Efficient Biosynthesis of N-Acetyllactosamine by a Metabolically Engineered Escherichia coli BL21(DE3) Strain

The biological synthesis of human milk oligosaccharides (HMOs) through the use of metabolically engineered strains represents a significant area of interest. However, comparatively less attention has been directed to the biological production of N -acetyllactosamine (LacNAc). LacNAc serves as a key intermediate in the synthesis of sialylated milk oligosaccharides (SMOs). In this study, we constructed the metabolic pathway for LacNAc in Escherichia coli BL21(DE3). To facilitate the design of artificial metabolic pathways, β-1,4-galactosyltransferase from Helicobacter pylori was identified as a critical glycosyltransferase for enhancing LacNAc production. The expression of essential gene clusters within the N -acetylglucosamine (GlcNAc) pathway was augmented and integrated into the chromosome. Subsequently, combinatorial optimization of the gene copy number was conducted to modulate the expression strength and translation rates of enzymes in the GlcNAc and UDP-Gal pathways. Glycerol was determined to be a more effective carbon source for LacNAc synthesis than glucose. The final engineered strain achieved LacNAc production levels of 6.32 g/L in shake-flask cultures and 38.26 g/L in a 5 L bioreactor using fed-batch fermentation. This work demonstrates the potential of engineered E. coli for LacNAc production via metabolic engineering and establishes a foundational pathway for SMO synthesis.