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

Background: The 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 alpha 1,2-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. Results: Here 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 lambda 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). Conclusions: By 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.