Reconstitution of human milk oligosaccharide biosynthesis in cultured mammalian cells

Human milk oligosaccharides (HMOs) are complex sugars that play crucial roles in infant health by supporting the development of the gut microbiota and the immune system. HMOs are structurally based on lactose, which serves as the core disaccharide. These HMOs are synthesized in mammary epithelial cells through sequential glycosylation reactions catalyzed by specific glycosyltransferases, whereas little to no HMO production has been observed in other cell types and commonly used cultured mammalian cell lines. In this study, we reconstituted the biosynthesis of HMOs in cultured cells by applying targeted glycoengineering strategies. HEK293 cells expressing α-lactalbumin (LALBA) gene produced various HMOs including 3'-sialyllactose, 2'-fucosyllactose, sialyllacto-N-tetraose a/d, and disialyllacto-N-tetraose. We showed that different cell lines, such as HCT116 and CHO-K1 cells, can produce HMOs when engineered to express LALBA. By manipulating genes like B4GALT1, B3GNT2, and B3GALT5, we altered the complexity and composition of the HMOs produced. By supplementing the culture medium with N-azidoacetylmannosamine, we achieved efficient metabolic incorporation of azido groups into 3'-sialyllactose. Furthermore, HMOs produced by HEK293 cells promoted the growth of Bifidobacterium, demonstrating prebiotic effects. Our findings highlight the potential to tailor HMO production in vitro, offering a platform for generating these bioactive molecules.