Infant Gut Microbial Metagenome Mining of α-L-Fucosidases with Activity on Fucosylated Human Milk Oligosaccharides and Glycoconjugates

The gastrointestinal microbiota members produce alpha-L-fucosidases that play key roles in mucosal, human milk, and dietary oligosaccharide assimilation. Here, 36 open reading frames (ORFs) coding for putative alpha-L-fucosidases belonging to glycosyl hydrolase family 29 (GH29) were identified through metagenome analysis of breast-fed infant fecal microbiome. Twenty-two of those ORFs showed a complete coding sequence with deduced amino acid sequences displaying the highest degree of identity with alpha-L-fucosidases from Bacteroides thetaiotaomicron, Bacteroides caccae, Phocaeicola vulgatus, Phocaeicola dorei, Ruminococcus gnavus, and Streptococcus parasanguinis. Based on sequence homology, 10 alpha-L-fucosidase genes were selected for substrate specificity characterization. The alpha-L-fucosidases Fuc18, Fuc19A, Fuc35B, Fuc39, and Fuc1584 showed hydrolytic activity on alpha 1,3/4-linked fucose present in Lewis blood antigens and the human milk oligosaccharide (HMO) 3-fucosyllactose. In addition, Fuc1584 also hydrolyzed fucosyl-alpha-1,6-N-acetylglucosamine (6FN), a component of the core fucosylation of N-glycans. Fuc35A and Fuc193 showed activity on alpha 1,2/3/4/6 linkages from H type-2, Lewis blood antigens, HMOs and 6FN. Fuc30 displayed activity only on alpha 1,6-linked L-fucose, and Fuc5372 showed a preference for alpha 1,2 linkages. Fuc2358 exhibited a broad substrate specificity releasing L-fucose from all the tested free histo-blood group antigens, HMOs, and 6FN. This latest enzyme also displayed activity in glycoconjugates carrying lacto-N-fucopentaose II (Le(a)) and lacto-N-fucopentaose III (Le(x)) and in the glycoprotein mucin. Fuc18, Fuc19A, and Fuc39 also removed L-fucose from neoglycoproteins and human alpha-1 acid glycoprotein. These results give insight into the great diversity of alpha-L-fucosidases from the infant gut microbiota, thus supporting the hypothesis that fucosylated glycans are crucial for shaping the newborn microbiota composition. IMPORTANCE alpha-L-Fucosyl residues are frequently present in many relevant glycans, such as human milk oligosaccharides (HMOs), histo-blood group antigens (HBGAs), and epitopes on cell surface glycoconjugate receptors. These fucosylated glycans are involved in a number of mammalian physiological processes, including adhesion of pathogens and immune responses. The modulation of L-fucose content in such processes may provide new insights and knowledge regarding molecular interactions and may help to devise new therapeutic strategies. Microbial alpha-L-fucosidases are exoglycosidases that remove alpha-L-fucosyl residues from free oligosaccharides and glycoconjugates and can be also used in transglycosylation reactions to synthesize oligosaccharides. In this work, alpha-L-fucosidases from the GH29 family were identified and characterized from the metagenome of fecal samples of breastfed infants. These enzymes showed different substrate specificities toward HMOs, HBGAs, naturally occurring glycoproteins, and neoglycoproteins. These novel glycosidase enzymes from the breast-fed infant gut microbiota, which resulted in a good source of alpha-L-fucosidases, have great biotechnological potential.