Construction and Systematic Optimization of Plasmid-free Engineered <i>Escherichia coli</i> for Efficient Lacto-<i>N</i>-Neotetraose Biosynthesis

Lacto-<i>N</i>-neotetraose (LNnT) is a key human milk oligosaccharide (HMO) with important prebiotic functions, supporting the growth of beneficial gut microbiota and contributing to infant health. Constructing plasmid-free strains via metabolic engineering for LNnT biosynthesis represents a feasible strategy for efficient industrial-scale production. This study integrates various strategies to construct plasmid-free strains capable of efficiently producing LNnT. Building on the previously developed <i>Escherichia coli</i> MG1655 strain for lacto-<i>N</i>-triose II (LNTri II) production, Hplex2B (encoding β1,4-galactosyltransferase) was incorporated, and its copy number was optimized to construct a complete and efficient biosynthetic pathway. By integrating an extra copy of the multidrug efflux pump gene <i>mdfA</i>, the strain tolerance was improved, resulting in a higher yield of LNnT. The integration of expression cassettes for key genes in the glycosyl donor synthesis pathway (including <i>glmS</i>, <i>glmM</i>, <i>glmU</i>, <i>galU</i>, and <i>galE</i>) with varying promoter strengths optimized the supply and balance. The subsequent removal of a key feedback inhibition circuit directed the reaction toward the synthesis of LNnT. The final strain, after successive optimization, produced LNnT at 7.76 g/L in shake flask culture and 34.24 g/L in a 5 L bioreactor, with precursor LNTri II concentrations of 0.58 g/L and 1.61 g/L, respectively.