Human milk oligosaccharides (HMOs) have been thought to play a role in the development of specific intestinal flora in breast-fed infants for many years. Nowadays it is known that they are also potent inhibitors of bacterial adhesion to epithelial surfaces (initial stage of the infection process). Oligosaccharides are not hydrolyzed in the upper small intestine and reach the large intestine intact, where they serve as substrates for bacterial metabolism. Thus, HMOs are considered as the ''dietary fiber''of human milk. Another characteristic of oligosaccharides is their proposed ''anti-infective effect''. This role is achieved thanks to their capacity to inhibit the adhesion of bacteria to the epithelial surfaces, thereby playing an important protective role against infection in the gastrointestinal, respiratory and urogenital tracts by direct and indirect mechanisms. Therefore, HMOs have antimicrobial activity and may be useful in treating and/or preventing specific enteric bacterial and viral infections. However, the road to convert HMOs into pharmaceuticals or nutritional substances has been blocked by the lack of pure, single component oligosaccharides from human milk in quantities large enough for scientific investigation, as well as preclinical tolerance and safety studies and for safety and clinical testing in populations that are exposed to gastrointestinal pathogens. Therefore, this proposed research program aims to develop practical processes to produce HMOs on multi-gram to kilo-gram scales. Since it was repeatedly reported that 2-linked fucosyloligosaccharides exhibited more antimicrobial activity than non-2-linked fucosyloligosaccharides, we will choose five 2-linked fucosyloligosaccharides such as 2'-FL, LNF-I, 2H-antigen, LDFH-I and Ley as our main targets. Moreover, non-2-linked fucosyloligosaccharides LNF-II and LNF-III will provide us the opportunity to confirm the observation of higher antimicrobial activity for 2-linked fucosyloligosaccharides. In addition, the non-fucosylated oligosaccharide LNT and LNnT will provide control experiments to evaluate the effect of fucose in oligosaccharides. Over the past 14 years, the Wang lab has been developing enzymatic oligosaccharides synthesis. We have invented and further developed the "superbeads" and "superbug" technology for large scale oligosaccharide production. The most efficient approach for oligosaccharide synthesis is to follow the natural carbohydrate biosynthetic pathway where oligosaccharides are assembled together by specific glycosyltransferases using individual sugar nucleotides as building blocks. These building blocks are themselves biosynthesized and recycled from individual monosaccharides through a series of biosynthetic enzymes. For small to medium scale synthesis of oligosaccharides, Wang has developed simple solid phase synthetic systems by immobilizing all the necessary biosynthetic enzymes onto a so-called "superbeads". These beads function as stable and versatile synthetic reagents, which can be used to synthesize a variety of glycoconjugates in cell-free systems. For large-scale production, Wang "superbug" essentially transfers the entire natural biosynthetic pathway into an E. coli strain. The approach includes cloning each enzyme along the biosynthetic pathway and connecting the genes of these enzymes together to produce an artificial gene cluster. A recombinant E. coli transformed with such a gene cluster is then used to produce the oligosaccharide through fermentation and purification. Thus, the "superbeads" and "superbug" approaches will be used in this program to produce the 9 oligosaccharides. Specifically, there are four aims: Aim I: Production of HMOs by immobilizing multiple enzymes (superbeads). This involves investigation on the necessary microbial glycosyltransferases, development of superbeads for UDP-GlcNAc, UDP-Gal and GDP-Fuc production, and combination of the glycosyltransferases with sugar nucleotide production to produce oligosaccharides. Aim II: Production of HMOs by recombinant E. coli (Superbug), which involves combination of the biosynthetic pathways of these HMOs into one or several recombinant E. coli strains. Aim III: Production of HMOs by GRAS (Generally Recognized as Safe) yeast cells. This new system will provide safer production system for HMOs synthesis. Aim IV: Characterization of the oligosaccharides through systematic biomedical and microbiome approaches in collaboration with other specialized laboratories, also in our own lab, with the advantage of multi-gram or kilo-gram scale neutral human milk oligosaccharides produced from this project. It is expected that the biosynthetic technology developed in Aim I - III will be transferred to biotech company(s) (such as the biotech startup Carbogene USA LLC which specializes in large scale oligosaccharide production) and GMP processes will be developed to produce the oligosaccharides in quantities large enough for preclinical studies of tolerance and safety, as well as for safety, dose-ranging, and efficacy trials in infants and children who are at high risk of exposure to gastrointestinal pathogens.