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

Abstract

Fucosylation of glycans on glycoproteins and -lipids requires the enzymatic activity of relevant fucosyltransferases and GDP-L-fucose as the donor. Due to the biological importance of fucosylated glycans, a readily accessible source of GDP-L-fucose would be required. Here we describe the construction of a stable recombinant S.cerevisiae strain expressing the E.coli genes gmd 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)) respectively, 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.cerevisiae cells we could easily produce 0.2 mg/l of GDP-L-fucose with this recombinant yeast strain without addition of any external GDP-mannose. The GDP-L-fucose product could be used as the fucose donor for alpha1,3fucosyltransferase to synthesize sialyl Lewis x (sLex), a glycan crucial for the selectin-dependent leukocyte traffic.

Substrate Specificities and Availability of Fucosyltransferase and β-Carotene Hydroxylase for Myxol 2′-Fucoside Synthesis in Anabaena sp. Strain PCC 7120 Compared with Synechocystis sp. Strain PCC 6803

“…strain PCC 7120. GDP-L-fucose is synthesized from GDP-4-dehydro-6-deoxy-Dmannose by GDP-fucose synthase, which is WcaG in E. coli (25,32). Anabaena sp.…”

“…The next two steps, epimerization and an NADPH-dependent reduction with resulting formation of GDP-fucose, are catalyzed by the bifunctional enzyme GDPfucose synthase (EC 1.1.1.271). The genes encoding GDPmannose 4,6-dehydratase and GDP-fucose synthase have been cloned from several organisms, and the genes from Escherichia coli are designated gmd and wcaG, respectively (25,32). Homologous gmd and wcaG genes have been found in Anabaena sp.…”

A Bifunctional 3,5-Epimerase/4-Keto Reductase for Nucleotide-Rhamnose Synthesis in Arabidopsis

“…The 4-keto-6-deoxy-Man is then converted to GDP-Fuc by a single bifunctional enzyme (GDP-4-keto-6-deoxy-mannose 3,5-epimerase/4-reductase) that is referred to as either GDP-Man epimerase-reductase (GER; Bonin and Reiter, 2000), GDP-fucose synthase (GFS; Menon et al, 1999), FX (Tonetti et al, 1996), or GDP-Man epimerase/reductase (GMER; Rizzi et al, 1998). The GDP-Fuc synthesizing enzymes have been cloned from humans (Tonetti et al, 1996;Sullivan et al, 1998), plants (Bonin et al, 1997;Bonin and Reiter, 2000), and bacteria (Sturla et al, 1997;Menon et al, 1999;Mattila et al, 2000). There is no primary amino acid sequence similarity between the bifunctional NRS/ER and the bifunctional GFS (GDP-Fuc Synthase).…”

Two Time-Resolved Fluorometric High-Throughput Assays for Quantitation of GDP--Fucose

“…The stable recombinant S. cerevisiae strain expressing the E. coli genes for GMD and GFS, needed to convert GDP-Man to GDP-Fuc, have been prepared and described in (10). As determined with the first, enzyme-based assay (Fig.…”

“…We constructed Saccharomyces cerevisiae strains expressing the Escherichia coli genes for the enzymes of the de novo pathway, GMD and GFS (10). The cytoplasm of yeast cells is a relatively rich source of GDPMan, the starting material for the de novo pathway (11).…”