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.
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Abstract |
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Key words: glycosyltransferase/high-cell-density cultivation/metabolic engineering/oligosaccharide
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Introduction |
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Recently, promising new strategies for oligosaccharides production have been developed using metabolically engineered bacteria (Endo and Koizumi, 2000). 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., 1998
), lactosamine (Endo et al., 1999
), and sialyllactose (Endo et al., 2000
). 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, 2000), 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 · L1of chitopentaose have been produced by a high-cell-density culture of an E. coli strain overexpressing the chitooligosaccharide synthase nodC (Samain et al., 1997
). 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., 1999
; Bettler et al., 1999
) 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.
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Results |
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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 · L1 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.
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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.
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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 · L1, 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 (NeuAc2-3 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 -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 · L1 in the intracellular fraction and 1.1 g · L1 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.
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Discussion |
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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.
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Materials and methods |
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The plasmid pBBlgtB (Bettler et al., 1999) was obtained by subcloning lgtB gene from pCWlgtB into pBBR1MCS (Kovach et al., 1995
). To obtain the plasmid pBBnsy, the NSY-01 plasmid was digested with XbaI, and the XbaI-fragment containing the CMPNeuAcsyntase 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., 1989).
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., 1997). 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 · L1; then a 5-h fed-batch phase with a high substrate feeding rate of 4.5 g · L1 · h1; and finally a 1020-h fed-batch phase with a lower feeding rate of 2.7 g · L1 · h1. 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 · L1) 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., 1999). 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., 1997) with 50% (v/v) aqueous ethanol. The purification yield was around 6070%.
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 · h1.
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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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