Metabolomics-Guided Integration of a “Push–Pull-Restrain” Strategy for Enhanced LNnT Production in Saccharomyces cerevisiae

Lacto-N-neotetraose (LNnT) is a prominent neutral human milk oligosaccharide (HMO) with limited availability. Here, Saccharomyces cerevisiae (GRAS strain, Generally Recognized as Safe) was metabolically engineered to enhance LNnT production. Metabolomics analysis revealed that competition from chitin synthesis and insufficient metabolic driving force for heterologous glycosyltransferases in yeast constituted major metabolic bottlenecks. To overcome these limitations, a "push-pull-restraint" strategy was applied, integrating glycosyltransferase screening, protein tag fusion evaluation, targeted chitin disruption of chitin biosynthetic genes, and balanced modulation of UDP-Gal/UDP-GlcNAc supply. These interventions increased extracellular LNnT titer in shake-flask cultures from 14.65 mg/L to 1.66 g/L, with total LNnT reaching 2.01 g/L. Further scale-up in a 5 L bioreactor under fed-batch conditions resulted in a total LNnT titer of 4.70 g/L. These results demonstrate the potential for the production of LNnT by S. cerevisiae and open up an innovative framework for the effective synthesis of LNnT.