Pathway Reinforcement and Chassis Optimization of Escherichia coli BL21 for Enhancing Lacto‑N‑tetraose Production.

Lacto‑N‑tetraose (LNT) is an important neutral non-fucosylated form of human milk oligosaccharides, widely recognized for its protective effects on infant health and enhancement of immune functions. In this study, to reinforce the fermentative synthesis of LNT, lactose utilization was enhanced for LNT production through overexpressing lacY (encoding the lactose permease gene) in Escherichia coli BL21, which can achieve the LNT titer and productivity of 1.55 g/L and 0.02 g/L/h, respectively. Moreover, the chassis was optimized through deleting byproduct genes: wecB (encoding UDP-N-acetyl glucosamine-2-epimerase), lacZ (encoding β-galactosidase), and the nagB gene (encoding glucosamine-6-phosphate deaminase), so we can obtain a 2.8 g/L LNT titer and 0.039 g/L/h productivity. Furthermore, we designed the UTP regeneration for precursor production through overexpressing the ndk gene (encoding nucleoside diphosphate kinase) and increased the LNT titer by 3.02 g/L. Finally, the LNT titer and productivity were improved to 3.22 g/L and 0.05 g/L/h by integrating the LgtA-WbgO expression cassette in the genome. This study establishes a stepwise optimization strategy in Escherichia coli BL21 for LNT biosynthesis, providing a foundation for industrial-scale production.