High-Efficiency Production of N -Acetyllactosamine (LacNAc) Using a Cell-Coupled Biocatalytic Strategy with Engineered Escherichia coli and Saccharomyces cerevisiae

N -Acetyllactosamine (LacNAc) serves as a key intermediate in the biosynthesis of 6′-sialyl- N -acetyllactosamine (6′-SLN) and 3′-sialyl- N -acetyllactosamine (3′-SLN), which are the predominant components of sialylated milk oligosaccharides (SMOs) in goat milk. Therefore, methods for high-efficiency and low-cost production of LacNAc are necessary for 6′-SLN and 3′-SLN production. In this study, a new strategy for LacNAc production through a whole-cell coupled fermentation approach using sucrose and N -acetylglucosamine (GlcNAc) as substrates was explored. First, the enzymatic activities of β-1,4-galactosyltransferase (Hpβ4GalT and HpGalT), which were involved in LacNAc biosynthesis, were evaluated; the results indicated that Hpβ4GalT exhibits a higher LacNAc synthesis capacity than HpGalT. Subsequently, LacNAc was synthesized using a whole-cell catalytic system based on multistrain coupling. Then, to further enhance the titer, Saccharomyces cerevisiae cells were introduced to enable energy regeneration, and a coexpression strategy was employed to minimize the cell density. Following the optimization of induction and coupled fermentation conditions, LacNAc production increased 6-fold. Finally, the fermentation optimization conditions were validated in a 5-L bioreactor, achieving a maximum LacNAc titer of 32.38 g/L and a substrate GlcNAc molar yield of 84.46%.