<i>De Novo</i> Synthesis of Lacto-<i>N</i>-Neotetraose in <i>Escherichia coli</i> through Metabolic Engineering with Glucose as the Sole Carbon Source

Lacto-<i>N</i>-neotetraose (LNnT), the predominant nonfucosylated human milk oligosaccharide (HMO), significantly enhances the immune function of intestinal epithelial cells and prevents necrotizing enterocolitis. However, current LNnT synthesis processes depend on exogenous lactose supplementation, leading to increased production costs and operational complexity. In this study, we established <i>de novo</i> LNnT synthesis by engineering an endogenous lactose biosynthesis pathway with glucose as the sole carbon source. First, we established the endogenous lactose synthesis pathway. Second, we optimized the metabolic network balance and reduced metabolic flux branching by modulating the glucose phosphorylation efficiency and fine-tuning the expression levels of key synthesis modules. Following this, LNnT production was increased through targeted enhancement of the glucose transport efficiency and UDP-Gal synthesis optimization. As a result, we developed a high-yielding <i>E. coli</i> engineered strain for LNnT production. The engineered strain SC16-14 achieved an LNnT titer of 25.4 g/L during 79 h of fermentation in a 3-L bioreactor. This study not only demonstrates the feasibility of endogenous lactose synthesis but also provides a novel strategy to reduce production costs and simplify the LNnT manufacturing process.