Sialylation method

Abstract

The present invention relates to a novel and efficient method for the sialylation of a glycoside, the method comprising mixing said glycoside with sialic acid, cytidine monophosphate, a nucleoside triphosphate and one or more cell-free extracts of a microorganism, said microorganism comprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein said one or more cell-free extracts comprise: at least one polypeptide having cytidine monophosphate kinase activity, at least one polypeptide having N / -acylneuraminate citydyltransferase activity, and at least one polypeptide having sialyltransferase activity, thereby sialylating said glycoside.

Description

[0001] DESCRIPTION SIALYLATION METHOD Field of the invention The present invention relates to a novel and efficient method for the sialylation of glycosides or analogues thereof. Background Glycosylation reactions are widespread in nature and are critical for physiological and pathological cellular functionality. In fact, glycosylation reactions often confer additional specific biological function to the glycosylated products. For example, certain carbohydrate moieties play important functional roles in the modulation of various biological processes, such as cell–cell recognition, communication, and intercellular adhesion. An important type of glycosylation reaction is the sialylation reaction, wherein one or more sialic acid units are added to a biomolecule such as an oligosaccharide, a lipid, or a protein. Sialic acid is a unique monosaccharide. It belongs to the family of nine-carbon atom sugars, and at physiological pH it possesses a negative charge. Furthermore, sialic acid can be modified at several positions by acetyl, sulfate, and other groups. Sialic acid occurs naturally at the non-reducing end of saccharide chains of biomolecules such as oligosaccharides, glycoproteins, glycolipids, and typically it is responsible for their in-vivo activity. In humans, sialic acid occurs in the brain wherein is an essential part of ganglioside structures. Particularly, gangliosides participate in synaptogenesis and neural transmission, as well as in neurological diseases especially Alzheimer’s, Parkinson’s, and Huntington’s diseases (Chiricozzi E. et al., Int. J. Mol. Sci.2020, 21, 868). Furthermore, certain gangliosides are found in the intestinal mucosa and can promote intestinal health, as well as function as anti-infective agents (E. J. Park et al., Glycobiology 2005, 15,.935–942). Human milk also contains sialic acid bound to the terminal end of free oligosaccharides or glycolipids such as lactose or lactosyl ceramide. Particularly, human milk oligosaccharides such as 3’-sialyl lactose or human milk gangliosides such as GM3 and GD3 contribute to the antiviral, anti-inflammatory and immunomodulatory properties of human milk (Quitadamo et al., Frontiers in Public Health 2021, 8, article 589736). Sialylated biomolecules, and oligosaccharides hold great potential as therapeutics, and as food ingredients. However, they are not readily available for fundamental and clinical research. In fact, they are characterized by a high structural complexity and their preparation represents a challenge. Numerous attempts have been made to develop methods for the sialylation of glycosides. For example, sialylated saccharides and glycosphingolipids may be obtained via chemical synthesis (J. A. Morales-25 Serna, Carbohydr. Res.2007, Yukishige et al., Tetrahedron 1990, 46, 89-102). Drawbacks connected to this approach are the control of stereo- and regiochemistry, the need of multiple protecting group manipulations, difficult purification and scale-up. Alternatively, enzymatic synthesis may be utilized for the sialylation of saccharides and glycolipids. Enzymatic synthesis offers many advantages over purely chemical routes, such as high regio- and stereo- chemical control, it does not require the use of protecting group manipulations, and it is typically performed under mild conditions. A common approach for the enzymatic sialylation of glycosides is based on the use of sialyltransferase enzymes, wherein sialic acid is transferred from CMP-sialic acid to a glycoside acceptor. CMP-sialic acid is a relatively expensive and unstable reagent and methods have been described wherein the sugar nucleotide is generated and / or regenerated in situ (Yu et al., Org Biomol Chem.2018, 4076–4080, WO9928491). Drawbacks connected to these approaches comprise the use of multiple purified enzymes, and expensive reagents such as cytidine triphosphate and phosphoenolpyruvate rendering the scale-up difficult. Accordingly, there is a demand for the development of novel methodologies characterized by high technological feasibility and low costs, which enable the efficient and large-scale production of sialylated biomolecules and their analogues. Summary of the invention In a first aspect the present invention relates to a method for the sialylation of a glycoside of formula (1), or a salt thereof: (1), wherein X is a glycosyl moiety, wherein the glycosyl moiety is preferably selected from the group consisting of Gal1-, or a glycosyl moiety carrying one or more terminal galactose units and / or one or more terminal N-acetyl-galactosamine units and / or one or more terminal sialic acid units; Y is selected from the group consisting of hydroxyl, fluoride, or a moiety of formula (2), or a salt thereof: wherein R1is hydrogen, aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17alkyl, more preferably a substituted or unsubstituted C10-17alkyl, R2is hydrogen or -OR5, wherein R5is selected from hydrogen, a substituted or unsubstituted C1-6alkyl, or a substituted or unsubstituted C2-6acyl, the bond may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -OR5, R3is hydrogen, a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C1-6 acyl, preferably hydrogen, R4is selected from hydrogen, a substituted or unsubstituted aryl, a heteroalkyl, a substituted or unsubstituted C2-32acyl, the method comprising: mixing the glycoside of formula (1) with sialic acid, cytidine monophosphate, a nucleoside triphosphate and one or more cell-free extracts of a microorganism, said microorganism comprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein said one or more cell-free extracts comprise: ─ at least one polypeptide having cytidine monophosphate kinase activity, ─ at least one polypeptide having N-acylneuraminate citydyltransferase activity, and ─ at least one polypeptide having sialyltransferase activity, thereby sialylating said glycoside. In a second aspect the present invention relates to a sialylating agent comprising one or more cell-free extracts of a microorganism, said microorganism comprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein said one or more cell-free extract comprise: ─ at least one polypeptide having cytidine monophosphate kinase activity, ─ at least one polypeptide having N-acylneuraminate citydyltransferase activity, and ─ at least one polypeptide having sialyltransferase activity. Brief Description of the figures Figure 1: Schematic diagram of a sialyltransferase cycle wherein CMP-Neu5Ac is generate / regenerated. Figure 2: Schematic diagram of a sialyltransferase cycle wherein CMP-Neu5Ac is generate / regenerated, and ATP is regenerated. Detailed Description of the invention The present invention describes a novel and efficient method for the in vitro sialylation of biomolecules, and analogues thereof catalysed by sialyl transferase enzymes and wherein the expensive nucleotide donor is generated in-situ and regenerated during the sialylation cycle. Particularly, the method is characterised by the use of one or more cell-free extracts of a microorganism which comprise all the enzymes required for the sialylation cycle, and wherein the microorganism endogenous enzymatic activities are harnessed. Advantages connected to method described herein comprise avoiding additional purification procedures targeted to isolate enzymes, and the reduction of the number of specific enzymes used during the sialylation cycle by exploiting the activity of those which are naturally present in the microorganism cell free extract. Furthermore, the method makes use of inexpensive reagents. Therefore, the method is suited for the large-scale production of sialylated glycosides and saccharides such gangliosides, sialylated glycosyl fluorides, and human milk oligosaccharides, the method comprising mixing a glycoside of formula (1), or a salt thereof: (1), wherein X is a glycosyl moiety, wherein the glycosyl moiety is preferably selected from the group consisting of Gal1-, or a glycosyl moiety carrying one or more terminal galactose units and / or one or more terminal N-acetyl-galactosamine units and / or one or more terminal sialic acid units; Y is selected from the group consisting of hydroxyl, fluoride, or a moiety of formula (2), or a salt thereof: (2), wherein R1is hydrogen, aryl, or a C1-50 alkyl, preferably a C1-17 alkyl, more preferably a C10-17 alkyl, which may be saturated or contain one or more double and / or triple bonds, and / or which may contain one or more functional groups, the functional group being preferably selected from the group consisting of a hydroxyl group, an amino group, an alkoxy group, an acyloxy group, an acylamido group, a thiol, a thioether or a phosphorus-containing functional group, R2is hydrogen or -OR5, wherein R5is selected from hydrogen, a substituted or unsubstituted C1-6alkyl, or a substituted or unsubstituted C2-6acyl, the bond may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -OR5, R3is hydrogen, a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C1-6 acyl, preferably hydrogen, R4is selected from hydrogen, a substituted or unsubstituted aryl, a heteroalkyl, a substituted or unsubstituted C2-32acyl, the method comprising: mixing the glycoside of formula (1) with sialic acid, cytidine monophosphate, a nucleoside triphosphate and one or more cell-free extracts of a microorganism, wherein said microorganism comprising one or more endogenous polypeptide having inorganic diphosphatase activity and one or more endogenous polypeptide having phosphotransferase activity, and wherein the said one or more cell-free extract(s) comprise: ─ at least one polypeptide having cytidine monophosphate kinase activity, ─ at least one polypeptide having N-acylneuraminate citydyltransferase activity, and ─ at least one polypeptide having sialyltransferase activity, thereby sialylating said glycoside. Non-limiting embodiments of different aspects of the invention are described below and illustrated by non-limiting examples. The terms, definitions and embodiments described throughout the specification of the invention relate to all aspects and embodiments of the invention. The term “a” grammatically is a singular, but it may as well mean the plural of e.g., the intended compound. For example, a skilled person would understand that in the expression “a glycoside”, the provision of not only one single glycoside, but of a variety of glycosides of the same type is meant. As used herein, the term “alkyl” refers to an acyclic straight or branched hydrocarbyl group having 1-50 carbon atoms which may be saturated or contain one or more double and / or triple bonds (so, forming for example an alkenyl or an alkynyl), and / or which may be substituted or unsubstituted, as herein further described. Examples of “alkyl” include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, n-pentyl, neo-pentyl, n-hexyl, ethenyl, propenyl, 1- butenyl, 2-butenyl, isobutenyl,1-pentenyl, 2-pentenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 2- methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, methylpentenyl, dimethylbutenyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, pentynyl, and hexynyl, each of which may be substituted or unsubstituted. Typically, the term alkyl refers to a straight saturated acyclic hydrocarbyl group having 1-31 carbons, which may be substituted or unsubstituted. As used herein, the term “aryl” refers to an aromatic cyclic hydrocarbyl group having 5-14 ring carbon atoms, which may be mono- or polycyclic, which may contain fused rings, preferably 1 to 3 fused or unfused rings, and which may contain one or more heteroatoms, and / or which may be substituted or unsubstituted, as herein further described. Examples of “aryl” include, but are not limited to, phenyl, naphtyl, anthracyl, phenantryl, pyrrolyl, imidazolyl, thiophenyl, furanyl, oxazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, and benzofuranyl, each of which may be substitute or unsubstituted. Typically, the term “aryl” refers to a substituted or unsubstituted phenyl. As used herein, the term “acyl” refers to a group derived by the removal of one or more hydroxyl group from an oxoacid, preferably from a carboxylic acid. The acyl group according to the present invention is typically a saturated or unsaturated C2-32 acyl, which may be substitute or unsubstituted. As used herein, the term “substituted” means that the group in question is substituted with a group which typically modifies the general chemical characteristics of the group in question. The substituents can be used to modify characteristics of the molecule, such as molecule stability, molecule solubility and the ability of the molecule to form crystals. The person skilled in the art will be aware of other suitable substituents of a similar size and charge characteristics, which could be used as alternatives in a given situation. In connection with the terms “alkyl”, “aryl”, and “acyl” the term substituted means that the group in question is substituted one or several times, preferably 1 to 3 times, with group(s) selected from hydroxy (which when bound to an unsaturated carbon atom may be present in the tautomeric keto form), oxo, C1-6-alkoxy (i.e. C1-6-alkyl-oxy), C2-6-alkenyloxy, carboxy, oxo, C1-6-alkoxycarbonyl, C1-6- alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylamino, arylcarbonyl, heteroaryl, heteroarylamino, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di (C1-6- alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)aminocarbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di (C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkylcarbonylamino, cyano, guanidino, carbamido, C1-6-alkyl-sulphonyl-amino, aryl-sulphonyl-amino, heteroaryl-sulphonyl-amino, C1-6-alkanoyloxy, C1-6- alkyl-sulphonyl, C1-6-alkyl-sulphinyl, C1-6-alkylsulphonyloxy, nitro, C1-6-alkylthio, halogen, where any alkyl, alkoxy, and the like representing substituents may be substituted with hydroxy, C1-6-alkoxy, C2-6- alkenyloxy, carboxy, C1-6-alkylcarbonylamino, halogen, C1-6-alkylthio, C1-6-alkyl-sulphonyl-amino, or guanidino. In connection with the term “alkyl” the term “substituted” preferably means that the group in question is substituted one or several times, preferably 1 to 3 times, with group(s) selected from a hydroxyl group, an alkoxy group, an acyloxy group, an acylamido group, a thiol, a thioether or a phosphorus- containing functional group. In connection with the term polypeptide the term “functional analogue” refers to a protein wherein the amino acid sequence has a certain percent homology compared to the amino acid sequence of a reference protein (i.e. about 30% homology, preferably 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology over a specified region, for example over a region of at least about 25, 50, 75, 100, 150, 200, 250, 500, 1000, or more amino acids, up to the full length sequence, when compared and aligned for maximum correspondence over a comparison window or designated region) and maintains the same functional activity of the reference protein. The percent homology may be determined using e.g. a BLAST sequence comparison algorithm, or by manual alignment and visual inspection (see e.g. NCBI website http: / / www.ncbi.nlm.nih.gov / BLAST / or the like). Such sequences may be termed “substantially identical”. Typically, the term functional analogue refers to a mutant protein, a truncated variant of the protein, or to a fusion protein which maintains the same functional activity of the reference protein. Amino acid sequences are herein typically defined by the commonly used one-letter code or by their three-letter code, as summarized in Table 1. Table 1 Amino acid codes: Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V The skilled person will understand that in formulas showing a specific compound, like for example compounds of formulas (2), or (3) unless the chemical formula expressly describes a carbon atom having a particular stereochemical configuration, the formula is intended to cover compounds where such a stereocenter has an R or an S configuration, or wherein a double bond has a cis or a trans configuration. The skilled person would understand that when speaking of position C-1, C-2, C-3, C-4, C-5 etc., reference is herein always made to the respective carbon atoms of compounds such as those represented by formula (3), or moieties such as those represented by formula (2). In the context of the present invention, the terms “about”, “around”, or “approximate” are applied interchangeably to a particular value (e.g. “a pH of about 7.0”, “a pH around 7.0”, or “a pH of approximate 7.0”), or to a range (e.g. “a conversion from about 10% to about 99%”, “a conversion from around 10% to around 99%”, or “a conversion from approximate 10% to approximate 99%” ), to indicate a deviation from 0.1% to 10% of that particular value or range. The term “isolating”, in the context of the present invention, refers to a procedure or a step of the procedure that is applied to separate the desired compound from a mixture comprising said desired compound and other compounds. In this context, the other compounds of the mixture are regarded as contaminants. The term "isolation” and “isolating” may be used interchangeably. The term “sialic acid” refers to any member of a family of nine-carbon carboxylated sugars. The most common member of the sialic acid family is N-acetyl-neuraminic acid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of Neu5Ac is hydroxylated. A third sialic acid family member is 2-keto-3-deoxy- nonulosonic acid (KDN). Also included are 9-substituted sialic acids such as a 9-O-C1-C6acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of the sialic acid family. Preferably, in the context of the present invention, the term sialic acid refers to N-acetyl-neuraminic acid (Neu5Ac). N-Acetyl-neuraminic acid can be synthesised by method known to skilled person such as for example the method described in US2011165626 (A1). As used herein, the term “cell-free extract” refers to a mixture of biomolecules (e.g. proteins, nucleic acids, etc.) and cell debris (e.g. membranes, organelles, etc.) and not to living cells. Preferably, cell-free extracts lack the genetic material and membranes inherent to the living cells and comprises the components necessary to carry out the desired biochemical process. Typically, cell-free extracts are prepared by destroying biological cells, e.g., by chemical or mechanical cell lysis. Cell lysis may be performed by methods known to the person skilled in the art, such as those for example described by Cole et al. Synthetic and Systems Biotechnology 2020, 5, 252–267. In connection with the term microorganism the term “genetically engineered” means that the microorganism comprises genetic material which does not constitute part of the organism genome in nature, i.e., wild-type genome. A genetically engineered microorganism is e.g., a microorganism comprising at least one alteration in the microorganism own DNA sequence which has been performed artificially, i.e., by genomic manipulation in a lab, in order to give that microorganism a desired specific phenotype. The alteration in the DNA may e.g., be an introduction or a deletion of a DNA fragment in the genome, or an introduction of an expression vector carrying an endogenous or heterologous gene in the cell. The alteration in the DNA sequence is herein especially achieved by the expression of a heterologous nucleic acid sequence, in particular a heterologous nucleic acid sequence encoding a specific polypeptide. Genome editing may be performed e.g., by commonly known recombinant nucleic acid techniques as e.g., described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The CRISPR technology may also be used to perform genetic modifications. The term "heterologous" is understood to mean, not naturally occurring within or not native to the specified host microorganism. When used herein to describe a polypeptide or a polypeptide sequence, the term heterologous includes, for example, polypeptides which are not naturally produced by a specific microorganism, synthetic or otherwise non-naturally occurring polypeptides, and / or sequences thereof, and any polypeptide sequence which is purposefully manipulated to achieve a non-naturally occurring level or activity within a defined host cell or microorganism. The term “glycoside” when used herein refers to a chemical compound wherein a glycosyl moiety is bound to a non-sugar chemical moiety via a glycosidic linkage. The glycosyl moiety may be referred to as “glycone”, and the non-sugar chemical moiety may be referred to as the “aglycone”. The “glycone” may consist of a single sugar unit (monosaccharide), two sugar units (disaccharide), or several sugar units (oligosaccharide). In the context of the present invention, a compound of formula (1) represents a “glycoside” wherein the glycosyl moiety (glycone) X is bound via a glycosidic linkage to the aglycone Y. The glycosidic linkage may be an alpha (α) or a beta (β) glycosidic linkage. In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety Gal1-. In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal galactose units. In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal N-acetyl-galactosamine units. In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal sialic acid units, or a salt thereof. In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal galactose units, one or more terminal N-acetyl-galactosamine units, and one or more terminal sialic acid units. In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal galactose units and one or more terminal N-acetyl-galactosamine units. In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal galactose units and one or more terminal sialic acid units. In some embodiments, X of the glycoside of formula (1) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal N-acetyl-galactosamine units and one or more terminal sialic acid units. In some embodiment Y of the glycoside of formula (1) is selected from the group consisting of a hydroxyl group, a fluoride, or a moiety of formula (2), or a salt thereof. In some embodiments, Y of the glycoside of formula (1) is a hydroxyl group. Accordingly, in some embodiments the glycoside of formula (1) is a saccharide. In some embodiments, Y of the glycoside of formula (1) is a fluoride. Accordingly, in some embodiments, the glycoside of formula (1) is a glycoside of formula (4): X-F (4), wherein X is a glycosyl moiety as defined as for the glycoside of formula (1). Glycosides of formula (4) may also be referred to as glycosyl fluorides. In some embodiments, the glycoside of formula (4) is a an α-glycosyl fluoride. In some preferred embodiments, Y of the glycoside of formula (1) is a moiety of formula (2), or a salt thereof. Accordingly in some preferred embodiments, the glycoside of formula (1) is a glycoside of formula (3), or a salt thereof: (3), wherein X is as defined as for the glycoside of formula (1); R1is hydrogen, aryl, or a substituted or unsubstituted C1-50alkyl, preferably a substituted or unsubstituted C1-17alkyl, more preferably a substituted or unsubstituted C10-17alkyl; R2is hydrogen or -OR5, wherein R5is selected from hydrogen, a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C2-6 acyl; the bond may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -OR5; R3is hydrogen, a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C1-6 acyl, preferably hydrogen; R4is selected from hydrogen, a substituted or unsubstituted aryl, a heteroalkyl, a substituted or unsubstituted C2-32acyl. In some embodiments, for the glycoside of formula (3) R1is a saturated unsubstituted C10-17 alkyl, R2is -OR5, wherein R5is hydrogen, R3and R4are hydrogen, and the bond is a single bond. In some embodiments, for the glycoside of formula (3) R1is a saturated unsubstituted C10-17 alkyl, R2, R3and R4are hydrogen, and the bond is a single bond. In some embodiments, for the glycoside of formula (3) R1is a C10-171-hydroxyalkyl, R2, R3and R4are hydrogen, and the bond is a double bond. In some embodiments, the glycoside of formula (3) is a glycoside selected from the group consisting of glycosides of formulas (5), (6), (7), and (8): wherein X is a glycosyl moiety defined as for the glycoside of formula (1). In some embodiments, X of the glycoside of formula (3) is a glycosyl moiety, wherein the glycosyl moiety Gal1-. In some embodiments, X of the glycoside of formula (3) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal galactose units. In some embodiments, X of the glycoside of formula (3) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal N-acetyl-galactosamine units. In some embodiments, X of the glycoside of formula (3) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal sialic acid units, or a salt thereof. In some embodiments, X of the glycoside of formula (3) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal galactose units, one or more terminal N-acetyl-galactosamine units, and one or more terminal sialic acid units. In some embodiments, X of the glycoside of formula (3) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal galactose units and one or more terminal N-acetyl-galactosamine units. In some embodiments, X of the glycoside of formula (3) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal galactose units and one or more terminal sialic acid units. In some embodiments, X of the glycoside of formula (3) is a glycosyl moiety, wherein the glycosyl moiety carrying one or more terminal N-acetyl-galactosamine units and one or more terminal sialic acid units. In some embodiments, for the glycoside of formula (3) R1is a saturated unsubstituted C10-C17 alkyl, R2, and R3are hydrogen, R4is a substituted or unsubstituted C16-32 acyl, and the bond is a double bond. In some embodiments, for the glycoside of formula (3) R1is a saturated unsubstituted C10-17 alkyl, R2is - OR5, wherein R5is hydrogen, R3is hydrogen, R4is a substituted or unsubstituted C16-32acyl, and the bond is a single bond. In some embodiments, for the glycoside of formula (3) R1is a saturated unsubstituted C10-C17alkyl, R2, and R3are hydrogen, R4is a substituted or unsubstituted C16-32 acyl, and the bond is a single bond. In some embodiments, for the glycoside of formula (3) R1is a C10-C171-hydroxyalkyl, R2, and R3are hydrogen, R4is a substituted or unsubstituted C16-32 acyl, and the bond is a double bond. In some embodiments, the glycoside of formula (3) is a glycoside selected from the group consisting of glycosides of formulas (9), (10), (11), and (12): wherein X is a glycosyl moiety as defined as for the glycoside of formula (1). Glycosides of formula (3), and of formula (5)-(12) may also be referred to as glycosphingolipids. The term “glycosphingolipid”, as used herein, refers to compounds that structurally consist of a glycosyl moiety and a sphingolipid moiety, or analogues thereof. The glycosyl moiety is typically linked to the sphingolipid moiety via a glycosidic bond between the anomeric carbon at the reducing end of the glycosyl moiety and the hydroxyl group at the C-1 position of the sphingolipid. The sphingolipid moiety of the glycosphingolipid of the present invention typically derives from an aliphatic amino alcohol such as a sphingoid base or a ceramide. Sphingoid bases denote in the context of the present invention naturally occurring sphingoid bases, analogues thereof or derivatives thereof. Naturally occurring sphingoid bases are D-erythro-sphingosine (S), 6-Hydroxy-D-erythro-sphingosine (H), D-ribo-phytosphingosine (P) or DL-erythro-dihydrosphingosine (DS), wherein the number of sphingoid carbons may be expressed in parenthesis following the letters S, H, P, and DS. The letters S, H, P, and DS refer to the shorthand nomenclature developed by Motta et al., Biochim Biophys Acta.1993, 1182:147-151 and expanded by Rabionet, Biochim Biophys Acta 2014, 1841:422-434 and by Masukawa et al., Journal of Lipid Research 2008, 49, 1466-1476.D-Erythro-dihydrosphingosine may also be represented by the letter G according to the INCI nomenclature. Ceramides denote in the context of the present invention naturally occurring ceramides, analogues thereof or derivatives thereof. Preferred ceramides are those naturally occurring in humans. Naturally occurring human ceramides [CER] include, but are not limited to, CER[NS], CER[AS], CER[EOS], CER[NH], CER[AH], or CER[EOH], CER[NP], CER[AP], or CER[EOP], CER[NDS], CER[ADS], or CER[EODS]. The letters in brackets refer to the shorthand nomenclature developed by Motta et al., Biochim Biophys Acta.1993, 1182, 147-151 and expanded by Rabionet, Biochim Biophys Acta 2014, 1841, 422-434 and by Masukawa et al., Journal of Lipid Research 2008, 49, 1466-1476. Particularly, the letters N, A, and EO represent non-hydroxy fatty acids (N), alpha-hydroxy fatty acids (A), and omega-linoleoyloxy fatty acids (EO), respectively, wherein the number of fatty acid carbons and unsaturations may be expressed in parentheses following the letters of N, A, E, and O. The letters, S, H, P, and DS represent D-erythro- sphingosine (S), 6-hydroxy-D-erythro-sphingosine (H),D-ribo-phytosphingosine (P),D-erythro- dihydrosphingosine (DS), respectively, wherein the number of sphingoid carbons may be expressed in parenthesis following the letters S, H, P, and DS. Ceramides, CER[NDS], CER[ADS], or CER[EODS], may also be referred to as CER[NG], CER[AG], or CER[EOG], respectively, wherein the letter G represents the INCI name for D-erythro-dihydrosphingosine. The glycosyl moiety of the glycoside according to the present invention may derive from a monosaccharide or from an oligosaccharide (more than one monosaccharide units), wherein the anomeric carbon of the monosaccharide or the anomeric carbon at the reducing end of the oligosaccharide is engaged in a glycosidic bond with another chemical entity, such as a sphingolipid, and the bond, if not further specified, may be an alpha or a beta glycosidic bond. A glycosyl moiety having more than one monosaccharide unit may represent a linear or a branched structure. The monosaccharide unit is preferably any 5-9 carbon atom sugar, comprising aldoses (e.g. D-glucose, D- galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), ketoses (e.g. D-fructose, D- sorbose, D-tagatose, etc.), deoxysugars (e.g. L-rhamnose, L-fucose, etc.), deoxy-aminosugars (e.g. N- acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acids, ketoaldonic acids (e.g. sialic acid). The monosaccharide unit can form different cyclic structures such as pyranose (six- membered) cyclic structures or furanose (five-membered) cyclic structures. The glycosyl moieties according to the present invention may be illustrated in the following style: Galβ1- 4Glc1-, wherein the dash (-) represents the point of attachment of the glycosyl moiety and wherein the glycosyl moiety, may be linked via an alpha or a beta glycosidic bond. In some embodiments, for a glycoside of formula (1), (3), or (4), or (5)-(12) the glycosyl moiety X is a glycosyl moiety selected from the group consisting of Gal1-, or a glycosyl moiety carrying one or more terminal galactose units and / or one or more terminal N-acetyl-galactosamine units and / or one or more terminal sialic acid units. In some preferred embodiments, for a glycoside of formula (1), (3), or (4), or (5)-(12) the glycosyl moiety X is a glycosyl moiety selected from the group consisting of Gal1-, Galβ1-4Glc1-. In some preferred embodiments, for a glycoside of formula (1), (3), or (4), or (5)-(12) the glycosyl moiety X is a glycosyl moiety selected from the group consisting of the following glycosyl moieties, or salts thereof Neu5Acα2-3Galβ1-4Glc-, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-. In some preferred embodiments, the glycoside of formula (1) is a glycoside of formula (3), and wherein the glycoside of formula (3) is a glycoside of formula (5). In some preferred embodiments, the glycoside of formula (1) is a glycoside of formula (5). In some embodiments, X of the glycoside of formula (5) is selected from the group consisting of Gal1-, or Galβ1-4Glc1-. Accordingly, in some preferred embodiments, the glycoside of formula (3) is a glycoside of formula (5), wherein the glycoside of formula (5) is selected form the group consisting of psychosine, or lactosyl sphingosine. In some preferred embodiments, X of the glycoside of formula (5) is selected from the group consisting of Neu5Acα2-3Galβ1-4Glc-, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc1-, or salts thereof. Accordingly, in some preferred embodiments, the glycoside of formula (3) is a glycoside of formula (5), wherein the glycoside of formula (5) is selected form the group consisting of N-lyso-GM3, and N-Lyso-GM1a. N-Lyso-GM3, and N-Lyso-GM1a, represent lysosphingolipids. Lysosphingolipids, are typically defined as sphingolipid breakdown products which lack the amide-linked fatty acyl group at the 2-position of the sphingoid base backbone. Accordingly, for each parental sphingolipid there is a corresponding lysosphingolipid that has an identical head group at the 1-position but lacks the amide-bound fatty acyl group at the 2-position (Hannun et al., Science 1989, 243, 500-507). In some embodiments, the sialylation of the glycoside according to the present invention is performed in the presence of a cyclodextrin. The term “cyclodextrin”, in the context of the present invention, refers to a cyclic oligosaccharide consisting of a macrocyclic ring of monosaccharide subunits (e.g., glucose). Cyclodextrins, typically contain 6-, 7- or 8-monosaccharide subunits and may be referred to as α-cyclodextrins, β-cyclodextrins, and γ-cyclodextrins, respectively. The cyclodextrin may be modified such that some or all of the primary or secondary hydroxyl groups of the macrocycle, or both, may be alkylated or acylated. Methods of modifying these alcohols are well known to the person skilled in the art and many derivatives are commercially available. Thus, some or all of the hydroxyl groups of the cyclodextrin may be substituted with an -OR6group and / or an O-C(=O)-R7group, wherein R6and R7are independently selected from a saturated or unsaturated C1-6 alkyl, a saturated or unsaturated C1-6 heteroalkyl, a saturated or unsaturated cycloalkyl, a saturated or unsaturated heterocycloalkyl, an aryl, or a heteroaryl, each of which may be substituted or unsubstituted. In some embodiments, R6and R7are independently selected from the group consisting of 2-hydroxyethyl, 2-hydroxypropyl, and sulfobutylether. In some embodiments, the cyclodextrin is α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or derivatives thereof. In some embodiments, the cyclodextrin is selected from the group consisting of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, randomly methylated β-cyclodextrin, or sulfobutylether-β-cyclodextrin. In some preferred embodiments, the cyclodextrin is β-cyclodextrin. The present invention describes a method for the sialylation of a glycoside, wherein the sialylation is typically carried out as part of a sialyltransferase cycle, which comprises a CMP-sialic acid recycling system, wherein CMP-sialic acid is generated / regenerated from sialic acid and CMP. CMP-sialic acid is a relative expensive sugar nucleotide. Therefore, the in-situ generation and regeneration of the sialic acid donor is of economic advantage and enables the scale up of the process. The sialyltransferase cycle described in the present invention, typically comprises sialic acid, cytidine monophosphate (CMP), a nucleoside triphosphate, and the use of one or more cell-free extract(s) of a microorganism, wherein the one or more cell-free extracts comprising the enzymatic activities needed for the sialyltransferase cycle. The enzymatic activities needed for the sialyltransferase cycle comprise: ─ at least one phosphotransferase enzymatic activity ─ at least one inorganic diphosphatase enzymatic activity, ─ at least one cytidine monophosphate kinase enzymatic activity, ─ at least one N-acylneuraminate cytidylyltransferase enzymatic activity, and ─ at least one sialyl transferase enzymatic activity. Nucleoside triphosphates suitable for use in the context of the present invention are adenosine-5'- triphosphate (ATP), uridine-5´-triphosphate (UTP), guanosine-5´-triphosphate (GTP), inosine triphosphate (ITP) and thymidine-5´-triphosphate (TTP). In some preferred embodiments, the nucleoside triphosphate is adenosine-5'-triphosphate (ATP). Accordingly in some preferred embodiments the sialyltransferase cycle comprises N-acetyl-neuraminic acid (Neu5Ac), cytidine monophosphate (CMP), adenosine 5'-triphosphate (ATP), and one or more cell- free extract of a microorganism, wherein the one or more cell-free extract comprising one polypeptide having cytidine monophosphate kinase activity (CMK) (for the phosphorylation of CMP), one polypeptide having phosphotransferase activity (for the phosphorylation of CDP), one polypeptide having N-acylneuraminate cytidyltransferase activity (CSS) (for the transfer of CMP from CTP to Neu5Ac), one polypeptide having sialyltransferase activity (for the transfer of Neu5Ac from CMP- Neu5Ac to the acceptor substrate), and one polypeptide having inorganic diphosphatase activity (PPase) (to degrade the inorganic pyrophosphate (PPi) formed during the cycle). And wherein, the polypeptide having phosphotransferase activity, and the polypeptide having inorganic diphosphatase activity (PPase) are endogenously expressed by the microorganism. The sialyltransferase cycle described in this preferred embodiment is depicted in Figure 1. In some embodiments, the sialyltransferase cycle further comprises the regeneration of ATP, wherein ATP is regenerated by using a source of phosphate and a polypeptide having kinase activity. Sources of phosphate that can be used for the regeneration of ATP include but are not limited to polyphosphate, phosphoenol pyruvate, and acetyl phosphate. The selection of a particular kinase for use in the regeneration of ATP depends upon the phosphate sourced employed. In some embodiments ATP is regenerated by using polyphosphate as the source of phosphate and a polypeptide having polyphosphatase kinase activity. Accordingly, in some embodiments the sialyltransferase cycle comprises N-acetyl-neuraminic acid (Neu5Ac), cytidine monophosphate (CMP), adenosine 5'-triphosphate (ATP), polyphosphate, and one or more cell-free extract(s) of a microorganism, wherein the one or more cell-free extract comprising a polypeptide having cytidine monophosphate kinase activity (CMK) (for the phosphorylation of CMP), a polypeptide having phosphotransferase activity (for the phosphorylation of CDP), a polypeptide having N-acylneuraminate cytidyltransferase activity (CSS) (for the transfer of CMP from CTP to Neu5Ac), a polypeptide having sialyltransferase activity (for the transfer of Neu5Ac from CMP-Neu5Ac to the acceptor substrate), a polypeptide having polyphosphatase kinase activity (PPK) (for the phosphorylation of ADP), and a polypeptide having inorganic diphosphatase activity (PPase) (to degrade the inorganic pyrophosphate (PPi) formed during the cycle. And wherein, the polypeptide having phosphotransferase activity, and the polypeptide having inorganic diphosphatase activity (PPase) are endogenously expressed by the microorganism. The sialyltransferase cycle described in this embodiment is depicted in Figure 2. In some preferred embodiments, the one or more cell-free extract(s) have reduced or no β- galactosidase activity. Typically, the one or more cell-free extracts are of a microorganism, wherein said microorganism comprising at least one endogenous polypeptide having phosphotransferase enzymatic activity, and at least one endogenous polypeptide having diphosphatase enzymatic activity, and wherein the said microorganism is genetically engineered for the expression of one or more polypeptides selected fromthe group consisting of: ─ at least one polypeptide having cytidine monophosphate kinase activity, ─ at least one polypeptide having N-acylneuraminate citydyltransferase activity, ─ at least one polypeptide having sialyl-transferase activity. In some embodiments, the one or more cell-free extract(s) are of a microorganism, wherein said microorganism is genetically engineered for the expression and / or overexpression of one or more polypeptides selected fromthe group consisting of: ─ at least one endogenous polypeptide having phosphotransferase enzymatic activity, ─ at least one endogenous polypeptide having diphosphatase enzymatic activity, ─ at least one polypeptide having cytidine monophosphate kinase activity, ─ at least one polypeptide having N-acylneuraminate citydyltransferase activity, ─ at least one polypeptide having sialyl-transferase activity. The at least one polypeptide having cytidine monophosphate kinase activity, the at least one polypeptide having N-acylneuraminate cytidyltransferase activity, and at the least one polypeptide having sialyl transferase activity may be endogenous polypeptides or heterologous polypeptides. In some embodiments, the one or more cell-free extract(s) are of a microorganism, wherein said microorganism comprising at least one endogenous polypeptide having phosphotransferase enzymatic activity, and at least one endogenous polypeptide having diphosphatase enzymatic activity, and wherein the said microorganism is genetically engineered for the expression of one or more polypeptides selected from: ─ at least one heterologous polypeptide having cytidine monophosphate kinase activity, ─ at least one heterologous polypeptide having N-acylneuraminate citydyltransferase activity, ─ at least one heterologous polypeptide having sialyl-transferase activity. In some embodiments, the one or more cell-free extract(s) are of a microorganism, wherein said microorganism is genetically engineered for the expression and / or overexpression of one or more polypeptides selected from: ─ at least one endogenous polypeptide having phosphotransferase enzymatic activity, ─ at least one endogenous polypeptide having diphosphatase enzymatic activity, ─ at least one heterologous polypeptide having cytidine monophosphate kinase activity, ─ at least one heterologous polypeptide having N-acylneuraminate citydyltransferase activity, ─ at least one heterologous polypeptide having sialyl-transferase activity. The microorganism according to the present invention preferably comprises reduced or no β- galactosidase activity. The reduction or knock-out of the β-galactosidase activity may be achieved e.g., by genetic manipulation of a gene encoding a polypeptide with β-galactosidase activity, e.g., by introduction of a mutation leading to expression of inactive enzyme, to a gene knock out or by other means. The microorganism according to the present invention may be a yest or a bacterium, preferably a bacterium. In some embodiments, the microorganism is Escherichia coli (E. coli). In some embodiments, the microorganism is an E. coli BL21 (DE3) strain. The person skilled in the art will understand that in the context of the present invention, the term “a microorganism” is meant to encompass a living cell, such as a bacterial or yeast cell, and may comprise one or more variations of said living cell (also called herein “strain(s)” of said microorganism. The term “strains” also includes variants of a microorganism artificially (recombinantly) created by genetic engineering of the said microorganism. In some embodiments, the one or more strain(s) of the microorganism are created by genetic engineering of the E. coli BL21 (DE3) strain. In some embodiments the microorganism comprises one strain created by genetic engineering of E. coli BL21 (DE3). In some embodiments the microorganism comprises two strains created by genetic engineering of E. coli BL21 (DE3). In some preferred embodiments, the microorganism comprises three strains created by genetic engineering of E. coli BL21 (DE3). In some embodiments, the microorganism comprises four strains created by genetic engineering of bacterial E. coli BL21 (DE3). E. coli BL21 (DE3) cells can be obtain from established manufacturer such as ThermoFischer Scientific. Typically, the one or more strain(s) of the microorganism are utilized for the production of the one or more cell-free extract(s) via methods known to the skilled person (e.g., Cole et al. Synthetic and Systems Biotechnology 2020, 5, 252–267). The use of one or more cell-free extract(s) provides several advantages such as avoiding additional purification procedures targeted to isolate enzymes, and the reduction of the number of specific enzymes used during the sialylation cycle by exploiting the activity of those which are naturally present in the microorganism, thus, rendering the sialylation process inexpensive and suitable for the industrial production of sialylated glycosides. In some embodiments, the method comprises the use of one cell-free extract of a microorganism, wherein the one cell-free extract comprises a polypeptide having cytidine monophosphate kinase activity, a polypeptide having N-acylneuraminate citydyltransferase activity, and one or more enzymes having sialyltransferase activity, and wherein the cell-free extract is of a microorganism which endogenously express a polypeptide having inorganic diphosphatase activity, and at least one polypeptide having phosphotransferase activity. In some embodiments, the method comprises the use of two cell-free extracts of a microorganism, wherein the first cell-free extract comprising a polypeptide having cytidine monophosphate kinase activity, and a polypeptide having N-acylneuraminate citydyltransferase activity, and the second cell- free extract comprises at least one polypeptide having sialyltransferase activity, and wherein both cell- free extract are of a microorganism which endogenously express a polypeptide having inorganic diphosphatase activity, and at least a polypeptide having phosphotransferase activity. In some embodiments, the method comprises the use of three cell-free extracts of a microorganism, wherein the first cell-free extract comprising a polypeptide having cytidine monophosphate kinase activity, the second cell-free extract comprising a polypeptide having N-acylneuraminate citydyltransferase activity, and the third cell-free extract comprising a polypetide having sialyltransferase activity, and wherein the three cell-free extracts are of a microorganism which endogenously express a polypeptide having inorganic diphosphatase activity, and at least a polypeptide having phosphotransferase activity. In some embodiments, the method comprises the use of four cell-free extracts of a microorganism, wherein the first cell-free extract comprising a polypeptide having cytidine monophosphate kinase activity, the second cell-free extract comprising a polypeptide having N-acylneuraminate citydyltransferase activity, the third cell-free extract comprising a first polypeptide having sialyltransferase activity, and the fourth cell-free extract comprising a second polypeptide having sialyltransferase activity, and wherein the four cell-free extracts are of a microorganism which endogenously express an polypeptide having inorganic diphosphatase activity, and at least one polypeptide having phosphotransferase activity. The term “a polypeptide having a sialyltransferase activity” may be interchangeably used with the term “sialyltransferase” and denotes, in the context of the present invention, an enzyme belonging to the EC class 2.4.99.-. Suitable sialyltransferases for use in the context of the present invention are sialyltransferases capable of catalyzing the transfer of a sialic acid residue to the O-3 of a β-linked galactose residue of a glycoside acceptor and / or to the O-8 of an α-2-3-linked sialic acid residue of a glycoside acceptor. The sialyltransferase, in its wild-type form, may originate from microorganisms such as bacteria, yeasts, ascomycete, actinomycetes, hyphomycetes, basidiomycotina, and the like, or mammals. The sialyltransferase, in its wild-type form, may originate from Bibersteinia trehalosi, Neisseria meningitidis, Vibrio sp., Pasteurella multocida, and / or Campilobacter jejuni. The sialyltransferase in its wild-type form may originate from any known sialyltransferase sequence or from any sialyltransferase sequence which has yet to be determined. Sialyltransferase yet to be determined can be identified using sequence databases and sequence alignment algorithms, for example, the publicly available GenBank database and the BLAST alignment algorithm. In some embodiments, the polypeptide having sialyltransferase activity is the wild-type α-2,3 / α-2,8- sialyltransferase originating from Campylobacter jejuni, strain OX=197. The amino acid sequence of the wild-type α-2,3 / α-2,8-sialyltransferase originating from Campylobacter jejuni, strain OX=197 corresponds to the amino acid sequence having Accession No: Q9LAK3 (https: / / www.ncbi.nlm.nih.gov / protein / ). In some embodiments, the polypeptide having sialyltransferase activity is a mutant of the wild-type α- 2,3 / α-2,8-sialyltransferase Q9LAK3, wherein the mutant preferably comprises or consists of an amino acid sequence of SEQ ID NO: 1, wherein the mutant comprising the following mutations / modifications compared to the wild-type: Ile53Ser, deletion of 32 amino acids at the C-terminus. In some embodiments, the polypeptide having sialyltransferase activity is a mutant of the wild-type α- 2,3 / α-2,8-sialyltransferase Q9LAK3, wherein the mutant preferably comprises or consists of an amino acid sequence of SEQ ID NO: 2, wherein the mutant comprising the following mutations / modifications compared to the wild-type: Ile53Ser, N-terminal-histidine tag, deletion of 32 amino acids at the C- terminus. In some embodiments, the polypeptide having sialyltransferase activity is a mutant of the wild-type α- 2,3 / α-2,8-sialyltransferase originating from Campylobacter jejuni, strain OX=197 (Q9LAK3), wherein the mutant preferably comprises or consists of an amino acid sequence of SEQ ID NO: 3, wherein the mutant comprising the following mutations / modifications compared to the wild-type: Ile53Gly, N- terminal-histidine tag, deletion of 32 amino acids at the C-terminus. The α-2,3 / α-2,8-sialyltransferase originating from Campylobacter jejuni, or the functional analogues thereof may also be referred to as CST-II. In some embodiments, the polypeptide having sialyltransferase activity is an α-2,3 sialyltransferase originating from Bibersteinia trehalosi, strain DSM 23101, or a functional analogue thereof. In some embodiments, the polypeptide having sialyltransferase activity is the wild-type α-2,3 sialyltransferase originating from Bibersteinia trehalosi, strain DSM 23101. The amino acid sequence of the wild-type Bibersteinia trehalosi α-2,3 sialyltransferase corresponds to the amino acid sequence having Accession No: WP_0252672561 (https: / / www.ncbi.nlm.nih.gov / protein). In some embodiments, the polypeptide having sialyltransferase activity is a mutant derived from the wild-type α-2,3 sialyltransferase WP_025267256, wherein the mutant comprising the following mutations / modifications compared to the wild-type: N-terminal histidine tag MGHHHHHH. The α-2,3-sialyltransferase originating from Bibersteinia trehalose, or the functional analogues thereof may also be referred to as BtSiaT. The term “a polypeptide having cytidine monophosphate kinase activity” may be interchangeably used with the term “CMP kinase” or “CMK” and denotes, in the context of the present invention, an enzyme of the EC class 2.7.4.25., which typically catalyses the phosphorylation of CMP (or dCMP), using ATP as the preferred phosphoryl donor. The CMK, in its wild-type form, may originate from Mycobacterium tuberculosis, Escherichia coli, Yersinia pseudotuberculosis, or Bacillus subtilis. The CMP kinase in its wild-type form may originate from any known CMP kinase sequence or from any CMP kinase sequence which has yet to be determined. CMP kinases yet to be determined can be identified using sequence databases and sequence alignment algorithms, for example, the publicly available GenBank database and the BLAST alignment algorithm. In some embodiments, the polypeptide having cytidine monophosphate kinase activity is a CMP kinase originating from Mycobacterium tuberculosis, or a functional analogue thereof. In some embodiments, the polypeptide having CMK kinase activity is the wild-type CMK kinase originating form Mycobacterium tuberculosis. The amino acid sequence of the wild-type CMP kinase originating from Mycobacterium tuberculosis corresponds to the amino acid sequence having Accession No: WP_129368399 (https: / / www.ncbi.nlm.nih.gov / genbank / ). In some embodiments, the polypeptide having cytidine monophosphate kinase activity is a mutant deriving from the wild-type CMP kinase WP_129368399, wherein the mutant comprising the following mutations / modifications compared to the wild-type: N-terminal histidine tag MGHHHHHH. The CMP kinase originating from Mycobacterium tuberculosis, or its functional analogues thereof may also be referred to as MtCMK. In some preferred embodiments, the enzyme having cytidine monophosphate kinase activity is a CMP kinase originating from Bacillus Subtilis, or a functional analogue thereof. In some embodiments, the enzyme having cytidine monophosphate kinase activity is a CMP kinase originating from Bacillus Subtilis, strain 168, or a functional analogue thereof. In some embodiments, the enzyme having cytidine monophosphate kinase activity is the wild-type CMP kinase originating from Bacillus Subtilis, strain 168. The amino acid sequence of the wild-type CMP kinase originating from Bacillus Subtilis, strain 168, corresponds to the amino acid sequence having Accession No: AAC83961 (https: / / www.ncbi.nlm.nih.gov / genbank / ). In some embodiments, the polypeptide having cytidine monophosphate kinase activity is a mutant derived from the wild-type CMP kinase AAC83961, wherein the mutant comprising the following mutations / modifications compared to the wild-type: N-terminal histidine tag MGHHHHHH. The CMP kinase originating from Bacillus Subtilis, or its functional analogues thereof may also be referred to as BsCMK. The term “a polypeptide having N-acylneuraminate cytidyltransferase activity” may be interchangeably used with the term “N-acylneuraminate cytidylyltransferase” or “CSS” and denotes, in the context of the present invention, an enzyme of the EC class 2.7.7.43, which catalyses the transfer of CMP from CTP to N-acetyl-neuraminic acid (Neu5Ac). The CSS in its wild-type form may originate from any known CSS sequence or from any CSS sequence which has yet to be determined. CSS yet to be determined can be identified using sequence databases and sequence alignment algorithms, for example, the publicly available GenBank database and the BLAST alignment algorithm. In some embodiments, the polypeptide having N-acylneuraminate cytidyltransferase activity is a CSS originating from Neisseria meningitidis, or a functional analogue thereof. In some embodiments, the polypeptide having N-acylneuraminate cytidyltransferase activity is the wild- type CSS originating from Neisseria meningitidis. The amino acid sequence of the wild-type CSS originating from Neisseria meningitidis corresponds to the amino acid sequence having Accession No: WP_061726245 (https: / / www.ncbi.nlm.nih.gov / genbank / ). In some embodiments, the polypeptide having N-acylneuraminate cytidyltransferase activity is a mutant derived from the wild-type CSS WP_061726245 comprising or consisting of an amino acid sequence of SEQ ID NO:4, wherein the mutant comprising the following mutations / modifications compared to the wild-type: N-terminal histidine tag MGHHHHHH. The N-acylneuraminate cytidyltransferase originating from Neisseria meningitidis, or its functional analogues thereof may also be referred to as NmCSS. The term a “polypeptide having inorganic diphosphatase activity” may be interchangeably used with the term “inorganic diphosphatase” or “PPase” and denotes, in the context of the present invention, an enzyme of the EC class 3.6.1.1., which catalyses the hydrolysis of pyrophosphate (PPi). In some preferred embodiments, the polypeptide having inorganic diphosphatase activity is the wild- type PPase originating from Escherichia coli. The amino acid sequence of the wild-type inorganic diphosphatase originating from Escherichia coli corresponds to the amino acid sequence having Accession No: WP_073849715 (https: / / www.ncbi.nlm.nih.gov / genbank / ). The inorganic diphosphatase originating from Escherichia coli may also be referred to as EcPPase. The “polypeptide having phosphotransferase activity” is selected from the group consisting of a polypeptide having nucleoside diphosphate kinase activity, and / or a polypeptide having myokinase activity. In some embodiments, the polypeptide having phosphotransferase activity” is a polypeptide having nucleoside diphosphate kinase activity. The term “a polypeptide having nucleoside diphosphate kinase activity” may be interchangeably used with the term “nucleoside-diphosphate kinase” or “NDK” and denotes, in the context of the present invention, an enzyme of the EC class 2.7.4.6., which catalyses the phosphorylation of a nucleoside diphosphate. In some preferred embodiments, the polypeptide nucleoside diphosphate kinase activity is the wild-type NDK originating from Escherichia coli, strain BL21(DE3). The amino acid sequence of the wild-type NDK originating from Escherichia coli, strain BL21(DE3), corresponds to the amino acid sequence having Accession No: ACT44230 (https: / / www.ncbi.nlm.nih.gov / genbank / ). The nucleoside diphosphatase originating from Escherichia coli, strain BL21(DE3) may also be referred to as EcNDK. In some embodiments, the polypeptide having phosphotransferase activity is a polypeptide having myokinase activity. The term “a polypeptide having myokinase activity” may be interchangeably used with the term “myokinase”, “adenylate kinase”, or “ADK” and denotes, in the context of the present invention, an enzyme of the EC class 2.7.4.3., which catalyses the interconversion of the various adenosine phosphates (e.g. ATP, ADP, and AMP). In some embodiments, the polypeptide having myokinase activity is the wild-type myokinase originating from Escherichia coli, strain BL21(DE3). The amino acid sequence of the wild-type ADK originating from Escherichia coli, strain BL21(DE3), corresponds to the amino acid sequence having Accession No: ACT42324 (https: / / www.ncbi.nlm.nih.gov / genbank / ). The myokinase originating from Escherichia coli, strain BL21(DE3) may also be referred to as EcADK. The term “an enzyme having amylase activity” may be interchangeably used with the term “amylase” and denotes, in the context of the present invention, an enzyme belonging to the EC class 3.2.1., which typically catalyses the hydrolysis of S- and / or O-glycosyl compounds. The amylase in its wild-type form, may originate from microorganisms such as bacteria, yeasts, ascomycete, actinomycetes, hyphomycetes, basidiomycotina, and the like. The amylase in its wild-type form, may originate from Geobacillus thermoleovorans, Anoxybacillus flavithermus, or Pyrococcus furiosus. The amylase in its wildtype form, may originate from a microorganism having a vector, to which a gene encoding a wildtype amylase has been ligated, or introduced. The amylase in its wildtype form may originate from any known amylase sequence or from any amylase sequence which has yet to be determined. Amylases yet to be determined can be identified using sequence databases and sequence alignment algorithms, for example, the publicly available GenBank database and the BLAST alignment algorithm. In some embodiments, the enzyme having amylase activity is a wild-type amylase originating from Geobacillus thermoleovorans, or a functional analogue thereof. The amino acid sequence of the wild- type amylase originating from Geobacillus thermoleovorans corresponds to the amino acid sequence having Accession No: AFM43699 (https: / / www.ncbi.nlm.nih.gov / protein / ). In the context of the present invention an amylase originating from Geobacillus thermoleovorans may also be referred to as maltogenic-amylase, or GtCDase. In some embodiments, the enzyme having amylase activity is a wild-type amylase originating from Anoxybacillus flavithermus, or a functional analogue thereof. The amino acid sequence of the wild-type amylase originating from Anoxybacillus flavithermus corresponds to the amino acid sequence having Accession No: AMB26774 (https: / / www.ncbi.nlm.nih.gov / protein / ). In the context of the present invention an amylase originating from Anoxybacillus flavithermus may also be referred to as cyclomaltodextrinase, or AfCDase. In some embodiments, the enzyme having amylase activity is a wild-type amylase originating from Pyrococcus furiosus, or a functional analogue thereof. The amino acid sequence of the wild-type amylase originating from Pyrococcus furiosus corresponds to the amino acid sequence having Accession No: WP_011013079 (https: / / www.ncbi.nlm.nih.gov / protein / ). In the context of the present invention an amylase originating from Pyrococcus furiosus may also be referred to as alpha amylase, or PfCDase. The term “an enzyme having polyphosphate kinase activity” may be interchangeably used with the term “polyphosphate kinase” or “PPK” and denotes, in the context of the present invention, an enzyme that catalyses the phosphorylation of ADP. In some embodiments, the enzyme having polyphosphate kinase activity is a PPK originating from Meiothermus ruber strain DSM 1279, or a functional analogue thereof. In some embodiments, the enzyme having polyphosphate kinase activity is the wild-type PPK originating from Meiothermus ruber strain DSM 1279. The amino acid sequence of the wild-type PPK originating from Meiothermus ruber strain DSM 1279 corresponds to the amino acid sequence having accession number Accession No: ADD29239 (https: / / www.ncbi.nlm.nih.gov / genbank / ). In some embodiments, the polypeptide having polyphosphate kinase activity is a mutant derived from the wild-type polyphosphate kinase ADD29239, wherein the mutant comprising the following mutations / modifications compared to the wild-type: N-terminal histidine tag MGHHHHHH. The polyphosphate kinase originating from Meiothermus ruber, or its functional analogues thereof may also be referred to as MrPPK. The one or more cell-free extracts according to the present invention, comprise polypeptides or enzymes having the activities required to perform the sialyltransferase cycle. Non limiting examples of polypeptides or enzymes having the activities required for the sialyltransferase cycle are summarized in Table 2, wherein for a specific enzymatic activity more than one polypeptide or enzyme may be suitable.

[0002] Table 2: Overview of suitable enzymes for sialylation cycle Mutation(s) / GenBank Accession Source Modifications Enzyme Abbreviation NO / compared to wild- SEQ ID NO type Nucleoside- Escherichia coli Accession NO: EcNDK - Diphosphate Kinase BL21(DE3) ACT44230 Escherichia coli Accession NO: Myokinase EcADK - BL21(DE3) ACT42324 Inorganic Accession NO: Escherichia coli EcPPase - Diphosphatase WP_073849715 Mycobacterium MtCMK Accession NO: - tuberculosis WP_129368399 Bacillus Subtilis, BsCMK - accession: AAC83961 strain 168 Cytidine Yersinia Monophosphate pseudotuberculosis Accession NO: Kinase YpCMK - serotype IB (strain ACC88489.1 PB1 / +) Escherichia coli BL21 Accession NO: EcCMK - (DE3) ACT29707.1 Accession NO: N-acylneuraminate Neisseria NmCSS - WP_061726245 cytidyltransferase meningitidis N-term. His-Tag SEQ ID NO: 4 Polyphosphate Meiothermus ruber PKK - ADD29239 kinase - Accession NO: Q9LAK3 Ile53Ser, C-term. SEQ ID NO: 1 ∆32 Ile53Ser, N-term α-2,3 / α-2,8- Campylobacter jejuni CSTII His Tag, C-term. SEQ ID NO: 2 sialyltransferase OX=197 Δ32, Ile53Gly, N-term. His Tag, C-term. SEQ ID NO: 3 ∆32, Accession NO: Bibersteinia trehalosi BtSiaT - WP_025267256 Neisseria Accession NO: NmSiaT - α-2,3- meningitidis WP_002244089 sialyltransferase Pasteurella Accession NO: PmSiaT - multocida AAK02272 Accession NO: Vibrio sp. VsSiaT - BAF91160 In some embodiments, the method comprises the use of three cell-free extracts of an Escherichia coli, strain BL21(DE3), wherein the first cell-free extract comprising the wild-type MtCMK (GenBank Accession NO: WP_129368399), the second cell-free extract comprising the NmCSS of SEQ ID NO: 4, and the third cell-free extract comprising a CSTII selected from a CSTII of SEQ ID NO:1, or a CSTII of SEQ ID NO: 2, or a CSTII of SEQ ID NO: 3 and wherein the Escherichia coli, strain BL21(DE3) endogenously express the following enzymes: EcPPase (GenBank Accession NO: WP_073849715), EcNDK (GenBank Accession NO: ACT44230), and EcADK (GenBank Accession NO: ACT42324). In some embodiments, the method comprises the use of three cell-free extracts of an Escherichia coli, strain BL21(DE3), wherein the first cell-free extract comprising the wild-type BsCMK (GenBank Accession NO: AAC83961), the second cell-free extract comprising the NmCSS of SEQ ID NO: 4, the third cell-free extract comprising a CSTII selected from a CSTII of SEQ ID NO:1, or a CSTII of SEQ ID NO: 2, or a CSTII of SEQ ID NO: 3, and wherein the Escherichia coli, strain BL21(DE3) endogenously express the following enzymes: EcPPase (GenBank Accession NO: WP_073849715), EcNDK (GenBank Accession NO: ACT44230), and EcADK (GenBank Accession NO: ACT42324). In some embodiments, the method comprises the use of three cell-free extracts of an Escherichia coli, strain BL21(DE3), wherein the first cell-free extract comprising the wild-type MtCMK (GenBank Accession NO: WP_129368399), the second cell-free extract comprising the NmCSS of SEQ ID NO: 4, and the third cell-free extract comprising the wild-type BtSiaT (GenBank Accession NO: WP_025267256), and wherein the Escherichia coli, strain BL21(DE3) endogenously express the following enzymes: EcPPase (GenBank Accession NO: WP_073849715), EcNDK (GenBank Accession NO: ACT44230), and EcADK (GenBank Accession NO: ACT42324). In some embodiments, the method comprises the use of three cell-free extracts of an Escherichia coli, strain BL21(DE3), wherein the first cell-free extract comprising the wild-type BsCMK (GenBank Accession NO: AAC83961), the second cell-free extract comprising the NmCSS of SEQ ID NO: 4, and the third cell- free extract comprising the wild-type BtSiaT (GenBank Accession NO: WP_025267256), and wherein the Escherichia coli, strain BL21(DE3) endogenously express the following enzymes: EcPPase (GenBank Accession NO: WP_073849715) , EcNDK (GenBank Accession NO: ACT44230), and EcADK (GenBank Accession NO: ACT42324). In some embodiments, the method comprises the use of four cell-free extracts of an Escherichia coli, strain BL21(DE3), wherein the first cell-free extract comprising the wild-type MtCMK ( GenBank Accession NO: WP_129368399), the second cell-free extract comprising the NmCSS of SEQ ID NO: 4, the third cell-free extract comprising a CSTII selected from a CSTII of SEQ ID NO:1, or a CSTII of SEQ ID NO: 2, or a CSTII of SEQ ID NO: 3, and the fourth cell-free extract comprising the wild-type BtSiaT (GenBank Accession NO: WP_025267256), and wherein the Escherichia coli, strain BL21(DE3) endogenously express the following enzymes: EcPPase (GenBank Accession NO: WP_073849715) , EcNDK (GenBank Accession NO: ACT44230), and EcADK (GenBank Accession NO: ACT42324). In some embodiments, the method comprises the use of four cell-free extracts of an Escherichia coli, strain BL21(DE3), wherein the first cell-free extract comprising the wild-type BsCMK (GenBank Accession NO: AAC83961), the second cell-free extract comprising the NmCSS of SEQ ID NO: 3, the third cell-free extract comprising the CSTII of SEQ ID NO: 1 or of SEQ ID NO: 2, and the fourth cell-free extract comprising the wild-type BtSiaT (GenBank Accession NO: WP_025267256), and wherein the Escherichia coli, strain BL21(DE3) endogenously express the following enzymes: EcPPase (GenBank Accession NO: WP_073849715) , EcNDK (GenBank Accession NO: ACT44230), and EcADK (accession: ACT42324). The selection of the sialyl transferase(s) to be comprised in the one or more cell-free extract, according to the present invention, depends on the selection of the glycosyl acceptor. Typically, for those glycosyl acceptors having a terminal galactosyl unit, an α-2,3-sialyltransferase or a combination of an α-2,3-sialyltransferase and an α-2,3 / α-2,8-sialyltransferase are selected, whereas typically, for those glycosyl acceptors having a glycosyl moiety carrying a terminal sialic acid unit an α- 2,3 / α-2,8-sialyltransferase is selected. In some embodiments, the glycosyl acceptor is psychosine, or lactosyl sphingosine and an α-2,3- sialyltransferase is selected, preferably the wild-type BtSiaT (Accession No: WP_025267256). In some embodiments, the glycosyl acceptor is psychosine, or lactosyl sphingosine and both an α-2,3- sialyltransferase and an α-2,3 / α-2,8-sialyltransferase are selected, preferably the wild-type BtSiaT (Accession No: WP_025267256), and a mutant CSTII selected from a CSTII of SEQ ID NO:1, or a CSTII of SEQ ID NO:2, or a CSTII of SEQ ID NO:3. In some embodiments, the glycosyl acceptor is N-lyso-GM3 or N-lyso-GM1a and an α-2,3 / α-2,8- sialyltransferase is selected, preferably the mutant CSTII selected from a CSTII of SEQ ID NO:1, or a CSTII of SEQ ID NO:2, or a CSTII of SEQ ID NO:3. For the sialyltransferase cycles, the concentrations or amounts of the various reactants used in the processes depend upon numerous factors including reaction conditions such as temperature and pH value, and the choice and amount of acceptor glycoside to be sialylated. Because the sialylation process permits regeneration of activating nucleotides, activated donor sugars, and scavenging of produced PPi in the presence of catalytic amounts of the enzymes, the process is limited by the concentrations or amounts of the stoichiometric substances. The upper limit for the concentrations of reactants that can be used in accordance with the method of the present invention is determined by the solubility of such reactants. Preferably, the concentrations of activating nucleotides, phosphate donor, the donor sugar and enzymes are selected such that glycosylation proceeds until the acceptor is consumed. The sialyl transferase cycle according to the method of the present invention, can also include other ingredients that facilitate the sialyltransferase activity. These ingredients can include a divalent cation (e.g., Mg+2or Mn+2), materials necessary for ATP regeneration, phosphate ions etc. The reaction medium may also comprise solubilizing detergents (e.g., Triton or SDS) and organic solvents such as methanol or ethanol, or a cyclodextrin. In a preferred embodiment the sialylation is performed in the presence of a cyclodextrin. In some embodiments, the cyclodextrin is selected from the group consisting of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, randomly methylated β-cyclodextrin, or sulfobutylether-β-cyclodextrin. In some preferred embodiments, the cyclodextrin is β-cyclodextrin. The cyclodextrin is typically used in an amount between about 0.1 equivalents to about 1 equivalent based on the amount of the glycosphingolipid acceptor. In some preferred embodiments the cyclodextrin is used in an amount between about 0.1 equivalents to about 0.5 equivalents based on the amount of the glycosphingolipid acceptor. Accordingly, in some preferred embodiments, the cyclodextrin is used in an amount of about 0.1, 0.2, 0.3, 0.4, or 0.5 equivalents based on the amount of the glycosphingolipid acceptor. The use of a cyclodextrin provides advantages such as high yields and eliminates the need for the use of a detergent or organic solvent to increase accessibility to the glycosyl moiety of the glycosphingolipid. However, detergents or organic solvents can also be used in the method of the invention. Preferably, when the sialylation is performed in the presence of a cyclodextrin the method further comprises the use of polypeptide having amylase activity. The polypeptide having amylase activity, is added to the sialylation mixture when a certain conversion of the glycoside is reached, preferably when a conversion of at least about 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the glycoside is reached. The conversion of glycoside can be determined by standard techniques known to the skilled person. Typically, the conversion of the glycoside is determined by HPLC and may be given in mol.% or wt.%. In some embodiments, the present invention describes a method for the sialylation of a glycoside of formula (1), or a salt thereof: (1), wherein X is a glycosyl moiety, wherein the glycosyl moiety is preferably selected from the group consisting of Gal1-, or a glycosyl moiety carrying one or more terminal galactose units and / or one or more terminal N-acetyl-galactosamine units and / or one or more terminal sialic acid units; Y is selected from the group consisting of hydroxyl, fluoride, or a moiety of formula (2), or a salt thereof: (2), wherein R1is hydrogen, aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl, R2is hydrogen or -OR5, wherein R5is selected from hydrogen, a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C2-6acyl, the bond may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -OR5, R3is hydrogen, a substituted or unsubstituted C1-6alkyl, or a substituted or unsubstituted C1-6acyl, preferably hydrogen, R4is selected from hydrogen, a substituted or unsubstituted aryl, a heteroalkyl, a substituted or unsubstituted C2-32 acyl, wherein the method comprising the steps of: ─ mixing the glycoside of formula (1) with sialic acid, cytidine monophosphate, a nucleoside triphosphate, a cyclodextrin, and one or more cell-free extracts of a microorganism, said microorganism comprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein said one or more cell-free extracts comprise: ─ at least one polypeptide having cytidine monophosphate kinase activity, ─ at least one polypeptide having N-acylneuraminate citydyltransferase activity, and ─ at least one polypeptide having sialyltransferase activity, thereby sialylating said glycoside, and sequentially: ─ adding a polypeptide having amylase activity. The polypeptide having amylase activity, may advantageously be utilized for the degradation of the cyclodextrin. Degrading the cyclodextrin facilitates the isolation of the sialylated glycosphingolipid product in high yield and purity. The polypeptide having amylase activity may be provided as purified polypeptide, as cell-free extract, or as lysate. In some embodiments, the polypeptide having amylase activity is provided as purified proteins, with a purity of about 50% to about 95%. In some embodiments, the polypeptide having amylase activity is provided as cell-free extract, wherein the cell-free extract contains from about 5 wt% to about 70 wt% of the enzyme. Preferably, the cell-free extract contains from about 20 wt% to about 70 wt% of the enzyme. In optimized reactions, the above ingredients can be combined by admixture in an aqueous reaction medium (solution) which has a pH value of about 6 to about 8.5. The medium is devoid of chelators that bind enzyme cofactors such as Mg+2or Mn+2. The selection of a medium is based on the ability of the medium to maintain pH value at the desired level. Thus, in some embodiments, the medium is buffered to a pH value at about 6.5 to about 8.5. If a buffer is not used, the pH of the medium should be maintained at about 6.5 to 8.0, preferably about 7.3 to 8.0, by the addition of base. A suitable base is NaOH. Accordingly, in some preferred embodiments the pH is buffered or kept at a value of about 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0 The temperature at which the above process is carried out can range from just above freezing to the temperature at which the most sensitive enzyme denaturates. That temperature range is preferably at about 0oC to about 45oC, and more preferably at about 20oC to 37oC. The reaction mixture so formed is maintained for a period of time sufficient for the sialyltransferase to sialylate a high percentage of the acceptors. Typically, the reaction will often be allowed to proceed for about 8 to about 240 hours, preferably between about 24 and 48 hours. The N-acetyl-neuraminic acid (Neu5Ac), the cytidine monophosphate (CMP), the adenosine 5'- triphosphate (ATP), polyphosphate, the cell-free extract(s), as well as any other component used during the cycle may be added to the reaction mixture either as a solid or dissolved in a solvent, and in any quantities and manner effective for the intended result of the process. In the context of the present invention, cell-free extract enzymatic activities are expressed in activity Units, which is a measure of the initial rate of catalysis. One activity Unit catalyses the formation of 1 μmol of product per minute at a given pH and temperature. The cell-free extract(s) enzymatic activities can be measured according to procedures described in the examples below. The sialylation of a glycoside of formula (1), according to the method of the present invention, results in the formation of a sialylated glycoside of formula (13): (13), wherein J is a glycosyl moiety carrying one or more sialic acid unit, Y is as defined as for the glycoside of formula (1). In some embodiments, Y of the sialylated glycoside of formula (13) is a hydroxyl group. Accordingly, in some embodiments the sialylated glycoside of formula (13) is a sialylated saccharide. In some embodiments, Y of the sialylated glycoside of formula (13) is a fluoride. Accordingly, in some embodiments, the sialylated glycoside of formula (13) is a sialylated glycoside of formula (14): J-F (14), wherein J is a glycosyl moiety as defined as for the sialylated glycoside of formula (13). In some embodiments, the sialylated glycoside of formula (14) is a an α-glycoside. In some preferred embodiments, Y of the sialylated glycoside of formula (13) is a moiety of formula (2), or a salt thereof. Accordingly in some preferred embodiments, the glycoside of formula (13) is a glycoside of formula (15), or a salt thereof: (15), wherein J is a glycosyl moiety as defined as for the sialylated glycoside of formula (13), R1is hydrogen, aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17alkyl, more preferably a substituted or unsubstituted C10-17alkyl, R2is hydrogen or -OR5, wherein R5is selected from hydrogen, a substituted or unsubstituted C1-6alkyl, or a substituted or unsubstituted C2-6acyl, the bond may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -OR5, R3is hydrogen, a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C1-6 acyl, preferably hydrogen, R4is selected from hydrogen, a substituted or unsubstituted aryl, a heteroalkyl, a substituted or unsubstituted C2-32acyl. In some embodiments, for the sialylated glycoside of formula (15) R1is a saturated unsubstituted C10-17alkyl, R2, R3and R4are hydrogen, and the bond is a double bond. In some embodiments, for the sialylated glycoside of formula (15) R1is a saturated unsubstituted C10-17alkyl, R2is -OR5, wherein R5is hydrogen, R3and R4are hydrogen, and the bond is a single bond. In some embodiments, for the glycoside of formula (15) R1is a saturated unsubstituted C10-17 alkyl, R2, R3and R4are hydrogen, and the bond is a single bond. In some embodiments, for the glycoside of formula (15) R1is a C10-171-hydroxyalkyl, R2, R3and R4are hydrogen, and the bond is a double bond. In some embodiments, the sialylated glycoside of formula (13) is a sialylated glycoside of formula (15), wherein the sialylated glycoside of formula (15) is a glycoside selected from the group consisting of glycosides of formulas (16), (17), (18), and (19): J is a glycosyl moiety as defined as for the sialylated glycoside of formula (13). In some embodiments, for the sialylated glycosides of formula (15) R1is a saturated unsubstituted C10- C17 alkyl, R2, and R3are hydrogen, R4is a substituted or unsubstituted C16-32 acyl, and the bond is a double bond. In some embodiments, for the glycoside of formula (15) R1is a saturated unsubstituted C10-17 alkyl, R2is - OR5, wherein R5is hydrogen, R3is hydrogen, R4is a substituted or unsubstituted C16-32acyl, and the bond is a single bond. In some embodiments, for the glycoside of formula (15) R1is a saturated unsubstituted C10-C17alkyl, R2, and R3are hydrogen, R4is a substituted or unsubstituted C16-32 acyl, and the bond is a single bond. In some embodiments, for the glycoside of formula (15) R1is a C10-C171-hydroxyalkyl, R2, and R3are hydrogen, R4is a substituted or unsubstituted C16-32 acyl, and the bond is a double bond. In some embodiments, the glycoside of formula (15) is a glycoside selected from the group consisting of glycosphingolipids of formulas (20), (21), (22), or (23): wherein J is a glycosyl moiety as defined as for the sialylated glycoside of formula (13). Sialylated glycosides of formula (15)-(23) may also be referred to as sialylated glycosphingolipids. In some embodiments, J of the sialylated glycoside of formula (13)-(23) is a glycosyl moiety selected from the following glycosyl moieties, or salts thereof: Neu5Acα2-3Gal1-, Neu5Acα2-3Galβ1-4Glc1-, Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc1-, or Neu5Acα2-8Neu5Acα2-3Galβ1-3GalNAcβ1-4Gal β1-4Glcβ1-. In some embodiments, J of the sialylated glycoside of formula (13)-(23), is Neu5Acα2-3Galβ1-4Glcβ1. In some embodiments, the sialylated glycoside of formula (13) is a sialylated glycoside of formula (16), and wherein J of the sialylated glycoside of formula (16) is Neu5Acα2-3Galβ1-4Glcβ1-. Accordingly, in some embodiments, the sialylated glycoside of formula (13) is a sialylated glycoside of formula (16), and wherein the sialylated glycoside of formula (16) is N-lyso-GM3. In some embodiments, J of the sialylated glycosphingolipid of formula (13)-(23), is Neu5Acα2- 8Neu5Acα2-3Galβ1-4Glcβ1-. In some embodiments, the sialylated glycoside of formula (13) is a sialylated glycosphingolipid of formula (16), and wherein J of the sialylated glycosphingolipid of formula (16) is Neu5Acα2-8Neu5Acα2- 3Galβ1-4Glcβ1-. Accordingly, in some embodiments, the sialylated glycoside of formula (13) is a sialylated glycoside of formula (16), and wherein the sialylated glycoside of formula (16) is N-lyso-GD3. In some embodiments, the sialylated glycoside of formula (13) is a sialylated glycoside of formula (16), and wherein J of the sialylated glycosphingolipid of formula (16) is Neu5Acα2-8Neu5Acα2-3Galβ1- 3GalNAcβ1-4Gal β1-4Glcβ1-. Accordingly, in some embodiments, the sialylated glycosphingolipid of formula (13) is a sialylated glycosphingolipid of formula (16), and wherein the sialylated glycosphingolipid of formula (16) is N-lyso-GD1a. In some embodiments, the sialylation of a glycoside of formula (1), according to the method of the present invention, results in the formation of a sialylated glycoside, wherein the sialylated glycoside is a mixture of more than one sialylated glycoside which differ in the degree of sialylation. In some embodiments, the sialylated glycoside is a mixture of N-lyso-GM3 and N-lyso-GD3. In some embodiments, the sialylated glycoside is a mixture of N-lyso-GM3 and N-lyso-GD3, wherein the weight ratio between N-lyso-GD3 and N-lyso-GM3 in said mixture is about 1:3. In some embodiments, the sialylated glycoside is a mixture of N-lyso-GM3 and N-lyso-GD3, wherein the weight ratio between N-lyso-GD3 and N-lyso-GM3 in said mixture is about 1:1. In some embodiments, the sialylated glycoside is a mixture of N-lyso-GM3 and N-lyso-GD3, wherein the weight ratio between N-lyso-GD3 and N-lyso-GM3 in said mixture is about 4:1. In some embodiments, the method further comprising isolating the sialylated glycoside. In some embodiments, the present invention describes a method for the sialylation of a glycoside of formula (1), or a salt thereof, the method comprising the steps of: ─ mixing the glycoside of formula (1) with sialic acid, cytidine monophosphate, a nucleoside triphosphate, a cyclodextrin, and one or more cell-free extracts of a microorganism, said microorganism comprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein said one or more cell-free extracts comprise: ─ at least one polypeptide having cytidine monophosphate kinase activity, ─ at least one polypeptide having N-acylneuraminate citydyltransferase activity, and ─ at least one polypeptide having sialyltransferase activity, thereby sialylating said glycoside, ─ isolating the sialylated glycoside produced in the preceding step. In some embodiments, the present invention describes a method for the sialylation of a glycoside of formula (1), or a salt thereof, the method comprising the steps of: ─ mixing the glycoside of formula (1) with sialic acid, cytidine monophosphate, a nucleoside triphosphate, a cyclodextrin, and one or more cell-free extracts of a microorganism, said microorganism comprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein said one or more cell-free extracts comprise: ─ at least one polypeptide having cytidine monophosphate kinase activity, ─ at least one polypeptide having N-acylneuraminate citydyltransferase activity, and ─ at least one polypeptide having sialyltransferase activity, thereby sialylating said glycoside, and sequentially: ─ adding a polypeptide having amylase activity, ─ isolating the sialylated glycoside produced in the preceding steps. The sialylated glycoside may be isolated from the reaction mixture. The isolation may be performed by standard methods known to the skilled person, such as for example extraction with organic solvents, chromatography and / or ion exchange chromatography. A method for the isolation of sialylated glycosphingolipid from enzymatic reaction mixtures is for example described in Bai et al., Current Protocols 2021, 1, e91. doi: 10.1002 / cpz1.91. A preferred method of isolation involves diafiltration (DF) of the reaction mixture, wherein the DF is used to remove ions, mainly monovalent ions, and / or to remove organic materials such as protein and organic molecules. In a preferred embodiment, the diafiltration is performed using a membrane having a MWCO of about 100-300 kDa, preferably of about 200-300 kDa. In some embodiments, the diafiltration membrane having a MWCO of about 100-150 kDa, 150-200 kDa, 200-250 kDa, or 250-300 kDa. It is noted that, even though a MWCO of about 100-300 kDa is well above the molecular weight of the sialylated glycosphingolipid, the sialylated glycosphingolipid is accumulated in the DF retentate (DFR). It has been described that ganglioside GM1, can form micellar aggregates in aqueous solutions having molecular weights between about 250 kDa and 450 kDa, wherein the size of the micellar aggregate will depend on the length of the fatty acid chain in the constituent molecule (D.B. Gammak, Biochem J 1963, 88, 373). This property may render gangliosides, such as GM1 not permeable through ultrafiltration membranes having a MWCO higher than that of GM1. However, surprisingly, the present inventors have found that also N-lyso forms of gangliosides, as well as glycosylated sphingoid bases, which lack a fatty acid chain are not permeable through diafiltration membranes having a MWCO higher than that of the lysosphingolipid. Accordingly, without being bound by theory, micellar aggregation may surprisingly occur independently from the presence of a fatty acid chain in the molecule. The person skilled in the art will understand that the DF step, according to the method of the present invention, results in the removal of any contaminant present in the aqueous media which is permeable through the diafiltration membrane. The DF step, according to the present invention, is conducted at a constant temperature, preferably between about 15-45oC, more preferably between about 20-35oC. The DF step is continued until reaching the desired concentration of the sialylated glycosphingolipid in the DFR. Other technical parameters like setting in the flux and pressure is a matter of routine skills. The DF step may optionally be followed by a concentration step. In some embodiments, the method further comprising a step of concentrating the DFR wherein the step of concentrating the DFR. The concentration of the DFR is typically performed using the same membrane used during the diafiltration step, and for a period of time required to reduce the volume of the DFR to the desired final volume. The DFR enriched with the sialylated glycosphingolipid, is spray dried or spray granulated in a subsequent step. In some preferred embodiments, the DFR comprising the glycosphingolipid of formula (1), is spray dried. The spray drying step, according to the present invention, is conducted with a fast-rotating disk or a nozzle which generates small particles. The particles can then fall, under gravity, towards the bottom of a spray drying tower. Here, a fluid bed may be provided, which can use hot air to effect drying (suitably at around 80° C to around 95° C). Here, agglomeration can take place, and the particles can stick together. Following this, the agglomerated (granular) particles are subjected to drying, for example on a belt drying bed or on a sub-fluidized bed. Another technique is to use a fluidized bed agglomeration. Here, powder can be fluidized in a gas flow. In the particle bed a fluid is sprayed with water that wets the powder and enhances the agglomeration. This combination of spray-drying in combination with a fluid bed after dryer is suited for the agglomeration of many different types of solutions. Drying can occur under air or under an inert gas, such as nitrogen. With fluidized and sub-fluidized bed drying, the temperature in the bed can be adjusted to pre-set values. These values can range widely, for example, from 35° to 120°C, such as 50 to 90°C, e. g. from 60 to 80°C. The spray-drying of the DFR retentate will result in the production of a spray-dried powder comprising a sialylated glycosphingolipids. The spray-dried powder, obtained following the method of the present invention, will typically have a median particles diameter between about 15 µm and about 30 µm. The Span of the particles will typically be less than about 3, preferably less than about 2. The Span of the particle is a dimensionless parameter indicative of the uniformity of the particle size distribution, and it is defined as: [D(0.9) – D(0.1)] / D(0.5), wherein D(0.9), D(0.1), and D(0.5) represent the cutoff size below which 10%, 50%, and 90% (by volume) of particles are distributed, respectively. Generally, a low Span (i.e., less than 3) is characteristic of a narrow particle size distribution, resulting in improved flow characteristics of the spray-dried powder. The spray-dried powder, obtained following the method of the present invention, will typically have a specific volume of less than about 4 mL / g, preferably less than about 3 ml / g. Spray-dried powders with such low specific volumes (i.e., less than 4 ml / g) are generally preferred as they have improved flow characteristics. The spray-dried powder, obtained following the method of the present invention, will typically have a glycosphingolipid content of at least about 65 wt.%, usually of at least about 70 wt.%, preferably of at least about 75 wt.%, more preferably of at least about 85 wt.%. In some embodiments, the spray-dried powder comprising at least about 50 wt.% of a mixture of N-lyso- GD3 and N-lyso-GM3, or at least about 60 wt.% of a mixture of N-lyso-GD3 and N-lyso-GM3, or at least about 70 wt.% of a mixture of N-lyso-GD3 and N-lyso-GM3, or at least about 80 wt.% of a mixture of N- lyso-GD3 and N-lyso-GM3, and wherein the weight ratio between N-lyso-GD3 and N-lyso-GM3 in said mixture is from about 1:10 to about 10:1. In some embodiments, the weight ratio between N-lyso-GD3 and N-lyso-GM3 in said mixture is about 1:3. In some embodiments, the weight ratio between N-lyso-GD3 and N-lyso-GM3 in said mixture is about 1:1. In some embodiments, the weight ratio between N-lyso-GD3 and N-lyso-GM3 in said mixture is about 4:1. In some embodiments, the spray-dried powder comprising about 40-55 wt.% of N-lyso-GD3 and about 10-15 wt.% of N-lyso-GM3, and wherein the spray-dried powder further comprising about 3-6 wt.% of N-lyso-GT3, about 4-6 wt.% of lactosyl D-erythro-sphingosine, and about 0.1-1.0 wt.% of glucosyl D- erythro-sphingosine. In some embodiments, the spray-dried powder comprising about 15-20 wt.% of N-lyso-GD3 and about 50-60 wt.% of N-lyso-GM3, and wherein the spray-dried powder further comprising about 0.1-0.5 wt.% of N-lyso-GT3, about 4-7 wt.% of lactosyl D-erythro-sphingosine, and about 0.1-1.0 wt.% of glucosyl D- erythro-sphingosine. In some embodiments, the spray-dried powder comprising about 35-40 wt.% of N-lyso-GD3 and about 25-40 wt.% of N-lyso-GM3, and wherein the spray-dried powder further comprising about 5-6 wt.% of lactosyl D-erythro-sphingosine, and about 0.5-1.0 wt.% of glucosyl D-erythro-sphingosine. In some embodiments, the glycoside and the sialylated glycoside according to the present invention may be utilized or produced in the form of salts, preferably in the form of pharmaceutical acceptable salts. In some embodiments, the salts comprising the following cations: Na+, K+, Mg2+, Ca2+, NH4+, Et3NH+. In some embodiments, the salts comprising the following anions: Cl-, Br-, CH3CO2-, CO32-, SO42-, HPO4-. In some embodiments, the present invention describes a sialylating agent comprising one or more cell- free extract(s) of a microorganism, said microorganism comprising or more endogenous polypeptide having inorganic diphosphatase activity and one or more endogenous polypeptide having phosphotransferase activity, and wherein the said one or more cell-free extract comprise: ─ at least one polypeptide having cytidine monophosphate kinase activity, ─ at least one polypeptide having N-acylneuraminate citydyltransferase activity, and ─ at least one polypeptide having sialyltransferase activity. In some embodiments, the sialylating agent further comprising sialic acid, cytidine monophosphate, a nucleoside triphosphate. Examples The working examples below describe non-limiting embodiments of the invention and are given only to illustrate the invention. General methods and materials LCMS analysis was performed with a Shimadzu ECO 2020 LC system coupled with a Shimadzu MS-2020 system. Samples (50 µL) were taken from reaction mixtures, mixed with DMSO (950 µL), subjected to centrifugation (16.000 rpm, 5 min) and analyzed with a Shimadzu ECO 2020 LC system coupled with a Shimadzu LCMS-2020 system. The HPLC analysis was performed using a Merck Ascentis Express RP- Amide column (15cm x 4.6mm, 2.7 µm). Amylases GtCDase, AfCDase, and PfCDase, were expressed from E. coli strains following methods described by Metha et al., PLOS ONE 2013, 8, e73612, Aliakbari et al., Starch 2019, 71, 1800133, and Yand et al., Applied and Environmental Microbiology 2004, 70, 5988 respectively. The following cell-free extracts were produced from Escherichia coli (E. coli) expression strains: i. Cell-free extract from E. coli BL21 (DE3) ΔLacZ genetically engineered for the expression of wild- type cytidine monophosphate kinase from Mycobacterium tuberculosis (MtCMK, accession: WP_129368399); ii. Cell-free extract from E. coli BL21 (DE3) ΔLacZ genetically engineered for the expression of the wild-type cytidine monophosphate kinase from Bacillus subtilis (BsCMK, accession: AAC83961); iii. Cell-free extract from E. coli BL21 (DE3) ΔLacZ genetically engineered for the expression of the mutant N-acylneuraminate citydyltransferase of SEQ ID NO: 4; iv. Cell-free extract from E. coli BL21 (DE3) ΔLacZ genetically engineered for expression of the wild- type sialyl transferase from Bibersteinia trehalosi, strain DSM 23101 (BtSiaT, accession: WP025267256); v. Cell-free extract from E. coli BL21 (DE3) ΔLacZ genetically engineered for expression the α-2,3 / α- 2,8-sialyltransferase of SEQ ID NO: 1. vi. Cell-free extract from E. coli BL21 (DE3) ΔLacZ genetically engineered for expression the α-2,3 / α- 2,8-sialyltransferase of SEQ ID NO: 2. vii. Cell-free extract from E. coli BL21 (DE3) ΔLacZ genetically engineered for expression the α-2,3 / α- 2,8-sialyltransferase of SEQ ID NO: 3. Cell-free extracts (i)-(vii) were produced and characterized following the procedures described in examples 1.1-1.3 and in examples 2.1-2.3 Example 1: Cloning Genes encoding the enzymes are usually ordered as codon-optimized synthetic genes for optimal expression in the E. coli host strain. The synthetic constructs contain overhangs with BsaI restriction sites for golden gate cloning into a pET28a-based expression vector (carrying introduced BsaI restriction sites and a fluorescent drop-out cassette). The resulting plasmids were used for transformation of E. coli BL21 (DE3) ΔLacZ. Example 2: Expression A preculture of the expression strain was prepared in 10mL LB medium supplemented with the respective antibiotic and incubated at 37°C shaking overnight. The culture of the expression strain was started by a 1:100-fold dilution of the preculture into TB medium supplemented with the respective antibiotic. The culture was incubated at 37°C until an OD600of 0.7-1.0 was reached. The culture was cooled to the desired expression temperature, induced with 0.5 mM IPTG, and incubated for the desired expression time. Example 3: Preparation of the cell-free extract Cells were harvested by centrifugation and resuspended in water. Cell lysis was achieved by sonication. The resulting lysed cell suspension was centrifuged to separate the cell-free extract, comprising soluble enzymes, from the debris. The supernatant, containing the cell-free extract, was freeze-dried to dryness. Example 4: Activity measurement of α-2,3-sialyl-transferase (α-2,3-SiaT) from cell-free extract To quantify the α-2,3-sialyl-transferase activity from a cell-free extract the synthesis of N-lyso-GM3 from lactosylsphingosine was assayed. Reaction progress was determined discontinuously by sampling the reaction at a given reaction time, quenching the samples through addition of DMSO and quantifying the amount of synthesized N-lyso-GM3 via LC / MS. One α-2,3-SiaT unit (U) corresponds to 1 μmol of lactosylsphingosine converted to N-lyso-GM3 per minute at 25 °C and pH=7.5 in a solution containing 20 mM Mg2+. Example 5: Activity measurement of α-2,8-sialyl-transferase (α-2,8-SiaT) from cell-free extract To quantify the α-2,8-sialyl-transferase activity from a cell-free extract the synthesis of N-lyso-GD3 from N-lyso-GM3 was assayed. Reaction progress was determined discontinuously by sampling the reaction at a given reaction time and quenching the samples through addition of DMSO and quantifying the amount of synthesized N-lyso-GD3 via HPLC. One α-2,8-SiaT unit (U) corresponds to 1 μmol of N-lyso- GM3 converted to N-lyso-GD3 per minute at 37°C and pH=8.0 in a buffered solution containing 20 mM Mg2+. Example 6: Measurement of cytidine monophosphate kinase (CMK) activity in a cell-free extract For the quantification of the cell free extract CMK activity the pyruvate kinase lactate dehydrogenase coupled enzymatic assay was utilized with spectrophotometric readout of NADH oxidation at 340 nm. Described by Blodin et al. Anal. Biochem.1994, 220, 219. One CMK unit (U) corresponds to 1 µmol of CMP converted to CDP per minute at 30°C and pH=7.5 in a buffered solution containing 20 mM Mg2+and 0.5 M KCl. Example 7: Measurement of N-acetylneuraminate cytidyltransferase (CSS) activity in cell-free extracts To quantify the activity of NmCSS (GenBank Accession NO: WP_061726245) in cell-free extracts the phosphate released from the hydrolysis of pyrophosphate (PPi) formed during the synthesis of CMP- sialic acid from sialic acid and cytidine triphosphate (CTP) was assayed. Cell-free extract of E. Coli overexpressing the endogeneous inorganic diphosphatase (EcPPase, GenBank Accession NO: WP_073849715.1) was added to the reaction mixture and a time course of the reaction was recorded. At selected time points, a sample was taken and mixed with the working reagent (4 parts of a 10 % ascorbic acid solution and 1 part of a 15 mM Zinc acetate 10 mM ammonium molybdate solution). After 10 min color development at 30°C the absorbance at 630 nm was measured. One CSS unit (U) corresponds to 1 µmol of PPi hydrolyzed per minute at 37°C in 50 mM TRIS buffer pH=7.2, which in turn represents 1 µmol CTP converted into CMP-sialic acid per minute under these conditions. Example 8: Measurement of endogenous inorganic diphosphatase (PPase) activity For the determination of the overexpressed or endogenous EcPPase (GenBank Accession NO: WP_073849715.1) activity in cell-free extract the phosphate released from the hydrolysis of sodium pyrophosphate was assayed by recording a time course of the reaction. At selected time points, a sample was taken and mixed with a working reagent comprising 4 parts of a 10 % ascorbic acid solution and 1 part of a 15 mM Zinc acetate 10 mM ammonium molybdate solution. After 10 min color development at 30°C the absorbance at 630 nm was measured. The reaction was performed in TRIS buffer pH=7.2. One inorganic diphosphatase unit (U) corresponds to 1 µmol of PPi hydrolyzed per minute at 37°C in 50 mM TRIS buffer pH=7.2. Example 9: Measurement of endogenous nucleoside diphosphate kinase (NDK) activity For the determination of the endogenous EcNDK (GenBank Accession NO: YP_490746) activity in cell- free extracts the phosphate released from the hydrolysis of the pyrophosphate (PPi) formed during the synthesis of CMP-sialic acid from sialic acid, cytidine diphosphate (CDP), and adenosine 5’-triphosphate (ATP) was assayed. Cell-free extracts of E. coli expressing NmCSS (GenBank Accession NO: WP_061726245), and EcPPase (GenBank Accession NO: WP_073849715) were added to the reaction mixture. A time course of the reaction was recorded. At selected time points, a sample was taken and mixed with a working reagent comprising 4 parts of a 10 % ascorbic acid solution and 1 part of a 15 mM Zinc acetate 10 mM ammonium molybdate solution. After 10 min color development at 30°C the absorbance at 630 nm was measured. The reaction was performed in TRIS buffer pH=7.2. One NDK unit (U) corresponds to 1 µmol of PPi hydrolized per minute at 37°C in 50 mM TRIS buffer pH=7.2, which in turn corresponds to 1 µmol of CDP converted into CTP per minute under these conditions. Example 10: General procedure for the sialylation of glycoside acceptors The sialyltransferase cycle was performed in an aqueous solution at a pH between about 7.0 to about 7.5, the temperature ranged between about 25oC to about 37oC. A typical reaction mixture contained the glycoside acceptor (1 eq.), N-acetylneuraminic acid (Neu5Ac, 1.2-2.5 eq.), β-cyclodextrin (0.5 eq.), ATP (2.0-3.5 eq.), CMP (0.1-0.3 eq.), MgCl2(0.5 M), and the cell-free extracts comprising the required enzymes. The sialylation cycle was monitored by LCMS (For method and conditions see example 22). Conversions were typically 10-99%. When the desired conversion was reached, GtCDase, or AfCDase, or PfCDase was added to the reaction mixture (1000 to 5000 U / L).and heated to 65 °C. Example 11: Production of α-N-acetylneuraminosyl-(2→3)-O-β-D-galactopyranosyl-(1→4)-β-D- glucopyranosyl-(1→1´)-D-erythro-sphingosine (N-lyso-GM3) N-lyso-GM3 was produced using lactosylsphingosine as the glycoside acceptor following the general procedure described in Example 10, wherein the following three cell-free extracts were utilized: cell free extract (i) or (ii) (3220-34250 U / L), cell-free extract (iii) (3750-14000 U / L), cell-free extract (iv) (2960 U / L). LC / MS: Rt = 4.38 min; ESI-MS calculated for [C41H74N2O20]: 914, found: 915 [M+H]+, 913 [M-H]-. Example 12: Production of α-N-acetylneuraminosyl-(2→8)-O-α-N-acetylneuraminosyl-(2→3)-O-β-D- galactopyranosyl-(1→4)-β-D-glucopyranosyl-(1→1´)-D-erythro-sphingosine (N-lyso-GD3) from lactosyl sphingosine N-lyso-GD3 was produced using lactosylsphingosine as the glycoside acceptor following the general procedure described in Example 10, and wherein the following four cell-free extracts were utilized: cell free extract (i) or (ii) (3220-34250 U / L), cell-free extract (iii) (3750-14000 U / L), cell-free extract (iv) (2960 U / L), and cell-free extracts (v), or (vi), or (vii) (163-1000 U / L). LC / MS: Rt = 4.74 min; ESI-MS calculated for [C52H91N3O28]: 1205, found 1206 [M+H]+, 1204 [M-H]-. Example 13: Production of α-N-acetylneuraminosyl-(2→8)-O-α-N-acetylneuraminosyl-(2→3)-O-β-D- galactopyranosyl-(1→4)-β-D-glucopyranosyl-(1→1´)-D-erythro-sphingosine (N-lyso-GD3) from N-lyso- GM3 N-lyso-GD3 was produced using N-lyso-GM3 as the glycoside acceptor following the general procedure described in Example 10, and wherein the following three cell-free extracts were utilized: cell free extract (i) or (ii) (3220-34250 U / L), cell-free extract (iii) (3750-14000 U / L), and cell-free extract (v), or(vi), or (vii) (163-500 U / L). LC / MS: Rt = 4.74 min; ESI-MS calculated for [C52H91N3O28]: 1205, found 1206 [M+H]+, 1204 [M-H]-. Example 14: Production of mixtures of N-lyso-GD3 and N-lyso-GM3 Mixtures comprising different ratios of N-lyso-GD3 and N-lyso-GM3 were produced using N-lyso-GM3 as the glycoside acceptor following the general procedure described in Example 10, and wherein the following three cell-free extracts were utilized: cell free extract (i) or (ii) (3220-34250 U / L), cell-free extract (iii) (3750-14000 U / L), and cell-free extract (v), or(vi), or (vii) (163-500 U / L). The sialylation cycle was monitored by LCMS, and the reaction was quenched by deactivation of the enzymes (preferably via heat treatment) when the desired N-lyso-GM3 conversion was reached, corresponding to a specific ratio. Three mixtures were obtained following the procedure described above, these are: ─ 4:1 mixture of N-lyso-GD3 and N-lyso-GM3, ─ 1:3 mixture of N-lyso-GD3 and N-lyso-GM3, ─ 1:1 mixture of N-lyso-GD3 and N-lyso-GM3. Example 15: Production of α-N-acetylneuraminosyl-(2→3)-O-β-D-galactopyranosyl-(1→4)-α-D- glucopyranosyl fluoride (3’SL-fluoride) 3’SL-fluoride was produced using α-D-lactosyl fluoride as the glycoside acceptor following the general procedure described in Example 10, without adding an amylase (GtCDase, or AfCDase, or PfCDase) and the final heating step. The following three cell-free extracts were utilized: cell free extract (i) or (ii) (3220-34250 U / L), cell-free extract (iii) (3750-14000 U / L), cell-free extract (iv) (2960 U / L). Example 16: Production of α-N-acetylneuraminosyl-(2→8)-O-α-N-acetylneuraminosyl-(2→3)-O-β-D- galactopyranosyl-(1→4)-β-D-glucopyranosyl fluoride The title compound was produced from α-D-lactosyl fluoride following the general procedure described in Example 10, without adding an amylase (GtCDase, or AfCDase, or PfCDase) and the final heating step. The following cell-free extracts were utilized: cell free extract (i) or (ii) (3220-34250 U / L), cell-free extract (iii) (3750-14000 U / L), cell-free extract (iv) (2960 U / L), and cell-free extracts (v), or (vi), or (vii) (163-500 U / L). Example 17: Isolation of sialylated glycosphingolipids Sialylated glycosphingolipids produced as described in Examples 10-14 were isolated from the reaction mixture via diafiltration (DF). The DF was performed by applying 250 kDa spiral-wound membranes having a membrane area of about 0.668 m2. During the DF a flow rate of about 10 l / h, a transmembrane pressure of about 8-10 bar, a temperature between about 20-25oC and a pH= 7.0-7.5 was maintained. Filtration was continued until around 2-10 DF volumes relative to the volume of the feed solution was passed. During diafiltration, a high flux of about 15.3-18.1 l / m2h was maintained. The DF retentate (DFR), containing the sialylated glycosphingolipid, was spray-dried on a Mobile Minor ® (GEA) spray drier under the following conditions: Inlet flow rate: 45-50 g / min Atomizer speed: 20,000 rpm Inlet temperature: 160oC Outlet temperature: 85oC Following this procedure, a spry-dried powder comprising about 70-90 wt.% of one or more sialylated glycosphingolipids was obtained. Example 18. Particle size analysis and water content of the spray-dried powder The mean particle diameter, as well as the D(0.1), D(0.5), and D(0.9) values were measured by laser diffraction particle size analysis using a Malvern Mastersizer 3000 (Malvern Instruments). The spray- dried powder obtained in Example 17 was dispersed in cyclohexane including 0.1 % soy lecithin. The samples were sonicated before size measurement to disperse the aggregated particles. The water content of the spray-dried powder obtained in Example 17 was determined by thermogravimetry (TG) and differential scanning calorimetry (DSC), or via Karl Fisher titration. TG and DSC measurements were performed on a Setaram LabsysEvo (Setaram). The spray-dried powder typically contains between about 2-3 wt.% of water. Example 19. Spray-dried powders comprising a 4:1 mixture of N-lyso-GD3 and N-lyso-GM3 A DFR comprising a 4:1 mixture N-lyso-GD3 and N-lyso-GM3, obtained following the procedure of Example 17, was spray-dried under the condition of Example 17 to afford a spray-dried powder having the following characteristics: Glycosphingolipid Content Water Specific Mean Span Content Volume Particle Diameter Compound wt.% Rt (min) N-lyso-GD3 52.0 5.1 N-lyso-GM3 12.6 5.6 N-lyso-GT3 4.8 4.8 4.1 wt.% 2.0 mL / g 16.7 µm 1.4 Lactosyl D-erythro- 5.4 6.3 sphingosine Glucosyl D-erythro- 0.2 7.0 sphingosine Example 20. Spray-dried powders comprising a 1:3 mixture of N-lyso-GD3 and N-lyso-GM3 A DFR comprising a 1:3 mixture N-lyso-GD3 and N-lyso-GM3, obtained following the procedure of Example 17, was spray-dried under the condition of Example 17 to afford a spray-dried powder having the following characteristics: Glycosphingolipid Content Water Specific Mean Particle Span Content Volume Diameter Compound wt.% Rt (min) N-lyso-GD3 19.0 5.1 N-lyso-GM3 58.9 5.6 N-lyso-GT3 0.3 4.8 2.5 wt.% 2.0 mL / g 16.7 µm 1.4 Lactosyl D-erythro- 7.0 6.3 sphingosine GlucosylD-erythro- 0.4 7.0 sphingosine Example 21. Spray-dried powders comprising a 1:1 mixture of N-lyso-GD3 and N-lyso-GM3 A DFR comprising a 1:1 mixture N-lyso-GD3 and N-lyso-GM3, obtained following the procedure of Example 17, was spray-dried under the condition of Example 17 to afford a spray-dried powder having the following characteristics: Glycosphingolipid Content Water Specific Mean Particle Span Content Volume Diameter Compound wt.% Rt (min) N-lyso-GD3 35.5 4.6 N-lyso-GM3 25.0 5.0 LactosylD-erythro- 5.4 5.9 2.5 wt.% 3.0 mL / g 16.7 µm 1.4 sphingosine Glucosyl D-erythro- 0.8 6.5 sphingosine Example 22: LC / MS Analysis Samples (50 µL) were taken from reaction mixtures of examples 5.1 and 5.2 , mixed with DMSO (950 µL), subjected to centrifugation (16.000 rpm, 5 min) and the supernatant was analysed. The eluent consisted of solvent D (2 mM ammonium formate, 0.2 % v / v formic acid, 75% v / v MeOH, 25% v / v ACN) – solvent C (2 mM formic acid in filtered water), and the following gradients were applied: N-lyso GM370-95% (D in C), N-lyso-GD370-98% (D in C). Sequence List Overview of the SEQ ID NOs of the present invention. SEQ ID NO: Description Amino acid sequence of mutant α-2,3 / α-2,8-sialyltransferase derived from Q9LAK3. 1 Mutations / modifications: Ile53Ser, deletion of 32 amino acids at the C-terminus. Amino acid sequence of mutant α-2,3 / α-2,8-sialyltransferase derived from Q9LAK3. 2 Mutations / modifications: Ile53Ser, deletion of 32 amino acids at the C-terminus, N- terminal histidine tag. Amino acid sequence of mutant α-2,3 / α-2,8-sialyltransferase derived from Q9LAK3. 3 Mutations / modifications: Ile53Gly, deletion of 32 amino acids at the C-terminus, N- terminal histidine tag. Amino acid sequence of a mutant N-acylneuraminate cytidyltransferase derived from 4 WP_061726245. Mutations / modifications: N-terminal histidine tag SEQ ID NO: 1 MKKVIIAGNGPSLKEIDYSRLPNDFDVFRCNQFYFEDKYYLGKKCKAVFYNPSLFFEQYYTLKHLIQNQEYETELIMCSNY NQAHLENENFVKTFYDYFPDAHLGYDFFKQLKDFNAYFKFHEIYFNQRITSGVYMCAVAIALGYKEIYLSGIDFYQNGSSY AFDTKQKNLLKLAPNFKNDNSHYIGHSKNTDIKALEFLEKTYKIKLYCLCPNSLLANFIELAPNLNSNFIIQEKNNYTKDILIP SSEAYGKFSKNIN SEQ ID NO: 2 MGHHHHHHMKKVIIAGNGPSLKEIDYSRLPNDFDVFRCNQFYFEDKYYLGKKCKAVFYNPSLFFEQYYTLKHLIQNQEY ETELIMCSNYNQAHLENENFVKTFYDYFPDAHLGYDFFKQLKDFNAYFKFHEIYFNQRITSGVYMCAVAIALGYKEIYLSG IDFYQNGSSYAFDTKQKNLLKLAPNFKNDNSHYIGHSKNTDIKALEFLEKTYKIKLYCLCPNSLLANFIELAPNLNSNFIIQE KNNYTKDILIPSSEAYGKFSKNIN SEQ ID NO: 3 MGHHHHHHMKKVIIAGNGPSLKEIDYSRLPNDFDVFRCNQFYFEDKYYLGKKCKAVFYNPGLFFEQYYTLKHLIQNQEY ETELIMCSNYNQAHLENENFVKTFYDYFPDAHLGYDFFKQLKDFNAYFKFHEIYFNQRITSGVYMCAVAIALGYKEIYLSG IDFYQNGSSYAFDTKQKNLLKLAPNFKNDNSHYIGHSKNTDIKALEFLEKTYKIKLYCLCPNSLLANFIELAPNLNSNFIIQE KNNYTKDILIPSSEAYGKFSKNIN SEQ ID NO: 4 MGHHHHHHMEKQNIAVILARQNSKGLPLKNLRKMNGISLLGHTINAAISSKCFDRIIISTDGGLIAEEAKNFGVEVVL RPAELASDTA SSISGVIHALETIGSNSGTVTLLQPTSPLRTGAHIREAFSLFDEKIKGSVVSACPMEHHP LKTLLQINNG EYAPMRHLSDLEQPRQQLPQAFRPNGAIYINDTASLIANNCFFIAPTKLYIMSHQDSIDI DTELDLQQAENILNHKES The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above described embodiments are combinable. The following claims further set out particular embodiments of the disclosure.

Claims

Claims 1. Method for the sialylation of a glycoside of formula (1), or a salt thereof:wherein X is a glycosyl moiety, wherein the glycosyl moiety is preferably selected from the group consisting of Gal1-, or a glycosyl moiety carrying one or more terminal galactose units and / or one or more terminal N-acetyl-galactosamine units and / or one or more terminal sialic acid units; Y is selected from the group consisting of hydroxyl, fluoride, or a moiety of formula (2), or a salt thereof:wherein R1is hydrogen, aryl, or a substituted or unsubstituted C1-50 alkyl, preferably a substituted or unsubstituted C1-17 alkyl, more preferably a substituted or unsubstituted C10-17 alkyl, R2is hydrogen or -OR5, wherein R5is selected from hydrogen, a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C2-6acyl, the bond may be a double or a single bond when R2is hydrogen, or is a single bond when R2is -OR5, R3is hydrogen, a substituted or unsubstituted C1-6 alkyl, or a substituted or unsubstituted C1-6 acyl, preferably hydrogen, R4is selected from hydrogen, a substituted or unsubstituted aryl, a heteroalkyl, a substituted or unsubstituted C2-32 acyl, the method comprising: mixing the glycoside of formula (1) with sialic acid, cytidine monophosphate, a nucleoside triphosphate and one or more cell-free extracts of a microorganism, said microorganismcomprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein said one or more cell-free extracts comprise: ─ at least one polypeptide having cytidine monophosphate kinase activity, ─ at least one polypeptide having N-acylneuraminate citydyltransferase activity, and ─ at least one polypeptide having sialyltransferase activity, thereby sialylating said glycoside.

2. The method according to claim 1, wherein the microorganism is genetically engineered for the expression of one or more polypeptides selected from the group consisting of: ─ at least one polypeptide having cytidine monophosphate kinase activity, ─ at least one polypeptide having N-acylneuraminate citydyltransferase activity, and ─ at least one polypeptide having sialyl-transferase activity.

3. The method according to claim 1 or 2 wherein the microorganism is Escherichia coli.

4. The method according to any one of claims 1 to 3, wherein the microorganism comprising one or two endogenous polypeptides having phosphotransferase activity, and one endogenous polypeptide having inorganic diphosphatase activity.

5. The method according to claims 3 or 4, wherein the one or two endogenous polypeptides having phosphotransferase activity are selected from the group consisting of an endogenous Escherichia coli nucleoside diphosphate kinase, and / or an endogenous Escherichia coli myokinase.

6. The method according to any one of claims 3 to 5, wherein the endogenous polypeptide having inorganic diphosphatase activity is an endogenous Escherichia coli inorganic diphosphatase.

7. The method according to any one of claims 3 to 6, wherein the one or more cell-free extracts comprise one polypeptide having cytidine monophosphate kinase activity, and wherein said one polypeptide is a heterologous cytidine monophosphate kinase of amino acid sequence originating from Mycobacterium tuberculosis, or from Bacillus subtilis, or a functional analogue thereof.

8. The method according to any one of claims 3 to 7, wherein the one or more cell-free extracts comprise one heterologous polypeptide having N-acylneuraminate citydyltransferase activity, and wherein said polypeptide is a heterologous N-acylneuraminate citydyltransferase of amino acid sequence originating from Neisseria meningitidis, or a functional analogue thereof.

9. The method according to any one of claims 3 to 8, wherein the one or more cell-free extracts comprise one polypeptide having sialyl-transferase activity, wherein said one polypeptide is a heterologous sialyl-transferase of amino acid sequence originating from Campylobacter jejuni, strain OX=197, or a functional analogue thereof.

10. The method according to any one of claims 3 to 8, wherein the one or more cell-free extracts comprise one polypeptide having sialyl-transferase activity, wherein said polypeptide is a heterologous sialyl-transferase of amino acid sequence originating from Bibersteinia trehalosi, strain DSM 23101, or a functional analogue thereof.

11. The method according to any one of claims 3 to 8, wherein the one or more cell-free extracts comprise two polypeptides having sialyl-transferase activity, and wherein said two polypeptides are heterologous sialyl-transferases according to claims 9 and 10.

12. The method according to any one of claims 1 to 11, wherein the nucleoside triphosphate is adenosine 5’-triphosphate (ATP).

13. The method according to any one of claims 1 to 12 wherein the method further comprising the use of a cyclodextrin.

14. The method according to claim 13, wherein the cyclodextrin is β-cyclodextrin.

15. The method according to claims 13 or 14, wherein the method further comprising the use of a polypeptide having amylase activity.

16. The method according to claim 15, wherein the polypeptide having amylase activity is an amylase of amino acid sequence originating from Geobacillus thermoleovorans, Anoxybacillus flavithermus, or Pyrococcus furiosus, or a functional analogue thereof.

17. The method according to any one of claims 1 to 16, wherein the glycoside of formula (1), is a glycoside of formula (3):(3), wherein X is as defined as for the glycoside of formula (1), the bond , R1, R2, R3, and R4are as defined as for the moiety of formula (2).

18. The method according to claim 17, wherein for the glycoside of formula (3) R2, R3and R4are hydrogen, and R1is a C13-17alkyl, preferably a C13alkyl.

19. The method according to any one of claims 1 to 18, wherein X is a glycosyl moiety selected from the group consisting of Gal1β-, Galβ1-4Glc1β-, Neu5Acα2-3Galβ1-4Glc1β-, or Galβ1-3GalNAcβ1- 4(Neu5Acα2-3)Galβ1-4Glcβ-.

20. The method according to any one of claims 17 to 19, wherein the glycoside of formula (3) N- lyso-GM3.

21. The method according to any one of claims 1 to 20, wherein the method further comprising isolating the sialylated glycoside.

22. A sialylating agent comprising one or more cell-free extracts of a microorganism, said microorganism comprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein said one or more cell-free extract comprise: ─ at least one polypeptide having cytidine monophosphate kinase activity, ─ at least one polypeptide having N-acylneuraminate citydyltransferase activity, and ─ at least one polypeptide having sialyltransferase activity.

23. The sialylating agent according to claim 22, wherein the microorganism is genetically engineered for the expression of one or more polypeptides selected from: ─ at least one polypeptide having cytidine monophosphate kinase activity, and ─ at least one polypeptide having N-acylneuraminate citydyltransferas activity, and ─ at least one polypeptide having sialyltransferase activity.

24. The sialylating agent according to claims 21 or 22, wherein the microorganism is Escherichia coli.

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