摘要：SummaryHuman 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 anin vitromultienzyme 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−1GlcNAc h−1of 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.Graphical abstractDisplay OmittedHighlights•Introducing an ATP recycling system improved LNB production efficiency•In situATP recycling fulfilled by combining pyruvate oxidase with acetate kinase•LNB yield reached 0.96 mol/mol GlcNAc, whereas ATP addition decreased to 50%Biological Sciences ; Biotechnology ; Biochemical Reactors
