Lacto-N-triose II (LNT II), a core structural unit of human milk oligosaccharides (HMOs), has attracted substantial attention for its nutraceutical potentials and applications in the production of complex HMOs. In this study, Escherichia coli was metabolically engineered to efficiently produce LNT II using glycerol as a carbon source and lactose as a substrate. The UDP-Nacetylglucosamine (UDP-GlcNAc) biosynthesis pathway was strengthened, and β-1,3-N-acetylglucosaminyltransferase (LgtA) was introduced to construct an LNT II-producing base strain. To increase the titer and yield of LNT II, combinatorial optimization of the copy number and the ribosomal binding site sequence was performed to tune the gene expression strength and translation rates of the pathway enzymes. Next, multipoint mutations were introduced to glucosamine-6-phosphatesynthase (GlmS) to relieve the feedback inhibition. Then, a series of engineered strains were constructed by blocking the futile pathways by the deletion of the relevant genes. Finally, the culture conditions were optimized. LNT II titer was improved step-by-step from 0.53 to 5.52 g/L in shake-flask cultivations. Fed-batch culture of the final engineered strain produced 46.2 g/L of LNT II, with an LNT II productivity and content of 0.77 g/(L•h) and 0.95 g/g dry cell weight, respectively.

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Human milk oligosaccharides (HMOs) attract particular attention because of their health benefits for infants. Lacto-N-neotetraose (LNnT) is one of the most abundant neutral core structures of HMOs. Bacterial β-1,4-galactosyltransferase (β-1,4-GalT) displays an irreplaceable role in the practical application of LNnT biosynthesis. In this study, a novel β-1,4-GalT from Histophilus somni was identified to efficiently synthesize LNnT from UDP-Gal and lacto-N-triose II (LNT II). The optimum pH and temperature were determined to be pH 6.0 and 30 °C, respectively. The enzyme showed both transgalactosylation and hydrolysis activity, with a specific activity of 3.7 and 6.6 U/mg, respectively. LNnT was synthesized using H. somni β-1,4-GalT via both enzymatic and cell factory approaches, and both approaches provided an LNnT ratio with the remaining LNT II at approximately 1:2 when reactions attained a balance. These findings indicated that H. somni β-1,4-GalT has a potential in biosynthesis of LNnT and derivatives in future.