Construction of an engineered Escherichia coli for effective synthesis of 2′-fucosyllactose via the salvage pathway

Abstract

2′-Fucosyllactose (2′-FL) is one of the important functional oligosaccharides in breast milk. So far, few attempts on biosynthesis of 2′-FL by the salvage pathway have been reported. Herein, the salvage pathway enzyme genes were introduced into the E. coli BL21star(DE3) for synthesis of 2′-FL. The 2′-FL titer increased from 1.56 to 2.13 g/L by deleting several endogenous genes on competitive pathways. The α-1,2-fucosyltransferase (WbgL) was selected, and improved the 2′-FL titer to 2.88 g/L. Additionally, the expression level of pathway enzyme genes was tuned through optimizing the plasmid copy number. Furthermore, the spatial distribution of WbgL was enhanced by fusing with the MinD C-tag. After optimizing the fermentation conditions, the 2′-FL titer reached to 7.13 g/L. The final strain produced 59.22 g/L of 2′-FL with 95% molar conversion rate of lactose and 92% molar conversion rate of fucose in a 5 L fermenter. These findings will contribute to construct a highly efficient microbial cell factory to produce 2′-FL or other HMOs.

Keywords

• 2′-fucosyllactose
• Human milk oligosaccharides
• Escherichia coli
• Fed-batch fermentation
• Salvage pathway