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

ABSTRACT

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 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. 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 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). 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.