alpha-1,2-fucosyltransferase function of wbsJ gene was unambiguously determined by enzymatic assay and NMR spectroscopy analysis.; Globotetraose and isoglobotetraose are common receptors in the adhesion of uropathogenic bacteria to urinary tract epithelial cells. The efficient synthesis of a variety of globotetraose and isoglobotetraose derivatives was successfully achieved in one-pot, enzymatic reactions catalyzed by coupled glycosyltransferases including a recombinant UDP-GlcNAc C4 epimerase/beta(1 → 3) N-acetylgalactosaminyltransferase fusion protein. An in situ enzymatic UDP-GlcNAc regeneration system was also utilized to further reduce the cost of reactions.; The potential of alpha-Gal epitopes for practical applications in clinical processes such as immunoadsorption has led to an increased demand for an efficient approach adaptable to their large-scale synthesis. A metabolically engineered Pichia pastoris strain was constructed three heterologous enzymes. The whole P. pastoris cells were used to synthesize alpha-Gal trisaccharide with in situ regeneration of UDP-galactose. Up to 28 mM of alpha-Gal was accumulated in a 200-ml reaction.; Finally, recombinant N-acetylglucosamine kinase, N-acetylglucosamine phosphate mutase, UDP-N-acetylglucosamine pyrophosphorylase, pyruvate kinase and inorganic pyrophosphatase were overexpressed in E. coli and co-immobilized on agarose beads for the practical synthesis of UDP-N-acetylglucosamine.