Simonsiella muelleri α2,6-sialyltransferase enables efficient biosynthesis of 6'-sialyllactose in both plasmid-dependent and plasmid-free engineered Escherichia coli.

6'-Sialyllactose (6'-SL) is the most abundant sialylated human milk oligosaccharide in breast milk. Its health effects have been widely confirmed, and its biological production attracts increasing attention. The metabolic pathway of 6'-SL synthesis is clear, and the typical metabolic engineering strategies for its production improvement have been reported. However, there are not many α2,6-sialyltransferase (α2,6-SiaT) candidates reported. In this study, α2,6-SiaT from Simonsiella muelleri ATCC 29453 was reported to be effective in vivo 6'-SL productivity in engineered Escherichia coli. Based on this α2,6-SiaT, a two-plasmid E. coli BL21(DE3) was constructed to produce 6'-SL. After plasmid combination optimization and RBS engineering, which optimizes gene expression and enhances target product production by designing and modifying ribosome binding site (RBS) sequences, the titer was enhanced to 3.405 and 21.020 g/L in shake-flask and fed-batch cultivation, respectively. In addition, E. coli MG1655 was engineered to produce 6'-SL by genomic multiple copy integration of pathway genes, and the fed-batch cultivation generated 17.220 g/L of 6'-SL without the use of a plasmid. This study provided a new α2,6-SiaT candidate for microbial synthesis of 6'-SL.