NMR Analyses of the Enzymatic Degradation End-Products of Diabolican: The Secreted EPS of Vibrio diabolicus CNCM I-1629.

Diabolican, or HE800, is an exopolysaccharide secreted by the non-pathogenic Gram-negative marine bacterium Vibrio diabolicus (CNCM I-1629). This polysaccharide was enzymatically degraded by the Bacteroides cellulosilyticus WH2 hyaluronan lyase. The end products were purified by size-exclusion chromatography and their structures were analyzed in depth by nuclear magnetic resonance (NMR). The oligosaccharide structures confirmed the possible site of cleavage of the enzyme showing plasticity in the substrate recognitions. The production of glycosaminoglycan-mimetic oligosaccharides of defined molecular weight and structure opens new perspectives in the valorization of the marine polysaccharide diabolican.

Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space.

Over the last two decades, the number of gene/protein sequences gleaned from sequencing projects of individual genomes and environmental DNA has grown exponentially. Only a tiny fraction of these predicted proteins has been experimentally characterized, and the function of most proteins remains hypothetical or only predicted based on sequence similarity. Despite the development of postgenomic methods, such as transcriptomics, proteomics, and metabolomics, the assignment of function to protein sequences remains one of the main challenges in modern biology. As in all classes of proteins, the growing number of predicted carbohydrate-active enzymes (CAZymes) has not been accompanied by a systematic and accurate attribution of function. Taking advantage of the CAZy database, which groups CAZymes into families and subfamilies based on amino acid similarities, we recombinantly produced 564 proteins selected from subfamilies without any biochemically characterized representatives, from distant relatives of characterized enzymes and from nonclassified proteins that show little similarity with known CAZymes. Screening these proteins for activity on a wide collection of carbohydrate substrates led to the discovery of 13 CAZyme families (two of which were also discovered by others during the course of our work), revealed three previously unknown substrate specificities, and assigned a function to 25 subfamilies.

Structure of the Polysaccharide Secreted by Vibrio alginolyticus CNCM I-5035 (Epidermist 4.0TM).

Vibrio alginolyticus (CNCM I-5035) secretes an exopolysaccharide used as ingredient in cosmetic industry under the trademark Epidermist 4.0TM. It is appreciated for its ability to improve the physical and chemical barrier functions of the skin by notably increasing the keratinocyte differentiation and epidermal renewal. Composition analyses and in depth characterization of the polysaccharides as well as oligosaccharides obtained by mild acid hydrolyses revealed that it was composed of a repetition unit of three residues: d-galactose (d-Gal), d-N-acetylglucosamine (GlcNAc) and l-N-acetylguluronic acid, of which 30% (M/M) was acetylated in position 3. The complete structure of the polysaccharide was resolved giving the repetition unit: [→3)-α-d-Gal-(1→4)-α-l-GulNAcA/α-l-3OAc-GulNAcA-(1→4)-ß-d-GlcNAc-(1→].

Structure of the Exopolysaccharide Secreted by a Marine Strain Vibrio alginolyticus.

Vibrio alginolyticus (CNCM I-4151) secretes an exopolysaccharide whose carbohydrate backbone is decorated with amino acids, likely conferring its properties that are appreciated in cosmetics. Here, the secreted polysaccharide of another strain of V. alginolyticus (CNCM I-5034) was characterized by chromatography and one- and two-dimensional NMR spectroscopy experiments. The structure was resolved and shows that the carbohydrate backbone is made of four residues: D-galactose (Gal), D-galacturonic acid (GalA) D-N-acetylglucosamine (GlcNAc) and D-glucuronic acid (GlcA), forming a tetrasaccharide repetition unit [→4)-ß-d-GlcA-(1→3)-α-d-Gal-(1→3)-α-d-GalA-(1→3)-ß-GlcNAc(1→]. GlcA is derivatized with a lactate group giving 'nosturonic acid', and GalA is decorated with the amino acid alanine.

Bacterial synthesis of polysialic acid lactosides in recombinant Escherichia coli K-12.

Bacterial polysialyltransferases (PSTs) are processive enzymes involved in the synthesis of polysialic capsular polysaccharides. They can also synthesize polysialic acid in vitro from disialylated and trisialylated lactoside acceptors, which are the carbohydrate moieties of GD3 and GT3 gangliosides, respectively. Here, we engineered a non-pathogenic Escherichia coli strain that overexpresses recombinant sialyltransferases and sialic acid synthesis genes and can convert an exogenous lactoside into polysialyl lactosides. Several PSTs were assayed for their ability to synthesize polysialyl lactosides in the recombinant strains. Fed-batch cultures produced α-2,8 polysialic acid or alternate α-2,8-2,9 polysialic acid in quantities reaching several grams per liter. Bacterial culture in the presence of propargyl-ß-lactoside as the exogenous acceptor led to the production of conjugatable polysaccharides by means of copper-assisted click chemistry.

Databases of Conformations and NMR Structures of Glycan Determinants.

The present study reports a comprehensive nuclear magnetic resonance (NMR) characterization and a systematic conformational sampling of the conformational preferences of 170 glycan moieties of glycosphingolipids as produced in large-scale quantities by bacterial fermentation. These glycans span across a variety of families including the blood group antigens (A, B and O), core structures (Types 1, 2 and 4), fucosylated oligosaccharides (core and lacto-series), sialylated oligosaccharides (Types 1 and 2), Lewis antigens, GPI-anchors and globosides. A complementary set of about 100 glycan determinants occurring in glycoproteins and glycosaminoglycans has also been structurally characterized using molecular mechanics-based computation. The experimental and computational data generated are organized in two relational databases that can be queried by the user through a user-friendly search engine. The NMR ((1)H and (13)C, COSY, TOCSY, HMQC, HMBC correlation) spectra and 3D structures are available for visualization and download in commonly used structure formats. Emphasis has been given to the use of a common nomenclature for the structural encoding of the carbohydrates and each glycan molecule is described by four different types of representations in order to cope with the different usages in chemistry and biology. These web-based databases were developed with non-proprietary software and are open access for the scientific community available at http://glyco3d.cermav.cnrs.fr.

Structure of an Amino Acid-Decorated Exopolysaccharide Secreted by a Vibrio alginolyticus Strain.

Vibrio alginolyticus (CNCM I-4994) secretes an exopolysaccharide that can be used as an ingredient in cosmetic applications. The structure was resolved using chromatography and one- and two-dimensional NMR spectroscopy experiments. The results show that the carbohydrate backbone is made of two residues: d-galacturonic acid and N-acetyl-d-glucosamine (GlcNac), which together constitute a tetrasaccharide repetition unit: [→3)-α-d-GalA-(1→4)-α-d-GalA-(1→3)-α-d-GalA-(1→3)-ß-GlcNAc(1→]. Two amino acids, alanine and serine, are linked to GalA residues via amido linkages. The position and the distribution of the amino acids were characterized by two-dimensional NMR spectroscopy. To our knowledge, this is the first description of a structure for a marine exopolysaccharide decorated with an amino acid.

Structure and enzymatic degradation of the polysaccharide secreted by Nostoc commune.

Noctoc commune is a cyanobacterium living in various and extreme environments. Its ability to survive in desert, on ice or high altitude is explained by its exceptional metabolism and its capacity to resist to desiccation. N. commune cells are embedded in a gelatinous matrix made of polysaccharides which fixes water and participates in maintaining the cells in hydrated conditions. The structure of the polysaccharide of N. commune harvested in Saint Martin d'Uriage (France) and the oligosaccharides obtained after its enzymatic degradation were determined. The repeating unit of the main chain is a tetra-saccharide: [→4)-ß-D-Glcp-(1 â†’ 4)-ß-D-Xylp-(1 â†’ 4)-ß-D-Glcp-(1 â†’ 4)-α-D-Galp-(1→], branched at position 6 of a glucose residue by a ß-linked pyruvated glucuronic acid residue. About 30% of the Xylp residues were branched with a Xylf residue. Comparison of this structure with the polysaccharides secreted by other Nostoc species and strains suggest a strong selection pressure on the structure in agreement with its important biological role.

Misincorporation of Galactose by Chondroitin Synthase of Escherichia coli K4: From Traces to Synthesis of Chondbiuronan, a Novel Chondroitin-Like Polysaccharide.

Chondroitin synthase KfoC is a bifunctional enzyme which polymerizes the capsular chondroitin backbone of Escherichia coli K4, composed of repeated ß3N-acetylgalactosamine (GalNAc)-ß4-glucuronic acid (GlcA) units. Sugar donors UDP-GalNAc and UDP-GlcA are the natural precursors of bacterial chondroitin synthesis. We have expressed KfoC in a recombinant strain of Escherichia coli deprived of 4-epimerase activity, thus incapable of supplying UDP-GalNAc in the bacterial cytoplasm. The strain was also co-expressing mammal galactose ß-glucuronyltransferase, providing glucuronyl-lactose from exogenously added lactose, serving as a primer of polymerization. We show by the mean of NMR analyses that in those conditions, KfoC incorporates galactose, forming a chondroitin-like polymer composed of the repeated ß3-galactose (Gal)-ß4-glucuronic acid units. We also show that when UDP-GlcNAc 4-epimerase KfoA, encoded by the K4-operon, was co-expressed and produced UDP-GalNAc, a small proportion of galactose was still incorporated into the growing chain of chondroitin.

Glucuronylation in Escherichia coli for the bacterial synthesis of the carbohydrate moiety of nonsulfated HNK-1.

We have previously reported the large-scale synthesis of neolactotetraose (Galbeta-4GlcNAcbeta-3Galbeta-4Glc) from lactose in engineered Escherichia coli cells (Priem B, Gilbert M, Wakarchuk WW, Heyraud A and Samain E. 2002. A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. Glycobiology. 12:235-240). In the present study we analyzed the adaptation of this system to glucuronylated oligosaccharides. The catalytic domain of mouse glucuronyl transferase GlcAT-P was cloned and expressed in an engineered strain which performed the in vivo synthesis of neolactotetraose. Under these conditions, efficient glucuronylation of neolactotetraose was achieved, but some residual neolactotetraose was still present. Although E. coli K-12 has an indigenous UDP-glucose dehydrogenase, the yield of glucuronylated oligosaccharides was greatly improved by the additional expression of the orthologous gene kfiD from E. coli K5. Glucuronylation of neolactohexaose and lactose was also observed. The final glucuronylated oligosaccharides are precursors of the brain carbohydrate motif HNK-1, involved in neural cell adhesion.

Ancient acquisition of "alginate utilization loci" by human gut microbiota.

In bacteria from the phylum Bacteroidetes, the genes coding for enzymes involved in polysaccharide degradation are often colocalized and coregulated in so-called "polysaccharide utilization loci" (PULs). PULs dedicated to the degradation of marine polysaccharides (e.g. laminaran, ulvan, alginate and porphyran) have been characterized in marine bacteria. Interestingly, the gut microbiome of Japanese individuals acquired, by lateral transfer from marine bacteria, the genes involved in the breakdown of porphyran, the cell wall polysaccharide of the red seaweed used in maki. Sequence similarity analyses predict that the human gut microbiome also encodes enzymes for the degradation of alginate, the main cell wall polysaccharide of brown algae. We undertook the functional characterization of diverse polysaccharide lyases from family PL17, frequently found in marine bacteria as well as those of human gut bacteria. We demonstrate here that this family is polyspecific. Our phylogenetic analysis of family PL17 reveals that all alginate lyases, which have all the same specificity and mode of action, cluster together in a very distinct subfamily. The alginate lyases found in human gut bacteria group together in a single clade which is rooted deeply in the PL17 tree. These enzymes were found in PULs containing PL6 enzymes, which also clustered together in the phylogenetic tree of PL6. Together, biochemical and bioinformatics analyses suggest that acquisition of this system appears ancient and, because only traces of two successful transfers were detected upon inspection of PL6 and PL17 families, the pace of acquisition of marine polysaccharide degradation system is probably very slow.

Synthesis of globopentaose using a novel beta1,3-galactosyltransferase activity of the Haemophilus influenzae beta1,3-N-acetylgalactosaminyltransferase LgtD.

We have previously described a bacterial system for the conversion of globotriaose (Gb3) into globotetraose (Gb4) by a metabolically engineered Escherichia coli strain expressing the Haemophilus influenzae lgtD gene encoding beta1,3-N-acetylgalactosaminyltransferase [Antoine, T., Bosso, C., Heyraud, A. Samain, E. (2005) Large scale in vivo synthesis of globotriose and globotetraose by high cell density culture of metabolically engineered Escherichia coli. Biochimie 87, 197-203]. Here, we found that LgtD has an additional beta1,3-galactosyltransferase activity which allows our bacterial system to be extended to the synthesis of the carbohydrate portion of globopentaosylceramide (Galbeta-3GalNAcbeta-3Galalpha-4Galbeta-4Glc) which reacts with the monoclonal antibody defining the stage-specific embryonic antigen-3. In vitro assays confirmed that LgtD had both beta1,3-GalT and beta1,3-GalNAcT activities and showed that differences in the affinity for Gb3 and Gb4 explain the specific and exclusive formation of globopentaose.

Chemo-bacterial synthesis of conjugatable glycosaminoglycans.

Conjugatable glycosaminoglycans hold promise for medical applications involving the vectorization of specific molecules. Here, we set out to produce bacterial chondroitin and heparosan from a conjugatable precursor using metabolically engineered Escherichia coli strains. The major barrier to this procedure was the glucuronylation of a lactosyl acceptor required for polymerization. To overcome this barrier, we designed E. coli strains expressing mouse ß-1,3-glucuronyl transferase and E. coli K4 chondroitin and K5 heparosan synthases. These engineered strains were cultivated at high density in presence of a lactose-furyl precursor. Enzymatic polymerization occurred on the lactosyl precursor resulting in small chains ranging from 15 to 30kDa that accumulated in the cytoplasm. Furyl-terminated polysaccharides were produced at a gram-per-liter scale, a yield similar to that reported for conventional strains. Their efficient conjugation using a Diels-Alder cycloaddition reaction in aqueous and catalyst-free conditions was also confirmed using N-methylmaleimide as model dienophile.

Aphanomyces euteiches cell wall fractions containing novel glucan-chitosaccharides induce defense genes and nuclear calcium oscillations in the plant host Medicago truncatula.

N-acetylglucosamine-based saccharides (chitosaccharides) are components of microbial cell walls and act as molecular signals during host-microbe interactions. In the legume plant Medicago truncatula, the perception of lipochitooligosaccharide signals produced by symbiotic rhizobia and arbuscular mycorrhizal fungi involves the Nod Factor Perception (NFP) lysin motif receptor-like protein and leads to the activation of the so-called common symbiotic pathway. In rice and Arabidopsis, lysin motif receptors are involved in the perception of chitooligosaccharides released by pathogenic fungi, resulting in the activation of plant immunity. Here we report the structural characterization of atypical chitosaccharides from the oomycete pathogen Aphanomyces euteiches, and their biological activity on the host Medicago truncatula. Using a combination of biochemical and biophysical approaches, we show that these chitosaccharides are linked to ß-1,6-glucans, and contain a ß-(1,3;1,4)-glucan backbone whose ß-1,3-linked glucose units are substituted on their C-6 carbon by either glucose or N-acetylglucosamine residues. This is the first description of this type of structural motif in eukaryotic cell walls. Glucan-chitosaccharide fractions of A. euteiches induced the expression of defense marker genes in Medicago truncatula seedlings independently from the presence of a functional Nod Factor Perception protein. Furthermore, one of the glucan-chitosaccharide fractions elicited calcium oscillations in the nucleus of root cells. In contrast to the asymmetric oscillatory calcium spiking induced by symbiotic lipochitooligosaccharides, this response depends neither on the Nod Factor Perception protein nor on the common symbiotic pathway. These findings open new perspectives in oomycete cell wall biology and elicitor recognition and signaling in legumes.

Production of intracellular heparosan and derived oligosaccharides by lyase expression in metabolically engineered E. coli K-12.

The cluster of genes of capsular K5 heparosan is composed of three regions, involved in the synthesis and the exportation of the polysaccharide. The region 2 possesses all the necessary genes involved in the synthesis of heparosan, namely kfiA, encoding alpha-4-N-acetylglucosaminyltransferase, kfiD, encoding ß-3-glucuronyl transferase, kfiC, encoding UDP-glucose dehydrogenase (UDP-glucuronic acid synthesis), and kfiB encoding a protein of unknown function. The cloning and expression of kfiADCB into Escherichia coli K-12 were found to be sufficient for the production of heparosan, which accumulates in the cells due to a lack of the exporting system. The concentration of recombinant heparosan reached one gram per liter under fed-batch cultivation. The cytoplasmic localization of heparosan inside the bacteria allowed subsequent enzymatic modifications such as a partial degradation with K5 lyase when expressed intracellularly. Under these conditions, the production of DP 2-10 oligosaccharides occurred intracellularly, at a concentration similar to that of recombinant intracellular heparosan.

Synthesis of α-galactosyl epitopes by metabolically engineered Escherichia coli.

The α-Gal epitope is a carbohydrate structure, Galα-3Galß-4GlcNAc-R, expressed on glycoconjuguates in many mammals, but not in humans. Species that do not express this epitope have present in their serum large amounts of natural anti-Gal antibodies, which contribute to organ hyperacute rejection during xenotransplantation. We first describe the efficient conversion of lactose into isoglobotriaose (Galα-3Galß-4Glc) using high cell density cultures of a genetically engineered Escherichia coli strain expressing the bovine gene for α-1,3-galactosyltransferase. Attempts to produce the Galili pentasaccharide (Galα-3Galß-4GlcNAcß-3Galß-4Glc) by additionally expressing the Neisseria meningitis lgtA gene for ß-1,3-N-acetylglucosaminyltransferase and the Helicobacter pylori gene for ß-1,4-galactosyltransferase were unsuccessful and led to the formation of a series of long chain oligosaccharides formed by the repeated addition of the trisaccharide motif [Galß-4GlcNAcß-3Galα-3] onto a lacto-N-neotetraose primer. The replacement of LgtA by a more specific ß-1,3-N-acetylglucosaminyltransferase from H. pylori, which was unable to glycosylate α-galactosides, prevented the formation of these unwanted compounds and allowed the successful formation of the Galili pentasaccharide and longer α-Gal epitopes.

Efficient synthesis of 6'-sialyllactose, 6,6'-disialyllactose, and 6'-KDO-lactose by metabolically engineered E. coli expressing a multifunctional sialyltransferase from the Photobacterium sp. JT-ISH-224.

We have previously reported the efficient conversion of lactose into 3'-sialyllactose by high cell density cultures of a genetically engineered Escherichia coli strain expressing the Neisseria meningitidis gene for alpha-(2-->3)-sialyltransferase [Fierfort, N.; Samain, E. J. Biotechnol. 2008, 134, 261-265.]. First attempts to use a similar strategy to produce 6'-sialyllactose with a strain expressing alpha-(2-->6)-sialyltransferase from the Photobacterium sp. JT-ISH-224 led to the production of a trisaccharide that was identified as KDO-lactose (2-keto-3-deoxy-manno-octonyllactose). This result showed that alpha-(2-->6)-sialyltransferase was able to use CMP-KDO as sugar donor and preferentially used CMP-KDO over CMP-Neu5Ac. By reducing the expression level of the sialyltransferase gene and increasing that of the neuABC genes, we have been able to favour the formation of 6'-sialyllactose and to prevent the formation of KDO-lactose. However, in this case, a third lactose derivative, which was identified as 6,6'-disialyllactose, was also produced. Formation of 6,6'-disialyllactose was mainly observed under conditions of lactose shortage. On the other hand, when the culture was continuously fed with an excess of lactose, 6'-sialyllactose was almost the only product detected and its final concentration was higher than 30g/L of culture medium.