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.

No abstract

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.

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.

MATERIALS AND METHODSThe oligosacchadde glycans mannosylal-6(mannosylal-3)mannosyla1-6(mannosyla1-3)mannosyl 1-4-N-acetylglucosamine and mannosylal-6(mannosylal-3)(xylosyl81-2)mannosyl#1-4-N-acetylglucosaminyl(fucosyla1-3)N-acetylglucosamine were infiltrated into mature green tomato fruit (Lycopersicon esculentum Mill., cv Rutgers). Coinfiltration of 1 nanogram per gram fresh weight of the glycans with 40 micrograms per gram fresh weight galactose, a level of galactose insufficient to promote ripening, stimulated ripening as measured by red coloration and ethylene production.Glycoproteins contain oligosaccharide chains, i.e. glycans, linked via an N-linkage through the amido group of asparagine or an 0-linkage through the hydroxyl groups of serine, threonine, or hydroxyproline. N-Glycoproteins, i.e. N-glycosylated, have been found in plant cell walls as well as in other organelles (10). It has been suggested that glycosylation is involved with protein stability, biological activity, and mobility (1).N-Glycans have a pentasaccharidic core structure Man3(GlcNAc)2, substituted with mannosyl residues (highmannose type) or by xylosyl, fucosyl, N-acetylglucosaminyl, and galactosyl residues (complex type). Free N-glycans can occur as precursors of glycosylation or by glycoprotein proteolysis. Occurrence of the free N-glycans Man5(Xyl)-GlcNAc(Fuc)GlcNAc and Man5GlcNAc has been demonstrated in a cell suspension culture of Silene alba (9). The authors hypothesized that their presence was due to proteolysis related to autophagy. The xylosyl-containing oligosaccharide purified from the suspension culture was described as a growth factor acting at nanomolar range during early development of flax (8). We report here further evidence for the biological activity of free N-glycans in plant metabolism. The oligosaccharides Man3(Xyl)GlcNAc(Fuc)GlcNAc and Man3GlcNAc stimulated ripening oftomato fruit as measured by red coloration and ethylene production. This is the first evidence for the potential involvement of free N-glycans in fruit ripening. It is also the first description of the biological activity of the N-glycan Man5GlcNAc. Plant MaterialTomato (Lycopersicon esculentum Mill. cv Rutgers) plants were grown in a greenhouse without supplemental lighting. Flowers were pollinated by a mechanical vibrator and tagged at anthesis. Fruit of uniform size (100 ± 10 g/fresh weight) were hand-harvested at the mature green stage of ripeness at 34 ± 2 d postpollination. OligosaccharidesThe oligomannoside Man5GlcNAc was obtained commercially (BioCarb,' Lund, Sweden) and was also generously provided by Dr.

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.

The concentration-dependent stimulatory and inhibitory effect of N-glycans on tomato (Lycopersicon esculentum Mill.) fruit ripening was recently reported (B. Priem and K.C. Cross [1992] Plant Physiol 98: 399-401). We report here the structure of 10 free Nglycans in mature green tomatoes. N-Glycans were purified from fruit pericarp by ethanolic extraction, desalting, concanavalin A-Sepharose chromatography, and amine-bonded d i c a high performance liquid chromatography. N-Glycan structures were determined using 500 MHz 'H-nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, and glycosyl linkage methylation analysis by gas chromatography-mass spectrometry. A nove1 arabinosyl-containing N-glycan, M a n a 1 4 6(Ara,a1+2)Man~14GIcNAc~l+4(Fucal+3)ClcNAc, was purified from a retarded concanavalin A fraction. The location of the arabinosyl residue was the same as the xylosyl residue in complex N-glycans. C~cNAc~5'1Man3(Xy~)ClcNAc(Fuc)ClcNAc and CIcNAc~5'1Man2ClcNAc(Fuc)ClcNAc were also purified from the weakly retained fraction. The oligomannosyl N-glycans Man,ClcNAc, Man6GlcNAc, Man,GlcNAc, and Man&lcNAc were purified from a strongly retained concanavalin A fraction. The finding of free MansClcNAc in situ was important physiologically because previously we had described it as a promoter of tomato ripening when added exogenously. Mature green pericarp tissue contained more than 1 pg of total free N-glycanlg fresh weight. Changes in N-glycan composition were determined during ripening by comparing glycosyl and glycosyl-linkage composition of oligosaccharidic extracts from fruit at different developmental stages. N-Clycans were present in pericarp tissue at all stages of development. However, the amount increased during ripening, as did the relative amount of xylosyl-containing N-glycans.Plant N-glycans, an integral part of N-glycoproteins, are classified into two types based on the presence (complex type) or absence (oligomannosidic type) of a pl-2-linked xylosyl in addition to mannosyl and N-acetylglucosaminyl residues. Research on N-glycoproteins has included structural analyses, biosynthesis, importance in protein conformation

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.