Engineering Cellobiose Phosphorylase toward Lactose with Its Application to the Biosynthesis of Lacto- <i>N</i> -Biose

Lacto- N -biose I (LNB), a core structural unit of human milk oligosaccharides, was usually fermented by microbial cell factories because its biosynthesis involved the supplies of ATP and UDP-sugars. Here we designed an in vitro ATP- and UDP-sugar-free enzymatic pathway for the biosynthesis of LNB from lactose and N -acetylglucosamine (GlcNAc). Thermoclostridium caenicola cellobiose phosphorylase was discovered to have a very high promiscuous activity of lactose phosphorylase. Its lactose phosphorylase activity was enhanced greatly by directed evolution, yielding the variant M2 (N654A/Y501A) having 1.56-fold higher lactose activity and doubled lactose/cellobiose specificity. The coenzyme-free three-enzyme molecular machine containing lactose phosphorylase M2, lacto- N -biose phosphorylase and polyphosphate glucokinase produced up to 115 g/L LNB from lactose and GlcNAc and exhibited the highest volumetric productivity of 14.4 g/L/h. This coenzyme-free multienzyme molecular machine could provide a cost-competitive platform for in vitro biomanufacturing of LNB.