title: Enhanced production of GDP-L-fucose by overexpression of NADPH regenerator in recombinant Escherichia coli.
abstract: Biosynthesis of guanosine 5'-diphosphate-L-fucose (GDP-L-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP(+)-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-L-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-L-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-L-fucose production. However, GDP-L-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-L-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-L-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-L-fucose concentration of 235.2 ± 3.3 mg l(-1), corresponding to a 21% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-L-fucose production in recombinant E. coli.
authors.affiliation: Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, South Korea.
authors.forename: Won-Heong
authors.initials: WH
authors.lastname: Lee
authors.forename: Young-Wook
authors.initials: YW
authors.lastname: Chin
authors.forename: Nam Soo
authors.initials: NS
authors.lastname: Han
authors.forename: Myoung-Dong
authors.initials: MD
authors.lastname: Kim
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Seo
mesh_terms: D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D015870:Gene Expression; D005818:Genetic Engineering; D005947:Glucose / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism*; D007521:Isocitrate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D050538:Malate Dehydrogenase (NADP+) / Q000235:genetics / Q000378:metabolism; D053858:Metabolic Networks and Pathways / Q000235:genetics; D009249:NADP / Q000235:genetics* / Q000378:metabolism*
chemical_list: D006154:Guanosine Diphosphate Fucose; D009249:NADP; D007521:Isocitrate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); D005947:Glucose