Modular Systems Engineering Enables Streamlined Development of Escherichia coli MG1655 Strains for Neutral Core Human Milk Oligosaccharide Synthesis

Human milk oligosaccharides (HMOs) are key functional components of human milk. This study reports a modular systems engineering framework in Escherichia coli MG1655 for the efficient synthesis of neutral core HMOs, including lacto- N -triose II (LNTri II), lacto- N -tetraose (LNT), and lacto- N -neotetraose (LNnT). The strategy involved the sequential optimization of three modules: glycosyltransferase expression, nucleotide sugar donor supply (via gene integration and feedback inhibition removal), and transport engineering. First applied to construct a high-yield LNTri II platform, the same modular logic was then adapted by swapping the terminal glycosyltransferase to generate LNT or LNnT producers. All strains were constructed via stable chromosomal integration, ensuring plasmid-free, inducer-free operation. In 5-L bioreactors, the engineered strains achieved high titers of 81.96 g/L LNTri II, 78.52 g/L LNT, and 43.17 g/L LNnT. This work provides a streamlined and adaptable paradigm for developing microbial cell factories for complex oligosaccharide synthesis.