WbgL: a novel bacterial α1,2-fucosyltransferase for the synthesis of 2′-fucosyllactose

Abstract

Fucosyltransferases (FucTs) are essential tools for the synthesis of fucosylated glycoconjugates. Multistep enzyme catalysis of fucosylated glycans is not limited as long as isolated and well-characterized FucTs are available. The present paper introduces a novel bacterial α1,2-FucT of the glycosyltransferase family 11 encoded by the gene wbgL in the E. coli O126 genome, which only displays 25-30% homology to previously published α1,2-FucTs. A tailor made cloning and expression strategy allowed the successful production of active soluble enzyme in the cytoplasm of E. coli BL21(DE3) and E. coli JM109(DE3), respectively. The lack of a DxD motif and its high activity without divalent metal ions suggests that WbgL belongs to the GT-B fold superfamily. Substrate screening revealed the highest activity for β4-linked galactoside acceptor substrates, such as lactose and lactulose, making WbgL unique among other characterized α1,2-FucTs. Based on its excellent kinetic efficiency for lactose, we present here a sequential reaction strategy for the synthesis of α1,2-fucosyllactose in one pot including the synthesis of the donor substrate 3,3'-Diaminobenzidine (GDP)-β-l-fucose by the bifunctional l-fucokinase/GDP-β-l-Fuc pyrophosphorylase of Bacteroides fragilis 9343.

Multiplexed Capillary Electrophoresis as Analytical Tool for Fast Optimization of Multi‐Enzyme Cascade Reactions – Synthesis of Nucleotide Sugars

“…In this way, N(M)DP‐sugar modules and GT‐modules are combined to generate production modules in assembly lines for the synthesis of glycoconjugates . In our approach to build up a ”Golgi Glycan Factory (GGF)“ sequential and one‐pot enzyme cascades were established for the synthesis of novel hybrid or natural glycosylated products . Enzyme cascade reactions require intensive investigations on parameters influencing enzyme performance such as temperature optima, pH‐optima, kinetics, cofactor dependency, inhibition by products or intermediate products, substrate spectrum, and enzyme ratios.…”

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

“…Using chemical-and/or enzymatic-methods, different HMOs have been synthesized, e.g. fucosyllactose [7][8][9][10][11][12][13], sialyllactose [14][15][16][17], or lacto-N-neotetraose [18][19][20].…”

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

“…The overall predominant oligosaccharides in human milk, found in previous studies which were dominated by milk from so-called secretor women are 2'-fucosyllactose (2'-FL), lacto-Nfucopentaose I (LNF I), lacto-N-difucohexaose I (LNDFH I), and lacto-N-tetraose (LNT). 7 Focusing on neutral HMOs, previous studies described chemical, enzymatic or whole-cell syntheses of fucosyllactoses [8][9][10][11][12][13][14] and core structures such as lacto-N-neotetraose (LNnT) and LNT. [15][16][17][18][19] Fucosylated derivatives of the core structure LNnT were synthesized in whole-cell cultivations, 10,11,20 while syntheses of fucosylated LNT structures so far have been only described from in vitro enzymatic conversions.…”