Highly efficient biosynthesis of 6'-sialyllactose in a metabolically engineered plasmid-free Escherichia coli using a novel α2,6-sialyltransferase from Nicoletella semolina.

6'-Sialyllactose (6'-SL), one of the most prevalent sialylated human milk oligosaccharides (HMOs), has recently garnered significant attention due to its promising health effects for infants. In this study, the 6'-SL biosynthetic pathway in EZAK (E. coli BL21(DE3) ΔlacZΔnanAΔnanK) was initially constructed by introducing a plasmid expressing the precursor CMP-Neu5Ac synthesis pathway genes neuBCA. A novel α2,6-sialyltransferase Ev6ST (NCBI Reference Sequence: WP_132500470) was selected by introducing plasmids expressing various α2,6-sialyltransferase-encoding genes and subsequent comparisons of the yields of 6'-SL. Subsequently, by integrating neuBCA and ev6st individually or in combination on the chromosome of EZAK, the high-yielding plasmid-free strain EZAKBEP with an extracellular yield of 5.68 g/L. In the 5 L bioreactor, fed-batch fermentation resulted in an extracellular yield of 15.35 g/L of 6'-SL. This work successfully screened a high-efficiency α2,6-sialyltransferase Ev6ST and constructed a high-yielding strain EZAKBEP under plasmid-free conditions, which is the highest yield of shake flask fermentation to date, and provides some reference significance for subsequent research.