Production of GDP-L-fucose, L-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli.
A recombinant Escherichia coli strain was developed to produce guanosine 5'-diphosphate (GDP)-L-fucose, donor of L-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-D: -mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-L-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25 degrees C and 0.1 mM isopropyl-beta-D-thioglucopyranoside. Maximum GDP-L-fucose concentration of 38.9 +/- 0.6 mg l(-1) was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 +/- 0.5 mg l(-1) GDP-L-fucose under the same cultivation condition.
Byun
Seong-Goo
SG
Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul, South Korea.
Kim
Myoung-Dong
MD
Lee
Won-Heong
WH
Lee
Kun-Jae
KJ
Han
Nam Soo
NS
Seo
Jin-Ho
JH
D002238:Carbohydrate Epimerases / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D005954:Glucosephosphate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D006154:Guanosine Diphosphate Fucose / Q000096:biosynthesis*; D006836:Hydro-Lyases / Q000235:genetics; D007218:Industrial Microbiology; D007658:Ketone Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C110682:wcaG protein, E coli; D006154:Guanosine Diphosphate Fucose; D005954:Glucosephosphate Dehydrogenase; D007658:Ketone Oxidoreductases; D006836:Hydro-Lyases; C044971:GDPmannose 4,6-dehydratase; D002238:Carbohydrate Epimerases