<i>De novo</i> production of lacto- <i>N</i> -neotetraose by metabolic engineering of <i>Saccharomyces cerevisiae</i> as a cell factory

Lacto-N-neotetraose (LNnT) is a crucial neutral core human milk oligosaccharide (HMO). In this study, we established a LNnT-producing <em>Saccharomyces cerevisiae</em> cell factory was established through comprehensive metabolic engineering. Specifically, the <em>de novo</em> biosynthetic pathway of LNnT was assembled by heterologously expressing the lactose permease (Lac12) from <em>Kluyveromyces lactis </em>and the glycosyltransferase from <em>Neisseria meningitid</em><em>is</em> in <em>Saccharomyces cerevisiae</em>. Subsequently, carbon source regulation based on the glucose-sensitive GAL regulatory system was employed to optimize the expression time of heterologous genes, achieving a production of 15.61 mg/L of LNnT in shake-flask fermentation. In addition, the key rate-limiting steps involved in LNnT synthesis pathway were identified and the corresponding genes were overexpressed to enhance LNnT production, resulting in an eight-fold increase in LNnT titer compared to that of parental strain. To our knowledge, this is the first report on LNnT biosynthesis in <em>Saccharomyces cerevisiae</em>, opening up the possibility of green production of LNnT using food-safe microorganisms.