biostudies-literatureUnknown24(3)Du ZChina Postdoctoral Science FoundationHuman milk oligosaccharides (HMOs), the third most abundant solid component of human milk, are reported to be beneficial to infant health. The biosynthesis of lacto-<i>N</i>-biose (LNB), the building block for HMOs, suffers from excessive addition of cofactors and intermediate inhibition. Here, we developed an <i>in vitro</i> 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.iScience102236https://www.ebi.ac.uk/biostudies/studies/S-EPMC7967015biostudies-literatureLacto-<i>N</i>-biose synthesis via a modular enzymatic cascade with ATP regeneration.PMC7967015Fang XLiu ZTan YGuo WJia YNiu KDu ZfalseLacto-<i>N</i>-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-<i>N</i>-biose (LNB), the building block for HMOs, suffers from excessive addition of cofactors and intermediate inhibition. Here, we developed an <i>in vitro</i> 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.2021-01-01T00:00:00Z2021 Mar2026-04-16T15:03:43.628Z2025-02-19T02:14:11.388ZS-EPMC79670153374871810.1016/j.isci.2021.102236