Engineering Escherichia coli for the High-Titer Biosynthesis of Lacto- N-tetraose

Lacto-N-tetraose (LNT), a member of the human milk oligosaccharides family, has received widespread attention because of its importance in infant health. We constructed a whole-cell biotransformation method in Escherichia coli BL21(DE3) for high-titer LNT synthesis. The approach was performed by using a systematic design and metabolic engineering based on the metabolic pathway of LNT. The lgtA (encoding β-1,3-N-acetylglucosaminyltransferase) and wbgO (encoding β-1,3-galactosyltransferase) genes were introduced into the engineered E. coli BL21(DE3) to construct an LNT-producing starting strain B1 (0.22 g/L). Then, the genes related to the LNT metabolic pathway were screened in two vectors to evaluate LNT synthesis. The lgtA-wbgO and galE-galT-galK genes were overexpressed through the two-plasmid system in E. coli BL21(DE3). The titer of LNT (3.42 g/L) had a gain of 14.55 times compared with that of B1. Furthermore, the ugd gene, which was associated with the UDP-Gal bypass pathway, was inactivated to further improve LNT production in shake-flask cultivation (4.14 g/L). The final fed-batch cultivation of the engineered strain produced 31.56 g/L of LNT. This study provided a strategy for the effective production of LNT in E. coli.