Combination of Metabolic Engineering and High-throughput Screening to Realize High-Producing Lacto-N-triose II in Escherichia coli

Lacto-N-triose II (LNT II) exhibits diverse biological activities. It serves as the core structural unit of complex human milk oligosaccharides (HMOs) and is an essential precursor for synthesizing other HMO derivatives. In this study, an Escherichia coli (E. coli) strain with a high capacity for LNT II production was engineered. The best suitable key enzyme β-1,3-N-acetylglycoaminotransferase (LgtA) for LNT II synthesis was selected from different sources and modified with the addition of appropriate solubilizing tags. The rate-limiting genes of the LNT II synthesis pathway were identified, and various ribosome-binding sites were used to further fine-tune the expression levels of GlmS* and LgtA. The mutant LgtA^{R13H, L24M, and R205C}, which exhibits improved LNT II production, was screened using a lactose biosensor. As a result, the production of LNT II reached 57.44 ± 3.5 g/L in a 5 L fermenter, which was the highest production among E. coli strains by now.