Biosynthesis of\nSialyllacto‑<i>N</i>‑tetraose c in Engineered Escherichia coli

Human milk oligosaccharides (HMOs) have attracted considerable\ninterest for their vital role in supporting infant health. Among these,\nsialyllacto-<i>N</i>-tetraose c (LST c), a pentasaccharide\nwith the structure Neu5Ac(α2,6)Gal(β1,4)GlcNAc(β1,3)Gal(β1,4)Glc,\nstands out due to its critical importance in the development and application\nof complex HMOs. In this study, we employed multivariate modular metabolic\nengineering (MMME) to screen for efficient sialyltransferases and\nbalance metabolic fluxes, successfully constructing strains capable\nof LST c biosynthesis. Additionally, by blocking competing pathway\ngenes, enhancing the supply of UDP-GlcNAc and UDP-Gal precursors,\nand establishing a CTP cofactor regeneration system, we developed\na high-yielding Escherichia coli strain,\nW15. This strain achieved an LST c titer of 220.9 mg/L in shake flask\ncultures. In a 3-L fed-batch fermentation, the LST c concentration\nreached 922.2 mg/L, with a productivity of 10.25 mg/L/h and a specific\nyield of 38.70 mg/g DCW. This research provides an effective strategy\nfor producing LST c in microbial cell factories.