Patent Description:
The fermentative syntheses of foreign or exogenous oligosaccharides using recombinant microorganisms have recently become of great commercial and industrial interest. In such syntheses, oligosaccharides of interest would be synthesized by enzymatic glycosylation of sugar acceptors mediated by one or more heterologous glycosyl transferases of the microorganisms, and the one or more activated sugar nucleotides necessary for glycosylation would be produced by the same microorganism through overexpressing one or more genes encoding endogenous activated sugar nucleotide producing enzymes. The metabolic pathways of such syntheses require a carbon source which is mainly a simple carbon building block, typically glycerol or glucose (see e.g. <CIT>, <NPL>),<NPL>), <NPL>), <CIT>, <CIT>, <CIT>, <CIT>). In some syntheses, lactose can be the carbon source if it also serves as an acceptor (<NPL>)). As the microorganisms have been genetically manipulated, antibiotic-resistance selection marker genes have been utilized to separate the transformed microorganisms from the non-transformed ones in the inoculum and the fermentation broth. However, the use of antibiotics has been avoided by integrating the genes coding for enzymes involved in the de novo biosynthesis of the donor sugar in the chromosome of the microorganisms (<NPL>)).

Around <NUM> % of wild-type E. coli are able to utilize sucrose as a carbon and energy source, but most of them are pathogenic. coli strains used mainly in industry to synthesize chemical materials cannot live and grow on sucrose (<NPL>)). However, in some cases, sucrose can be a cheaper carbon and energy source. For this reason, attempts have been made to create suc+ strains of E. coli that can live and grow on sucrose (e.g. <NPL>)) and produce industrially profitable products by them such as amino acids, biofuel, carotenoids etc. (e.g. <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>,<NPL>)). However, these suc+ transformants have generally been less productive than suc-strains (<NPL>)).

<CIT> describes E. coli transformants that express either a sucrose phosphorylase or a sucrose invertase in combination with a fructokinase. Thereby, the microorganism is able to produce <NUM>'-fucosyllactose, utilizing sucrose as its main carbon source. Furthermore, <CIT> describes an E. coli transformant comprising a csc-gene cluster that enables it to grow on sucrose and produces fucose.

<CIT> relates to a method for the large scale in vivo synthesis of sialylated oligosaccharides, culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as lactose, wherein said microorganism comprises heterologous genes encoding a CMP-Neu5Ac synthetase, a sialic acid synthase, a GlcNAc-<NUM>-phosphate <NUM> epimerase and a sialyltransferase, and wherein the endogenous genes coding for sialic acid aldolase (NanA) and for ManNac kinase (NanK) have been deleted or inactivated. D1 is silent about the synthesis of any non-sialylated HMOs.

There has been, however, a continuing need for alternative processes for making recombinant oligosaccharides, particularly HMOs, using transformed microorganisms that are able to utilize more effectively sucrose as a carbon and energy source.

A first aspect of the invention relates to a process for making an oligosaccharide, or a glycoside of said oligosaccharide, by glycosylating a carbohydrate acceptor which is a monosaccharide or disaccharide and which is not sucrose, or a glycoside of said acceptor, comprising the steps of:.

said process being characterized in that said cell also comprises one or more genes encoding a heterologous PTS-dependent sucrose utilization transport system, so that said cell can use sucrose as a carbon source for making said activated sugar nucleotide and as an energy source for making said oligosaccharide,
wherein the glycosyl transferase is selected from the group consisting of β-<NUM>,<NUM>-N-acetylglucosaminyl transferase, β-<NUM>,<NUM>-N-acetylglucosaminyl transferase, β-<NUM>,<NUM>-galactosyl transferase, β-<NUM>,<NUM>-galactosyl transferase, α-<NUM>,<NUM>-fucosyl transferase, α-<NUM>,<NUM>-fucosyl transferase and α-<NUM>,<NUM>-fucosyl transferase.

A second aspect of the invention relates to a genetically modified microorganism for making an oligosaccharide, or a glycoside of said oligosaccharide, comprising:.

wherein the glycosyl transferase is selected from the group consisting of β-<NUM>,<NUM>-N-acetylglucosaminyl transferase, β-<NUM>,<NUM>-N-acetylglucosaminyl transferase, β-<NUM>,<NUM>-galactosyl transferase, β-<NUM>,<NUM>-galactosyl transferase, α-<NUM>,<NUM>-fucosyl transferase, α-<NUM>,<NUM>-fucosyl transferase and α-<NUM>,<NUM>-fucosyl transferase.

The Figure is intended to illustrate the invention further. It is not intended to limit the subject matter of the invention thereto.

The engineered microorganism is a fully metabolically active cell in which the growth and the oligosaccharide synthesis may proceed simultaneously. The cell comprises a heterologous PTS-dependent sucrose utilization transport system containing a sucrose specific porin (facilitates the sucrose diffusion through the outer membrane), a sucrose transport protein (provides intracellular sucrose-<NUM>-phosphate from extracellular sucrose) and a sucrose-<NUM>-phosphate hydrolase (provides glucose-<NUM>-phosphate and fructose). The oxidation of glucose-<NUM>-phosphate and fructose provides biological energy source by the organism's own metabolic system. Also, glucose-<NUM>-phosphate and fructose serve as carbon source for producing sugar nucleotides in the cell's natural biosynthetic pathway. The so-produced sugar nucleotides are donors for glycosylating carbohydrate acceptors (e.g. lactose), internalized through a specific permease by the cell, and thereby manufacturing oligosaccharides of interest. The glycosylation is mediated by one or more glycosyl transferases which are directly produced by expressing heterologous genes. The organism lacks any enzyme degrading either the acceptor or the oligosaccharide product in the cell.

It has been surprisingly discovered that an exogenous mono- or disaccharide acceptor, preferably lactose, can be internalized in a suitable genetically transformed microorganism, particularly E. coli, by a transport mechanism involving permeases of the microorganism, that this carbohydrate acceptor can be glycosylated in the microorganism using sucrose as its carbon and energy source, and that an exogenous oligosaccharide can be produced and separated in good yield. Thereby, an efficient, cheap and easily up-scalable process for producing oligosaccharides can be obtained. In order to make the process successful, a special oligosaccharide-producing microorganism is needed that can live on sucrose, utilize sucrose for the metabolic syntheses of the necessary nucleotide sugar donors for glycosylation, can internalize simple carbohydrate acceptors and perform glycosylation reactions on them for synthesizing more complex oligosaccharides.

The process features the cell being transformed with one or more foreign genes encoding a sucrose utilization system that allows the cell to use sucrose as a carbon source, preferably the main carbon source, more preferably the sole carbon source, for the biosynthesis of the activated sugar nucleotide by the cell. The sucrose utilization system, with which the cell is transformed, also preferably allows the cell to use sucrose as an energy source, preferably the main energy source, more preferably the sole energy source, for the biosynthesis of the oligosaccharide.

In accordance with this invention, the term "carbohydrate acceptor" or "acceptor" preferably means a mono- or disaccharide other than sucrose and its glycosides. A monosaccharide acceptor or a monosaccharide part of a disaccharide acceptor can comprise any <NUM>-<NUM> carbon atom sugar moiety that is an aldose (e.g. D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), ketose (e.g. D-fructose, D-sorbose, D-tagatose, etc.), deoxysugar (e.g. L-rhamnose, L-fucose, etc.) or deoxy-aminosugar (e.g. N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.). In a glycoside-type carbohydrate acceptor the sugar moiety is attached to a non-sugar residue (aglycon) by either a covalent bond, which is a direct linkage between the glycosidic carbon atom of the sugar residue and any atom of the non-sugar moiety, or by a linker, which consists of one, two, three or four atoms such as -O-, -C-, -NH-, -N(OH)-, -S-, -C(=O)-, -C(=S)-, -C(=NH)-, -C(=N-OH)-, -C(=O)-O-, -O-C(=O)-, -C(=O)-S-, -S-C(=O)-, -C(=S)-O-, -O-C(=S)-, -C(=S)-S-, -S-C(=S)-, -C(=O)-NH-, -NH-C(=O)-, -C(=NH)-O-, -O-C(=NH)-, - C(=S)-NH-, -NH-C(=S)-, -C(=NH)-S- and -S-C(=NH). Thus, the C-<NUM> (in case of aldoses) or C-<NUM> (in the case of ketoses) anomeric carbon atom at the reducing end of the mono- or disaccharide residue is linked to the non-sugar moiety by a covalent bond or a linker forming a O-, N-, S- or C-glycoside. Preferably, the aglycon of these glycosidic derivatives, with or without a linker, is one of the following groups:.

Preferably, the carbohydrate acceptor is a galactosyl disaccharide, particularly lactose.

Also in accordance with this invention, the term "oligosaccharide product" or "oligosaccharide" preferably means a glycosylated derivative of a carbohydrate acceptor disclosed above wherein a glycosyl residue is attached to the carbohydrate moiety of the carbohydrate acceptor by interglycosidic linkage. Preferably, an oligosaccharide product is of <NUM>-<NUM> monosaccharide units, particularly of <NUM>-<NUM> monosaccharide units. The oligosaccharide product of this invention is a recombinant product, i.e., it is made by a genetically transformed microorganism and is foreign or heterologous to the microorganism.

Further in accordance with this invention, the term "microorganism" or "cell" preferably means a cell of a microorganism, especially an E. coli cell, in which there is at least one alteration in its DNA sequence. The alteration can result in a change in the original characteristics of the wild type cell, e.g., the modified cell is able to perform additional chemical transformation due to the introduced new genetic material that encodes the expression of an enzymes not being in the wild type cell, or is not able to carry out transformation like degradation due to removal of gene/genes (knockout). A genetically modified cell can be produced in a conventional manner by genetic engineering techniques that are well-known to those skilled in the art.

The genetically modified microorganism or cell used in the process of this invention can be selected from the group consisting of bacteria and yeasts, preferably a bacterium. Bacteria are preferably selected from the group of: Escherichia coli, Bacillus spp. (e.g. 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, among which E. coli is preferred.

The process of this invention also involves transporting the exogenous carbohydrate acceptor, preferably lactose, into the genetically modified microorganism for glycosylation to produce a foreign oligosaccharide of interest, preferably without adversely affecting the basic functions of the cell or destroying its integrity. In one embodiment, the transport takes place via a passive mechanism, during which the exogenous acceptor diffuses passively across the plasma membrane of the cell. Diffusion of the acceptor into the microorganism is a function of the concentration differences between the fermentation broth and the extra- and intracellular space of the cell with respect to the acceptor, whereby the acceptor passes from the place of higher concentration to the place of lower concentration. In another embodiment, the acceptor is internalized with the aid of an active transport. In such a case, the genetically modified microorganism contains transporter proteins, called permeases, which act as enzymes and with which the microorganism is able to admit exogenous substances and to concentrate them in the cytoplasm. Specifically, lactose permease (LacY) acts specifically on galactose, simple galactosyl disaccharides such as lactose and their glycosides. The specificity towards the sugar moiety of the exogenous carbohydrate acceptor to be internalized can be altered by mutation of the microorganism by means of conventional recombinant DNA manipulation techniques. In a preferred embodiment, the internalization of exogenous lactose or its derivative takes place via an active transport mechanism mediated by a lactose permease. The genetically modified microorganism preferably lacks any enzyme activity (such as LacZ) that would degrade the acceptor. Likewise, the microorganism is not able to hydrolyze or degrade the oligosaccharide product.

Moreover, the genetically modified cell used in the process of the 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 the internalized acceptor. The gene or an equivalent DNA sequence thereof, if it is recombinant, can be introduced into the cell by conventional techniques, e.g. using an expression vector or by chromosomal intergration. 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 or from algae, prokaryotic cells such as those originated from E. coli, Bacteroides fragilis, Photo bacterium sp. , Bacillus subtilis, Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Rhizobium meliloti, Neisseria gonorrhoeae and Neisseria meningitidis, or virus. The glycosyl transferases are selected from β-<NUM>,<NUM>-N-acetylglucosaminyl transferase, β-<NUM>,<NUM>-N-acetylglucosaminyl transferase, β-<NUM>,<NUM>-galactosyl transferase, β-<NUM>,<NUM>-galactosyl transferase, α-<NUM>,<NUM>-fucosyl transferase, α-<NUM>,<NUM>-fucosyl transferase and α-<NUM>,<NUM> fucosyl transferase, that is from those involved in the construction of HMO core structures as well as fucosylated HMOs and its glycosidic derivatives, wherein the aglycon is a moiety defined above at the group of carbohydrate acceptors. The genes encoding the above-mentioned transferases have been described in the literature.

In the glycosyl transferase mediated glycosylation processes of this invention, activated sugar nucleotides serve as donors. Each activated sugar nucleotide generally comprises a phosphorylated glycosyl residue attached to a nucleoside, and the specific glycosyl transferase enzyme accepts only the specific sugar nucleotide. Thus, preferably the following activated sugar nucleotides are involved in the glycosyl transfer: UDP-Glc, UDP-Gal, UDP-GIcNAc, UDP-GaINAc, UDP-glucuronic acid, UDP-Xyl, GDP-Man, and GDP-Fuc, particularly those selected from the group consisting of UDP-Gal, UDP-GIcNAc, and GDP-Fuc.

The genetically modified microorganism used in the process of this invention possesses a biosynthetic pathway to the above-mentioned activated sugar nucleotides, that is, it has one or more sets of genes encoding one or more enzymes responsible for the synthesis of one or more activated glycosyl nucleotides, ready for glycosylation in glycosyl transferase mediated reaction in the cell, when cultured. The sets of genes are either naturally present in the cell or introduced into the cell by means of recombinant DNA manipulation techniques. The production of the activated glycosyl nucleotides by the cell takes place under the action of enzymes involved in the biosynthetic pathway of that respective sugar nucleotide stepwise reaction sequence starting from a carbon source (for a review for monosaccharide metabolism see e.g. <NPL>)).

It should be emphasized, that the production of the activated sugar nucleotides by the genetically modified microorganism via its own biosynthetic pathway is advantageous compared to in vitro versions of transfer glycosylation, as it avoids using the very expensive sugar nucleotide type donors added exogenously, hence the donors are formed by the cell in situ and the phosphatidyl nucleoside leaving groups are recycled in the cell.

In addition, the microorganism used in the process of the invention comprises genes encoding a sucrose utilization system, that is the cell has a capability to catabolically utilize sucrose as a carbon source, as well as an energy source. The system that enables the cell to utilize sucrose can be one normally found in the gene pool of that cell but preferably is a heterologous system (i.e. derived from a different organism and transferred to the host cell by conventional recombinant DNA manipulation techniques, preferably via an expression vector). Typically two kinds of sucrose catabolism can be used. According to the phosphoenolpyruvate (PEP)-dependent phosphotransferase system ("PTS"), sucrose is transported into the microorganism and concomitantly phosphorylated to generate intracellular sucrose-<NUM>-phosphate which is hydrolysed to glucose-<NUM>-phosphate and fructose that are then involved in the central carbon metabolism of the cell. PTS can be encoded by scr or sac genes. According to non-phosphotransferase-dependent system ("non-PTS"), extracellular sucrose enters the cell with the aid of a proton symport transport system (sucrose permease) and, after transport, is hydrolysed by an invertase enzyme to glucose and fructose followed by phosphorylation. In this regard, the csc regulon consists of genes encoding the enzymes that are responsible for the non-PTS sucrose utilization.

During fermentation in the process of this invention, the oligosaccharide-producing microorganism is fed with sucrose that provides energy via glycolysis for growing, reproducing and maintaining its structure. In addition, the sucrose taken up by the cell provides, via sucrose catabolism, precursors for the synthesis of the activated sugar nucleotide(s) necessary for the glycosylation process that takes place in the cell. The internalized carbohydrate acceptor participates in the glycosyl transferase induced glycosylation reaction, in which a glycosyl residue of an activated nucleotide donor produced by the cell is transferred so that the acceptor is glycosylated. Optionally, when more than one glycosyl transferase is expressed by the cell, additional glycosylation reactions can occur resulting in the formation of the target oligosaccharide. Of course, the cell preferably lacks any enzyme activity which would degrade the oligosaccharide derivatives produced in the cell.

The sucrose utilization system is heterologous. This is the case when the microorganism, preferably a bacterium, more preferably an E. coli, is a strain that is optimized for an industrially profitable transformation like oligosaccharide production, because such a strain generally has no ability to utilize sucrose. Therefore, a sucrose uptake cassette should be introduced, using an appropriate expression plasmid or via chromosome integration, in the sucrose minus cell to make it be sucrose plus. The sucrose pathway genes comprise a PTS-dependent sucrose utilization system. More preferably, the source regulon is scr. Microorganisms having scrgenes are for example Salmonella ssp. , Klebsellia pneumonia, Bacteroides fragilis, Vibrio alginolyticus.

The scr genes comprise the following: scrY, scrA, scrB and scrR. The gene scrA codes for the sucrose transport protein Enzyme IIScr that provides intracellular sucrose-<NUM>-phosphate from extracellular sucrose via an active transport through the cell membrane and the concomitant phosphorylation. The sucrose specific ScrY porin (encoded by scrY) facilitate the sucrose diffusion through the outer membrane. The ScrB invertase enzyme (encoded by scrB) splits the accumulated sucrose-<NUM>-phosphate by hydrolysis to glucose-<NUM>-phosphate and fructose. Optionally, a fructokinase ScrK (encoded by scrK) phosphorylates fructose to fructose-<NUM>-phosphate, however the presence of this enzyme is not crucial because the fructose can be phosphorylated by other mechanisms owned by the cell. The repressor protein ScrR (encoded by scrR) negatively controls the expression of the scrYAB genes and is induced by sucrose or fructose. The expression of the sucrose genes are repressed in the presence of glucose.

In a preferred embodiment, the heterologous scr genes are introduced into the microorganism using plasmids, more preferably by a two-plasmid system where one contains the scrA gene and the other does the scrB gene. The scrY, scrR and optionally the scrK gene can be carried by either plasmids.

Also preferably, antibiotics are not added to the fermentation broth in the process of this invention.

The carbohydrate acceptor to be glycosylated by the microorganism in the process of the invention can be a mono- or disaccharide selected from galactose, N-acetyl-glucosamine, a galactosylated monosaccharide, an N-acetyl-glucosaminylated monosaccharide, and glycosidic derivatives thereof defined above. All these carbohydrate derivatives can be easily taken up by a cell having a LacY permease by means of an active transport and accumulate in the cell before being glycosylated (<CIT>, <NPL>), <CIT>, <CIT>, <CIT>). This is because the cell is able to transport these carbohydrate acceptors into the cell using its LacY permease, and the cell lacks any enzymes that could degrade these acceptors, especially LacZ. Preferably the cell has a deleted or deficient lacA gene on the lac operon.

According to another preferred embodiment, the lacI gene for the lac repressor is also deleted in the microorganism. In the absence of the functioning repressor, no inducer is needed for expressing LacY.

According to another preferred embodiment, a genetically modified cell, particularly a LacZ-Y+ E. coli cell, is cultured in an aqueous culture medium in the following phases:.

During the feeding phase, the exogenous carbohydrate acceptor, preferably lactose, to be internalized by and glycosylated in the cell, can be added to the culture medium at once, sequentially or continuously. The acceptor can be added in this second phase as a pure solid/liquid or in a form of a concentrated aqueous solution or suspension. The oligosaccharide production takes place in this second phase and can take up to <NUM>-<NUM> days. Preferably, this feeding phase is performed under conditions allowing the production of a culture with a high cell density.

A feature of the process of this invention is that there is no need to change the carbon source and/or the energy source between the growth phase and the production phase of the microorganism.

Optionally, the process further comprises the addition of an inducer to the culture medium to induce the expression in the cell of enzyme(s) and/or of protein(s) involved in the transport of the acceptor and/or in the glycosylation of the internalized acceptor and/or in the biosynthesis of the activated sugar nucleotide donors. The inducer is preferably isopropyl β-D-thiogalactoside (IPTG) and is added to the culture medium in the beginning of the feeding phase. However, the use of inducer is not necessary if the cell is of LacI- phenotype.

It is believed that the microorganisms described above are highly stable under the process conditions of this invention described above. As a result, it is believed that these microorganisms can be used to produce oligosaccharides using sucrose as their carbon and energy sources at least at the same production rate as, and in a more reliable and reproducible manner than, like microorganisms using glycerol and/or glucose as their carbon and/or energy sources. In this regard, one or more plasmids, preferably one or two plasmids, containing the scrgenes (needed by the microorganisms for utilizing sucrose) plus one or more glycosyl transferase genes (needed by the microorganisms for making an exogenous oligosaccharide) are particularly stable in the above described microorganisms over fermentation periods of more than <NUM> days, preferably <NUM> to <NUM> days.

At the end of the second phase, the oligosaccharide product has accumulated both in the intraand the extracellular matrix of the microorganism. The product is then preferably transported out of the cell to the supernatant in a passive way, i.e., it can diffuse outside across the cell membrane. This transport can be facilitated by one or more sugar efflux transporters in the cell, i.e. proteins that promote the effluence of sugar derivatives from the cell to the supernatant. The sugar efflux transporter(s) can be present exogenously or endogenously and can be 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 of the cell by means of conventional recombinant DNA manipulation techniques. Preferably, the oligosaccharide accumulates in the extracellular matrix. Alternatively, the oligosaccharide can be transported out of the cell to the supernatant by disrupting the cell walls in a conventional manner.

The oligosaccharide product can then be separated in a conventional manner from the aqueous culture medium, in which it was made by the cell.

A first step of separating the oligosaccharide from the culture medium preferably involves separating the oligosaccharide from the microorganism which produced it. This preferably involves clarifying the culture medium to remove suspended particulates and contaminants, particularly cells, cell components, insoluble metabolites and debris produced by culturing the genetically modified microorganism. In this step, the aqueous culture medium, which contains the oligosaccharide product, can be clarified in a conventional manner. Preferably, the culture medium is clarified by centrifugation and/or filtration.

A second step of separating the oligosaccharide from the culture medium preferably involves removing substantially all the proteins, as well as peptides, amino acids, RNA and DNA and any endotoxins and glycolipids that could interfere with the subsequent separation step, from the aqueous culture medium, preferably after it has been clarified. In this step, proteins and related impurities can be removed from the culture medium in a conventional manner. Preferably, proteins and related impurities are removed from the culture medium by ultrafiltration, tangential flow highperformance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography. With the exception of size exclusion chromatography, proteins and related impurities are retained by a chromatography medium or a selected membrane, while the oligosaccharide product remains in the aqueous culture medium.

If desired, the oligosaccharide product in the aqueous culture medium can then be separated from sugar-like by-product(s) and from the culture medium, after proteins and related impurities have been removed from the culture medium. This can be suitably done by subjecting the culture medium to chromatographic separation. This separation can be carried out, in case of a neutral oligosaccharide product, in a chromatographic separation column, filled with a conventional acidic cationic ion exchange resin. The acidic cationic ion exchange resin can be in monovalent or divalent cationic form and is preferably in H+, K+, Na+, Mg<NUM>+ or Ca<NUM>+ form, particularly Ca<NUM>+. The chromatographic separation can be carried out in a conventional manner at a pH of the solution of <NUM> to <NUM>. The eluent used in the chromatographic separation is preferably water, especially demineralized water, but aqueous salt solutions can also be used. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used.

According to a preferred embodiment, the process of this invention for producing an oligosaccharide, preferably having a lactose unit at the reducing end or a glycoside thereof, comprises the steps of:.

characterized in that the cell also comprises a heterologous sucrose utilization system which is a PTS-dependent sucrose utilization system, especially where the source regulon is scr, to provide sucrose as a carbon source for biosynthesis of said activated sugar nucleotide by said cell,
wherein the glycosyl transferase is selected from the group consisting of β-<NUM>,<NUM>-N-acetylglucosaminyl transferase, β-<NUM>,<NUM>-N-acetylglucosaminyl transferase, β-<NUM>,<NUM>-galactosyl transferase, β-<NUM>,<NUM>-galactosyl transferase, α-<NUM>,<NUM>-fucosyl transferase, α-<NUM>,<NUM>-fucosyl transferase and α-<NUM>,<NUM>-fucosyl transferase.

The genetically modified cell, used in this preferred process, can have more than one recombinant gene, encoding more than one glycosyl transferase enzyme which is able to transfer the glycosyl residue of an activated sugar nucleotide to an internalized acceptor molecule or the previously glycosylated acceptor made by the same cell, so the oligosaccharide product is formed from the internalized acceptor by multiple glycosyl transfer mediated by multiple glycosyl transferases expressed by the cell. Accordingly, the resulting oligosaccharide product can be a glycosylated lactose or a glycoside thereof. The glycosylated lactose is preferably an N-acetyl-glucosaminylated, galactosylated, and/or fucosylated lactose. In order to produce these derivatives the cell comprises one or more recombinant genes encoding an N-acetyl-glucosaminyl transferase, a galactosyl transferase, and/or a fucosyl transferase, and also comprise a biosyntethic pathway to the corresponding activated sugar type nucleotides, that is UDP-GIcNAc, UDP-Gal, and/or GDP-Fuc.

More preferably, the oligosaccharide product made by this process is characterized by formula <NUM>
<CHM>
wherein Y is OH or a non-sugar aglycon defined above, preferably OH,.

Even more preferably, the compound of formula <NUM> made by this process can be characterized by formula 1a, 1b or 1c
<CHM>
<CHM>
wherein Y, R<NUM> and R<NUM> are as defined above, preferably OH,.

Still more preferably, the compounds according to formulae 1a or 1b made by this process are characterized in that:.

Yet more preferably, the compounds according to formulae 1a, 1b and 1c made by the process are human milk oligosaccharides (when Y is OH) or glycosides thereof (when Y is non-sugar aglycon).

The preferred compounds of formula 1a made by the process are selected from lacto-N-neotetraose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-neohexaose, para-lacto-N-octaose, lacto-N-neooctaose and glycosides thereof, all of which can optionally be substituted with one or more fucosyl residue. The preferred compounds of formula 1b made by the process is selected from lacto-N-tetraose, lacto-N-hexaose, lacto-N-octaose, iso-lacto-N-octaose, lacto-N-decaose, lacto-N-neodecaose and glycosides thereof, all of them can optionally be substituted with one or more fucosyl residue.

Particularly preferred compounds of formula 1a or 1b are characterized in that:.

According to the most preferred aspect, the compounds of subformulae 1a, 1b or 1c are selected from the group of: <NUM>'-fucosyllactose, <NUM>-fucosyllactose, <NUM>',<NUM>-difucosyllactose, 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, FDS-LNT II and their glycosides, or salts thereof. The glycosides can be alpha or beta-anomers, but preferably beta-anomers.

The preferred carbohydrate acceptor, exogenously added to the culture medium, is lactose, and the preferred oligosaccharide product is a human milk oligosaccharide (HMO). The HMOs consist of a lactose unit at the reducing end and one or more from the following monosaccharide units: N-acetyl-glucosamine, galactose, and fucose (see Urashima et al. : Milk Oligosaccharides, Nova Biomedical Books, New York, <NUM>, ISBN: <NUM>-<NUM>-<NUM>-<NUM>-<NUM>). In order to produce HMOs the cell then comprises one or more recombinant genes encoding β-<NUM>,<NUM>-N-acetyl-glucosaminyl transferase, β-<NUM>,<NUM>-N-acetyl-glucosaminyl transferase, β-<NUM>,<NUM>-galactosyl transferase, β-<NUM>,<NUM>-galactosyl transferase, α-<NUM>,<NUM>-fucosyl transferase, α-<NUM>,<NUM>-fucosyl transferase and/or α-<NUM>,<NUM> fucosyl transferase, and also comprise a biosyntethic pathway to the corresponding activated sugar type nucleotides, that is UDP-GIcNAc, UDP-Gal, and/or GDP-Fuc.

According to another preferred embodiment, the process of this invention for producing an N-acetyled HMO, preferably of <NUM>-<NUM> monosaccharide units, comprises the steps of:.

characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of UDP-GIcNAc and optionally UDP-Gal by the cell.

The heterologous PTS-dependent sucrose utilization system preferably comprises scr genes, more preferably scrY, scrA, scrB and scrR, and particularly does not contain scrK.

If the N-acetyl-glucosaminyl transferase is a β-<NUM>,<NUM>-N-acetyl-glucosaminyl transferase and no recombinant gene encoding a galactosyl transferase is present in the cell, the product is preferably lacto-N-triose, and if a β-<NUM>,<NUM>- or a β-<NUM>,<NUM>-galactosyl transferase is also present in the cell, the product is preferably LNT or LNnT, respectively.

According to yet another preferred embodiment, the process of this invention for producing a fucosylated HMO, preferably of <NUM>-<NUM> monosaccharide units, comprises the steps of:.

characterized in that the cell also comprises a heterologous PTS-dependent sucrose utilization system to provide sucrose as a carbon source, as well as an energy source, for biosynthesis of GDP-Fuc by the cell. Preferably, in this process, the culturing step comprises a two-step feeding, with a second feeding phase by continuously adding to the culture an amount of sucrose that is less than that added continuously in a first feeding phase so as to slow the cell growth and increase the content of product produced in the high cell density culture. The feeding rate of sucrose added continuously to the cell culture during the second feeding phase is around <NUM>-<NUM> % less than that of sucrose added continuously during the first feeding phase. During both feeding phases, lactose can be added continuously, preferably with sucrose in the same feeding solution, or sequentially. Optionally, the culturing further comprises a third feeding phase when considerable amount of unused acceptor remained after the second phase in the extracellular fraction. Then the addition is sucrose is continued without adding the acceptor, preferably with around the same feeding rate set for the second feeding phase until consumption of the acceptor.

The heterologous PTS-dependent sucrose utilization system preferably comprises scrgenes, more preferably scrY, scrA, scrB and scrR, and particularly does not contain scrK.

If the fucosyl transferase is an α-<NUM>,<NUM>-fucosyl transferase, the product is preferably <NUM>'-fucosyllactose, if the fucosyl transferase is an α-<NUM>,<NUM>-fucosyl transferase, the product is preferably <NUM>-fucosyllactose, and if both α-<NUM>,<NUM>- and α-<NUM>,<NUM>-fucosyl transferases are expressed in the cell, the product is preferably difucosyllactose.

The genetically modified microorganism of the second aspect can be selected from the group consisting of bacteria and yeasts, preferably a bacterium. Bacteria are preferably selected from the group of: Escherichia coli, Bacillus spp. (e.g. 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, among which E. coli is preferred.

Moreover, the genetically modified cell of the second aspect 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 the internalized acceptor. 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 or from algae, prokaryotic cells such as those originated from E. coli, Bacteroides fragilis, Photo bacterium sp. , Bacillus subtilis, Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Rhizobium meliloti, Neisseria gonorrhoeae and Neisseria meningitidis, or virus. The glycosyl transferase enzyme/enzymes expressed by the protein(s) encoded by the gene(s) or equivalent DNA sequence(s) are selected from β-<NUM>,<NUM>-N-acetylglucosaminyl transferase, β-<NUM>,<NUM>-N-acetylglucosaminyl transferase, β-<NUM>,<NUM>-galactosyl transferase, β-<NUM>,<NUM>-galactosyl transferase, α-<NUM>,<NUM>-fucosyl transferase, α-<NUM>,<NUM>-fucosyl transferase and α-<NUM>,<NUM> fucosyl transferase, that is from those involved in the construction of HMO core structures as well as fucosylated HMOs and its glycosidic derivatives, wherein the aglycon is a moiety defined above at the group of carbohydrate acceptors.

Furthermore, the genetically modified microorganism of the second aspect of the invention involves a transporting system that internalizes the exogenous carbohydrate acceptor, preferably lactose, into the microorganism for glycosylation and to produce a foreign oligosaccharide of interest, preferably without adversely affecting the basic functions of the microorganism or destroying its integrity. Preferably, the carbohydrate acceptor is internalized with the aid of an active transport, mediated by a transporter protein, called permease, which act as enzymes and with which the microorganism is able to admit exogenous substances and to concentrate them in the cytoplasm. Specifically, lactose permease (LacY) acts specifically on galactose, simple galactosyl disaccharides such as lactose and their glycosides. The genetically modified microorganism preferably lacks any enzyme activity (such as LacZ) that would degrade the acceptor. Likewise, the microorganism is not able to hydrolyze or degrade the oligosaccharide product.

In addition, the genetically modified microorganism of the second aspect comprises genes encoding the phosphoenolpyruvate (PEP)-dependent phosphotransferase (PTS) sucrose utilization system, that is the cell has a capability to catabolically utilize sucrose as a carbon source, as well as an energy source. The PTS-system is heterologous (i.e. derived from a different organism and transferred to the host cell by conventional recombinant DNA manipulation techniques, preferably via an expression vector) and can be encoded by scr or sac genes, preferably scr genes.

Preferably scr genes comprised by the genetically modified microorganism, preferably E. coli, are the following: scrY, scrA, scrB and scrR. The gene scrA codes for the sucrose transport protein Enzyme IIScr that provides intracellular sucrose-<NUM>-phosphate from extracellular sucrose via an active transport through the cell membrane and the concomitant phosphorylation. The sucrose specific ScrY porin (encoded by scrY) facilitate the sucrose diffusion through the outer membrane. The ScrB invertase enzyme (encoded by scrB) splits the accumulated sucrose-<NUM>-phosphate by hydrolysis to glucose-<NUM>-phosphate and fructose. The presence of a fructokinase ScrK (encoded by scrK) is not crucial because the fructose can be phosphorylated by other mechanisms owned by the cell. The repressor protein ScrR (encoded by scrR) negatively controls the expression of the scrYAB genes and is induced by sucrose or fuctose.

In a preferred embodiment, the heterologous scr genes are introduced into the microorganism using plasmids, more preferably by a two-plasmid system where one contains the scrA gene and the other does the scrB gene. The scrY and scrR can be carried by either plasmids.

Preferably the genetically modified microorganism of the second aspect has a deleted or deficient lacA gene on the lac operon.

Also preferably, the lacl gene for the lac repressor is also deleted in the genetically modified microorganism.

The genetically modified microorganism disclosed above is suitable for preparing, from lactose or lactosides having an aglycon disclosed above, an oligosaccharide of formula <NUM>
<CHM>
wherein Y is OH or a non-sugar aglycon defined above, preferably OH,.

Preferably, the oligosaccharide of formula <NUM> is a human milk oligosaccharide (when Y is OH) or a glycoside thereof (when Y is non-sugar aglycon), more preferably a human milk oligosaccharide.

According to a preferred embodiment, the genetically modified microorganism is an E. coli cell of LacZ-, LacY+ genotype or LacZ-, LacY+, LacI- genotype, and comprises:.

The heterologous scr cluster particularly does not contain scrK.

More preferably, the N-acetyl-glucosaminyl transferase is a β-<NUM>,<NUM>-N-acetyl-glucosaminyl transferase and no recombinant gene encoding a galactosyl transferase is present in the cell. In this case the genetically modified E. coli produces primarily lacto-N-triose.

Also more preferably, the N-acetyl-glucosaminyl transferase is a β-<NUM>,<NUM>-N-acetyl-glucosaminyl transferase and galactosyl transferase is a β-<NUM>,<NUM>-galactosyl transferase. In this case the genetically modified E. coli produces primarily LNT.

Yet more preferably, the N-acetyl-glucosaminyl transferase is a β-<NUM>,<NUM>-N-acetyl-glucosaminyl transferase and galactosyl transferase is a β-<NUM>,<NUM>-galactosyl transferase. In this case the genetically modified E. coli produces primarily LNnT.

According to another preferred embodiment, the genetically modified microorganism is an E. coli cell of LacZ-, LacY+ genotype or LacZ-, LacY+, LacI- genotype, and comprises:.

More preferably, the fucosyl transferase is an α-<NUM>,<NUM>-fucosyl transferase. In this case the genetically modified E. coli produces primarily <NUM>'-FL.

Also more preferably, the fucosyl transferase is an α-<NUM>,<NUM>-fucosyl transferase. In this case the genetically modified E. coli produces primarily <NUM>-FL.

Yet more preferably, there are two fucosyl transferases present in the cell: an α-<NUM>,<NUM>-fucosyl transferase and an α-<NUM>,<NUM>-fucosyl transferase. In this case the genetically modified E. coli produces primarily DFL.

Both strains were constructed from Escherichia coli K12 strain DH1 which was obtained from the Deutsche Sammlung von Mikroorganismen (reference DSM <NUM>) by deleting the genes: lacZ nanKETA lacA melA wcaJ mdoH, by inserting a Plac promoter, and maintaining genes involved in the UDP-GIcNAc and UDP-Gal biosynthesis. The glycerol utilizing strain (strain I) contains a pBBR3 -IgtA-tet plasmid carrying N. meningitidis IgtA gene for β-<NUM>,<NUM>-N-acetylglucosaminyl transfearse and the tetracycline resistant gene, and a pBS-galT-amp plasmid carrying Helicobacter pylori galT gene for β-<NUM>-<NUM>-galactosyl transferase and the ampicillin resistant gene. The sucrose utilizing strain (strain II) contains the two following plasmids:.

Glucose, glycerol, sucrose and lactose were each sterilized at <NUM>. Isopropyl thio-β-D-galactopyranoside (IPTG) was filter sterilized.

The culture was carried out in a <NUM> I fermenter containing ≈<NUM> I of mineral culture medium (<NPL>); does not contain antibiotics for the sucrose system). The temperature was kept at <NUM> and the pH regulated at <NUM> with <NUM>% NH<NUM>OH. The inoculum of the producing strain consisted in a LB medium (<NUM>) supplemented with ampicillin and tetracycline for strain I or M9 medium (<NUM>) supplemented with sucrose for strain II was added to the fermenter. The exponential growth phase started with the inoculation and stopped until exhaustion of the carbon source (glucose for strain I or sucrose for strain II, ≈<NUM>/l) initially added to the medium. A lactose solution (<NUM> of lactose/<NUM> of water) was then added before starting the feeding with the carbon source (<NUM>/l solution, <NUM>/h of glycerol for strain I and <NUM>/l of sucrose for strain II). The inducer (isopropyl thio-β-D-galactopyranoside, IPTG, <NUM>-<NUM> of a <NUM>/ml solution) was also added. The glycerol-fed fermentation (strain I) lasted for <NUM> hours after which the cells died (LNnT titre: <NUM>/l). The sucrose-fed fermentation (strain II) produced an LNnT concentration of <NUM>/l after <NUM> hours.

The strain was constructed from Escherichia coli K12 strain DH1 which was obtained from the Deutsche Sammlung von Mikroorganismen (reference DSM <NUM>) by deleting the genes: lacZ nanKETA lacA melA wcaJ mdoH and by inserting a Plac promoter to upstream the gmd gene. In addition the starin contains the two following plasmids:.

Sucrose and lactose were each sterilized at <NUM>. Isopropyl thio-β-D-galactopyranoside (IPTG) was filter sterilized.

Claim 1:
A process for making an oligosaccharide, or a glycoside of said oligosaccharide, by glycosylating a carbohydrate acceptor which is a monosaccharide or disaccharide and which is not sucrose, or a glycoside of said acceptor, comprising the steps of:
a) providing a cell that can internalize said acceptor into said cell and comprises
- a recombinant gene encoding a glycosyl transferase which is able to transfer a glycosyl residue of an activated sugar nucleotide to said acceptor, internalized in said cell, and
- a biosynthetic pathway to make said activated sugar nucleotide in said cell,
b) culturing said cell in the presence of said acceptor and sucrose, and
c) separating said oligosaccharide from said cell, from the culture medium or from both,
said process being characterized in that said cell also comprises one or more genes encoding a heterologous PTS-dependent sucrose utilization transport system, so that said cell can use sucrose as a carbon source for making said activated sugar nucleotide and as an energy source for making said oligosaccharide,
wherein the glycosyl transferase is selected from the group consisting of β-<NUM>,<NUM>-N-acetylglucosaminyl transferase, β-<NUM>,<NUM>-N-acetylglucosaminyl transferase, β-<NUM>,<NUM>-galactosyl transferase, β-<NUM>,<NUM>-galactosyl transferase, α-<NUM>,<NUM>-fucosyl transferase, α-<NUM>,<NUM>-fucosyl transferase and α-<NUM>,<NUM>-fucosyl transferase.