WO2013182206A1 - Procédé de production d'oligosaccharides et d'oligosaccharide glycosides par fermentation - Google Patents

Procédé de production d'oligosaccharides et d'oligosaccharide glycosides par fermentation Download PDF

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WO2013182206A1
WO2013182206A1 PCT/DK2013/050182 DK2013050182W WO2013182206A1 WO 2013182206 A1 WO2013182206 A1 WO 2013182206A1 DK 2013050182 W DK2013050182 W DK 2013050182W WO 2013182206 A1 WO2013182206 A1 WO 2013182206A1
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transferase
cell
oligosaccharide
derivative
aglycon
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PCT/DK2013/050182
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English (en)
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Pauline Peltier-Pain
Gyula Dekany
Remy Dureau
Christian Risinger
Markus Hederos
Elise Champion
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Glycom A/S
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Priority to EP13800288.6A priority Critical patent/EP2859112A4/fr
Priority to US14/406,379 priority patent/US20150133647A1/en
Publication of WO2013182206A1 publication Critical patent/WO2013182206A1/fr

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    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/14—Acyclic radicals, not substituted by cyclic structures attached to a sulfur, selenium or tellurium atom of a saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • 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
    • C12P19/28—N-glycosides
    • 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
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • 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
    • C12P19/64—Preparation of S-glycosides, e.g. lincomycin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a method of making glycosides of oligosaccharides or glycosidic derivatives of oligosaccharides, particularly of human milk oligosaccharides
  • HMOs Human milk oligosaccharides
  • para-lacto-/V-octaose para-LNO
  • HMOs Low cost ways have been sought for making industrial quantities of as many as possible of the HMOs, so that their uses in nutritional and therapeutic formulations for infants, as well as possibly children and adults, could be discovered, developed and exploited by researchers worldwide.
  • a few HMOs have recently been chemically or enzymatically synthesized, for example, by hydrogenating their benzyl glycoside precursors after removing other protecting groups from such precursors and then isolating (e.g. by crystallization) the HMOs (WO 2011/100979, WO 2011/100980, WO 2012/007585, WO 2012/007588, WO 2012/113405, WO 2012/127410, WO 2012/155916).
  • HMOs like benzyl glycosides or glycosidic HMOs
  • Simpler and cheaper alternative can be the in vivo microbial production of HMO derivatives comprising glycosylation of an appropriate simple acceptor like lactose derivatives.
  • said method comprising the step of culturing, in a culture medium containing a lactose acceptor having the aglycon R, wherein R is as defined above, a genetically modified cell having a recombinant gene that encodes an enzyme capable of modifying said lactose acceptor or one of the intermediates in the biosynthetic pathway of said oligosaccharide derivative from said lactose acceptor and that is necessary for the synthesis of said oligosaccharide derivative from said lactose acceptor.
  • said recombinant gene encodes a glycosyl transferase that can transfer a glycosyl residue of an activated sugar nucleotide to said lactose acceptor.
  • said oligosaccharide derivative having said aglycon R is separated from said culture medium, particularly after separating said cell from said culture medium.
  • said culturing step comprises: (i) a first phase of exponential cell growth ensured by said carbon-based substrate, and
  • An embodiment of the first aspect of the invention relates to using the method for the production of said oligosaccharide derivative having said aglycon R, wherein its
  • oligosaccharide moiety is a human milk oligosaccharide selected from the group consisting of 2'-FL, 3-FL, difucosyllactose, 3'-SL, 6'-SL, sialyl-fucosyl lactose, LNT, LNnT, sialylated and/or fucosylated LNT and sialylated and/or fucosylated LNnT.
  • the second aspect of this invention relates to an oligosaccharide derivative having an aglycon R, wherein R is as defined above, particularly a human milk oligosaccharide benzyl glycoside, quite particularly a benzyl glycoside of 2'-FL, LNnT or LNT, produced by the method.
  • the third aspect of the invention relates to a method for producing an oligosaccharide, preferably an HMO, comprising the steps of: a) carrying out the method according to the first aspect to obtain an oligosaccharide derivative having an aglycon R, wherein R is as defined above, then b) removing/deprotecting said aglycon R to obtain said oligosaccharide.
  • the fourth aspect of the invention relates to a method for producing a compound of formula
  • R 3 is fucosyl or H
  • R 4 is fucosyl or H
  • R 5 is selected from H, sialyl, N-acetyl- lactosaminyl and lacto-N-biosyl groups, wherein the N-acetyl lactosaminyl group may carry a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N- biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue
  • R 6 is selected from H, sialyl and N-acetyl- lactosaminyl groups optionally substituted with a glycosyl residue comprising one or more N- acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each of the N-acetyl
  • said method comprising the step of culturing, in a culture medium containing allyl lactoside, a genetically modified cell having a recombinant gene that encodes an enzyme capable of modifying allyl lactoside or one of the intermediates in the biosynthetic pathway of a compound of formula 3 from allyl lactoside and that is necessary for the synthesis of compound of formula 3 from allyl lactoside.
  • the fifth aspect of the invention relates to providing a compound of formula 3 defined above.
  • exogenous carbohydrates with altered, preferably limited water solubility due to the presence of a hydrophobic aglycon thereon wherein the aglycon can be a bulky group as well, namely exogenous carbohydrate precursors having an aglycon R, wherein R is as defined above, preferably carbohydrates containing a galactose residue, more preferably lactose derivatives can be internalized into a genetically modified cell by a transport mechanism involving permeases, allowing thus this carbohydrate precursors to be glycosylated in a genetically modified cell able to act so.
  • glycosylated products having an aglycon R, wherein R is as defined above, made by the genetically modified cell are able to be secreted to the extracellular space, and thus they can be isolated from the fermentation broth.
  • the term "genetically modified cell” preferably means a cell in which at least one DNA sequence has been added to, deleted from or changed in its genome, so that the cell has a changed phenotype. This change in phenotype alters the characteristics of the genetically modified cell from that of the wild type cell.
  • the genetically modified cell can perform at least an additional chemical transformation, when cultured or fermented, due to the added or changed DNA that encodes the expression of at least one enzyme not found in the wild type cell, or the genetically modified cell cannot perform a chemical transformation due to the deleted, added or changed DNA that encodes the expression of an enzyme found in the wild type cell.
  • the genetically modified cell can be produced by well-known, conventional genetic engineering techniques.
  • the genetically modified cell can be bacteria or a yeast but preferably is a bacterium. Preferred bacteria include Escherichia coli, Bacillus spp. (e.g.
  • Bacillus subtilis Bacillus subtilis), Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Neisseria gonorrhoeae, Neisseria meningitis, Lactobacillus spp., Lactococcus spp., Enterococcus spp., Bifidobacterium spp., Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., Pseudomonas, particularly E. coli.
  • oligosaccharide preferably means a sugar polymer containing at least two monosaccharide units, i.e. a di-, tri-, tetra- or higher oligosaccharide.
  • the oligosaccharide can have a linear or branched structure containing monosaccharide units that are linked to each other by interglycosidic linkages.
  • the oligosaccharide comprises a lactose residue at the reducing end and one or more naturally occurring monosaccharides of 5-9 carbon atoms selected from aldoses (e.g. glucose, galactose, ribose, arabinose, xylose, etc.), ketoses (e.g.
  • the oligosaccharide is a HMO.
  • protecting group that is removable by hydrogenolysis or “group removable by hydrogenolysis” preferably means a group having a C-0 bond to the anomeric OH that can be cleaved by addition of hydrogen in the presence of catalytic amounts of palladium, Raney nickel or another appropriate metal catalyst known for use in
  • protecting groups are well known to the skilled man and are discussed in Protective Groups in Organic Synthesis, PGM Wuts and TW Greene, John Wiley & Sons 2007.
  • Suitable protecting groups include benzyl, diphenylmethyl (benzhydryl), 1-naphthylmethyl, 2-naphthylmethyl or triphenylmethyl (trityl) groups, each of which can be optionally substituted by one or more groups selected from: alkyl, alkoxy, phenyl, amino, acylamino, alkylamino, dialkylamino, nitro, carboxyl, alkoxycarbonyl, carbamoyl, /V-alkylcarbamoyl, ⁇ ,/V-dialkylcarbamoyl, azido, halogenalkyl or halogen.
  • protecting groups are benzyl or 1- or 2-naphthylmethyl groups optionally substituted with one or more groups selected from phenyl, alkyl or halogen. More preferably, the protecting group is selected from unsubstituted benzyl, unsubstituted 1-naphthylmethyl, unsubstituted 2-naphthylmethyl, 4-chlorobenzyl, 3-phenylbenzyl, 4-methylbenzyl and 4- nitrobenzyl.
  • alkyl preferably means a linear or branched chain saturated hydrocarbon group with 1-6 carbon atoms, such as methyl, ethyl, n-propyl, / ' -propyl, n-butyl, / ' -butyl, s-butyl, t-butyl, n-hexyl, etc. ;
  • aryl preferably means a homoaromatic group such as phenyl or naphthyl; and the term “optionally substituted” preferably means a chemical group that can either carry a substituent or can be unsubstituted.
  • alkyl preferably a compound having the following properties: hydroxy, alkoxy, carboxy, oxo, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylamino, arylcarbonyl, amino, mono- and dialkylamino, carbamoyl, mono- and dialkyl-aminocarbonyl, alkylcarbonylamino, cyano, alkanoyloxy, nitro, alkylthio and halogens.
  • group(s) selected from alkyl (only for aryl and benzyl), hydroxy, alkoxy, carboxy, oxo, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylamino, arylcarbonyl, amino, mono- and dialkylamino, carbamoyl, mono- and dialkyl-amin
  • the genetically modified cell used in the method of this invention comprises one or more endogenous or recombinant genes encoding one or more glycosyl transferase enzymes that are able to transfer the glycosyl residue of an activated sugar nucleotide to an internalized acceptor molecule.
  • the gene or an equivalent DNA sequence thereof, if it is recombinant, is introduced into the cell by known techniques, using an expression vector.
  • the origin of the heterologous nucleic acid sequence can be any animal (including human) or plant, eukaryotic cells such as those from Saccharomyces cerevisae, Saccharomyces pombe, Candida albicans and the like, prokaryotic cells such as those originated from E. coli, Bacillus subtilis,
  • Campylobacter pylori Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Rhizobium meliloti, Neisseria gonorrhoeae and Neisseria meningitis, or virus.
  • glycosyl transferase enzyme/enzymes expressed by the protein(s) encoded by the gene(s) or equivalent DNA sequence(s) are preferably glucosyl transferases, galactosyl transferases, N- acetylglucosaminyl transferases, N-acetylgalactosaminyl transferases, glucuronosyl transferases, xylosyl transferases, mannosyl transferases, fucosyl transferases, sialyl transferases and the like.
  • the glycosyl transferases are selected from the group consisting of B-l,3-N-acetylglucosaminyl transferase, B-l,3-galactosyl transferase, B-l,3-N-acetylgalactosaminyl transferase, B-l,3-glucuronosyl transferase, ⁇ -1,6- N-acetylglucosaminyl transferase, B-l,4-N-acetylgalactosaminyl transferase, B-l,4-galactosyl transferase, a-l,3-galactosyl transferase, a-l,4-galactosyl transferase, a-2,3-sialyl transferase, a-2,6-sialyl transferase, a-2,8-sialyl transferase,
  • the glycosyl transferases are selected from those involved in the construction of HMO core structures nos. 2-13 shown in Table 1, fucosylated and/or sialylated HMOs and glycosidic derivatives thereof, particularly those having an aglycon R, that is B-l,3-N-acetylglucosaminyl transferase, ⁇ -1,6- N-acetylglucosaminyl transferase, B-l,3-galactosyl transferase, B-l,4-galactosyl transferase, a-2,3-sialyl transferase, a-2,6-sialyl transferase, a-l,2-fucosyl transferase, a-l,3-fucosyl transferase and/or a-1,4 fucosyl transferase.
  • the genes encoding the above-mentioned transferases have been described in the literature.
  • glycosylation reaction preferably takes place in which an activated sugar nucleotide serves as donor.
  • An activated sugar nucleotide generally has a phosphorylated glycosyl residue attached to a nucleoside, a specific glycosyl transferase enzyme accept only a specific sugar nucleotide.
  • activated sugar nucleotides are involved in the glycosyl transfer: UDP-GIc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, UDP-glucuronic acid, GDP- Fuc and CMP-sialic acid, particularly those selected from the group consisting of UDP-Gal, UDP-GlcNAc, GDP-Fuc and CMP-sialic acid.
  • the genetically modified cell is able to produce one or more activated sugar nucleotide mentioned above by a de novo pathway.
  • an activated sugar nucleotide is made by the cell under the action of enzymes involved in the de novo biosynthetic pathway of that respective sugar nucleotide in a stepwise reaction sequence starting from a simple carbon source like glycerol, fructose or glucose (for a review for monosaccharide metabolism see e.g. H. H. Freeze and A. D. Elbein : Chapter 4:
  • the enzymes involved in the de novo biosynthetic pathway of an activated sugar nucleotide can be naturally present in the cell or introduced into the cell by means of gene technology or recombinant DNA techniques, all of them are parts of the general knowledge of the skilled person.
  • the genetically modified cell can utilize salvaged monosaccharide for producing activated sugar nucleotide.
  • oligosaccharides derived from degraded oligosaccharides are phosphorylated by kinases, and converted to nucleotide sugars by pyrophosphorylases.
  • the enzymes involved in the procedure can be heterologous ones, or native ones of the cell used for genetic modification.
  • the synthesis of GDP-fucose or CMP-sialic acid can be accomplished using the salvage pathway, when exogenous fucose or sialic acid is also added to the culture.
  • the genetically modified cell is cultured in the presence of a carbon-based substrate such as glycerol, glucose, glycogene, fructose, maltose, starch, cellulose, pectin, chitin, etc.
  • a carbon-based substrate such as glycerol, glucose, glycogene, fructose, maltose, starch, cellulose, pectin, chitin, etc.
  • the cell is cultured on glycerol and/or glucose and/or fructose.
  • the method of the invention also involves initially transporting an exogenous lactose derivative having the aglycon R, as an acceptor molecule, from the culture medium into the genetically modified cell for glycosylation where it can be glycosylated to produce the oligosaccharide derivative.
  • the acceptor can be added exogenously in a conventional manner to the culture medium, from which it can then be transported into the cell.
  • the internalization of the acceptor should not, of course, affect the basic and vital functions or destroy the integrity of the cell. In one embodiment the internalization can take place via a passive transport mechanism during which the exogenous acceptor diffuses passively across the plasma membrane of the cell.
  • the flow is directed by the concentration difference in the extra- and intracellular space with respect to the acceptor molecule to be internalized, which acceptor is supposed to pass from the place of higher concentration to the zone of lower concentration tending towards equilibrium.
  • the exogenous acceptor can be internalized in the cell with the aid of an active transport mechanism, during which the exogenous acceptor diffuses across the plasma membrane of the cell under the influence of a transporter protein or permease of the cell.
  • Lactose permease (LacY) has specificity towards galactose and simple galactosyl disaccharides like lactose.
  • the specificity towards the sugar moiety of the substrate to be internalized can be altered by mutation by means of known recombinant DNA techniques.
  • the internalization of the exogenous lactose derivative acceptor takes place via an active transport mechanism mediated by lactose permease.
  • Culturing or fermenting the genetically modified cell according to the method of this invention can be carried out in a conventional manner.
  • the exogenous lactose derivative acceptor is internalized into, and accumulates in, the genetically modified cell.
  • the internalized substrate, acting as acceptor participates in a glycosyl transferase induced glycosylation reaction, in which a glycosyl residue of an activated nucleotide donor is transferred so that the acceptor is glycosylated giving thus a trisaccharide derivative.
  • the cell when more than one glycosyl transferase is expressed by the cell, additional glycosylation reactions can occur resulting in the formation of tetra- or higher oligosaccharide derivatives.
  • the cell preferably lacks any enzyme activity which would degrade the acceptor or the oligosaccharide derivatives produced in the cell.
  • the oligosaccharide glycoside as product can be accumulated both in the intra- and the extracellular matrix.
  • the product can be transported to the supernatant in a passive way, i.e. it diffuses outside across the cell membrane.
  • the transport can be facilitated by sugar efflux transporters, proteins that promote the effluence of sugar derivatives from the cell to the supernatant.
  • the sugar efflux transporter can be present exogenously or endogenously and is overexpressed under the conditions of the fermentation to enhance the export of the oligosaccharide derivative produced.
  • the specificity towards the sugar moiety of the product to be secreted can be altered by mutation by means of known recombinant DNA techniques.
  • the method also comprises the addition of an inducer to the culture medium.
  • the role of the inducer is to promote the expression of enzymes involved in the de novo or salvage pathway and/or of permeases involved in the active transport and/or of sugar efflux transporters of the cell.
  • the inducer is isopropyl ⁇ - D-thiogalactoside (IPTG) .
  • the oligosaccharide derivative having the aglycon R formed can be collected from the culture or fermentation broth in a conventional manner.
  • the supernatant containing the oligosaccharide glycoside can be separated from the cells by centrifugation.
  • the separated cells can be resuspended in water and subjected to heat and/or acid treatment in order to permeabilize them for releasing the oligosaccharide glycoside accumulated intracellular ⁇ .
  • the product can be separated from the treated cell by centrifugation.
  • the two supernatants containing the extra- and intracellular products, respectively, are combined and the products can be purified and isolated by means of standard separation, purification and isolation techniques such as gel and/or cationic ion exchange resin (H + form) chromatography.
  • the oligosaccharide derivative is collected only from the supernatant.
  • concentration of the oligosaccharide derivative, particularly a trisaccharide derivative, especially a glycosidic 2'-FL derivative in the extracellular fraction of the culture is surprisingly high using only the normal secreting mechanism of the cell.
  • the lactose derivative acceptors used in the method are known compounds that can be prepared by conventional methods.
  • the lactose acceptors having aglycon R wherein R is ORi, which Ri is a group removable by catalytic hydrogenolysis, or R is -SR 2 , which R 2 is selected from optionally substituted alkyl, optionally substituted aryl and optionally substituted benzyl, or R is azido
  • R is ORi, which Ri is a group removable by catalytic hydrogenolysis
  • R is -SR 2
  • R 2 is selected from optionally substituted alkyl, optionally substituted aryl and optionally substituted benzyl, or R is azido
  • Ri-OH or R 2 -SH preferably a benzyl/substituted benzyl alcohol or alkyl-, benzyl- or phenyl-SH in an organic solvent such as DCM, toluene or THF, or followed by a treatment with sodium azide.
  • the vinylogous glycosyl amine acceptors can be synthesized by the treatment of lactose with aqueous ammonium hydrogen carbonate followed by the reaction of the resulting lactosyl amine with an activated vinyl reagent, such as an alkoxymethylenated or dialkylaminomethylenated malonic acid derivative, in the presence or absence of a base (Ortiz Mellet et al. J. Carbohydr. Chem. 12, 487 ( 1993) ; WO 2007/104311) .
  • an oligosaccharide derivative having an aglycon R can be produced by fermenting a genetically modified cell starting with at least one internalized exogenous precursor consisting of a lactose derivative having an aglycon R, the method comprises the steps of:
  • the Lac Z " Y + E. coli cell is cultured in the following way:
  • said enzyme capable of modifying the exogenous precursor or one of the intermediates in the biosynthetic pathway is an enzyme capable of performing a glycosylation by means of, preferably exogenous, glycosyl transferases.
  • said carbon-based substrate is selected from the group consisting of glycerol and glucose. More preferably, the carbon-based substrate added during the second phase glycerol.
  • said culturing is performed under conditions allowing the production of a culture with a high cell density. Also preferably, said culturing further comprises a third phase of slowed cell growth obtained by continuously adding to the culture an amount of said carbon-based substrate that is less than the amount of the carbon-based substrate added in said second phase so as to increase the content of the oligosaccharide derivative having an aglycon R produced in the high cell density culture.
  • the amount of the carbon-based substrate added continuously to the cell culture during said third phase is at least 30% less than the amount of the carbon-based substrate added continuously during said second phase.
  • the method further comprises the addition of an inducer to said culture medium to induce the expression in said cell of said enzyme and/or of a protein involved in said transport.
  • the inducer is preferably isopropyl ⁇ -D-thiogalactoside (IPTG) and the protein is lactose permease.
  • the exogenous lactose derivative having the aglycon R to be internalized by and glycosylated in the fermented cell can be added to the culture medium at once or continuously. If added at once, it is done at the end of the first phase of exponential cell growth.
  • a concentrated aqueous solution of the acceptor is added to reach a concentration of not more than 15 g/l, preferably of about 3-5 g/l calculated on the volume of the culture, then the fermentation is continued by addition of the carbon-based substrate as described above.
  • the continuous addition is beneficial when higher amount exogenous acceptor is intended to be used at a given volume.
  • the exogenous acceptor is dissolved in the feeding solution to be added during the second (and optionally the third) phase, therefore a continuous addition of the acceptor (with the carbon-based substrate) is realized.
  • the method is able to produce an oligosaccharide derivative having an aglycon R, wherein the oligosaccharide is a human milk oligosaccharide selected from the group consisting of 2'-FL, 3-FL, difucosyllactose, 3'-SL, 6'-SL, sialyl-fucosyl lactose, LNT, LNnT, sialylated and/or fucosylated LNT and sialylated and/or fucosylated LNnT, and R is as defined above.
  • the oligosaccharide is a human milk oligosaccharide selected from the group consisting of 2'-FL, 3-FL, difucosyllactose, 3'-SL, 6'-SL, sialyl-fucosyl lactose, LNT, LNnT, sialylated and/or fucosylated LNT and sialylated and/or fucosylated LNn
  • R t is a group removable by catalytic hydrogenolysis, preferably optionally substituted benzyl, more preferably benzyl, is used in the method to obtain an oligosaccharide having an aglycon -ORi, wherein Ri is defined above.
  • R 2 is selected from optionally substituted alkyl, optionally substituted aryl and optionally substituted benzyl, preferably alkyl and phenyl, is used in the method to obtain an oligosaccharide having an aglycon -SR 2 , wherein R 2 is defined above.
  • lactosyl azide as an exogenous precursor is used in the method to obtain an oligosaccharide having an azido aglycon.
  • the method can be carried out as described in US patent 7 521 212 and PCT publication WO 01/04341 Al, which are incorporated herein by reference, by adding a lactoside precursor having an aglycon R, preferably that of formula 1 or 2, or lactosyl azide, to the fermentation broth of the LacZ " Y + E. coli, described above.
  • the resulting oligosaccharide derivative obtainable by the method described above is selected from LNT, LNnT and 2'-FL having an aglycon R, fermenting a genetically modified LacZ " Y + E. coli having genes expressing p-l,3-N-acetyl-glucosaminyl transferase and ⁇ -1,3- galactosyl transferase for making an LNT derivative, p-l,3-N-acetyl-glucosaminyl transferase and p-l,4-galactosyl transferase for making a LNnT derivative, or a-l,2-fucosyl transferase for making a 2'-FL derivative.
  • the resulting oligosaccharide having the aglycon R preferably having the aglycon -ORi, -SR 2 or azido, more preferably having the aglycon O-benzyl/O-substituted benzyl, -S-alkyl, -S-phenyl or azido, can be isolated in a conventional manner from the aqueous fermentation broth, in which the LacZ " Y + E. coli cell was cultured.
  • the aqueous fermentation broth is preferably separated (for example, by centrifugation) from the fermented E. coli, cells, filtered and then contacted with cationic and anionic ion exchange resins to remove proteins and ionic compounds.
  • the resulting aqueous medium can then be dried (for example, by freeze drying).
  • the resulting supernatant after fermentation preferably contains no more than about 8-10 wt% and at least about 15 wt%, especially at least about 25 wt % of the glycoside.
  • the dry, preferably protein-free glycoside powder can then be treated with a hot, preferably boiling, solvent, such as a Ci-C 6 alcohol solvent, and the resulting solution can be filtered while still hot, then kept hot to concentrate it by partial evaporation down to at least about 60-70 % of its original volume and then allowed to cool somewhat. Seed crystals of the desired glycoside can then be added to the concentrated solution while it is still warm, the solution can then be allowed to cool to room temperature, and precipitated crystals of the glycoside can then be filtered from the solution.
  • a hot, preferably boiling, solvent such as a Ci-C 6 alcohol solvent
  • lactose derivatives having an aglycon R are surprising. No glycosidic lactosides have been internalized successfully in preparative scale so far except for allyl and propargyl lactosides (Fort et al. Chem. Comm. 2558 (2005), EP-A-1911850). However, the efficient transportation of compounds of formula 1 by a lactose permease unexpected due to the significant difference in bulkiness, conformation and hydrophobicity of an allyl/propargyl moiety vs a benzyl/substituted benzyl group.
  • the internalization of thiolactosides and their transformation by means of glycosylation in a living cell under culturing is also surprising and the present invention is the first example to show this.
  • Fort et al. reported on the unsuccessful utilization of a lactose N-glycoside, and another lactoside having an azido group in the anomeric substituent gave poor result in a fermentation process; in this view the internalization of the lactosyl azide and its transformation by means of glycosylation in a living cell under culturing can be considered non-expected.
  • the second aspect of the invention relates to an
  • an oligosaccharide derivative defined above made by the method comprising the step of: culturing, in a culture medium containing a lactose acceptor having the aglycon R, wherein R is as defined above, a genetically modified cell having a recombinant gene that encodes an enzyme capable of modifying said lactose acceptor or one of the intermediates in the biosynthetic pathway of said oligosaccharide derivative from said lactose acceptor and that is necessary for the synthesis of said oligosaccharide derivative from said lactose acceptor.
  • R is selected from -ORi, -SR 2 and azido, wherein Ri is a group removable by catalytic hydrogenolysis, particularly benzyl or substituted benzyl, R 2 is selected from alkyl, aryl and benzyl, particularly alkyl and phenyl.
  • the oligosaccharide derivative obtainable by the method is a HMO, particularly 2'-FL, 3-FL, difucosyllactose, 3'-SL, 6'-SL, sialyl-fucosyl lactose, LNT, LNnT, sialylated and/or fucosylated LNT and sialylated and/or fucosylated LNnT derivatives having an aglycon R.
  • the oligosaccharide derivatives obtainable by the method is selected from -O-benzyl glycoside, -O-substituted benzyl glycoside, -S-alkyl thioglycoside, -S-phenyl thioglycoside and 1-azido-l-deoxy derivative of 2'-FL, 3-FL, difucosyllactose, 3'-SL, 6'-SL, sialyl-fucosyl lactose, LNT, LNnT, sialylated and/or fucosylated LNT and sialylated and/or fucosylated LNnT, particularly -O-benzyl glycoside, -S-alkyl thioglycoside, -S-phenyl thioglycoside and 1-azido-l-deoxy derivative of 2'-FL, 3-FL, difucosyllactose, 3'-SL, 6'-SL,
  • oligosaccharides having an aglycon R preferably when R means - O-benzyl or -O-substituted benzyl, or R means -S-alkyl or -S-phenyl, or R means azido, over preparing the free oligosaccharides directly lies upon the fact, that these derivatives has limited water solubility due to the presence of the more hydrophobic group R, thus allowing the practitioner to enlarge the repertoire of e.g. chromatographic separations. For example, due to the different polarity of the glycoside derivatives vs.
  • Crystallization or recrystallization is one of the simplest and cheapest methods to isolate a product from a reaction mixture, separate it from contaminations and obtain pure substance. Isolation or purification that uses crystallization makes the whole technological process robust and cost-effective, thus it is advantageous and attractive compared to other procedures (see above). Fourthly, removal of the anomeric protective group from a glycosidic oligosaccharide derivate obtained in the method claimed takes place under delicate conditions nearly quantitatively. For example benzyl/substituted benzyl protective groups in
  • -ORi can be converted exclusively into toluene/substituted toluene under the hydrogenolysis condition and they can easily be removed even in multi ton scales from water soluble oligosaccharide products via evaporation and/or extraction processes.
  • the compounds having R as -SR 2 can be converted into the corresponding reducing oligosaccharides in the following way: the thioglycoside is dissolved in water or a dipolar aprotic solvent containing water followed by the addition of a thiophilic activator such as mercury(II) salts, Br 2 , I 2 , NBS, NIS, triflic acid or triflate salts, or a mixture thereof.
  • a thiophilic activator such as mercury(II) salts, Br 2 , I 2 , NBS, NIS, triflic acid or triflate salts, or a mixture thereof.
  • the activated intermediate reacts easily with the water present in the reaction milieu and a deprotected oligosaccharide can be produced.
  • Oligosaccharides having an aglycon R can be subjected to catalytic hydrogenolysis or reduced by complex metal hydrides like NaBH 4 , or by PPh 3 . Both types of reactions yield amine functionality at the anomeric position, the hydrolysis of which under neutral or slightly acidic pH (pH « 4-7) readily provides the deprotected
  • oligosaccharides oligosaccharides.
  • Suitable solvents for this reaction include methanol, ethanol, water, acetic acid, or ethyl acetate, and mixtures thereof.
  • Amino compounds for this reaction are the aqueous and anhydrous primary amines, such as ethylamine, propylamine and butylamine, the hydrazines, such as hydrazine hydrate and hydrazine acetate, hydroxylamine derivatives, an aqueous ammonia solution and ammonia gas.
  • the acyclic vinylogous amine can also be cleaved with a halogen such as chlorine gas or bromine. Both types of reactions yield amine functionality at the anomeric position, the hydrolysis of which under neutral or slightly acidic pH (pH « 4-7) readily provides the deprotected oligosaccharides.
  • oligosaccharide derivative having an aglycon R wherein R is as above, from said lactose acceptor and that is necessary for the synthesis of said oligosaccharide derivative having an aglycon R from said lactose acceptor, b) separating said oligosaccharide derivative having an aglycon R from the cell, from the culture medium or from both, and then c) removing/deprotecting aglycon R to obtain said oligosaccharide.
  • this method comprises carrying out the method disclosed in the first aspect of the present invention including the preferred and the more preferred embodiments, followed by removing/deprotecting the aglycon R from the product so-obtained by means of catalytic hydrogenolysis, when R is -ORi, wherein Ri is a group removable by catalytic hydrogenolysis, - treating the product with a thiophilic activator such as mercury(II) salts, Br 2 , I 2 , NBS,
  • R is -SR 2 , wherein R 2 is optionally substituted alkyl, optionally substituted aryl or optionally substituted benzyl, followed by hydrolysis, reducing the azido group, when R is azide, followed by hydrolysis, or - treating the product with ammonia, amino compound or halogen, R is -NH-
  • the method according to the third aspect comprises culturing a genetically modified cell, which cell can be originated from a bacterium or yeast, more preferably a bacterium, particularly E. coli, having a gene encoding a glycosyl transferase that can transfer a glycosyl residue of an activated sugar nucleotide to a lactose acceptor having an aglycon R, wherein R is as described above, to prepare an oligosaccharide derivative having an aglycon R, and removing/deprotecting the aglycon R from them by one of the ways disclosed above.
  • a genetically modified cell which cell can be originated from a bacterium or yeast, more preferably a bacterium, particularly E. coli, having a gene encoding a glycosyl transferase that can transfer a glycosyl residue of an activated sugar nucleotide to a lactose acceptor having an aglycon R, wherein R is as described above
  • the method according to the third aspect comprises culturing a genetically modified cell having a glycosyl transferase selected from the group consisting of ⁇ - 1,3- ⁇ - acetyl-glucosaminyl transferase, B- l,3-galactosyl transferase, B- l,3-N-acetyl-galactosaminyl transferase, B- l,3-glucuronosyl transferase, B- l,3-N-acetyl-galactosaminyl transferase, ⁇ - 1,4-N-acetyl-galactosaminyl transferase, B- l,4-galactosyl transferase, a- l,3-galactosyl transferase, a- l,4-galactosyl transferase, a-2,3-sialyl transferase,
  • the method according to the third aspect comprises culturing a genetically modified cell having a glycosyl transferase, wherein the culturing is characterized by (a) a first phase of exponential cell growth ensured by a carbon-based substrate, and
  • the method according to the third aspect comprises the preparation of an oligosaccharide derivative having an aglycon R by a genetically modified cell, wherein the oligosaccharide is a human milk oligosaccharide, preferably a HMO selected from the group consisting of 2'-FL, 3-FL, difucosyllactose, 3'-SL, 6'-SL, sialyl-fucosyl lactose, LNT, LNnT, sialylated and/or fucosylated LNT and sialylated and/or fucosylated LNnT, and
  • a human milk oligosaccharide preferably a HMO selected from the group consisting of 2'-FL, 3-FL, difucosyllactose, 3'-SL, 6'-SL, sialyl-fucosyl lactose, LNT, LNnT, sialylated and/or fucosylated LNT and sialylated and/or fucosylated LNnT.
  • HMO selected from the group consisting of 2'-FL, 3-FL, difucosyllactose, 3'-SL, 6'-SL, sialyl-fucosyl lactose, LNT, LNnT, sialylated and/or fucosylated LNT and sialylated and/or fucosylated LNnT.
  • the method according to the third aspect comprises the preparation of an oligosaccharide derivative having an aglycon -ORi, -SR 2 and azido, by a genetically modified cell from a precursor: a) of formula 1
  • Rt is a group removable by catalytic hydrogenolysis, preferably optionally substituted benzyl, more preferably benzyl, or b) of formula 2
  • R 2 is selected from optionally substituted alkyl, optionally substituted aryl and optionally substituted benzyl, preferably alkyl and phenyl, or c) lactosyl azide, and removing/deprotecting the aglycon a) -ORi by catalytic hydrogenolysis, b) -SR 2 by treatment with a thiophilic activator such as mercury(II) salts, Br 2 , I 2 , NBS, NIS, triflic acid or triflate salts, or a mixture thereof, followed by hydrolysis, or c) azido by reducing it to amino group followed by hydrolysis, to prepare an oligosaccharide, preferably an HMO.
  • a thiophilic activator such as mercury(II) salts, Br 2 , I 2 , NBS, NIS, triflic acid or triflate salts, or a mixture thereof, followed by hydrolysis, or c) azido by reducing it to amino group followed by hydro
  • the method according to the third aspect comprises the fermentation a genetically modified cell in the presence of a precursor of formula 1 depicted above, preferably from that having an aglycon -ORi, more preferably benzyl lactoside, to prepare an oligosaccharide derivative, preferably a LNT, LNnT and 2'-FL derivative, having an aglycon -OF ! , preferably benzyloxy, which is then subjected to catalytic hydrogenolysis to remove the Ri group and to make an oligosaccharide, preferably LNT, LNnT and 2'-FL.
  • a precursor of formula 1 depicted above preferably from that having an aglycon -ORi, more preferably benzyl lactoside, to prepare an oligosaccharide derivative, preferably a LNT, LNnT and 2'-FL derivative, having an aglycon -OF ! , preferably benzyloxy, which is then subjected
  • the fourth aspect of the invention relates to a method for producing a compound of formula 3
  • R 3 is fucosyl or H
  • R 4 is fucosyl or H
  • R 5 is selected from H, sialyl, N-acetyl- lactosaminyl and lacto-N-biosyl groups, wherein the N-acetyl lactosaminyl group may carry a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N- biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue
  • R 6 is selected from H, sialyl and N-acetyl- lactosaminyl groups optionally substituted with a glycosyl residue comprising one or more N- acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each of the N-acetyl
  • said method comprising the step of culturing, in a culture medium containing allyl lactoside, a genetically modified cell having a recombinant gene that encodes an enzyme capable of modifying allyl lactoside or one of the intermediates in the biosynthetic pathway of a compound of formula 3 from allyl lactoside and that is necessary for the synthesis of compound of formula 3 from allyl lactoside.
  • a method for producing a compound of formula 3 defined above using a genetically modified cell starting with an internalized allyl lactoside the method comprises the steps of:
  • a compound of formula 3 is separated from said culture medium, particularly after separating said cell from said culture medium.
  • heterologous nucleic acid sequence can be any animal (including human) or plant, eukaryotic cells, prokaryotic cells or virus as described above.
  • glycosyl transferase enzyme/enzymes expressed by the protein(s) encoded by the gene(s) or equivalent DNA sequence(s) are as described above, with the proviso that when the genetically modified cell contains only one recombinant gene expressing a glycosyl transferase, then this glycosyl transferase is different from a-2,3-sialyl transferase.
  • a glycosyl transferase mediated glycosylation reaction preferably takes place in which an activated sugar nucleotide serves as donor.
  • An activated sugar nucleotide generally has a phosphorylated glycosyl residue attached to a nucleoside, a specific glycosyl transferase enzyme accept only a specific sugar nucleotide.
  • activated sugar nucleotides are involved in the glycosyl transfer: UDP-GIc, UDP-Gal, UDP-GlcNAc, UDP-GalNAc, UDP-glucuronic acid, GDP-Fuc and CM P-sialic acid, particularly those selected from the group consisting of UDP- Gal, UDP-GlcNAc and GDP-Fuc.
  • the genetically modified cell used in the method according to the fourth aspect is able to produce one or more activated sugar nucleotide mentioned above by a de novo pathway, or can utilize salvaged monosaccharide for producing activated sugar nucleotide (see above) .
  • the method according to the fourth aspect also involves initially transporting the exogenous allyl lactoside, as an acceptor molecule, from the culture medium into the genetically modified cell for glycosylation where it can be glycosylated to produce an oligosaccharide derivative of formula 3.
  • the allyl lactoside can be added exogenously in a conventional manner to the culture medium, from which it can then be transported into the cell.
  • the internalization of the acceptor should not, of course, affect the basic and vital functions or destroy the integrity of the cell.
  • the internalization can take place via a passive transport mechanism during which the exogenous acceptor diffuses passively across the plasma membrane of the cell. The flow is directed by the concentration difference in the extra- and intracellular space with respect to the acceptor molecule to be internalized, which acceptor is supposed to pass from the place of higher concentration to the zone of lower concentration tending towards equilibrium.
  • the exogenous acceptor can be internalized in the cell with the aid of an active transport mechanism, during which the exogenous acceptor diffuses across the plasma membrane of the cell under the influence of a transporter protein or permease of the cell.
  • Lactose permease has specificity towards galactose and simple galactosyl disaccharides like lactose.
  • the specificity towards the sugar moiety of the substrate to be internalized can be altered by mutation by means of known recombinant DNA techniques.
  • the internalization of the exogenous lactose derivative acceptor takes place via an active transport mechanism mediated by lactose permease.
  • Culturing or fermenting the genetically modified cell according to the method of the fourth aspect can be carried out in a conventional manner.
  • the exogenous allyl lactoside is internalized into, and accumulates in, the genetically modified cell.
  • the internalized substrate acting as acceptor, participates in a glycosyl transferase induced glycosylation reaction, in which a glycosyl residue of an activated nucleotide donor is transferred so that the acceptor is glycosylated giving thus a trisaccharide derivative.
  • the cell when more than one glycosyl transferase is expressed by the cell, additional glycosylation reactions can occur resulting in the formation of tetra- or higher oligosaccharide derivatives.
  • the cell preferably lacks any enzyme activity which would degrade the acceptor or the oligosaccharide derivatives produced in the cell.
  • the oligosaccharide glycoside of formula 3 as product can be accumulated both in the intra- and the extracellular matrix.
  • the product can be transported to the supernatant in a passive way, i.e. it diffuses outside across the cell membrane.
  • the transport can be facilitated by sugar efflux transporters, proteins that promote the effluence of sugar derivatives from the cell to the supernatant.
  • the sugar efflux transporter can be present exogenously or endogenously and is overexpressed under the conditions of the fermentation to enhance the export of the oligosaccharide derivative produced .
  • the specificity towards the sugar moiety of the product to be secreted can be altered by mutation by means of known recombinant DNA techniques.
  • the method also comprises the addition of an inducer to the culture medium.
  • the role of the inducer is to promote the expression of enzymes involved in the de novo or salvage pathway and/or of permeases involved in the active transport and/or of sugar efflux transporters of the cell.
  • the inducer is isopropyl ⁇ - D-thiogalactoside (IPTG) .
  • the oligosaccharide derivative of formula 3 formed can be collected from the culture or fermentation broth in a conventional manner.
  • supernatant containing the product can be separated from the cells by centrifugation.
  • the separated cells can be resuspended in water and subjected to heat and/or acid treatment in order to permeabilize them for releasing the oligosaccharide glycoside accumulated intracellular ⁇ .
  • the product can be separated from the treated cell by centrifugation.
  • the two supernatants containing the extra- and intracellular products, respectively, are combined and the products can be purified and isolated by means of standard separation, purification and isolation techniques such as gel and/or cationic ion exchange resin (H + form)
  • the oligosaccharide derivative is collected only from the supernatant.
  • a compound of formula 3 can be produced by fermenting a genetically modified cell starting with an internalized exogenous allyl lactoside, the method comprising the steps of:
  • the Lac Z " Y + E. coli cell is cultured in the following way:
  • said enzyme capable of modifying the exogenous allyl lactoside or one of the intermediates in the biosynthetic pathway is an enzyme capable of performing a glycosylation by means of, preferably exogenous, glycosyl transferases.
  • said carbon-based substrate is selected from the group consisting of glycerol and glucose. More preferably, the carbon-based substrate added during the second phase glycerol.
  • said culturing is performed under conditions allowing the production of a culture with a high cell density.
  • said culturing further comprises a third phase of slowed cell growth obtained by continuously adding to the culture an amount of said carbon-based substrate that is less than the amount of the carbon-based substrate added in said second phase so as to increase the content of the oligosaccharide derivative of formula 3 produced in the high cell density culture.
  • the amount of the carbon-based substrate added continuously to the cell culture during said third phase is at least 30% less than the amount of the carbon-based substrate added continuously during said second phase.
  • the method further comprises the addition of an inducer to said culture medium to induce the expression in said cell of said enzyme and/or of a protein involved in said transport.
  • the inducer is preferably isopropyl ⁇ -D-thiogalactoside (IPTG) and the protein is lactose permease.
  • the exogenous allyl lactoside to be internalized by and glycosylated in the fermented cell can be added to the culture medium at once or continuously. If added at once, it is done at the end of the first phase of exponential cell growth.
  • a concentrated aqueous solution of the acceptor is added to reach a concentration of not more than 15 g/l, preferably of about 3-5 g/l ca lculated on the volume of the culture, then the fermentation is continued by addition of the carbon-based substrate as described above.
  • the continuous addition is beneficial when higher amount exogenous acceptor is intended to be used at a given volume.
  • the exogenous acceptor is dissolved in the feeding solution to be added during the second (and optionally the third) phase, therefore a continuous addition of the acceptor (with the carbon- based substrate) is realized.
  • the method is able to produce an oligosaccharide derivative of formula 3 characterized by formula 3a, 3b or 3c
  • R 3 and R 4 are as defined above,
  • R 5a is an N-acetyl-lactosaminyl group optionally substituted with a glycosyl residue comprising one N-acetyl-lactosaminyl and/or one lacto-N-biosyl group; each of the N-acetyl- lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,
  • R 6a is H or an N-acetyl-lactosaminyl group optionally substituted with a lacto-N-biosyl group; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,
  • R 5b is a lacto-N-biosyl group optionally substituted with one or more sialyl and/or fucosyl residue(s),
  • R 6b is H or an N-acetyl-lactosaminyl group optionally substituted with one or two N-acetyl- lactosaminyl and/or one lacto-N-biosyl groups; each of the N-acetyl-lactosaminyl and lacto- N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residues,
  • R 7 and R 8 are, independently, H or sialyl, provided that at least one of R 3 , R 4 , R 7 and R 8 is not H, and further provided that when R 7 is sialyl then at least one of R 3 , R 4 and R 8 is not H.
  • the compounds according to formulae 3a or 3b obtainable by the method of the fourth aspect are characterized in that: the N-acetyl-lactosaminyl group in the glycosyl residue of R 5a is attached to another N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage, the lacto-N-biosyl group in the glycosyl residue of R 5a is attached to the N-acetyl- lactosaminyl group with a 1-3 interglycosidic linkage, the lacto-N-biosyl group in the glycosyl residue of R 6a is attached to the N-acetyl- lactosaminyl group with a 1-3 interglycosidic linkage, - the N-acetyl-lactosaminyl group in the glycosyl residue of R 6b is attached to
  • the lacto-N-biosyl group in the glycosyl residue of R 6b is attached to the N-acetyl- lactosaminyl group with a 1-3 interglycosidic linkage.
  • a compound of formula 3a obtainable by the method of the fourth aspect is allyl glycoside of lacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N- neohexaose, lacto-N-neohexaose, para-lacto-N-octaose or lacto-N-neooctaose optionally substituted with one or more sialyl and/or fucosyl residue
  • a compound of formula 3b obtainable by the method of the fourth aspect is allyl glycoside of lacto-N-tetraose, lacto-N- hexaose, lacto-N-octaose, iso-lacto-N-octaose, lacto-N-decaose or lacto-N-neodecaose optionally substituted with one or more sialyl and
  • the compounds of formula 3a or 3b obtainable by the method of the fourth aspect are characterized in that: the fucosyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked to o the galactose of the lacto-N-biosyl group with 1-2 interglycosidic linkage and/or o the N-acetyl-glucosamine of the lacto-N-biosyl group with 1-4 interglycosidic linkage and/or o the N-acetyl-glucosamine of the N-acetyl-lactosaminyl group with 1-3
  • the sialyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked to o the galactose of the lacto-N-biosyl group with 2-3 interglycosidic linkage and/or o the N-acetyl-glucosamine of the lacto-N-biosyl group with 2-6 interglycosidic linkage and/or o the galactose of the N-acetyl-lactosaminyl group with 2-6 interglycosidic linkage.
  • a compound according to subformulae 3a, 3b or 3c obtainable by the method of the fourth aspect may be selected from the group of: allyl glycoside of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, 6'-sialyllactose, 3'- sialyl-3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LST-a, LST-b, LST-C, FLST-a, FLST-b, FLST-C, LNDFH-I, LNDFH-II, LNDFH-III, DS- LNT, FDS-LNT I and FDS-LNT II, or salts thereof.
  • the glycosides may be alpha or
  • the method according to the fourth aspect can be carried out as described in WO 01/04341 Al and Fort et al. Chem. Comm. 2558 (2005), which are incorporated herein by reference, by adding allyl lactoside to the fermentation broth of the LacZ " Y + E. coli, described above.
  • the resulting oligosaccharide derivative obtainable by the method described above is selected from LNT, LNnT and 2'-FL allyl glycoside, fermenting a genetically modified LacZ " Y + E. coli having genes expressing p-l,3-N-acetyl-glucosaminyl transferase and ⁇ -1,3- galactosyl transferase for making LNT allyl glycoside, p-l,3-N-acetyl-glucosaminyl transferase and p-l,4-galactosyl transferase for making LNnT allyl glycoside, or a- l,2-fucosyl transferase for making 2'-FL allyl glycoside.
  • the fifth aspect of the invention provides a compound of formula 3
  • R 3 is fucosyl or H
  • R 4 is fucosyl or H
  • R 5 is selected from H, sialyl, N-acetyl- lactosaminyl and lacto-N-biosyl groups, wherein the N-acetyl lactosaminyl group may carry a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N- biosyl groups; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue
  • R 6 is selected from H, sialyl and N-acetyl- lactosaminyl groups optionally substituted with a glycosyl residue comprising one or more N- acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; each of the N-acetyl
  • an oligosaccharide derivative of formula 3 is characterized by formula 3a, 3b or 3c
  • R 3 and R 4 are as defined above,
  • R 5a is an N-acetyl-lactosaminyl group optionally substituted with a glycosyl residue comprising one N-acetyl-lactosaminyl and/or one lacto-N-biosyl group; each of the N-acetyl- lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue,
  • R 6a is H or an N-acetyl-lactosaminyl group optionally substituted with a lacto-N-biosyl group; each of the N-acetyl-lactosaminyl and lacto-N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residue, R 5b is a lacto-N-biosyl group optionally substituted with one or more sialyl and/or fucosyl residue(s),
  • R 6b is H or an N-acetyl-lactosaminyl group optionally substituted with one or two N-acetyl- lactosaminyl and/or one lacto-N-biosyl groups; each of the N-acetyl-lactosaminyl and lacto- N-biosyl groups can be substituted with one or more sialyl and/or fucosyl residues,
  • R 7 and R 8 are, independently, H or sialyl, provided that at least one of R 3 , R 4 , R 7 and R 8 is not H, and further provided that when R 7 is sialyl then at least one of R 3 , R 4 and R 8 is not H.
  • the compounds according to formulae 3a or 3b are characterized in that: the N-acetyl-lactosaminyl group in the glycosyl residue of R 5a is attached to another N-acetyl-lactosaminyl group with a 1-3 interglycosidic linkage, the lacto-N-biosyl group in the glycosyl residue of R 5a is attached to the N-acetyl- lactosaminyl group with a 1-3 interglycosidic linkage, the lacto-N-biosyl group in the glycosyl residue of R 6a is attached to the N-acetyl- lactosaminyl group with a 1-3 interglycosidic linkage, the N-acetyl-lactosaminyl group in the glycosyl residue of R 6b is attached to another N-acetyl-lactosaminyl group with a 1-3 or a 1-6 interglycos
  • a compound of formula 3a is allyl glycoside of lacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-neohexaose, para-lacto-N-octaose or lacto-N-neooctaose optionally substituted with one or more sialyl and/or fucosyl residue
  • a compound of formula 3b is allyl glycoside of lacto-N-tetraose, lacto-N-hexaose, lacto- N-octaose, iso-lacto-N-octaose, lacto-N-decaose or lacto-N-neodecaose optionally substituted with one or more sialyl and/or fucosyl residue.
  • the compounds of formula 3a or 3b are characterized in that: the fucosyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked to o the galactose of the lacto-N-biosyl group with 1-2 interglycosidic linkage and/or o the N-acetyl-glucosamine of the lacto-N-biosyl group with 1-4 interglycosidic linkage and/or o the N-acetyl-glucosamine of the N-acetyl-lactosaminyl group with 1-3
  • the sialyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked to o the galactose of the lacto-N-biosyl group with 2-3 interglycosidic linkage and/or o the N-acetyl-glucosamine of the lacto-N-biosyl group with 2-6 interglycosidic linkage and/or o the galactose of the N-acetyl-lactosaminyl group with 2-6 interglycosidic linkage.
  • a compound according to subformulae 3a, 3b or 3c may be selected from the group of: allyl glycoside of 2'-fucosyllactose, 3-fucosyllactose, 2',3- difucosyllactose, 6'-sialyllactose, 3'-sialyl-3-fucosyllactose, lacto-N-tetraose, lacto-N- neotetraose, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LST-a, LST-b, LST-C, FLST-a, FLST-b, FLST- c, LNDFH-I, LNDFH-II, LNDFH-III, DS-LNT, FDS-LNT I and FDS-LNT II, or salts thereof.
  • the glycosides may be alpha or beta-anomers
  • a compound of formula 3 is selected from LNT, LNnT and 2'-FL allyl glycoside.
  • a compound of formula 3 is a useful functionalized intermediate.
  • the double bond can be used in e.g. cycloaddition reaction to bind a compound of formula 3 to other species.
  • the chemical transformation of the double bond to aldehyde or amine also allows subsequent chemical or enzymatic conjugation to another species (solid support, protein, oligonucleotide, peptide).
  • Bacterial strains and inoculum preparation Bacterial strains and inoculum preparation :
  • Engineered E. coli used in Examples 1 to 7 was constructed from E. coli K strain in accordance with WO 01/04341 and Drouillard et al. Angew. Chem. Int. Ed. Eng. 45, 1778
  • Engineered E. coli used in Example 8 was constructed from E. coli K strain JM 109 in accordance with WO 01/04341, Dumon et al. Glycoconj. J. 18, 465 (2001) and Priem et al. Glycobiology 12, 235 (2002), by deleting genes that are liable to degrade the acceptor, the oligosaccharide product and its metabolic intermediates, inter alia the lacZ, lacA and wcaJ genes, maintaining genes involved in the UDP-GlcNAc and UDP-Gal biosynthesis, and inserting N. meningitidis IgtA gene for p- l,3-N-acetylglucosaminyl transfearse and N.
  • Example 9 meningitidis IgtB gene for ⁇ - ⁇ -4-galactosyl transferase.
  • Engineered E. coli used in Example 9 was constructed from E. coli K strain in accordance with WO 01/04341 and M .
  • Randriantsoa Synthese microbiologique des antigenes glucidiques des sexuals, These de Doctoratopathice le 30/03/2008 a ⁇ Universite Joseph Fourier, Grenoble, pp 64-66, by deleting genes that are liable to degrade the acceptor, the oligosaccharide product and its metabolic intermediates, inter alia the lacZ, lacA and wcaJ genes, maintaining genes involved in the UDP-GlcNAc and UDP-Gal biosynthesis, and inserting N. meningitidis IgtA gene for p- l,3-N-acetylglucosaminyl transfearse and H. pylori galTK gene for ⁇ - ⁇ -3-galactosyl transferase.
  • the culture was carried out in a 2 I fermenter (except if noted otherwise) containing 1.5 I of mineral culture medium (Samain et al. J. Biotechnol. 72, 33 ( 1999)) .
  • the temperature was kept at 33 °C and the pH regulated at 6.8 with 28% NH 4 OH .
  • the inoculum ( 1 % of the volume of the basal medium) consisted in a LB medium and the culture of the producing strain .
  • the exponential growth phase started with the inoculation and stopped until exhaustion of the carbon source (glucose 17.5 g/l) initially added to the medium.
  • the acceptor (various amount, given in the examples) and the inducer (isopropyl thio-p-D- galactopyranoside, IPTG, 1-2 ml of a 50 mg/ml solution) was added at the end of the exponential phase. Then a fed-batch was realized, using a 500 g/l aqueous glycerol solution, with a high substrate feeding rate of 4.5 g/h of glycerol for 1 I of culture for 5-6 hours followed by a lower glycerol feeding rate of 3 g/h for 1 I culture for a time indicated in the examples.
  • the culture was centrifuged for 25-40 min at 4500-6000 rpm at 20-25 °C.
  • the supernatant was kept and acidify to pH 3 using a H + form resin. This resulted in the precipitation of the proteins.
  • the resin was recovered by decantation and precipitated proteins removed by centrifugation for 25-40 min at 4500-6000 rpm at 20-25 °C.
  • the supernatant was passed through a H + form ion-exchange resin column and immediately neutralized by passing through a free base form anion exchange resin column.
  • the compounds were eluted with water or aqueous ethanol, the flow rate was about 20 ml/min and the final pH was 6.0.
  • the fractions containing the product were collected, concentrated and freeze-dried/crystallized/precipitated.
  • Example 1 - l-Q-Benzyl-p-2'-FL The fermentation was carried out using benzyl ⁇ -lactoside (Matsuoka et al. Carbohydr.
  • the fermentation was carried out using benzyl ⁇ -lactoside (62 g) that was added to the glycerol feeding solution and thus continuously added to the fermentation broth during the fermentation which lasted 66 hours altogether. After usual work-up 71 g of product could be isolated.
  • the fermentation was carried out using ⁇ -lactosyl azide (60 g) that was added to the glycerol feeding solution and thus continuously added to the fermentation broth during the fermentation which lasted 60 hours altogether. After usual work-up 51 g of product could be isolated .
  • the fermentation was carried out in a 1 I fermenter containing 0.75 I of mineral culture medium using phenyl l-thio-B-lactoside (Guilbert et al. Tetrahedron: Asymmetry 5, 2163
  • Example 6 - l-deoxy- l-thiomethyl-B-2'-FL
  • the fermentation was carried out using methyl l-thio-B-lactoside (Leontein et al. Carbohydr. Res. 144, 231 ( 1985), 4.8 g dissolved in about 25 ml of water) which was added at once at the end of the exponential phase.
  • the second feeding phase lasted 30 hours. After usual work-up 5.7 g of product could be isolated .
  • allyl ⁇ -lactoside prepared from allyl heptaacetyl- ⁇ - lactoside [Mereyala et al. Carbohydr. Res. 307, 351 ( 1998)] by Zemplen deacetylation, 62 g
  • Zemplen deacetylation, 62 g was added to the glycerol feeding solution and thus continuously added to the fermentation broth during the fermentation which lasted 60 hours altogether.
  • 54 g of product could be isolated.
  • LC-MS 527.1994 Da [M-H] ⁇ .
  • the fermentation was carried out using benzyl ⁇ -lactoside (20 g) that was added to the glycerol feeding solution and thus continuously added to the fermentation broth during the fermentation which lasted 36 hours altogether. After usual work-up 18 g of product could be isolated which was identical to the sample prepared according to WO 2011/100980.
  • the fermentation was carried out using benzyl ⁇ -lactoside (40 g) that was added to the glycerol feeding solution and thus continuously added to the fermentation broth during the fermentation which lasted 36 hours altogether. After usual work-up 24 g of product could be isolated.

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Abstract

L'invention concerne un procédé de production de dérivés oligosaccharides glycosidiques protégés de façon anomérique, comprenant l'étape de culture, dans un milieu de culture contenant un accepteur de lactose protégé de façon anomérique, une cellule génétiquement modifiée ayant un gène recombinant qui code pour une glycosyl transférase qui peut transférer un résidu glycosyl d'un nucléotide sucré activé audit accepteur de lactose. L'invention concerne en outre un procédé de production d'un oligosaccharide comprenant les étapes de : (a) culture, dans un milieu de culture contenant un accepteur de lactose protégé de façon anomérique, une cellule génétiquement modifiée ayant un gène recombinant qui code pour une glycosyl transférase qui peut transférer un résidu glucosyl d'un nucléotide sucré activé audit accepteur de lactose pour produire un dérivé olygosaccaharidique glycosidique protégé de façon anomérique, puis (b) élimination/déprotection du groupe protecteur anomérique.
PCT/DK2013/050182 2012-06-08 2013-06-07 Procédé de production d'oligosaccharides et d'oligosaccharide glycosides par fermentation WO2013182206A1 (fr)

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US14/406,379 US20150133647A1 (en) 2012-06-08 2013-06-07 Method for Producing Oligosaccharides and Oligosaccharide Glycosides by Fermentation

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WO2015032412A1 (fr) * 2013-09-06 2015-03-12 Glycom A/S Production d'oligosaccharides par fermentation
WO2015197082A1 (fr) 2014-06-27 2015-12-30 Glycom A/S Production d'oligosaccharides
WO2016008602A1 (fr) 2014-07-14 2016-01-21 Basf Se Production biotechnologique de lnt, lnnt et leurs dérivés fucosylés
JP2017509346A (ja) * 2014-03-31 2017-04-06 イェンネワイン バイオテクノロジー ゲーエムベーハーＪｅｎｎｅｗｅｉｎ Ｂｉｏｔｅｃｈｎｏｌｏｇｉｅ ＧｍｂＨ オリゴ糖の全発酵
WO2017101958A1 (fr) 2015-12-18 2017-06-22 Glycom A/S Production d'oligosaccharides par fermentation
WO2017152918A1 (fr) 2016-03-07 2017-09-14 Glycom A/S Séparation d'oligosaccharides dans un bouillon de fermentation
EP2896628B1 (fr) 2014-01-20 2018-09-19 Jennewein Biotechnologie GmbH Procédé permettant de purifier efficacement des oligosaccharides du lait humain neutre (HMO) à partir de la fermentation microbienne
EP3456836A1 (fr) 2017-09-13 2019-03-20 Glycom A/S Séparation d'oligosaccharides sialylés d'un bouillon de fermentation
DE202017007248U1 (de) 2016-04-19 2020-04-23 Glycom A/S Abtrennung von Oligosacchariden aus der Fermentationsbrühe
WO2021061991A1 (fr) 2019-09-24 2021-04-01 Prolacta Bioscience, Inc. Compositions et procédés de traitement de maladies inflammatoires et immunitaires
WO2021122687A1 (fr) 2019-12-19 2021-06-24 Basf Se Augmentation du rendement spatio-temporel, de l'efficacité de conversion du carbone et de la flexibilité des substrat carbonés dans la production de produits chimiques fins
WO2021123113A1 (fr) 2019-12-18 2021-06-24 Inbiose N.V. Production d'oligosaccharide sialylé dans des cellules hôtes
WO2022013143A1 (fr) 2020-07-13 2022-01-20 Glycom A/S Production d'oligosaccharide
WO2022036225A1 (fr) 2020-08-14 2022-02-17 Prolacta Bioscience, Inc. Compositions d'oligosaccharide de lait humain destinées à être utilisées avec des bactériothérapies
WO2022155201A1 (fr) 2021-01-12 2022-07-21 Prolacta Bioscience, Inc. Régimes de traitement symbiotique
WO2022243310A1 (fr) 2021-05-17 2022-11-24 Dsm Ip Assets B.V. Nouvelle technologie pour permettre l'utilisation de saccharose dans des souches pour la production biosyntétique
US11548908B2 (en) 2017-12-29 2023-01-10 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3
US11845771B2 (en) 2018-12-27 2023-12-19 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3
US11873317B2 (en) 2018-12-27 2024-01-16 Glycomimetics, Inc. Galectin-3 inhibiting c-glycosides
US11926858B2 (en) 2014-06-27 2024-03-12 Glycom A/S Oligosaccharide production

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US11597740B2 (en) 2014-01-20 2023-03-07 Chr. Hansen HMO GmbH Process for efficient purification of neutral human milk oligosaccharides (HMOs) from microbial fermentation
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JP2017509346A (ja) * 2014-03-31 2017-04-06 イェンネワイン バイオテクノロジー ゲーエムベーハーＪｅｎｎｅｗｅｉｎ Ｂｉｏｔｅｃｈｎｏｌｏｇｉｅ ＧｍｂＨ オリゴ糖の全発酵
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US11845771B2 (en) 2018-12-27 2023-12-19 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3
WO2021061991A1 (fr) 2019-09-24 2021-04-01 Prolacta Bioscience, Inc. Compositions et procédés de traitement de maladies inflammatoires et immunitaires
WO2021123113A1 (fr) 2019-12-18 2021-06-24 Inbiose N.V. Production d'oligosaccharide sialylé dans des cellules hôtes
WO2021122687A1 (fr) 2019-12-19 2021-06-24 Basf Se Augmentation du rendement spatio-temporel, de l'efficacité de conversion du carbone et de la flexibilité des substrat carbonés dans la production de produits chimiques fins
WO2022013143A1 (fr) 2020-07-13 2022-01-20 Glycom A/S Production d'oligosaccharide
WO2022036225A1 (fr) 2020-08-14 2022-02-17 Prolacta Bioscience, Inc. Compositions d'oligosaccharide de lait humain destinées à être utilisées avec des bactériothérapies
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