A Novel β‑1,4-Galactosyltransferase from <i>Histophilus somni</i> Enables Efficient Biosynthesis of Lacto‑<i>N</i>‑Neotetraose via Both Enzymatic and Cell Factory Approaches

Human milk oligosaccharides (HMOs) attract particular attention because of their health benefits for infants. Lacto-<i>N</i>-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 <i>Histophilus somni</i> was identified to efficiently synthesize LNnT from UDP-Gal and lacto-<i>N</i>-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 <i>H. somni</i> 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 <i>H. somni</i> β-1,4-GalT has a potential in biosynthesis of LNnT and derivatives in future.