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

Sialyllacto-<i>N</i>-tetraose a (LST-a, Neu5Ac<i>α</i>2,3Gal<i>β</i>1,3GlcNAc<i>β</i>1,3Gal<i>β</i>1,4Glc) 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 <i>β</i>1,3-<i>N</i>-acetylglucosaminyltransferase (<i>β</i>1,3-GlcNAcT), <i>β</i>1,3-galactosyltransferase (<i>β</i>1,3-GalT), and <i>α</i>2,3-sialyltransferase (<i>α</i>2,3-SiaT). In this study, <i><i>Escherichia coli</i></i> BL21(DE3) was engineered to produce LST-a using multilevel metabolic engineering strategies efficiently. <i>β</i>1,3-GlcNAcT and <i>β</i>1,3-GalT genes were integrated into the chromosome with multiple copies to produce LNT free of plasmids efficiently. Cytidine 5'-monophospho-<i>N</i>-acetylneuraminic acid and uridine 5'-diphospho-<i>N</i>-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.