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

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.