Lacto--neotetraose (LNnT) and lacto--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 BL21 star (DE3) strain for efficient synthesis of LNnT and LNT using β-1,4-galactosyltransferase (HpgalT) from and β-1,3-galactosyltransferase (SewbdO) from subsp. , respectively. Further, we optimized the expression of three key genes, , , and (), to synthesize LNnT or LNT and deleted several genes (, , , , , and ) 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.
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