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

Abstract

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, Helicobacter pylori α1,3-fucosyltransferase was employed to fucosylate para-nitrophenyl β-lactoside, resulting in the production of para-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.

Keywords: CDP-tyvelose 2-epimerase; GDP-fucose biosynthesis; enzyme repurposing; recombinant enzymes; sucrose utilization.

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GDP-fucose generation via (a) the de novo pathway from glucose and (b) the fucose salvage pathway; (c) known substrate scope of Thermodesulfatator atlanticus tyvelose 2-epimerase; (d) proposed fucosylation pathway starting from sucrose as sugar donor substrate, showing the final transformation product para-nitrophenyl 3-fucosyllactoside (pNP-3FL). The gray panel on the right shows the structures of the cytidine diphosphate (CDP) and guanosine diphosphate (GDP) moieties.

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(a) SDS-PAGE of the purified enzymes used in the biocatalytic fucosylation cascade. (b) Activity screen of the CDP-tyvelose 2-epimerase candidates. (c) Fucosylation activity of reaction mixtures lacking required components of the fucosylation cascade. (d) SDS-PAGE of SeTyvE wild-type and mutant variants. (e) Relative activity of SeTyvE wild-type and mutant variants. (f) Temperature optimum of SeTyvE. (g) Temperature optimum of SlSUS. (h) Reverse-phase HPLC chromatogram (top) and corresponding mass spectrometric signal intensities (bottom) for the expected m/z ratios of pNP-Lac and pNP-3FL. (i) Mass spectra of reaction mixtures at 5.9 and 6.2 min retention time.