WO2001004341A1 - Procede de production d'oligosaccharides - Google Patents
Procede de production d'oligosaccharides Download PDFInfo
- Publication number
- WO2001004341A1 WO2001004341A1 PCT/FR2000/001972 FR0001972W WO0104341A1 WO 2001004341 A1 WO2001004341 A1 WO 2001004341A1 FR 0001972 W FR0001972 W FR 0001972W WO 0104341 A1 WO0104341 A1 WO 0104341A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- transferase
- cell
- precursor
- oligosaccharide
- lacto
- Prior art date
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Classifications
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
- A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
Definitions
- oligosaccharides play an important biological role, in particular at the level of protein activity and function; they thus serve to modulate the duration of the half-life of proteins, sometimes they intervene in the structure of the protein. Oligosaccharides play a critical role in antigenic variability (blood group for example), and in certain bacterial infections such as those caused by Neisseria menincfitidis.
- oligosaccharides are usually obtained in low yield by purification from natural sources, the synthesis of oligosaccharides has become a major challenge in the chemistry of carbohydrates, in order to provide sufficient quantities of well characterized oligosaccharides, necessary for research fundamental or for all other potential applications (Boons et al, 1996).
- the synthesis of complex oligosaccharides of biological interest can be carried out chemically, enzymatically or microbiologically.
- the enzyme substrates for these reactions are readily available, but these enzyme reactions are not very versatile.
- Another enzymatic method developed uses the glycosyl-transferases of the Leloir biochemical pathway which exhibit a high regiospecificity for the precursor as well as for the donor substrate; these glycosyl transferases are not as readily available as glycosyl hydrolases.
- the recombinant DNA technique has recently made it possible to clone and produce a certain number of them.
- the main limitation of this enzymatic method lies in the very high cost of sugar nucleotides which are the donors of sugar used by these enzymes.
- microbiological pathway for the production of recombinant oligosaccharides in vivo is the most attractive of the synthetic pathways since the bacterium is responsible for the biosynthesis of enzymes, the regeneration of nucleotide-sugars and ultimately the production of the oligosaccharide.
- the first descriptions of the synthesis of oligosaccharides by the microbiological route using recombinant bacteria can be considered to a certain extent as the work which led to the elucidation of the biosynthetic pathways of nodulation factors; these factors are signal molecules secreted by the rhizobia to allow recognition by legumes in the nodulation process.
- the nodulation factors consist of a chitooligosaccharide skeleton carrying different substitutions.
- the present invention therefore relates to a process for the production of an oligosaccharide of interest by a genetically modified cell from at least one exogenous precursor internalized by said cell, said precursor intervening in the biosynthetic pathway of said oligosaccharide, said process comprising the steps (i) of obtaining a cell which comprises at least one recombinant gene coding for an enzyme capable of effecting a modification of said exogenous precursor or of one of the intermediates of the biosynthesis pathway of said oligosaccharide from said exogenous precursor necessary for the synthesis of said oligosaccharide from said precursor, as well as the elements allowing the expression of said gene in said cell, said cell being devoid of enzymatic activity capable of degrading said oligosaccharide, said precursor and said intermediates; (ii) culturing said cell in the presence of at least one said exogenous precursor, under conditions allowing internalization according to a passive and / or active transport mechanism said exogenous precursor by said cell and
- the present invention relates to a method as described above, characterized in that said cell further comprises at least one gene coding for an enzyme capable of effecting a modification of an endogenous precursor involved in the biosynthetic pathway of said oligosaccharide, said enzyme being identical to or different from the enzyme used in the process described above, as well as the elements allowing the expression of said gene in said cell and characterized in that said cell is devoid of activity enzymatic likely to degrade said precursor.
- oligosaccharides is intended to denote linear or branched polymers with a variable number of residues, bonds and subunits; the number of residues being greater than 1.
- Oligosaccharides are carbohydrates which transform on hydrolysis into several molecules of monosaccharides; monosaccharides being sugars which cannot be transformed by hydrolysis into a simpler substance. The monosaccharides are subdivided into trioses, tetroses, pentoses, hexoses, heptoses according to the number of carbon atoms in their hydrocarbon chain and also into aldoses and ketoses according to the presence of an aldehyde function or of a ketone function in their molecule.
- exogenous precursor is intended to denote a compound intervening in the biosynthetic pathway of the oligosaccharide according to the invention which is internalized by said cell.
- endogenous precursor is intended to denote a compound intervening in the biosynthetic pathway of the oligosaccharide according to the invention which is naturally present in said cell.
- genetically modified cell is intended to denote a microorganism into which at least one alteration of the DNA sequence has been introduced into its genome in order to confer a particular phenotype on said cell. Such alterations can thus confer, for example, the ability of the cell not to degrade or not to modify a compound according to the invention or not to decrease the frequency of DNA rearrangement.
- the method according to the invention is characterized in that said cell is a cell chosen from bacteria and yeasts.
- the bacterium is chosen from the group composed of Escherichia coli, Bacillus subtilis, Campylobacter pylori, Helicobacter pylori, Agrobacterium tuméfaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobiutn caulinodans, Rhizobium leguminosarum, Neisseria geaisseria Neisseria meningitis.
- the bacterium is Escherichia coli.
- the cell is a yeast which is preferably Saccharomyces cerevisiae, Saccharomyces pombe, Candida albicans.
- the cell according to the invention is devoid of enzymatic activity capable of degrading said oligosaccharide, said precursor or said metabolic intermediates.
- the nucleic acid sequence coding for the enzyme according to the invention is either naturally present in said cell or is introduced into said cell by recombinant DNA techniques known to those skilled in the art.
- the term “nucleic acid” is intended to denote a DNA fragment, both double-stranded and single-stranded, as transcripts of said DNAs, and / or an RNA fragment.
- the nucleic acid sequence introduced into said cell by recombinant DNA techniques and which codes for an enzyme intervening in the biosynthesis pathway of the oligosaccharide of interest is heterologous.
- heterologous nucleic acid sequence is intended to denote a nucleic acid sequence which is not naturally present in the cell according to the invention.
- the heterologous nucleic acid sequence according to the invention can come from any animal or plant, eukaryotic or prokaryotic cell type and can come from virus.
- bacteria in particular Escherichia coli, Bacillus subtilis, Campylobacter pylori, Helicobacter pylori, Agrobacterium tuméfaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium legumin , Rhizobium meliloti, Neisseria gonorrhoeae, Neisseria meningitis.
- bacteria in particular Escherichia coli, Bacillus subtilis, Campylobacter pylori, Helicobacter pylori, Agrobacterium tuméfaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium legumin , Rhizobium meliloti, Neisseria gonorrhoeae, Neisseria meningitis.
- heterologous nucleic acid sequence originates from plant or animal eukaryotic cells.
- the heterologous nucleic acid sequence originates from mammalian cells and preferably from human cells.
- the cell according to the invention is the bacterium Escherichia coli and the nucleic acid sequence introduced into the bacterium and coding for the enzyme according to the invention preferably comes from the bacterium chosen from the group cited above.
- the nucleic acid sequence coding for the enzyme according to the invention is introduced into said cell in the form of an expression vector.
- the vector must include a promoter, translation initiation and termination signals, as well as appropriate regions for transcription regulation.
- the vector must be able to be maintained stably in the cell during successive generations and may possibly have specific signals specifying the secretion of the translated enzyme. These different control signals are chosen according to the cellular host used.
- the nucleic acid sequences can be inserted into vectors with autonomous replication within the chosen host or into integrative vectors which integrates into the genome of the chosen host.
- Such vectors are prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate cell host by standard methods, such as for example thermal shock or electroporation.
- the invention further relates to the above cells, characterized in that they are transformed by at least one isolated recombinant nucleic acid encoding the enzyme according to the invention or by at least one recombinant vector as defined above.
- the method according to the invention is characterized in that said modification carried out by said enzyme is chosen from glycosylation, sulfation, acetylation, phosphorylation, succinylation, methylation and the addition of an enolpyruvate group, sialylation , fucosylation.
- the method according to the invention is characterized in that said enzyme is an enzyme capable of carrying out a glycosylation chosen from glycosyl-transferases, glycosyl-hydrolases, glycosyl-phosphorylases.
- the enzyme capable of carrying out glycosylation is a glycosyl-transferase.
- the glycosyl-transferase according to the invention is chosen from ⁇ -1,3-N-acetyl-glucosarninyl-transferase, ⁇ -1,3 galactosyl-transferase, ⁇ -1,3 N-acetyl-galactosaminyl-transferase, ⁇ -1,3 glucuronosyl-transferase, ⁇ -1,3 N-acetyl-galactosaminyl-transferase, ⁇ -1,4 N-acetyl-galactosaminyl-transferase, ⁇ -1 , 4-galactosyl-transferase, a- 1-3- galactosyl-transferase, a- 1,4-galactosyl-transferase, a-2,3-sialyl-transferase, ⁇ -2,6 sialyl-
- the glycosyl transferases used in the present invention are capable of stereospecific unit conjugation of specific activated saccharides on a specific acceptor molecule.
- Activated saccharides generally consist of saccharide derivatives uridine-, guanosine- and cytidine- diphosphate.
- the activated saccharides can be a UDP-saccharide, a GDP-saccharide, a CMP-saccharide.
- the enzyme capable of performing acetylation is encoded by the NodL gene of the bacterium Azorhizobium caulinodans.
- the enzyme capable of carrying out sulfation is coded by the NodH gene of the bacterium Rhizobium meliloti.
- the method according to the invention is characterized in that said cell culture is carried out preferably on a carbon substrate; according to a particular embodiment of the invention, said carbon substrate is chosen from glycerol and glucose. Other carbon substrates can also be used; mention should be made of maltose, starch, cellulose, pectin, chitin. According to another embodiment, the cell culture is carried out on a substrate composed of amino acids and / or protein and / or lipids.
- the method according to the invention is characterized in that the said culturing step is carried out under conditions allowing a high cell density culture to be obtained; this cultivation step comprises a first phase of exponential cell growth ensured by said carbon substrate, a second phase of cell growth limited by said carbon substrate which is added continuously and finally a third phase of slowed cell growth obtained by adding continuously in the culture an amount of said substrate decreased relative to the amount of substrate added in step b) so as to increase the content of oligosaccharides produced in the high cell density culture.
- the method according to the invention is characterized in that the amount of substrate added continuously in the cell culture during said phase c) is reduced by at least 30%, preferably 50%, preferably 60% by relative to the amount of substrate added continuously during said phase b).
- the method according to the invention is also characterized in that said exogenous precursor is added during phase b).
- the method is characterized in that said exogenous precursor is of a carbohydrate nature, preferably of an oligosaccharide nature.
- said exogenous precursor is of a carbohydrate nature, preferably of an oligosaccharide nature.
- the originality and the feasibility of the process according to the invention rests on the use of two modes of internalization of the exogenous precursor which neither destroy the integrity of the cell nor reach its vital functions. This notably excludes conventional techniques of permeabilization of the membrane by organic solvents which will block growth and energy metabolism.
- the two possible modes of internalization of the exogenous precursor use a passive or active transport mechanism.
- the invention firstly relates to a method characterized in that said exogenous precursor is internalized according to a passive transport mechanism.
- the term “internalization by passive transport” is intended to denote the passive diffusion of one of the exogenous precursor through the plasma membrane, the molecular flux being directed from the most concentrated zones towards the least concentrated zones in order to finally tend towards a state of equilibrium.
- Internalization by passive transport consists in using an exogenous precursor which is small enough and hydrophobic to passively diffuse through the membrane.
- a precursor, a monosaccharide having the anomeric position blocked by an alkyl substituent constitutes an example of a precursor capable of being internalized in this way.
- the present invention therefore relates to a process characterized in that said exogenous precursor is a monosaccharide whose anomeric carbon is linked to an alkyl group; preferably said alkyl group is an allyl group.
- oligosaccharides which have a functionalizable group such as the allyl group and which can therefore be used as a precursor for the chemical synthesis of glycoconjugates (neoglycoprotein or neoglycolipids) or glycopolymers.
- the double bond of the allyl group is capable of being opened by ozonolysis to form an aldehyde and allow the conjugation of the oligosaccharide on a protein by reductive amination (Roy et al., 1997).
- the method according to the invention relates to the production of [ ⁇ -D-Gal- [1-4] - ⁇ -D-GlcNac-1-O-allyl); the method is characterized in that said cell is a bacterium of the LacZ- genotype, said enzyme is ⁇ -1,4-galactosyl transferase, said substrate is glycerol and said precursor is allyl-N-acetyl- ⁇ - D-glucosaminide ( ⁇ -D-GlcNac-1-> O-allyl).
- the method according to the invention is characterized in that the double bond of the allyl group of said ( ⁇ -D-Gal- [l-> 4] - ⁇ -D-GlcNac-l- 0-allyl) is chemically modified by addition, oxidation or ozonolysis reactions.
- the present invention also relates to a method characterized in that said precursor is internalized according to an active transport mechanism.
- active transport is intended to denote the ability of cells and preferably bacteria to admit and selectively concentrate certain exogenous substances or precursors in their cytoplasm. This transport is carried out by transporters of protein nature called permeases which act like enzymes; permeases are inducible catalysts, that is to say synthesized in the presence of substrate or of the precursor.
- permeases which act like enzymes
- permeases are inducible catalysts, that is to say synthesized in the presence of substrate or of the precursor.
- lactose and ⁇ -galactosides constitute precursors which are actively transported in the cytoplasm of the bacterium Escherichia coli by lactose permease also called galactoside permease.
- the invention therefore relates to a method according to the invention characterized in that said active transport of said precursor is carried out by lactose permease.
- Lactose permease has a fairly broad specificity which allows it to transport, in addition to lactose, other molecules. It is in fact capable of transporting various natural or synthetic ⁇ -galactosides, ⁇ -galactosides and sucrose. It is therefore one of the objects of the invention to provide, according to a preferred embodiment, a process characterized in that said precursor is lactose which constitutes the basic motif of very many biologically active oligosaccharides.
- said precursor is chosen from the group consisting of: (i) natural or synthetic ⁇ -galactosides, preferably in 4-O- ⁇ -D-galactopyranosyl-D-fructofuranose (lactulose), 3-O- ⁇ -D- galactopyranosyl-D-arabinose and allyl- ⁇ -D-galactopyranoside, (ii) ⁇ -galactosides, preferably the melibiose and raffinose, sucrose allyl- ⁇ - D-galactopyranoside (iii).
- lactose permease can even be modified by mutation and allow the transport of other compounds such as maltose and cellobiose. All these compounds can therefore be used as a precursor for the synthesis of oligosaccharides. It is also within the scope of this invention to use as precursors lactose analogs having a chemically reactive group for a subsequent functionalization of the product preferably one of these analogs is allyl ⁇ -D-galactopyranoside. It is also within the scope of this invention to use other permeases modified or not by recombinant DNA techniques to allow the internalization of different types of precursors.
- ⁇ -galactosides are normally hydrolyzed in the cytoplasm of the bacterium by ⁇ -galactosidase encoded by the LacZ gene.
- a lacZ- bacterial mutant lacking ⁇ -galactosidase activity is used when the precursor used is lactose and / or a ⁇ -galactoside. It is therefore also one of the objects of the invention to provide the method according to the invention characterized in that said cell is devoid of enzymatic activity capable of degrading said precursor as well as said metabolic intermediates.
- the invention relates to a process described above characterized in that said precursor is sialic acid.
- said active transport of said precursor is carried out by the NanT permease.
- the invention relates to a process described above characterized in that said precursor is sialic acid and lactose. In this case, said active transport of said precursor is carried out by lactose permease and NanT permease.
- said cell can be devoid of enzymatic activity capable of degrading said precursor or said precursors.
- the method is characterized in that said cell has a genotype chosen from LacZ- and / or NanA-.
- the method is characterized in that it further comprises the addition of an inducer in said culture medium to induce the expression in said cell of said enzyme and / or of a protein involved in said active transport; according to a preferred embodiment, the method according to the invention is characterized in that said inducer is isopropyl ⁇ -D-thiogalactoside (IPTG) and said protein is lactose permease.
- IPTG is isopropyl ⁇ -D-thiogalactoside
- the invention makes it possible for the first time to produce complex oligosaccharides with yields of the order of grams per liter. Depending on its size, the oligosaccharide either accumulates in the bacterial cytoplasm, either is secreted in the culture medium.
- the method according to the invention is used for the production of the trisaccharide 4- O- [3-O- (2-acetamido-2deoxy- ⁇ -D-glucopyranosyl) - ⁇ -D- galactopyranosyl ] -D-glucopyranose, ( ⁇ -D-GlcNac- [l-> 3] - ⁇ -D-Gal- [l-> 4] -D-Glc); it is characterized in that said cell is a bacterium of genotype LacZ-, LacY + , said enzyme is ⁇ - 1,3-N-acetyl-glucosaminyl-transferase, said substrate is glycerol, said inducer is isopropyl ⁇ -D-thiogalactoside (IPTG) and said precursor is lactose.
- IPTG isopropyl ⁇ -D-thiogalactoside
- the process according to the invention is used for the production of lacto-N-neo-tetraose and of polylactosamine; it is characterized in that said cell is a bacterium of genotype Lac Z; Lac Y + , said enzymes are ⁇ - 1,3-N-acetyl-glucosaminyl-transferase and ⁇ -1,4-galactosyl-transferase, said substrate is glucose, said inducer is isopropyl- ⁇ -D- thiogalactoside (IPTG), said precursor is lactose.
- said cell is a bacterium of genotype Lac Z; Lac Y + , said enzymes are ⁇ - 1,3-N-acetyl-glucosaminyl-transferase and ⁇ -1,4-galactosyl-transferase, said substrate is glucose, said inducer is is isopropyl- ⁇ -D- thiogalactoside (IPTG), said precursor is lac
- the method according to the invention is used for the production of allyl 3-O- (2-acetamido-2deoxy- ⁇ -D-glucopyranosyl) - ⁇ -D-galactopyranoside, ( ⁇ - D-GlcNac- [1 ⁇ 3] - ⁇ -D-Gal-1- »O-allyl); it is characterized in that said cell is a bacterium of the LacZ genotype; LacY + , said enzyme is ⁇ - 1,3-N-acetyl-glucosaminyl-transferase, said substrate is glycerol, said inducer is isopropyl ⁇ -D- thiogalactoside (IPTG), said precursor is allyl- ⁇ -D- galactopyranoside.
- the method according to the invention is used for the production of lacto analogs N-neo-tetraose and polylactosamines in which the glucose residue is replaced by an allyl group; it is characterized in that said cell is a bacterium of genotype LacZ-, LacY +, said enzymes are ⁇ - 1,3-N-acetyl-glucosaminyl-transferase and ⁇ -1,4-galactosyl-transferase, said substrate is glucose, said inducer is is isopropyl ⁇ -D-thiogalactoside (IPTG) and said precursor is rallyl- ⁇ -D-galactopyranoside.
- said cell is a bacterium of genotype LacZ-, LacY +
- said enzymes are ⁇ - 1,3-N-acetyl-glucosaminyl-transferase and ⁇ -1,4-galactosyl-transferase
- said substrate is glucose
- said inducer
- the method according to the invention is used for the production of allyl- ⁇ -D- lactosamine ( ⁇ -D-Gal- [l-> 4] - ⁇ -D-GlcNac- l- >O-allyl); it is characterized in that said cell is a bacterium of genotype LacZ-, LacY +, said enzyme is ⁇ - 1,4-galactosyl-transferase, said substrate is glycerol, said precursor is aHyl-N-acetyl ⁇ - D- glucosaminide ( ⁇ -D-GlcNac- [l-> O-allyl)).
- the invention also relates to a method which makes it possible to envisage the production of a large number of different oligosaccharides obtained by glycosylation of lactose.
- IgtA and IgtB genes which code respectively for ⁇ - 1,3-N-acetyl-glucosaminyl-transferase and ⁇ -1,4-galactosyl-transferase
- several bacterial glycosyl-transferase genes using lactose as precursor have been recently cloned.
- the method according to the invention also makes it possible to obtain a large number of different oligosaccharides obtained by glycosylation of exogenous precursors other than lactose and transported by lactose permease or by other permeases.
- the process according to the invention makes it possible to obtain a large number of different oligosaccharides obtained by modification (sulfation, acetylation, phosphorylation, succinylation, methylation, addition of an enolpyruvate group) in vivo of precursors.
- modification sulfation, acetylation, phosphorylation, succinylation, methylation, addition of an enolpyruvate group
- the synthesis of certain oligosaccharides may require, in addition to the modification of exogenous precursors, the modification of endogenous precursors.
- UDP-GalNAc can be produced from UDP-GlcNAc if the epimerase gene is introduced into a cell according to the invention.
- Another subject of the invention relates to a process described above for the production of 3'-sialyllactose ( ⁇ -NeuAc- [2- »3] - ⁇ -D- Gal- [l ⁇ 4] - ⁇ -D- Glc) or 6'-sialyllactose ( ⁇ -NeuAc- [2 ⁇ 6] - ⁇ -D-Gal- [l- 4] - ⁇ -D-Glc) characterized in that: • said cell is a bacterium of genotype LacZ-, LacY +,
- said substrate is glycerol; • said inducer is isopropyl- ⁇ -D-thiogalactoside (IPTG);
- said precursors are lactose and sialic acid.
- the method according to the invention is used for the production of a sialylated derivative of lacto-N-neotetraose and of polylactosamine (lacto-N-neo-hexaose , lacto-N-neo-octaose, lacto-N-neo-decaose) characterized in that it also comprises a said enzyme chosen from ⁇ -2,3 sialyl-transferase, ⁇ -2,6 sialyl - transferase, and that said cell also has a NanA-, NanT + genotype and expresses the CMP-NeuAc synthase gene, said acceptors are lactose and sialic acid.
- Another subject of the invention relates to a process described above for the production of lacto-N-neotetraose, ⁇ -D- Gal [l ⁇ 4] - ⁇ -D-GlcNac [l- 3] - ⁇ -D -Gal [l ⁇ 4] - ( ⁇ -L-Fuc- [l ⁇ 3]) ⁇ -D-Glc, ⁇ -D-Gal- [l ⁇ 4] - ( ⁇ -L-Fuc- [l ⁇ 3]) - ⁇ -D-GlcNac- [l ⁇ 3] - ⁇ -D-Gal- [l ⁇ 4] - ( ⁇ -L-Fuc- [l ⁇ 3]) - ⁇ -D-Glc, ⁇ -D-Gal- [l ⁇ 4] - ( ⁇ -L-Fuc- [l ⁇ 3]) - ⁇ -D-Glc, ⁇ -D-Gal- [l
- said enzymes are ⁇ -1,3-N-acetyl-glucosaminyl transferase, ⁇ -1,4-galactosyl transferase, ⁇ -1,3 fucosyl transferase;
- said inducer is isopropyl- ⁇ -D-thiogalactoside (IPTG);
- the method according to the invention is used for the production of 3'fucosyllactose ( ⁇ -D-Gal- [l ⁇ 4] - ( ⁇ -L-Fuc- [l-> 3] - D-Glc) or 2'fucosyllactose ( ⁇ -D- Gal- [l- 2] - ( ⁇ -L-Fuc- [l ⁇ 3] -D-Glc) characterized in that it comprises a said enzyme chosen among a-1,3 fucosyltransferase or a-1,2 fucosyltransferase, and that the cell has a wcaj lacZ genotype and overexpresses the rcsA gene and that said precursor is lactose.
- the method according to the invention is used for the production of a fucosylated derivative of lacto-N-neotetraose and of polylactosamine (lacto-N-neo-hexaose, lacto-N-neo-octaose, lacto-N-neo-decaose) characterized in that it further comprises a said enzyme chosen from ⁇ -1,2 fucosyltransferase, ⁇ -1,3 fucosyl-transferase, and that said cell also has a Wca J- genotype and overexpress the Rcs A gene, said acceptor being lactose.
- the method according to the invention is used for the production of a sialylated and fucosylated derivative of lacto-N-neotetraose, lacto-N-neo-decaose) characterized in that it further comprises a said enzyme chosen from ⁇ -2,3 sialyl-transferase, ⁇ -2,6 sialyl-transferase, and further said enzyme chosen from ⁇ -1,2 fucosyl-transferase, has - 1.3 fucosyl- transferase, and that said cell also has a NanA-, NanT +, Wca J- genotype and overexpresses the Rcs A gene and the CMP- NeuAc synthase gene, said acceptors are lactose and sialic acid.
- Another object of the invention is to provide a process for producing oligosaccharides labeled or enriched with radioisotopes; such oligosaccharides are extremely valuable for basic studies of biology or conformational analysis.
- the invention therefore relates to a method for producing an oligosaccharide labeled with at least one radioisotope, characterized in that said cell is cultured on said carbon substrate marked with said radioisotope and / or in the presence of a said precursor labeled with said radioisotope.
- the radioisotopes are preferably chosen from the group composed of: 14 C, 13 C, 3 H, 35 S, 3 P, 33 P.
- the invention also relates to an oligosaccharide capable of being obtained by a process according to the invention.
- the invention relates to an activated oligosaccharide usable for the chemical synthesis of glycoconjugates or glycopolymers capable of being obtained by a process as described above, said oligosaccharide being characterized in that the double bond of the allyl group is chemically modified by addition, oxidation, or ozonolysis reactions.
- the oligosaccharide according to the invention is useful in a wide range of therapeutic and diagnostic applications; it can for example be used as a blocking agent for cell surface receptors in the treatment of multiple diseases involving cell adhesion or be used as nutritional supplements, antibacterials, anti-metastatic agents, anti-inflammatory agents.
- the invention therefore relates to an oligosaccharide according to the invention as a medicament and in particular as a medicament intended to selectively prevent the adhesion of biological molecules.
- the oligosaccharide according to the invention is also used as a medicament intended for the treatment of cancer, inflammation, heart diseases, diabetes, bacterial infections, viral infections, neurological diseases and as medicament intended for transplants.
- the invention also relates to a pharmaceutical composition characterized in that it comprises an oligosaccharide according to the invention and a pharmaceutically acceptable vehicle.
- the invention also relates to the use of an oligosaccharide according to the invention in agriculture and agronomy, in particular for the growth and defense of plants.
- oligosaccharides play a predominant role in the Rhizobium / legume symbiosis.
- certain oligosaccharides originating from the hydrolysis of walls or plant or fungal glycoproteins can act as phytohormones or as elicitors of defense reactions in plants.
- the industrial interest of the process according to the invention is obvious because it allows for the first time to achieve a production of the order of the kilogram of complex oligosaccharides of biological interest. All the oligosaccharides of biological interest which we consider synthesis on an industrial scale are currently only available on a mg scale and at extremely high costs (up to 1 million francs per gram); the cost price of these compounds produced by the present microbiological route are infinitely lower.
- Figure 1 Principle of the process for producing the trisaccharide 4-O- [3-O- (2-acetamido-2deoxy- ⁇ -D- glucopyranosyl) - ⁇ -D-galacto-pyranosyl] -D-glucopyranose, ( ⁇ -D - GlcNac- [1 ⁇ 3] - ⁇ -D-Gal- [l- 4] -D-Glc)
- Lactose ( ⁇ -D-Gal- [1-4] - ⁇ -D-Glc) is transported into the cell by lactose permease (Lac permease). Lactose cannot be hydrolyzed in the cell because the strain is a LacZ- mutant.
- Expression of the IgtA gene allows the production of the enzyme LgtA which transfers a GlcNAc from UDP-GlcNAc to a lactose molecule.
- the trisacharide formed ( ⁇ -D-GlcNAc- [l-3] - ⁇ -D-Gal- [l-4] - ⁇ -D- Glc) is excreted in the medium.
- FIG. 1 High cell density culture of the control strain JM109 and of the strain JM109 (pCWlgtA) having the glycosyl transferase gene LgtA.
- lactose is added continuously and the residual lactose is determined enzymatically.
- concentration of hydrolyzable GlcNAc in the culture medium is measured colorimetrically after acid hydrolysis. Lactose added represents the total cumulative amount of lactose that has been continuously added.
- Figure 4 Spectrum of the trisaccharide 4-O- [3-O- (2-acetamido-2deoxy- ⁇ -D-glucopyranosyl) - ⁇ -D-galactopyranosyl] -D- glucopyranose, ( ⁇ -D-GlcNac- [l- »3] - ⁇ -D-Gal- [l-» 4] -D-Glc) in proton NMR at 323 ° K.
- the signal at 1.4 ppm is due to the protons of the isopropyl group of the glycosylated derivative of IPTG.
- Figure 5 13 C NMR spectrum of trisaccharide 4-O- [3-O- (2-acetamido-2deoxy- ⁇ -D-glucopyranosyl) - ⁇ -D-galactopyranosyl] -D- glucopyranose, ( ⁇ -D-GlcNac- [1 ⁇ 3] - ⁇ -D-Gal- [l- »4] -D-Glc).
- Figure 6 Principle of the production process for lacto-N- neo-tetraose ( ⁇ -D-Gal- [l-4] - ⁇ -D-GlcNAc- [l-3] - ⁇ -D-Gal- [l- 4] - ⁇ -D- Glc). Lactose ( ⁇ -D-Gal- [1-4] - ⁇ -D-Glc) is transported into the cell by Lac permease. Lactose cannot be hydrolyzed in the cell because the strain is a LacZ- mutant. Expression of the IgtA gene allows the production of the enzyme LgtA which transfers a GlcNAc from lTJDP-GlcNAc to a lactose molecule.
- JM109 (pCWlgtA, pBBlgtB).
- Figure 8 Separation on Biogel P4 of the oligosaccharides produced by the JM109 strain (pCWlgtA, pBBlgtB) in the presence of lactose at an initial concentration of 5 gl 1 (A) or 1 gl-MB).
- Peaks 1, 2, 3, 4 correspond respectively to lacto-N-neo-tetraose, lacto-N-neo-hexaose, lacto-N-neo-octaose and lacto-N-neo-decaose.
- Lactose and sialic acid are internalized in the cell by lactose permease (lacY) and sialic acid permease (nanT). These two compounds are not degraded in the cell because the strain is a lacZ- and nanA- mutant.
- lacY lactose permease
- nanT sialic acid permease
- the expression of CMP-NeuA synthase and of ⁇ -2,3 sialyltransferase allows the activation of sialic acid internalized in CMP-NeuAc and its transfer to intracellular lactose.
- the JM107 and JM109 strains of Escherichia coli K12 were used as host cells for all the examples of production of oligosaccharides described.
- the strains were obtained from DSM (Deutsche Sammlung von Mikroorganismen)
- the genotype of strain JM109 is as follows: F- traD36 lacP ⁇ acZ) M15 proA + B + / el4- (McrA-) ⁇ ac-proAB) supE44 recAl endAl gyrA96 (Nal r ) thi hsdR17 relAl.
- the genotype of the JM107 strain is identical to that of the JM 109 strain except that the recAl gene is not inactivated.
- the Neisseria meningitis MC58 IgtA and IgtB genes were supplied by Dr W. Wakarchuk (Institute for Biological Sciences, National Research council of Canada, 100shire Drive, Ottawa, Ontario, K1A OR6, Canada) as two plasmids pCW, one containing the IgtA gene (here called pCWlgtA) and the other containing the IgtB gene (here called pCWlgtB).
- the sequences of these two genes are available in the GenBank database under the number U25839.
- the plasmid pLitmus28 was purchased from the company New Englands Biolabs.
- the plasmid pBBRIMCS was supplied by Dr M. Kovach (Department of Microbiology and Immunology, Louisiana State University, Shreveport, LA 71130-3932, USA.)
- Plasmid NSY-01 is a derivative of plasmid pT7-7 which contains the gene (GenBank U60146) of CMP-sialic acid synthase (Gilbert et al 1997).
- the plasmid NST-01 is a derivative of the plasmid pBluescript Sk- which contains the gene (GenBank n ° U60660) of the ⁇ -2,3 sialyltransferase (Gilbert et al. 1996)
- the fucT gene for Helicobacter pylori ⁇ -1,3 fucosyltransferase was supplied by Dr S. Martin (Glaxo Wellcome Research and Development, Gunnels Wood Road, Stevenage, Hertfordshire, SGI 2NY, UK) a plasmid pHP0651 derived from pET-21a. The sequence is available at Genbank (AE000578, gene HP0651).
- Plasmid pBBlgtB The DNA fragment of 0.835 kb containing the IgtB gene was obtained by digestion of the plasmid pCWlgtB with BamHI and HindIII. This fragment was subcloned into the vector pLitmus28 previously digested with BamH I and HindIII to form the plasmid pLitlgtB.
- the 0.9 kb fragment containing the IgtB gene was excised from the plasmid pLitlgtB by a digestion with Xhol and HindIII and subcloned in the plasmid pBBRIMCS previously digested with Xhol and HindIII to form the plasmid pBBlgtB.
- Construction of the plasmid pBBns The fragment containing the gene for CMP-sialic acid synthase was excised from the plasmid NSY-01 by digestion with Xbal and subcloned in the plasmid pBBRIMCS previously digested with Xbal to form the plasmid pBBnsy.
- the IgtA gene present in the construction pCWlgtA was amplified by PCR at the same time as the UV5 tactac promoter of the plasmid using the primers CTTTAAGCTTCCGGCTCGTATAA (sense, upstream promoter) and GACAGCTTATCATCGATAAGCTT ( antisense, late IgtA) both containing a HindIII site.
- the amplified fragment of 1.3 kb was then subcloned into the HindIII site of the vector pBBlgtB.
- the rcsA gene (Stout et al., 1991) was first amplified by PCR from genomic DNA of JM109 with the primers AGGGTACCCATGTTGTTCCGTTTAG (Kpnl site, left rcsA) and AATCTAGAGTAATCTTATTCAGCCTG (right site Xbal rcsA), then cloned into the Kpnl-Xbal sites of the vector pBBRl-MCS.
- the vector pBBRl-MCS-rcsA was then opened upstream of the gene by digestion with Kpnl, blunted (Amersham kit), released by Xbal, and inserted into the Smal-Xbal sites of the construction pBBLnt, allowing cloning downstream of the lgtB-OV5 tactac-IgtA, placing rcsA under the control of the promoter UV5 tactac. 1.3. Culture conditions
- the MgSO 4 is autoclaved separately and the thiamine is sterilized by filtration.
- the trace element solution contains: nitrilotriacetate (70 mM, pH 6.5), ferric citrate (7.5 gF), MnCl 2 . 4H 2 O (1.3 gF), C0CI2 6H 2 O (0.21 gF),
- High cell density cultures are inoculated at 2%. During the whole culture, the dissolved oxygen level is maintained at 20% saturation by manually regulating the air flow and automatically adjusting the stirring speed. The pH is automatically regulated to 6.8 by the addition of aqueous ammonia (15% w / v). The temperature is maintained at 34 ° C for the strain JM109 (pCWlgtA) and at 28 ° C for the strain JM109 (pCWlgtA, pBBlgtB).
- the high density culture strategy generally includes 3 phases: a first phase of exponential growth which is provided by the carbon substrate (glycerol or glucose) initially present in the medium; a second phase which begins when growth becomes limited by the carbon source which is then added continuously at a rate of 4.5 gh- 1 . H of glycerol or 3.6 gh 1. ! - 1 of glucose. In a third phase, this rate is reduced by 60% to slow growth so as to increase the content of oligosaccharides.
- a first phase of exponential growth which is provided by the carbon substrate (glycerol or glucose) initially present in the medium
- a second phase which begins when growth becomes limited by the carbon source which is then added continuously at a rate of 4.5 gh- 1 .
- H of glycerol or 3.6 gh 1. ! - 1 of glucose In a third phase, this rate is reduced by 60% to slow growth so as to increase the content of oligosaccharides.
- the samples (1 ml) are taken during the culture and immediately centrifuged in microtubes. The supernatant is kept for the determination of extracellular oligosaccharides.
- the bacterial pellet is resuspended in 1 ml of water and then is incubated in a water bath at 100 ° C for 30 min to burst the cells. After a second centrifugation, the supernatant is kept for the determination of intracellular oligosaccharides.
- the lactose concentration is measured using an enzymatic determination kit (Roche diagnostic).
- the N-acetyl-glucosamine residues present in the oligosaccharides are released by acid hydrolysis as previously described (Samain et al, 1997) and then quantified colorimetrically by the method of Reissig et al, (1955); in the description, the term hydrolyzable GlcNAc is understood to mean the amount of GlcNAc dosed in this way.
- lactose with and without treatment with a neuraminidase makes it possible to estimate the concentration of sialyl-lactose.
- Total fucose is measured colorimetrically by the cysteine hydrochloride method of Dische and Shettles (1948).
- the bacterial cells are harvested by centrifugation. The supernatant is kept for purification extracellular oligosaccharides.
- the bacterial cells are resuspended in 1 liter of water, then are permeabilized by heat treatment (30 min at 100 ° C) to release the intracellular oligosaccharides. After a second centrifugation these oligosaccharides are recovered in the supernatant.
- the first and second supernatants containing the extra- and intracellular oligosaccharides respectively are adsorbed on activated carbon (100 g per liter of supernatant). After rinsing with distilled water, the oligosaccharides are eluted with 50% ethanol (v / v), concentrated by evaporation and lyophilized.
- oligosaccharides are separated by steric exclusion chromatography on a column (4.5 cm x 95 cm) of Biogel P4 allowing the injection of approximately 300 mg of mixture of oligosaccharides. Elution is carried out with distilled water with a flow rate of 40 ml. h- 1 .
- the non-fucosylated oligosaccharides are separated by steric exclusion chromatography on a column (4.5 cm x 95 cm) of Biogel P4 allowing the injection of approximately 300 mg of mixture of oligosaccharides. Elution is carried out with distilled water with a flow rate of 40 ml.tr 1
- the fucosylated oligosaccharides are separated by size exclusion chromatography on a column (1.5 cm x
- Biogel P2 thermostatically controlled at 60 ° C allowing the injection of about 30 mg of oligosaccharide mixture. Elution is carried out with distilled water with a flow rate of 30 ml.h 1
- Sialyllactose is separated from neutral oligosacchariodes by fixation on a Dowex 1X4-400 resin (in HCO3 form). and eluted with a NaHC ⁇ 3 gradient (0 to 100 mM). Bicarbonnate is then eliminated by treating the eluate with a Dowex 50X4-400 resin in H + form.
- the mass spectra were carried out with a mass spectrometer (Nermag R-1010C). For each experiment the initial volume of matrix is 4 ⁇ l. The products were analyzed in FAB + mode. NMR spectra were obtained with a spectrometer
- a JM 107 strain incapable of metabolizing sialic acid was prepared by insertional inactivation of the nanA gene (Nan operon) coding for NeuAc aldolase (Plumbridge et al., 1999). Two PCR amplification reactions were carried out on either side of the nanA gene center so as to insert a BamHI restriction site there. A first 1.6 kb BamHI-Xbal fragment comprising the right part of nanA was amplified from genomic DNA of JM109 using the primers
- GCTCTAGAATGGTAATGATGAGGCAC and clone between sites BamHI and Xbal of the vector pUC19, forming the vector pUC-nanl, 6.
- a second 2.1 kb Kpnl-BamHI fragment comprising the left side of nanA was amplified using the primers AAAGGATCCGCGTAGGTGCGCTGAAAC and AAAGGTACCTCAGGCCACCGTTAGCAG and clone between the Kpnl and BamHI sites of the vector pUC-nanl, 6 forming the vector pUC-nan- 7.
- the kanamycin resistance gene (pUC-4K, Pharmacia cassette) was then cloned into the BamHI site of pUC-nan-3,7.
- the 4.9 kb Sacl-Xb ⁇ l fragment containing nanA :: kan was inserted into the same sites of the suicide vector pCVD442 (Donnenberg and Kaper 1991). This plasmid was used to obtain, by homologous recombination, JM107 nanAv.kan mutants, selected for their resistance to kanamycin and their inability to metabolize sialic acid (strain JM107-nanA).
- the vector thus obtained was subjected to a treatment with an EcoRI methylase, allowing the subsequent addition of the kanamycin resistance gene in the Apol site present at the center of wcaJ.
- the wcaJr.kan recombinant DNA has finally been transferred to the suicide vector pCVD442 allowing, by homologous recombination, the obtaining of genomic mutants JM 107 containing the inactivated gene, selected by PCR using the primers having served for cloning (strain JM107-col).
- the strain JM107-col- was made lysogenic for the phage ⁇ DE3 using the lysogenization kit from Novagen.
- Example 2 Production of the trisaccharide 4-O- [3-O- (2-acetamido-2deoxy- ⁇ -D-glucopyranosyl) - ⁇ -D-galactopyranosyl] -D- glucopyranose, ( ⁇ -D-GlcNac- [l- "3] - ⁇ -D-Gal- [l-» 4] -D-Glc).
- the principle is illustrated in FIG. 1.
- the JM 109 strain of Escherichia coli K12 into which we have introduced the plasmid pCWlgtA gene IgtA.
- the JM 109 strain is lacZ-, ie it is incapable of hydrolyzing lactose. On the other hand, it is lacY +, which means that it can synthesize lactose permease.
- the IgtA gene codes for a ⁇ -1,3-N-acetyl-glucosaminyl transferase (LgtA) transferring a ⁇ -acetyl-glucosamine unit on the lactose galactose.
- the JM 109 strain pCWlgtA as well as the control strain
- JM109 were grown at high cell density (Samain et al, 1997) on glycerol as a source of carbon and energy. After a first phase of exponential growth ensured by the glycerol initially present in the medium (17.5 g / 1), the growth becomes limited by the glycerol which is then added continuously at a rate of 4.5 gh- 1 .! 1 . During this second phase of the culture, 90 mg.hM- 1 of lactose is continuously introduced.
- ÎTPTG isopropyl- ⁇ -D-thiogalactoside (0.5 mM) is also injected at the start of this phase to induce the expression of the lactose permease and the ⁇ -l, 3-N-acetyl-glucosaminyl-transferase.
- the added lactose practically does not accumulate in the medium, indicating that the lactose is well internalized by the bacterial cells.
- the strain JM 109 pCWlgtA
- a significant accumulation in the culture medium of a compound containing ⁇ -acetylglucosamine hydrolyzable GlcNAc
- the cells are eliminated by centrifugation and the oligosaccharides present in the supernatant are purified by adsorption on activated carbon and elution with ethanol.
- the oligosaccharides present are then separated according to their molecular weight on a column of Biogel P4. Only one majority compound is found.
- the mass spectrometry and RM ⁇ data indicate that this compound is indeed the trisaccharide ( ⁇ -D- Glc ⁇ Ac- [l-> 3] - ⁇ -D-Gal- [l-> 4] - ⁇ -D-Glc) formed by the addition of a GlcNAc residue on a lactose molecule.
- the mass spectrum in FAB + mode indeed shows the presence of a quasi-molecular ion [M + H] + at m / z 546 ( Figure 3).
- the * H NMR spectrum confirms the trisaccharide structure, the presence of an acetyl group and the ⁇ configuration of the two O-glycosidic bonds (FIG. 4).
- the 13 C NMR spectrum also specifies that the bond between GlcNAc and galactose is indeed of the 1.3 type (FIG. 5).
- EXAMPLE 3 Production of Lacto-N-Neo-Tetraose and Polylactosamine The principle is described in FIG. 6.
- the strain of E. coli JM 109 was cotransformed with the two plasmids pCWlgtA and pBBlgtB carrying the genes IgtA (used previously) and IgtB respectively (coding for a ⁇ -1,4-Galactosyl - transferase called LgtB).
- the JM109 strain (pCWlgtA, pBBlgtb) was grown at high cell density using glucose as the growth substrate.
- lacto- ⁇ -neo-tetraose polylactosamines The formation of higher homologous lactyl-N-neo-tetraose polylactosamines is explained by the fact that LgtA is capable of using lacto-N-neo-tetraose to form an intermediate pentasaccharide which is glycosylated by LgtB to give lacto- ⁇ - neo-hexaose. The latter is itself a precursor for a new glycosylation cycle leading to the formation of lacto- ⁇ -neo-octaose and so on until lacto- ⁇ -neo-decaose.
- the JM 109 strain (pCWlgtA) was cultured at high cell density on glycerol. At the start of the second culture phase, 0.75 g ⁇ l of allyl- ⁇ -D-galactopyranoside and 0.1 mM of IPTG are added. A total internalization of the allyl- ⁇ -D-galactopyranoside is observed after 9 h with a stoichiometric appearance of hydrolyzable GlcNAc in the medium. extracellular. The oligosaccharides present in the extracellular medium are purified as in Example 2.
- the strain JM109 (pBBlgtB) was cultured at high cell density on glycerol.
- 0.5 g -1 of allyl-N-acetyl- ⁇ -D-glucosaminide ( ⁇ -D-Glc ⁇ Ac- l-> Oallyl) is added. It is observed for the first 5 hours an approximately 30% decrease in the amount of extracellular hydrolyzable GlcNAc, which demonstrates a partial internalization of allyl-N-acetyl- ⁇ -D-glucosaminide.
- E. coli K12 is capable of degrading sialic acid (Plumbridge et. Vimr 1999) and has a permease (NanT) which allows exogenous sialic acid to enter the cell. This sialic acid is then normally catabolized by an aldolase (NanA).
- the strain JM107-nanA- (Nst-01, pBBnsy) and the control strain JM107 (Nst-01, pBBnsy) having the NanA activity were cultured at high cell density on glycerol. Lactose (1.5 gl- 1 ) of IPTG (0.1 mM) and sialic acid (0.6 gl- 1 ) are added at the start of the second culture phase of duration 5 h. Throughout the duration (17 h) of the third phase of the culture, 100 mg.h ⁇ .L- 1 of sialic acid and 200 mg.lF.L- l of lactose are introduced continuously.
- the intracellular and extracellular oligosaccharides are purified by adsorption on activated carbon and elution with ethanol. After purification on anion exchange resin, only one product is detected by HPLC.
- the mass spectrum in FAB + mode shows the presence of two quasi-molecular ions [M + H] + at m / z 656 and [M + Na] at m / z + 678 corresponding to the sodium salt of sialyllactose.
- the GDP-fucose biosynthesis genes are part of the operon responsible for the biosynthesis of an extracellular polysaccharide, colanic acid (Stevenson et al 1996).
- the expression of this operon is controlled by a complex regulatory network in which the RcsA protein is involved (Stout et al 1991).
- the overexpression of the rcsA gene thus results in an overproduction of colanic acid (Russo and Singh 1993) and consequently of the genes for biosynthesis of GDP fucose.
- Plasmid pHP0651 contains the fucT gene for Helicobacter pylori a-1,3 fucosyltransferase. This fucosyltransferase uses N-acetyllactosamine and lacto-N-neotetraose as acceptor but not lactose (Martin et al 1997).
- the plasmid pBBLnt contains the IgtA and IgtB genes.
- the plasmid pBBLntRcsA contains the IgtA, IgtB and rcsA genes.
- the two strains JM107-col DE3 pHP0651, pBBLnt) and
- JM107-col DE3 (pHP0651, pBBLntRcsA) were cultured as in Example 3 in the presence of 5 gH of lactose.
- the quantity of hydrolyzable GlcNAc produced by the two strains (1.7 gl 1 ) was comparable to that obtained by the strain JM109 (pCWlgtA, pBBlgtb) in Example 3.
- the colorimetric determination of fucose at the end of culture shows a significant difference between the two strains with a fucose production of lg.l 1 for the strain JM107-col DE3 (pHP0651, pBBLntRcsA) and only 0.25 gl 1 for the strain JM107-col- DE3 (pHP0651, pBBLnt). More than 70% of the fucosylated oligosaccharides are found in the intracellular fraction.
- the mass spectrum of compound 2 shows the presence of a quasi-molecular ion [M + H] + at m / z at 854 corresponding to the molar mass of lacto-N-fucopentaose.
- the presence of a secondary ion at 327 indicates that the molecule is fucosylated on the glucose residue and has the following structure ⁇ -D-Gal- [l ⁇ 4] ⁇ -D-Glc ⁇ Ac- [l- »3] - ⁇ - D-Gal- [l- 4] - ( ⁇ -L-Fuc- [l ⁇ 3]) - ⁇ - D-Glc
- the mass spectrum of the majority compound 3 shows the presence of 3 quasi-molecular ions at m / z 1000, 1022 and 1038 corresponding to the three forms [M + H] + , [M + Na] + and [M + K] + of the lacto-N-difucohexaose molecule having the following structure ⁇
- the mass spectrum of compound 4 makes it possible to identify two quasi-molecular ions at m / z 1365 and 1388 corresponding to the forms [M + H] + and [M + Na] + of a lacto-N-difucooctaose molecule.
- the presence of a secondary ion at m / z 512 indicates that the GlcNAc residue of the non-reducing end carries a fucose.
- the RM ⁇ data show that the proton J H of a fucose residue is sensitive to anomerism and that this fucose residue is therefore fixed on the glucose.
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Priority Applications (9)
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CA2378562A CA2378562C (fr) | 1999-07-07 | 2000-07-07 | Procede de production d'oligosaccharides |
NZ516808A NZ516808A (en) | 1999-07-07 | 2000-07-07 | Method for producing oligopolysaccharides |
US10/019,954 US7521212B1 (en) | 1999-07-07 | 2000-07-07 | Method for producing oligopolysaccharides |
MXPA02000240A MXPA02000240A (es) | 1999-07-07 | 2000-07-07 | Metodo para producir oligosacaridos. |
EP00949678A EP1194584B1 (fr) | 1999-07-07 | 2000-07-07 | Procede de production d'oligosaccharides |
DE60026142T DE60026142T2 (de) | 1999-07-07 | 2000-07-07 | Verfahren zur herstellung von oligosaccharide |
AU62961/00A AU780290B2 (en) | 1999-07-07 | 2000-07-07 | Method for producing oligopolysaccharides |
JP2001509544A JP5058420B2 (ja) | 1999-07-07 | 2000-07-07 | オリゴポリサッカライドの製造法 |
US11/930,663 US8586332B2 (en) | 1999-07-07 | 2007-10-31 | Method for producing oligopolysaccharides |
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FR99/08772 | 1999-07-07 | ||
FR9908772A FR2796082B1 (fr) | 1999-07-07 | 1999-07-07 | Procede de production d'oligosaccharides |
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US10/019,954 A-371-Of-International US7521212B1 (en) | 1999-07-07 | 2000-07-07 | Method for producing oligopolysaccharides |
US11/930,663 Continuation US8586332B2 (en) | 1999-07-07 | 2007-10-31 | Method for producing oligopolysaccharides |
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CA (1) | CA2378562C (fr) |
DE (2) | DE60044310D1 (fr) |
FR (1) | FR2796082B1 (fr) |
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WO2022263406A1 (fr) | 2021-06-15 | 2022-12-22 | Dsm Ip Assets B.V. | Séparation d'oligosaccharides de lait humain à partir d'un bouillon de fermentation |
WO2022263425A1 (fr) | 2021-06-15 | 2022-12-22 | Dsm Ip Assets B.V. | Séparation d'oligosaccharides de lait humain d'un bouillon de fermentation |
WO2023242194A1 (fr) | 2022-06-14 | 2023-12-21 | Dsm Ip Assets B.V. | Séparation d'oligosaccharides de lait humain à partir d'un bouillon de fermentation |
WO2023242184A1 (fr) | 2022-06-14 | 2023-12-21 | Dsm Ip Assets B.V. | Séparation d'oligosaccharides de lait humain d'un bouillon de fermentation |
WO2023247483A1 (fr) | 2022-06-20 | 2023-12-28 | Dsm Ip Assets B.V. | Mélange de hmo fucosylés |
DE202023103382U1 (de) | 2022-06-20 | 2023-11-29 | Dsm Ip Assets B.V. | Gemisch fucosylierter HMOs |
WO2023247577A1 (fr) | 2022-06-20 | 2023-12-28 | Dsm Ip Assets B.V. | Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de lactobacilles |
WO2023247578A1 (fr) | 2022-06-20 | 2023-12-28 | Dsm Ip Assets B.V. | Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de bifidobactéries |
WO2023247579A1 (fr) | 2022-06-20 | 2023-12-28 | Dsm Ip Assets B.V. | Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de lactobacillus rhamnosus |
Also Published As
Publication number | Publication date |
---|---|
EP1637611B1 (fr) | 2010-04-28 |
MXPA02000240A (es) | 2002-06-21 |
EP1194584B1 (fr) | 2006-02-22 |
DE60026142T2 (de) | 2006-11-23 |
AU6296100A (en) | 2001-01-30 |
US8586332B2 (en) | 2013-11-19 |
ATE466094T1 (de) | 2010-05-15 |
JP5058420B2 (ja) | 2012-10-24 |
US7521212B1 (en) | 2009-04-21 |
NZ516808A (en) | 2004-07-30 |
ATE318324T1 (de) | 2006-03-15 |
EP1194584A1 (fr) | 2002-04-10 |
FR2796082B1 (fr) | 2003-06-27 |
DE60044310D1 (de) | 2010-06-10 |
AU780290B2 (en) | 2005-03-17 |
US20090082307A1 (en) | 2009-03-26 |
EP1637611A1 (fr) | 2006-03-22 |
FR2796082A1 (fr) | 2001-01-12 |
CA2378562C (fr) | 2013-11-26 |
CA2378562A1 (fr) | 2001-01-18 |
DE60026142D1 (de) | 2006-04-27 |
JP2003504072A (ja) | 2003-02-04 |
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