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Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli

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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Acknowledgements

This work was supported by Korea Ministry of Education through the BK21 program and by Korea Research Foundation Grant (KRF-2005-042-F00051). M.D. Kim was supported by a 2006 research grant from Kangwon National University.

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Correspondence to Jin-Ho Seo.

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Byun, S., Kim, M., Lee, W. et al. Production of GDP-l-fucose, l-fucose donor for fucosyloligosaccharide synthesis, in recombinant Escherichia coli . Appl Microbiol Biotechnol 74, 768–775 (2007). https://doi.org/10.1007/s00253-006-0730-x

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Keywords

  • Nucleotide Sugar
  • G6PDH Activity
  • IPTG Concentration
  • Glycosyl Donor
  • Human Milk Oligosaccharide