Cellular Coupling Fermentation Utilizing Engineered Escherichia coli and Yeast for Efficient Biosynthesis of...

Sialyllacto-N-tetraose a (LSTa) is a representative member of sialylated human milk oligosaccharides (HMOs) that exhibits distinctive physiological properties, including antiviral defense and modulation of intestinal immunity. Efficient synthesis of LSTa remains a central challenge in the research on complex sialylated HMOs. To address the challenges of large-scale production, this study screened a highly efficient α-2,3-sialyltransferase to establish a glycosyltransferase module in Escherichia coli, and it was functionally coupled with Saccharomyces cerevisiae for endogenous cytidine-5'-triphosphate (CTP) cofactor regeneration. LSTa was successfully synthesized via a modular biosynthesis strategy, utilizing N-acetylneuraminic acid (Neu5Ac) and lacto-N-tetraose (LNT) as substrates. Then through systematic optimization of induction parameters and catalytic reaction conditions, LSTa productivity and substrate conversion efficiency were substantially enhanced. Ultimately, a whole-cell catalytic process was implemented in a 5 L bioreactor, achieving a maximum titer of 38.03 g/L for LSTa, demonstrating the scalability and potential for the biosynthesis of complicated sialylated HMOs.