Highly efficient production of lacto-N-tetraose in plasmid-free Escherichia coli through chromosomal integration of multicopy key glycosyltransferase genes.

Lacto-N-tetraose (LNT) is a functional human milk oligosaccharide (HMO) commercially added to infant formula. Metabolically engineered strains for efficient production of LNT have been widely constructed. However, most of them rely on the use of plasmids, which might bring metabolic burden and the antibiotic issue. In this study, we attempted to construct a plasmid-free Escherichia coli MG1655 for LNT biosynthesis. Firstly, lacZ gene was disrupted and lacY expression was enhanced to improve the bioavailability of lactose as the initial substrate. Three copies of lgtA (encoding for β1,3-N-acetylglucosaminyltransferase) were integrated into the chromosome, enabling the highly efficient production of lacto-N-triose II (LNTri II) as the direct precursor of LNT. Efficient production of LNT was then optimized by multicopy integration of wbgO (encoding for β1,3-galactosyltransferase), disruption of the competitive pathways, and strengthening of UDP-galactose supply and oligosaccharide efflux. The final titer reached 6.99 and 42.38 g/L in shake-flask and fed-batch cultivation, respectively.