Metabolic Engineering of Saccharomyces cerevisiae for the Efficient Production of Human Milk Oligosaccharide, Lacto‑N‑neotetraose (LNnT).

Lacto-N-neotetraose (LNnT) is one of the important ingredients of human milk oligosaccharides, with various promising health effects for infants. In this study, the biosynthetic pathway of LNnT was constituted in GRAS (generally recognized as safe) host Saccharomyces cerevisiae by coexpressing β1,3-N-acetylglucosaminyltransferase (LgtA), β1,4-galactosyltransferase (LgtB), lactose permease (Lac12), and UDP galactose 4-epimerase (Gal10). The engineered yeast strain designated LN1, coexpressing all these enzymes, achieved an LNnT production level of 1.54 g/L. To further enhance LNnT yield, the flux through the pathway was improved by employing both protein fusion and modular assembly strategies. The assembly of LgtA and LgtB using short peptide tags resulted in a significant increase in LNnT titers, reaching 2.42 g/L. Finally, the LNnT titer reached 6.25 g/L under fed-batch fermentation conditions in a 500 mL flask, representing the highest reported production level of LNnT in S. cerevisiae to date.