Building a Microbial Factory for Human Milk Oligosaccharides: Efficient Production of 3′-Sialyllactosamine in <i>Escherichia coli</i> BL21 (DE3)

The biosynthetic production of human milk oligosaccharides through metabolic engineering has gained increasing attention in recent years. Yet, limited research has focused on the microbial synthesis of 3'-sialyllactosamine (3'-SLN), a critical intermediate for assembling sialyl Lewis X (sLe^x). In this work, a pathway enabling 3'-SLN biosynthesis was constructed in <i>Escherichia coli</i> BL21(DE3). A key α2,3sialyltransferase (α2,3SiaT) from <i>Bibersteinia trehalosi</i> was identified and utilized to enhance the sialylation step toward 3'-SLN formation. To improve flux through the <i>N</i>-acetyllactosamine branch, essential genes were chromosomally integrated and overexpressed, while a CMP-<i>N</i>-acetylneuraminic acid synthesis module was coexpressed to secure an adequate donor supply. Furthermore, the expression balance was optimized through combinatorial adjustment of gene copy numbers and translational strength across modules. The engineered <i>E. coli</i> strain produced 0.78 g/L of 3'-SLN in shake-flask cultivation and further accumulated up to 7.75 g/L during 5-L fed-batch fermentation conditions, confirming the feasibility of microbial synthesis of 3'-SLN.