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. 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.
Mattila
P
P
Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Finland.
Räbinä
J
J
Hortling
S
S
Helin
J
J
Renkonen
R
R
D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods; D006154:Guanosine Diphosphate Fucose / Q000096:biosynthesis* / Q000737:chemistry; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism*; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D007658:Ketone Oxidoreductases; D009097:Multienzyme Complexes; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013237:Stereoisomerism; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C110682:wcaG protein, E coli; D006154:Guanosine Diphosphate Fucose; D006155:Guanosine Diphosphate Mannose; D013401:Sugar Alcohol Dehydrogenases; D007658:Ketone Oxidoreductases; D006836:Hydro-Lyases; C044971:GDPmannose 4,6-dehydratase; D002238:Carbohydrate Epimerases