Biosynthesis of Lacto-N-biose I from starch and N-acetylglucosamine via an in vitro synthetic enzymatic biosystem

Human milk oligosaccharides (HMOs) are very distinctive components in human milk and are beneficial for infant health. Lacto-N-biose I (LNB) is the core structural unit of HMOs, which could be used for the synthesis of other HMOs. In this study, an ATP-free in vitro synthetic enzymatic biosystem contained four thermostable enzymes (alpha-glucan phosphorylase from Thermococcus kodakarensis, UDP-glucose-hexose-1-phosphate uri-dylyltransferase from Thermotoga maritima, UDP-glucose 4-epimerase from T. maritima, lacto-N-biose phos-phorylase from Clostridium thermobutyricum) were constructed for the biosynthesis of LNB from starch and N-acetylglucosamine (GlcNAc). Under the optimal conditions, 0.75 g/L and 2.23 g/L LNB was produced from 1.1 g/ L and 4.4 g/L GlcNAc and excess starch, with the molar yield of 39% and 29% based on the GlcNAc concen-tration, respectively, confirming the feasibility of this in vitro synthetic enzymatic biosystem for LNB synthesis and shedding light on the biosynthesis of other HMOs using LNB as the core structural unit from low cost polysaccharides.

Automated summary

A non-ATP-dependent in vitro synthetic enzymatic biosystem was constructed to produce LNB, a core structural unit of HMOs, from starch and GlcNAc. The study demonstrated the feasibility of this biosystem for the synthesis of LNB and shed light on the biosynthesis of other HMOs using low-cost polysaccharides.