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.

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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.