Efficient Production of 3′-Sialyllactose by Single Whole-Cell in One-Pot Biosynthesis

Sialyllactose (SL) is one of the most important acidic oligosaccharides in human milk, which plays an important role in the health of infants. In this work, an efficient multi-enzyme cascade was developed in a single whole cell to produce 3′-SL. We constructed two compatible plasmids with double cloning sites to co-express four genes. Different combinations were assessed to verify the optimal catalytic ability. Then, the conversion temperature, pH, and stability under the optimal temperature and pH were investigated. Moreover, the optimal conversion conditions and surfactant concentration were determined. By using the optimal conditions (35 °C, pH 7.0, 20 mM polyphosphate, 10 mM cytidine monophosphate (CMP), 20 mM MgCl2), 25 mL and 4 L conversion systems were carried out to produce 3′-SL. Similar results were obtained between different volume conversion reactions, which led the maximum production of 3′-SL to reach 53 mM from 54.2 mM of sialic acid (SA) in the 25 mL system and 52.8 mM of 3′-SL from 53.8 mM of SA in the 4 L system. These encouraging results demonstrate that the developed single whole-cell multi-enzyme system exhibits great potential and economic competitiveness for the manufacture of 3′-SL.