A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria

Bernard Priem2, Michel Gilbert3, Warren W. Wakarchuk3, Alain Heyraud2 and Eric Samain1,2

2Centre de Recherches sur les Macromolécules Végétales, CNRS (affiliated with the Joseph Fourier University) BP 53, 38041 Grenoble cedex 9 France, and 3Institute for Biological Sciences, NRCC, Ottawa, Ontario, K1A 0R6, Canada

Received on June 3, 2001; revised on August 2, 2001; accepted on August 8, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
When fed to a ß-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 ß1,3 N acetyl glucosaminyltransferase lgtA gene of Neisseria meningitidis resulted in the synthesis of 6 g · L–1 of the expected trisaccharide (GlcNAcß1-3Galß1-4Glc). When the ß1,4 galactosyltransferase lgtB gene of N. meningitidis was coexpressed with lgtA, the trisaccharide was further converted to lacto-N-neotetraose (Galß1-4GlcNAcß1-3Galß1-4Glc) and lacto-N-neoheaxose with a yield higher than 5 g · 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 {alpha}2,3 sialyltransferase and of the CMP-NeuAc synthase efficiently produced sialyllactose (NeuAc{alpha}2-3Galß1-4Glc) from exogenous NeuAc and lactose.

Key words: glycosyltransferase/high-cell-density cultivation/metabolic engineering/oligosaccharide


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Human milk differs from milk of most of other species by its high content of various complex lactose-derived oligosaccharides (more than 10 g · L–1). The terminal nonreducing ends of human milk oligosaccharides (HMOs) are identical to structures found in glycoconjugate and HMOs have been shown to act as competitive ligands preventing the attachment of pathogens to intestinal epithelial cells (Newburg, 1997Go). HMOs are not digested in the small intestine and are important prebiotics that stimulate the growth of a specific Bifidus flora (Beerens et al., 1980Go). Intact HMOs have been shown to be slightly absorbed in the intestinal track and are believed to have immunomodulatory effects (Kunz et al., 2000Go). Despite their unanimously recognized importance in infants’ health, research on HMOs has been hindered by the by the fact that it is still very difficult to obtain large quantities of them either by purification from human fluids or by chemical or enzymatic synthesis (Chen et al., 2000Go).

Recently, promising new strategies for oligosaccharides production have been developed using metabolically engineered bacteria (Endo and Koizumi, 2000Go). These methods are based on the utilization of whole cells that overexpress the glycosyltransferase genes that are naturally involved in the synthesis of oligosaccharides from sugar-nucleotides and on systems that allow a sugar-nucleotide recycling by these whole cells. In the technology developed by the Kyowa Hakko Kogyo company, a Corynebacterium ammoniagenes strain has been engineered to produce and regenerate UTP from inexpensive substrate; by coupling this strain with two recombinant Escherichia coli, one overexpressing the genes of sugar nucleotides biosynthesis and the other overexpressing a glycosyltransferase gene, the Kyowa researchers were able to produce in very high yield different oligosaccharides, such as globotriose (Koizumi et al., 1998Go), lactosamine (Endo et al., 1999Go), and sialyllactose (Endo et al., 2000Go). It has to be pointed out that this method requires a permeabilization of the bacteria with xylene to allow a passive circulation of the substrate between the different types of cells and that the reaction is carried out by nongrowing cells.

As an alternative in the living factory approach (Geremia and Samain, 2000Go), the oligosaccharides are produced in a single growing bacterium that overexpresses the recombinant glycosyltransferase genes and that maintains the pool level of the sugar nucleotides by its enzymatic cellular machinery. By this method, more than 2 g · L–1of chitopentaose have been produced by a high-cell-density culture of an E. coli strain overexpressing the chitooligosaccharide synthase nodC (Samain et al., 1997Go). Up to now the range of oligosaccharides that could be produced by this in vivo method has been limited to chitooligosaccharide derivatives (Samain et al., 1999Go; Bettler et al., 1999Go) because most of the glycosyltransferases of interest require specific acceptors that are not naturally present in the bacterial cytoplasm.

The main problem to address to extend the living factory method was to find a way to provide the bacterium with a suitable acceptor for other glycosyltransferases. We have investigated the possibility of using sugar permeases to introduce acceptors in bacteria, and we have first focused on lactose because it can serve as initial acceptor for the synthesis of number of high-value oligosaccharides, such as those of human milk. We showed here that lactose can be efficiently converted by metabolically engineered E. coli into different human milk oligosaccharides, such as lacto-N-neotetraose and sialyllactose.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The basic strategy was the following: (1) use a ß-galactosidase-negative (lacZ) E. coli strain that was still able to express the lacY gene of the ß-galactoside permease; (2) transform this strain with a gene of a glycosyltransferase that use lactose as acceptor; (3) cultivate this strain at high cell density on an alternative carbon source, such as glycerol, in conditions that allow the expression of both glycosyltransferase and ß-galactoside permease genes; (4) feed the culture with lactose that should be actively internalized by the permease and glycosylated by the transferase.

Production of the trisaccharide LNT-2 (GlcNAcß1-3Galß1-4Glc)
The glycosyltransferase gene used in this first example was the Neisseria meningitidis lgtA gene that encode a ß1,3 N-acetylglucosaminyltransferase (Figure 1). The strain JM109, which is lacY+ lacZ, was transformed with the plasmid pCWlgtA carrying the lgtA gene. The resulting strain JM109 (pCWlgtA) and the control strain JM109 were both cultivated at high cell density with glycerol as the carbon and energy source using a classical fed-batch strategy. As shown in Figure 2A, the lactose, which was added at a concentration of 5 g · L–1 at the beginning of the fed-batch phase, was rapidly internalised by the control strain and accumulated intracellularly. Surprisingly, bacterial growth was not significantly affected by this accumulation. Although the JM109 was lacZ, lactose was slowly degraded, and only one-fifth of the initially added lactose was still present at the end of the culture.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1. Production scheme of the trisaccharide LNT-2 (GlcNAcß1-3 Galß1-4 Glc) by E. coli JM109 expressing the N. meningitidis lgtA gene that encodes a ß1,3 N-acetylglucosaminyltransferase. Lactose is transported into the cell by the ß-galactoside permease LacY. Intracellular lactose, which cannot be degraded because the cell is devoid of ß-galactosidase activity, is glycosylated into LNT-2 by LgtA using the endogenous pool of UDP-N-acetylglucosamine. Glycerol provides the carbon and energy source required for bacterial growth, lactose transport, and LNT-2 synthesis.

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. High-cell-density cultivation of (A) E. coli JM109 and (B) E. coli JM109 (pCWLgtA). (diamonds) bacterial growth; (open squares) extracellular and (filled squares) intracellular lactose; (open triangles) extracellular and (filled triangles) intracellular acid-hydrolyzable N-acetylglucosamine. Glycerol was the growth substrate and lactose (5 g · L–1) was added at the beginning of the fed-batch.

 
Instead of accumulating lactose, the strain JM109(pCWlgtA) produced a high amount of an N-acetylglucosamine (GlcNAc) containing compound (Figure 2B). This compound accumulated transiently in the cell and was then released in the extracellular medium, where it was almost entirely found at the end of the fermentation time course. After purification by charcoal adsorption and size exclusion chromatography on Biogel P4, the structure of the expected trisaccharide LNT-2 GlcNAcß1-3 Galß1-4 Glc was confirmed by nuclear magnetic resonance (NMR) analysis and mass spectrometry, which showed in the FAB+ mode the presence of a quasi-molecular ions [M+H]+ at m/z 546. By colorimetric quantification of GlcNAc, a final production yield of 6 g · L–1 was estimated, indicating that 73% of the initially added lactose has been converted into LNT-2.

Production of the tetrasaccharide LNnT (Galß1-4GlcNAcß1-3Galß1-4Glc)
In this second example, an other N. meningitidis gene, lgtB, encoding a ß1,4 galactosyltransferase was coexpressed with lgtA in a JM109 strain. The strain JM109 (pCWlgtA,pBBlgtB) could not be grown on glycerol medium, probably because of a toxicity problem due to the overexpression of the two lgtAB genes. This was overcome by growing the cells with glucose and by lowering the cultivation temperature at 28°C. As in the first example this strain converted lactose into GlcNAc-containing compounds (Figure 3). However, in that case, these compounds remained mainly intracellular, suggesting that the expression of lgtB had resulted in the synthesis of the expected tetrasaccharide LNnT, which had not been released in the extracellular medium because of its longer size. After purification of the oligosaccharide intracellular fraction by charcoal adsorption, the chromatography on Biogel P4 revealed the presence of several GlcNAc containing compounds of different molecular weights (Figure 4). The mass spectrometry data of the purified three main compounds revealed the presence of quasi-molecular ions [M+H]+ at m/z 708 (compound A), m/z 1073 (compound B), and [M+Na]+ at m/z 1460 (compound C) corresponding to lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), and lacto-N-neooctaose (LNnO) respectively. The identification of compound A as LNnT was confirmed by NMR analysis.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3. High-cell-density cultivation of E. coli JM109 (pCWLgtA, pBBlgtB). (diamonds) bacterial growth; (squares) extracellular lactose; (open triangles) extracellular and (filled triangles) intracellular acid-hydrolyzable N-acetylglucosamine. Glucose was the growth substrate and lactose (5 g · L–1) was added at the beginning of the fed-batch. The cultivation temperature was 28°C.

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. Separation on Biogel P4 of oligosaccharides produced by E. coli JM109 (pCWLgtA, pBBlgtB) when lactose was added at an initial concentation of 5 g · L–1 (solid line) or 1 g · l–1 (dashed line). The peaks A, B, C, and D were identified as LNnT, LNnH, LNnO, and lacto-N-neodecaose (LNnD), respectively.

 
The production of poly-N-acetylactosamine results from the fact that LNnT was used as an acceptor by ß1,3 N-acetylglucosaminyltransferase to form an intermediate pentasaccharide that can be converted to LNnH by the galactosyltransferase LgtB. In a similar way LNnH can be used as acceptor for another cycle resulting in the addition of one additional N-acetyllactosamine unit and leading to the synthesis of LNnO. There was no accumulation of tri-, penta-, or heptasaccharide harboring GlcNAc as terminal nonreducing sugar, suggesting that the LgtA activity (rather than the LgtB activity) was the limiting factor in the synthesis of these oligosaccharides. However it is also possible that the UDP-GlcNAc pool level was the limiting factor.

The relative distribution of LNntT and its longer derivatives obviously depends on the availability of lactose, which is the initial acceptor. Indeed, when the quantity of lactose added to the fermentation medium was reduced from 5 to 1 g · L–1, the distribution was shifted toward higher-molecular-weight compounds, and an additional longer poly-N-acetyllactosamine, lacto-N-neodecaose (compound D) was produced (Figure 4).

Production of the 3' sialyllactose (NeuAc{alpha}2-3 Galß1-4 Glc)
The genes for the biosynthesis of sialic acid and CMP-NeuAc are not normally present in E. coli K12. The in vivo synthesis of sialylated oligosaccharides in such bacteria thus required a metabolic engineering of the CMP-NeuAc biosynthesis pathway. It is known that E. coli K12 can grow with sialic acid as the sole carbon and energy source: sialic acid is first transported in the cytoplasm by a specific permease NanT, then it is degraded by the aldolase encoded by the nanA gene into pyruvate and N-acetylmannosamine. Our idea was to take advantage of this catabolic pathway and to engineer it into an anabolic pathway leading to the synthesis of CMP-NeuAc. The strategy was the following: (1) construct a strain, which would be devoid of sialic acid aldolade activity, by inactivating the nanA gene; (2) genetically modify this strain by adding the gene of the CMP-NeuAc synthase; (3) grow the strain on an alternative carbon source in conditions that allow the expression of both sialic acid permease and CMP-NeuAc synthase gene; and (4) feed the culture with exogelous sialic acid that should be transported into the cell and converted into CMP-NeuAc.

This strategy was applied to the synthesis of 3' sialyllactose (Figure 5). A lacY+, lacZ, nanT+, nanA derivative of strain JM107 was transformed with the two plasmids NST-01 and pBBnsy that contained the N. meningitidis genes for the {alpha}-2,3sialyltransferase and for the CMP-NeuAc synthase, respectively. This strain was cultivated at high cell density on glycerol, and the culture was supplemented with lactose and sialic acid at the beginning of the fed-batch phase (Figure 6). Lactose accumulated transiently into the cell and was then converted into sialyllactose, which accumulated first in the intracellular fraction before being partly released in the intracellular mediu. After 22 h of production, the final yield of sialyllactose was estimated by enzymatic assay to be 1.5 g · L–1 in the intracellular fraction and 1.1 g · L–1 in the extracellular fraction. Both fractions were purified by charcoal adsorption and ion exchange chromatography, and a final yield of 49% was obtained. The identity of sialyllactose was confirmed by mass spectrometry, which indicated the presence of two quasi-molecular ions [M+H]+ at m/z 656 and [M+Na]+ at m/z 678 corresponding to sodium salt of sialyllactose.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5. Production scheme of the 3' sialyllactose (NeuNAc{alpha}2-3 Galß1-4 Glc) by E. coli JM107-nanA expressing the N. meningitidis genes for {alpha}-2,3 sialyltransferase and CMP-NeuAc synthase. Sialic acid and lactose are transported into the cell by the sialic acid (NanT) and ß-galactoside(LacY) permeases. None of these compounds can be degraded by the cells that are nanA and lacZ. The expression of both CMP-NeuAc synthase and {alpha}-2,3 sialyltransferase allow the intracellular activation of NeuAc into CMP-NeuAc and its subsequent transfer on lactose. Glycerol provides the carbon and energy source required for bacterial growth, lactose and sialic acid transport, and sialyllactose synthesis.

 


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 6. High-cell-density cultivation of E. coli JM107-nanA (NST-01, pBBnsy). (diamonds) bacterial growth; (open squares) extracellular and (filled squares) intracellular lactose; (open triangles) extracellular and (filled triangles) intracellular sialyllactose. Glycerol was the growth substrate and lactose (1.5 g · L–1) and sialic acid (0.6 g · L–1) were added at the beginning of the fed-batch. During the last 17 h of cultivation the culture was continuously fed with lactose and sialic acid with a rate of 0.2 and 0.1 g · L–1 · h1, respectively.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
These results demonstrate that biologically important oligosaccharides such as human milk lactose derivatives can be produced by direct fermentation of inexpensive carbon source with a yield of several grams per liter. The availability of large quantities of oligosaccharides will be useful to investigate the different beneficial effects of HMOs on infants’ health. It would also make possible the development of therapeutic applications of free oligosaccharides as anti-infective agent (Zopf and Roth, 1996Go)

The idea of using sugar permeases to internalize the acceptors for recombinant glycosyltransferases opens new perspectives in the field of carbohydrate synthesis. The process can certainly be extended to the synthesis of a large variety of oligosaccharides. First the broad specificity of the lactose permease could be used to transport various analogues of lactose. Then other sugar permeases either from E. coli or even from other organisms could be used to transport different types of acceptors.

The variety of structures that can be produced can also be extended by using some of the many glycosyltransferase genes of different specificities that have been identified. So far, only the glycosyltransferases that use UDP-GlcNAc, UDP-Gal, and CMP-NeuAc have been shown to work in our process. The next step will be to demonstrate that other important sugar nucleotides, such as GDP-fucose, UDP-GalNAc, and UDP-GlcU, can be used as precursor for an in vivo glycosylation in growing E. coli. That may require the metabolic engineering of their biosynthesis pathway, such as was done for the synthesis of CMP-NeuAc.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bacterial strains, plasmids, and cloning procedure
E. coli JM107 and JM109 were obtained from the Deutsche Sammlung von Mikroorganismen. Due to the {Delta}(lacZ)M15 deletion in the lacZ gene, these strains are unable to produce a complete active ß-galactosidase. The N. meningitidis MC58 genes for ß-1,3 N-acetylglucosaminyltransferase (plasmid pCWlgtA, GenBank no. 25839) (Wakarchuk et al., 1996Go), ß-1,4 galactosyltransferase (plasmid pCWlgtB, GenBank no. 25839) (Wakarchuk et al., 1996Go), {alpha}-2,3 sialyltransferase (plasmid NST-01, GenBank no. U60660) (Gilbert et al., 1996Go), and CMP-NeuAc-synthase (plasmid NSY-01, GenBank no. U60146) (Gilbert et al., 1997Go) were from the collection of the Institute for Biological Science.

The plasmid pBBlgtB (Bettler et al., 1999Go) was obtained by subcloning lgtB gene from pCWlgtB into pBBR1MCS (Kovach et al., 1995Go). To obtain the plasmid pBBnsy, the NSY-01 plasmid was digested with XbaI, and the XbaI-fragment containing the CMPNeuAc–syntase gene was cloned in the XbaI site pBBR1MCS.

The strain JM107-nanA was prepared from strain JM107 by inactivation of nanA encoding for NeucAc aldolase. This was done by insertion of the kanamycin-resistance gene from pUC-4K cassette (Pharmacia) into nanA by homologous recombination using an appropriately designed suicide vector derived from the plasmid pMAK705 (Hamilton et al., 1989Go).

High-cell-density cultivation
They were carried out in 2-L reactors containing 1 L of mineral culture medium as previously described (Samain et al., 1997Go). Unless otherwise indicated, the temperature was 34°C and the pH was 6.8. The cultivation strategy included three phases: a first exponential growth phase, which started at the inoculation of the fermentor and lasted until exhaustion of the carbon substrate (glucose or glycerol) initially added to the medium at a concentration of 17.5 g · L–1; then a 5-h fed-batch phase with a high substrate feeding rate of 4.5 g · L–1 · h–1; and finally a 10–20-h fed-batch phase with a lower feeding rate of 2.7 g · L–1 · h–1. The acceptor (lactose and sialic acid) were added at the beginning of the first fed-batch phase, at the same time as the inducer (IPTG 50 mg · L–1) of the ß-galactosidase permease and the recombinant glycosyltransferase genes that were under the control of the Plac promoter.

Quantification of oligosaccharides
Culture samples (1 ml) were immediately centrifuged in microfuge tubes (5 min, 12,000 x g). The supernatants were saved for quantification of extracellular oligosaccharides. The pellets were resuspended in 1 ml of distilled water, boiled for 30 min, and centrifuged as above. The second supernatant was kept to quantify the intracellular oligosaccharides.

After acid hydrolysis N-acetylglucosamine content was quantified colorometrically using the Ehrlich reagent (Samain et al., 1999Go). Lactose concentration was quantified using the Kit from Roche Diagnostic for the enzymatic determination of lactose and glucose. Because the lactose unit of sialyllactose is not detected by the enzymatic kit, the sialyllactose concentration was calculated by enzymatically determining the lactose concentration in samples treated with a neuraminidase from Clostridium perfringens (Type 5, Sigma N2876) and by subtracting the value obtained with the same samples untreated with the neuraminidase.

Purification of oligosaccharides
At the end of the fermentation time course, the bacterial cells were recovered by centrifugation (20 min at 12,000 x g). The supernatants were saved for purification of extracellular oligosaccharides. The pellets were resuspended in a volume of distilled water equal to that of the original culture medium, and the cells were permeabilized by being autoclaved at 100°C for 30 min. After another centrifugation (30 min at 12,000 x g) the cells debris were discarded and the supernatants were saved to purify the intracellular oligosaccharides.

The oligosaccharides were adsorbed on activated charcoal and, after a thorough washing with distilled water, they were eluted as previously described (Samain et al., 1997Go) with 50% (v/v) aqueous ethanol. The purification yield was around 60–70%.

The oligosaccharides produced by the strain JM109(pCWlgtA) and JM109(pCWlgtA, pBBlgtB) were further purified by size exclusion chromatography on a Biogel P4 (4.5 cm x 95 cm) column, with distilled water and the flow rate was 40 ml · h–1.

Sialyllactose was separated from neutral oligosaccharides by being fixed on a Dowex 1X4-400 (HCO3 form) resin, and elution with a linear NaHCO3 gradient (0 to 100 mM). The sodium bicarbonate was then eliminated by treating the eluate with a Dowex 50X4-400 (H+ form) resin.

Structural analysis
1H-NMR spectra were recorded at 30°C with a 300 MHz Bruker AM300 spectrometer using D2O as solvent. Mass spectra in the FAB(+) mode were recorded with a Nermag R 10 10C spectrometer.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank C. Bosso of CERMAV for performing the mass spectrometry measurement and Dr S.D. Kushner of University of Georgia for kindly providing the pMAK705 suicide vector.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
HMO, human milk oligosaccharide; GlcNAc, N-acetylglucosamine; LNnH, lacto-N-neohexaose; LNnO, lacto-N-neooctaose; LNnT, lacto-N-neotetraose; NeuAc, N-acetylneuraminic acid; NMR, nuclear magnetic resonance.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Beerens, H., Romond, C., and Neut, C. (1980) Influence of breast-feeding on the bifid flora of the newborn intestine. Am. J. Clin. Nutr., 33, 2434–2439.[Abstract]

Bettler, E., Samain, E., Chazalet, V., Bosso, C., Heyraud, A., Joziasse, D.H., Wakarchuk, W.W., Imberty, A., and Geremia, R.A. (1999) The living factory: in vivo production of N-acetyllactosamine containing carbohydrates in E. coli. Glycoconj. J., 6, 205–212.

Chen, X., Kowal, P., and Wang, P.G. (2000) Large scale enzymatic synthesis of oligosaccharides. Curr. Opin. Drug Discov. Develop., 3, 756–763.

Endo, T. and Koizumi, S. (2000) Large-scale production of oligosaccharides using engineered bacteria. Curr. Opin. Struct. Biol., 10, 536–541.[CrossRef][ISI][Medline]

Endo, T., Koizumi, S., Tabata, K., Kakita, S., and, Ozaki, A. (1999) Large scale production of N-acetyllactosamine through bacterial coupling. Carbohydr. Res., 316, 179–183.[CrossRef][ISI][Medline]

Endo, T., Koizumi, S., Tabata, K., and Ozaki, A. (2000) Large scale production of CMP-NeuAc and sialylated oligosaccharides through bacterial coupling. Appl. Microbiol. Biotechnol., 53, 257–261.[CrossRef][ISI][Medline]

Geremia, R.A. and Samain, E. (2000) Production of heterologous oligosaccharides by recombinant bacteria. In Carbohydrate in chemistry and biology. Wiley VCH Verlag, Weinhem, Germany, vol. 2, pp. 845–859

Gilbert, M, Watson, D.C., and Wakarchuk, W.W (1997) Purification and characterization of the recombinant CMP-sialic acid synthetase from Neisseria meningitidis. Biotechnol. Lett., 19, 417–420.[CrossRef][ISI]

Gilbert, M., Watson, D.C., Cunningham, A.M., Jennings, M.P., Young, N.M., and Wakarchuk, WW. (1996) Cloning of the lipooligosaccharide {alpha}-2, 3-sialyltransferase from the bacterial pathogen Neisseria meningitidis and Neisseria gonorrhoea. J. Biol. Chem., 271, 28271–28276.[Abstract/Free Full Text]

Hamilton, C.M., Aldea, M., Washburn, B.K., Babitze, P., and Kushner S.R. (1989) New method for generating deletions and gene replacements in Escherichia coli. J. Bacteriol., 171, 4617–4622.[ISI][Medline]

Koizumi, S., Endo, T., Tabata, K., and Ozaki A. (1998) Large-scale production of UDP-galactose and globotriose by coupling metabolically engineered bacteria. Nat. Biotechnol., 16, 847–850.[ISI][Medline]

Kovach, M.E., Elzer, P.H., Hill, D.S., Robertson, G.T., Farris, M.A., Roop, R.M., and Peterson, K.M. (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic–resistance cassettes. Gene, 166, 175–176.[CrossRef][ISI][Medline]

Kunz, C., Rudloff, S., Baier, W., Klein, N., and Strobel, S. (2000) Oligosaccharides in human milk: structural, functional, and metabolic aspects. Ann. Rev. Nutr., 20, 699–722.[CrossRef][ISI][Medline]

Newburg, D.S (1997) Do the binding properties of oligosaccharides in milk protect human infants from gastrointestinal bacteria? J. Nutr., 125, 980S–984S.

Samain, E., Chazalet, V., and Geremia, R.A. (1999) Production of O-acetylated and sulfated chitooligosaccharides by recombinant Escherichia coli strains harboring different combinations of nod genes. J. Biotechnol., 72, 33–47.[CrossRef][ISI][Medline]

Samain, E., Drouillard, S., Heyraud, A., Driguez, H., and Geremia, R. (1997) Gram scale synthesis of recombinant chitooligosaccharides in Escherichia coli. Carbohydr. Res., 302, 35–42.[CrossRef][ISI][Medline]

Sears, P. and Wong, CH. (1998) Enzyme action in glycoprotein synthesis. Cell Mol. Life Sci., 54, 223–252.[ISI]

Wakarchuk, W.W., Martin, A., Jenning, M.P., Moxon, E.R., and Richards, J.C. (1996) Functional relationships of the genetic locus encoding the glycosyltransferase enzymes involved in expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis. J. Biol. Chem., 271, 19166–19173.[Abstract/Free Full Text]

Zopf, D. and Roth, S. (1996) Oligosaccharides anti-infective agents. Lancet, 347, 1017–1021[ISI][Medline]





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (18)
Disclaimer
Request Permissions
Google Scholar
Articles by Priem, B.
Articles by Samain, E.
PubMed
PubMed Citation
Articles by Priem, B.
Articles by Samain, E.