Synthesis of the Human Milk Oligosaccharide Lacto‐N‐Tetraose in Metabolically Engineered, Plasmid‐Free E. coli

Abstract Human milk oligosaccharides (HMOs) constitute the third most abundant solid component of human milk. HMOs have been demonstrated to show positive effects on the infant’s well‐being. Despite numerous studies, more physiological analyses of single compounds are needed to fully elucidate these effects. Although being one of the most abundant core structures in human milk, the HMO lacto‐ N ‐tetraose (LNT) is not available at reasonable prices. In this study, we demonstrate the construction of the first E. coli strain capable of producing LNT in vivo. The strain was constructed by chromosomally integrating the genes lgtA and wbgO , encoding β‐1,3‐ N ‐acetylglucosaminyltransferase and β‐1,3‐galactosyltransferase. In shake‐flask cultivations, the strain yielded a total concentration of 219.1±3.5 mg L −1 LNT (LNT in culture broth and the cell pellet). After recovery of LNT, structural analysis by NMR spectroscopy confirmed the molecule structure.