Abstract
Background2'-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′-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.ResultsThe gene for FucT2 from Helicobacter pylori was introduced to the GDP- l-fucose producing recombinant E. coli BL21 star(DE3) strain. However, only small amount of 2-FL was produced in a batch fermentation because the E. coli BL21star(DE3) strain assimilated lactose instead of converting to 2-FL. As an alternative host, the E. coli JM109(DE3) strain which is incapable of assimilating lactose was chosen as a 2-FL producer. Whole cell biosynthesis of 2-FL from lactose was investigated in a series of batch fermentations using various concentrations of lactose. The results of batch fermentations showed that lactose was slowly assimilated by the engineered E. coli JM109(DE3) strain and 2-FL was synthesized without supplementation of another auxiliary sugar for cell growth. A maximum 2-FL concentration of 1.23 g/l was obtained from a batch fermentation with 14.5 g/l lactose. The experimentally obtained yield (g 2-FL/g lactose) corresponded to 20% of the theoretical maximum yield estimated by the elementary flux mode (EFM) analysis.ConclusionsThe experimental 2-FL yield in this study corresponded to about 20% of the theoretical maximum yield, which suggests further modifications via metabolic engineering of a host strain or optimization of fermentation processes might be carried out for improving 2-FL yield. Improvement of microbial production of 2-FL from lactose by engineered E. coli would increase the feasibility of utilizing 2-FL as a prebiotic in various foods.
Highlights
Human milk oligosaccharides (HMOs) are known to be the most relevant factor for the development of intestinal microbiota in breast-fed infants [1]
Expression of α-1,2-fucosyltransferase (FucT2) in recombinant E. coli The expression pattern of FucT2 was investigated during a batch fermentation of recombinant E. coli harboring plasmid pHfucT2
Batch fermentations From the preliminary experiments (whole cell bioconversion of 2 g/l lactose with E. coli BL21star(DE3) strain), we concluded that the E. coli BL21star(DE3) strain is not beneficial for 2-FL production because it consumed lactose for growth and maintenance instead of converting to 2-FL
Summary
Human milk oligosaccharides (HMOs) are known to be the most relevant factor for the development of intestinal microbiota in breast-fed infants [1]. HMOs have been reported to play important roles in preventing adhesion of pathogens and toxins to epithelial surfaces [2]. N-fucopentaose and lacto-N-difucohexaose, are common HMOs. Fucosylated oligosaccharides act as growth stimulating factors for select Bifidobacteria and soluble analogs of receptors for pathogenic bacteria, thereby protecting infants against infection from enteric pathogens and binding of toxins [3,4]. 2-FL is a promising oligosaccharide for nutraceutical and pharmaceutical purposes
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