Functional expression of Escherichia coli enzymes synthesizing GDP-L-fucose from inherent GDP-D-mannose in Saccharomyces cerevisiae

Fucosylation of glycans on glycoproteins and -lipids requires the enzymatic activity of relevant fucosyltransferases and GDP-L-fucose as the donor. Du e to the biological importance of fucosylated glycans, a readily accessible source of GDP-L-fucose would be required. Here we describe the constructio n of a stable recombinant S.cerevisiae strain expressing the E.coli genes g md and wcaG encoding the two enzymes, GDP-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GMER(FX)) respecti vely, needed to convert GDP-mannose to GDP-fucose via the de novo pathway. Taking advantage of the rich inherent cytosolic GDP-mannose pool in S.cerev isiae cells we could easily produce 0.2 mg/l of GDP-L-fucose with this reco mbinant yeast strain without addition of any external GDP-mannose, The GDP- L-fucose product could be used as the fucose donor for alpha1,3-fucosyltran sferase to synthesize sialyl Lewis x (sLex), a glycan crucial for the selec tin-dependent leukocyte traffic.