Lacto-N-tetraose biosynthesis from lactose via metabolically rewired Escherichia coli

Human milk oligosaccharides (HMOs), such as lacto-N-tetraose (LNT), play critical roles in infant health by shaping gut microbiota and modulating immune function. While LNT is already produced at industrial scales, efficient microbial routes to more complex HMOs derived from LNT remain limited. Here, we established a simplified microbial platform in Escherichia coli that produces LNT directly from lactose as the sole carbon and precursor source. A key innovation was construction of a strain library with tunable β-galactosidase (LacZ) activity, enabling controlled lactose hydrolysis to generate glucose and galactose for UDP-sugar biosynthesis while preserving sufficient intact lactose as the scaffold for LNT assembly. Quantitative profiling of intracellular UDP-sugars further guided identification of metabolic bottlenecks. The optimized strain achieved co-production of 2.4 g/L LNT and 2.0 g/L lacto-N-triose II (LNT II) from 10 g/L lactose. This streamlined strategy demonstrates the feasibility of producing LNT from a single substrate and provides a versatile foundation for scalable microbial biosynthesis of more complex HMOs.