Mechanism of 2'-fucosyllactose degradation by human-associated Akkermansia.

Among the first microorganisms to colonize the human gut of breastfed infants are bacteria capable of fermenting human milk oligosaccharides (HMOs). One of the most abundant HMOs, 2'-fucosyllactose (2'-FL), may specifically drive bacterial colonization of the intestine. Recently, differential growth has been observed across multiple species of Akkermansia on various HMOs including 2'-FL. In culture, we found growth of two species, A. muciniphila MucT and A. biwaensis CSUN-19,on HMOs corresponded to a decrease in the levels of 2'-FL and an increase in lactose, indicating that the first step in 2'-FL catabolism is the cleavage of fucose. Using phylogenetic analysis and transcriptional profiling, we found that the number and expression of fucosidase genes from two glycoside hydrolase (GH) families, GH29 and GH95, vary between these two species. During the mid-log phase of growth, the expression of several GH29 genes was increased by 2'-FL in both species, whereas the GH95 genes were induced only in A. muciniphila. We further show that one putative fucosidase and a ß-galactosidase from A. biwaensis are involved in the breakdown of 2'-FL. Our findings indicate that the plasticity of GHs of human-associated Akkermansia sp. enables access to additional growth substrates present in HMOs, including 2'-FL. Our work highlights the potential for Akkermansia to influence the development of the gut microbiota early in life and expands the known metabolic capabilities of this important human symbiont.IMPORTANCEAkkermansia are mucin-degrading specialists widely distributed in the human population. Akkermansia biwaensis has recently been observed to have enhanced growth relative to other human-associated Akkermansia on multiple human milk oligosaccharides (HMOs). However, the mechanisms for enhanced growth are not understood. Here, we characterized the phylogenetic diversity and function of select genes involved in the growth of A. biwaensis on 2'-fucosyllactose (2'-FL), a dominant HMO. Specifically, we demonstrate that two genes in a genomic locus, a putative ß-galactosidase and α-fucosidase, are likely responsible for the enhanced growth on 2'-FL. The functional characterization of A. biwaensis growth on 2'-FL delineates the significance of a single genomic locus that may facilitate enhanced colonization and functional activity of select Akkermansia early in life.

Characterization of Bifidobacterium kashiwanohense that utilizes both milk- and plant-derived oligosaccharides.

Bifidobacteria are prominent members of the human gut microbiota throughout life. The ability to utilize milk- and plant-derived carbohydrates is important for bifidobacterial colonization of the infant and adult gut. The Bifidobacterium catenulatum subspecies kashiwanohense (B. kashiwanohense) was originally isolated from infant feces. However, only a few strains have been described, and the characteristics of this subspecies have been poorly investigated. Here, we characterized genotypes and phenotypes of 23 B. kashiwanohense-associated strains, including 12 newly sequenced isolates. Genome-based analysis clarified the phylogenetic relationship between these strains, revealing that only 13 strains are genuine B. kashiwanohense. We defined specific marker sequences and investigated the worldwide prevalence of B. kashiwanohense based on metagenome data. This revealed that not only infants but also adults and weaning children harbor this subspecies in the gut. Most B. kashiwanohense strains utilize long-chain xylans and possess genes for extracellular xylanase (GH10), arabinofuranosidase and xylosidase (GH43), and ABC transporters that contribute to the utilization of xylan-derived oligosaccharides. We also confirmed that B. kashiwanohense strains utilize short- and long-chain human milk oligosaccharides and possess genes for fucosidase (GH95 and GH29) and specific ABC transporter substrate-binding proteins that contribute to the utilization of a wide range of human milk oligosaccharides. Collectively, we found that B. kashiwanohense strains utilize both plant- and milk-derived carbohydrates and identified key genetic factors that allow them to assimilate various carbohydrates.

Synthesis of 2'-fucosyllactose from apple pomace-derived xyloglucan oligosaccharides by an α-L-fucosidase from Pedobacter sp. CAU209.

2'-Fucosyllactose (2'-FL) is known for its ability to provide various health benefits to infants, such as gut maturation, pathogen resistance, improved immunity, and nervous system development. However, the production of 2'-FL using α-L-fucosidases is hindered by the lack of low-cost natural fucosyl donors and high-efficiency α-L-fucosidases. In this work, a recombinant xyloglucanase from Rhizomucor miehei (RmXEG12A) was applied to produce xyloglucan-oligosaccharide (XyG-oligos) from apple pomace. Then, an α-L-fucosidase gene (PbFucB) was screened from the genomic DNA of Pedobacter sp. CAU209 and expressed in Escherichia coli. The capability of purified PbFucB to catalyze XyG-oligos and lactose to synthesize 2'-FL was further evaluated. The deduced amino acid sequence of PbFucB shared the highest identity (38.4%) with that of other reported α-L-fucosidases. PbFucB showed the highest activity at pH 5.5 and 35 °C. It catalyzed the hydrolysis of 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc, 20.3 U mg-1), 2'-FL (8.06 U mg-1), and XyG-oligos (0.43 U mg-1). Furthermore, PbFucB demonstrated a high enzymatic conversion rate in 2'-FL synthesis with pNP-Fuc or apple pomace-derived XyG-oligos as donors and lactose as acceptor. Under the optimized conditions, PbFucB converted 50% of pNP-Fuc or 31% of the L-fucosyl residue in XyG-oligos into 2'-FL. This work elucidated an α-L-fucosidase that mediates the fucosylation of lactose and provided an efficient enzymatic strategy to synthesize 2'-FL either from artificial pNP-Fuc or natural apple pomace-derived XyG-oligos. KEY POINTS: • Xyloglucan-oligosaccharide (XyG-oligos) was produced from apple pomace by a xyloglucanase from Rhizomucor miehei. • An α-L-fucosidase (PbFucB) from Pedobacter sp. CAU209 shared the highest identity (38.4%) with reported α-L-fucosidases. •PbFucB synthesized 2'-FL using apple pomace-derived XyG-oligos and lactose with a conversion ratio of 31%.

Substrate specificity and transglycosylation capacity of α-L-fucosidases across GH29 assessed by bioinformatics-assisted selection of functional diversity.

Glycoside hydrolase family 29 (GH29) encompasses α-L-fucosidases, i.e. enzymes that catalyze the hydrolytic release of fucose from fucosylated glycans, including N- and O-linked glycans on proteins, and these α-L-fucosidases clearly play important roles in biology. GH29 enzymes work via a retaining exo-action mechanism, and some can catalyze transfucosylation. There is no formal subfamily division of GH29 α-L-fucosidases, but they are nonetheless divided into two subfamilies: GH29A having a range of substrate specificities and GH29B having narrower substrate specificity. However, the sequence traits that determine the substrate specificity and transglycosylation ability of GH29 enzymes are not well characterized. Here, we present a new functional map of family GH29 members based on peptide-motif clustering via CUPP (conserved unique peptide patterns) and compare the substrate specificity and transglycosylation activity of 21 representative α-L-fucosidases across the 53 CUPP groups identified. The 21 enzymes exhibited different enzymatic rates on 8 test substrates, CNP-Fuc, 2'FL, 3FL, Lewisa, Lewisx, Fuc-α1,6-GlcNAc, Fuc-α1,3-GlcNAc, and Fuc-α1,4-GlcNAc. Certain CUPP groups clearly harbored a particular type of enzymes, e.g. the majority of the enzymes having activity on Lewisa or Lewisx categorized in the same CUPP clusters. In general, CUPP was useful for resolving GH29 into functional diversity subgroups when considering hydrolytic activity. In contrast, the transglycosylation capacity of GH29 α-L-fucosidases was distributed across a range of CUPP groups. Transglycosylation thus appears to be a common trait among these enzymes and not readily predicted from sequence comparison.

Sialidases and fucosidases of Akkermansia muciniphila are crucial for growth on mucin and nutrient sharing with mucus-associated gut bacteria.

The mucolytic human gut microbiota specialist Akkermansia muciniphila is proposed to boost mucin-secretion by the host, thereby being a key player in mucus turnover. Mucin glycan utilization requires the removal of protective caps, notably fucose and sialic acid, but the enzymatic details of this process remain largely unknown. Here, we describe the specificities of ten A. muciniphila glycoside hydrolases, which collectively remove all known sialyl and fucosyl mucin caps including those on double-sulfated epitopes. Structural analyses revealed an unprecedented fucosidase modular arrangement and explained the sialyl T-antigen specificity of a sialidase of a previously unknown family. Cell-attached sialidases and fucosidases displayed mucin-binding and their inhibition abolished growth of A. muciniphila on mucin. Remarkably, neither the sialic acid nor fucose contributed to A. muciniphila growth, but instead promoted butyrate production by co-cultured Clostridia. This study brings unprecedented mechanistic insight into the initiation of mucin O-glycan degradation by A. muciniphila and nutrient sharing between mucus-associated bacteria.

Structure and function of microbial α-l-fucosidases: a mini review.

Fucose is a monosaccharide commonly found in mammalian, insect, microbial and plant glycans. The removal of terminal α-l-fucosyl residues from oligosaccharides and glycoconjugates is catalysed by α-l-fucosidases. To date, glycoside hydrolases (GHs) with exo-fucosidase activity on α-l-fucosylated substrates (EC 3.2.1.51, EC 3.2.1.-) have been reported in the GH29, GH95, GH139, GH141 and GH151 families of the Carbohydrate Active Enzymes (CAZy) database. Microbes generally encode several fucosidases in their genomes, often from more than one GH family, reflecting the high diversity of naturally occuring fucosylated structures they encounter. Functionally characterised microbial α-l-fucosidases have been shown to act on a range of substrates with α-1,2, α-1,3, α-1,4 or α-1,6 fucosylated linkages depending on the GH family and microorganism. Fucosidases show a modular organisation with catalytic domains of GH29 and GH151 displaying a (ß/α)8-barrel fold while GH95 and GH141 show a (α/α)6 barrel and parallel ß-helix fold, respectively. A number of crystal structures have been solved in complex with ligands, providing structural basis for their substrate specificity. Fucosidases can also be used in transglycosylation reactions to synthesise oligosaccharides. This mini review provides an overview of the enzymatic and structural properties of microbial α-l-fucosidases and some insights into their biological function and biotechnological applications.

Enzymatic production of a suite of human milk oligosaccharides directly in milk.

Human milk oligosaccharides (HMOs) denote specific glycans in human breast milk. They function as prebiotics, immune modulating, and antimicrobial agents in the gut of breastfed infants, and certain HMOs even promote the cognitive development of the baby. HMOs are virtually absent in cow's milk and hence in infant formula, which provides a huge incentive for identifying ways in which HMOs can be produced to improve infant formulas. Here, we show that different sialylated and fucosylated HMOs can be generated in cow's milk via different simultaneous enzymatic transglycosylation reactions catalyzed by an engineered sialidase (EC 3.2.1.18, from Trypanosoma rangeli) and an 1,2-α-L-fucosidase (EC 3.2.1.63, from Tannerella forsinthia) acting on the lactose in the milk and on casein glycomacropeptide, two types of commercially available HMOs, i.e. 2'-fucosyllactose and lacto-N-neotetraose, added to the milk. We also outline the details of the individual reactions in aqueous systems, demonstrate that the enzymatic reactions can be accomplished at 5 °C, and validate the products formed by LC-MS and NMR analysis. Enzymatic production of HMOs directly in milk provides opportunities for enriching milk and infant formulas and extends the use of enzymatic transglycosylation reactions to synthesis of HMOs in milk and eventually in other beverages.

Enhanced antibody-defucosylation capability of α-L-fucosidase by proximity-based protein fusion.

Up to date, the reported fucosidases generally show poor activities toward the IgG core-fucose, which limits the efficiency of ENGase-catalyzed glycoengineering process. However, EndoS or EndoS2 owns excellent activity and great selectivity towards the N-glycosylation of IgGs, and their non-catalytic domains are deduced to have specific interactions to IgG Fc domain that result in the great activity and selectivity. Herein, we constructed a series fusion protein of AlfC (an α-l-fucosidase from Lactobacillus casei BL23) with EndoS/S2 non-catalytic domain by replacing the catalytic GH (glycan hydrolase) domain of EndoS/S2 with the AlfC. We found that all these fused AlfCs showed significantly enhanced defucosylation activity toward the deglycosylated IgGs (Fucα1,6GlcNAc-IgG). We also performed the kinetic study of these fusion enzymes, and our results tend to tell that the EndoS-based fusion proteins have higher kcat values while the EndoS2-based ones possess lower Km values other than higher kcat. Conclusively, our research provides an effective approach to improve the activity of AlfC and remarkably shortened the defucosylation process within several minutes, which will significantly promote the development of glycoengineered antibodies in the future.

FUCA2 and TSTA3 expression in gastric cancer: candidate biomarkers of malignant transformation.

INTRODUCTION: Aberrant fucosylation is closely related to malignant transformation, cancer detection, and evaluation of treatment efficacy. The fucosylation process requires GDP-L-fucose, fucosyltransferases, and fucosidases. In gastric cancer (GC), fucosylation alterations were associated with tumor formation, metastasis inhibition, and multi-drug resistance. It is not clear whether tissue-specific transplantation antigen P35B (TSTA3) and alpha-L-fucosidase 2 (FUCA2) have any effect on the development of GC. MATERIALS AND METHODS: We used immunohistochemistry to assess the expression of TSTA3 and FUCA2 in 71 gastric adenocarcinoma samples and their relationship with clinicopathological parameters. RESULTS: TSTA3 expression was associated with lower histological grade I and II (P = 0.0120) and intestinal type Lauren classification (P = 0.0120). TSTA3 immunopositivity could predict Lauren's classification. Analysis of mRNA expression in GC validation cohorts corroborates the significant TSTA3 association with histological grade observed in our study. However, no associations were found between TSTA3 staining and overall survival. FUCA2 expression was markedly increased in GC tissues compared with non-tumoral tissues (P < 0.0001) and was associated with surgical staging III and IV (P = 0.0417) and advanced histological grade tumor states (P = 0.0125). CONCLUSIONS: Alterations of FUCA2 and TSAT3 immunoexpression could lay the basis for future studies using cell glycosylation as a biomarker for the planning of therapeutic strategy in primary gastric cancer.

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

The gastrointestinal microbiota members produce α-l-fucosidases that play key roles in mucosal, human milk, and dietary oligosaccharide assimilation. Here, 36 open reading frames (ORFs) coding for putative α-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 α-l-fucosidases from Bacteroides thetaiotaomicron, Bacteroides caccae, Phocaeicola vulgatus, Phocaeicola dorei, Ruminococcus gnavus, and Streptococcus parasanguinis. Based on sequence homology, 10 α-l-fucosidase genes were selected for substrate specificity characterization. The α-l-fucosidases Fuc18, Fuc19A, Fuc35B, Fuc39, and Fuc1584 showed hydrolytic activity on α1,3/4-linked fucose present in Lewis blood antigens and the human milk oligosaccharide (HMO) 3-fucosyllactose. In addition, Fuc1584 also hydrolyzed fucosyl-α-1,6-N-acetylglucosamine (6FN), a component of the core fucosylation of N-glycans. Fuc35A and Fuc193 showed activity on α1,2/3/4/6 linkages from H type-2, Lewis blood antigens, HMOs and 6FN. Fuc30 displayed activity only on α1,6-linked l-fucose, and Fuc5372 showed a preference for α1,2 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 (Lea) and lacto-N-fucopentaose III (Lex) and in the glycoprotein mucin. Fuc18, Fuc19A, and Fuc39 also removed l-fucose from neoglycoproteins and human α-1 acid glycoprotein. These results give insight into the great diversity of α-l-fucosidases from the infant gut microbiota, thus supporting the hypothesis that fucosylated glycans are crucial for shaping the newborn microbiota composition. IMPORTANCE α-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 α-l-fucosidases are exoglycosidases that remove α-l-fucosyl residues from free oligosaccharides and glycoconjugates and can be also used in transglycosylation reactions to synthesize oligosaccharides. In this work, α-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 α-l-fucosidases, have great biotechnological potential.

Structural features of free N-glycans in α1,3/4-fucosidase-deficient Arabidopsis thaliana: deletion of α1,3/4-fucosidase activity induced accumulation of plant complex type GN1 free N-glycans.

Deletion of α-1,3/4-fucosidase activity in Arabidopsis thaliana resulted in the accumulation of GN1-type free N-glycans with the Lewis a epitope (GN1-FNG). This suggests that the release of α-fucose residue(s) may trigger rapid degradation of the plant complex-type (PCT) GN1-FNG. The fact that PCT-GN1-FNG has rarely been detected to date is probably due to its easier degradation compared with PCT-GN2-FNG.

Glycan degradation promotes macroautophagy.

Macroautophagy promotes cellular homeostasis by delivering cytoplasmic constituents to lysosomes for degradation [Mizushima, Nat. Cell Biol. 20, 521-527 (2018)]. However, while most studies have focused on the mechanisms of protein degradation during this process, we report here that macroautophagy also depends on glycan degradation via the glycosidase, α-l-fucosidase 1 (FUCA1), which removes fucose from glycans. We show that cells lacking FUCA1 accumulate lysosomal glycans, which is associated with impaired autophagic flux. Moreover, in a mouse model of fucosidosis-a disease characterized by inactivating mutations in FUCA1 [Stepien et al., Genes (Basel) 11, E1383 (2020)]-glycan and autophagosome/autolysosome accumulation accompanies tissue destruction. Mechanistically, using lectin capture and mass spectrometry, we identified several lysosomal enzymes with altered fucosylation in FUCA1-null cells. Moreover, we show that the activity of some of these enzymes in the absence of FUCA1 can no longer be induced upon autophagy stimulation, causing retardation of autophagic flux, which involves impaired autophagosome-lysosome fusion. These findings therefore show that dysregulated glycan degradation leads to defective autophagy, which is likely a contributing factor in the etiology of fucosidosis.

Crystal structure of metagenomic ß-glycosidase MeBglD2 in complex with various saccharides.

Metagenomic MeBglD2 is a glycoside hydrolase family 1 (GH1) ß-glycosidase that has ß-glucosidase, ß-fucosidase, and ß-galactosidase activities, and is highly activated in the presence of monosaccharides and disaccharides. The ß-glucosidase activity of MeBglD2 increases in a cellobiose concentration-dependent manner and is not inhibited by a high concentration of D-glucose or cellobiose. Previously, we solved the crystal structure of MeBglD2 and designed a thermostable mutant; however, the mechanism of substrate recognition of MeBglD2 remains poorly understood. In this paper, we report the X-ray crystal structures of MeBglD2 complexed with various saccharides, such as D-glucose, D-xylose, cellobiose, and maltose. The results showed that subsite - 1 of MeBglD2, which contained two catalytic glutamate residues (a nucleophilic Glu356 and an acid/base Glu170) was common to other GH1 enzymes, but the positive subsites (+ 1 and + 2) had different binding modes depending on the type of sugar. Three residues (Glu183, Asn227, and Asn229), located at the positive subsites of MeBglD2, were involved in substrate specificity toward cellobiose and/or chromogenic substrates in the presence of additive sugars. The docking simulation of MeBglD2-cellobiose indicated that Asn229 and Trp329 play important roles in the recognition of + 1 D-glucose in cellobiose. Our findings provide insights into the unique substrate recognition mechanism of GH1, which can incorporate a variety of saccharides into its positive subsites. KEY POINTS: • Metagenomic glycosidase, MeBglD2, recognizes various saccharides • Structures of metagenomic MeBglD2 complexed with various saccharides are determined • MeBglD2 has a unique substrate recognition mechanism at the positive subsites.

Detection of Bacterial α-l-Fucosidases with an Ortho-Quinone Methide-Based Probe and Mapping of the Probe-Protein Adducts.

Fucosidases are associated with several pathological conditions and play an important role in the health of the human gut. For example, fucosidases have been shown to be indicators and/or involved in hepatocellular carcinoma, breast cancer, and helicobacter pylori infections. A prerequisite for the detection and profiling of fucosidases is the formation of a specific covalent linkage between the enzyme of interest and the activity-based probe (ABP). The most commonly used fucosidase ABPs are limited to only one of the classes of fucosidases, the retaining fucosidases. New approaches are needed that allow for the detection of the second class of fucosidases, the inverting type. Here, we report an ortho-quinone methide-based probe with an azide mini-tag that selectively labels both retaining and inverting bacterial α-l-fucosidases. Mass spectrometry-based intact protein and sequence analysis of a probe-labeled bacterial fucosidase revealed almost exclusive single labeling at two specific tryptophan residues outside of the active site. Furthermore, the probe could detect and image extracellular fucosidase activity on the surface of live bacteria.

The first structure-function study of GH151 α-l-fucosidase uncovers new oligomerization pattern, active site complementation, and selective substrate specificity.

Fucosylated compounds are abundantly present in nature and are associated with many biological processes, therefore carrying great potential for use in medicine and biotechnology. Efficient ways to modify fucosylated compounds are still being developed. Promising results are provided by glycosyl hydrolases with transglycosylating activities, such as α-l-fucosidase isoenzyme 2 from Paenibacillus thiaminolyticus (family GH151 of Carbohydrate-Active enZYmes). Currently, there is no 3D structure representing this glycoside hydrolase family and only a few members have been investigated. Here, we present the first structure-function study of a GH151 member, providing the key insights into its specific oligomerization and active site properties. According to the crystal structure, small-angle X-ray scattering data and catalytic investigation, this enzyme functions as a tetramer of a new type and represents the second known case of active site complementation among all α-l-fucosidases. Mutation of the active site-complementing residue histidine 503 to alanine confirmed its influence on α-l-fucosidase activity and, specifically, on substrate binding. Several unique features of GH151 family α-l-fucosidases were revealed, including the oligomerization pattern, active site accessibility and complementation, and substrate selectivity. Some common properties of GH151 glycosyl hydrolases then would be the overall three-domain structure and conservation of the central domain loop 2 function, including its complementation role and the formation of the carbohydrate-binding platform in the active site vicinity.

Cancer-associated adipocytes release FUCA2 to promote aggressiveness in TNBC.

Cancer-associated adipocytes (CAAs) have been suggested to promote tumor progression. Yet, the role of CAAs in triple-negative breast cancer (TNBC) is poorly investigated. We compared the expression of secretory protein-encoding genes in CAAs and control adipocytes. The effect of key secretory protein(s) on TNBC cell behaviors was explored. CAAs expressed and secreted FUCA2 at greater levels than control adipocytes. When FUCA2 activity was blocked with a neutralizing antibody, TNBC cell proliferation and migration induced by CAA-conditioned medium was impaired. In contrast, supplement of exogenous FUCA2 protein reinforced the proliferation, colony formation, and migration of TNBC cells. In vivo studies confirmed that FUCA2 exposure enhanced tumorigenesis and metastasis of TNBC cells. Mechanistic investigation revealed that FUCA2 induced TNBC aggressiveness through TM9SF3-dependent signaling. Depletion of TM9SF3 blocked CAA- and FUCA2-induced TNBC cell proliferation and migration. Compared to adjacent breast tissues, TNBC tissues had increased expression of TM9SF3. Moreover, high TM9SF3 expression was associated with advanced TNM stage, lymph node metastasis, and shorter overall survival of TNBC patients. Altogether, CAAs secrete FUCA2 to promote TNBC growth and metastasis through interaction with TM9SF3. Inhibition of TM9SF3 may represent a potential therapeutic strategy in the treatment of TNBC.

Fucosylated Human Milk Oligosaccharide Foraging within the Species Bifidobacterium pseudocatenulatum Is Driven by Glycosyl Hydrolase Content and Specificity.

Human milk enriches members of the genus Bifidobacterium in the infant gut. One species, Bifidobacterium pseudocatenulatum, is found in the gastrointestinal tracts of adults and breastfed infants. In this study, B. pseudocatenulatum strains were isolated and characterized to identify genetic adaptations to the breastfed infant gut. During growth on pooled human milk oligosaccharides (HMOs), we observed two distinct groups of B. pseudocatenulatum, isolates that readily consumed HMOs and those that did not, a difference driven by variable catabolism of fucosylated HMOs. A conserved gene cluster for fucosylated HMO utilization was identified in several sequenced B. pseudocatenulatum strains. One isolate, B. pseudocatenulatum MP80, which uniquely possessed GH95 and GH29 α-fucosidases, consumed the majority of fucosylated HMOs tested. Furthermore, B. pseudocatenulatum SC585, which possesses only a single GH95 α-fucosidase, lacked the ability to consume the complete repertoire of linkages within the fucosylated HMO pool. Analysis of the purified GH29 and GH95 fucosidase activities directly on HMOs revealed complementing enzyme specificities with the GH95 enzyme preferring 1-2 fucosyl linkages and the GH29 enzyme favoring 1-3 and 1-4 linkages. The HMO-binding specificities of the family 1 solute-binding protein component linked to the fucosylated HMO gene cluster in both SC585 and MP80 are similar, suggesting differential transport of fucosylated HMO is not a driving factor in each strain's distinct HMO consumption pattern. Taken together, these data indicate the presence or absence of specific α-fucosidases directs the strain-specific fucosylated HMO utilization pattern among bifidobacteria and likely influences competitive behavior for HMO foraging in situ. IMPORTANCE Often isolated from the human gut, microbes from the bacterial family Bifidobacteriaceae commonly possess genes enabling carbohydrate utilization. Isolates from breastfed infants often grow on and possess genes for the catabolism of human milk oligosaccharides (HMOs), glycans found in human breast milk. However, catabolism of structurally diverse HMOs differs between bifidobacterial strains. This study identifies key gene differences between Bifidobacterium pseudocatenulatum isolates that may impact whether a microbe successfully colonizes an infant gut. In this case, the presence of complementary α-fucosidases may provide an advantage to microbes seeking residence in the infant gut. Such knowledge furthers our understanding of how diet drives bacterial colonization of the infant gut.

Discovery and characterization of a novel α-l-fucosidase from the marine-derived Flavobacterium algicola and its application in 2'-fucosyllactose production.

2'-Fucosyllactose (2'-FL) is one of the nutrient ingredients in human milk, which has various beneficial health effects. α-l-fucosidase is a biotechnological tool for 2'-FL preparation. Here, a novel and efficient α-l-fucosidase OUC-Jdch16 from the fucoidan-digesting strain Flavobacterium algicola 12076 was heterologously expressed and applied to produce 2'-FL in vitro. OUC-Jdch16 belongs to glycoside hydrolases (GH) family 29 and exhibits the highest 4-nitrophenyl-α-l-fucopyranoside-hydrolyzing activity at 25 °C and pH 6.0. OUC-Jdch16 could catalyze the synthesis of 2'-FL via transferring the fucosyl residue from pNP-α-fucose to lactose. Under the optimal transfucosylation conditions, the yield of the transfucosylation product reached 84.82% and 92.15% (mol/mol) from pNP-α-fucose within 48 h and 120 h, respectively. Moreover, OUC-Jdch16 was capable of transferring the fucosyl residue to other glycosyl receptors with the generation of novel fucosylated compounds. This study demonstrated that OUC-Jdch16 could be a promising tool to prepare 2'-FL and other novel glycosides.

Identification of Allobaculum mucolyticum as a novel human intestinal mucin degrader.

The human gut microbiota plays a central role in intestinal health and disease. Yet, many of its bacterial constituents are functionally still largely unexplored. A crucial prerequisite for bacterial survival and proliferation is the creation and/or exploitation of an own niche. For many bacterial species that are linked to human disease, the inner mucus layer was found to be an important niche. Allobaculum mucolyticum is a newly identified, IBD-associated species that is thought be closely associated with the host epithelium. To explore how this bacterium is able to effectively colonize this niche, we screened its genome for factors that may contribute to mucosal colonization. Up to 60 genes encoding putative Carbohydrate Active Enzymes (CAZymes) were identified in the genome of A. mucolyticum. Mass spectrometry revealed 49 CAZymes of which 26 were significantly enriched in its secretome. Functional assays demonstrated the presence of CAZyme activity in A. mucolyticum conditioned medium, degradation of human mucin O-glycans, and utilization of liberated non-terminal monosaccharides for bacterial growth. The results support a model in which sialidases and fucosidases remove terminal O-glycan sugars enabling subsequent degradation and utilization of carbohydrates for A. mucolyticum growth. A. mucolyticum CAZyme secretion may thus facilitate bacterial colonization and degradation of the mucus layer and may pose an interesting target for future therapeutic intervention.

Architecture Insight of Bifidobacterial α-L-Fucosidases.

Fucosylated carbohydrates and glycoproteins from human breast milk are essential for the development of the gut microbiota in early life because they are selectively metabolized by bifidobacteria. In this regard, α-L-fucosidases play a key role in this successful bifidobacterial colonization allowing the utilization of these substrates. Although a considerable number of α-L-fucosidases from bifidobacteria have been identified by computational analysis, only a few of them have been characterized. Hitherto, α-L-fucosidases are classified into three families: GH29, GH95, and GH151, based on their catalytic structure. However, bifidobacterial α-L-fucosidases belonging to a particular family show significant differences in their sequence. Because this fact could underlie distinct phylogenetic evolution, here extensive similarity searches and comparative analyses of the bifidobacterial α-L-fucosidases identified were carried out with the assistance of previous physicochemical studies available. This work reveals four and two paralogue bifidobacterial fucosidase groups within GH29 and GH95 families, respectively. Moreover, Bifidobacterium longum subsp. infantis species exhibited the greatest number of phylogenetic lineages in their fucosidases clustered in every family: GH29, GH95, and GH151. Since α-L-fucosidases phylogenetically descended from other glycosyl hydrolase families, we hypothesized that they could exhibit additional glycosidase activities other than fucosidase, raising the possibility of their application to transfucosylate substrates other than lactose in order to synthesis novel prebiotics.