A Novel De Novo Biosynthetic Pathway for Efficient Synthesis of 6′-Sialyllactose in Escherichia coli

6'-Sialyllactose (6′-SL) is an essential human milk oligosaccharide (HMO) that plays a crucial role in infant development with significant industrial potential. In this study, a novel biosynthetic pathway was employed to produce 6′-SL through de novo synthesis from glycerol in E. coli. First, the key enzymes for 6′-SL biosynthesis were expressed to achieve a yield of 6′-SL of 9.72 mg/L in E. coli. Next, to further increase the yield of 6′-SL, lactose degradation was prevented and genes involved in competing pathways were knocked out. Afterwards, by eliminating carbon catabolite repression (CCR), together with previous genetic modifications, 1.92 g/L of 6′-SL was successfully produced. Finally, by alleviating metabolic pressure from growth production interference and knocking out GlcNAc degradation genes, the yield of 6′-SL reached 6.21 g/L, which was higher than previously reported shake-flask yields. Additionally, in a 3-L fermenter, a yield of 14.33 g/L of 6′-SL was successfully achieved. This study successfully developed a novel plasmid-free, high-yield 6′-SL strain, demonstrating the strong potential of the GlcNAc pathway for 6′-SL biosynthesis.