Identification and Engineering of a β-1,3-Galactosyltransferase for Efficient Lacto- N -Tetraose Production

Lacto-N-tetraose (LNT), a major human milk oligosaccharide, is of growing interest for functional food applications. Microbial production of LNT is limited by the low catalytic efficiency of β-1,3-galactosyltransferase (β-1,3-galT) and insufficient precursor supply. Here, we combined key enzyme screening and engineering with metabolic pathway optimization to enhance LNT biosynthesis in Escherichia coli (E. coli). Among the 3 identified β-1,3-galTs, PmgalT from Proteus mirabilis exhibited the highest activity and was further improved through computationally guided rational and semirational engineering. Genomic integration of the PmgalTV27I/V39I/Y147F-galE-galU module established the LNTII-to-LNT conversion pathway, while prsA integration enhanced PRPP supply, resulting in a plasmid-free production strain. In 3-L fed-batch fermentation, the engineered strain achieved 68.19 g/L LNT, representing the highest reported titer to date in E. coli using glucose as the sole carbon source. This work provides an effective strategy for high-level microbial production of LNT.