Sialo-oligosaccharides are important products of emerging biotechnology for complex carbohydrates as nutritional ingredients. Cascade bio-catalysis is central to the development of sialo-oligosaccharide production systems, based on isolated enzymes or whole cells. Multienzyme transformations have been established for sialooligosaccharide synthesis from expedient substrates, but systematic engineering analysis for the optimization of such transformations is lacking. Here, we show a mathematical modeling-guided approach to 3ʹ-sialyllactose (3SL) synthesis from N-acetyl-D-neuraminic acid (Neu5Ac) and lactose in the presence of cytidine 5ʹ-triphosphate, via the reactions of cytidine 5ʹ-monophosphate-Neu5Ac synthetase and α2,3-sialyltransferase. The Neu5Ac was synthesized in situ from N-acetyl-Dmannosamine using the reversible reaction with pyruvate by Neu5Ac lyase or the effectively irreversible reaction with phosphoenolpyruvate by Neu5Ac synthase.We show through comprehensive time-course study by experiment and modeling that, due to kinetic rather than thermodynamic advantages of the synthase reaction, the 3SL yield was increased (up to 75%; 10.4 g/L) and the initial productivity doubled (15 g/L/h), compared with synthesis based on the lyase reaction. We further show model-based optimization to minimize the total loading of protein (saving: up to 43%) while maintaining a suitable ratio of the individual enzyme activities to achieve 3SL target yield (61%-75%; 7-10 g/L) and overall productivity (3-5 g/L/h).Collectively, our results reveal the principal factors of enzyme cascade efficiency for 3SL synthesis and highlight the important role of engineering analysis to make multienzyme-catalyzed transformations fit for oligosaccharide production.