Engineering the Metabolic Pathway of Escherichia coli for Efficient Biosynthesis of Lacto- N -tetraose

Abstract Lacto-N-tetraose (LNT), a core component of human milk oligosaccharides (HMOs), plays essential biological roles; however, its production remains challenging due to low conversion efficiency and the accumulation of intermediates. In this study, a systematic metabolic engineering strategy was applied to Escherichia coli BL21(DE3) to enhance LNT production. Key approaches included CRISPR-Cas9-mediated host optimization, chromosomal integration of critical pathway genes, and systematic optimization of fermentation conditions. As a result, the engineered strain achieved an LNT titer of 36.29 g/L, with a molar conversion rate of 95.6% from the intermediate lacto-N-triose II to LNT, representing the highest conversion efficiency reported to date for LNT biosynthesis in a single microbial strain. This integrated strategy significantly improves production efficiency while reducing the burden of downstream purification, providing a robust technological framework for the industrial biosynthesis of LNT and other high-value oligosaccharides.