摘要Lacto-N-neotetraose(LNnT)is a crucial neutral core human milk oligosaccharide(HMO).In this study,we established a LNnT-producing Saccharomyces cerevisiae cell factory through comprehensive metabolic engineering.Specifically,the de novo biosynthetic pathway of LNnT was assembled by heterologously expressing the lactose permease(lac 12)from Kluyveromyces lactis and the glycosyltransferase from Neisseria meningitidis in S.cerevisiae.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 8-fold increase in LNnT titer compared to that of parental strain.To our knowledge,this is the first report on LNnT biosynthesis in S.cerevisiae,opening up the possibility of green production of LNnT using food-safe microorganisms.