WO2018167259A1

WO2018167259A1 - Production of glycoconjugates and multivalent carbohydrate structures and uses thereof

Info

Publication number: WO2018167259A1
Application number: PCT/EP2018/056637
Authority: WO
Inventors: Eric Samain
Original assignee: Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2017-03-16
Filing date: 2018-03-16
Publication date: 2018-09-20

Abstract

The invention relates to a method of transglycosylation for producing glycoconjugates and multivalent carbohydrates structures, comprising the transfer via a 4-α-glucanotransferase (EC 2.4.1.25) of at least one terminal non-reducing end of a modified maltooligosaccharide as donor substrate on at least one glycosidic acceptor according to the following reaction R-Glcα1-4[Glcα1-4]n-Glc + glycosidic acceptor --> R-Glcα1-4[Glcα1-4]x-acceptor +[Glcα1-4](n-x)-Glc wherein - R-Glcα1-4[Glcα1-4]n-Glc is said modified maltooligosaccharide, - n is comprised between 1 and 5, - x is comprised between 1 and 5, - x is inferior or equal to n, - R is a substituent attached on the terminal non-reducing glucosyl group of a modified maltooligosaccharide, thereby constituting terminal non-reducing end of a modified maltooligosaccharide, and - glycosidic acceptor is selected from the group consisting of a monosaccharide, an oligosaccharide, a polysaccharide, a monovalent glycoside and a multivalent glycoside. and their uses thereof.

Description

Production of glycoconjugates and multivalent carbohydrate structures

and uses thereof

FIELD OF THE INVENTION

The present invention concerns new method of transglycosylation for the production of glycoconjugates and multivalent carbohydrates structures. BACKGROUND OF THE INVENTION

Cell surface carbohydrates, which are found in glycolipids and glycoproteins, play a major role in many biological and pathophysiological processes. Due to their high and universal biological importance, these complex carbohydrates have numerous potential therapeutic applications in fields as diverse as neurodegeneration, anti-cancer vaccines, anti-infectious, glycotargeting, diagnosis, cosmetology and functional food.

The synthesis of complex carbohydrates has thus become a challenge for the chemistry community. Following the pioneering work of Lemieux on the synthesis of human blood group determinants in the 1970s, the chemical synthesis of the major carbohydrate ligands was successfully achieved during the 1980s. However these syntheses required multiple protection/deprotection steps and the resulting compounds were generally only obtained in low yields on the mg scale.

In nature, the formation of glycosyl bonds is catalyzed in a very specific manner by the glycosyltransferases of the Leloir pathway, which use activated sugar nucleotides as substrate donors. The majority of genes encoding individual glycosyltransferases were cloned and characterized during the 1990s, thus enabling their production as recombinant proteins for enzymatic synthesis purposes. The major drawbacks of this approach were the cost of the sugar nucleotides and the inhibition by nucleoside diphosphate generated in the reaction. Over the past 20 years, new strategies have emerged to solve these problems by carrying out the glycosylation reactions in whole bacterial cells, which can provide the biological energy to power oligosaccharide synthesis and can be genetically engineered to produce the necessary enzymes (Samain 1999). The high production yield of these systems and the low cost of the substrate they use offer the possibility of producing complex oligosaccharides on the multi-kilogram or even multi-ton scale at a reasonable price. However, in spite of their availability, complex free oligosaccharides have surprisingly found very little practical applications in the various promising fields of research in which protein-carbohydrate interactions play a central role. One of the reasons may lie in the fact that individual interactions between carbohydrate and proteins are actually very weak and that free oligosaccharides are therefore quite inefficient ligands for protein receptors. Nature has overcome this by presenting multiple copies of both carbohydrate ligands and protein (lectin) receptors. The overall interaction observed is significantly enhanced with respect to the sum of the individual interactions and this phenomenon is often referred to as the "cluster glycoside effect or "multivalent effect" (Linquist and Toone 2002). Natural cell surface carbohydrates are covalently linked to proteins or lipids to constitute scaffolds of high affinity multivalent ligands. Designing and synthesizing efficient multivalent carbohydrate architecture is therefore crucial to develop efficient glycotherapies and technologies based on carbohydrate protein interactions.

Plethora of synthetic multivalent carbohydrate architecture have been described: dendrimer, polymer, nanoparticle, glycoliposome, self-assembled monolayers etc. However, in most cases these architectures were constructed as model systems with simple monosaccharidic structure and the chemical synthesis of similar architecture with complex carbohydrate represents an immense challenging task. It is thus the main object of the invention to provide a new biotechnological method of producing glycoconjugates and multivalent carbohydrate structures.

Indeed, the Applicant developed a method of transglycosylation allowing the enzymatic transfer of substituted galactosylated maltooligosaccharides ligands (MD-ligands) on a glycosyl residue linked to various molecular structures. The process is based on:

- the synthesis of modified maltooligosaccharides in particular substituted galactosylated maltooligosaccharides ligands (MD-ligands), in particular via microbiological process, the said modified maltooligosaccharides being produced by modification of the terminal non- reducing end of maltodextrin (maltooligosaccharide) with various carbohydrate affinity ligands;

- the use of a 4-a-glucanotransferase (EC 2.4.1.25) to transfer the carbohydrate affinity motif attached to a 1 ,4- a -D-glucan segment of the modified maltooligosaccharide as donor substrate to a glycosyl residue of an acceptor. SUMMARY OF THE INVENTION

A first object of the invention is a method of transglycosylation for producing glycoconjugates and multivalent carbohydrates structures, comprising the transfer via a 4-a- glucanotransferase (EC 2.4.1.25) of at least one terminal non-reducing end of a modified maltooligosaccharide as donor substrate on at least one glycosidic acceptor according to the following reaction

R-Glca1 -4[Glca1 -4]_n-Glc + glycosidic acceptor -> R-Glca1 -4[Glca1-4]_x-acceptor +[Glca1-4]_(n-_X)-Glc wherein

R-Glca1-4[Glca1 -4]_n-Glc is said modified maltooligosaccharide

- n is comprised between 1 and 5,

- x is comprised between 1 and 5,

- x is inferior or equal to n

R is a substituent attached on the terminal non-reducing glucosyl group of a modified maltooligosaccharide, thereby constituting terminal non-reducing end of modified maltooligosaccharide, and

- glycosidic acceptor is selected from the group consisting of a monosaccharide, an oligosaccharide, a polysaccharide, a monovalent glycoside and a multivalent glycoside. In a particular embodiment, the donor substrate of transglycosylation is a maltooligosaccharide modified at its non-reducing end, in particular a substituted galactosylated maltooligosaccharide (MD-ligands), susceptible to be obtained by a method of production comprising the step of culturing a microorganism in a culture medium comprising an exogenous precursor selected from maltooligosaccharides with a degree of polymerization (DP) ranging from 2 to 7, preferably selected from the group consisting of maltotriose (M3) and maltotetraose (M4), wherein said microorganism:

internalizes said maltooligosaccharide by an active transport,

comprises at least one heterologous gene encoding an enzyme modifying said internalized maltooligosaccharide, and

- is devoid of enzymatic activities degrading maltooligosaccharides and modified maltooligosaccharides. In a particular embodiment, the donor substrate used in the transglycosylation method is a substituted g rmula (I) :

wherein

. n is comprised between 0 and 5,

. R1 , R2, R3, R4, and R5 represent independently a hydrogen atom, a monosaccharide or an oligosaccharide, and

. at least one of R1 , R2, R3, R4, and R5 is a monosaccharide or an oligosaccharide, preferably a monosaccharide selected from the group consisting of fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine,

N-acetylneuraminic acid, and derivatives thereof.

Another object of the invention is a method for producing maltooligosaccharides modified at their non-reducing end in particular substituted galactosylated maltooligosaccharide (MD- ligands) of formula (I) as further defined, susceptible to be used as donor substrate in the method of transglycosylation according to the invention, comprising the step of culturing a microorganism in a culture medium comprising an exogenous precursor selected from maltooligosaccharides with a degree of polymerization (DP) ranging from 2 to 7, preferably selected from the group consisting of maltotriose (M3) and maltotetraose (M4), wherein said microorganism :

internalizes the said maltooligosaccharide by an active transport

comprises at least one heterologous genes encoding an enzyme modifying said internalized maltooligosaccharide, and

is devoid enzymatic activities degrading maltooligosaccharides and modified maltooligosaccharides.

Another object of the invention is substituted galactosylated maltooligosaccharides (MD- ligands), susceptible to be used as donor substrate in the method of transglycosylation according to the invention, of the following formula (I):

wherein

. n is comprised between 0 and 5,

. at least one of R1 , R2, R3, R4, and R5 is a monosaccharide or an oligosaccharide, preferably a monosaccharide selected from the group consisting of fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and derivatives thereof.

The invention also concerns glycoconjugates and multivalent carbohydrate structures as produced according to the invention for uses for at least one application selected from the group consisting of therapeutical applications, nutritional supplements, bioactive detergent, diagnostic, medical devices, cosmetic, affinity purification process, and combinations thereof.

DEFINITIONS

The term 'glycoconjugates' is the general classification for carbohydrates covalently linked with other chemical species such as proteins, peptides, lipids and saccharides. They are very important compounds in biology and consist of many different categories such as glycoproteins, glycopeptides, peptidoglycans, glycolipids, glycosides and lipopolysaccharides. They are involved in cell-cell interactions, cell-cell recognition, and detoxification process. The term 'multivalent carbohydrate structure' encompasses a structure with multiple binding groups (e.g carbohydrates) interacting with multiple chemical species (e.g, a protein).

The terms 'modified maltooligosaccharide' and 'modified maltodextrin' will be used interchangeably in the description.

The 'microorganisms' referred herein to practice the invention are recombinant cells. Recombinant cells are generally made by creating or otherwise obtaining a polynucleotide that encodes the particular enzyme(s) of interest, placing the polynucleotide in an expression cassette under the control of a promoter and other appropriate control signals, and introducing the expression cassette into a cell. More than one of the enzymes can be expressed in the same host cells using a variety of methods. For example, a single extrachromosomal vector can include multiple expression cassettes or more than one compatible extrachromosomal vector can be used maintain an expression cassette in a host cell. Expression cassettes can also be inserted into a host cell chromosome, using methods known to those of skill in the art. Those of skill will recognize that combinations of expression cassettes in extrachromosomal vectors and expression cassettes inserted into a host cell chromosome can also be used. Other modification of the host cell, described in detail below, can be performed to enhance production of the desired MD-ligand.

The recombinant cells of the invention are generally microorganisms, such as, for example, yeast cells, bacterial cells, or fungal cells. In a preferred embodiment, the recombinant microorganism is a bacterium, preferably E. coli.

A 'heterologous gene', as used herein, is one that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form. Thus, a heterologous gene encoding galactosyltransferase or glycosyltransferase in a cell includes a gene that is endogenous to the particular host cell but has been modified. Modification of the heterologous sequence may occur, e.g., by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of being operably linked to a promoter. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous sequence.

An 'expression cassette' or 'recombinant expression cassette' is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of affecting expression of a structural gene in hosts compatible with such sequences. Expression cassettes include at least promoters and optionally, transcription termination signals. Typically, the recombinant expression cassette includes a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors necessary or helpful in effecting expression may also be used. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in an expression cassette. When more than one heterologous protein is expressed in a microorganism, the genes encoding the proteins can be expressed on a single expression cassette or on multiple expression cassettes that are compatible and can be maintained in the same cell. As used herein, expression cassette also encompasses nucleic acid constructs that are inserted into the chromosome of the host microorganism. Those of skill are aware that insertion of a nucleic acid into a chromosome can occur, e.g., by homologous recombination. An expression cassette can be constructed for production of more than one protein. The proteins can be regulated by a single promoter sequence, as for example, an operon. Or multiple proteins can be encoded by nucleic acids with individual promoters and ribosome binding sites. Non limitative examples of genes and plasmids used herein are depicted in Table 2 further disclosed.

The construction of polynucleotide constructs generally requires the use of vectors able to replicate in bacteria. A plethora of kits are commercially available for the purification of plasmids from bacteria. For their proper use, follow the manufacturer's instructions (see, for example, EasyPrepJ, FlexiPrepJ, both from Pharmacia Biotech; StrataCleanJ, from Stratagene; and, QIAexpress Expression System, Qiagen). The isolated and purified plasmids can then be further manipulated to produce other plasmids, and used to transfect cells.

Construction of suitable vectors containing one or more of the above listed components employs standard ligation techniques as described in the references cited above. Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligated in the form desired to generate the plasmids required. To confirm correct sequences in plasmids constructed, the plasmids can be analyzed by standard techniques such as by restriction endonuclease digestion, and/or sequencing according to known methods. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids are well-known to persons of skill.

A 'culture medium refers to any liquid, semi-solid or solid media that can be used to support the growth of a microorganism used in the methods of the invention. In some embodiments, the microorganism is a bacterium, e.g., E. coli. Media for growing microorganisms are well known, see, e.g., Sambrook et al. and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1998 Supplement) (Ausubel). The growth medium according to the invention comprises an exogenous precursor selected from maltooligosaccharides with a degree of polymerization (DP) ranging from 2 to 7, in particular maltotriose (M3) or maltotetraose (M4). The culturing step is performed under conditions allowing the production of a culture with a high cell density; this culturing step comprises a first phase of exponential cell growth ensured by said carbon-based substrate, a second phase of cell growth limited by said carbon-based substrate which is added continuously, and finally a third phase of slowed cell growth obtained by continuously adding to the culture an amount of said substrate that is less than the amount of substrate added in step b) so as to increase the content of modified maltooligosaccharides produced in the high cell density culture. The method according to the invention is characterized in that the amount of substrate added continuously to the cell culture during said phase c) is at least 30% less, preferentially 50% and preferably 60% less than the amount of substrate added continuously during said phase b). The method according to the invention is also characterized in that said exogenous precursor is added during phase b).

The general culture conditions (time, temperature...) are conventional and well known from the man skilled in the art. The term 'commercial scale' refers to gram scale production of MD-ligand or complex carbohydrate structure in a single reaction. In a particular embodiment, commercial scale refers to production of some micrograms to several kilograms of MD-ligands or complex carbohydrate structure in a single reaction. In preferred embodiments, commercial scale refers to production of greater than about 50, 75, 80, 90 or 100, 125, 150, 175, or 200 grams.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Production of glycoconiugates and multivalent carbohydrate structures

A first object of the invention is a method of transglycosylation for producing glycoconjugates and multivalent carbohydrates structures comprising the transfer via a 4-a-glucanotransferase (EC 2.4.1.25) of the terminal non-reducing end of a modified maltodextrin (maltooligosaccharide) on a glycosidic acceptor.

In particular, the method of transglycosylation for producing glycoconjugates and multivalent carbohydrates structures comprises the transfer via a 4-a-glucanotransferase (EC 2.4.1.25) of at least one terminal non-reducing end of a modified maltooligosaccharide as donor substrate on at least one glycosidic acceptor according to the following reaction

R-Glca1-4[Glca1 -4]_n-Glc is said modified maltooligosaccharide,

n is comprised between 1 and 5,

- x is comprised between 1 and 5,

- x is inferior or equal to n

- glycosidic acceptor is selected from the group consisting of a monosaccharide, an oligosaccharide, a polysaccharide, a monovalent glycoside and a multivalent glycoside.

As 'donor substrate' according to the invention, mention may be made of a modified maltooligosaccharide at its non-reducing end, in particular a substituted galactosylated maltooligosaccharides ligans (MD-ligands) as further disclosed in the description.

These modified maltooligosaccharides, also named modified maltodextrins according to the invention, may be produced by chemical synthesis process, or advantageously by microbiological synthesis process. As 'glycosidic acceptor' according to the invention, mention may be made in particular of:

- a monosaccharide such as mannose, glucosamine, xylose and A/-acetylglucosamine,

- an oligosaccharide such as isomaltose or highly branched maltodextrin and pyrodextrin,

- a polysaccharide such as glycogen amylopectin or enzymatically produced hyperbranched glucan of various molecular weight and degree of branching;

- a monovalent glycoside which could be linked to an aglycone group by an O-glycosidic bound, a N-glycosidic bound (glycosylamine, glycosylamide, azidoglycoside), a S- glycosidic bound (thioglycoside), a C-glycosidic bound . The aglycone group can be coupled to a biological, a colorimetric or fluorescent maker or linked to functionalizable chemical group (propargyl, allyl azide, amino...). The aglycone group can also be a hydrophobic compound resulting in the synthesis of amphiphilic glycoconjugate which can self-assemble in glycoliposomes or glycovesicules displaying a multivalent presentation of carbohydrate ligands; multivalent glycosides: several glycosidic acceptors can also be linked to the same molecular or macromolecular structure to create multivalent acceptors on which multiple terminal non-reducing end of modified maltooligosaccharides can be transferred, producing multivalent carbohydrate structures.

In a particular embodiment, the monosaccharide can be free or linked to an oligosaccharide, a polysaccharide, or to any aglycone group by a O-glycosidic , N-glycosidic, S-glycosidic or a C- linkage. In a particular embodiment, the glycosidic acceptor is a multivalent glycoside comprising several glycosidic acceptors, on which several terminal non-reducing ends of modified maltooligosaccharides can be transferred.

In a preferred embodiment, the multivalent glycoside is a branched glucan.

A 'branched glucan' according to the invention is a multibranched polysaccharide consisting of linear chains of glucose residues with further chains branching off every 8 to 12 glucoses such as in glycogen, by a1 ,6 glycosidic bonds.

In another preferred embodiment, at least one glycosidic acceptor is linked to at least one lipid chain.

A 'lipid chain' is in general a fatty acid chain constituted by a hydrocarbon chain that terminates with a carboxylic acid group. The carbon chain may be typically between 4 to 24 carbons long, may be saturated or unsaturated and may be attached to functional groups containing oxygen, halogen, nitrogen or sulfur group.

The 4-a-glucanotransferase normally catalyzes glucan transfer from one a-1 ,4-glucan to another a-1 ,4-glucan or to glucose. The smallest substrate which amylomaltase recognizes is maltotriose (Palmer et al 1976). Acting on maltotriose, it releases glucose from the reducing end, forms a maltosyl-enzyme complex, and transfers the maltosyl residue onto the non-reducing end of an acceptor, be it glucose, maltose and larger maltodextrin or any glycosyl group which would be recognized as an acceptor. When using longer maltodextrin as donor substrate, amylomaltase, is able to release not only glucose but also longer dextrins from the reducing end of its maltodextrin substrates. Maltodextrin can therefore serve as both donor and acceptor substrate for 4-a- glucanotransferase. It is an object of the invention to use a maltodextrin which is modified at its non-reducing end in such way that it cannot be used as an acceptor substrate by 4-a- glucanotransferase but which still contains at its reducing end a maltooligosaccharide motif which is large enough to be recognized by the 4-a-glucanotransferase as a donor substrate in order to allow the terminal non reducing part of the modified maltodextrin to be transferred on a glycosidic acceptor according to the following reaction.

R-Glca1-4[Glca1 -4]_n-Glc + Acceptor -> R-Glca1-4[Glca1-4]_x-Acceptor +[Glca1 -4](_n-x)-Glc wherein

R-Glca1-4[Glca1 -4]_n-Glc is said modified maltooligosaccharide

n is comprised between 1 and 5,

- x is comprised between 1 and 5,

- x is inferior or equal to n

R is a substituant attached on the terminal non reducing glucosyl group of maltooligosaccharide, thereby constituting terminal non-reducing end of modified maltooligosaccharide and

In a preferred embodiment, the 4-a-glucanotransferase used in the method of transglycosylation according to the invention is the amylomaltase MalQ from Escherichia coli. Interestingly the amylomaltase from E. coli strain has a broad acceptor specificity and is able to use as acceptor the following compounds: methyl-a-glucoside, methyl-p-glucoside, phenyl-a-glucoside, methyl-p-glucoside, isomaltose, mannose, glucosamine and N- acetylglucosamine (Kitahata et 1989).

Production of a 4-a-glucanotransferase

4-a-glucanotransferase; EC 2.4.1.25) was first found in Escherichia coli and called amylomaltase. This enzyme has since been found in many bacterial species including thermophilic organisms. A similar enzyme is also present in plants and is called disproportionating enzyme (D-enzyme) (EC 2.4.1.25). Analyses of the activity of the E. coli enzyme (Palmer et al 1976) and of the potato enzyme (Jones and Whelan 1969) indicated that amylomaltase and D-enzyme catalyze similar reactions.

The E. coli amylomaltase is encoded by the malQ gene which can overexpressed in E.coli as described in example 2

Use of monovalent glycoside acceptor

As shown in the example 10, the amylomaltase MalQ uses M3-Gal as donor substrate in presence of various alkyl-p-glucoside but does not modify M3-Gal in the control reaction run without any acceptor. This indicates that the terminal Galactose residue of M3-Gal is not recognized as an acceptor by MalQ. The MD ligand functions exclusively as a donor substrate for the amylomaltase, which can catalyse an exclusive transfer of the carbohydrate ligand to a glucosyl acceptor without forming any autocondensation product. As illustrated in Figure 2, the reaction of a M3 ligand with an alkyl-glucoside in presence of MalQ result in the formation of the expected allyl-M3-ligand with the concomitant liberation of glucose. In order to displace the equilibrium toward the maximum formation of desired transglycosylation product, the reaction can be conducted in presence of yeast that will efficiently consume glucose as soon as it is formed. Alternatively glucose removal could be achieved by an enzymatic system based on the glucose oxidase as described by Mislovicova et al (2009). Cristal structure has shown that amylomaltase has several binding subsites located on both sides of the catalytic site (WeiB et al 2015). The fact that M3-Gal, M3-LNT and M3-Gb3 are recognized as donor substrate indicate that the subsite -3 of MalQ has a relatively large specificity that allows it to accommodate different glycosyl residues attached with a a1 -3 β1 -3 or β1-4 linkage to the glucose bound into the -2 site. On the contrary the presence of an a1-2 linked fucosyl residue seems to completely prevent the binding of M3-H and M3-A and the transfer of ligands containing a1-2 linked fucose requires a longer a-glucan chain to fit into the active site as shown by the formation of octyl-M4-H from MD-H in example 12.

Use of multivalent glucosyl acceptor

Neopentyl Glycol class detergents are commercial glycolipids which have been developed for membrane protein studies. Theses amphiphilic molecules consist of a central quaternary carbon with two lipophilic tails and two hydrophilic heads which can be glucosyl or maltosyl group. In example 10, two of these detergents, octyl glucose neopentyl glycol (OGNG) and decyl maltose neopentyl glycol (DMNG) have been tested as MalQ acceptor in presence of M3-Gal as a donor substrate. Only DMNG, the one having two maltose group was successfully used as acceptor leading to an amphiphilic molecule bearing two galactose terminal groups as shown in figure 3. This result demonstrate the possibility of creating multivalent carbohydrate structures by enzymatic transfer of MD-ligands

Self-assembling of amphiphilic carbohydrate ligand

Amphiphilic carbohydrate ligands can undergo self-assembly in aqueous media to form glycoliposomes or glycovesicles valuable structures to study carbohydrate-protein interactions (Jayaraman et al 2013). They also can form self-assembled monolayer on hydrophobic solid supports to create model system of carbohydrate affinity ligands (Imura et al 2007). In example 13 we showed that octyl-M4-A can organize as a self-assembled monolayer at the surface of octadecyl-bonded silica particles to form an affinity chromatography support (Torres et al 1987) for blood group anti-A antibodies.

Transfer of carbohydrate ligands on glycogen and hyperbranched glucans

Glycogen is a branched biopolymer consisting of 6,000-20,000 glucose units. It is made of linear chains of a1 ,4 glucose residues with further chains branching off every 8 to 12 glucoses by a1 ,6 glycosidic bonds. Example 15 demonstrates that MD-ligand can be transferred on the terminal glucose residue of glycogen to create a macromolecular structure displaying multivalent carbohydrate ligands. The molecular weight of glycogen is 10⁵-10⁷ but it is possible to enzymatically produce taylored hyperbranched glucan of various molecular weight and degree of branching (Grimaud et al 2013). Other interesting hyperbranched glucan acceptors include resistant dextrins produced by heat which are also known as pyrodextrins. During roasting under acid condition the starch hydrolyses and short chained starch parts partially rebranch with nondigestible linkages, e.g., linear and/or branched a-1 ,2 and/or β-1 ,2, β-1 ,4, α-1 ,3 and/or β-1 ,3 linkages and β-1 ,6 linkages (Bai and Shi 2016). Modified maltooligosaccharides (MD-ligands) as donor substrate

The invention also concerns modified maltooligosaccharides and in particular substituted galactosylated maltooligosaccharides ligands (MD-ligands), susceptible to be used as donor substrate in the transglycosylation method according to the invention, in particular of the following formula (I) :

wherein

. n is comprised between 0 and 5,

■ Ri , F½, R3, R₄, and R₅ represent independently a hydrogen atom, a monosaccharide or an oligosaccharide, and

. at least one of R-i , R₂, R₃, R₄, and R₅ is a monosaccharide or an

oligosaccharide. The general term 'MD-ligand' refers to an oligosaccharide structure derived from a maltooligosaccharide with a degree of polymerization (DP) which can be between 2 and 7. The term 'degree of polymerization' (DP) is generally defined as the number of monomeric units in a macromolecule, for example three glucose units for maltotriose (M3).

These maltooligosaccharides are modified at their non-reducing end with a Galactose residue which is attached with β1 -4 linkage to form a terminal lactosyl motif. This lactosyl motif can be further modified by addition of different glycosyl residues to form various carbohydrate ligand structures such as the examples listed hereunder in table 1 . When speaking of a particular ligand such as Galactose (Gal) or Blood group A antigen (A), the general term MD- ligand can be advantageously replaced by the two letters MD- followed by the ligand structure abbreviation. For example MD-Gal corresponds to the structure Gal3-4[Glco4]_nGlc.

The terms M2-ligands, M3-ligands and M4 ligands refer to oligosaccharide structures derived from maltose, maltotriose and maltotetraose respectively. Like MD-ligands, these structures are modified at their non-reducing end by the addition of various carbohydrate affinity ligand. For example M3-Gal corresponds to the structure Gal3-4Glca-4Glca-4Glc.

M3-3S corresponds to the structure Neu5Aca-3Gaip-4Glca-4Glca-4Glc and M3-6S corresponds to the structure Neu5Aca-6Gaip-4Glca-4Glca-4Glc. Table 1 : Examples of substituted galactosylated maltooligosaccharides (MD-ligands) which can be synthetized or produced by engineered E. coli

Ligand name MD-ligand structure (1 < n < 6) Abbreviation

Galactose Gaip-4[Glca-4]_nGlc MD-Gal

Blood group H Fuca-2Gaip-4[Glca-4]_nGlc MD-H

antigen

Blood group A GalNAca-3Gaip-4[Glca-4]_nGlc MD-A

antigen I

Fuca-2

Blood group B Gala-3Gaip-4[Glca-4]_nGlc MD-B

antigen I

Fuca-2

Lacto-N-neotetraose Gaip-3GlcNAcp-3Gaip-4[Glca-4]_nGlc MD-LNnT

Lewis X (LeX) Gaip-4GlcNAcp-3Gaip-4[Glca-4]_nGlc MD-LeX

I

Fuca-3

Lewis Y (LeY) Fuca-2Gaip-4GlcNAcp-3Gaip-4[Glca-4]_nGlc MD-LeY

I

Fuca-3

Lacto-N-tetraose Gaip-4GlcNAcp-3Gaip-4[Glca-4]_nGlc MD-LNT

Lewis A Gaip-3GlcNAcp-3Gaip-4[Glca-4]_nGlc MD-LeA

I

Fuca-4

Lewis B Fuca-2Gaip-3GlcNAcp-3Gaip-4[Glca-4]_nGlc MD-LeB

I

Fuca-4

Globoside Gala-4Gaip-4[Glca-4]_nGlc MD-Gb3

xeno antigen Gala-3Gaip-4[Glca-4]_nGlc MD-iGb3

3'sialyl, Neu5Aca-3Gaip-4[Glca-4]_nGlc MD-3S

Ganglioside GM3

Ganglioside GD3 Neu5Aca-8Neu5Aca-3Gaip-4[Glca-4]_nGlc MD-GD3

Ganglioside GM2 GalNAcp-4Gaip-[Glca-4]_nGlc MD-GM2

I

Neu5Aca-3

Ganglioside GMla Gaip-3GalNAcp-4Gaip-4[Glca-4]_nGlc MD-GMla

I

Neu5Aca-3

6'sialyl Neu5Aca-6Gaip-4[Glca-4]_nGlc MD-6S

Glucuronyl lactose GlcAp-3Gaip-4[Glca-4]_nGlc MD-GIcA In a particular embodiment, the invention concerns substituted galactosylated maltooligosaccharides ( MD-ligands) as defined above, wherein at least one of R-i , R₂, R3, R4, and R₅ is a monosaccharide selected from the group consisting of fucose, galactose, N- acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and derivatives thereof. In a particular embodiment, the invention concerns substituted galactosylated maltooligosaccharides (MD-ligands) as defined above, wherein at least one of R-i , R₂, R3, R4, and R₅ is a galactose.

In a particular embodiment, the invention concerns substituted galactosylated maltooligosaccharides (MD-ligands) as defined above, wherein at least one of R-i , R₂, R3, R4, and R₅ is a N-acetylneuraminic acid.

In a preferred embodiment, the invention concerns the substituted galactosylated maltooligosaccharides (MD-ligands), wherein at least one of Ri , R₂, R3, R4, and R₅ is a monosaccharide or an oligosaccharide selected from the groups consisting respectively of :

- for R3 : Gala- , GalNAcp-3Gala- , Galp-3GalNAcp-3Gala- , Fuca-2Galp- 3GalNAcp-3Gala-, GalNAca- , GlcNAcp- , Galp-4GlcNAcp- , Galp-4[Fuca- 3]GlcNAcp- , Fuca-2Galp-4[Fuca-3]GlcNAcp- , NeuAca-3Galp-4[Fuca- 3]GlcNAcp- , Fuca-2Galp^lGlcNAcp-, Gala-3Galp-4GlcNAcp- , Galp-4GlcNAcp- 3Galp-4GlcNAcp-Galp-3GlcNAcp- , Galp-3[Fuca-4]GlcNAcp- , Fuca-2Galp-

3[Fuca-4]GlcNAcp- , NeuAca-3Galp-3[Fuca-4]GlcNAcp- , Fuca-2Galp- 3GlcNAcp-, Gala-3Gaip-3GlcNAcp-, NeuAca- , NeuAc-8NeuAca-3, NeuAc- 8[NeuAc-8]_nNeuAca- (1 < n <400), GlcAp- , [GlcAp-4GlcNAca-4]_nGlcAp- with 1 < n <10000 (heparosan), [GlcAp-3GalNAcp-4]_nGlcAp- with 1 < n <10000 (chondroitin), or [GlcAp-3GlcNAcp-4]_nGlcAp- with 1 < n <10000 (hyaluronic acid)

- for R₄ : Gala- , GalNAcp-3Gala- , Galp-3GalNAcp-3Gala- , GalNAca-3Galp- 3GalNAcp-3Gala-, Fuca-2Galp-3GalNAcp-3Gala-, GalNAcp- , Galp-3GalNAcp-, NeuAca-3Galb-3GalNAcp-, or NeuAc-8NeuAca-3Galb-3GalNAcp-,

- for R_{5 :} NeuAca- . In a particular embodiment, the substituted galactosylated maltooligosaccharides (MD- ligands) according to the invention have two substitutions, in particular selected from the group consisting of:

R₂ is Fuca- and R₃ is GalNAca- (MD- Blood group A antigen)

- R₂ is Fuca- and R₃ is Gala- (MD- Blood group B antigen)

R₃ is selected from the group consisting of Neu5Aca-3, Neu5Aca-8Neu5Aca- or Neu5Aca-8 Neu5Aca-8Neu5Aca- and R₄ is selected from the group consisting of GalNAcp-, Galp-3GalNAcp- NeuAca-3Galb-3GalNAcp-, or NeuAc-8NeuAca- 3Galb-3GalNAcp- (MD- Ganglioside sugars)

- Ri is Fuca- and R₂ is Fuca- (MD-3 fucosyllactose derivative), or

Ri is Fuca- and R₃ is NeuAc- or Gala- (MD-3 fucosyllactose derivative).

These substituted galactosylated maltooligosaccharides or sialylated maltooligosaccharides (MD-ligands) may be obtained by a synthetic process according to usual methods known by the man skilled in the art. For example galactosyl maltooligosaccharides have already been prepared by enzymatic modification of the nonreducing end glucosyl residues of maltotetraose and maltotriose using lactose as galactosyl donor in a transgalactosylation reaction catalysed by the Bacillus circulans beta galactosidase (Takada et al 1998). In a preferred embodiment, these substituted galactosylated maltooligosaccharides or sialylated maltooligosaccharides (MD-ligands) are obtained by a biotechnological process according to the invention, as disclosed hereunder.

Production of modified maltooligosaccharides (MD-ligands) by microbial process

Another object of the invention is a method for producing maltooligosaccharides modified at their non-reducing end in particular substituted galactosylated maltooligosaccharide (MD- ligands) of formula (I) as defined above, susceptible to be used as donor substrate in the method of transglycosylation according to the invention, comprising the step of culturing a microorganism in a culture medium comprising an exogenous precursor selected from maltooligosaccharides with a degree of polymerization (DP) ranging from 2 to 7, preferably selected from the group consisting of maltotriose (M3) and maltotetraose (M4), wherein said microorganism :

internalizes said maltooligosaccharide by an active transport

comprises at least one heterologous genes encoding an enzyme modifying terminal non-reducing end of internalized maltooligosaccharide, and is devoid of enzymatic activities degrading maltooligosaccharides and modified maltooligosaccharides.

The expression 'internalization by an active transport' is intended to denote the ability of cells and preferably of bacteria to selectively admit and concentrate certain exogenous substances or precursors into their cytoplasm. This transport is performed by transporters of protein nature known as permeases, which act as enzymes; permeases are inducible catalysts, that is to say catalysts that are synthesized in the presence of the substrate or the precursor. According to one particular embodiment of the invention, maltooligosaccharides constitute precursors that are actively transported into the cytoplasm of the bacterium Escherichia coli by the maltose/maltodextrin transport system which includes the translocation complex MalFGK2.

The expression 'exogenous precursor' is intended to denote a compound involved in the biosynthetic pathway of the maltooligosaccharides modified at their non-reducing end in particular substituted galactosylated maltooligosaccharides (MD-ligands) according to the invention that is internalized by the cells.

The genes, plasmids and microorganisms which can be used in the method for producing maltooligosaccharides modified at their non-reducing end in particular substituted galactosylated maltooligosaccharides or sialylated maltooligosaccharides (MD-ligands) by Escherichia Coli are disclosed hereunder in the table 2:

Table 2 Genes, plasmids and Escherichia coli strains used

Description Reference or source

Genes

IgtEm pi ,4-galactosyltransferase from Neisseria meningitidis AAB48387.1

126E

In a preferred embodiment, the maltooligosaccharide as exogenous precursor is selected from the group consisting of maltotriose (M3) and maltotetraose (M4). Maltotriose is a trisaccharide (three-part sugar) consisting of three glucose molecules linked with

Maltotetraose is a tetrasaccharide (four-part sugar) consisting of four glucose molecules linke

In a particular embodiment, the said microorganism further encodes a protein that facilitates uptake of maltooligosaccharides (internalization with active transport). In a preferred embodiment, the said microorganism is E.coli strain which is preferably malFG+, malK+, and malPQZ-, lacZ-, and optionally nanKA-, lacA- and melA-, wcaJ and contains the gene coding IgtE for β1 ,4 galactosyltransferase or derivative thereof.

In a particular embodiment, the said microorganism contains an additional glycosyltransferase, in particular selected from a-1 ,2-fucosyltransferase, a-1 ,3- fucosyltransferase a1 ,4-fucosyltransferase, 3-1 ,3-N-acetylglucosaminyltransferase, β-1 ,4-Ν- acetyl-glucosaminyltransferase, β-1 ,6-N-acetylglucosaminyltransferase, β-1 ,3-galactosyl- transferase, β-1 ,4-galactosyltransferase, a-1 ,4-galactosyltransferase a-1 ,3-galactosyl- transferase, a-1 ,3-N-acetyl-galactosaminyltransferase, p-1 ,3-N-acetyl-galactosaminyl- transferase, a-1 ,4-N-acetylgalactosaminyltransferase, β-1 ,4-N-acetyl-galactosaminyl- transferase, a-2,3-sialyl-transferase, a-2,6-sialyl-transferase, a-2,8-sialyl-transferase, β-1 ,3- glucuronosyl-transferase and combinations thereof. In a particular embodiment, the said microorganism contains an additional glycosyltransferase selected from a-2,3-sialyl-transferase, a-2,6-sialyl-transferase.

According to a preferred embodiment, the microorganism is cultivated at high cell density on carbon substrate, such as glucose or preferably glycerol, and fed with precursor selected from maltotriose or maltotetraose which is internalize by the malFG/malK and glycosylated by at least a β1 ,4 galactosyltransferase and an additional glycosyltransferase.

The invention also concerns a recombinant microorganism as defined above. Another object of the invention is a cell culture medium comprising (i) a precursor selected from maltotriose and maltotetraose, and (ii) a microorganism.

MD-ligands are preferably produced by metabolically engineered Escherichia coli cells as described in Figure 1. In particular, the bacteria are cultured at high cell density with glycerol (or other cheap carbon source) as the carbon and energy source, while exogenous maltooligosaccharides are supplied as precursors for MD-ligand synthesis. During the culture, maltooligosaccharides are actively internalized in the cytoplasm by the maltose/maltodextrin transport system which includes the maltoporin lamB, the soluble periplasmic maltose binding protein MalE and the translocation complex MalFGK2 (Boos and Shuman 1998). In wild type E. coli strains, maltodextrin are normally converted into glucose and glucose-1-P by the combined action of the amylomaltase MalQ, the maltodextrin phosphorylase MalP and the maltodextrin glucosidase MalZ (Boos and Shuman 1998). To prevent intracellular catabolism of matodextrins, the malPQZ genes were therefore knocked out in MD-ligand producing strains as described in example 1.

To produce substituted galactosylated maltooligosaccharides (MD-ligands or MD-Gal oligosaccharides), the strains must be devoid of the indigenous β-galactosidase LacZ and express a recombinant β1 ,4 galactosyltransferase able to galactosylate the terminal non reducing glucosyl residue of maltodextrins. The IgtE gene have been shown to encode a β1 ,4 galactosyltransferase activity responsible for the synthesis of the lactosyl motif in Neisseria sp. lipopolysaccharide (Gotschlich 1994) and therefore represents a good candidate. As shown in example 4 the two IgtE gene candidates from Neisseria gonorrhoeae and N. meningitis were both able of producing M2-Gal from maltose. The results also indicate that galactosylation of maltose was more efficient in strain MS-2 expressing the N. meningitidis IgtE than in strain MS-1 expressing the N. gonorrhoeae IgtE gene. Consequently the N. meningitidis IgtE was chosen for the construction of others MD-ligand producing strains.

By additionally expressing the appropriate glycosyltransferase genes, the terminal lactosyl motif of substituted galactosylated maltooligosaccharides (MD-Gal oligosaccharides) can serve as a precursor for the synthesis of various structure of complex carbohydrate ligand such as those presented in table 1. These ligand structures have already been produced from lactose by using our previously described process of microbial fermentation (FR2796082) and it should therefore be possible to produce these structures as MD- ligands by using the same glycosyltransferase genes and the same strategy for the metabolic engineering of sugar-nucleotide biosynthetic pathways. For this purpose, a host strain MS was constructed as a general platform for the production of the largest diversity of MD-ligand structure. In addition of malPQZ and lacZ knockout, the host strain MS contains advantageously null mutations in the following genes: wcaJ nanKETA lacA melA

The wcaJ gene encodes the colanic biosynthesis UDP-glucose lipid carrier transferase. Colanic acid is an extracellular polysaccharide which contains fucose and the gene for GDP- fucose biosynthesis are located within the colanic acid gene cluster. To ensure an efficient GDP-fucose production rate under normal physiological conditions, the strategy developed by Dumon et al (2001 ) consisted in overexpressing the positive regulator rcsA of the colanic acid cluster, while blocking the colanic production by inactivating the wcaJ gene. The same strategy was used in the present invention : the wcaJ was inactivated in strain MS and the rcsA gene was coexpressed with the a1 ,2 fucosyltransferase gene in the plasmid pWKS-rcsA-futC which served for the construction of MD-H and MD-A producing strains.

The nanKETA genes were disrupted to prevent the catabolism of sialic acid and N- acetylmannosamine which serve as precursor for the synthesis of the of the nucleotide sugar (CMP-NeuAc ) used in the synthesis of sialylated oligosaccharides (Fierfort and Samain 2008). The melA gene encodes an a-galactosidase. The knockout of melA was carried out to prevent the hydrolysis of oligosaccharides containing a terminal alpha-linked galactose (Bettler et al 2003)

The lacA gene encodes a galactoside O-acetyltransferase which was shown to acetylate galactose residus in oligosaccharides produced in Escherichia coli (Dumon et al 2006). Knockout of lacA was performed to prevent this acetylation reaction.

The MD-ligand producing strains can be cultured in presence of mixture of maltodextrin or in presence of individual maltodextrins with a defined degree of polymerization. Mixtures of maltodextrins can be easily prepared by enzymatic hydrolysis of starch and are commercially available at very low cost. However, maltoheptaose (M7) is the largest compound that can be transported into cytoplasm (Ferenci 1980) and commercial maltodextrin generally contain a large proportion of larger maltodextrin which can therefore not be utilized as precursor for MD-ligand production.

Maltose is a readily available inexpensive precursor for M2-ligands production. However maltose is unable to function as a donor substrate for a 4-a-glucanotransferase (Palmer et al 1976) and our results confirmed that M2-ligand are not be used as donor substrate by the E. coli a mylomaltase MalQ.

Maltotriose (M3) can advantageously prepared by hydrolyzing pullulan with pullulanase (Wu et al 2009). Pullulan is an exocellular homopolysaccharide produced by Aureobasidium pullulans. It is a linear mixed linkage a-D -glucan consisting mainly of maltotriose repeating units interconnected by a 1 ,6 linkages. Pullulanase (EC 3.2.1.41 ) hydrolyzes the a1 , 6 glucosidic linkages in pullulan and produces maltotriose as the end product.

Maltotetraose (M4) can be conveniently obtained by starch hydrolysis with the maltotetraose producing amylase from Pseudomonas stutzeri (Robyt and Ackerman 1971 ).

Uses- Applications

The invention also relates to glycoconjugates or multivalent carbohydrate structures as produced according to the transglycosylation method of the invention and/or MD-ligands as produced according to the method of the invention or as defined above, for use in at least one application selected from the fields of therapeutical applications, nutritional supplements, bioactive detergent, diagnostic, medical device, cosmetic, affinity purification process and combinations thereof.

They may be used, for example, as an agent for blocking cell surface receptors in the treatment of a host of diseases involving cellular adhesion, or may be used as nutritional supplements, antibacterial agents, anti-metastatic agents and anti-inflammatory agents. The invention thus relates to glyconjugates or multivalent structures according to the invention for use as pharmaceutical product, and especially as a pharmaceutical product intended for selectively preventing the adhesion of biological molecules. In a particular embodiment, they may be used as pharmaceutical product intended for treating cancer, inflammation, heart diseases, diabetes, bacterial infections, viral infections and neurological diseases and as a medicinal product intended for grafts. Pharmaceutical composition

The invention also relates to a pharmaceutical composition comprising at least one glycoconjugate or multivalent carbohydrate structure as produced according to the invention and/or at least one MD-ligand as produced or defined according to the invention, and a pharmaceutically acceptable vehicle.

Commercial scale

The invention also concerns a commercial scale composition comprising one or several MD- ligands as defined above or multivalent carbohydrate structures as defined above. The industrial advantage of the method according to the invention is obvious since it makes it possible to achieve a production of kilograms of glycoconjugates of biological interest, and the cost price of these compounds produced by the present microbiological route are infinitely lower than compounds obtained by chemical synthesis.

The invention will be illustrated with the non-limitative figures and examples disclosed hereunder.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Production of MD-ligands by metabolically engineered E. coli strain

Figure 2 Production of alkyl-M3-Gal from alkyl-glucoside and M3-Gal in amylomatase MalQ and baker yeast.

Figure 3. Divalent galactosylated structure obtained by MalQ transfer of M3-Gal on Decyl Maltose Neopentyl Glycol

Figure 4. TLC analysis of oligosaccharides produced by high cell density culture of strains MS1 and MS-2 in presence of maltose. Lanes 1 and 16 standard solution (2 mg.ml^" each) of lactose lacto-A/-neotetraose (LNnT), lacto-A/-neohexaose (LNnH). Lanes 2,3,4,5: extratracellular fractions of strain MS-1 withdrawn 0, 3, 6 and 20 hours after maltose addition. Lanes 6, 7 and 8 : intracellular fractions of strain MS-1 withdrawn 3, 6, and 20 hours after maltose addition. Lanes 9, 10 , 11 , 12 : extratracellular fractions of strain MS-2 withdrawn 0, 3, 6 and 20 hours after maltose addition . Lanes 13, 14, 15 : intracellular fractions of strain MS-1 withdrawn 3, 6, and 20 hours after maltose addition.

Figure 5. TLC analysis of intracellular oligosaccharides produced from maltotriose (M3) by high cell density culture of M3-Gal producing strain MS-2 (Lane 3), M3-H producing strain MS-5 (lane 4), M3-A producing strain MS-7 (lane 5), M3-Gb3 producing strain MS-10 (lane 6) and M3-LNT producing strain MS-6 (lane 7). Lanes 1 and 10 : standard solution of maltodextrins. Lane 2 and 9 : standard solution of maltotriose obtained by hydrolysis of pullulan. Lane 8 : standard solution of maltotetraose obtained by hydrolysis of starch by Pseudomonas stutzeri amylase.

Figure 6. MALDI-TOF mass spectrum of the intracellular fraction of strain MS-2 cultured in presence of maltotriose obtained by hydrolysis of pullulan Figure 7. TLC analysis of MalQ transglycosylation reaction products obtained by using M3- Gal as donor and different alkyl-glucosides acceptors : control without acceptor; methyl-GIc; octyl-GIc, dodecyl-GIc; B, blank (before malQ addition); R, after 20 hours of reaction with malQ; S dodecyl-GIc standard (10 mM) without Yeast.

Figure 8. MALDI-TOF mass spectrum of reaction product formed by MalQ transfert of M3- Gal on dodecyl-GIc. The Peak at m/z 873 and 1035 correspond to the quasi-molecular ion [M+K]⁺ derived from dodecyl-M3-Gal and dodecyl-M4-Gal

Figure 9. TLC analysis of MalQ transglycosylation reaction products obtained by using M3- Gal as donor and two Neopentyl Glycol (NG) class detergents as acceptor: control without acceptor, OGNG, octyl glucose neopentyl glycol; DMNG, decyl maltose neopentyl glycol. B, blank (before malQ addition); R, after 20 hours of reaction with malQ.

Figure 10. MALDI-TOF mass spectrum of reaction products formed by MalQ transfert of M4- A on decyl maltose neopentyl glycol (decyl-NG-[M2,M2]). The peaks at m/z 1473 and 1635 corresponds to the quasi-molecular ion [M+K]⁺ derived from decyl-NG-[M2,M4-Gal] and decyl-NG-[M2,M5-Gal] respectively. The peaks at m/z 1959, 2121 and 2284 corresponds to the quasi-molecular ion [M+K]⁺ derived from decyl-NG-[M4-Gal,M4-Gal], decyl-NG-[M4-Gal,M5-Gal] and decyl-NG-[M5-Gal,M5-Gal].

Figure 11. MALDI-TOF mass spectrum of reaction product formed by MalQ transfert of M3- LNT on octyl-GIc. the Peak at m/z 1 166 corresponds to the quasi-molecular ion [M+Na]+ derived from C8-M3-LNT. Peak at m/z 801 is explained by the presence of residual M3-Gal which has been converted into C8-M3-Gal

Figure 12. TLC analysis of : 1 Mixture of maltooligosaccharides (DP 2-7) used as acceptor for the production of MD-H mixture by strain MS-5, 2 MD-H mixture produced by strain MS- 5 ; 3 MalQ transglycosylation reaction products obtained by using the mixture of MD-H oligosaccharide as donor and octyl-GIc as acceptor, 4 M3-H standard

Figure 13. MALDI-TOF mass spectrum of reaction product formed by MalQ transfert of MD- H on octyl-GIc. The peak at m/z 1 109 corresponds to the quasi-molecular ion [M+Na]+ derived from octyl-M4-H.

Figure 14. MALDI-TOF mass spectrum of reaction products formed by MalQ transfert of M4- A on dodecyl-GIc. The peaks at m/z 1368 and 1530 corresponds to the quasi-molecular ion [M+Na]+ derived from docecyl-M4-A and dodecyl-M5-A respectively

Figure 15 Binding of Anti-A antibodies on octadecyl silica particles coated with mixtures of dodecyl-maltose and dodecyl-M4-A. The absorbance at 405 nm correspond to the amount of 4-nitrophenyl-phosphate hydrolysed by the secondary antibody bound to the anti-A antibody after 1 hour of incubation at room temperature

Figure 16. MALDI-TOF mass spectrum in negative mode of reaction products formed by MalQ transfert of M3-3S and M4-3S on octyl-Glc. The peak at m/z 1068 and 1230 corresponds to the quasi-molecular ion [M-H]^" derived from octyl-M3-3S and octyl-M4-3S respectively.

Figure 17. MALDI-TOF mass spectrum in negative mode of reaction products formed by MalQ transfert of M4-6S on dodecyl maltoside. The peaks at m/z 1086, 1448, and 1610 correspond to the quasi-molecular ions [M-H]- derived from dodecyl-M4-6S, dodecyl-M5-6S and dodecyl-M6-6S respectively.

EXAMPLES

Example 1 : Construction of the host strain MS

The strain MS was constructed from the Escherichia coli K12 collection strain DH1 (DSM 4235) by disrupting the genes lacZ nanKETA lacA melA wcaJ malPQ and malZ.

The knockout of lacZ was performed by deleting a 1.545 kb DNA segment located between nucleotides 814 and 2358 of the lacZ gene using the method of Hamilton et al (1989). The construction of the suicide plasmid pMAK705 carrying the truncated lacZ sequence is described in Dumon et al (2004).

The nanKETA genes were disrupted by removing a 3.339-kb segment in the chromosomal DNA using a one-step procedure that employs PCR primers to provide the homology to the targeted sequence (Datsenko and Wanner, 2000). Primers sequences are listed in Fierfort and Samain (2008).

The lacA gene was disrupted by removing a 0.513 kb segment in the chromosomal DNA using the procedure of Datsenko and Wanner (2000). Primers sequences are listed in Dumon et al (2006).

The knockout of melA was performed by deleting a 0.852 DNA segment located between nucleotides 451 and 1302 of the melA gene using the method of Hamilton et al (1989). The construction of the suicide plasmid pMAK705 carrying the truncated melA sequence is described in Bettler et al et al (2003)

To disrupt the wcaJ gene, a 0.522 kb segment located between nucleotides 607 and 1 128 of the wcaJ gene was deleted using the method of Hamilton et al (1989). The construction of the suicide plasmid pMAK705 carrying the truncated wcaJ sequence is described) in Dumon et al (2001 ).

To knockout the malPQ genes, a 3.1 10 kb segment located between nucleotides 371 of malP and 1097 of malQ was deleted and replaced by the 5TCTAG sequence as follows: two DNA segments flanking the deleted sequence were amplified by PCR. The upstream 1.080 kb segment was amplified with primers 5' GTCGACTCGACTCAATGGCAACTGTC (SEQ ID N°1 ) and 5' TCTAGAGTGGGGTAATACCGTTGGTG (SEQ ID N°2) and the downstream 0.958 kb segment was amplified with primers 5' TCTAGAATGCCGATTGGCTTGTATC (SEQ ID N° 3) and 5'. GGATCCTCTGTCCAAATCCTTCAGCA (SEQ ID N°4). The two amplified fragments were ligated at their terminal Xbal restriction site and cloned together into the BamHI Sail sites of the suicide vector pK03. The deletion was then carried out according to the pK03 gene replacement protocol of Link et al (1997).

To knockout the malZ gene, a 0.491 kb segment located between nucleotides 594 and 1084 of malZ was deleted as follows: two DNA segments flanking the deleted sequence were amplified by PCR. The upstream 0.847 kb segment was amplified with primers 5'GGATCCGCGTATCTCGCTGTATGTCGGTTTC (SEQ ID N°5) and 5'GGATCCGCGTATCTCGCTGTATGTCGGTTTC (SEQ ID N° 6) and the downstream 0.843 kb segment was amplified with primers 5' AAGCTTGATCACCGAAGCGGCGAAAGAAAC (SEQ ID N° 7) and 5'GTCGACGATTTAGACGCTCATTATGACGCCCTC (SEQ ID N° 8). The two amplified fragments were ligated at their terminal Hindlll restriction site and cloned together into the BamHI Sail sites of the suicide vector pK03. The deletion was then carried out according to the pK03 gene replacement protocol of Link et al (1997).

The complete genotype of the host strain MS is: endA 1 recA 1 gyrA96 thi-1 glnV44 relA 1 hsdR17 lacZ- nanKETA lacA melA wcaJ malPQ malZ.

Example 2 : Production of the amylomaltase MalQ

A 2.090 kb DNA fragment containing the sequence of the malQ gene was amplified by PCR using the genomic DNA of Escherichia coli K12 as a template and the following primers : 5'CCATGGAAAGCAAACGTCTGGATAATG (SEQ ID N° 9) and 5' ACTCTACTTCTTCTTCGCTGCAG (SEQ ID N° 10). The amplified fragment was first cloned into pCR4Blunt-TOPO vector (Invitrogen) and then sub-cloned into the Ncol and EcoRII sites of expression vector pProEX-HTb (Invitrogen) to form pPro-malQ.

For the production of recombinant MalQ, the strain MS was transformed with pPro-malQ plasmid and cultivated in a 3 L fermenter containing 1.5 I of Terrific broth (rich medium) at 30°C. MalQ production was induced by adding IPTG (150 m/L) when the Optical Density of the culture reached 5.0. Cells were harvested by centrifugation 4 hours after the induction and the cell pellet was resuspended in 150 ml of potassium phosphate buffer (10 mM, pH 7.0). Cells were disrupted in a high pressure cell homogenizer (Constant System Ltd) and the crude extract containing 50 units/ml of MalQ was aliquoted and freezed at -80°C until its direct utilization as a MalQ source in enzymatic synthetic reactions. MalQ activity was determined by measuring the initial rate of glucose formation from maltotriose using a Glucose enzymatic assay kit from Megazyme. One MalQ unit was defined as the amount of enzyme required to produce one μηιοΐβ per min of glucose from maltotriose at 25 °C. Example 3 : Construction of MP -Gal producing strains

The MD-Gal producing strain MS-1 was constructed by transforming the host strain MS with the pBBR3-lgtEg plasmid (llg et al 2010) containing the Neisseria gonorrhoeae IgtE gene coding a β1 -4 galactosyltransferase activity.

The pBBR3-lgtEm plasmid containing the Neisseria meningitis IgtE gene was constructed as follow: a 0.942 kb DNA fragment containing the sequence of the IgtE gene (GenBank: AAB48387.1 ) was amplified by PCR using the genomic DNA of Neisseria meningitis 126E as a template. A Xbal site was added to the left primer (5' TCTAGATACCGGGGCTATTGAAACC) (SEQ ID N° 1 1 ) and a Sacl site was added to the right primer (GAGCTCGCGGGAATGACAGTGTATC) (SEQ ID N°12). The amplified fragment was first cloned into pCR4Blunt-TOPO vector (Invitrogen) and then sub-cloned into the Xbal and Sacl sites of plasmid pBBR1 -MCS3 plasmid (Kovach et al 1995) to form pBBR3-lgtEm. The MD-Gal producing strain MS2 was constructed by transforming the host strain MS with pBBR3-lgtEm_m

Example 4 : Production of M2-Gal

Strain MS-1 and MS-2 were grown at high cell density as previously described (Priem et al. 2002). Cultures were carried out in 3-liter reactors containing 1.5 liter of mineral culture medium, the temperature was maintained at 34°C and the pH was regulated to 6.8 with 14 % NH40H. The high cell density culture consisted of three phases: an exponential growth phase, which started with the inoculation of the fermenter and lasted until exhaustion of the carbon substrate (glycerol 17.5g.L-1 ), a 5 h fed-batch with a high glycerol feeding rate of 5 g.L-1 h-1 and a 20 h fed-batch phase with a glycerol feeding rate of 3 g.L-1 h-1 and. Maltose (M2) was added at the beginning of the feeding phase at a concentration of 10 g/l.

The oligosaccharide production was followed during fermentation process by TLC-plate analysis. Culture samples (1 ml.) were centrifuged into microfuge tubes (2 min, 12,000g) after collection. The supernatants were saved for the analysis of extracellular oligosaccharides. The pellets were re-suspended in distilled water (1 ml_), boiled for 20 minutes, and centrifuged (2 min, 12,000g). The supernatants were kept for the analysis of the intracellular oligosaccharides.

TLC-plate analyses were carried out on silica gels and the oligosaccharides were eluted with n-butanol/acetic acid/water buffer (2:1 : 1 ). Sugars were detected after dipping the plate in orcinol sulfuric reagent and heating. Migration profiles were compared with a standard sample containing a mix of the disaccharide lactose, the tetrasaccharide lacto-N-neotetraose and the hexasaccharide lacto-N-neohexaose. TLC analysis indicated that, after 20 hours of culture, maltose has been entirely consumed by both strain MS-1 and MS-2 and converted into a longer oligosaccharide which migrated as the expected M2-Gal product (Figure 4). Transient intracellular accumulation of M2 in strain MS-1 (Fig 4 lane 6,7) was more important than in strain MS-2 (Fig 4 lane 6,7). Conversely the initial rate of M2-Gal production appears higher in strain MS-2 than MS-1 .

Example 5 : Construction of MD-H producing strains

The plasmid pWKS-futC was constructed by subcloning the 0.972 DNA fragment obtained by Xbal Sail digestion of plasmid pET-21 a-futC (Drouillard et al 2006) into the Xbal Sail sites of plasmid pWKS130. The 0.757 DNA fragment containing the rcsA gene was then excised from pBBR1 -rcsA (Priem et al 2003) by a Kpnl Xbal digestion and sub-cloned into the Kpnl Xbal sites of pWKS-futC to form pWKS-rcsA-futC.

The strain MS-5 was constructed by transforming the MS host strain with the two plasmids pBBR3-lgtEm and pWKS-rcsA-futC. Example 6 : Construction of MD-A producing strains

The E. coli codon optimized sequence of truncated Blood group A glycosyltransferase ( Seto et al 1997) was synthesized with a deletion of the N-terminal 63 amino acids. The ATG start codon was flanked with the upstream sequence G GATCC ATC GATGCTTAG GAG GTCAT (SEQ ID N°13) containing a ribosome biding site and a BamHI site. The synthetic sequence also contained a Hindlll site downstream of the stop codon and was cloned into the BamHI Hindlll sites of the pSU27-18 plasmid yielding pSU-gtA. To construct the plasmid pSU-gtA- gne, the 1.1 kb DNA fragment containing the gne gene was excised with EcoRI from the pCR4blunt vector containing the gne gene (Randriantsoa et al 2007) and blunt-end cloned into the Hindlll site of pSU-gtA.

The strain MS-7 was constructed by transforming the MS host strain with the three plasmids pBBR3-lgtEm pWKS-rcsA-futC and pSU-gtA-gne.

Example 7 construction of MD-Gb3 producing strains

The strain MS-10 was constructed by co-transforming the host strain MS with pBBR3- IgtEm and the plasmid pBS-lgtC containing the a1 ,4-galactosyltransferase from Neisseria meningitidis 126E (Antoine et al 2005)

Example 8 : Construction of MD-LNT producing strains

A 1.34 kb DNA containing the sequence of a β-3 galactosyltransferase gene was amplified by PCR using the genomic DNA of Helicobacter pylori ATCC43504 a template. A Sail site was added to the left primer

(5'GGTCGACGGTAAGGAGATATACATATGATTTCTGTTTATATCATTTCTTTAAAAG) (SEQ ID N°14) and a Pstl site was added to the right primer (5'CTGCAGTTAAACCTCTTTAGGGGTTTTTAAAGG) (SEQ ID N°15). The amplified fragment was first cloned into pCR4Blunt-TOPO vector and then sub-cloned into the Sail and Pstl sites of pBluescript-KS plasmid to form pBS-3-3GalT.

Plasmid pLNTIT (Dumon et al 2001 ) was digested with Ndel to remove IgtB in order to give after ligation the pBBR3-lgtA plasmid. The 1.15 kb DNA fragment containing IgtA was then excised from pBBR3-lgtA by a Kpnl Xbal digestion and sub-cloned into the same sites of pWKS130 vector to form pWKS-lgtA.

The strain MS-6 was constructed by transforming the host strain MS with the three plasmids pBBR3-lgtEm, pWKS-lgtA and pBS-3-3GalT. Example 9 : Production of M3-ligands

For the production of Maltotriose (M3), 50 g of polysaccharide pullulan (TCI chemicals) were dissolved in 400 ml of autoclaved water. Hydrolysis was carried out by adding 1 ml of pullulanase microbial solution (Sigma Aldrich E2412). After 48 hours of incubation at 30°C, the complete conversion of pullulan into M3 was checked by TLC and the M3 solution was autoclaved and kept at room temperature prior its utilization as acceptor for the synthesis of M3-ligands.

The strains MS-2, MS-5, MS-6, MS-7, and MS-10 were cultivated at high cell density as in example 4, except that M3 was used acceptor instead of M2. M3 was continuously added at a rate of 0.5 g.L-1 h-1 throughout the third culture phase of glycerol feeding. At the end of the fermentation period, bacterial cells were recovered by centrifugation (7000 x g, 30 min) and the cell pellets were re-suspended in 1.5 L of distilled. The cells were permeabilized by autoclaving at 100°C for 50 min. After cooling and a second centrifugation step (7,000 x g, 30 min) the supernatants were recovered and analyzed by TLC using a n-butanol/formic acid/water (4:8:1 ) solvent mixture. As shown in Figure 5, TLC analysis indicates that M3 has been entirely consumed in all the cultures and that longer oligosaccharides which migrated as the expected M3-Gal (Iane3) M3-H (lane 4) M3-A (lane 5) M3-Gb3 (lane 6) and M3-LNT (lane 7) products were present in intracellular fractions of strains MS-2 MS-5 MS-7 MS-10 MS-6 respectively. Oligosaccharides were purified by charcoal adsorption as previously described (Priem et al 2003) and their identification was confirmed by MALDI-TOF mass spectroscopy. The spectrum of oligosaccharide purified from strain MS-2 showed a major molecular peak at m/z 689 corresponding to the quasi-molecular ions[M+Na]⁺ derived from the tetrasaccharide M3-Gal (Figure 6). However this spectrum also showed minor peaks at m/z 851 , 1013 1 175 and 1337 which must originate from M4-Gal and longer oligomer. The formation of these minor compounds is explained by the irregular structure of the pullulan in which some of maltotriose units can be been replaced maltotetraose and longer units (Carolan et al 1982).

Example 10 : Synthesis of M3-Gal amphiphiles

1 g of dehydrated commercial baker yeast was suspended in 50 ml of sterile 10 mM sodium phosphate buffer (pH 7.0). After centrifugation at 2000 g for 5 min, the supernatant was removed and the yeast pellet was re-suspended in 50 ml of the same buffer containing 15 mg/ml of charcoal purified M3-Gal. Transglycosylation reactions were carried in 50 ml falcon tubes containing 5 ml of the yeast suspension supplemented with 10 mM of the following acceptor: methyl β-D-glucopyranoside (methyl-GIc, Sigma M0779), octyl β - D-glucopyranoside (octyl-GIc, Carbosynth DO05161 ), dodecyl β -D-glucopyranoside (dodecyl-GIc, Carbosynth DD06359), octyl glucose neopentyl glycol ( OGNG, Carbosynth DO14034), decyl maltose neopentyl glycol ( DMNG, Carbosynth DD14033). Reaction was started by the addition of MalQ (2/units/ml) and the tubes were incubated at room temperature for 20 hours under mild agitation. Yeast were removed by centrifugation and the supernatant were analyzed by TLC using butanol/acetic acid/water buffer (2: 1 :1 ). As shown in Figure 7, M3-Gal was not modified in the control tube run without any acceptor indicating that the terminal Galactose residue of M3-Gal is not used acceptor by MalQ. Methyl-GIc and octyl-GIc were modified by MalQ and this consumption was correlated with the disappearance of M3-Gal and the formation of compounds that migrated as the expected methyl-M3-Gal (2) and octyl-M3-Gal (3). Dodecyl-GIc adsorbed on the yeast surface and was not detected in the blank reaction without MalQ (lane 4 B). In spite of this adsorption, dodecyl-GIc was used as acceptor and converted into two compounds which were identified by mass spectrometry as dodecyl-M3-Gal and dodecyl-M4-Gal (Figure 8). Surprisingly dodecyl-M4-Gal was the major product, whereas its precursor M4-Gal was only present in trace amount in the pullulan hydrolysate. This indicates that, in comparison with M3-Gal, M4-Gal is preferentially used as a donor when the acceptor is in limiting concentration.

As shown in Figure 9, migration profile of octyl glucose neopentyl glycol (OGNG) was not modified by malQ. On the contrary decyl maltose neopentyl glycol (DMNG, decyl-NG- [M2,M2]) was almost completely modified by MalQ leading to the formation of at least two new compounds. MALDI-TOF mass spectrum shown the presence of two groups of peaks (Figure 10). A first group of two main peaks at m/z 1473 and 1635 correspond to the quasi- molecular ions [M+K]+ derived DNMG mono-substituted with M3-Gal and M4-Gal respectively. A second group of peaks correspond to the quasi-molecular ions [M+K]+ derived from DNMG di-substituted with either two M3-Gal (peak at m/z 1959) or one M3- Gal and one M4-Gal (peak at m/z 2121 ) or two M4-Gal (peak at m/z 2284). Example 11 : Production of octyl-M3-ligands

Octyl-Glc was chosen as an acceptor to test the enzymatic transfer of the other M3-ligands produced in example 9. The oligosaccharides M3-H, M3-A, M3-LNT and M3-Gb3 were thus incubated in presence of octyl-GIc using the experimental procedure described in example 10. TLC analysis of reaction products indicated that M3-LNT and M3-Gb3 have been modified by malQ and the reaction products were identified by MALDI-TOF mass spectroscopy as octyl-M3-LNT (Figure 1 1 ) and octyl-M3-Gb3. On the contrary M3-H and M3-A were absolutely not modified by malQ even after prolonged incubation. These results indicate the transfer of M3-ligands is blocked by the presence of a fucosyl residue attached on the carbon 2 of Gal but not by the presence of a glycosyl residue attached on the carbon 3 of Gal.

Example 12 Production of octyl-M4-H

The strain MS-5 was cultivated at high cell density as in example 9, except that a mixture of maltodextrin (DP2- 7) was used as acceptor for the synthesis of a mixture of MD-H oligosaccharides.

At the end of the fermentation, TLC analysis showed that the maltooligosaccharide series (Figure 12 lane 1 ) has been converted in to a series of longer oligosaccharides that migrated as the expected MD-H series (Figure 12, lane 2). After treatment with malQ in presence of octyl-GIc, the shorter M2-H and M3-H oligosaccharide remained unmodified whereas the longer MD-H oligosaccharides completely disappeared. The disappearance of long MD-H oligosaccharide was associated with the formation of a major product which was identified as octyl-M4-H by MALDI-TOF mass spectroscopy (figure 13 ). This result indicates that MD- H oligosaccharides must have a size equal or higher to that of M4-H to be used as donor by MalQ.

Example 13 Production of dodecyl-M4-A

The strain of Pseudomonas stutzeri NRRL B-3389 was obtained from the DSMZ collection (DSM 13627) and cultured on starch for the production of maltotetraose producing amylase as described by Robyt and Ackerman (1971 ). After cell removal by centrifugation, 100 ml of the culture supernatant were filtered-sterilized through a 0.22 μηι filter and added to 1 liter of a 100 g/l autoclaved gelatinized starch solution. After 48 hours of incubation at 30°C, TLC analysis confirmed the formation M4 as the major starch hydrolysis product (Figure 5, lane 8). The mixture was centrifuged ad the M4 containing supernatant was autoclaved and kept at room temperature prior its utilization as acceptor for the synthesis of M4-ligands.

The MD-A producing strain MS-7 was cultivated at high cell density as in example 9, except that a M4:M3 mixture (90:10) was used as acceptor instead of M3. At the end of the culture, TLC analysis indicates that M4 has been converted into the expected longer M4-A heptasaccharide.

For the production of dodecyl-M4-A, 200 ml of the intracellular fraction of the MS-7 culture were incubated for 24 hours in presence of dodecyl-GIc (200 mg), dehydrated baker yeast (0.5 g) and 50 units of MalQ. After centrifugation half of the supernatant (100 ml) was loaded on a small C18 - modified silica column (1 x 2.5 cm). TLC analysis indicated that all the dodecyl-M4-A glycolipid was adsorbed on the column and was recovered after elution with 50 % aqueous methanol. After freeze drying, 180 mg of purified dodecyl-M4-A were obtained and the structural identification of dodecyl-M4-A was confirmed by MALDI- TOF mass spectroscopy (Figure 14). The mass spectrum also showed a peak at m/z 1530 due to the presence of dodecyl-M5-A. The presence of significant amount of dodecyl-M5-A is explained by the presence of small amount of M5 in the starch hydrolysate.

Example 14 Immobilization of dodecyl-M4-A to silica support for affinity binding of anti-A antibodies

Silica micro-particles samples were prepared by mixing 5 mg of octadecyl-bonded silica (LiChroprep RP18, 4-20 μηι, Merck Darmstadt) with 1 ml of different mixture of dodecyl- maltose (1.5 g/ 1) and crude dodecyl-M4-A solution prepared as in example 13. Once coated with dodecyl-oligosaccharides, octadecyl-bonded silica particles formed stable aqueous suspensions that were washed by centrifugation and resuspended in 1 ml of a solution of 1 % BSA (Bovine Serum Albumin) in PBS (Phosphate buffer saline pH 7.4). After 1 hour of incubation at 30°C under mild agitation, each sample was mixed with 1 μΙ of anti-A monoclonal mouse antibody (HE-193) solution (Pierce MA1 -19693), and incubated again for 45 min at 30°C. Particles were then washed twice by centrifugation and resuspension in 1 ml of 1 % BSA in PBS before adding 1 μΙ of anti-mouse secondary antibody alkaline phosphatase conjugate (Pierce PA1 29587). After 45 min of incubation at 30°C, samples were washed twice with PBS. Binding of anti-A antibodies on coated silica particles was quantified by mixing 10 μΙ of coated particle suspension with 1 ml of 0.1 % 4-nitrophenyl phosphate in diethanolamine buffer( 1 %, pH 9.5) and by measuring the increase in absorbance at 405 nm resulting from the hydrolysis of p-nitrophenyl-phosphate by the secondary antibody bound to the anti-A antibody. As shown in figure 15, anti-A antibody specifically bound to particle coated with dodecyl-M4-A and no binding could be detected in absence of dodecyl-M4-A. Interestingly substitution of up to 90 % of dodecyl-M4-A with dodecyl-maltose did not affect the binding.

Example 15 transfer of M3-Gal on glycogen

0.1 g of dehydrated commercial baker yeast was suspended in 10 ml of sterile 10 mM sodium phosphate buffer (pH 7.0). After centrifugation at 2000 g for 5 min, the supernatant was removed and the yeast pellet was re-suspended in 10 ml of the same buffer containing 5 mg/ml of charcoal purified M3-Gal. Transglycosylation reactions were carried in 50 ml falcon tubes containing 5 ml of the yeast suspension supplemented with 12.5 mg/l of glycogen from oyster (Sigma G4751 ). A control reaction tube was also run without glycogen. Reaction was started by the addition of MalQ (2 units/ml). Samples (0.5 ml) were withdrawn just before malQ addition and after 20 hours of incubation. Samples were immediately centrifuged to pellet the baker yeasts and 200 μΙ of each supernatant were mixed with 0.8 ml of a 75% aqueous ethanol solution to precipitate the glycogen. After centrifugation glycogen pellet was suspended in 100 μΙ of distilled water and galactose concentration was determined spectrophotometrically after hydrolysis with a beta galactosidase using a lactose/galactose assay kit (Biosentec). The results showed that the glycogen which had been incubated with M3-Gal and MalQ, contained 0.75 % of galactose which could be specifically hydrolyzed by a beta-galactosidase.

Example 16 Production of octyl-MD-3S

The pSU-nst plasmid containing the Neisseria meningitis a-2,3 sialyltransferase gene was constructed by subcloning the 2.15 kb DNA fragment obtained by Sacl Kpnl digestion of plasmid NST-01 (Priem et al 2002) into the Sacl Kpnl sites of pSU27-18. The strain MS-11 was then constructed by transforming the host strain MS with the three plasmids pBBR3- IgtEm, pBS-SS and pSU-nst. The M4-3S and M3-3S oligosaccharides were produced as described in example 13 by culturing the strain MS11 at high cell density in presence of mixture of maltotriose and maltotretaose.

Octyl-MD-3S glycolipides were produced by incubating the M4-3S and M3-3S oligosaccharides in presence of MalQ and octyl-GIc as described in example 10. The reaction products were identified by MALDI-TOF mass spectroscopy as octyl-M3-3S and octyl-M4-3S as shown in figure 16.

Example 17 Production of dodecyl-MD-6S

The strain MS-12 was constructed by transforming the host strain MS with the three plasmids pBBR3-lgtEm, pBS-SS and pSU-6ST carrying the gene for a-2,6-sialyltransferase. The M4- 6S oligosaccharides were produced as described in example 13 by culturing the strain MS-12 at high cell density in presence of maltotetraose.

Dodecyl-MD-6S glycolipides were produced by incubating the M4-6S oligosaccharide in presence of MalQ and dodecyl β-D-maltopyranoside (Carbosynth DD06199) as described in example 10. The reaction products were identified by MALDI-TOF mass spectroscopy as dodecyl-M4-6S, dodecyl-M5-6S and dodecyl-M6-6S as shown in figure 17.

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Claims

1. Method of transglycosylation for producing glycoconjugates and multivalent carbohydrates structures, comprising the transfer via a 4-a-glucanotransferase (EC 2.4.1.25) of at least one terminal non-reducing end of a modified maltooligosaccharide as donor substrate on at least one glycosidic acceptor according to the following reaction R-Glca1 -4[Glca1 -4]_n-Glc + glycosidic acceptor -> R-Glca1 -4[Glca1-4]_x-acceptor +[Glca1-4]_(n-_X)-Glc wherein

R-Glca1-4[Glca1 -4]_n-Glc is said modified maltooligosaccharide,

n is comprised between 1 and 5,

- x is comprised between 1 and 5,

- x is inferior or equal to n

R is a substituent attached on the terminal non-reducing glucosyl group of a modified maltooligosaccharide, thereby constituting terminal non-reducing end of said modified maltooligosaccharide, and

2. Method of transglycosylation for producing glycoconjugates and multivalent carbohydrates structures according to claim 1 , wherein the glycosidic acceptor is a multivalent glycoside comprising several glycosidic acceptors, on which several terminal non-reducing ends of said modified maltooligosaccharide can be transferred.

3. Method of transglycosylation for producing glycoconjugates and multivalent carbohydrates structures according to claim 2, wherein the multivalent acceptor is a branched glucan.

4. Method of transglycosylation for producing glycoconjugates and multivalent carbohydrates structures according to anyone of claims 1 to 3, wherein at least one glycosidic acceptor is linked to at least one lipid chain.

5. Method of transglycosylation for producing glycoconjugates and multivalent carbohydrates structures according to anyone of claims 1 to 4, wherein the donor substrate of transglycosylation is a maltooligosaccharide modified at its terminal non- reducing end in particular a substituted galactosylated maltooligosaccharide (MD-ligands), susceptible to be obtained by a method of production comprising the step of culturing a microorganism in a culture medium comprising an exogenous precursor selected from maltooligosaccharides with a degree of polymerization (DP) ranging from 2 to 7, wherein said microorganism:

internalizes said maltooligosaccharide by an active transport,

comprises at least one heterologous gene encoding an enzyme modifying the terminal non-reducing end of said internalized maltooligosaccharide, and is devoid of enzymatic activities degrading maltooligosaccharides and modified maltooligosaccharides.

Method of transglycosylation for producing glycoconjugates and multivalent carbohydrates structures according to anyone of claims 1 to 5, wherein the donor substrate of transglycosylation is a substituted galactosylated maltooligosaccharide (MD- ligands)

wherein

. n is comprised between 0 and 5,

7. Method of transglycosylation for producing glycoconjugates and multivalent carbohydrates structures according to anyone of claims 1 to 6, wherein the 4-a- glucanotransferase is the amylomaltase MalQ from Escherichia coli.

8. Method for producing maltooligosaccharides modified at their terminal non-reducing end, susceptible to be used as donor substrate in the method of transglycosylation according to anyone of claims 1 to 7, in particular substituted galactosylated maltooligosaccharide (MD-ligands) of formula (I) as defined in claim 6, comprising the step of culturing a microorganism in a culture medium comprising an exogenous precursor selected from maltooligosaccharides with a degree of polymerization (DP) ranging from 2 to 7, preferably selected from the group consisting of maltotriose (M3) and maltotetraose (M4), wherein said microorganism :

internalizes said maltooligosaccharide by an active transport

- comprises at least one heterologous genes encoding an enzyme modifying terminal non-reducing end of said internalized maltooligosaccharide, and is devoid enzymatic activities degrading maltooligosaccharides and modified maltooligosaccharides. 9. Method for producing modified maltooligosaccharides according to claim 8, wherein said microorganism comprises heterologous genes encoding at least a β1 ,4 galactosyltransferase and at least an additional glycosyltransferase, in particular selected from the group consisting of a-1 ,2-fucosyltransferase, a-1 ,3-fucosyltransferase a1 ,4- fucosyltransferase, β-1 ,3-N-acetylglucosaminyltransferase, β-1 ,4-N-acetyl- glucosaminyltransferase, β-1 ,6-N-acetylglucosaminyltransferase, β-1 ,3-galactosyl- transferase, β-1 ,4-galactosyltransferase, a-1 ,4-galactosyltransferase a-1 ,3-galactosyl- transferase, a-1 ,3-N-acetyl-galactosaminyltransferase, β-1 ,3-N-acetyl-galactosaminyl- transferase, a-1 ,4-N-acetylgalactosaminyltransferase, β-1 ,4-N-acetyl-galactosaminyl- transferase, a-2,3-sialyl-transferase, a-2,6-sialyl-transferase, a-2,8-sialyl-transferase, β- 1 ,3-glucuronosyl-transferase and combinations thereof.

10. Method according to claims 8 or 9, wherein said microorganism is E. coli strain which is preferably malFG+, malK+, and malPQZ-, lacZ- and contains the IgtE gene for β1 ,4 galactosyltransferase or derivative thereof, and is optionally nanKA-, lacA- and melA-, wcaJ-. Substituted galactosylated maltooligosaccharides (MD-ligands), susceptible to be used as donor substrate in the method of transglycosylation according to anyone of claims 1 to 7, of the foll

wherein

. n is comprised between 0 and 5,

. at least one of R1 , R2, R3, R4, and R5 is a monosaccharide or an

oligosaccharide, preferably a monosaccharide selected from the group consisting of fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and derivatives thereof. Substituted galactosylated maltooligosaccharides ( MD-ligands) according to claim 1 1 , wherein at least one of R1 , R2, R3, R4, and R5 is a monosaccharide selected from the groups consisting respectively of :

- for R3 : Gala- , GalNAcp-3Gala- , Galp-3GalNAcp-3Gala- , Fuca-2Galp- 3GalNAcp-3Gala-, GalNAca- , GlcNAcp- , Galp-4GlcNAcp- , Galp-4[Fuca- 3]GlcNAcp- , Fuca-2Galp-4[Fuca-3]GlcNAcp- , NeuAca-3Galp-4[Fuca- 3]GlcNAcp- , Fuca-2Galp^lGlcNAcp-, Gala-3Galp-4GlcNAcp- , Galp-4GlcNAcp- 3Galp-4GlcNAcp-Galp-3GlcNAcp- , Galp-3[Fuca-4]GlcNAcp- , Fuca-2Galp- 3[Fuca-4]GlcNAcp- , NeuAca-3Galp-3[Fuca-4]GlcNAcp- , Fuca-2Galp- 3GlcNAcp-, Gala-3Gaip-3GlcNAcp-, NeuAca- , NeuAc-8NeuAca-3, NeuAc- 8[NeuAc-8]_nNeuAca- (1 < n <400), GlcAp- , [GlcAp-4GlcNAca-4]_nGlcAp- with 1 < n <10000 (heparosan), [GlcAp-3GalNAcp-4]_nGlcAp- with 1 < n <10000 (chondroitin), or [GlcAp-3GlcNAcp-4]_nGlcAp- with 1 < n <10000 (hyaluronic acid) - for R₄ : Gala- , GalNAcp-3Gala- , Galp-3GalNAcp-3Gala- , GalNAca-3Galp- 3GalNAcp-3Gala-, Fuca-2Galp-3GalNAcp-3Gala-, GalNAcp- , Galp-3GalNAcp-, NeuAca-3Galb-3GalNAcp-, or NeuAc-8NeuAca-3Galb-3GalNAcp-,

- for R_{5 :} NeuAca- .

13. Substituted galactosylated maltooligosaccharides (MD-ligands) according to claim 12, wherein

R₂ is Fuca- and R₃ is GalNAca-

R₂ is Fuca- and R₃ is Gala- - R₃ is selected from the group consisting of Neu5Aca-3, Neu5Aca-8Neu5Aca- or

Neu5Aca-8 Neu5Aca-8Neu5Aca- and R₄ is selected from the group consisting of

GalNAcp-, Galp-3GalNAcp- NeuAca-3Galb-3GalNAcp-, or NeuAc-8NeuAca-

3Galb-3GalNAcp-

Ri is Fuca- and R₂ is Fuca- - R_! is Fuca- and R₃ is NeuAc- or Gala- .

14. Glycoconjugates or multivalent carbohydrates structures obtained by a method according to anyone of claims 1 to 7 and/or MD ligands as produced according to the method of claims 8 to 10 or as defined according to method of claims 1 1 to 13, for use in at least one application selected from the group consisting of therapeutical applications, nutritional supplements, bioactive detergent, diagnostic, medical device, cosmetic, affinity purification process, and combinations thereof. 15. Pharmaceutical composition comprising at least one glycoconjugate or multivalent carbohydrate structure obtained by a method according to anyone of claims 1 to 7 and/or at least one MD ligand as produced according to the method of claims 8 to 10 or as defined according to method of claims 1 1 to 13 and a pharmaceutically acceptable vehicle.