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

Byun, Seong-Goo; Kim, Myoung-Dong; Lee, Won-Heong; Lee, Kun-Jae; Han, Nam Soo; Seo, Jin‐Ho

doi:10.1007/s00253-006-0730-x

Cited by 35 publications

(22 citation statements)

References 27 publications

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“…The constructed plasmid was confirmed by DNA sequencing. The conditions for PCR reaction, DNA manipulation and bacterial transformation followed the descriptions in the previous study [21]. …”

Section: Methodsmentioning

confidence: 99%

Whole cell biosynthesis of a functional oligosaccharide, 2′-fucosyllactose, using engineered Escherichia coli

et al. 2012

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Background: 2 0 -Fucosyllactose (2-FL) is a functional oligosaccharide present in human milk which protects against the infection of enteric pathogens. Because 2-FL can be synthesized through the enzymatic fucosylation of lactose with guanosine 5 0 -diphosphate (GDP)-L-fucose by α-1,2-fucosyltransferase (FucT2), an 2-FL producing Escherichia coli can be constructed through overexpressing genes coding for endogenous GDP-L-fucose biosynthetic enzymes and heterologous fucosyltransferase.

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Section: Methodsmentioning

confidence: 99%

Whole cell biosynthesis of a functional oligosaccharide, 2′-fucosyllactose, using engineered Escherichia coli

et al. 2012

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“…Recently, the productivity of GDP-fucose fermentation has been improved tremendously in recombinant E. coli via several modulation schemes including enhanced production of GDP-fucose by modulation of the biosynthesis pathways for NADPH and guanosine nucleotides [20][21][22][23]. The improvement of GDP-l-fucose production by manipulating the biosynthetic pathway for guanosine nucleotides could also be applicable for the processes based on the salvage pathway since GTP is also the direct substrate for GDP-fucose biosynthesis in the salvage pathway (Fig.…”

Section: Introductionmentioning

confidence: 98%

Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

Zhai

Han

Pan

et al. 2015

Enzyme and Microbial Technology

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“…The de novo synthesis pathway, found in bacteria, mammals, and plants, transforms mannose-6-phosphate via mannose-1-phosphate, GDP-mannose, and GDP-4-keto-6-deoxymannose to GDP-L-fucose. Recombinant expression of the de novo pathway biosynthesis genes has been used for the in vitro and in vivo synthesis of GDP-L-fucose [19-22]. …”

Section: Introductionmentioning

confidence: 99%

Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2′-fucosyllactose

et al. 2013

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BackgroundThe trisaccharide 2′-fucosyllactose (2′-FL) is one of the most abundant oligosaccharides found in human milk. Due to its prebiotic and anti-infective properties, 2′-FL is discussed as nutritional additive for infant formula. Besides chemical synthesis and extraction from human milk, 2′-FL can be produced enzymatically in vitro and in vivo. The most promising approach for a large-scale formation of 2′-FL is the whole cell biosynthesis in Escherichia coli by intracellular synthesis of GDP-L-fucose and subsequent fucosylation of lactose with an appropriate α1,2-fucosyltransferase. Even though whole cell approaches have been demonstrated for the synthesis of 2′-FL, further improvements of the engineered E. coli host are required to increase product yields. Furthermore, an antibiotic-free method of whole cell synthesis of 2′-FL is desirable to simplify product purification and to avoid traces of antibiotics in a product with nutritional purpose.ResultsHere we report the construction of the first selection marker-free E. coli strain that produces 2′-FL from lactose and glycerol. To construct this strain, recombinant genes of the de novo synthesis pathway for GDP-L-fucose as well as the gene for the H. pylori fucosyltransferase futC were integrated into the chromosome of E. coli JM109 by using the λ-Red recombineering technique. Strains carrying additional copies of the futC gene and/or the gene fkp (from Bacteroides fragilis) for an additional salvage pathway for GDP-L-fucose production were used and shown to further improve production of 2′-FL in shake flask experiments. An increase of the intracellular GDP-L-fucose concentration by expression of fkp gene as well as an additional copy of the futC gene lead to an enhanced formation of 2′-FL. Using an improved production strain, feasibility of large scale 2′-FL production was demonstrated in an antibiotic-free fed-batch fermentation (13 l) with a final 2′-FL concentration of 20.28 ± 0.83 g l-1 and a space-time-yield of 0.57 g l-1 h-1.ConclusionsBy chromosomal integration of recombinant genes, altering the copy number of these genes and analysis of 2′-FL and intracellular GDP-L-fucose levels, we were able to construct and improve the first selection marker-free E. coli strain which is capable to produce 2′-FL without the use of expression plasmids. Analysis of intracellular GDP-L-fucose levels identified the de novo synthesis pathway of GDP-L-fucose as one bottleneck in 2′-FL production. In antibiotic-free fed-batch fermentation with an improved strain, scale-up of 2′-FL could be demonstrated.

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

Cited by 35 publications

References 27 publications

Whole cell biosynthesis of a functional oligosaccharide, 2′-fucosyllactose, using engineered Escherichia coli

Whole cell biosynthesis of a functional oligosaccharide, 2′-fucosyllactose, using engineered Escherichia coli

Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2′-fucosyllactose

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