Multi-Enzymatic Cascade One-Pot Biosynthesis of 30-Sialyllactose Using Engineered Escherichia coli

Among the human milk oligosaccharides (HMOs), one of the most abundantoligosaccharides and has great benefits for human health is 3'-sialyllactose (3'-SL). Given its importantphysiological functions and the lack of cost-effective production processes, we constructed an in vitromulti-enzymatic cofactor recycling system for the biosynthesis of 3'-SL from a low-cost substrate.First, we constructed the biosynthetic pathway and increased the solubility of cytidine monophosphatekinase (CMK) with chaperones. We subsequently identified that -galactosidase (lacZ) affects theyield of 3'-SL, and hence with the lacZ gene knocked out, a 3.3-fold increase in the production of30-SL was observed. Further, temperature, pH, polyphosphate concentration, and concentrationof divalent metal ions for 30-SL production were optimized. Finally, an efficient biotransformationsystem was established under the optimized conditions. The maximum production of 3'-SL reached38.7 mM, and a molar yield of 97.1% from N-acetylneuraminic acid (NeuAc, sialic acid, SA) wasobtained. The results demonstrate that the multi-enzymatic cascade biosynthetic pathway withcofactor regeneration holds promise as an industrial strategy for producing 3'-SL.