Two-step ProductionMethod for Lacto‑<i>N</i>‑Triose II via Cell-CoupledBiocatalytic Strategy

Lacto-<i>N</i>-triose II (LNT II) represents a significant neutral human milk oligosaccharide (HMO) that functions as a fundamental structural framework for the synthesis of more complex HMOs. In this study, we utilize GlcNAc and lactose as substrates, employing a whole-cell catalytic approach for the synthesis of LNT II. First, we have identified and successfully expressed three genes associated with the production of LNT II: Nahk, EcGlmU, and LgtA, synthesized LNT II through a whole-cell catalytic system utilizing multistrain coupling. Then, to further enhance the yield, we incorporated yeast cells for energy regeneration, employed coexpression strategies to minimize cell density, following a series of systematic optimizations resulting in a 7-fold increase in LNT II production. Finally, a two-step catalytic process was conducted in a 5L bioreactor, and the maximum LNT II production reached 52.34 g/L with a conversion rate of lactose 95.95%.