Human milk oligosaccharides (HMOs) have been reported to exhibit numerous health
benefits, such as enhancing immune and nerve system developments in breastfed infants.
Additionally, HMOs can be used as food safety additives because they have been shown
to inhibit Norovirus. Among more than 100 identified HMOs, 2’-Fucosyllactose (2-FL) is
prevalent in human milk and exhibits benefits for developing healthy gut microbiota. While
2-FL can be synthesized by engineered microorganisms, economic and large-scale
production of 2-FL has been hampered by low yields of 2-FL production.
We undertook a metabolic engineering approaches to enhance the production of 2-FL by
microbial fermentation. Specifically, we hypothesized that a limiting factor of the efficient
2-FL production is the low intracellular GDP-L-fucose concentration, a precursor of 2-FL.
Prior studies have mainly focused on producing more GDP-L-fucose via overexpressing
target genes (manB, manC, gmd, wcaG) by strong induction system. However, the strong
induction system has several considerable drawbacks, including cell stability and
reproducibility. Since strong induction system physiologically can be a significant burden
for cell, resulting in growth inhibition, cell lysis, or even death during fermentation, which
limits its use for large-scale production of 2-FL.
Alternatively, in this study, Escherichia coli was engineered through deletion of two
genetic targets (waaF, wcaJ) instead of overexpressing target genes to stably synthesize
more GDP-L-fucose and 2-FL. As a result, the strain with deletion of genes produced 1.3-
fold more GDP-L-fucose and 3-fold more 2-FL in shaking flask than the stain with the
strong induction system, respectively. Finally, fed-batch fermentation of the strain with
deletion of genes resulted in 14.7 g/L, yield of 2-FL were 0.25 g/g lactose and the
productivity was 0.31 g/L/h. Overall, improvement of microbial production of 2-FL by
engineered E.coli increased the feasibility of utilizing 2-FL as a prebiotic in various foods.
benefits, such as enhancing immune and nerve system developments in breastfed infants.
Additionally, HMOs can be used as food safety additives because they have been shown
to inhibit Norovirus. Among more than 100 identified HMOs, 2’-Fucosyllactose (2-FL) is
prevalent in human milk and exhibits benefits for developing healthy gut microbiota. While
2-FL can be synthesized by engineered microorganisms, economic and large-scale
production of 2-FL has been hampered by low yields of 2-FL production.
We undertook a metabolic engineering approaches to enhance the production of 2-FL by
microbial fermentation. Specifically, we hypothesized that a limiting factor of the efficient
2-FL production is the low intracellular GDP-L-fucose concentration, a precursor of 2-FL.
Prior studies have mainly focused on producing more GDP-L-fucose via overexpressing
target genes (manB, manC, gmd, wcaG) by strong induction system. However, the strong
induction system has several considerable drawbacks, including cell stability and
reproducibility. Since strong induction system physiologically can be a significant burden
for cell, resulting in growth inhibition, cell lysis, or even death during fermentation, which
limits its use for large-scale production of 2-FL.
Alternatively, in this study, Escherichia coli was engineered through deletion of two
genetic targets (waaF, wcaJ) instead of overexpressing target genes to stably synthesize
more GDP-L-fucose and 2-FL. As a result, the strain with deletion of genes produced 1.3-
fold more GDP-L-fucose and 3-fold more 2-FL in shaking flask than the stain with the
strong induction system, respectively. Finally, fed-batch fermentation of the strain with
deletion of genes resulted in 14.7 g/L, yield of 2-FL were 0.25 g/g lactose and the
productivity was 0.31 g/L/h. Overall, improvement of microbial production of 2-FL by
engineered E.coli increased the feasibility of utilizing 2-FL as a prebiotic in various foods.