Metabolism Of Human Milk Oligosaccharides Research Articles

Genome-scale metabolic modeling of the human milk oligosaccharide utilization by Bifidobacterium longum subsp. infantis.

Bifidobacterium longum subsp. infantis is a representative and dominant species in the infant gut and is considered a beneficial microbe. This organism displays multiple adaptations to thrive in the infant gut, regarded as a model for human milk oligosaccharides (HMOs) utilization. These carbohydrates are abundant in breast milk and include different molecules based on lactose. They contain fucose, sialic acid, and N-acetylglucosamine. Bifidobacterium metabolism is complex, and a systems view of relevant metabolic pathways and exchange metabolites during HMO consumption is missing. To address this limitation, a refined genome-scale network reconstruction of this bacterium is presented using a previous reconstruction of B. infantis ATCC 15967 as a template. The latter was expanded based on an extensive revision of genome annotations, current literature, and transcriptomic data integration. The metabolic reconstruction (iLR578) accounted for 578 genes, 1,047 reactions, and 924 metabolites. Starting from this reconstruction, we built context-specific genome-scale metabolic models using RNA-seq data from cultures growing in lactose and three HMOs. The models revealed notable differences in HMO metabolism depending on the functional characteristics of the substrates. Particularly, fucosyl-lactose showed a divergent metabolism due to a fucose moiety. High yields of lactate and acetate were predicted under growth rate maximization in all conditions, whereas formate, ethanol, and 1,2-propanediol were substantially lower. Similar results were also obtained under near-optimal growth on each substrate when varying the empirically observed acetate-to-lactate production ratio. Model predictions displayed reasonable agreement between central carbon metabolism fluxes and expression data across all conditions. Flux coupling analysis revealed additional connections between succinate exchange and arginine and sulfate metabolism and a strong coupling between central carbon reactions and adenine metabolism. More importantly, specific networks of coupled reactions under each carbon source were derived and analyzed. Overall, the presented network reconstruction constitutes a valuable platform for probing the metabolism of this prominent infant gut bifidobacteria.IMPORTANCEThis work presents a detailed reconstruction of the metabolism of Bifidobacterium longum subsp. infantis, a prominent member of the infant gut microbiome, providing a systems view of its metabolism of human milk oligosaccharides.

Infant microbiome cultivation and metagenomic analysis reveal Bifidobacterium 2’-fucosyllactose utilization can be facilitated by coexisting species

The early-life gut microbiome development has long-term health impacts and can be influenced by factors such as infant diet. Human milk oligosaccharides (HMOs), an essential component of breast milk that can only be metabolized by some beneficial gut microorganisms, ensure proper gut microbiome establishment and infant development. However, how HMOs are metabolized by gut microbiomes is not fully elucidated. Isolate studies have revealed the genetic basis for HMO metabolism, but they exclude the possibility of HMO assimilation via synergistic interactions involving multiple organisms. Here, we investigate microbiome responses to 2’-fucosyllactose (2’FL), a prevalent HMO and a common infant formula additive, by establishing individualized microbiomes using fecal samples from three infants as the inocula. Bifidobacterium breve, a prominent member of infant microbiomes, typically cannot metabolize 2’FL. Using metagenomic data, we predict that extracellular fucosidases encoded by co-existing members such as Ruminococcus gnavus initiate 2’FL breakdown, thus critical for B. breve’s growth. Using both targeted co-cultures and by supplementation of R. gnavus into one microbiome, we show that R. gnavus can promote extensive growth of B. breve through the release of lactose from 2’FL. Overall, microbiome cultivation combined with genome-resolved metagenomics demonstrates that HMO utilization can vary with an individual’s microbiome.

Molecular strategies for the utilisation of human milk oligosaccharides by infant gut-associated bacteria.

A number of bacterial species are found in high abundance in the faeces of healthy breast-fed infants, an occurrence that is understood to be, at least in part, due to the ability of these bacteria to metabolize human milk oligosaccharides (HMOs). HMOs are the third most abundant component of human milk after lactose and lipids, and represent complex sugars which possess unique structural diversity and are resistant to infant gastrointestinal digestion. Thus, these sugars reach the infant distal intestine intact, thereby serving as a fermentable substrate for specific intestinal microbes, including Firmicutes, Proteobacteria, and especially infant-associated Bifidobacterium spp. which help to shape the infant gut microbiome. Bacteria utilising HMOs are equipped with genes associated with their degradation and a number of carbohydrate-active enzymes known as glycoside hydrolase enzymes have been identified in the infant gut, which supports this hypothesis. The resulting degraded HMOs can also be used as growth substrates for other infant gut bacteria present in a microbe-microbe interaction known as 'cross-feeding'. This review describes the current knowledge on HMO metabolism by particular infant gut-associated bacteria, many of which are currently used as commercial probiotics, including the distinct strategies employed by individual species for HMO utilisation.

Role of human milk oligosaccharide metabolizing bacteria in the development of atopic dermatitis/eczema.

Despite affecting up to 20% of infants in the United States, there is no cure for atopic dermatitis (AD), also known as eczema. Atopy usually manifests during the first six months of an infant's life and is one predictor of later allergic health problems. A diet of human milk may offer protection against developing atopic dermatitis. One milk component, human milk oligosaccharides (HMOs), plays an important role as a prebiotic in establishing the infant gut microbiome and has immunomodulatory effects on the infant immune system. The purpose of this review is to summarize the available information about bacterial members of the intestinal microbiota capable of metabolizing HMOs, the bacterial genes or metabolic products present in the intestinal tract during early life, and the relationship of these genes and metabolic products to the development of AD/eczema in infants. We find that specific HMO metabolism gene sets and the metabolites produced by HMO metabolizing bacteria may enable the protective role of human milk against the development of atopy because of interactions with the immune system. We also identify areas for additional research to further elucidate the relationship between the human milk metabolizing bacteria and atopy. Detailed metagenomic studies of the infant gut microbiota and its associated metabolomes are essential for characterizing the potential impact of human milk-feeding on the development of atopic dermatitis.

Comparative genomic analysis revealed genetic divergence between Bifidobacterium catenulatum subspecies present in infant versus adult guts

BackgroundThe two subspecies of Bifidobacterium catenulatum, B. catenulatum subsp. kashiwanohense and B. catenulatum subsp. catenulatum, are usually from the infant and adult gut, respectively. However, the genomic analysis of their functional difference and genetic divergence has been rare. Here, 16 B. catenulatum strains, including 2 newly sequenced strains, were analysed through comparative genomics.ResultsA phylogenetic tree based on 785 core genes indicated that the two subspecies of B. catenulatum were significantly separated. The comparison of genomic characteristics revealed that the two subspecies had significantly different genomic sizes (p < 0.05) but similar GC contents. The functional comparison revealed the most significant difference in genes of carbohydrate utilisation. Carbohydrate-active enzymes (CAZyme) present two clustering patterns in B. catenulatum. The B. catenulatum subsp. kashiwanohense specially including the glycoside hydrolases 95 (GH95) and carbohydrate-binding modules 51 (CBM51) families involved in the metabolism of human milk oligosaccharides (HMO) common in infants, also, the corresponding fucosylated HMO gene clusters were detected. Meanwhile, B. catenulatum subsp. catenulatum rich in GH3 may metabolise more plant-derived glycan in the adult intestine.ConclusionsThese findings provide genomic evidence of carbohydrate utilisation bias, which may be a key cause of the genetic divergence of two B. catenulatum subspecies.

How far is it from infant formula to human milk? A look at the human milk oligosaccharides

Human milk is the gold standard for the nutrition of infants. Human milk oligosaccharides (HMOs) are structurally diverse sugars highly abundant in human milk but not present in infant formula. The interest in HMOs has gradually increased in recent years due to their beneficial functions on infants. Here we summarize the structures and composition of HMOs, and compare the differences of oligosaccharides in human and other mammals. The absorption and metabolism of HMOs are discussed. Then we discuss existing studies on the functional biology of HMOs. Additionally, the structure-function relationships of HMOs are presented. Finally, this review summarizes the oligosaccharides in recent infant formula as well as limitations and future perspectives of HMOs research. The structures and composition of HMOs are affected by many factors. Large amounts of HMOs are metabolized by gut microbiota, and few are absorbed into circulation to perform physiological functions. Evidences from in vitro and in vivo studies, as well as clinical trials suggest that HMOs modulate gut microbiota, reduce pathogen adhesion, modulate epithelial cell and immune responses, reduce the risk of necrotizing enterocolitis and promote brain development and cognition. The specific structure determines different functions. Currently two HMOs, 2′-fucosyllactose and Lacto- N -Neotetraose, have been demonstrated safe and well-tolerated to be added into infant formula. However, the application in HMOs has been limited due to their insufficient availability. Thus, basic and clinical studies of HMOs need to be further elucidated. • The structures and composition of human milk oligosaccharides are summarized. • The absorption and metabolism of human milk oligosaccharides are discussed. • The biological functions of human milk oligosaccharides are reviewed. • The structure-function relationships of human milk oligosaccharides are presented. • The limitations and future perspectives of HMOs research are outlined.

Dynamic Changes in Human Milk Oligosaccharides in Chinese Population: A Systematic Review and Meta-Analysis.

The aim of this systematic review was to summarize concentrations of human milk oligosaccharides (HMOs) in the Chinese population. We searched articles originally published in both Chinese and English. When compiling data, lactation was categorized into five stages. We found that 6′-sialyllactose, lacto-N-tetraose, and lacto-N-neotetraose decreased over lactation. Conversely, 3′-fucosyllactose increased over lactation. Our study represents the first systematic review to summarize HMO concentrations in Chinese population. Our findings not only provide data on HMO profiles in Chinese population but suggest future directions in the study of the metabolism of HMOs.

The association between breastmilk oligosaccharides and faecal microbiota in healthy breastfed infants at two, six, and twelve weeks of age

Several factors affect gut microbiota development in early life, among which breastfeeding plays a key role. We followed 24 mother-infant pairs to investigate the associations between concentrations of selected human milk oligosaccharides (HMOs) in breastmilk, infant faeces, and the faecal microbiota composition in healthy, breastfed infants at two, six and 12 weeks of age. Lactation duration had a significant effect on breastmilk HMO content, which decreased with time, except for 3-fucosyllactose (3FL) and Lacto-N-fucopentaose III (LNFP III). We confirmed that microbiota composition was strongly influenced by infant age and was associated with mode of delivery and breastmilk LNFP III concentration at two weeks, with infant sex, delivery mode, and concentrations of 3′sialyllactose (3′SL) in milk at six weeks, and infant sex and Lacto-N-hexaose (LNH) in milk at 12 weeks of age. Correlations between levels of individual breastmilk HMOs and relative abundance of OTUs found in infant faeces, including the most predominant Bifidobacterium OTUs, were weak and varied with age. The faecal concentration of HMOs decreased with age and were strongly and negatively correlated with relative abundance of OTUs within genera Bifidobacterium, Parabacteroides, Escherichia-Shigella, Bacteroides, Actinomyces, Veillonella, Lachnospiraceae Incertae Sedis, and Erysipelotrichaceae Incertae Sedis, indicating the likely importance of these taxa for HMO metabolism in vivo.

Breast milk-derived human milk oligosaccharides promote Bifidobacterium interactions within a single ecosystem

Diet-microbe interactions play an important role in modulating the early-life microbiota, with Bifidobacterium strains and species dominating the gut of breast-fed infants. Here, we sought to explore how infant diet drives distinct bifidobacterial community composition and dynamics within individual infant ecosystems. Genomic characterisation of 19 strains isolated from breast-fed infants revealed a diverse genomic architecture enriched in carbohydrate metabolism genes, which was distinct to each strain, but collectively formed a pangenome across infants. Presence of gene clusters implicated in digestion of human milk oligosaccharides (HMOs) varied between species, with growth studies indicating that within single infants there were differences in the ability to utilise 2′FL and LNnT HMOs between strains. Cross-feeding experiments were performed with HMO degraders and non-HMO users (using spent or ‘conditioned’ media and direct co-culture). Further 1H-NMR analysis identified fucose, galactose, acetate, and N-acetylglucosamine as key by-products of HMO metabolism; as demonstrated by modest growth of non-HMO users on spend media from HMO metabolism. These experiments indicate how HMO metabolism permits the sharing of resources to maximise nutrient consumption from the diet and highlights the cooperative nature of bifidobacterial strains and their role as ‘foundation’ species in the infant ecosystem. The intra- and inter-infant bifidobacterial community behaviour may contribute to the diversity and dominance of Bifidobacterium in early life and suggests avenues for future development of new diet and microbiota-based therapies to promote infant health.

Novel Genes and Metabolite Trends in Bifidobacterium longum subsp. infantis Bi-26 Metabolism of Human Milk Oligosaccharide 2\u2032-fucosyllactose

Human milk oligosaccharides (HMOs) function as prebiotics for beneficial bacteria in the developing gut, often dominated by Bifidobacterium spp. To understand the relationship between bifidobacteria utilizing HMOs and how the metabolites that are produced could affect the host, we analyzed the metabolism of HMO 2′-fucosyllactose (2′-FL) in Bifidobacterium longum subsp. infantis Bi-26. RNA-seq and metabolite analysis (NMR/GCMS) was performed on samples at early (A600 = 0.25), mid-log (0.5–0.7) and late-log phases (1.0–2.0) of growth. Transcriptomic analysis revealed many gene clusters including three novel ABC-type sugar transport clusters to be upregulated in Bi-26 involved in processing of 2′-FL along with metabolism of its monomers glucose, fucose and galactose. Metabolite data confirmed the production of formate, acetate, 1,2-propanediol, lactate and cleaving of fucose from 2′-FL. The formation of acetate, formate, and lactate showed how the cell uses metabolites during fermentation to produce higher levels of ATP (mid-log compared to other stages) or generate cofactors to balance redox. We concluded that 2′-FL metabolism is a complex process involving multiple gene clusters, that produce a more diverse metabolite profile compared to lactose. These results provide valuable insight on the mode-of-action of 2′-FL utilization by Bifidobacterium longum subsp. infantis Bi-26.

Inefficient Metabolism of the Human Milk Oligosaccharides Lacto-N-tetraose and Lacto-N-neotetraose Shifts Bifidobacterium longum subsp. infantis Physiology.

Human milk contains a high concentration of indigestible oligosaccharides, which likely mediated the coevolution of the nursing infant with its gut microbiome. Specifically, Bifidobacterium longum subsp. infantis (B. infantis) often colonizes the infant gut and utilizes these human milk oligosaccharides (HMOs) to enrich their abundance. In this study, the physiology and mechanisms underlying B. infantis utilization of two HMO isomers lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) was investigated in addition to their carbohydrate constituents. Both LNT and LNnT utilization induced a significant shift in the ratio of secreted acetate to lactate (1.7–2.0) in contrast to the catabolism of their component carbohydrates (~1.5). Inefficient metabolism of LNnT prompts B. infantis to shunt carbon toward formic acid and ethanol secretion. The global transcriptome presents genomic features differentially expressed to catabolize these two HMO species that vary by a single glycosidic linkage. Furthermore, a measure of strain-level variation exists between B. infantis isolates. Regardless of strain, inefficient HMO metabolism induces the metabolic shift toward formic acid and ethanol production. Furthermore, bifidobacterial metabolites reduced LPS-induced inflammation in a cell culture model. Thus, differential metabolism of milk glycans potentially drives the emergent physiology of host-microbial interactions to impact infant health.

Compositional Analysis and Metabolism of Human Milk Oligosaccharides in Infants.

It is a great success that biotechnological means are available today to produce amounts of single human milk oligosaccharides (HMOs) in a purity which allows performing metabolic and functional studies even in humans. As recent data indicate that there is a link between the Lewis blood group and the secretor status of an individual and certain inflammatory diseases, this review will also focus on the metabolic fate of secretor- and Lewis blood group-specific components. We conclude that there is no simple urinary or fecal excretion pattern of HMOs, although the pattern in urine often reflects the mother's secretor/nonsecretor status. However, there are deviations for single HMOs which deserve special attention. In feces, the variation in excretion is much higher than in urine, which may be caused by variations in the infant's intestinal microbiota. A gradual decrease in HMO excretion with time as proposed earlier does not take place as even after 7 months of exclusive breastfeeding often intact HMOs can be detected in feces and urine. In addition, we found that whenever oligosaccharides were detected in feces, LNT, the major core structure of HMOs, was present. Hence, our data do not support speculations that LNT is a preferable source for the microbiota.

Bosom Buddies: The Symbiotic Relationship Between Infants and Bifidobacterium longum ssp. longum and ssp. infantis. Genetic and Probiotic Features.

The intestinal microbiota is a complex community that plays an important role in human health from the initial steps of its establishment. Its microbial composition has been suggested to result from selective pressures imposed by the host and is modulated by competition among its members. Bifidobacterium longum is one of the most abundant species of the Bifidobacterium genus in the gut microbiota of healthy breast-fed infants and adults. The recent advancements of 'omics techniques have facilitated the genetic and functional studies of different gut microbiota members. They have revealed the complex genetic pathways used to metabolize different compounds that likely contribute to the competitiveness and persistence of B. longum in the colon. The discovery of a genomic island in B. longum ssp. infantis that encodes specific enzymes for the metabolism of human milk oligosaccharides suggests a specific ecological adaptation. Moreover, B. longum is widely used as probiotic, and beneficial effects in infant health have been reported in several studies.

Metabolic fate of neutral human milk oligosaccharides in exclusively breast-fed infants.

Various biological effects have been postulated for human milk oligosaccharides (HMO), as deduced from in vitro, animal, and epidemiological studies. Little is known about their metabolic fate in vivo in the breast-fed infant, which is presented here. Human milk and infant urine and feces were collected from ten mother-child pairs and analyzed by MALDI-TOF MS (/MS), accompanied by high-performance anion-exchange chromatography with pulsed amperometric detection. Previously, we detected intact small and complex HMO in infant urine, which had been absorbed from gut, as verified via intrinsic (13) C-labeling. Our current work reveals the presence of novel HMO metabolites in urine and feces of breast-fed infants. The novel metabolites were identified as acetylated HMOs and other HMO-like structures, produced by the infants or by their gut microbiota. The finding of secretor- or Lewis-specific HMO in the feces/urine of infants fed with nonsecretor or Lewis-negative milk suggested a correspondent modification in the infant. Our study reveals new insights into the metabolism of neutral HMO in exclusively breast-fed infants and provides further indications for multiple factors influencing HMO metabolism and functions that should be considered in future in vivo investigations.

13C-labeled oligosaccharides in breastfed infants' urine: Individual-, structure- and time-dependent differences in the excretion

Human milk oligosaccharides (HMOs) have been paid much attention due to their beneficial effects observed in vitro, e.g., prebiotic, anti-infective and anti-inflammatory properties. However, in vivo investigations with regard to HMO metabolism and functions are rare. The few data available indicate that HMOs are absorbed to a low extent and excreted via urine without noteworthy modifications, whereas the major proportion reaches infant's colon undigested. Via intrinsic (13)C-labeling of HMOs during their biosynthesis in the mammary gland of 10 lactating women, we were able to follow the fate of (13)C-labeled oligosaccharides (OSs) from their secretion in milk to the excretion in the urine of their breastfed infants. To a certain extent, we could therefore discriminate between original HMOs and non-labeled OSs derived from degradation of HMOs or endogenous glycoconjugates. By means of our novel, rapid, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based approach, we found a homogeneous time pattern of isotopomer enrichment in milk among all subjects and between single OS species. In contrast, the time curves from infants' urine varied strongly between individuals and OS species, though the overall MALDI-TOF MS profile resembled those of the mothers' milk. Our data suggest that neutral HMOs might be processed and/or utilized differentially after or upon absorption from the gut, as deduced from their structure-dependent variation in the extent of tracer enrichment and in the retention times in infant's organism. This sheds new light on the role of HMOs within infant's body, beyond the intestine and its microbiota alone.

Characterization of the Cytosolic β-N-Acetylglucosaminidase from Bifidobacterium longum subsp. longum

The BLLJ_1391 protein from Bifidobacterium longum subsp. longum JCM1217, a cytosolic β-N-acetylglucosaminidase belonging to glycoside hydrolase family (GH) 20, hydrolyzed lacto-N-triose II (LNTri) as well as chitin oligosaccharides. Its reaction was found to follow a substrate-assisted mechanism with anomeric retention, which is common for GH 20 enzymes. Homologous enzymes are found in genomic sequences of B. longum subsp. infantis, Bifidobacterium bifidum, and Bifidobacteium breve, all of which are infant gut-associated species of Bifidobacterium. The distribution resembles that of 1,3-β-galactosyl-N-acetylhexosamine phosphorylase, suggesting that the enzyme plays a role in metabolism of human milk oligosaccharides by hydrolyzing LNTri generated via the cytosolic hydrolysis of lacto-N-tetraose (LNT) by LNT 1,3-β-galactosidase.

Bifidobacteria Isolated From Infants and Cultured on Human Milk Oligosaccharides Affect Intestinal Epithelial Function

Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk. Our laboratory has previously revealed gene clusters specifically linked to HMO metabolism in selected bifidobacteria isolated from fecal samples of infants. Our objective was to test the hypothesis that growth of selected bifidobacteria on HMO stimulates the intestinal epithelium. Caco-2 and HT-29 cells were incubated with lactose (LAC)- or HMO-grown Bifidobacterium longum subsp infantis (B infantis) or B bifidum. Bacterial adhesion and translocation were measured by real-time quantitative polymerase chain reaction. Expression of pro- and anti-inflammatory cytokines and tight junction proteins was analyzed by real-time reverse transcriptase. Distribution of tight junction proteins was measured using immunofluorescent microscopy. We showed that HMO-grown B infantis had a significantly higher rate of adhesion to HT-29 cells compared with B bifidum. B infantis also induced expression of a cell membrane glycoprotein, P-selectin glycoprotein ligand-1. Both B infantis and B bifidum grown on HMO caused less occludin relocalization and higher expression of anti-inflammatory cytokine, interleukin-10 compared with LAC-grown bacteria in Caco-2 cells. B bifidum grown on HMO showed higher expression of junctional adhesion molecule and occludin in Caco-2 cells and HT-29 cells. There were no significant differences between LAC or HMO treatments in bacterial translocation. The study provides evidence for the specific relation between HMO-grown bifidobacteria and intestinal epithelial cells. To our knowledge, this is the first study describing HMO-induced changes in the bifidobacteria-intestinal cells interaction.

Human milk oligosaccharide consumption by intestinal microbiota

Human milk oligosaccharides (HMO) constitute the third most abundant class of molecules in breast milk. Since infants lack the enzymes required for milk glycan digestion, this group of carbohydrates passes undigested to the lower part of the intestinal tract, where they can be consumed by specific members of the infant gut microbiota. We review proposed mechanisms for the depletion and metabolism of HMO by two major bacterial genera within the infant intestinal microbiota, Bifidobacterium and Bacteroides.

Bifidobacterial Enzymes Involved in the Metabolism of Human Milk Oligosaccharides

Intestinal colonization of bifidobacteria is important for the health of infants. Human milk oligosaccharides (HMO) have been identified as growth factors for bifidobacteria. Recently, a bifidobacterial enzymatic system to metabolize HMO was identified. 1,3-β-Galactosyl-N-acetylhexosamine phosphorylase (GLNBP, EC 2.4.1.211), which catalyzes the reversible phosphorolysis of galacto-N-biose (GNB) (Galβ1→3GalNAc)] and lacto-N-biose I (LNB) (Galβ1→3GlcNAc), is a key enzyme to explain the metabolism of HMO. Infant-type bifidobacteria possess the intracellular pathway to specifically metabolize GNB and LNB (GNB/LNB pathway). Bifidobacterium bifidum possesses extracellular enzymes to liberate LNB from HMO. However, Bifidobacterium longum subsp. infantis imports intact HMO to be hydrolyzed by intracellular enzymes. Bifidobacterial enzymes related to the metabolism of HMO are useful tools for preparing compounds related to HMO. For instance, LNB and GNB were produced from sucrose and GlcNAc/GalNAc in 1 pot using 4 bifidobacterial enzymes, including GLNBP. LNB is expected to be a selective bifidus factor for infant-type strains.

Bifidobacterium longum subsp. infantis ATCC 15697 α-Fucosidases Are Active on Fucosylated Human Milk Oligosaccharides

Bifidobacterium longum subsp. infantis ATCC 15697 utilizes several small-mass neutral human milk oligosaccharides (HMOs), several of which are fucosylated. Whereas previous studies focused on endpoint consumption, a temporal glycan consumption profile revealed a time-dependent effect. Specifically, among preferred HMOs, tetraose was favored early in fermentation, with other oligosaccharides consumed slightly later. In order to utilize fucosylated oligosaccharides, ATCC 15697 possesses several fucosidases, implicating GH29 and GH95 α-L-fucosidases in a gene cluster dedicated to HMO metabolism. Evaluation of the biochemical kinetics demonstrated that ATCC 15697 expresses three fucosidases with a high turnover rate. Moreover, several ATCC 15697 fucosidases are active on the linkages inherent to the HMO molecule. Finally, the HMO cluster GH29 α-L-fucosidase possesses a crystal structure that is similar to previously characterized fucosidases.