Lacto-N-biose synthesis via a modular enzymatic cascade with ATP regeneration

Human milk oligosaccharides (HMOs), the third most abundant solid component of human milk, are reported to be beneficial to infant health. The biosynthesis of lacto-N-biose (LNB), the building block for HMOs, suffers from excessive addition of cofactors and intermediate inhibition. Here, we developed an in vitro multienzyme cascade composed of LNB module, ATP regeneration, and pyruvate oxidase-driven phosphate recycling to produce LNB. The integration between ATP regeneration and Pi alleviation increased the LNB conversion ratio and resulted in a ΔG'° decrease of 540 KJ/mol. Under optimal conditions, the LNB conversion ratio was improved from 0.34 to 0.83 mol/mol GlcNAc and the ATP addition decreased to 50%. Finally, 0.96 mol/mol GlcNAc and 71.6 mg LNB g^-1 GlcNAc h^-1 of LNB yield was achieved in a 100-mL reaction system. The synergistic strategy not only paves the way for producing LNB but also facilitates other chemicals with multienzyme cascades.