Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli |
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Authors: | Seong-Goo Byun Myoung-Dong Kim Won-Heong Lee Kun-Jae Lee Nam Soo Han Jin-Ho Seo |
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Institution: | (1) Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul, 151-921, South Korea;(2) School of Bioscience and Biotechnology, Kangwon National University, Chuncheon, 200-701, South Korea;(3) Department of Food Science and Technology, Chungbuk National University, Cheongju, 361-763, South Korea |
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Abstract: | 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°C and 0.1 mM isopropyl-β-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. |
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