Repurposing CDP-Tyvelose 2-Epimerase Enables a GDP-Fucose-Based Fucosylation Pathway Starting from Sucrose

This study introduces a novel enzymatic cascade featuring five recombinant enzymes for the efficient synthesis of fucosylated glycosides, using sucrose exclusively as the sugar donor substrate. In our approach, we employed a sucrose synthase sourced from tomato to generate GDP-glucose from sucrose and GDP. By repurposing CDP-tyvelose 2-epimerase from Salmonella enterica, chosen for its catalytic efficiency from a panel of 27 CDP-tyvelose 2-epimerase candidates, it was possible to epimerize GDP-glucose into GDP-mannose. The subsequent transformation of GDP-d-mannose to GDP-l-fucose was achieved by GDP-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-d-mannose epimerase/reductase, also derived from S. enterica. In the final stage, <i>Helicobacter pylori</i> α1,3-fucosyltransferase was employed to fucosylate <i>para</i>-nitrophenyl β-lactoside, resulting in the production of <i>para</i>-nitrophenyl 3-fucosyllactoside with a conversion of more than 40%. Analysis of the synthesized compound by LC-MS and NMR analyses substantiated its structure. This investigation not only highlights the utility of this five-enzyme fucosylation cascade but also establishes a novel methodological paradigm for the biocatalytic production of α-l-fucosides for biochemical research and for biotechnological applications.