Characterization and synthetic application of a novel β1,3-galactosyltransferase from Escherichia coli O55:H7

Galactose-limited fed-batch cultivation of Escherichia coli for the production of lacto-N-tetraose

“…The K m -value of WbgO for UDP-galactose has been determined to be 3.4 mmol L −1 [40]. Under the assumption that the total cellular volume of the E. coli cells in a culture is 3.6 L OD 600 −1 mL −1 [41], the K m -value is significantly higher than the intracellular UDP-galactose concentration measured in this study.…”

“…Therefore the WbgO-catalyzed reaction might not only compete with the activity of GalE for the available UDP-galactose, but also with the secretion, causing LNT II to appear in the culture supernatant. Considering the relatively low K m -value of WbgO for LNT II, determined to be 0.055 mmol L −1 [40], we assume that the intracellular concentration of LNT II is not ratelimiting in the WbgO-catalyzed reaction. Therefore, we suggest that a higher WbgO activity, attainable by an increased expression of wbgO, might lead to a faster intracellular conversion of LNT II and a more competitive utilization of UDP-galactose by WbgO, resulting in higher product concentrations of LNT.…”

Characterization of Two UDP-Gal:GalNAc-Diphosphate-Lipid β1,3-Galactosyltransferases WbwC from Escherichia coli Serotypes O104 and O5

“…Human core 1 Gal-transferase (C1GalT1), which also forms a Gal␤1-3GalNAc linkage, shows very low sequence identity to WbwC ECO104 (12%) and to WbwC ECO5 (11%). WbwC is predicted to form a GT-A glycosyltransferase fold and has been classified in the CAZy GT2 family, which includes many other bacterial and mammalian inverting ␤-glycosyltransferases (22)(23)(24)(37)(38)(39). All of the WbwC homologs have a DxD sequence that might be involved in catalysis (40).…”

Synthesis of fucosylated lacto-N-tetraose using whole-cell biotransformation

“…This GDP-L-fucose was thus available as donor substrate for a Leloir fucosyltransferase. Another recent publication 27 demonstrates the in vivo synthesis of LNT from lactose and glucose or glycerol, employing the enzymes of the two recombinant genes lgtA andwbgO, encoding for a β1,3-N-acetylglucosaminyltransferase (LgtA)28 from Neisseria meningitidis and a β1,3-galactosyltransferase (WbgO)29 from E. coli O55:H7, respectively. In the present work, we combined the capabilities of enhanced GDP-L-fucose synthesis and LNT formation and added α1,2-and α1,4-fucosyltransferase activity for the in vivo synthesis of fucosylated LNTs(Fig.…”