Production of 2-fucosyllactose, a human milk oligosaccharide by metabolically engineered escherichia coli

Abstract

Human milk has many biological functions including prebiotic effects, prevention of pathogenic infection, modulation of immune systems and anti-inflammatory effects since it contains a high dose of unique oligosaccharides which are relatively rare in other mammalian milks. Especially, 2-fucosyllactose (2-FL) which is one of the most abundant ones among 200 different oligosaccharides has received great attention in terms of nutraceutical and pharmaceutical purpose. As biosynthesis of 2-FL with microbial systems has been proven to be more efficient than chemical or enzymatic synthesis, several studies on microbial production of 2-FL have been reported recently. Biosynthesis of 2-FL is influenced by several factors including intracellular lactose availability, activity of α-1,2-fucosyltransferase and supply of guanosine 5-diphosphate (GDP)-L-fucose as a donor of fucose. In this study, Escherichia coli BL21star(DE3) was engineered by focusing on the key factors for efficient production of 2-FL. Specifically, the activity of β-galactosidase was attenuated through deletion of the whole endogenous lactose operon and introduction of the modified lactose operon containing lacZ△M15. Expression of α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori along with the genes for the de novo pathway of GDP-L-fucose (manB, manC, gmd and wcaG) allowed engineered E. coli BL21star(DE3) to produce 2-FL with 3-times enhanced yield of 0.091 g 2-FL/g lactose than the non-engineered E. coli BL21star(DE3) strain. The titer and yield of 2-FL were improved by adding the three aspartate molecules at the N-terminal of FucT2. 6.4 g/L of a maximum concentration of 2-FL was obtained in fed-batch fermentation of the β-galactosidase attenuated E. coli strain (△L M15) expressing three aspartate tagged FucT2 with the genes involved in the de novo pathway of GDP-L-fucose. Moreover, expression of putative α-1,2-fucosyltransferase (WcfB) from Bacteroides fragilis in the △L M15 strain led to a 4-fold enhancement in 2-FL concentration compared to that of △L M15 exspressing FucT2. However, an unknown by-product also accumulated in the media together with 2-FL. Since the unknown by-product was composed of galactose, complete elimination of the residual β-galactosidase activity was attempted. Finally, 2-FL titer was further increased up to 15.4 g/L with 2-FL yield of 0.858 g/g lactose and productivity of 0.530 g/L/h by fed-batch fermentation of the β-galactosidase deleted E. coli strain (△L YA) expressing WcfB and the enzymes involved in the de novo pathway for GDP-L-fucose biosynthesis. An engineered E. coli strain able to produce 2-FL from fucose, lactose and glycerol was also constructed by introducing the fkp gene coding for fucokinase/GDP-L-fucose pyrophosphorylase from B. fragilis involved in the salvage GDP-L-fucose biosynthetic pathway and expressing FucT2 from H. pylori. Employment of the LacZ-attenuation mutant (△L M15) and LacZ-deletion mutant (△L YA) allowed a significant improvement of 2-FL titer and yield based on lactose. In addition, 2-FL titer and yield based on fucose were further increased by deletion of the fucI-fucK gene cluster encoding fucose isomerase and fuculose kinase in the chromosomes of the LacZ mutants. Fed-batch fermentation of the △L YA strain deleted fucI-fucK and expressing Fkp and FucT2 resulted in 23.1 g/L of 2-FL concentration with yields of 0.37 mole 2-FL/mole lactose, 0.36 mole 2-FL/mole fucose and productivity of 0.39 g/L/h. These results suggest that microbial production of 2-FL might be a promising method for mass production of 2-FL.