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-enzymecascade was developed in a single whole cell to produce 3'-SL. We constructed two compatibleplasmids with double cloning sites to co-express four genes. Different combinations were assessed toverify the optimal catalytic ability. Then, the conversion temperature, pH, and stability under theoptimal temperature and pH were investigated. Moreover, the optimal conversion conditions andsurfactant concentration were determined. By using the optimal conditions (35 C, pH 7.0, 20 mMpolyphosphate, 10 mM cytidine monophosphate (CMP), 20 mM MgCl2), 25 mL and 4 L conversionsystems were carried out to produce 3'-SL. Similar results were obtained between different volumeconversion reactions, which led the maximum production of 3'-SL to reach 53 mM from 54.2 mMof 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 systemexhibits great potential and economic competitiveness for the manufacture of 3'-SL.