Bernard Priem

Large-Scale In Vivo Synthesis of the Carbohydrate Moieties of Gangliosides GM1 and GM2 by Metabolically Engineered Escherichia coli

Two metabolically engineered Escherichia coli strains have been constructed to produce the carbohydrate moieties of gangliosides GM2 (GalNAcβ‐4(NeuAcα‐3)Galβ‐4Glc; Gal=galactose, Glc=glucose, Ac=acetyl) and GM1 (Galβ‐3GalNAcβ‐4(NeuAcα‐3)Galβ‐4Glc. The GM2 oligosaccharide‐producing strain TA02 was devoid of both β‐galactosidase and sialic acid aldolase activities and overexpressed the genes for CMP‐NeuAc synthase (CMP=cytidine monophosphate), α‐2,3‐sialyltransferase, UDP‐GlcNAc (UDP=uridine diphosphate) C4 epimerase, and β‐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 β‐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.

In vivo fucosylation of lacto-N-neotetraose and lacto-N-neohexaose by heterologous expression of Helicobacter pylori α-1,3 fucosyltransferase in engineered Escherichia coli

We report here the in vivo production of type 2 fucosylated-N-acetyllactosamine oligosaccharides in Escherichia coli. Lacto-N-neofucopentaose Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc, lacto-N-neodifucohexaose Galβ1-4(Fucα1-3)Glc-NAcβ1-3Galβ1-4(Fucα1-3)Glc, and lacto-N-neodifucooctaose Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)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 α-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 Galβ1-4Fru 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.

Sulphated exopolysaccharides produced by two unicellular strains of cyanobacteria, Synechocystis PCC 6803 and 6714

The exopolysaccharides (EPS) of two unicellular strains of cyanobacteria Synechocystis PCC 6803 and 6714, formed labile, radial structures, uniformly distributed on the cell surface, and stainable by specific dyes for acidic polysaccharides. The two strains produced EPS at similar rates, which depended, along with the duration of the producing phase, on the incubation conditions. The exopolysaccharides from both strains were constituted of at least 11–12 mono-oses, probably forming several types of polymers. They contained about 15–20% (w/w) uronic derivatives and 10–15% (w/w) osamines. Proteins represented 20–40% of total weight. A most interesting feature was the presence of 7–8% (molar ratio) sulphate residues, a characteristic that is otherwise limited to exopolysaccharides produced by eukaryotic algae.

Assessment of the Two Helicobacter pylori α‐1,3‐Fucosyltransferase Ortholog Genes for the Large‐Scale Synthesis of LewisX Human Milk Oligosaccharides by Metabolically Engineered Escherichia coli

We previously described a bacterial fermentation process for the in vivo conversion of lactose into fucosylated derivatives of lacto‐ N‐neotetraose Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (LNnT). The major product obtained was lacto‐ N‐neofucopentaose‐V Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)[Fuc(α1–3)]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 (LeX) was also produced. We report here a fermentation process for the large‐scale production of LeX 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 LeX 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 LeX carrying oligosaccharides. Lacto‐ N‐neodifucohexaose‐II Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)[Fuc(α1–3)]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 futBled to equal amounts of both lacto‐ N‐neofucopentaose‐V and lacto‐ N‐neofucopentaose‐II Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)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(β1–4)[Fuc(α1–3)]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.

Isolation and characterization of free glycans of the oligomannoside type from the extracellular medium of a plant cell suspension

The oligosaccharides Man5GlcNAc and Man3(Xyl)GlcNAc(Fuc)GlcNAc presumed to originate fromN-glycosyl proteins have been purified from an extracellular medium (concentration: 2–5 mg/l of 14 day cultures) of white campion (Silene alba) suspension culture. Their primary structures have been determined by1H-400-MHz NMR spectroscopy and FAB-MS spectrometry. They are probably the result of an autophagic process including protein catabolism due to sucrose starvation. Additional identification of digalactosylglycerol (galactolipid breakdown) argues for this hypothesis.

Glycomimicry: Display of the GM3 sugar epitope on Escherichia coli and Salmonella enterica sv Typhimurium

Oligosaccharides present on the surface of pathogenic bacteria play an important role in their interaction with their host. Bacteria with altered cell surface structures can be used to study these interactions, and glycoengineering represents a tool to display a glycoepitope on a different bacterium. Here, we present non-pathogenic Escherichia coli and Salmonella enterica serovar Typhimurium expressing the sialyllactose oligosaccharide epitope of the ganglioside GM3. By expression of the galactosyltransferase LgtE and the sialic acid transferase Lst as well as the CMP-sialic acid synthetase SiaB from Neisseria gonorrhoeae and Neisseria meningitidis in engineered strains devoid of the sialic acid catabolism, the GM3 sugar epitope was displayed on these bacteria as demonstrated by live cell immunostaining and a detailed analysis of their lipooligosaccharides. These strains offer the possibility to investigate the role of sialic acid in the recognition of bacteria by the immune system in a non-pathogenic background.

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 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.

Supported Lipopolysaccharide Bilayers

In this report, the formation of supported lipopolysaccharide bilayers (LPS-SLBs) is studied with extracted native and glycoengineered LPS from Escherichia coli ( E. coli ) and Salmonella enterica sv typhimurium ( S. typhimurium ) to assemble a platform that allows measurement of LPS membrane structure and the detection of membrane tethered saccharide-protein interactions. We present quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP) characterization of LPS-SLBs with different LPS species, having, for example, different molecular weights, that show successful formation of SLBs through vesicle fusion on SiO(2) surfaces with LPS fractions up to 50 wt %. The thickness of the LPS bilayers were investigated with AFM force-distance measurements which showed only a slight thickness increase compared to pure POPC SLBs. The E. coli LPS were chosen to study the saccharide-protein interaction between the Htype II glycan epitope and the Ralstonia solanacearum lectin (RSL). RSL specifically recognizes fucose sugars, which are present in the used Htype II glycan epitope and absent in the epitopes LPS1 and EY2. We show via fluorescence microscopy that the specific, but weak and multivalent interaction can be detected and discriminated on the LPS-SLB platform.

Synthesis of allyl 2-O-(α-l-arabinofuranosyl)-6-O-(α-d-mannopyranosyl)-β-d-mannopyranoside, a unique plant N-glycan motif containing arabinose

Abstract The synthesis of the trisaccharide allyl 2- O -(α- l -arabinofuranosyl)-6- O -(α- d -mannopyranosyl)-β- d -mannopyranoside is reported. Stereoselective glycosylation at C-6 of a non-protected allyl β-mannoside with the acetylated α- d -mannosyl bromide gave the α-(1→6)-disaccharide as the main product and the crystalline 3,6-branched trisaccharide as minor compound. Further glycosylation of the 2,3 diol (1→6) disaccharide with l -arabinofuranosyl bromide furnished a mixture of 3- O - and 2- O -α- l -Ara-trisaccharides from which the title compound was isolated.

Glycomimicry: display of fucosylation on the lipo-oligosaccharide of recombinant Escherichia coli K12

We recently described the design of Escherichia coli K12 and Salmonella enterica sv Typhimurium to display the gangliomannoside 3 (GM3) antigen on the cell surface [1]. We report here the fucosylation of modified lipooligosaccharide in a recombinant E.coli strain with a truncated lipid A core due to deletion of the core glycosyltransferases genes waaO and waaB. This truncated structure was used as a scaffold to assemble the Lewis Y motif by consequent action of the heterologously expressed β-1,4 galactosyltransferase LgtE (Neisseria gonorrheae), the β-1,3 N-acetylglucosaminyltransferase LgtA and the β-1,3 galactosyltransferase LgtB from Neisseria meningitidis, as well as the α-1,2 and α-1,3 fucosyltransferases FutC and FutA from Helicobacter pylori. We show the display of the Lewis Y structure by immunological and chemical analysis.