Construction and Systematic Optimization of Plasmid-free Engineered Escherichia coli for Efficient Lacto-N-Neotetraose Biosynthesis.

Lacto-N-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 Escherichia coli MG1655 strain for lacto-N-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 mdfA, 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 glmS, glmM, glmU, galU, and galE) 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.