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Glycan-protein interactions play a crucial role in physiological and pathological events. Hence, improving the isolation of carbohydrate-binding proteins (i.e., lectins and anti-glycan antibodies) from complex media might not only lead to a better understanding of their function, but also provide solutions for public health issues, such as water contamination or the need for universal blood plasma. Here we report a rapid and efficient method for producing carbohydrate-based affinity adsorbents combining enzymatic synthesis and metal-free click chemistry. Both simple and complex glycans (maltose, blood group antigens A, B, and H) were readily modified by the addition of a furyl group at the reducing end without the need for protecting groups and were then efficiently conjugated to maleimide-activated Sepharose particles through Diels-Alder cycloaddition. These neoglycoconjugates showed high efficiency for the purification of lectins (concanavalin A and Ulex europaeus agglutinin), as well as for the capture of anti-A and anti-B blood group antibodies, opening new prospects for glycoproteomics and for the development of universal blood plasma., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A fast chemoenzymatic synthesis of sialylated oligosaccharides containing C5-modified neuraminic acids is reported. Analogues of GM3 and GM2 ganglioside saccharidic portions where the acetyl group of NeuNAc has been replaced by a phenylacetyl (PhAc) or a propanoyl (Prop) moiety have been efficiently prepared with metabolically engineered E. coli bacteria. GM3 analogues were either obtained by chemoselective modification of biosynthetic N-acetyl-sialyllactoside (GM3 NAc) or by direct bacterial synthesis using C5-modified neuraminic acid precursors. The latter strategy proved to be very versatile as it led to an efficient synthesis of GM2 analogues. These glycomimetics were assessed against hemagglutinins and sialidases. In particular, the GM3 NPhAc displayed a binding affinity for Maackia amurensis agglutinin (MAA) similar to that of GM3 NAc, while being resistant to hydrolysis by Vibrio cholerae (VC) neuraminidase. A preliminary study with influenza viruses also confirmed a selective inhibition of N1 neuraminidase by GM3 NPhAc, suggesting potential developments for the detection of flu viruses and for fighting them., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

While chitooligosaccharides (COs) derived from fungal chitin are potent elicitors of defense reactions, structurally related signals produced by certain bacteria and fungi, called lipo-chitooligosaccharides (LCOs), play important roles in the establishment of symbioses with plants. Understanding how plants distinguish between friend and foe through the perception of these signals is a major challenge. We report the synthesis of a range of COs and LCOs, including photoactivatable probes, to characterize a membrane protein from the legume Medicago truncatula. By coupling photoaffinity labeling experiments with proteomics and transcriptomics, we identified the likely LCO-binding protein as LYR3, a lysin motif receptor-like kinase (LysM-RLK). LYR3, expressed heterologously, exhibits high-affinity binding to LCOs but not COs. Homology modeling, based on the Arabidopsis CO-binding LysM-RLK AtCERK1, suggests that LYR3 could accommodate the LCO in a conserved binding site. The identification of LYR3 opens up ways for the molecular characterization of LCO/CO discrimination.

Functionalized oligosaccharides are useful intermediates to prepare products for biological research or for the development of advanced functional materials. Here, we report the unprecedented use of aniline as an efficient organocatalyst reaction with "clickable" (azide or alkyne) amine for the transimination-mediated reductive amination of a chitooligosaccharide. Moreover, we demonstrate that alkyne-bearing aniline constitutes an excellent tool for the easy derivatization of chitosan oligosaccharides. Evidence for such improvement has been illustrated by the straightforward design of a FRET substrate to probe chitinase activity and of amphiphilic polycaprolactone-grafted-chitosan. This efficient methodology paves the way to the preparation of novel chitosan oligosaccharide-based advanced materials.

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 β-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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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.

Two tetrasaccharides carrying the trisaccharidic Lewis x motif on a GlcNAc or a Gal residue were produced on the gram-scale by high-cell-density cultures of metabolically engineered Escherichia coli strains that overexpressed the Helicobacter pylori futA gene for alpha-3 fucosyltransferase and the Neisseria meningitidis lgtB gene for beta-4 galactosyltransferase. The first compound Galbeta-4(Fucalpha-3)GlcNAcbeta-4GlcNAc was produced by glycosylation of chitinbiose, which was endogenously generated in the bacterial cytoplasm by the successive action of the rhizobial chitin-synthase NodC and the Bacillus circulans chitinase A1, whose genes were additionally expressed in the E. coli strain. The second compound, Galbeta-4(Fucalpha-3)GlcNAcbeta-3Gal, was produced from exogenously added Gal by a strain that was deficient in galactokinase activity and overexpressed the additional N. meningitidis lgtA gene for beta-3 N-acetylglucosaminyltransferase.

Oligosaccharidic moieties of GM(2) and GM(3) gangliosides bearing an allyl or a propargyl aglycon, are efficiently biosynthesized on the gram scale by growing metabolically engineered Escherichia coli cells in the presence of the corresponding lactoside acceptors and sialic acid.

Sulfonated and non-sulfonated lipo-chitooligosaccharides involved in Sinorhizobium meliloti-legume symbiosis are efficiently obtained on a multi mg scale by a 2-step procedure combining biotechnological and chemical approaches.

We previously described a bacterial fermentation process for the in vivo conversion of lactose into fucosylated derivatives of lacto-N-neotetraose Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LNnT). The major product obtained was lacto-N-neofucopentaose-V Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, carrying fucose on the glucosyl residue of LNnT. Only a small amount of oligosaccharides fucosylated on N-acetylglucosaminyl residues and thus carrying the LewisX group (Le(X)) was also produced. We report here a fermentation process for the large-scale production of Le(X) oligosaccharides. The two fucosyltransferase genes futA and futB of Helicobacter pylori (strain 26695) were compared in order to optimize fucosylation in vivo. futA was found to provide the best activity on the LNnT acceptor, whereas futB expressed a better Le(X) activity in vitro. Both genes were expressed to produce oligosaccharides in engineered Escherichia coli (E. coli) cells. The fucosylation pattern of the recombinant oligosaccharides was closely correlated with the specificity observed in vitro, FutB favoring the formation of Le(X) carrying oligosaccharides. Lacto-N-neodifucohexaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc represented 70% of the total oligosaccharide amount of futA-on-driven fermentation and was produced at a concentration of 1.7 g/L. Fermentation driven by futB led to equal amounts of both lacto-N-neofucopentaose-V and lacto-N-neofucopentaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, produced at 280 and 260 mg/L, respectively. Unexpectedly, a noticeable proportion (0.5 g/L) of the human milk oligosaccharide 3-fucosyllactose Gal(beta1-4)[Fuc(alpha1-3)]Glc was produced in futA-on-driven fermentation, underlining the activity of fucosyltransferase FutA in E. coli and leading to a reassessment of its activity on lactose. All oligosaccharides produced by the products of both fut genes were natural compounds of human milk.

Two metabolically engineered Escherichia coli strains have been constructed to produce the carbohydrate moieties of gangliosides GM2 (GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc; Gal = galactose, Glc = glucose, Ac = acetyl) and GM1 (Galbeta-3GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc. The GM2 oligosaccharide-producing strain TA02 was devoid of both beta-galactosidase and sialic acid aldolase activities and overexpressed the genes for CMP-NeuAc synthase (CMP = cytidine monophosphate), alpha-2,3-sialyltransferase, UDP-GlcNAc (UDP = uridine diphosphate) C4 epimerase, and beta-1,4-GalNAc transferase. When this strain was cultivated on glycerol, exogenously added lactose and sialic acid were shown to be actively internalized into the cytoplasm and converted into GM2 oligosaccharide. The in vivo synthesis of GM1 oligosaccharide was achieved by taking a similar approach but using strain TA05, which additionally overexpressed the gene for beta-1,3-galactosyltransferase. In high-cell-density cultures, the production yields for the GM2 and GM1 oligosaccharides were 1.25 g L(-1) and 0.89 g L(-1), respectively.

When fed to a beta-galactosidase-negative (lacZ(-)) Escherichia coli strain that was grown on an alternative carbon source (such as glycerol), lactose accumulated intracellularly on induction of the lactose permease. We showed that intracellular lactose was efficiently glycosylated when genes of glycosyltransferase that use lactose as acceptor were expressed. High-cell-density cultivation of lacZ(-) strains that overexpressed the beta 1,3 N acetyl glucosaminyltransferase lgtA gene of Neisseria meningitidis resulted in the synthesis of 6 g x L(-1) of the expected trisaccharide (GlcNAc beta 1-3Gal beta 1-4Glc). When the beta 1,4 galactosyltransferase lgtB gene of N. meningitidis was coexpressed with lgtA, the trisaccharide was further converted to lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) and lacto-N-neoheaxose with a yield higher than 5 g x L(-1). In a similar way, the nanA(-) E. coli strain that was devoid of NeuAc aldolase activity accumulated NeuAc on induction of the NanT permease and the lacZ(-) nanA(-) strain that overexpressed the N. meningitidis genes of the alpha2,3 sialyltransferase and of the CMP-NeuAc synthase efficiently produced sialyllactose (NeuAc alpha 2-3Gal beta 1-4Glc) from exogenous NeuAc and lactose.

We report here the in vivo production of type 2 fucosylated-N-acetyllactosamine oligosaccharides in Escherichia coli. Lacto-N-neofucopentaose Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)Glc, lacto-N-neodifucohexaose Galbeta1-4(Fucalpha1-3)Glc-NAcbeta1-3Galbeta1-4(Fucalpha1-3)Glc, and lacto-N-neodifucooctaose Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)Glc were produced from lactose added in the culture medium. Two of them carry the Lewis X human antigen. High cell density cultivation allowed obtaining several grams of fucosylated oligosaccharides per liter of culture. The fucosylation reaction was catalyzed by an alpha-1,3 fucosyltransferase of Helicobacter pylori overexpressed in E. coli with the genes lgtAB of N. meningitidis. The strain was genetically engineered in order to provide GDP-fucose to the system, by genomic inactivation of gene wcaJ involved in colanic acid synthesis and overexpression of RcsA, positive regulator of the colanic acid operon. To prevent fucosylation at the glucosyl residue, lactulose Galbeta1-4Fru was assayed in replacement of lactose. Lactulose-derived oligosaccharides carrying fucose were synthesized and characterized. Fucosylation of the fructosyl residue was observed, indicating a poor acceptor specificity of the fucosyltransferase of H. pylori.

High cell density cultivation of recombinant Escherichia coli strains harboring the nodBC genes (encoding chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively) from Azorhizobium caulinodans has been previously described as a practical method for the preparation of gram-scale quantities of penta-N-acetyl-chitopentaose and tetra-N-acetylchitopentaose (Samain, E., Drouillard, S., Heyraud, A., Driguez, H., Geremia, R.A., 1997. Carbohydr. Res. 30, 235-242). We have now extended this method to the production of sulfated and O-acetylated derivatives of these two compounds by coexpressing nodC or nodBC with nodH and/or nodL that encode chitooligosaccharide sulfotransferase and chitooligosaccharide O-acetyltransferase, respectively. In addition, these substituted chitooligosaccharides were also obtained as tetramers by using nodC from Rhizobium meliloti instead of nodC from A. caulinodans. These compounds should be useful precursors for the preparation of Nod factor analogues by chemical modification.

Scientific and commercial interest in oligosaccharides is increasing, but their availability is limited as production relies on chemical or chemo-enzymatic synthesis. In search for a more economical, alternative procedure, we have investigated the possibility of producing specific oligosaccharides in E. coli that express the appropriate glycosyltransferases. The Azorhizobium chitin pentaose synthase NodC (a beta(1,4)GlcNAc-oligosaccharide synthase), and the Neisseria beta(1,4)galactosyltransferase LgtB, were co-expressed in E. coli. The major oligosaccharide isolated from the recombinant strain, was subjected to LC-MS, FAB-MS and NMR analysis, and identified as betaGal(1,4)[betaGlcNAc(1,4)]4GlcNAc. High cell density culture yielded more than 1.0 gr of the hexasaccharide per liter of culture. The compound was found to be an acceptor in vitro for betaGal(1,4)GlcNAc alpha(1,3)galactosyltransferase, which suggests that the expression of additional glycosyltransferases in E. coli will allow the production of more complex oligosaccharides.

Cultivation of Escherichia coli harbouring heterologous genes of oligosaccharide synthesis is presented as a new method for preparing large quantities of high-value oligosaccharides. To test the feasibility of this method, we successfully produced in high yield (up to 2.5 g/L) penta-N-acetyl-chitopentaose (1) and its deacetylated derivative tetra-N-acetyl-chitopentaose (2) by cultivating at high density cells of E. coli expressing nodC or nodBC genes (nodC and nodB encode for chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively). These two products were easily purified by charcoal adsorption and ion-exchange chromatography. One important application of compound 2 could be its utilisation as a precursor for the preparation of synthetic nodulation factors by chemical acylation.

A successful chemoenzymatic synthesis of oligosaccharides with an interglucosidic sulfur atom as inhibitors of 1,3:1,4-D-glucanases is described. The key compound 3a was synthesized from acetylated 1-thio-beta-laminaribiose 4 and the methyl 4'-O-triflyl-lactoside 5. After de-O-acylation, the tetrasaccharide 3b was used as an acceptor and glucose-1-P as a donor in a phosphorolytic elongation catalysed by cellodextrin phosphorylase from Clostridium thermocellum. The expected pentasaccharide 2a and hexasaccharide 1 were isolated in 56% and 13% yield, respectively. As expected, the thiooligosaccharides 1, 2a, and 3b were resistant to enzymatic cleavage by 1,3:1,4-beta-D-glucanase isolated from Bacillus licheniformis. Furthermore, they have been shown to act as competitive inhibitors of the hydrolysis of the chromophoric trisaccharide substrate 11 by this enzyme.