Human milk oligosaccharides (HMOs), the third most abundant solid component of human milk, are reported to be beneficial to infant health (Eriksen et al., 2018; Han et al., 2012). The biosynthesis of Lacto-
N-biose (LNB), the building block for HMOs, suffers from excessive cofactors addition and intermediate inhibition (Nishimoto and Kitaoka, 2007; Nishimoto 2020). 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 ÎG'° decrease of 540 KJ/mol. Under the 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, about 0.96 mol/mol GlcNAc and 71.6 mg LNB g
-1 GlcNAc h
-1 of LNB yield were achieved in 100 mL reaction system. The synergistic strategy not only paves the way for producing LNB but also facilitates other chemicals with multienzyme cascades.
Reference:
Du, Z. Q.; Liu, Z. Y.; Tan, Y. S.; Niu, K. L.; Guo, W.; Jia, Y.Y.; Fang, X. (2021). Lacto-N-biose synthesis via a modular enzymatic cascade with ATP regeneration. iScience, 24(3), 102236.
Eriksen, K. G.; Christensen, S. H.; Lind, M. V.; and Michaelsen, K. F. (2018). Human milk composition and infant growth. Curr. Opin. Clin. Nutr. 21 (3), 200-206.
Han, N. S.; Kim, T. J.; Park, Y. C.; Kim, J.; and Seo, J. H. (2012). Biotechnological production of human milk oligosaccharides. Biotechnol. Adv. 30 (6), 1268-1278.
Nishimoto, M. (2020). Large scale production of lacto-N-biose I, a building block of type I human milk oligosaccharides, using sugar phosphorylases. Biosci. Biotech. Bioch. 84, 17-24.
Nishimoto, M. and Kitaoka, M. (2007). Practical preparation of lacto-N-biose I, a candidate for the Bifidus factor in human milk. Biosci. Biotech. Bioch. 71 (8), 2101-2104.
