Enhancement of Sialyllacto-N-tetraose a Biosynthesis in Escherichia coli BL21(DE3) by Combinatorial Metabolic Engineering Strategies

Sialyllacto-N-tetraose a (LST-a, Neu5Acα2,3Galβ1,3GlcNAcβ1,3Galβ1,4Glc) is a typical sialylated human milk oligosaccharide (HMO). Metabolic engineering synthesis of LTS-a should require the introduction of three key glycosyltransferases for the sequential glycosylation extension of lactose, including β1,3-N-acetylglucosaminyltransferase (β1,3-GlcNAcT), β1,3-galactosyltransferase (β1,3-GalT), and α2,3-sialyltransferase (α2,3-SiaT). In this study, Escherichia coli BL21(DE3) was engineered to produce LST-a using multilevel metabolic engineering strategies efficiently. β1,3-GlcNAcT and β1,3-GalT genes were integrated into the chromosome with multiple copies to produce LNT free of plasmids efficiently. Cytidine 5'-monophospho-N-acetylneuraminic acid and uridine 5'-diphospho-N-acetylglucosamine pathway genes were introduced via plasmids to improve the glycosyl donor supply. Ribosome Binding Site engineering and cytidine triphosphate regeneration were used to enhance LST-a production. Finally, the maximum titer of LST-a reached 2.238 and 8.178 g/L by shake-flask and fed-batch cultivation, respectively.