Lacto-N-neotetraose (LNnT) and lacto-N-tetraose (LNT) are important oligosaccharides found in breast milk and are commonly used as nutritional supplements in infant formula. We used metabolic engineering techniques to optimize the modified Escherichia coli BL21 star (DE3) strain for efficient synthesis of LNnT and LNT using β-1,4-galactosyltransferase (HpgalT) from Helicobacter pylori and β-1,3-galactosyltransferase (SewbdO) from Salmonella enterica subsp. salamae serovar, respectively. Further, we optimized the expression of three key genes, lgtA, galE, and HpgalT (SewbdO), to synthesize LNnT or LNT and deleted several genes (ugd, ushA, agp, wcaJ, otsA, and wcaC) to block competition in the UDP-galactose synthesis pathway. The optimized strain produced LNnT or LNT with a titer of 22.07 or 48.41 g/L, respectively, in a supplemented batch culture, producing 0.41 or 0.73 g/L/h, respectively. The strategies used in this study contribute to the development of cell factories for high-level LNnT and LNT and their derivatives.
Keywords: Escherichia coli BL21 star (DE3); galactosyltransferase; human milk oligosaccharide; lacto-N-neotetraose; lacto-N-tetraose.