Improving 3′-sialyllactose biosynthesis in Escherichia coli by engineering Neisseria meningitidis 406Y α2,3-sialyltransferase

Abstract

3′-Sialyllactose (3′-SL, Neu5Acα2,3 Galβ1,4Glc), a prominent sialylated human milk oligosaccharide (HMO), has attracted significant attention due to its diverse physiological properties. The efficient α2,3-sialyltransferase (α2,3-SiaT) is crucial for the biosynthesis of 3′-SL. In this study, the 3′-SL biosynthetic pathway was constructed in EZAK (E. coli BL21(DE3) ΔlacZΔnanAΔnanK). 406NST, which exhibits a high 3′-SL yield and low by-product formation, was chosen for molecular modification. Five amino acid differences between 406NST and NST were targeted for site-directed mutagenesis. Subsequently, saturation mutagenesis was carried out at the D40 position, followed by superimposed multipoint mutagenesis to generate the optimal strain Z28 (406NST-D40R-N113D-P120H), resulting in an extracellular 3′-SL yield of 4.67 g/L in shake flasks. In a 5 L bioreactor, the extracellular 3′-SL yield reached 29.54 g/L, achieving an overall 3′-SL yield of 0.52 g/L/h and a lactose conversion yield of 0.62 mol 3′-SL/mol. In conclusion, a highly active and specific α2,3-SiaT was successfully constructed, significantly improving the yield of 3′-SL.