Metabolic Engineering of <i>Escherichia coli</i> for High-Level Production of Lacto-<i>N</i>-neotetraose and Lacto-<i>N</i>-tetraose

Abstract

Lacto-<i>N</i>-neotetraose (LNnT) and lacto-<i>N</i>-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 <i>Escherichia coli</i> BL21 star (DE3) strain for efficient synthesis of LNnT and LNT using β-1,4-galactosyltransferase (HpgalT) from <i>Helicobacter pylori</i> and β-1,3-galactosyltransferase (SewbdO) from <i>Salmonella enterica</i> subsp. <i>salamae serovar</i>, respectively. Further, we optimized the expression of three key genes, <i>lgtA</i>, <i>galE</i>, and <i>HpgalT</i> (<i>SewbdO</i>), to synthesize LNnT or LNT and deleted several genes (<i>ugd</i>, <i>ushA</i>, <i>agp</i>, <i>wcaJ</i>, <i>otsA</i>, and <i>wcaC</i>) 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.