Elimination of Residual Lacto-N-triose II for Lacto-N-tetraose Biosynthesis in Engineered Escherichia coli

Lacto-N-tetraose (LNT) is an important neutral human milk oligosaccharide (HMO) and acts as a significant core structure for complex HMO biosynthesis. We previously achieved high-yield LNT biosynthesis (57.5 g/L) using fed-batch fermentation; however, residual lacto-N-triose II (LNTri II) was also found (21.58 g/L). Here, we re-engineered an efficient LNT-producing Escherichia coli with low LNTri II accumulation using genetically stable LNTri II-producing strains with a genomic insertion of lgtA (encoding β1,3-N-acetylglucosaminyltransferase). Comparable and low titers of LNT (3.73-4.61 g/L) and LNTri II (0.33-0.63 g/L), respectively, were obtained by introducing β1,3-galactosyltransferase. To reduce residual LNTri II, the E. coli transporter gene setA was disrupted, obviously reducing the accumulation of LNTri II and LNT. Next, the gene encoding β-N-acetylhexosaminidase (BbhI) was introduced into LNT-producing strains or E. coli BL21(DE3) for single- or mixed-strain cultivation, respectively. Finally, LNT was obtained (30.13 g/L) in a cocultivation system of mixed engineered strains without undesired LNTri II.