Identification and characterization of a novel α2,3-sialyltransferase from Basilea psittacipulmonis for...

3'-Sialyllactose (3'-SL), a key sialylated human milk oligosaccharide (HMO), possesses considerable potential in infant nutrition. Its efficient enzymatic synthesis relies on α2,3-sialyltransferases (α2,3-SiaTs), yet many reported enzymes are limited by poor soluble expression or low catalytic efficiency. In this study, a novel α2,3-sialyltransferase (designated IX) was identified from Basilea psittacipulmonis DSM 24701. The enzyme was heterologously expressed in Escherichia coli, purified, and biochemically characterized. IX exhibited optimal activity at pH 8.0 and 35 °C, with Mg²⁺ significantly enhancing its performance. In vitro, it achieved a 63% conversion of CMP-Neu5Ac to 3'-SL within 12 h. In vivo, IX supported a 3'-SL titer of 3.87 g/L, representing a 72% increase over a widely used enzyme (NST). Further knockout of competing pathway genes increased 3'-SL titers to 4.25 g/L in shake-flask culture and to 35.79 g/L in a 5-L fed-batch fermentation. These results establish IX as a promising and efficient biocatalyst for the scalable biosynthesis of 3'-SL.