Sialyltransferases for the production of sialylated oligosaccharides

Abstract

The present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation. The present invention relates to newly identified sialyltransferases having alpha-2,3- sialyltransferase activity on an acceptor which is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide. The invention also describes methods for the production of a 3'sialylated oligosaccharide using any one of said newly identified sialyltransferases as well as the purification of said 3'sialylated oligosaccharide. The present invention also provides a cell for production of said 3'sialylated oligosaccharide and the use of said cell in a cultivation or incubation.

Description

[0001] Sialyltransferases for the production of sialylated oligosaccharides

[0002] Field of the invention

[0003] The present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation. The present invention relates to newly identified sialyltransferases having alpha-2, 3- sialyltransferase activity on an acceptor which is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide. The invention also describes methods for the production of a 3'sialylated oligosaccharide using any one of said newly identified sialyltransferases as well as the purification of said 3'sialylated oligosaccharide. The present invention also provides a cell for production of said 3'sialylated oligosaccharide and the use of said cell in a cultivation or incubation.

[0004] Background

[0005] More than 150 structurally distinct human milk oligosaccharides (HMOs) have been identified to date. Although HMOs represent only a minor amount of total human milk nutrients, their beneficial effects on the development of breast-fed infants became evident over the past decades.

[0006] Among the HMOs, sialylated HMOs (SHMOs) were observed to support several beneficial effects as described in the art. Among the sialylated oligosaccharides in human milk, 3'sialyllactose, 5'sialyllactose, sialyllacto-N-tetraose a, sialyl lacto-N-tetraose b, sialyllacto-N-tetraose c and disialyllacto-N-tetraose are the most prevalent members.

[0007] Sialylated oligosaccharides are found to be a complex structure and their chemical or (chemo-)enzymatic syntheses has been proven challenging: there are extensive difficulties, e.g. control of stereochemistry, formation of specific linkages, availability of feedstocks, etc. As a consequence, alternative production methods have been developed, amongst which efforts in metabolic engineering of microorganisms to produce sialylated oligosaccharides have been made.

[0008] Several sialyltransferases have been identified and characterized to date, from bacterial species e. g. from Neisseria, Campylobacter, Pasteurella, Helicobacter and Photobacterium, as well as from mammals and viruses. Sialyltransferases have been generally classified into six glycosyltransferase (GT) families, based on protein sequence similarities. Sialyltransferases are distinguished due to the glycosidic linkages that they form, e. g. into a-2,3-, a-2,6- and a-2,8-sialyltransferases. All of these sialyltransferases transfer the sialic acid residue from cytidine 5'-monophosphate sialic acid (e. g. CMP-NeuNAc) to a variety of acceptor molecules, usually a galactose (Gal) moiety, an N-acetylgalactosamine (GalNAc) moiety or an N- acetylglucosamine (GIcNAc) moiety or another sialic acid (Sia) moiety. Description

[0009] Summary of the invention

[0010] It is an object of the present invention to provide for tools and methods by means of which a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, can be produced, preferably in an efficient, time and cost-effective way and which yields high amounts of the desired oligosaccharide.

[0011] According to the invention, this and other objects are achieved by providing newly identified alpha-2, 3- sialyltransferases as described herein, each of which can be used in a method for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide. Such method comprising contacting a sialyltransferase with a mixture comprising a donor comprising a sialic acid residue, and an acceptor, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from the donor to the acceptor, thereby producing said 3'sialylated oligosaccharide, wherein said acceptor is a saccharide comprising at least one N- acetylglucosamine monosaccharide and a galactose monosaccharide, chosen from the list consisting of an oligosaccharide or a disaccharide.

[0012] Furthermore, any one of said newly identified alpha-2, 3-sialyltransferases can be used in a cell for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.

[0013] More preferably, the invention provides newly identified alpha-2, 3-sialyltransferases having alpha-2, 3- sialyltransferase activity on a galactose (Gal) residue, preferably a terminal Gal residue, of an acceptor wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide chosen from the list consisting of an oligosaccharide or disaccharide, and wherein any one or more of said alpha-2, 3-sialyltransferase comprise an amino acid sequence that is at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30, 31, 25, 18, 26 or 22, or that is at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28, or 29.

[0014] The invention also provides methods and a cell for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide. The present invention also provides methods for the purification of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide. Furthermore, the present invention provides a cell which is metabolically engineered with any one of said newly identified alpha-2, 3-sialyltransferases as described herein and which comprises a pathway for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide. The present invention also provides for newly identified alpha-2, 3-sialyltransferases for use in the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.

[0015] Definitions

[0016] The words used in this specification to describe the invention and its various embodiments are to be understood not only in the sense of their commonly defined meanings, but to include by special definition in this specification structure, material or acts beyond the scope of the commonly defined meanings. Thus, if an element can be understood in the context of this specification as including more than one meaning, then its use in a claim must be understood as being generic to all possible meanings supported by the specification and by the word itself.

[0017] The various aspects and embodiments of the invention disclosed herein are to be understood not only in the order and context specifically described in this specification, but to include any order and any combination thereof. Each embodiment as identified herein may be combined together unless otherwise indicated. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0018] Whenever the context requires, unless specifically stated otherwise, all words used in the singular number shall be deemed to include the plural and vice versa. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described herein are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications.

[0019] In the specification, there have been disclosed embodiments of the invention, and although specific terms are employed, the terms are used in a descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims. It must be understood that the illustrated embodiments have been set forth only for the purposes of example and that it should not be taken as limiting the invention. It will be apparent to those skilled in the art that alterations, other embodiments, improvements, details and uses can be made consistent with the letter and spirit of the disclosure herein and within the scope of this disclosure, which is limited only by the claims, construed in accordance with the patent law, including the doctrine of equivalents. In the claims that follow, reference characters used to designate claim steps are provided for convenience of description only and are not intended to imply any particular order for performing the steps, unless specifically stated otherwise.

[0020] Throughout the application, unless explicitly stated otherwise, the features "synthesize", "synthesized" and "synthesis" are interchangeably used with the features "produce", "produced" and "production", respectively.

[0021] Throughout the application, unless explicitly stated otherwise, the expressions "capable of...<verb>" and "capable to...<verb>" are preferably replaced with the active voice of said verb and vice versa. For example, the expression "capable of expressing" is preferably replaced with "expresses" and vice versa, i.e. "expresses" is preferably replaced with "capable of expressing".

[0022] In this document and in its claims, the verbs "to comprise", "to have" and "to contain" and their conjugations are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Throughout the application, the verb "to comprise" may be replaced by "to consist of" or "to consist essentially of" and vice versa. In addition, the verb "to consist of" may be replaced by "to consist essentially of" meaning that a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In this document and in its claims, unless specifically stated otherwise, the verbs "to comprise", "to have" and "to contain", and their conjugations, may be replaced by "to consist of"(and its conjugations) or "to consist essentially of"(and its conjugations) and vice versa.

[0023] In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0024] Throughout the application, unless explicitly stated otherwise, the articles "a" and "an" are preferably replaced by "at least two", more preferably by "at least three", even more preferably by "at least four", even more preferably by "at least five", even more preferably by "at least six", most preferably by "at least two".

[0025] The word "about" or "approximately" when used in association with a numerical value (e.g. "about 10") or with a range (e.g. "about x to approximately y") preferably means that the value or range is interpreted as being as accurate as the method used to measure it. If no error margins are specified, the expression "about" or "approximately" when used in association with a numerical value is interpreted as having the same round-off as the given value. Throughout this document and its claims, unless otherwise stated, the expression "from x to y", wherein x and y represent numerical values, refers to a range of numerical values wherein x is the lower value of the range and y is the upper value of the range. Herein, x and y are also included in the range.

[0026] According to the present invention, the term "polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triplestranded regions, or a mixture of single- and double-stranded regions. In addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" according to the present invention. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, are to be understood to be covered by the term "polynucleotides". It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. The term "polynucleotide(s)" also embraces short polynucleotides often referred to as oligonucleotide(s).

[0027] "Polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. "Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to the skilled person. The same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Furthermore, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid sidechains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulphide bond formation, demethylation, formation of covalent cross-links, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation, selenoylation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.

[0028] The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions that also may contain coding and / or non-coding sequences.

[0029] "Isolated" means altered "by the hand of man" from its natural state, i.e. if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Similarly, a "synthetic" sequence, as the term is used herein, means any sequence that has been generated synthetically and not directly isolated from a natural source. "Synthesized", as the term is used herein, means any synthetically generated sequence and not directly isolated from a natural source.

[0030] "Recombinant" means genetically engineered DNA prepared by transplanting or splicing genes from one species into the cells of a host organism of a different species. Such DNA becomes part of the host's genetic makeup and is replicated.

[0031] The terms "recombinant" or "transgenic" or "metabolically engineered" or "genetically engineered" as used herein with reference to a cell or host cell are used interchangeably and indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e. a sequence "foreign to said cell" or a sequence "foreign to said location or environment in said cell"). Such cells are described to be transformed with at least one heterologous or exogenous gene or are described to be transformed by the introduction of at least one heterologous or exogenous gene. Metabolically engineered or recombinant or transgenic or genetically engineered cells can contain genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means. The terms also encompass cells that contain a nucleic acid endogenous to the cell that has been modified or its expression or activity has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, replacement of a promoter; site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis and related techniques as known to a person skilled in the art. Accordingly, a "recombinant polypeptide" is one which has been produced by a recombinant cell. The terms also encompass cells that have been modified by removing a nucleic acid endogenous to the cell by means of common well-known technologies for a skilled person (like e.g. knocking-out genes).

[0032] A "heterologous sequence" or a "heterologous nucleic acid", as used herein, is one that originates from a source foreign to the particular cell (e.g. from a different species), or, if from the same source, is modified from its original form or place in the genome. Thus, a heterologous nucleic acid operably linked to a promoter is from a source different from that from which the promoter was derived, or, if from the same source, is modified from its original form or place in the genome. The heterologous sequence may be stably introduced, e.g. by transfection, transformation, conjugation or transduction, into the genome of the host microorganism cell, wherein techniques may be applied which will depend on the cell and the sequence that is to be introduced. Various techniques are known to a person skilled in the art and are, e.g. disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The term "mutant" or "engineered" cell or microorganism as used within the context of the present invention refers to a cell or microorganism which is genetically engineered.

[0033] The term "endogenous," within the context of the present disclosure refers to any polynucleotide, polypeptide or protein sequence that is a natural part of a cell and is occurring at its natural location in the cell chromosome and of which the control of expression has not been altered compared to the natural control mechanism acting on its expression. The term "exogenous" refers to any polynucleotide, polypeptide or protein sequence that originates from outside the cell under study and not a natural part of the cell or that is not occurring at its natural location in the cell chromosome or plasmid.

[0034] The term "heterologous" when used in reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme refers to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is from a source or derived from a source other than the host organism species. In contrast a "homologous" polynucleotide, gene, nucleic acid, polypeptide, or enzyme is used herein to denote a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is derived from the host organism species. When referring to a gene regulatory sequence or to an auxiliary nucleic acid sequence used for maintaining or manipulating a gene sequence (e.g. a promoter, a 5' untranslated region, 3' untranslated region, poly A addition sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genome homology region, recombination site, etc.), "heterologous" means that the regulatory sequence or auxiliary sequence is not naturally associated with the gene with which the regulatory or auxiliary nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome. Thus, a promoter operably linked to a gene to which it is not operably linked to in its natural state (i.e. in the genome of a non- genetically engineered organism) is referred to herein as a "heterologous promoter," even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked.

[0035] The term "modified expression" of a gene relates to a change in expression compared to the wild-type expression of said gene in any phase of the production process of the desired 3'sialylated oligosaccharide. Said modified expression is either a lower or higher expression compared to the wild-type, wherein the term "higher expression" is also defined as "overexpression" of said gene in the case of an endogenous gene or "expression" in the case of a heterologous gene that is not present in the wild-type strain. Lower expression is obtained by means of common well-known technologies for a skilled person (such as the usage of siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon mutagenesis, etc.) which are used to change the genes in such a way that they are less able (i.e. statistically significantly 'less able' compared to a functional wild-type gene) or completely unable (such as knocked-out genes) to produce functional final products. The term "riboswitch" as used herein is defined to be part of the messenger RNA that folds into intricate structures that block expression by interfering with translation. Binding of an effector molecule induces conformational change(s) permitting regulated expression post- transcriptionally. Next to changing the gene of interest in such a way that lower expression is obtained as described above, lower expression can also be obtained by changing the transcription unit, the promoter, an untranslated region, the ribosome binding site, the Shine Dalgarno sequence or the transcription terminator. Lower expression or reduced expression can for instance be obtained by mutating one or more base pairs in the promoter sequence or changing the promoter sequence fully to a constitutive promoter with a lower expression strength compared to the wild-type or an inducible promoter which result in regulated expression or a repressible promoter which results in regulated expression.

[0036] Overexpression or expression is obtained by means of common well-known technologies for a skilled person (such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids), wherein said gene is part of an "expression cassette” that relates to any sequence in which a promoter sequence, untranslated region sequence (containing either a ribosome binding sequence, Shine Dalgarno or Kozak sequence), a coding sequence (for instance a sialyltransferase gene sequence) and optionally a transcription terminator is present, and leading to the expression of a functional active protein. Said expression is either constitutive or conditional or regulated or tuneable.

[0037] The term "constitutive expression" is defined as expression that is not regulated by transcription factors other than the subunits of RNA polymerase (e.g. the bacterial sigma factors like o70, <J54, or related o- factors and the yeast mitochondrial RNA polymerase specificity factor MTF1 that co-associate with the RNA polymerase core enzyme) under certain growth conditions. Non-limiting examples of such transcription factors are CRP, Lacl, ArcA, Cra, IcIR in E. coli, or Aft2p, Crzlp, Skn7 in Saccharomyces cerevisiae, or DeoR, GntR, Fur in B. subtilis. These transcription factors bind on a specific sequence and may block or enhance expression in certain growth conditions. The RNA polymerase is the catalytic machinery for the synthesis of RNA from a DNA template. RNA polymerase binds a specific DNA sequence to initiate transcription, for instance via a sigma factor in prokaryotic hosts or via MTFl in yeasts. Constitutive expression offers a constant level of expression with no need for induction or repression.

[0038] The term "regulated expression" is defined as a facultative or regulatory or tuneable expression of a gene that is only expressed upon a certain natural condition of the host (e.g. mating phase of budding yeast, stationary phase of bacteria), as a response to an inducer or repressor such as but not limited to glucose, allo-lactose, lactose, galactose, glycerol, arabinose, rhamnose, fucose, IPTG, methanol, ethanol, acetate, formate, aluminium, copper, zinc, nitrogen, phosphates, xylene, carbon or nitrogen depletion, or substrates or the produced product or chemical repression, as a response to an environmental change (e.g. anaerobic or aerobic growth, oxidative stress, pH shifts, temperature changes like e.g. heat-shock or cold-shock, osmolarity, light conditions, starvation) or dependent on the position of the developmental stage or the cell cycle of said host cell including but not limited to apoptosis and autophagy. Regulated expression allows for control as to when a gene is expressed.

[0039] The term "inducible expression by a natural inducer" is defined as a facultative or regulatory expression of a gene that is only expressed upon a certain natural condition of the host (e.g. organism being in labour, or during lactation), as a response to an environmental change (e.g. including but not limited to hormone, heat, cold, pH shifts, light, oxidative or osmotic stress / signalling), or dependent on the position of the developmental stage or the cell cycle of said host cell including but not limited to apoptosis and autophagy.

[0040] The term "inducible expression upon chemical treatment" is defined as a facultative or regulatory expression of a gene that is only expressed upon treatment with a chemical inducer or repressor, wherein said inducer and repressor comprise but are not limited to an alcohol (e.g. ethanol, methanol), a carbohydrate (e.g. glucose, galactose, glycerol, lactose, arabinose, rhamnose, fucose, allo-lactose), metal ions (e.g. aluminium, copper, zinc), nitrogen, phosphates, IPTG, acetate, formate, xylene.

[0041] The term "control sequences" refers to sequences recognized by the cells transcriptional and translational systems, allowing transcription and translation of a polynucleotide sequence to a polypeptide. Such DNA sequences are thus necessary for the expression of an operably linked coding sequence in a particular host cell, cell or organism. Such control sequences can be, but are not limited to, promoter sequences, ribosome binding sequences, Shine Dalgarno sequences, Kozak sequences, transcription terminator sequences. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. DNA for a presequence or secretory leader may be operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Said control sequences can furthermore be controlled with external chemicals, such as, but not limited to, IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose via an inducible promoter or via a genetic circuit that either induces or represses the transcription or translation of said polynucleotide to a polypeptide.

[0042] Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.

[0043] The term "wild-type" refers to the commonly known genetic or phenotypical situation as it occurs in nature.

[0044] The term "modified expression of a protein" as used herein refers to i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein, iii) expression and / or overexpression of a variant protein that has a higher activity compared to the wild-type (i.e. native in the expression host) protein, iv) reduced expression of an endogenous protein or v) expression and / or overexpression of a variant protein that has a reduced activity compared to the wild-type (i.e. native in the expression host) protein. Preferably, the term "modified expression of a protein" as used herein refers to i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein or iii) expression and / or overexpression of a variant protein that has a higher activity compared to the wild-type (i.e. native in the expression host) protein.

[0045] The term "modified activity" of a protein relates to a non-native activity of the protein in any phase of the production process of the desired 3'sialylated oligosaccharide. The term "non-native", as used herein with reference to the activity of a protein indicates that the protein has been modified to have an abolished, impaired, reduced, delayed, higher, accelerated or improved activity compared to the native activity of said protein. A modified activity of a protein is obtained by modified expression of said protein or is obtained by expression of a modified, i.e. mutant form of the protein. A mutant form of the protein can be obtained by expression of a mutant form of the gene encoding the protein, e.g. comprising a deletion, an insertion and / or a mutation of one or more nucleotides compared to the native gene sequence. A mutant form of a gene can be obtained by techniques well-known to a person skilled in the art, such as but not limited to site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis.

[0046] The term "non-native", as used herein with reference to a cell producing a 3'sialylated oligosaccharide, indicates that the 3'sialylated oligosaccharide is i) not naturally produced or ii) when naturally produced not in the same amounts by the cell; and that the cell has been genetically engineered to be able to produce said 3'sialylated oligosaccharide or to have a higher production of the 3'sialylated oligosaccharide.

[0047] "Variant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to the persons skilled in the art.

[0048] In some embodiments, the present invention contemplates making functional variants by modifying the structure of an enzyme as used in the present invention. Variants can be produced by amino acid substitution, deletion, addition, or combinations thereof. For instance, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (e.g. conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Whether a change in the amino acid sequence of a polypeptide of the invention results in a functional homolog can be readily determined by assessing the ability of the variant polypeptide to produce a response in cells in a fashion similar to the wild-type polypeptide.

[0049] "Fragment", with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule, particularly a part of a polynucleotide that retains a usable, functional characteristic of the full-length polynucleotide molecule. Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A "polynucleotide fragment" refers to any subsequence of a polynucleotide SEQ ID NO, typically, comprising or consisting of at least about 9, 10, 11, 12 consecutive nucleotides from said polynucleotide SEQ ID NO, for example at least about 30 nucleotides or at least about 50 nucleotides of any of the polynucleotide sequences provided herein. Exemplary fragments can additionally or alternatively include fragments that comprise, consist essentially of, or consist of a region that encodes a conserved family domain of a polypeptide. Exemplary fragments can additionally or alternatively include fragments that comprise a conserved domain of a polypeptide. As such, a fragment of a polynucleotide SEQ ID NO preferably means a nucleotide sequence which comprises or consists of said polynucleotide SEQ ID NO wherein no more than about 200, 150, 100, 50 or 25 consecutive nucleotides are missing, preferably no more than about 50 consecutive nucleotides are missing, and which retains a usable, functional characteristic (e.g. activity) of the full-length polynucleotide molecule which can be assessed by the skilled person through routine experimentation. Alternatively, a fragment of a polynucleotide SEQ ID NO preferably means a nucleotide sequence which comprises or consists of an amount of consecutive nucleotides from said polynucleotide SEQ ID NO and wherein said amount of consecutive nucleotides is at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0% 91.0% 92.0% 93.0% 94.0% 95.0% 95.5% 96.0% 96.5% 97.0% 97.5% 98.0% 98.5% 99.0% 99.5%, 100%, preferably at least 80.0%, more preferably at least 85.0%, even more preferably at least 87.0%, even more preferably at least 90.0%, even more preferably at least 95.0%, most preferably at least 97.0%, of the full-length of said polynucleotide SEQ ID NO and retains a usable, functional characteristic (e.g. activity) of the full-length polynucleotide molecule which can be routinely assessed by the skilled person. As such, a fragment of a polynucleotide SEQ ID NO preferably means a nucleotide sequence which comprises or consists of said polynucleotide SEQ ID NO, wherein an amount of consecutive nucleotides is missing and wherein said amount is no more than 50.0%, 40.0%, 30.0% of the full-length of said polynucleotide SEQ ID NO, preferably no more than 20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, more preferably no more than 15.0%, even more preferably no more than 10.0%, even more preferably no more than 5.0%, most preferably no more than 2.5%, of the full-length of said polynucleotide SEQ ID NO and wherein said fragment retains a usable, functional characteristic (e.g. activity) of the full-length polynucleotide molecule which can be routinely assessed by the skilled person.

[0050] "Fragment", with respect to a polypeptide, refers to a subsequence of the polypeptide that performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. A "subsequence of the polypeptide" or "a stretch of amino acid residues" as described herein refers to a sequence of contiguous amino acid residues derived from the polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription. Fragments can vary in size from as few as 3 amino acid residues to the full length of the intact polypeptide, for example at least about 10 amino acid residues in length, for example at least about 20 amino acid residues in length, for example at least about 30 amino acid residues in length, for example at least about 150 amino acid residues in length, for example at least about 200 amino acid residues in length. As such, a fragment of a polypeptide SEQ ID NO (or UniProt ID) preferably means a polypeptide sequence which comprises or consists of said polypeptide SEQ ID NO (or UniProt ID) wherein no more than about 200, 150, 125, 100, 80, 60, 50, 40, 30, 20 or 15 consecutive amino acid residues are missing, preferably no more than about 100 consecutive amino acid residues are missing, more preferably no more than about 50 consecutive amino acid residues are missing, even more preferably no more than about 40 consecutive amino acid residues are missing, and performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent, as does the intact polypeptide which can be routinely assessed by the skilled person. Alternatively, a fragment of a polypeptide SEQ ID NO (or UniProt ID) preferably means a polypeptide sequence which comprises or consists of an amount of consecutive amino acid residues from said polypeptide SEQ ID NO (or UniProt ID) and wherein said amount of consecutive amino acid residues is at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0% 85.0% 86.0% 87.0% 88.0% 89.0% 90.0% 91.0% 92.0% 93.0% 94.0% 95.0% 95.5% 96.0% 96.5% 97.0% 97.5% 98.0% 98.5% 99.0% 99.5°% 100% preferably at least 80.0% more preferably at least 85.0%, even more preferably at least 87.0%, even more preferably at least 90.0%, even more preferably at least 95.0%, most preferably at least 97.0% of the full-length of said polypeptide SEQ ID NO (or UniProt ID) and that performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent, as does the intact polypeptide which can be routinely assessed by the skilled person. As such, a fragment of a polypeptide SEQ ID NO (or UniProt ID) preferably means a polypeptide sequence which comprises or consists of said polypeptide SEQ ID NO (or UniProt ID), wherein an amount of consecutive amino acid residues is missing and wherein said amount is no more than 50.0%, 40.0%, 30.0% of the full-length of said polypeptide SEQ ID NO (or UniProt ID), preferably no more than 20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, more preferably no more than 15.0%, even more preferably no more than 10.0%, even more preferably no more than 5.0%, most preferably no more than 2.5%, of the full-length of said polypeptide SEQ ID NO (or UniProt ID) and that performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent, as does the intact polypeptide which can be routinely assessed by the skilled person.

[0051] Throughout the application, the sequence of a polypeptide can be represented by a SEQ ID NO or alternatively by a UniProt ID. Therefore, the terms "polypeptide SEQ ID NO" and "polypeptide UniProt ID" can be interchangeably used, unless explicitly stated otherwise.

[0052] A "functional fragment" of a polypeptide has at least one property or activity of the polypeptide from which it is derived, preferably to a similar or greater extent. A functional fragment can, for example, include a functional domain or conserved domain of a polypeptide. It is understood that a polypeptide or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the polypeptide's activity. By conservative substitutions is intended substitutions of one hydrophobic amino acid for another or substitution of one polar amino acid for another or substitution of one acidic amino acid for another or substitution of one basic amino acid for another etc. Preferably, by conservative substitutions is intended combinations such as glycine by alanine and vice versa; valine, isoleucine and leucine by methionine and vice versa; aspartate by glutamate and vice versa; asparagine by glutamine and vice versa; serine by threonine and vice versa; lysine by arginine and vice versa; cysteine by methionine and vice versa; and phenylalanine and tyrosine by tryptophan and vice versa.

[0053] Homologous sequences as used herein describes those nucleotide sequences that have sequence similarity and encode polypeptides that share at least one functional characteristic such as a biochemical activity. More specifically, the term "functional homolog" as used herein describes those polypeptides that have sequence similarity (in other words, homology) and at the same time have at least one functional similarity such as a biochemical activity (Altenhoff et al., PLoS Comput. Biol. 8 (2012) el002514). Homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of the nucleotides or polypeptides of interest. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI- BLAST analysis of non-redundant databases using the amino acid sequence of a reference polypeptide sequence. The amino acid sequence is, in some instances, deduced from the nucleotide sequence. Typically, those polypeptides in the database that have greater than 40% sequence identity to a polypeptide of interest are candidates for further evaluation for suitability as a homologous polypeptide, amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another or substitution of one acidic amino acid for another or substitution of one basic amino acid for another etc. Preferably, by conservative substitutions is intended combinations such as glycine by alanine and vice versa; valine, isoleucine and leucine by methionine and vice versa; aspartate by glutamate and vice versa; asparagine by glutamine and vice versa; serine by threonine and vice versa; lysine by arginine and vice versa; cysteine by methionine and vice versa; and phenylalanine and tyrosine by tryptophan and vice versa. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated.

[0054] A domain can be characterized, for example, by a Pfam (El-Gebal i et al., Nucleic Acids Res. 47 (209) D427- D432), an IPR (InterPro domain) (http: / / ebi.ac.uk / interpro) (Mitchell et al., Nucleic Acids Res. 47 (2019) D351-D360), a protein fingerprint domain (PRINTS) (Attwood et al., Nucleic Acids Res. 31 (2003) 400-402), a SUBFAM domain (Gough et al., J. Mol. Biol. 313 (2001) 903-919), a TIGRFAM domain (Selengut et al., Nucleic Acids Res. 35 (2007) D260-D264), a Conserved Domain Database (CDD) designation (https: / / www.ncbi.nlm.nih.gov / cdd) (Lu et aL, Nucleic Acids Res. 48 (2020) D265-D258), a PTHR domain (http: / / www.pantherdb.org) (Mi et al., Nucleic Acids. Res. 41 (2013) D377-D386; Thomas et aL, Genome Research 13 (2003) 2129-2141) or a PATRIC identifier or PATRIC DB global family domain (https: / / www.patricbrc.org / ) (Davis et aL, Nucleic Acids Res. 48 (DI) (2020) D606-D612). Protein or polypeptide sequence information and functional information can be provided by a comprehensive resource for protein sequence and annotation data like e.g. the Universal Protein Resource (UniProt) (www.uniprot.or ) (Nucleic Acids Res. 2021, 49 (DI), D480-D489). UniProt comprises the expertly and richly curated protein database called the UniProt Knowledgebase (UniProtKB), together with the UniProt Reference Clusters (UniRef) and the UniProt Archive (UniParc). The UniProt identifiers (UniProt ID) are unique for each protein present in the database. Throughout the application, the sequence of a polypeptide is represented by a SEQ ID NO or a UniProt ID. Unless stated otherwise, the UniProt IDs of the proteins described correspond to their sequence version 01 as present in the UniProt Database (www.uniprot.org) version release 2021_03 and consulted on 09 June 2021.

[0055] InterPro provides functional analysis of proteins by classifying them into families and predicting domains and important sites. To classify proteins in this way, InterPro uses predictive models, known as signatures, provided by several different databases (referred to as member databases) that make up the InterPro consortium. Protein signatures from these member databases are combined into a single searchable resource, capitalizing on their individual strengths to produce a powerful integrated database and diagnostic tool.

[0056] It should be understood for those skilled in the art that for the databases used herein, comprising Pfam 32.0 (released Sept 2018), CDD v3.17 (released 3rdApril 2019), EggNOG 5.0.0 (released November 2018), InterPro 86.0 (released 3rd June 2021) and PATRIC 3.6.9 (released March 2020), the content of each database is fixed at each release and is not to be changed. When the content of a specific database is changed, this specific database receives a new release version with a new release date. All release versions for each database with their corresponding release dates and specific content as annotated at these specific release dates are available and known to those skilled in the art.

[0057] The terms "identical" or "percent identity" or "% identity" in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms or by visual inspection. For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are inputted into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the % sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. The percentage of sequence identity can be, preferably is, determined by alignment of the two sequences and identification of the number of positions with identical residues divided by the number of residues in the shorter of the sequences x 100. Percent identity may be calculated globally over the full-length sequence of a given SEQ ID NO, i.e. the reference sequence, resulting in a global % identity score. Alternatively, % identity may be calculated over a partial sequence of the reference sequence, resulting in a local percent identity score. A partial sequence preferably means at least about 50%, 60%, 70%, 80%, 90% or 95% of the full-length reference sequence. In another more preferred embodiment, a partial sequence of a reference polypeptide sequence means a stretch of at least 150 amino acid residues up to the total number of amino acid residues of a reference polypeptide sequence. In a most preferred embodiment, a partial sequence of a reference polypeptide sequence means a stretch of at least 200 amino acid residues up to the total number of amino acid residues of a reference polypeptide sequence. Using the full-length of the reference sequence in a local sequence alignment results in a global percent identity score between the test and the reference sequence.

[0058] Percent identity can be determined using different algorithms like for example BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403- 410; Altschul et al., 1997, Nucleic Acids Res 25: 17, 3389-402), the Clustal Omega method (Sievers et al., 2011, Mol. Syst. Biol. 7:539), the MatGAT method (Campanella et al., 2003, BMC Bioinformatics, 4:29) or EMBOSS Needle.

[0059] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 60.0% or more sequence identity over a stretch of at least 150 amino acid residues of a reference polypeptide sequence is to be understood as that the amino acid sequence has 60.0%, 61.0%, 62.0% 63.0%, 64.0%, 65.0%, 66.0%, 67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72.0%, 73.0%, 74.0%, 75.0%, 76.0%, 77.0%, 78.0%, 79.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 100% sequence identity over a stretch of at least 150 amino acid residues of the reference polypeptide sequence.

[0060] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 60.0% or more sequence identity over a stretch of at least 200 amino acid residues of a reference polypeptide sequence is to be understood as that the amino acid sequence has 60.0%, 61.0%, 62.0% 63.0%, 64.0%, 65.0%, 66.0%, 67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72.0%, 73.0%, 74.0%, 75.0%, 76.0%, 77.0%, 78.0%, 79.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 100% sequence identity over a stretch of at least 200 amino acid residues of the reference polypeptide sequence.

[0061] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 50.0% or more sequence identity to the full-length sequence of a reference polypeptide sequence is to be understood as that the amino acid sequence has 50.0%, 51.0%, 52.0%, 53.0%, 54.0%, 55.0%, 56.0%, 57.0%, 58.0%, 59.0%, 60.0%, 61.0%, 62.0%, 63.0%, 64.0%, 65.0%, 66.0%, 67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72.0%, 73.0%, 74.0%, 75.0%, 76.0%, 77.0%, 78.0%, 79.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 100% sequence identity to the full-length of the amino acid sequence of the reference polypeptide sequence.

[0062] Throughout the application, unless explicitly specified otherwise, a polypeptide comprising, consisting of or having an amino acid sequence having 50.0% or more sequence identity to the full-length amino acid sequence of a reference polypeptide, usually indicated with a SEQ. ID NO or UniProt ID, preferably has 50.0%, 55.0%, 60.0%, 65.0%, 70.0%, 75.0%, 80.0%, 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, more preferably has at least 50.0%, even more preferably has at least 55.0%, even more preferably has at least 60.0%, even more preferably has at least 65.0%, even more preferably has at least 70.0%, even more preferably has at least 80.0%, even more preferably has at least 85.0%, most preferably has at least 90.0%, sequence identity to the full length reference sequence. As used herein, a polypeptide comprising or consisting of an amino acid sequence having 65.0% or more sequence identity to the full-length sequence of a reference polypeptide sequence is to be understood as that the amino acid sequence has 65.0%, 66.0%, 67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72.0%, 73.0%, 74.0%, 75.0%, 76.0%, 77.0%, 78.0%, 79.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 100% sequence identity to the full-length of the amino acid sequence of the reference polypeptide sequence.

[0063] Throughout the application, unless explicitly specified otherwise, a polypeptide comprising, consisting of or having an amino acid sequence having 65.0% or more sequence identity to the full-length amino acid sequence of a reference polypeptide, usually indicated with a SEQ. ID NO or UniProt ID, preferably has 65.0%, 70.0%, 75.0%, 80.0%, 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, more preferably has at least 65.0%, even more preferably has at least 70.0%, even more preferably has at least 80.0%, even more preferably has at least 85.0%, most preferably has at least 90.0%, sequence identity to the full length reference sequence.

[0064] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 85.0% or more sequence identity over a stretch of at least 150 amino acid residues of a reference polypeptide sequence is to be understood as that the amino acid sequence has 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 100% sequence identity over a stretch of at least 150 amino acid residues of the reference polypeptide sequence.

[0065] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 85.0% or more sequence identity over a stretch of at least 200 amino acid residues of a reference polypeptide sequence is to be understood as that the amino acid sequence has 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 100% sequence identity over a stretch of at least 200 amino acid residues of the reference polypeptide sequence.

[0066] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 85.0% or more sequence identity to the full-length sequence of a reference polypeptide sequence is to be understood as that the amino acid sequence has 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 100% sequence identity to the full-length of the amino acid sequence of the reference polypeptide sequence.

[0067] Throughout the application, unless explicitly specified otherwise, a polypeptide comprising, consisting of or having an amino acid sequence having 80.0% or more sequence identity to the full-length amino acid sequence of a reference polypeptide, usually indicated with a SEQ ID NO or UniProt ID, preferably has 80.0%, 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, more preferably has at least 80.0%, even more preferably has at least 85.0%, most preferably has at least 90.0%, sequence identity to the full length reference sequence.

[0068] For the purposes of this invention, percent identity is determined using MatGAT2.01 (Campanella et aL, 2003, BMC Bioinformatics 4:29). The following default parameters for protein are employed: (1) Gap cost Existence: 12 and Extension: 2; (2) The Matrix employed was BLOSUM50. In a preferred embodiment, sequence identity is calculated based on the full-length sequence of a given SEQ ID NO, i.e. the reference sequence, or a part thereof. Part thereof preferably means at least 50 %, 60 %, 70 %, 80 %, 90 % or 95 % of the complete reference sequence.

[0069] The terms "sialic acid", "N-acetylneuraminate", "N-acylneuraminate", "N-acetylneuraminic acid" are used interchangeably and refer to an acidic sugar comprising but not limited to Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulonic acid (KDO).

[0070] Neu4Ac is also known as 4-O-acetyl-5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid or 4-O-acetyl neuraminic acid and has C11H19NO9 as molecular formula. Neu5Ac is also known as 5- acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, D-glycero-5-acetamido-3,5- dideoxy-D-galacto-non-2-ulo-pyranosonic acid, 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2- nonulopyranosonic acid, 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid, 5- (acetylamino)-3,5-dideoxy-D-glycero-D-galacto-non-2-nonulosonic acid or 5-(acetylamino)-3,5-dideoxy- D-glycero-D-galacto-non-2-ulopyranosonic acid and has C11H19NO9 as molecular formula. Neu4,5Ac2 is also known as N-acetyl-4-O-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 4-O-acetyl-N- acetylneuraminate, 4-acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonate, 4-acetate 5- (acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonate, 4-acetate-5-acetamido-3,5-dideoxy-D- glycero-D-galacto-nonulosonic acid or 4-acetate 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2- nonulosonic acid and has C13H21NO10 as molecular formula. Neu5,7Ac2 is also known as 7-O-acetyl-N- acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, 7-O-acetyl-N-acetylneuraminate, 7-acetate 5- acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonate, 7-acetate-5-(acetylamino)-3,5-dideoxy-D- glycero-D-galacto-2-nonulosonate, 7-acetate-5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonic acid or 7-acetate 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid and has C13H21NO10 as molecular formula. Neu5,8Ac2 is also known as 5-N-acetyl-8-O-acetyl neuraminic acid and has C13H21NO10 as molecular formula. Neu5,9Ac2 is also known as N-acetyl-9-O-acetylneuraminic acid, 9-anana, 9-O-acetylsialic acid, 9-O-acetyl-N-acetylneuraminic acid, 5-N-acetyl-9-O-acetyl neuraminic acid, N,9-O-diacetylneuraminate or N,9-O-diacetylneuraminate and has C13H21NO10 as molecular formula. Neu4,5,9Ac3 is also known as 5-N-acetyl-4,9-di-O-acetylneuraminic acid. Neu5,7,9Ac3 is also known as 5-N-acetyl-7,9-di-O-acetylneuraminic acid. Neu5,8,9Ac3 is also known as 5-N-acetyl-8,9-di-O- acetylneuraminic acid. Neu4,5,7,9Ac4 is also known as 5-N-acetyl-4,7,9-tri-O-acetylneuraminic acid. Neu5,7,8,9Ac4 is also known as 5-N-acetyl-7,8,9-tri-O-acetylneurarriinic acid. Neu4,5,7,8,9Ac5 is also known as 5-N-acetyl-4,7,8,9-tetra-O-acetylneuraminic acid. Neu5Gc is also known as N-glycolyl- neuraminic acid, N-glycolylneuraminicacid, N-glycolylneuraminate, N-glycoloyl-neuraminate, N-glycoloyl- neuraminic acid, N-glycoloylneuraminic acid, 3,5-dideoxy-5-((hydroxyacetyl)amino)-D-glycero-D-galacto- 2-nonulosonic acid, 3,5-dideoxy-5-(glycoloylamino)-D-glycero-D-galacto-2-nonulopyranosonic acid, 3,5- dideoxy-5-(glycoloylamino)-D-glycero-D-galacto-non-2-ulopyranosonic acid, 3,5-dideoxy-5- [(hydroxyacetyl)amino]-D-glycero-D-galacto-non-2-ulopyranosonic acid, D-glycero-5-glycolylamido-3,5- dideoxy-D-galacto-non-2-ulo-pyranosonic acid and has C11H19NO10 as molecular formula. 2-keto-3- deoxymanno-octulonic acid is also known as KDO, Kdo, kdo, 2-keto-3-deoxy-D-mannooctanoic acid, 2- oxo-3-deoxy-D-mannooctonic acid, 3-deoxy-D-manno-2-octulosonic acid, 3-deoxy-D-manno-oct-2-ulo- pyranosonic acid, 3-deoxy-D-manno-oct-2-ulosonic acid, 3-deoxy-D-manno-octulosonic acid, 3-deoxy-D- manno-oct-2-ulopyranosonic acid, ketodeoxyoctonic acid, ketodeoxyoctulonic acid, (6R)-6- (hydroxymethyl)-l-carboxy-2-deoxy-D-lyxo-hexopyranose, keto-deoxy-octulonic acid and has C8H14O8 as molecular formula.

[0071] The term "glycosyltransferase" as used herein refers to an enzyme capable to catalyse the transfer of a sugar moiety of a donor to a specific acceptor, forming glycosidic bonds. Said donor can be a precursor as defined herein. A classification of glycosyltransferases using nucleotide diphospho-sugar, nucleotide monophospho-sugar and sugar phosphates and related proteins into distinct sequence-based families has been described (Campbell et al., Biochem. J. 326, 929-939 (1997)) and is available on the CAZy (CArbohydrate-Active EnZymes) website (www.cazy.org).

[0072] As used herein the glycosyltransferase can be selected from the list comprising but not limited to: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N- acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N- acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases.

[0073] Sialyltransferases are glycosyltransferases that transfer a sialic acid (like Neu5Ac) from a donor (like CMP- Neu5Ac) onto an acceptor. Sialyltransferases comprise alpha-2, 3-sialyltransferases, alpha-2, 6- sialyltransferases and alpha-2, 8-sialyltransferases that catalyse the transfer of a sialic acid onto an acceptor via alpha-glycosidic bonds. Sialyltransferases can be found but are not limited to the GT29, GT42, GT52, GT80, GT97 and GT100 CAZy families. The terms "alpha-2, 3-sialyltransferase", "alpha 2,3 sialyltransferase", "3-sialyltransferase", "a-2,3- sialyltransferase", "a 2,3 sialyltransferase", "3 sialyltransferase", "3-ST", "3ST" or "a23-ST" as used in the present invention, are used interchangeably and refer to a glycosyltransferase that catalyzes the transfer of sialic acid from the donor CMP-sialic acid, to the acceptor molecule in an alpha-2, 3-linkage.

[0074] The term "monosaccharide" as used herein refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed either an aldose a ketose, a deoxysugar, a deoxy-aminosugar, a uronic acid, an aldonic acid, a ketoaldonic acid, an aldaric acid or a sugar alcohol,, and contains one or more hydroxyl groups per molecule. Monosaccharides are saccharides containing only one simple sugar. With the term polyol is meant an alcohol containing multiple hydroxyl groups. For example, glycerol, sorbitol, or mannitol.

[0075] The term "phosphorylated monosaccharide" as used herein refers to a monosaccharide, which is phosphorylated. Examples of phosphorylated monosaccharides include but are not limited to glucose-1- phosphate, glucose-6-phosphate, glucose-l,6-bisphosphate, galactose-l-phosphate, fructose-6- phosphate, fructose-l,6-bisphosphate, fructose-l-phosphate, glucosamine-l-phosphate, glucosamine-6- phosphate, N-acetylglucosamine-l-phosphate, mannose-l-phosphate, mannose-6-phosphate or fucose- 1-phosphate. Some, but not all, of these phosphorylated monosaccharides are precursors or intermediates for the production of activated monosaccharide.

[0076] The terms "activated monosaccharide", "nucleotide-activated sugar", "nucleotide-sugar", "activated sugar", "nucleoside" or "nucleotide donor” are used herein interchangeably and refer to activated forms of monosaccharides. Examples of activated monosaccharides include but are not limited to UDP-N- acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), UDP- glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy--L-arabino-4-hexulose, UDP-2- acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2- acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2- acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L- QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose, CMP-sialic acid, GDP-fucose (GDP- Fuc), GDP-rhamnose and UDP-xylose. Nucleotide-sugars act as glycosyl donors in glycosylation reactions. Glycosylation reactions are reactions that are catalysed by glycosyltransferases.

[0077] The term "CMP-sialic acid" as used herein refers to a nucleotide-activated form of sialic acid comprising but not limited to CMP-Neu5Ac, CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP- Neu5,9Ac2, CMP-Neu5,7 (8,9) Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) and CMP-KDO.

[0078] The term "disaccharide" as used herein refers to a saccharide polymer containing two simple sugars, i.e. monosaccharides. Such disaccharides contain monosaccharides preferably selected from the list of monosaccharides as used herein above. Examples of disaccharides comprise lactose (Gal-pi,4-Glc), lacto- N-biose (Gal-pi,3-GlcNAc), N-acetyllactosamine (Gal-pi,4-GlcNAc), LacDiNAc (GalNAc-pi,4-GlcNAc), N- acetylgalactosaminylglucose (GalNAc-pi,4-Glc), Neu5Ac-ot2,3-Gal, Neu5Ac-o.2,6-Gal, fucopyranosyl-(l- 4)-N-glycolylneuraminic acid (Fuc-(l-4)-Neu5Gc), sucrose (Glc-al,2-Fru), maltose (Glc-al,4-Glc) and melibiose (Gal-ocl,6-Glc).

[0079] The term "oligosaccharide" as used in the context of the present invention preferably refers to a saccharide containing 2 up to and including 20 monosaccharides, i.e. the degree of polymerization (DP) is 2-20, more preferably refers to a saccharide containing 3 up to and including 20 monosaccharides, i.e. the degree of polymerization (DP) is 3-20. In other words, the term "oligosaccharide" preferably refers to a saccharide consisting of 2-20, more preferably 3-20, monosaccharide units which are linked to each other via glycosidic bonds in a linear or in a branched structure. The linkage (e.g., glycosidic linkage, galactosidic linkage, glucosidic linkage, etc.) between two sugar units can be expressed, for example, as 1,4, l->4, or (1-4), used interchangeably herein. For example, the terms "Gal-bl,4-Glc", "Gal-pi,4-Glc", "b-Gal-(l->4)- Glc", "P-Gal-(l->4) -Glc", "Galbetal-4-Glc", "Gal-b(l-4)-Glc" and "Gal-P(l-4)-Glc" have the same meaning, i.e. a beta-glycosidic bond links carbon-1 of galactose (Gal) with the carbon-4 of glucose (Glc). Each monosaccharide can be in the cyclic form (e.g., pyranose or furanose form). Linkages between the individual monosaccharide units may include alpha l->2, alpha l->3, alpha l->4, alpha l->6, alpha 2->l, alpha 2->3, alpha 2->4, alpha 2->6, beta l->2, beta l->3, beta l->4, beta l->6, beta 2->l, beta 2->3, beta 2->4, and beta 2->5. An oligosaccharide can contain both alpha- and beta-glycosidic bonds or can contain only alpha-glycosidic or only beta-glycosidic bonds. The term "polysaccharide" refers to a compound consisting of a large number, typically more than twenty, of monosaccharides linked glycosidically.

[0080] Examples of oligosaccharides include but are not limited to Lewis-type antigen oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigen, enterobacterial common antigen (EGA), the glycan chain present in lipopolysaccharides (LPS), the oligosaccharide repeats present in capsular polysaccharides, peptidoglycan (PG), amino-sugars, antigens of the human ABO blood group system, an animal oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans, a plant oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans, sialylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, fucosylated oligosaccharide, N-acetylglucosamine containing oligosaccharides, lacto-N-biose containing oligosaccharides, N-acetyllactosamine-containing oligosaccharides, N-acetylglucosamine containing sialylated oligosaccharides, N-acetylglucosamine containing neutral (non-charged) oligosaccharides, N- acetylglucosamine containing negatively charged oligosaccharides, N-acetylglucosamine containing fucosylated oligosaccharides, N-acetylglucosamine containing non-fucosylated oligosaccharides, lacto-N- biose containing sialylated oligosaccharides, lacto-N-biose containing neutral (non-charged) oligosaccharides, lacto-N-biose containing negatively charged oligosaccharides, lacto-N-biose containing fucosylated oligosaccharides, lacto-N-biose containing non-fucosylated oligosaccharides, N- acetyllactosamine containing sialylated oligosaccharides, N-acetyllactosamine containing neutral (noncharged) oligosaccharides, N-acetyllactosamine containing negatively charged oligosaccharides, N- acetyllactosamine containing fucosylated oligosaccharides, N-acetyllactosamine containing non- fucosylated oligosaccharides, chitosan, chitosan comprising oligosaccharide, heparosan, chondroitin sulphate, glycosaminoglycan oligosaccharide, heparin, heparan sulphate, dermatan sulphate, hyaluronan, hyaluronic acid and keratan sulphate.

[0081] "Charged oligosaccharides" are oligosaccharide structures that contain one or more negatively charged monosaccharide subunits including sialic acid, glucuronate and galacturonate. Charged oligosaccharides are also referred to as acidic oligosaccharides. In contrast, neutral (non-charged) oligosaccharides are non- sialylated oligosaccharides, and thus do not contain an acidic monosaccharide subunit. Neutral oligosaccharides comprise non-charged fucosylated oligosaccharides that contain one or more fucose subunits in their glycan structure as well as non-charged non-fucosylated oligosaccharides that lack any fucose subunit. Other examples of charged oligosaccharides are sulphated chitosans and deacetylated chitosans.

[0082] The terms "negatively charged oligosaccharide" or "acidic oligosaccharide" are used interchangeably and refer to an oligosaccharide with a negative charge. In a preferred embodiment, the negatively charged oligosaccharide is a sialylated oligosaccharide.

[0083] As used herein, a 'sialylated oligosaccharide' is to be understood as a negatively charged sialic acid containing oligosaccharide, i.e., an oligosaccharide having a sialic acid residue. It has an acidic nature. Such sialylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of said monosaccharide subunit is a sialic acid residue. A sialylated oligosaccharide can contain more than one sialic acid residue, e.g., two, three or more. Said more than one sialic acid residue can be two, three or more identical sialic acid residues. Said more than one sialic acid residue can also be two, three or more different sialic acid residues. For example, a sialylated oligosaccharide can contain one or more Neu5Ac residues and one or more KDO residues.

[0084] Some examples are 3'-SL (3'-sialyllactose or 3'SL or Neu5Ac-a2,3-Gal-pi,4-Glc), 3'-sialyllactosamine, 6-SL (6'sialyllactose, 6'-sialyllactose or 6'SL or Neu5Ac-a2,6-Gal-pi,4-Glc), 3,6-disialyllactose (Neu5Ac-a2,3- (Neu5Ac-a2,6)-Gal-pi,4-Glc), 6,6'-disialyllactose (Neu5Ac-a2,6-Gal-pi,4-(Neu5Ac-a2,6)-Glc), 8,3- disialyllactose (Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-pi,4-Glc), 6'-sialyllactosamine, oligosaccharides comprising 6'sialyllactose (also known as 6'sialyllactose, 6'SL and 6'-SL), SGG hexasaccharide (Neu5Aca- 2,3Gaip -l,3GalNac -l,3Gala-l,4Gaip-l,4Gal), sialylated tetrasaccharide, sialylated pentasaccharide, sialylated lacto-N-triose, sialylated lacto-N-tetraose, sialyllacto-N-neotetraose, LSTc (Neu5Ac-ot2,6-Gal- pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), LSTd (Neu5Ac-a2,3-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto- N-neohexaose II, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a (LSTa, Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc), disialyllacto-N-hexaose I, sialyllacto-N-tetraose b (LSTb, Gal-|31,3-(Neu5Ac-a2,6)-GlcNAc-pi,3-Gal-pi,4-Glc), 3'-sialyl-3- fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyllacto-N-fucohexaose II, disialyllacto-N- fucopentaose II, monofucosyldisialyllacto-N-tetraose, Neu5Ac-a2,3-Gal-bl,4-GlcNAc-bl,3-Gal, Neu5Ac- a2,3-Gal-bl,3-GlcNAc-bl,3-Gal, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'-KDO-lacto-N-biose, 3'-KDO- 5'sialyllactose, 3'KDO-8-sialyllactose, KDO-2,3Gaip-l,3GalNacp-l,3Gala-l,4Gaip-l,4Gal, KDO-2,3Gaip- l,3GlcNacP-l,3Gaip-l,4Glc, KDO-2,3Gaip-l,4GlcNacp-l,3Gaip-l,4Glc, 3'-KDO-3-fucosyllactose, Neu5Ac- a2,8-Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal, 3'-Sialyl-2'-fucosyllactose, 6'-Sialyl-2'-fucosyllactose, 6'- Sialyl-3-fucosyllactose, Neu5Ac-a2,6-(Neu5Ac-a2,3-)Gal-bl,4-Glc, 3'-Sialyl-3-fucosyllactosamine, Fuc- al,4-(Neu5Ac-a2,3-Gal-bl,3-)GlcNAc, 6'-Sialyllacto-N-biose, 3’-Sialyllacto-N-biose, Neu5Ac-a2,6-(GlcNAc- bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,3-Gal-bl,4- (Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-)Gal- bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,4-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,3-GlcNAc-bl,3-)Gal- bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,6-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,3-Gal-bl,3- GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,3-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Fuc-al,2-Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Fuc-al,2-)Gal-bl,3-GlcNAc- bl,3-Gal-bl,4-Glc, Fuc-al,4-(Neu5Ac-a2,3-Gal-bl,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac- a2,6-Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Fuc-al,2-)Gal-bl,3- GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-Gal-bl,3- GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6- (Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-bl,3-GlcNAc-bl,3)- Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-bl,3-GlcNAc-bl,3)-Gal-bl,4-Glc, Neu5Ac- a2,6-(Neu5Ac-a2,3-Gal-bl,4-GlcNAc-bl,3)-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-bl,4-GlcNAc- bl,3)-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-bl,4-GlcNAc-bl,3)-Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-GlcNAc-bl,3)-Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,4- GlcNAc-bl,3)-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-GlcNAc- bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-bl,4-GlcNAc-bl,3)-Gal-bl,4-GlcNAc- bl,3)-Gal-bl,4-Glc, Neu5Ac-a2,3-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc, 6'-KDO- lactose, 6'-KDO-lactosamine, 6'-KDO-lacto-N-biose, KDO-lacto-N-triose, KDO-lacto-N-tetraose, KDO-lacto- N-tetraose, and oligosaccharides bearing one or several sialic acid residue(s), including but not limited to: oligosaccharide moieties of the gangliosides selected from GM3 (3'sialyllactose, Neu5Aca-2,3Gaip-4Glc) and oligosaccharides comprising the GM3 motif, GD3 Neu5Aca-2,8Neu5Aca-2,3Gaip-l,4Glc GT3 (Neu5Aca-2,8Neu5Aca-2,8Neu5Aca-2,3Gaip-l,4Glc); GM2 GalNAcP-l,4(Neu5Aca-2,3)Gaip-l,4Glc, GM1 Gaip-l,3GalNAcp-l,4(Neu5Aca-2,3)Gaip-l,4Glc, GDla Neu5Aca-2,3Gaip-l,3GalNAcp-l,4(Neu5Aca- 2,3)Gaip-l,4Glc, GTla Neu5Aca-2,8Neu5Aca-2,3Gaip-l,3GalNAcP-l,4(Neu5Aca-2,3)Gaip-l,4Glc, GD2 GalNAcP-l,4(Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GT2 GalNAcP-l,4(Neu5Aca-2,8Neu5Aca- 2,8Neu5Aca2,3)Gaip-l )Gai -l,4Glc, GTlb Neu5Aca-2,3Gaip-l,3GalNAcP-l,4(Neu5Aca-2,8Neu5Aca2,3)Gaip-l;4Glc, GQlb Neu5Aca-2,8Neu5Aca- 2,3Gaip-l,3GalNAc P -l,4(Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GTlc Gaip-l,3GalNAcP-l,4(Neu5Aca- 2,8Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GQlc Neu5Aca-2,3Gaip-l,3GalNAc P -l,4(Neu5Aca- 2,8Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GPlc Neu5Aca-2,8Neu5Aca-2,3Gaip-l,3GalNAcP- l,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GDla Neu5Aca-2,3Gaip-l,3(Neu5Aca- 2,6)GalNAcp -l,4Gaip-l,4Glc, Fucosyl-GMl Fuca-l,2Gaip-l,3GalNAcP -l,4(Neu5Aca-2,3)Gal -l,4Glc; all of which may be extended to the production of the corresponding gangliosides by reacting the above oligosaccharide moieties with ceramide or synthetizing the above oligosaccharides on a ceramide.

[0085] As used herein, a '3'sialylated oligosaccharide' is to be understood as a negatively charged sialic acid containing oligosaccharide comprising an oligosaccharide or disaccharide which is alpha-2, 3-glycosidically linked to a sialic acid residue. It has an acidic nature. Such sialylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of said monosaccharide subunit is an alpha-2, 3-glycosydically linked sialic acid residue. A 3'sialylated oligosaccharide can contain more than one sialic acid residue, e.g., two, three or more. Said more than one sialic acid residue can be two, three or more identical sialic acid residues. Said more than one sialic acid residue can also be two, three or more different sialic acid residues. For example, a 3'sialylated oligosaccharide can contain one or more Neu5Ac residues and one or more KDO residues. Some examples are 3'-SL (3'-sialyllactose or 3'SL or Neu5Ac-a2,3-Gal-pi,4-Glc), 3'-sialyllactosamine, 3,6- disialyllactose (Neu5Ac-a2,3-(Neu5Ac-a2,6)-Gal-pi,4-Glc), 8,3-disialyllactose (Neu5Ac-a2,8-Neu5Ac-a2,3- Gal-pi,4-Glc), SGG hexasaccharide (Neu5Aca-2,3Gai -l,3GalNac -l,3Gala-l,4Gai -l,4Gal), sialylated tetrasaccharide (Neu5Aca-2,3Gaip-l,4GlcNac -l,4GlcNAc), pentasaccharide LSTD (Neu5Aca-2,3Gaip- l,4GlcNacP-l,3Gaip-l,4Glc), 3'sialylated lacto-N-biose (3'sLNB), 3'sialylated N-acetyllactosamine (3'sLacNAc), 3'sialylated lacto-N-triose (3'sLN3 or 3'sLNTII) , 3'sialylated lacto-N-tetraose (e.g. Neu5Aca- 2,3Gai -l,3GlcNAc -l,3Gaip-l,4Glc (LSTa)); KDOa-2,3Gai -l,3GlcNAc -l,3Gaip-l,4Glc), sialyl lacto-N- neotetraose (e.g. Neu5Aca-2,3Gaip-l,4GlcNac -l,3Gaip-l,4Glc (LSTd)), monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto- N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a, disialyllacto-N- hexaose I, 3'-sialyl-3-fucosyllactose, disialomonofucosyllacto-N-neohexaose, monofucosyl- monosialyllacto-N-octaose (sialyl Lea), sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'-KDO-6'sialyllactose, 3'KDO- 8-sialyllactose, KDO-2,3Gaip-l,3GalNacP-l,3Gala-l,4Gaip-l,4Gal, KDO-2,3Gaip-l,3GlcNacP-l,3Gaip- l,4Glc, KDO-2,3Gaip-l,4GlcNac -l,3Gaip-l,4Glc, 3'-KDO-3-fucosyllactose and oligosaccharides bearing one or several sialic acid residue(s), including but not limited to: oligosaccharide moieties of the gangliosides selected from GM3 (3'sialyllactose, Neu5Aca-2,3Gaip-4Glc) and oligosaccharides comprising the GM3 motif, GD3 (Neu5Aca-2,8Neu5Aca-2,3Gaip-l,4Glc), GT3 (Neu5Aca-2,8Neu5Aca-2,8Neu5Aca- 2,3Gaip-l,4Glc); GM2 GalNAcP-l,4(Neu5Aca-2,3) Gaip-l,4Glc, GM1 Gaip-l,3GalNAcp-l,4 (Neu5Aca-2,3) Gaip-l,4Glc, GDla Neu5Aca-2,3Gaip-l,3GalNAcP-l,4 (Neu5Aca-2,3) Gaip-l,4Glc, GTla Neu5Aca- 2,8Neu5Aca-2,3Gaip-l,3GalNAcP-l,4 (Neu5Aca-2,3) Gaip-l,4Glc, GD2 GalNAcP-1,4 (Neu5Aca- 2,8Neu5Aca2,3) Gaip-l,4Glc, GT2 GalNAcP-1,4 (Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3) Gaip-l,4Glc, GDlb, Gaip-l,3GalNAcP-l,4 (Neu5Aca-2,8Neu5Aca2,3) Gaip-l,4Glc, GTlb Neu5Aca-2,3Gaip- l,3GalNAcP-l,4 (Neu5Aca-2,8Neu5Aca2,3) Gaip-l,4Glc, GQlb Neu5Aca-2,8Neu5Aca-2,3Gaip-l,3GalNAc P-l,4(Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GTlc Gaip-l,3GalNAcp-l,4(Neu5Aca-2,8Neu5Aca- 2,8Neu5Aca2,3)Gaip-l,4Glc, GQlc Neu5Aca-2,3Gaip-l,3GalNAc- -l,4(Neu5Aca-2,8Neu5Aca-

[0086] 2,8Neu5Aca2,3)Gaip-l,4Glc, GPlc Neu5Aca-2,8Neu5Aca-2,3Gaip-l,3GalNAc-P-l,4(Neu5Aca-

[0087] 2,8Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GDla Neu5Aca-2,3Gaip-l,3(Neu5Aca-2,6)GalNAcP-l,4Gaip- l,4Glc, Fucosyl-GMl Fuca-l,2Gaip-l,3GalNAcp-l,4(Neu5Aca-2,3)Gaip-l,4Glc; all of which may be extended to the production of the corresponding gangliosides by reacting the above oligosaccharide moieties with ceramide or synthetizing the above oligosaccharides on a ceramide.

[0088] The terms "3'sialylated lactosamine comprising oligosaccharide" or "3'sialylated LacNAc comprising oligosaccharide" "3'sLacNAc comprising oligosaccharide" are used interchangeably and refer to an oligosaccharide with a negative charge comprising one or more sialic acid groups and one or more lactosamine disaccharides. Some examples are 3'-sialyllactosamine, LSTd, sialyl lewis x, 3'-KDO- lactosamine.

[0089] The terms "3'sialylated lacto-N-biose comprising oligosaccharide" or "3'sialylated LNB comprising oligosaccharide" "3'sLNB comprising oligosaccharide" are used interchangeably and refer to an oligosaccharide with a negative charge comprising one or more sialic acid groups and one or more lacto- N-biose disaccharides. Some examples are LSTa, sialyl-Lewis a, 3'-sialyllacto-N-biose.

[0090] The terms "LNB" and "Lacto-N-biose" are used interchangeably and refer to the disaccharide Gal-pi,3- GIcNAc.

[0091] The terms "LacNAc" and "N-acetyllactosamine" are used interchangeably and refer to the disaccharide Gal-pi,4-GlcNAc.

[0092] The terms "LNT II", "LNT-II", "LN3", "lacto-N-triose II", "lacto-N-triose II", "lacto-N-triose", "lacto-N-triose" or "GlcNAc i-3Gaipi-4Glc" as used in the present invention, are used interchangeably.

[0093] The terms "LNT", "lacto-N-tetraose", "lacto-W-tetraose" or "Gaipi-3GlcNAcpi-3Gaipi-4Glc" as used in the present invention, are used interchangeably.

[0094] The terms "LNnT", "lacto-N-neotetraose", "lacto-W-neotetraose", "neo-LNT" or "Gaipi-4GlcNAcpi- 3Gaipi-4Glc" as used in the present invention, are used interchangeably.

[0095] The terms "LSTa", "LS-tetrasaccharide a", "sialyl-lacto-N-tetraose a", "sialyllacto-N-tetraose a", or "Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc" as used in the present invention, are used interchangeably. The terms "LSTb", "LS-Tetrasaccharide b", "Sialyl-lacto-N-tetraose b", "sialyllacto-N-tetraose b" or "Gal- pi,3-(Neu5Ac-a2,6)-GlcNAc-pi,3-Gal-pi,4-Glc" as used in the present invention, are used interchangeably.

[0096] The terms "LSTc", "LS-Tetrasaccharide c", "Sialyl-lacto-N-tetraose c", "sialyllacto-N-tetraose c", "sialyllacto-N-neotetraose c", or "Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc" as used in the present invention, are used interchangeably.

[0097] The terms "LSTd", "LS-tetrasaccharide d", "sialyl-lacto-N-tetraose d", "sialyllacto-N-tetraose d", "sialyllacto-N-neotetraose d", or "Neu5Ac-a2,3-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc" as used in the present invention, are used interchangeably.

[0098] The terms "DSLNT", "DS-LNT" and "disialyllacto-N-tetraose" as used in the present invention, are used interchangeably and refer to Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,3-)GlcNAc-pi,3-Gal-pi,4-Glc.

[0099] The terms "DS'LNnT" and "disialyllacto-N-neotetraose analog" as used in the present invention, are used interchangeably and refer to Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,4-GlcNAc-pi,3-)Gal-pi,4-Glc.

[0100] The term "sialylated tetraose type 1" as used in the present invention refers to Neu5Ac-a2,3-Gal-pi,3- GlcNAc-pi,3-Gal.

[0101] The term "sialylated tetraose type 2" as used in the present invention refers to Neu5Ac-a2,3-Gal-pi,4- GlcNAc-pi,3-Gal.

[0102] The term "sialyl-Lewis a" as used in the present invention refers to Fuc-al,4-(Neu5Ac-a2,3-Gal-pi,3-) GIcNAc.

[0103] The terms "sialyl-Lewis x" and "3'-sialyl-3-fucosyllactose" as used in the present invention, are used interchangeably and refer to Neu5Ac-a2,3-Gal-pi,4-(Fuc-al,3-)GlcNAc.

[0104] A 'neutral oligosaccharide' or a 'non-charged oligosaccharide' as used herein and as generally understood in the state of the art is an oligosaccharide that has no negative charge originating from a carboxylic acid group. Examples of such neutral oligosaccharide are 2'-fucosyllactose (2'FL), 3-fucosyl lactose (3FL), 2', 3- difucosyllactose (diFL), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto- N-fucopentaose I (LNFP I), lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose II (LNFP II), lacto- N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-fucopentaose VI, lacto-N- neofucopentaose V (LNnFP V), lacto-N-difucohexaose I (LNDFH I), lacto-N-difucohexaose II (LNDFH II), 6'- galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, difucosyl-lacto-N-hexaose and difucosyl-lacto-N-neohexaose, difucosyl-lacto- N-neohexaose (LNnDFH II), difucosyl-para-lacto-N-neohexaose, trifucosyllacto-N-hexaose, para-lacto-N- fucohexaose and lacto-N-trifucoheptaose.

[0105] The term "amino-sugar" as used herein refers to a sugar molecule in which a hydroxyl group has been replaced with an amine group. As used herein, an antigen of the human ABO blood group system is an oligosaccharide. Such antigens of the human ABO blood group system are not restricted to human n structures. Said structures involve the A determinant GalNAc-alphal,3 (Fuc-alphal,2) -Gal-, the B determinant Gal-alphal,3(Fuc-alphal,2)-Gal- and the H determinant Fuc-alphal,2-Gal- that are present on disaccharide core structures comprising Gal-betal,3-GlcNAc, Gal-betal,4-GlcNAc, Gal-betal,3-GalNAc and Gal-betal,4-Glc.

[0106] Mammalian milk oligosaccharides or MMOs comprise oligosaccharides present in milk found in any phase during lactation including colostrum milk from humans (i.e. human milk oligosaccharides or HMOs) and mammals including but not limited to cows (Bos Taurus), sheep (Ovisaries), goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus), horses (Equus ferus caballus), pigs (Sus scropha), dogs (Canis lupus familiaris), ezo brown bears (Ursus arctos yesoensis), polar bear (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephant (Loxodonta africana), giant anteater (Myrmecophaga tridactyla), common bottlenose dolphins (Tursiops truncates), northern minke whales (Balaenoptera acutorostrata), tammar wallabies (Macropus eugenii), red kangaroos (Macropus rufus), common brushtail possum (Trichosurus Vulpecula), koalas (Phascolarctos cinereus), eastern quolls (Dasyurus viverrinus), platypus (Ornithorhynchus anatinus).

[0107] As used herein, "mammalian milk oligosaccharide" refers to oligosaccharides such as but not limited to 3- fucosyllactose, 2'-fucosyllactose, 6-fucosyl lactose, 2',3-difucosyllactose, 2',2-difucosyllactose, 3,4- difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, 3,6-disialyllactose, 6,6'-disialyllactose, 8,3- disialyllactose, 3,6-disialyllacto-N-tetraose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto- N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, lacto-N- difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosylmonosialyllacto-N-tetraose c, monofucosyl para-lacto-N-hexaose, monofucosyllacto-N- hexaose III, isomeric fucosylated lacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose I, sialyllacto- N-hexaose, sialyllacto-N-neohexaose II, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, difucosyllacto-N-hexaose a, difucosyllacto-N-hexaose c, galactosylated chitosan, fucosylated oligosaccharides, neutral oligosaccharide and / or sialylated oligosaccharides.

[0108] Human milk oligosaccharides are also known as human identical milk oligosaccharides which are chemically identical to the human milk oligosaccharides found in human breast milk, but which are biotechnologically produced (e.g. using cell free systems or cells and organisms comprising a bacterium, a fungus, a yeast, a plant, animal, or protozoan cell, preferably metabolically engineered cells and organisms). Human identical milk oligosaccharides are marketed under the name HiMO. HMOs comprise fucosylated oligosaccharides, non-fucosylated neutral oligosaccharides and sialylated oligosaccharides (see e.g. Chen X., Chapter Four: Human Milk Oligosaccharides (HMOS): Structure, Function, and Enzyme- Catalyzed Synthesis in Adv. Carbohydr. Chem. Biochem. 72, 113 (2015) ). Examples of HMOs comprise 3- fucosyllactose, 2'-fucosyllactose, 2',3-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, LN3, lacto-N- tetraose, lacto-N-neotetraose, lacto-N-fucopentaose ll7lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, difucosyllacto-N-tetraose, lacto-N-hexaose, lacto-N-difucohexaose I, lacto-N- difucohexaose II, disialyllacto-N-tetraose, fucosyllacto-N-hexaose, difucosyllacto-N-hexaose, fucodisialyllacto-N-hexaose, disialyllacto-N-hexaose.

[0109] As used herein, the term "mammary cell (s)" generally refers to mammalian mammary epithelial cell (s), mammalian mammary-epithelial luminal cell(s), or mammalian epithelial alveolar cell(s), or any combination thereof. As used herein, the term "mammary-like cell(s)" generally refers to mammalian cell(s) having a phenotype / genotype similar (or substantially similar) to natural mammalian mammary cell(s) but is / are derived from mammalian non-mammary cell source(s). Such mammalian mammary-like cell(s) may be engineered to remove at least one undesired genetic component and / or to include at least one predetermined genetic construct that is typical of a mammalian mammary cell. Non-limiting examples of mammalian mammary-like cell (s) may include mammalian mammary epithelial-like cell(s), mammalian mammary epithelial luminal-like cell(s), mammalian non-mammary cell(s) that exhibits one or more characteristics of a cell of a mammalian mammary cell lineage, or any combination thereof. Further nonlimiting examples of mammalian mammary-like cell(s) may include mammalian cell(s) having a phenotype similar (or substantially similar) to natural mammalian mammary cell (s), or more particularly a phenotype similar (or substantially similar) to natural mammalian mammary epithelial cell (s). A mammalian cell with a phenotype or that exhibits at least one characteristic similar to (or substantially similar to) a natural mammalian mammary cell or a mammalian mammary epithelial cell may comprise a mammalian cell (e.g. derived from a mammary cell lineage or a non-mammary cell lineage) that exhibits either naturally, or has been engineered to, be capable of expressing at least one milk component.

[0110] As used herein, the term "non-mammary cell(s)" may generally include any mammalian cell of non- mammary lineage. In the context of the invention, a non-mammary cell can be any mammalian cell capable of being engineered to express at least one milk component. Non-limiting examples of such non- mammary cell(s) include hepatocyte(s), blood cell(s), kidney cell(s), cord blood cell(s), epithelial cell(s), epidermal cell(s), myocyte(s), fibroblast(s), mesenchymal cell(s), or any combination thereof. In some instances, molecular biology and genome editing techniques can be engineered to eliminate, silence, or attenuate myriad genes simultaneously.

[0111] The terms "cell genetically modified for the production of a sialylated oligosaccharide" or "cell metabolically engineered for the production of a sialylated oligosaccharide" within the context of the present disclosure refers to a cell of a microorganism which is genetically manipulated to comprise at least one sialyltransferase combined with any one or more of i) a gene encoding a glycosyltransferase necessary for the synthesis of said sialylated oligosaccharide, ii) a biosynthetic pathway to produce a nucleotide donor suitable to be transferred by said glycosyltransferase to a carbohydrate precursor, and / or iii) a biosynthetic pathway to produce a precursor or a mechanism of internalization of a precursor from the culture medium into the cell where it is glycosylated to produce the sialylated oligosaccharide.

[0112] The term "pathway for production of a sialylated oligosaccharide" as used herein is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of a sialylated oligosaccharide as defined herein. Said pathway for production of a sialylated oligosaccharide can comprise but is not limited to pathways involved in the synthesis of a nucleotide-activated sugar and the transfer of said nucleotide-activated sugar to an acceptor to create a sialylated oligosaccharide of the present invention. An example of such pathway is a sialylation pathway. Further examples of such pathway comprise but are not limited to a fucosylation, galactosylation, N-acetylglucosaminylation, N- acetylgalactosaminylation, mannosylation, N-acetylmannosaminylation pathway.

[0113] A 'sialylation pathway' is a biochemical pathway consisting of at least one of the enzymes and their respective genes chosen from the list comprising an L-glutamine— D-fructose-6-phosphate aminotransferase, a phosphoglucosamine mutase, an N-acetylglucosamine-6-P deacetylase, an N- acylglucosamine 2-epimerase, a UDP-N-acetylglucosamine 2-epimerase, an N-acetylmannosamine-6- phosphate 2-epimerase, a UDP-GIcNAc 2-epimerase / kinase, a glucosamine 6-phosphate N- acetyltransferase, an N-acetylglucosamine-6-phosphate phosphatase, a phosphoacetylglucosamine mutase, an N-acetylglucosamine 1-phosphate uridylyltransferase, a glucosamine-l-phosphate acetyltransferase, an Neu5Ac synthase, an N-acetylneuraminate lyase, an N-acylneuraminate-9- phosphate synthase, an N-acylneuraminate-9-phosphatase, a sialic acid transporter and a CMP-sialic acid synthase, combined with a sialyltransferase leading to a 2,3; a 2,6 and / or a 2,8 sialylated oligosaccharides. The terms "L-glutamine— D-fructose-6-phosphate aminotransferase", "glutamine — fructose-6-phosphate transaminase (isomerizing)", "hexosephosphate aminotransferase", "glucosamine-6-phosphate isomerase (glutamine-forming)", "glutamine-fructose-6-phosphate transaminase (isomerizing)", "D- fructose-6-phosphate amidotransferase", "fructose-6-phosphate aminotransferase", "glucosaminephosphate isomerase", "glucosamine 6-phosphate synthase", "GlcN6P synthase", "GFA", "glms", "glmS" and "glmS*54" are used interchangeably and refer to an enzyme that catalyses the conversion of D-fructose-6-phosphate into D-glucosamine-6-phosphate using L-glutamine.

[0114] The terms "phosphoglucosamine mutase" and "glmM" are used interchangeably and refer to an enzyme that catalyses the conversion of glucosamine-6-phosphate to glucosamine-l-phosphate. Phosphoglucosamine mutase can also catalyse the formation of glucose-6-P from glucose-l-P, although at a 1400-fold lower rate.

[0115] The terms "N-acetylglucosamine-6-P deacetylase", "N-acetylglucosamine-6-phosphate deacetylase" and "nagA" are used interchangeably and refer to an enzyme that catalyses the hydrolysis of the N-acetyl group of N-acetylglucosamine-6-phosphate (GlcNAc-6-P) to yield glucosamine-6-phosphate (GlcN6P) and acetate. An N-acylglucosamine 2-epimerase is an enzyme that catalyses the reaction N-acyl-D-glucosamine = N- acyl-D-mannosamine. Alternative names for this enzyme comprise N-acetylglucosamine 2-epimerase, N- acetyl-D-glucosamine 2-epimerase, GIcNAc 2-epimerase, N-acyl-D-glucosamine 2-epimerase and N- acetylglucosamine epimerase.

[0116] A UDP-N-acetylglucosamine 2-epimerase is an enzyme that catalyses the reaction N-acetyl-D-glucosamine = N-acetylmannosamine. Alternative names for this enzyme comprise UDP-N-acylglucosamine 2- epimerase, UDP-GlcNAc-2-epimerase, "neuC" and UDP-N-acetyl-D-glucosamine 2-epimerase.

[0117] An N-acetylmannosamine-6-phosphate 2-epimerase is an enzyme that catalyses the reaction N-acetyl-D- glucosamine 6-phosphate = N-acetyl-D-mannosamine 6-phosphate.

[0118] A bifunctional UDP-GIcNAc 2-epimerase / kinase is a bifunctional enzyme that catalyses the reaction UDP- N-acetyl-D-glucosamine = N-acetyl-D-mannosamine and the reaction N-acetyl-D-mannosamine + ATP = ADP + N-acetyl-D-mannosamine 6-phosphate.

[0119] A glucosamine 6-phosphate N-acetyltransferase is an enzyme that catalyses the transfer of an acetyl group from acetyl-CoA to D-glucosamine-6-phosphate thereby generating a free CoA and N-acetyl-D- glucosamine 6-phosphate. Alternative names comprise aminodeoxyglucosephosphate acetyltransferase, D-glucosamine-6-P N-acetyltransferase, glucosamine 6-phosphate acetylase, glucosamine 6-phosphate N-acetyltransferase, glucosamine-phosphate N-acetyltransferase, glucosamine-6-phosphate acetylase, N-acetylglucosamine-6-phosphate synthase, phosphoglucosamine acetylase, phosphoglucosamine N- acetylase phosphoglucosamine N-acetylase, phosphoglucosamine transacetylase, GNA and GNA1.

[0120] The term "N-acetylglucosamine-6-phosphate phosphatase" refers to an enzyme that dephosphorylates N-acetylglucosamine-6-phosphate (GlcNAc-6-P) hereby synthesizing N-acetylglucosamine (GIcNAc).

[0121] The terms "phosphoacetylglucosamine mutase", "acetylglucosamine phosphomutase", "acetylaminodeoxyglucose phosphomutase", "phospho-N-acetylglucosamine mutase" and "N-acetyl-D- glucosamine 1,6-phosphomutase" are used interchangeably and refer to an enzyme that catalyses the conversion of N-acetyl-glucosamine 1-phosphate into N-acetylglucosamine 6-phosphate.

[0122] The terms "N-acetylglucosamine 1-phosphate uridylyltransferase", "N-acetylglucosamine-l-phosphate uridyltransferase", "UDP-N-acetylglucosamine diphosphorylase", "UDP-N-acetylglucosamine pyrophosphorylase", "uridine diphosphoacetylglucosamine pyrophosphorylase", "UTP:2-acetamido-2- deoxy-alpha-D-glucose-l-phosphate uridylyltransferase", "UDP-GIcNAc pyrophosphorylase”, "GlmU uridylyltransferase", "Acetylglucosamine 1-phosphate uridylyltransferase", "UDP-acetylglucosamine pyrophosphorylase", "uridine diphosphate-N-acetylglucosamine pyrophosphorylase", "uridine diphosphoacetylglucosamine phosphorylase", and "acetylglucosamine 1-phosphate uridylyltransferase" are used interchangeably and refer to an enzyme that catalyses the conversion of N-acetylglucosamine 1- phosphate (GlcNAc-1-P) into UDP-N-acetylglucosamine (UDP-GIcNAc) by the transfer of uridine 5- monophosphate (from uridine 5-triphosphate (UTP) ).

[0123] The term glucosamine-l-phosphate acetyltransferase refers to an enzyme that catalyses the transfer of the acetyl group from acetyl coenzyme A to glucosamine-l-phosphate (GlcN-1-P) to produce N- acetylglucosamine-l-phosphate (GlcNAc-1-P).

[0124] The term "glmll" refers to a bifunctional enzyme that has both N-acetylglucosamine-l-phosphate uridyltransferase and glucosamine-l-phosphate acetyltransferase activity and that catalyses two sequential reactions in the de novo biosynthetic pathway for UDP-GIcNAc. The C-terminal domain catalyses the transfer of acetyl group from acetyl coenzyme A to GlcN-1-P to produce GlcNAc-1-P, which is converted into UDP-GIcNAc by the transfer of uridine 5-monophosphate, a reaction catalysed by the N- terminal domain.

[0125] The terms "Neu5Ac synthase", "N-acetylneuraminic acid synthase", "N-acetylneuraminate synthase", "sialic acid synthase", "NeuAc synthase", "NeuB”, "NeuBl", "NANA condensing enzyme", "N- acetylneuraminate lyase synthase", "N-acetylneuraminic acid condensing enzyme" as used herein are used interchangeably and refer to an enzyme capable to synthesize sialic acid (Neu5Ac) from N- acetylmannosamine (ManNAc) in a reaction using phosphoenolpyruvate (PEP).

[0126] The terms "N-acetylneuraminate lyase", "Neu5Ac lyase", "N-acetylneuraminate pyruvate-lyase", "N- acetylneuraminic acid aldolase", "NALase", "sialate lyase", "sialic acid aldolase", "sialic acid lyase" and "nanA" are used interchangeably and refer to an enzyme that degrades N-acetylneuraminate into N- acetylmannosamine (ManNAc) and pyruvate.

[0127] The terms "N-acylneuraminate-9-phosphate synthase", "N-acylneuraminate-9-phosphate synthetase", "NANA synthase", "NANAS", "NANS", "NmeNANAS", "N-acetylneuraminate pyruvate-lyase (pyruvate- phosphorylating)" as used herein are used interchangeably and refer to an enzyme capable to synthesize N-acylneuraminate-9-phosphate from N-acetylmannosamine-6-phosphate (ManNAc-6-phosphate) in a reaction using phosphoenolpyruvate (PEP).

[0128] The term "N-acylneuraminate-9-phosphatase" refers to an enzyme capable to dephosphorylate N- acylneuraminate-9-phosphate to synthesise N-acylneuraminate.

[0129] The terms "CMP-sialic acid synthase", "N-acylneuraminate cytidylyltransferase", "CMP-sialate synthase", "CMP-NeuAc synthase", "NeuA" and "CMP-N-acetylneuraminic acid synthase" as used herein are used interchangeably and refer to an enzyme capable to synthesize CMP-N-acetylneuraminate from N- acetylneuraminate using CTP in the reaction.

[0130] A 'fucosylation pathway' as used herein is a biochemical pathway comprising at least one of the enzymes and their respective genes chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase combined with a fucosyltransferase leading to a 1,2; a 1,3; a 1,4 and / or a 1,6 fucosylated compounds.

[0131] A 'galactosylation pathway' as used herein is a biochemical pathway comprising at least one of the enzymes and their respective genes chosen from the list comprising galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase combined with a galactosyltransferase leading to a galactosylated compound comprising a mono-, di-, or oligosaccharide having an alpha or beta bound galactose on any one or more of the 2, 3, 4 and 5 hydroxyl group of said mono-, di-, or oligosaccharide.

[0132] An 'N-acetylglucosaminylation pathway' as used herein is a biochemical pathway comprising at least one of the enzymes and their respective genes chosen from the list comprising L-glutamine— D-fructose-6- phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-l-phosphate uridylyltransferase / glucosamine-l-phosphate acetyltransferase combined with a glycosyltransferase leading to a GIcNAc-modified compound comprising a mono-, di-, or oligosaccharide having an alpha or beta bound N-acetylglucosamine (GIcNAc) on any one or more of the 3, 4 and 6 hydroxyl group of said mono-, di- or oligosaccharide.

[0133] An 'N-acetylgalactosaminylation pathway' as used herein is a biochemical pathway comprising at least one of the enzymes and their respective genes chosen from the list comprising L-glutamine— D-fructose- 5-phosphate aminotransferase, phosphoglucosamine mutase, / V-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, UDP-N-acetylglucosamine 4-epimerase, UDP-glucose 4-epimerase, N-acetylgalactosamine kinase and UDP-GalNAc pyrophosphorylase combined with a glycosyltransferase leading to a GalNAc-modified compound comprising a mono-, di- or oligosaccharide having an alpha or beta bound N-acetylgalactosamine on said mono-, di- or oligosaccharide.

[0134] A 'mannosylation pathway' as used herein is a biochemical pathway comprising at least one of the enzymes and their respective genes chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase and mannose-l-phosphate guanylyltransferase combined with a glycosyltransferase leading to a mannosylated compound comprising a mono-, di- or oligosaccharide having an alpha or beta bound mannose on said mono-, di- or oligosaccharide.

[0135] An 'N-acetylmannosaminylation pathway' as used herein is a biochemical pathway comprising at least one of the enzymes and their respective genes chosen from the list comprising L-glutamine— D-fructose- 5-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N- acetylglucosamine-6-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase, N- acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, UDP- GIcNAc 2-epimerase and ManNAc kinase combined with a glycosyltransferase leading to a ManNAc- modified compound comprising a mono-, di- or oligosaccharide having an alpha or beta bound N- acetylmannosamine on said mono-, di- or oligosaccharide.

[0136] The terms "pyruvate dehydrogenase", "pyruvate oxidase", "POX", "poxB" and "pyruvate:ubiquinone-8 oxidoreductase" are used interchangeably and refer to an enzyme that catalyses the oxidative decarboxylation of pyruvate to produce acetate and CO2.

[0137] The terms "lactate dehydrogenase", "D-lactate dehydrogenase", "IdhA", "hsll", "htpH", "D-LDH", "fermentative lactate dehydrogenase" and "D-specific 2-hydroxyacid dehydrogenase" are used interchangeably and refer to an enzyme that catalyses the conversion of lactate into pyruvate hereby generating NADH.

[0138] The term "purified" refers to material that is substantially or essentially free from components that interfere with the activity of the biological molecule. For cells, saccharides, nucleic acids, and polypeptides, the term "purified" refers to material that is substantially or essentially free from components that normally accompany the material as found in its native state. Typically, purified saccharides, oligosaccharides, proteins or nucleic acids of the invention are at least about 50%, 55%, 50%, 55%, 70%, 75%, 80% or 85% pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, or 99.0% pure as measured by band intensity on a silver-stained gel or other method for determining purity. Purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized. For di- and / or oligosaccharides, purity can be determined using methods such as but not limited to thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis or mass spectroscopy. Further herein, the terms "contaminants" and "impurities" preferably mean particulates, cells, cell components, metabolites, cell debris, proteins, peptides, amino acids, nucleic acids, glycolipids and / or endotoxins which can be present in an aqueous medium like e.g. a cultivation or an incubation.

[0139] The term "clarifying" as used herein refers to the act of treating an aqueous medium like e.g. a cultivation or an incubation, to remove suspended particulates and contaminants from the production process, like e.g. cells, cell components, insoluble metabolites and debris, that could interfere with the eventual purification of the one or more bioproduct(s). Such treatment can be carried out in a conventional manner by centrifugation, flocculation, flocculation with optional ultrasonic treatment, gravity filtration, microfiltration, foam separation or vacuum filtration (e.g. through a ceramic filter which can include a Celite™ filter aid).

[0140] The term "cultivation" refers to the culture medium wherein the cell is cultivated, or fermented, the cell itself, and a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, that is produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell. The terms "culture medium" and "cultivation medium" as used herein are used interchangeably and refer to the medium wherein the cell is cultivated.

[0141] The term "incubation" refers to a mixture wherein said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, is produced. Said mixture can comprise one or more enzyme(s), one or more precursor(s) and one or more acceptor(s) as defined herein present in a buffered solution and incubated for a certain time at a certain temperature enabling production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine and a galactose monosaccharide, catalysed by said one or more enzyme(s) using said one or more precursor(s) and said one or more acceptor(s) in said mixture. Said mixture can also comprise i) the cell obtained after cultivation or incubation, optionally said cell is subjected to cell lysis, ii) a buffered solution or the cultivation or incubation medium wherein the cell was cultivated or fermented, and iii) said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, that is produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracell ularly) of the cell. Said incubation can also be the cultivation as defined herein.

[0142] The terms "reactor" and "incubator" refer to the recipient filled with the cultivation or incubation. Examples of reactors and incubators comprise but are not limited to microfluidic devices, well plates, tubes, shake flasks, fermenters, bioreactors, process vessels, cell culture incubators, CO2 incubators.

[0143] As used herein, the term "cell productivity index (CPI)" refers to the mass of the sialylated oligosaccharide produced by the cells divided by the mass of the cells produced in the culture or cultivation.

[0144] The term "precursor" as used herein refers to substances that are taken up or synthetized by the cell for the specific production of a sialylated oligosaccharide according to the present invention. In this sense a precursor can be an acceptor as defined herein, but can also be another substance, metabolite, that is first modified within the cell as part of the biochemical synthesis route of a sialylated oligosaccharide, preferably a 3'sialylated oligosaccharide, said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide. The term "precursor" as used herein is also to be understood as a chemical compound that participates in a chemical or enzymatic reaction to produce another compound like e.g. an intermediate or an acceptor as defined herein, as part in the metabolic pathway of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide. The term "precursor" as used herein is also to be understood as a donor that is used by a glycosyltransferase to modify an acceptor as defined herein with a sugar moiety in a glycosidic bond, as part in the metabolic pathway of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.

[0145] Examples of such precursors comprise the acceptors as defined herein, and / or dihydroxyacetone, glucosamine, N-acetylglucosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyllactose, phosphorylated sugars or sugar phosphates like e.g. but not limited to glucose-1- phosphate, galactose-l-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6- bisphosphate, mannose-6-phosphate, mannose-l-phosphate, glycerol-3-phosphate, glyceraldehyde-3- phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N-acetylglucosamine-l-phosphate, N-acetylneuraminic acid-9- phosphate and nucleotide-activated sugars like nucleotide diphospho-sugars and nucleotide monophospho-sugars as defined herein like e.g. UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, CMP-sialic acid, GDP-mannose, GDP-4-dehydro-6-deoxy-a-D-mannose, GDP-fucose.

[0146] Optionally, the cell is transformed to comprise and to express at least one nucleic acid sequence encoding a protein selected from the group consisting of lactose transporter, N-acetylneuraminic acid transporter, fucose transporter, glucose transporter, galactose transporter, transporter for a nucleotide-activated sugar wherein said transporter internalizes a to the medium added precursor for the synthesis of the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, of present invention.

[0147] The term "acceptor" as used herein refers to a mono-, di- or oligosaccharide, which can be modified by a glycosyltransferase. Examples of such acceptors comprise glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, lacto-N-biose, N-acetyllactosamine, lacto-N-triose, lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-pentaose (LNP), lacto-N-neopentaose, para lacto-N- pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N-hexaose (LNH), lacto-N- neohexaose (LNnH), para lacto-N-neohexaose (pLNnH), para lacto-N-hexaose (pLNH), lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N- neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose, iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose, lacto-N-decaose, iso lacto-N-decaose, novo lacto-N-decaose, lacto-N-neodecaose, and oligosaccharide containing 1 or more N-acetyllactosamine units and / or 1 or more lacto-N-biose units or an intermediate into oligosaccharide, fucosylated and sialylated versions thereof, ceramide, N-acylated sphingoid, glucosylceramide, lactosylceramide, sphingosine, phytosphingosine, sphingosine synthons, peptide backbones with beta-GIcNAc-Asn residues, glycoproteins with terminal GIcNAc and Gal residues, immunoglobulins.

[0148] Detailed description of the invention

[0149] In a first aspect, the present invention provides a method for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide. The method comprises contacting a sialyltransferase with a mixture comprising a donor comprising a sialic acid residue, and an acceptor, preferably in a medium, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from the donor to the acceptor, thereby producing said 3'sialylated oligosaccharide. The acceptor used in the method is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide and the saccharide is chosen from the list consisting of an oligosaccharide or a disaccharide. The sialyltransferases used in the present invention have alpha-2, 3-sialyltransferase activity on an acceptor, and comprise an amino acid sequence that is i) at least 60.0 % identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 or ii) at least 85.0 % identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequence as represented by SEQ ID NO: 17, 14, 16, 28 or 29.

[0150] As used herein the wording "a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide" preferably is a disaccharide-containing 3’sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably is a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide, even more preferably is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-Lewis x.

[0151] As used herein the acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably said acceptor is LacNAc, LNB, a LacNAc comprising oligosaccharide or an LNB comprising oligosaccharide, more preferably an LNT or an LNnT.

[0152] Alternatively, the present invention provides a method for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide wherein the method comprises providing i) CMP-sialic acid, ii) an acceptor as defined herein, and iii) a sialyltransferase as defined herein. The method further comprises contacting the sialyltransferase and CMP-sialic acid with the acceptor, under conditions where the sialyltransferase catalyses the transfer of a sialic acid residue from said CMP-sialic acid to the acceptor resulting in the production of said 3'sialylated oligosaccharide. Preferably, the method further comprises separating said produced 3’sialylated oligosaccharide from the medium.

[0153] In a preferred aspect, the present invention provides a method for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide wherein the method comprises contacting a cell extract comprising a sialyltransferase as defined herein with a mixture comprising a donor comprising a sialic acid residue, and an acceptor as defined herein, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from the donor to the acceptor, thereby producing said 3'sialylated oligosaccharide. Preferably, said 3'sialylated oligosaccharide is separated. Herein, preferably the sialyltransferase has alpha-2, 3- sialyltransferase activity on the acceptor and comprises an amino acid sequence that is at least 80.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

[0154] In a preferred embodiment of the method of the present invention, the 3'sialylated oligosaccharide is produced in a cell-free system.

[0155] In another aspect, the present invention provides a method for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said method comprises the steps of: i. providing a cell, preferably a single cell, expressing, preferably heterologously expressing, more preferably overexpressing, even more preferably heterologously overexpressing, a sialyltransferase as defined herein; ii. providing CMP-sialic acid, optionally said CMP-sialic acid is produced by said cell, and iii. providing an acceptor being a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, optionally said acceptor is produced by said cell, and iv. cultivating and / or incubating said cell under conditions permissive to express said sialyltransferase, optionally permissive to produce said CMP-sialic acid and / or said acceptor as defined herein and preferably wherein said sialyltransferase catalyses the transfer of a sialic residue from said CMP-sialic acid to said acceptor resulting in the production of said 3'siaylated oligosaccharide, preferably, separating said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide e as defined herein from said cultivation or incubation.

[0156] In another aspect, the present invention provides a method for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said method comprises the steps of: i. providing a cell, preferably a single cell, expressing, preferably heterologously expressing, more preferably overexpressing, even more preferably heterologously overexpressing, a sialyltransferase as defined herein, ii. providing CMP-sialic acid, optionally said CMP-sialic acid is produced by said cell, and iii. providing an acceptor being a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, optionally said acceptor is produced by said cell, and iv. cultivating and / or incubating said cell under conditions permissive to express said sialyltransferase, optionally permissive to produce said CMP-sialic acid and / or said acceptor as defined herein, and preferably wherein said sialyltransferase catalyses the transfer of a sialic residue from said CMP-sialic acid to said acceptor resulting in the production of said 3'siaylated oligosaccharide, preferably, separating said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein from said cultivation or incubation.

[0157] In a preferred embodiment of the method of the present invention, the sialylated oligosaccharide is produced by a cell, preferably a single cell, wherein said cell expresses a sialyltransferase as defined herein. In a preferred embodiment of the method of the present invention, the 3'sialylated oligosaccharide is produced by a cell, preferably a single cell, wherein said cell expresses a sialyltransferase as described herein. The cell used in the present invention is preferably a metabolically engineered cell as described herein. Preferably, said cell is metabolically engineered for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.

[0158] In the methods and / or cell of present invention, the sialyltransferase as described herein comprises an amino acid sequence that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues.

[0159] In a more preferred embodiment, said sialyltransferase comprises an amino acid sequence that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22.

[0160] In a more preferred embodiment, said sialyltransferase comprises an amino acid sequence that is at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full- length amino acid sequences as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or 27.

[0161] In another preferred embodiment of the method and / or cell of present invention, the sialyltransferase as described herein comprises an amino acid sequence that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues.

[0162] In a more preferred embodiment, said sialyltransferase comprises an amino acid sequence that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 17, 10, 14, 16, 18, 28 or 29.

[0163] Alternatively, the sialyltransferase as described herein comprises an amino acid sequence comprising a fragment of any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 and having alpha-2, 3-sialyltransferase activity on the acceptor as defined herein. Most preferably, said sialyltransferase comprises an amino acid sequence as represented by any one of the SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

[0164] In the scope of the present invention, permissive conditions are understood to be conditions relating to physical or chemical parameters including but not limited to temperature, pH, pressure, osmotic pressure and product / donor / precursor / acceptor concentration.

[0165] In a particular embodiment, the permissive conditions may include a temperature-range of about 30 + / - 20 degrees centigrade, a pH-range of 2.0 - 10.0, preferably a pH range of 3.0 - 7.0.

[0166] In another and / or additional preferred embodiment of the method and / or cell of present invention, the sialic acid residue is at least one chosen from the list consisting of Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulonic acid (KDO). In a more preferred embodiment, the sialic acid residue is Neu5Ac.

[0167] In another and / or additional preferred embodiment of the method and / or cell of present invention, the donor comprising a sialic acid residue is CMP-sialic acid. In a more preferred embodiment, the donor comprising a sialic acid residue is chosen from the list consisting of CMP-Neu5Ac, CMP-Neu4Ac, CMP- Neu5Ac9N3, CMP-Neu4,5Acz, CMP-Neu5,7Acz, CMP-Neu5,9Acz, CMP-Neu5,7(8,9)Acz, CMP-N- glycolylneuraminic acid (CMP-Neu5Gc) and CMP-KDO. In an even more preferred embodiment, the donor comprising a sialic acid residue is CMP-Neu5Ac.

[0168] Preferably said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is a disaccharide-containing 3'sialylated oligosaccharide as defined herein, and said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.

[0169] In an embodiment of the method of present invention, the cultivation medium contains at least one carbon source selected from the group consisting of glucose, fructose, sucrose, and glycerol.

[0170] In another embodiment of the method of present invention, the cultivation or incubation medium contains at least one compound selected from the group consisting of lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP- GIcNAc), sialic acid and CMP-sialic acid.

[0171] Preferably, the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is recovered from the medium, the cultivation medium or incubation medium and / or from the cell or separated from the cultivation or incubation as explained herein. According to a preferred embodiment of the method of the invention, the method comprises the use of a cultivation or incubation medium comprising at least one precursor and / or acceptor for the production of the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as described herein and / or the method comprises adding to the cultivation or incubation medium at least one precursor and / or acceptor feed for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide. Preferably, said precursor is selected from the group comprising a monosaccharide like e.g. galactose, fucose, sialic acid, GIcNAc, GalNAc; a nucleotide-activated sugar like e.g. CMP-sialic acid, UDP-Gal, UDP-GIcNAc, GDP-fucose; a disaccharide like e.g. lactose, LNB or LacNAc; and an oligosaccharide like e.g. lacto-N-triose (LN3), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT). Additionally or preferably, said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, more preferably a LacNAc comprising oligosaccharide, an LNB comprising oligosaccharide, even more preferably an LNT or an LNnT. In a more preferred embodiment of the method of the invention, said precursor is chosen from the list comprising sialic acid, CMP-sialic acid and lactose. In another more preferred embodiment of the method of the invention, said acceptor is LNT or LNnT.

[0172] According to an alternative and / or additional embodiment of the method of the invention, the conditions permissive to produce said 3'sialylated oligosaccharide comprise adding to the cultivation or incubation medium at least one precursor and / or acceptor feed for the production of said 3'sialylated oligosaccharide.

[0173] According to an alternative embodiment of the method of the invention, the conditions permissive to produce said 3'sialylated oligosaccharide comprise the use of a cultivation or incubation medium wherein said cultivation or incubation medium lacks any precursor and / or acceptor for the production of said 3'sialylated oligosaccharide and is combined with a further addition to said cultivation or incubation medium of at least one precursor and / or acceptor feed for the production of said 3'sialylated oligosaccharide.

[0174] According to an embodiment of the method of the invention, the cultivation or incubation is contained in a reactor or incubator, as defined herein. The volume of said reactor or incubator ranges from microlitre (p.L) scale to 10.000 m3 (cubic meter). In a preferred embodiment, the volume of said reactor or incubator ranges from 250 mL (millilitre) to 10.000 m3 (cubic meter).

[0175] In an alternative or preferred embodiment, the method for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide comprises at least one of the following steps: i) use of a cultivation or incubation medium comprising at least one precursor and / or acceptor; ii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and / or acceptor feed wherein the total reactor or incubator volume ranges from 250 ml (millilitre) to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor and / or acceptor feed; iii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and / or acceptor feed wherein the total reactor or incubator volume ranges from 250 ml (millilitre) to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor and / or acceptor feed and wherein preferably, the pH of said precursor and / or acceptor feed is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor and / or acceptor feed is kept between 20°C and 80°C; iv) adding at least one precursor and / or acceptor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor and / or acceptor feeding solution; v) adding at least one precursor and / or acceptor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor and / or acceptor feeding solution and wherein preferably the concentration of said precursor and / or acceptor feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably, 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably, 550 g / L, most preferably 600 g / L; wherein preferably, the pH of said precursor and / or acceptor feeding solution is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor and / or acceptor feeding solution is kept between 20°C and 80°C; said method resulting in said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide with a concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the cultivation or incubation. In a more preferred embodiment of the method of the invention, said precursor is chosen from the list comprising lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP- GIcNAc), sialic acid and CMP-sialic acid. In another more preferred embodiment of the method of the invention, said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, as defined herein.

[0176] In another and / or additional preferred embodiment, the method for the production of 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide comprises at least one of the following steps: i) use of a cultivation or incubation medium comprising at least one precursor and / or acceptor; ii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and / or acceptor in one pulse or in a discontinuous (pulsed) manner wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m3(cubic meter), preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor and / or acceptor feed pulse(s); iii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and / or acceptor feed in one pulse or in a discontinuous (pulsed) manner wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m3(cubic meter), preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor and / or acceptor feed and wherein preferably, the pH of said precursor and / or acceptor feed pulse(s) is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor and / or acceptor feed pulse(s) is kept between 20°C and 80°C; iv) adding at least one precursor and / or acceptor feed in a discontinuous (pulsed) manner to the cultivation or incubation medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor and / or acceptor feeding solution; v) adding at least one precursor and / or acceptor feed in a discontinuous (pulsed) manner to the cultivation or incubation medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor and / or acceptor feeding solution and wherein preferably, the pH of said precursor and / or acceptor feeding solution is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor and / or acceptor feeding solution is kept between 20°C and 80°C; said method resulting in said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, with a concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the cultivation or incubation. In a more preferred embodiment of the method of the invention, said precursor is chosen from the list comprising lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N- acetylglucosamine (UDP-GIcNAc), sialic acid and CMP-sialic acid. In another more preferred embodiment of the method of the invention, said acceptor is a saccharide as defined herein.

[0177] In another and / or additional preferred embodiment, the method for the production of 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as described herein comprises at least one of the following steps: i) use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter); ii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor feed; iii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor feed and wherein preferably, the pH of said precursor feed is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor feed is kept between 20°C and 80°C; iv) adding at least one precursor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor feeding solution; v) aciding at least one precursor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor feeding solution and wherein the concentration of said precursor feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably, 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably, 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of said precursor feeding solution is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor feeding solution is kept between 20°C and 80°C; said method resulting in said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, with a concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the cultivation or incubation. In a more preferred embodiment of the method of the invention, said precursor is chosen from the list comprising lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N- acetylglucosamine (UDP-GIcNAc), sialic acid and CMP-sialic acid.

[0178] In another and / or additional preferred embodiment, the method for the production of a 3’sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as described herein comprises at least one of the following steps: i) use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter); ii) adding to the cultivation or incubation medium in a reactor or incubator at least one acceptor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said acceptor feed; iii) adding to the cultivation or incubation medium in a reactor or incubator at least one acceptor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 ml (millilitre) to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said acceptor feed and wherein preferably, the pH of said acceptor feed is set between 2.0 and 10.0 and wherein preferably, the temperature of said acceptor feed is kept between 20°C and 80°C; iv) adding at least one acceptor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of an acceptor feeding solution; v) adding at least one acceptor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of an acceptor feeding solution and wherein the concentration of said acceptor feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably, 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably, 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of said acceptor feeding solution is set between 2.0 and 10.0 and wherein preferably, the temperature of said acceptor feeding solution is kept between 20°C and 80°C; said method resulting in said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, with a concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the cultivation or incubation. In a more preferred embodiment of the method of the invention, said acceptor is a saccharide comprising at least one N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.

[0179] In another and / or additional preferred embodiment, the method for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as described herein comprises at least one of the following steps: i) use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter); ii) aciding to the cultivation or incubation medium in a reactor or incubator at least one precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m3(cubic meter) in one pulse or in a discontinuous (pulsed), preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor feed pulse(s); iii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m3(cubic meter) in one pulse or in a discontinuous (pulsed) manner, preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor feed and wherein preferably, the pH of said precursor feed pulse(s) is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor feed pulse(s) is kept between 20°C and 80°C; iv) adding at least one precursor feed in a discontinuous (pulsed) manner to the cultivation or incubation medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor feeding solution; v) adding at least one precursor feed in a discontinuous (pulsed) manner to the cultivation or incubation medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor feeding solution and wherein the concentration of said precursor feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably, 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably, 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of said precursor feeding solution is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor feeding solution is kept between 20°C and 80°C; said method resulting in said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, with a concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the cultivation or incubation. In a more preferred embodiment of the method of the invention, said precursor is chosen from the list comprising lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N- acetylglucosamine (UDP-GIcNAc), sialic acid and CMP-sialic acid.

[0180] In another and / or additional preferred embodiment, the method for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as described herein comprises at least one of the following steps: i) use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter); ii) adding to the cultivation or incubation medium in a reactor or incubator at least one acceptor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m3(cubic meter) in one pulse or in a discontinuous (pulsed), preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said acceptor feed pulse(s); iii) adding to the cultivation or incubation medium in a reactor or incubator at least one acceptor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m3(cubic meter) in one pulse or in a discontinuous (pulsed) manner, preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said acceptor feed and wherein preferably, the pH of said acceptor feed pulse(s) is set between 2.0 and 10.0 and wherein preferably, the temperature of said acceptor feed pulse(s) is kept between 20°C and 80°C; iv) adding at least one acceptor feed in a discontinuous (pulsed) manner to the cultivation or incubation medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of an acceptor feeding solution; v) adding at least one acceptor feed in a discontinuous (pulsed) manner to the cultivation or incubation medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of an acceptor feeding solution and wherein the concentration of said acceptor feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably, 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably, 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of said acceptor feeding solution is set between 2.0 and 10.0 and wherein preferably, the temperature of said acceptor feeding solution is kept between 20°C and 80°C; said method resulting in said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, with a concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the cultivation or incubation. In a more preferred embodiment of the method of the invention, said acceptor is a saccharide comprising at least one N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.

[0181] In a more preferred embodiment, the method for the production of a 3'sialylated oligosaccharide, as described herein comprises at least one of the following steps: i) use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter); ii) adding to the cultivation or incubation medium in a reactor or incubator a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than twofold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said lactose feed; iii) adding to the cultivation or incubation medium in a reactor or incubator a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than twofold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said lactose feed and wherein preferably, the pH of said lactose feed is set between 2.0 and 10.0, preferably between 3.0 and 7.0, and wherein preferably, the temperature of said lactose feed is kept between 20°C and 80°C; iv) adding a lactose feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; v) adding a lactose feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein the concentration of said lactose feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably, 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably, 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of said lactose feed is set between 2.0 and 10.0, preferably between 3.0 and 7.0 and wherein preferably, the temperature of said lactose feed is kept between 20°C and 80°C; said method resulting in said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide wherein preferably said 3'sialylated oligosaccharide is a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, preferably is a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide, more preferably is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl- Lewis x, with a concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the cultivation or incubation.

[0182] Preferably the lactose feed is accomplished by adding lactose from the beginning of the cultivation or incubation in a concentration of at least 5 mM, preferably in a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably in a concentration > 300 mM.

[0183] In another embodiment of the methods, the lactose feed is accomplished by adding lactose to the cultivation or incubation medium in a concentration, such that throughout the production phase of the cultivation or incubation a lactose concentration of at least 5 mM, preferably 10 mM or 30 mM is obtained. In a further embodiment of the methods described herein the cells are cultivated or incubated for at least about 60, 80, 100, or about 120 hours or in a continuous manner.

[0184] In a preferred embodiment, a carbon source is provided, preferably sucrose, in the cultivation medium for 3 or more days, preferably up to 7 days; and / or provided, in the cultivation medium, at least 100, advantageously at least 105, more advantageously at least 110, even more advantageously at least 120 grams of sucrose per litre of initial cultivation volume in a continuous manner, so that the final volume of the cultivation medium is not more than three-fold, advantageously not more than two-fold, more advantageously less than two-fold of the volume of the cultivation medium before the cultivation. Preferably, when performing the method as described herein, a first phase of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the cultivation medium before the lactose is added to the cultivation medium in a second phase.

[0185] In an alternative preferable embodiment, in the method as described herein, the lactose is added already in the first phase of exponential growth together with the carbon-based substrate.

[0186] As used herein, the sialyltransferases or alpha-2, 3-sialyltransferases according to the invention preferably have alpha-2, 3-sialyltransferase activity on a galactose (Gal) residue, preferably a terminal Gal residue, of an acceptor as defined herein.

[0187] According to another aspect, the present invention provides a metabolically engineered cell for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said cell has been metabolically engineered to possess, preferably to express, more preferably to heterologously express, even more preferably to overexpress, most preferably to heterologously overexpress, a sialyltransferase which has alpha-2, 3-sialyltransferase activity, and comprises an amino acid sequence that is i) at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22, or ii) at least 80.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 . Preferably, said sialyltransferase used in the cell is a sialyltransferase as described herein.

[0188] In another and / or additional aspect, the present invention provides a metabolically engineered cell for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as described herein, wherein said cell has been metabolically engineered to possess, preferably to express, more preferably to heterologously express, even more preferably to overexpress, most preferably to heterologously overexpress, a sialyltransferase which has alpha-2, 3-sialyltransferase activity and comprises an amino acid sequence that is at least 80.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8,

[0189] 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

[0190] In a preferred embodiment of the cell or method of present invention, the sialyltransferase comprises an amino acid sequence that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ. ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31. In an alternative preferred embodiment of the cell or method of present invention, the sialyltransferase comprises an amino acid sequence as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

[0191] Alternatively or preferably, the cell contains a nucleic acid molecule which comprises a polynucleotide sequence that encodes any one of the sialyltransferases as described herein.

[0192] Herein, a metabolically engineered cell comprising a pathway for production of said 3'sialylated oligosaccharide, is provided. Examples of such pathways comprise but are not limited to pathways involved in the synthesis of monosaccharide, phosphorylated monosaccharide, nucleotide-activated sugar, and / or glycosylation pathways like e.g., a fucosylation, sialylation, galactosylation, N- acetylglucosaminylation, N-acetylgalactosaminylation, mannosylation and / or N- acetylmannosaminylation pathway. Said pathway for production of a sialylated oligosaccharide preferably comprises at least one sialyltransferase as described herein.

[0193] In a preferred embodiment of the method and / or cell of present invention, the cell comprises one or more pathway(s) for monosaccharide synthesis. Said pathways for monosaccharide synthesis comprise enzymes like e.g. carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases, dehydrogenases, enzymes involved in the synthesis of one or more nucleoside triphosphate(s) like UTP, GTP, ATP and CTP, enzymes involved in the synthesis of any one or more nucleoside mono- or diphosphates like e.g. UMP and UDP, respectively, and enzymes involved in the synthesis of phosphoenolpyruvate (PEP).

[0194] In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell comprises one or more pathway(s) for phosphorylated monosaccharide synthesis. Said pathways for phosphorylated monosaccharide synthesis comprise enzymes involved in the synthesis of one or more monosaccharide(s), one or more nucleoside mono-, di- and / or triphosphate(s) and enzymes involved in the synthesis of phosphoenolpyruvate (PEP) like e.g., but not limited to PEP synthase, carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases and dehydrogenases. In another and / or additional preferred embodiment of the method and / or cell of present invention, the cell comprises one or more pathways for the synthesis of one or more nucleotide-activated sugars. Said pathways for nucleotide-activated sugar synthesis comprise enzymes like e.g. PEP synthase, carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases, dehydrogenases, mannose-6-phosphate isomerase, phosphomannomutase, mannose-1- phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, L- fucokinase / GDP-fucose pyrophosphorylase, L-glutamine— D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N- acetylglucosamine-6P 2-epimerase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine- 5-phosphate phosphatase, N-acetylmannosamine-6-phosphate 2-epimerase, N-acetylmannosamine-6- phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N- acetylglucosamine-l-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N- acylneuraminate-9-phosphatase, CMP-sialic acid synthase, d-arabinose 5-phosphate isomerase, KDO-8P synthase, KDO 8-phosphate phosphatase, CMP-KDO synthetase, galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase and / or phosphoglucomutase.

[0195] Said cell may further comprise and express at least one further glycosyltransferase that is involved in the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.

[0196] In a preferred embodiment of the method and / or cell of present invention, the cell is metabolically engineered to comprise a pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein. In an alternative preferred embodiment of the method and / or cell of present invention, the cell is metabolically engineered to comprise a pathway for production said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, and to have modified expression or activity of a sialyltransferase of present invention.

[0197] In a further preferred embodiment of the method and / or cell of present invention, the cell comprises a recombinant sialyltransferase capable of modifying said acceptor and / or wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein or another acceptor as defined herein with one or more sialic acid molecules that is / are synthesized by any one or more sialic acid synthases like e.g. Neu5Ac synthases expressed in the cell, into said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.

[0198] In a preferred embodiment of the method and / or cell of the invention, the metabolically engineered cell is modified with one or more expression modules.

[0199] Said expression modules are also known as transcriptional units and comprise polynucleotides for expression of recombinant genes including coding gene sequences and appropriate transcriptional and / or translational control signals that are operably linked to the coding genes. Said control signals comprise promoter sequences, untranslated regions, ribosome binding sites, terminator sequences. Said expression modules can contain elements for expression of one single recombinant gene but can also contain elements for expression of more recombinant genes or can be organized in an operon structure for integrated expression of two or more recombinant genes. Said polynucleotides may be produced by recombinant DNA technology using techniques well-known in the art. Methods which are well known to those skilled in the art to construct expression modules include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989 and yearly updates).

[0200] The expression of each of said expression modules can be constitutive or is created by a natural or chemical inducer. As used herein, constitutive expression should be understood as expression of a gene that is transcribed continuously in an organism. Expression that is created by a natural inducer should be understood as a facultative or regulatory expression of a gene that is only expressed upon a certain natural condition of the host (e.g. organism being in labour, or during lactation), as a response to an environmental change (e.g. including but not limited to hormone, heat, cold, pH shifts, light, oxidative or osmotic stress / signalling), or dependent on the position of the developmental stage or the cell cycle of said host cell including but not limited to apoptosis and autophagy. Expression that is created by a chemical inducer should be understood as a facultative or regulatory expression of a gene that is only expressed upon sensing of external chemicals (e.g. IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose) via an inducible promoter or via a genetic circuit that either induces or represses the transcription or translation of said polynucleotide to a polypeptide.

[0201] The expression modules can be integrated in the genome of said cell or can be presented to said cell on a vector. Said vector can be present in the form of a plasmid, cosmid, phage, liposome, or virus, which is to be stably transformed / transfected into said metabolically engineered cell. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. These vectors may contain selection markers such as but not limited to antibiotic markers, auxotrophic markers, toxinantitoxin markers, RNA sense / antisense markers. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and / or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et aL, see above. For recombinant production, cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the cell can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986), and Sambrook et aL, 1989, supra.

[0202] As used herein an expression module comprises polynucleotides for expression of at least one recombinant gene. Said recombinant gene is involved in the expression of a polypeptide acting in the synthesis of said 3'sialyl ated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein; or said recombinant gene is linked to other pathways in said cell that are not involved in the synthesis of a sialylated oligosaccharide. Said recombinant genes encode endogenous proteins with a modified expression or activity, preferably said endogenous proteins are overexpressed; or said recombinant genes encode heterologous proteins that are heterogeneously introduced and expressed in said modified cell, preferably overexpressed. The endogenous proteins can have a modified expression in the cell which also expresses a heterologous protein.

[0203] In a preferred embodiment of the method and / or cell of the invention, the expression of each of said expression modules present in said metabolically engineered cell is constitutive or tuneable as described herein.

[0204] In a further embodiment of the method and / or cell of the invention, the cell is modified in the expression or activity of at least one of said sialyltransferases. In a preferred embodiment, said sialyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous sialyltransferase is overexpressed; alternatively said sialyltransferase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed. Said endogenous sialyltransferase can have a modified expression in the cell which also expresses a heterologous sialyltransferase.

[0205] According to a preferred embodiment of the method and / or cell of the invention, the cell comprises a pathway for production of a sialylated oligosaccharide, preferably a 3'sialylated oligosaccharide, comprising at least one sialyltransferase according to present invention. According to another preferred embodiment of the method and / or cell of the invention, said pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine and a galactose monosaccharide as defined herein, further comprises at least one enzyme chosen from the list comprising L-glutamine— D-fructose- 6-phosphate aminotransferase, a phosphoglucosamine mutase, an N-acetylglucosamine-6-P deacetylase, an N-acylglucosamine 2-epimerase, a UDP-N-acetylglucosamine 2-epimerase, an N-acetylmannosamine- 6-phosphate 2-epimerase, a UDP-GIcNAc 2-epimerase / kinase, a glucosamine 6-phosphate N- acetyltransferase, an N-acetylglucosamine-6-phosphate phosphatase, a phosphoacetylglucosamine mutase, an N-acetylglucosamine 1-phosphate uridylyltransferase, a glucosamine-l-phosphate acetyltransferase, an Neu5Ac synthase, an N-acetylneuraminate lyase, an N-acylneuraminate-9- phosphate synthase, an N-acylneuraminate-9-phosphatase, a sialic acid transporter and a CMP-sialic acid synthase.

[0206] In a preferred embodiment, the cell comprises a pathway for production said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said cell expresses at least one enzyme chosen from the list comprising an N- acylglucosamine 2-epimerase like is known e.g. from several species including Bacteroides ovatus, E. coli, Homo sapiens, Rattus norvegicus, a Neu5Ac synthase, a CMP sialic acid synthase like is known e.g. from Neisseria meningitidis, and a sialyltransferase according to present invention, wherein the enzymes are as defined herein. N-acetylglucosamine (GIcNAc) can be added to the cell and / or can be provided by an enzyme expressed in the cell or by the mechanism of the cell. Such cell producing GIcNAc can express a phosphatase converting GlcNAc-6-phosphate into GIcNAc, like any one or more of e.g. the E. coli HAD-like phosphatase genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and Ybill, PsMupP from Pseudomonas putida, ScDOGl from 5. cerevisiae and BsAraL from Bacillus subtilis as described in WO 2018 / 122225. Preferably, the cell is modified to produce GIcNAc. More preferably, the cell is modified for enhanced GIcNAc production. Said modification can be any one or more chosen from the group comprising knockout of a glucosamine-5-phosphate deaminase, an N-acetylglucosamine-6-phosphate deacetylase and / or an N-acetyl-D-glucosamine kinase and over-expression of an L-glutamine— D- fructose-6-phosphate aminotransferase and / or a glucosamine 6-phosphate N-acetyltransferase.

[0207] In an alternative and / or additional preferred embodiment, the cell comprises a pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said cell expresses at least one enzyme chosen from the list comprising an UDP-N-acetylglucosamine 2-epimerase like is known e.g. from several species including Campylobacter jejuni, E. coli, Neisseria meningitidis, Bacillus subtilis, Citrobacter rodentium, a Neu5Ac synthase, a CMP sialic acid synthase like is known e.g from Neisseria meningitidis, and a sialyltransferase according to present invention, wherein the enzymes are as defined herein. UDP-N- acetylglucosamine (UDP-GIcNAc) can be added to the cell and / or can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing an UDP-GIcNAc can express enzymes converting, e.g. GIcNAc, which is to be added to the cell, to UDP-GIcNAc. These enzymes may be any one or more enzymes chosen from the list comprising an N-acetyl-D-glucosamine kinase, an N- acetylglucosamine-5-phosphate deacetylase, a phosphoglucosamine mutase, and an N- acetylglucosamine-l-phosphate uridylyltransferase / glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli. Preferably, the cell is modified to produce UDP- GIcNAc. More preferably, the cell is modified for enhanced UDP-GIcNAc production. Said modification can be any one or more chosen from the group comprising knock-out of an N-acetylglucosamine-6-phosphate deacetylase, over-expression of an L-glutamine— D-fructose-6-phosphate aminotransferase, over- expression of a phosphoglucosamine mutase, and over-expression of an N-acetylglucosamine-1- phosphate uridylyltransferase / glucosamine-l-phosphate acetyltransferase.

[0208] In an alternative and / or additional preferred embodiment, the cell comprises a pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said cell expresses at least one enzyme chosen from the list comprising an N-acetylmannosamine-6-phosphate 2-epimerase like is known e.g. from several species including E. coli, Haemophilus influenzae, Enterobacter sp., Streptomyces sp., an N- acylneuraminate-9-phosphate synthetase, an N-acylneuraminate-9-phosphatase like is known e.g. from Candidatus Magnetomorum sp. HK-1 or Bacteroides thetaiotaomicron, a Neu5Ac synthase, a CMP sialic acid synthase like is known e.g. from Neisseria meningitidis, and a sialyltransferase according to present invention, wherein the enzymes are as defined herein. N-acetyl-D-glucosamine 6-phosphate (GlcNAc-6P) can be added to the cell and / or can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing GlcNAc-6P can express an enzyme converting, e.g., GlcN6P, which is to be added to the cell, to GlcNAc-6P. This enzyme may be a glucosamine 6-phosphate N-acetyltransferase from several species including Saccharomyces cerevisiae, Kluyveromyces lactis, Homo sapiens. Preferably, the cell is modified to produce GlcNAc-6P. More preferably, the cell is modified for enhanced GlcNAc-6P production. Said modification can be any one or more chosen from the group comprising knockout of a glucosamine-6-phosphate deaminase, an N-acetylglucosamine-6-phosphate deacetylase and overexpression of an L-glutamine— D-fructose-6-phosphate aminotransferase and / or a glucosamine 6- phosphate N-acetyltransferase.

[0209] In an alternative and / or additional preferred embodiment, the cell comprises a pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said cell expresses at least one enzyme chosen from the list comprising a bifunctional UDP-GIcNAc 2-epimerase / kinase like is known e.g. from several species including Homo sapiens, Rattus norvegicus and Mus musculus, an N-acylneuraminate-9-phosphate synthetase, an N-acylneuraminate-9-phosphatase like is known e.g. from Candidatus Magnetomorum sp. HK-1 or Bacteroides thetaiotaomicron, a Neu5Ac synthase, a CMP sialic acid synthase like is known e.g. from Neisseria meningitidis, and a sialyltransferase according to present invention, wherein the enzymes are as defined herein. UDP-N-acetylglucosamine can be added to the cell and / or can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing an UDP-GIcNAc can express enzymes converting, e.g. GIcNAc, which is to be added to the cell, to UDP-GIcNAc. These enzymes may be an N-acetyl-D-glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, and an N-acetylglucosamine-l-phosphate uridylyltransferase / glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli. Preferably, the cell is modified to produce UDP-GIcNAc. More preferably, the cell is modified for enhanced UDP-GIcNAc production. Said modification can be any one or more chosen from the group comprising knock-out of an N-acetylglucosamine-6-phosphate deacetylase, over-expression of an L-glutamine— D-fructose-6-phosphate aminotransferase, over-expression of a phosphoglucosamine mutase, and over-expression of an N-acetylglucosamine-l-phosphate uridylyltransferase / glucosamine-1- phosphate acetyltransferase.

[0210] Additionally, or alternatively, the cell used herein is optionally genetically engineered to import a precursor and / or an acceptor in the cell, by the introduction and / or overexpression of a transporter able to import the respective precursor and / or acceptor in the cell. Such transporter is for example a membrane protein belonging to the major facilitator superfamily (MFS), the ATP-binding cassette (ABC) transporter family or the PTS system involved in the uptake of e.g. mono-, di- and / or oligosaccharides.

[0211] Additionally, or alternatively, the cell used herein is optionally genetically engineered to produce polyisoprenoid alcohols like e.g. phosphorylated dolichol that can act as lipid carrier.

[0212] Additionally, or alternatively, the cell used herein is optionally genetically engineered to import lactose in the cell, by the introduction and / or overexpression of a lactose permease. Said lactose permease is for example encoded by the lacY gene or the Iacl2 gene.

[0213] Additionally, or alternatively, the cell expresses a membrane protein that is a transporter protein involved in transport of compounds and / or a sialylated oligosaccharide as defined in present invention out of the cell. In the context of present invention, it should be understood that said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is preferably produced intracellularly. The skilled person will further understand that a fraction or substantially all of said produced 3'sialylated oligosaccharide, remains intracellularly and / or is excreted outside the cell either passively or through active transport.

[0214] Preferably the cell is transformed to comprise at least one nucleic acid sequence encoding a protein selected from the group comprising a lactose transporter like e.g. the LacY or Iacl2 permease, a glucose transporter, a galactose transporter, a transporter for a nucleotide-activated sugar like for example a transporter for UDP-GIcNAc, a transporter protein involved in transport of said 3’sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide a and a galactose monosaccharide s defined herein, out of the cell.

[0215] According to another preferred aspect of the method and / or cell of the present invention, the cell is capable to synthesize N-acetylmannosamine (ManNAc), N-acetylmannosamine-6-phosphate (ManNAc-6- phosphate) and / or phosphoenolpyruvate (PEP).

[0216] In a preferred embodiment, the cell comprises a pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, comprising a pathway for production of ManNAc. ManNAc can be provided by an enzyme expressed in the cell or by the mechanism of the cell. Such cell producing ManNAc can express an N-acylglucosamine 2-epimerase like is known e.g. from several species including Bacteroides ovatus, E. coli, Homo sapiens, Rattus norvegicus that converts GIcNAc into ManNAc. Alternatively, and / or additionally, the cell producing ManNAc can express an UDP-N-acetylglucosamine 2-epimerase like is known e.g. from several species including Campylobacter jejuni, E. coli, Neisseria meningitidis, Bacillus subtilis, Citrobacter rodentium that converts UDP-GIcNAc into ManNAc. GIcNAc and / or UDP-GIcNAc can be added to the cell and / or provided by an enzyme expressed in the cell or by the mechanism of the cell as described herein.

[0217] In a more preferred embodiment, the cell is modified for enhanced ManNAc production. Said modification can be any one or more chosen from the group comprising knock-out of N-acetylmannosamine kinase, over-expression of N-acetylneuraminate lyase.

[0218] In another preferred embodiment, the cell comprises a pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, comprising a pathway for production of ManNAc-6-phosphate. ManNAc-6-phosphate can be provided by an enzyme expressed in the cell or by the mechanism of the cell. Such cell producing ManNAc-6-phosphate can express a bifunctional UDP-GIcNAc 2- epimerase / kinase like is known e.g. from several species including Homo sapiens, Rattus norvegicus and Mus musculus that converts UDP-GIcNAc into ManNAc-6-phosphate. Alternatively, and / or additionally, the cell producing ManNAc-6-phosphate can express an N-acetylmannosamine-6-phosphate 2-epimerase that converts GlcNAc-6-phosphate into ManNAc-6-phosphate. UDP-GIcNAc and / or GlcNAc-6-phosphate can be added to the cell and / or provided by an enzyme expressed in the cell or by the mechanism of the cell as described herein. In a more preferred embodiment, the cell is modified for enhanced ManNAc-6- phosphate production. Said modification can be any one or more chosen from the group comprising overexpression of N-acetylglucosamine-6-phosphate deacetylase, over-expression of N-acetyl-D-glucosamine kinase, over-expression of phosphoglucosamine mutase, over-expression of N-acetylglucosamine-1- phosphate uridylyltransferase / glucosamine-l-phosphate acetyltransferase.

[0219] According to another embodiment of the method and / or cell of the invention, the cell is further capable to synthesize any one or more nucleotide-activated sugars. In a preferred embodiment of the method and / or cell of the invention, the cell is capable to synthesize one or more nucleotide-activated sugars chosen from the list comprising UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP- Gal), GDP-mannose (GDP-Man), UDP-glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy— L- arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N- acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L- pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), CMP-sialic acid (e.g. CMP-Neu5Ac, CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP- Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-Neu5Gc or CMP-KDO), GDP-fucose (GDP-Fuc), GDP-rhamnose and UDP-xylose. In a more preferred embodiment of the method and / or cell of the invention, the cell is capable to synthesize at least the nucleotide-activated sugar CMP-Neu5Ac. In an even more preferred embodiment of the method and / or cell of the invention, the cell uses at least one of the synthesized nucleotide-activated sugars in the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.

[0220] The cell used herein is optionally genetically engineered to express the de novo synthesis of UDP-GIcNAc. UDP-GIcNAc can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing an UDP-GIcNAc can express enzymes converting, e.g. GIcNAc, which is to be added to the cell, to UDP-GIcNAc. These enzymes may be any one or more of the list comprising an N-acetyl-D- glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, and an N-acetylglucosamine-l-phosphate uridylyltransferase / glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli. Preferably, the cell is modified to produce UDP-GIcNAc. More preferably, the cell is modified for enhanced UDP-GIcNAc production. Said modification can be any one or more chosen from the group comprising knock-out of an N-acetylglucosamine-6-phosphate deacetylase, over-expression of an L-glutamine— D-fructose-6- phosphate aminotransferase, over-expression of a phosphoglucosamine mutase, and over-expression of an N-acetylglucosamine-l-phosphate uridylyltransferase / glucosamine-l-phosphate acetyltransferase.

[0221] Additionally, or alternatively, the cell used herein is optionally genetically engineered to express the de novo synthesis of CMP-Neu5Ac. CMP-Neu5Ac can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing CMP-Neu5Ac can express an enzyme converting, e.g., sialic acid to CMP-Neu5Ac. This enzyme may be a CMP-sialic acid synthetase, like the N-acylneuraminate cytidylyltransferase from several species including Homo sapiens, Neisseria meningitidis, and Pasteurella multocida. Preferably, the cell is modified to produce CMP-Neu5Ac. More preferably, the cell is modified for enhanced CMP-Neu5Ac production. Said modification can be any one or more chosen from the group comprising knock-out of an N-acetylglucosamine-6-phosphate deacetylase, knock-out of a glucosamine- 6-phosphate deaminase, over-expression of a CMP-sialic acid synthetase, and over-expression of an N- acetyl-D-glucosamine-2-epimerase encoding gene.

[0222] Additionally, or alternatively, the cell used herein is optionally genetically engineered to express the de novo synthesis of GDP-fucose. GDP-fucose can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing GDP-fucose can express an enzyme converting, e.g., fucose, which is to be added to the cell, to GDP-fucose. This enzyme may be, e.g., a bifunctional fucose kinase / fucose-l-phosphate guanylyltransferase, like Fkp from Bacteroidesfragilis, or the combination of one separate fucose kinase together with one separate fucose-l-phosphate guanylyltransferase like they are known from several species including Homo sapiens, Sus scrofa and Rattus norvegicus. Preferably, the cell is modified to produce GDP-fucose. More preferably, the cell is modified for enhanced GDP-fucose production. Said modification can be any one or more chosen from the group comprising knock-out of an UDP-glucose:undecaprenyl-phosphate glucose-l-phosphate transferase encoding gene, over-expression of a GDP-L-fucose synthase encoding gene, over-expression of a GDP-mannose 4,6-dehydratase encoding gene, over-expression of a mannose-l-phosphate guanylyltransferase encoding gene, over-expression of a phosphomannomutase encoding gene and over-expression of a mannose-6-phosphate isomerase encoding gene.

[0223] Additionally, or alternatively, the cell used herein is optionally genetically engineered to express the de novo synthesis of UDP-Gal. UDP-Gal can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing UDP-Gal can express an enzyme converting, e.g. UDP-glucose, to UDP-Gal. This enzyme may be, e.g., the UDP-glucose-4-epimerase GalE like as known from several species including Homo sapiens, Escherichia coli, and Rattus norvegicus. Preferably, the cell is modified to produce UDP-Gal. More preferably, the cell is modified for enhanced UDP-Gal production. Said modification can be any one or more chosen from the group comprising knock-out of a bifunctional 5'- nucleotidase / UDP-sugar hydrolase encoding gene, knock-out of a galactose-l-phosphate uridylyltransferase encoding gene and over-expression of a UDP-glucose-4-epimerase encoding gene.

[0224] Additionally, or alternatively, the cell used herein is optionally genetically engineered to express the de novo synthesis of UDP-GalNAc. UDP-GalNAc can be synthesized from UDP-GIcNAc by the action of a single-step reaction using a UDP-N-acetylglucosamine 4-epimerase like e.g. wbgU from Plesiomonas shigelloides, gne from Yersinia enterocolitica or wbpP from Pseudomonas aeruginosa serotype 06. Preferably, the cell is modified to produce UDP-GalNAc. More preferably, the cell is modified for enhanced UDP-GalNAc production.

[0225] Additionally, or alternatively, the cell used herein is optionally genetically engineered to express the de novo synthesis of UDP-ManNAc. UDP-ManNAc can be synthesized directly from UDP-GIcNAc via an epimerization reaction performed by a UDP-GIcNAc 2-epimerase (like e.g. cap5P from Staphylococcus aureus, RffE from E. coli, Cpsl9fK from S. pneumoniae, and RfbC from S. enterica). Preferably, the cell is modified to produce UDP-ManNAc. More preferably, the cell is modified for enhanced UDP-ManNAc production.

[0226] According to another embodiment of the method and / or cell of the invention, the cell expresses at least one further glycosyltransferase chosen from the list comprising fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N- acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N- glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino- 4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases.

[0227] In a preferred embodiment of the method and / or cell of the invention, the fucosyltransferase is chosen from the list comprising alpha-1, 2-fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-1,3 / 4- fucosyltransferase, alpha-1, 4-fucosyltransferase and alpha-1, 6-fucosyltransferase.

[0228] In an alternative and / or additional embodiment of the method and / or cell of the invention, the further sialyltransferase is chosen from the list comprising alpha-2, 3-sialyltransferase, alpha-2, 5-sialyltransferase, and alpha-2, 8-sialyltransferase.

[0229] In an alternative and / or additional embodiment of the method and / or cell of the invention, the galactosyltransferase is chosen from the list comprising beta-1, 3-galactosyltransferase, N- acetylglucosamine beta-1, 3-galactosyltransferase, beta-1, 4-galactosyltransferase, N-acetylglucosamine beta-1, 4-galactosyltransferase, alpha-1, 3-galactosyltransferase and alpha-1, 4-galactosyltransferase.

[0230] In an alternative and / or additional embodiment of the method and / or cell of the invention, the glucosyltransferase is chosen from the list comprising alpha-glucosyltransferase, beta-1, 2- glucosyltransferase, beta-1, 3-glucosyltransferase and beta-1, 4-glucosyltransferase.

[0231] In an alternative and / or additional embodiment of the method and / or cell of the invention, the mannosyltransferase is chosen from the list comprising alpha-1, 2-mannosyltransferase, alpha-1, 3- mannosyltransferase and alpha-1, 6-mannosyltransferase.

[0232] In an alternative and / or additional embodiment of the method and / or cell of the invention, the N- acetylglucosaminyltransferase is chosen from the list comprising galactoside beta-1, 3-N- acetylglucosaminyltransferase and beta-1, 6-N-acetylglucosaminyltransferase.

[0233] In an alternative and / or additional embodiment of the method and / or cell of the invention, the N- acetylgalactosaminyltransferase is chosen from the list comprising alpha-1, 3-N- acetylgalactosaminyltransferase.

[0234] In a further embodiment of the method and / or cell of the invention, the cell is modified in the expression or activity of at least one of said glycosyltransferases. In a preferred embodiment, said glycosyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous glycosyltransferase is overexpressed; alternatively said glycosyltransferase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed. Said endogenous glycosyltransferase can have a modified expression in the cell which also expresses a heterologous glycosyltransferase.

[0235] According to another and / or alternative preferred embodiment of the method and / or cell of the invention, the cell comprises a fucosylation pathway comprising at least one enzyme chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, fucosyltransferase.

[0236] According to another and / or alternative preferred embodiment of the method and / or cell of the invention, the cell comprises a galactosylation pathway comprising at least one enzyme chosen from the list comprising galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase, galactosyltransferase.

[0237] According to another and / or alternative preferred embodiment of the method and / or cell of the invention, the cell comprises an N-acetylglucosaminylation pathway comprising at least one enzyme chosen from the list comprising L-glutamine— D-fructose-6-phosphate aminotransferase, N- acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-1- phosphate uridylyltransferase / glucosamine-l-phosphate acetyltransferase, N- acetylglucosaminyltransferase.

[0238] In an alternative and / or additional further embodiment of the method and / or cell of the invention, the cell is modified in the expression or activity of at least one pyruvate dehydrogenase like e.g. from E. coli, S. cerevisiae, H. sapiens and R. norvegicus. In a preferred embodiment, the cell has been modified to have at least one partially or fully knocked out or mutated pyruvate dehydrogenase encoding gene by means generally known by the person skilled in the art resulting in at least one protein with less functional or being disabled for pyruvate dehydrogenase activity. In a more preferred embodiment, the cell has a full knock-out in the poxB encoding gene resulting in a cell lacking pyruvate dehydrogenase activity.

[0239] In an alternative and / or additional further embodiment of the method and / or cell of the invention, the cell is modified in the expression or activity of at least one lactate dehydrogenase like e.g. from E. coli, S. cerevisiae, H. sapiens and R. norvegicus. In a preferred embodiment, the cell has been modified to have at least one partially or fully knocked out or mutated lactate dehydrogenase encoding gene by means generally known by the person skilled in the art resulting in at least one protein with less functional or being disabled for lactate dehydrogenase activity. In a more preferred embodiment, the cell has a full knock-out in the IdhA encoding gene resulting in a cell lacking lactate dehydrogenase activity.

[0240] According to another preferred embodiment of the method and / or cell of the invention, the cell comprises a lower or reduced expression and / or abolished, impaired, reduced or delayed activity of any one or more of the proteins comprising beta-galactosidase, galactoside O-acetyltransferase, N- acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphate glucose-l-phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N- acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, El I D- Man, ushA, galactose-l-phosphate uridylyltransferase, glucose-l-phosphate adenylyltransferase, glucose-l-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6- phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IcIR, Ion protease, glucose-specific translocating phosphotransferase enzyme I IBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme I IAGlc, beta-glucoside specific PTS enzyme II, fructose-specific PTS multiphosphoryl transfer protein FruA and FruB, ethanol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphate acetyltransferase, pyruvate decarboxylase.

[0241] According to another preferred embodiment of the method and / or cell of the invention, the cell is using a precursor for the synthesis of said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein Herein, the precursor is fed to the cell from the cultivation or incubation medium. In another preferred embodiment, the cell is producing a precursor for the synthesis of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.

[0242] In a preferred embodiment of the method and / or cell of present invention, the method results in the production of 45 g / L or more, preferably 50 g / L or more, more preferably 60 g / L or more, of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide. In a more preferred embodiment, the method results in the production of 45 g / L or more, preferably 50 g / L or more, more preferably 60 g / L or more of a 3'sLacNAc comprising oligosaccharide or a 3'sLNB comprising oligosaccharide.

[0243] According to another preferred embodiment of the method and / or cell of the invention, the cell produces 90 g / L or more of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, in the whole broth and / or supernatant. In a more preferred embodiment said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, produced in the whole broth and / or supernatant has a purity of at least 80% measured on the total amount of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, and its precursor produced by the cell in the whole broth and / or supernatant, respectively.

[0244] According to another preferred embodiment of the method and / or cell of the invention, the method results in the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, with a purity equal to or greater than 80% measured on the total amount of said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, and its precursor. In a more preferred embodiment, the method results in the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein with a purity equal to or greater than 85% measured on the total amount of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined hereinx, and its precursor. In an even more preferred embodiment, the method results in the production of said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, with a purity equal to or greater than 90% measured on the total amount of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, and its precursor. In another even more preferred embodiment, the method results in the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, with a purity equal to or greater than 91%, equal to or greater than 92%, equal to or greater than 93%, equal to or greater than 94%, equal to or greater than 95%, equal to or greater than 96%, equal to or greater than 97%, equal to or greater than 98%, equal to or greater than 99% measured on the total amount of said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, and its precursor. According to another preferred embodiment of the method and / or cell of the invention, the method results in the production of a mixture comprising said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, together with lactose and sialic acid, wherein said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, has a purity equal to or greater than 80% measured on the total amount of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, an acceptor as defined herein, and sialic acid in said mixture and wherein said mixture comprises less than 10% of said saccharide as defined herein, and / or less than 5% sialic acid. In a more preferred embodiment, said mixture comprises less than 9% of said saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein. In an even more preferred embodiment, said mixture comprises less than 8% of said saccharide comprising at least one N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein. In another even more preferred embodiment, said mixture comprises less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of said saccharide comprising at least one N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein. In an additional and / or alternative more preferred embodiment, said mixture comprises less than 5% sialic acid. In an even more preferred additional and / or alternative embodiment, said mixture comprises less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1% sialic acid.

[0245] According to another embodiment of the method and / or cell of the invention, the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is chosen from the list comprising a milk oligosaccharide, O-antigen, the oligosaccharide repeats present in capsular polysaccharides, an oligosaccharide present in lipopolysaccharides and aminosugars. In a more preferred embodiment, the milk oligosaccharide is a mammalian milk oligosaccharide. In an even more preferred embodiment, the milk oligosaccharide is a human milk oligosaccharide.

[0246] According to another embodiment of the method and / or cell of the invention, the cell is capable to synthesize a mixture of oligosaccharides. In an alternative and / or additional embodiment, the cell is capable to synthesize a mixture of di- and / or oligosaccharides, alternatively, the cell is capable to synthesize a mixture of sialic acid, di- and / or oligosaccharides.

[0247] Another aspect of the invention provides for a method and a cell wherein said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, is produced in and / or by a cell which is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell. The latter bacterium preferably belongs to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus or the phylum of Actinobacteria. The latter bacterium belonging to the phylum Proteobacteria belongs preferably to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacterium preferably relates to any strain belonging to the species Escherichia coli such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultivated Escherichia coli strains - designated as E. coli K12 strains - which are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Well-known examples of the E. coli K12 strains are K12 Wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Hence, the present invention specifically relates to a mutated and / or transformed Escherichia coli cell or strain as indicated above wherein said E. coli strain is a K12 strain. More preferably, the Escherichia coli K12 strain is E. coli MG1655. The latter bacterium belonging to the phylum Firmicutes belongs preferably to the Bacilli, preferably Lactobacilliales, with members such as Lactobacillus lactis, Leuconostoc mesenteroides, or Bacillales with members such as from the genus Bacillus, such as Bacillus subtilis or, B. amyloliquefaciens. The latter Bacterium belonging to the phylum Actinobacteria, preferably belonging to the family of the Corynebacteriaceae, with members Corynebacterium glutamicum or C. afermentans, or belonging to the family of the Streptomycetaceae with members Streptomyces griseus or 5. fradiae. The latter bacterium belonging to the phylum Proteobacteria, preferably belonging to the family of the Vibrionaceae, with member Vibrio natriegens. The latter yeast preferably belongs to the phylum of the Ascomycota or the phylum of the Basidiomycota or the phylum of the Deuteromycota or the phylum of the Zygomycetes. The latter yeast belongs preferably to the genus Saccharomyces (with members like e.g. Saccharomyces cerevisiae, S. bayanus, S. boulardii), Zygosaccharomyces, Pichia (with members like e.g. Pichia pastoris, P. anomala, P. kluyveri), Komagataella, Hansenula, Kluyveromyces (with members like e.g. Kluyveromyces lactis, K. marxianus, K. thermotolerans), Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces, Torulaspora, Yarrowia (like e.g. Yarrowia lipolytica) or Starmerella (like e.g. Starmerella bombicola). The latter yeast is preferably selected from Pichia pastoris, Yarrowia lipolitica, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha, Kluyveromyces marxianus, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Zygosaccharomyces rouxii, and Zygosaccharomyces bailii. The latter fungus belongs preferably to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus. Plant cells include cells of flowering and non-flowering plants, as well as algal cells, for example Chlamydomonas, Chlorella, etc. Preferably, said plant is a tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant. The latter animal cell is preferably derived from non-human mammals (e.g. cattle, buffalo, pig, sheep, mouse, rat, primate (e.g., chimpanzee, orangutan, gorilla, monkey (e.g., Old World, New World), lemur), dog, cat, rabbit, horse, cow, goat, ox, deer, musk deer, bovid, whale, dolphin, hippopotamus, elephant, rhinoceros, giraffe, zebra, lion, cheetah, tiger, panda, red panda, otter), birds (e.g. chicken, duck, ostrich, turkey, pheasant), fish (e.g. swordfish, salmon, tuna, sea bass, trout, catfish), invertebrates (e.g. lobster, crab, shrimp, clams, oyster, mussel, sea urchin), reptiles (e.g. snake, alligator, turtle), amphibians (e.g. frogs) or insects (e.g. fly, nematode) or is a genetically engineered cell line derived from human cells excluding embryonic stem cells. Both human and non-human mammalian cells are preferably chosen from the list comprising an epithelial cell like e.g., a mammary epithelial cell, an embryonic kidney cell (e.g., HEK293 or HEK 293T cell), a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell like e.g. an N20, SP2 / 0 or YB2 / 0 cell, an NIH-3T3 cell, a non-mammary adult stem cell or derivatives thereof such as described in WO 2021 / 067641, a lactocyte derived from mammalian induced pluripotent stem cells, preferably human induced pluripotent stem cells, a lactocyte as part of mammary-like gland organoids, a post-parturition mammary epithelium cell, a polarized mammary cell, preferably a polarized mammary cell selected from the group comprising live primary mammary epithelial cells, live mammary myoepithelial cells, live mammary progenitor cells, live immortalized mammary epithelial cells, live immortalized mammary myoepithelial cells, live immortalized mammary progenitor cells, a non- mammary adult stem cell or derivatives thereof as well-known to the person skilled in the art from e.g., WO 2021 / 219634, WO 2022 / 054053, WO 2021 / 141762, WO 2021 / 142241, WO 2021 / 067641 and WO 2021 / 242866. The latter insect cell is preferably derived from Spodoptera frugiperda like e.g., Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae, Trichoplusia ni like e.g., BTI-TN-5B1-4 cells or Drosophila melanogaster Wke e.g., Drosophila S2 cells. The latter protozoan cell preferably is a Leishmania tarentolae cell.

[0248] More preferably, the cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably from the group consisting of yeast cells, bacterial cells, archaebacterial cells, algae cells, and fungal cells as described herein.

[0249] According to another embodiment of the method and / or cell of the invention, the cell as described herein comprises a nucleic acid molecule comprising a polynucleotide sequence encoding a sialyltransferase as described herein and operably linked to control sequences recognized by the cell, wherein said sequence is foreign to the cell, said sequence further i) being integrated in the genome of said cell and / or ii) presented to said cell on a vector.

[0250] According to another preferred embodiment of the method and / or cell of the invention, the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and / or required for the synthesis of a sialylated oligosaccharide.

[0251] A further aspect of the present invention provides for an isolated nucleic acid molecule encoding a sialyltransferase wherein said sialyltransferase is an alpha-2, 3-sialyltransferase as defined herein.

[0252] In a preferred embodiment, the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues.

[0253] In another preferred embodiment, the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22.

[0254] In another preferred embodiment, the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence that is at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or 27.

[0255] In another preferred embodiment, the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues.

[0256] In another preferred embodiment, the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29.

[0257] In another preferred embodiment, the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31. Another further aspect of the present invention provides for an isolated nucleic acid molecule encoding a sialyltransferase wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity on the galactose (Gal) residue of said saccharide as defined herein, and comprises an amino acid sequence that is at least 80.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

[0258] In a preferred embodiment, the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full- length amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

[0259] In another preferred embodiment, the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

[0260] Another aspect of the present invention provides for a vector comprising an isolated nucleic acid molecule encoding a sialyltransferase as described herein.

[0261] Another aspect provides for a cell to be stably cultured in a medium, wherein said medium can be any type of growth medium comprising minimal medium, complex medium or growth medium enriched in certain compounds like, for example, but not limited to, vitamins, trace elements, amino acids.

[0262] The microorganism or cell as used herein is capable to grow on a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium or a mixture thereof as the main carbon source. With the term main is meant the most important carbon source for the microorganism or cell for the production of the sialylated oligosaccharide of interest, biomass formation, carbon dioxide and / or by-products formation (such as acids and / or alcohols, such as acetate, lactate, and / or ethanol), i.e. 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99% of all the required carbon is derived from the aboveindicated carbon source. In one embodiment of the invention, said carbon source is the sole carbon source for said organism, i.e. 100% of all the required carbon is derived from the above-indicated carbon source. Common main carbon sources comprise but are not limited to glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate. As used herein, a precursor as defined herein cannot be used as a carbon source for the production of the 3'sialylated oligosaccharide, of present invention. According to the present invention, the methods as described herein preferably comprises a step of separating said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide of present invention from said cultivation or incubation, otherwise said recovering the 3'sialylated oligosaccharide, from the cultivation or incubation medium and / or the cell.

[0263] The terms "separating from said cultivation or incubation" means harvesting, collecting, or retrieving said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide from the cell and / or the medium of its cultivation or incubation.

[0264] The 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide can be separated in a conventional mannerfrom the aqueous culture medium, in which the cell was cultivated or incubated. In case said 3'sialylated oligosaccharide, is still present in the cells producing the 3'sialylated oligosaccharide, conventional manners to free or to extract said 3'sialylated oligosaccharide, out of the cells can be used, such as cell destruction using high pH, heat shock, sonication, French press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergent, hydrolysis, etc. The cultivation or incubation medium and / or cell extract together and separately can then be further used for separating said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.

[0265] This preferably involves clarifying said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide to remove suspended particulates and contaminants, particularly cells, cell components, insoluble metabolites and debris produced by culturing or incubating the genetically engineered cell. In this step, said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide can be clarified in a conventional manner. Preferably, said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide is clarified by centrifugation, flocculation, decantation and / or filtration. Another step of separating said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide preferably involves removing substantially all the eventually remaining proteins, peptides, amino acids, RNA and DNA, and any endotoxins and glycolipids that could interfere with the subsequent separation step, from said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide preferably after it has been clarified. In this step, remaining proteins and related impurities can be removed from said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide in a conventional manner. Preferably, remaining proteins, salts, by-products, colour, endotoxins and other related impurities are removed from said 3'sialylated oligosaccharide, by ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, electrophoresis (e.g. using slab-polyacrylamide or sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands including e.g. DEAE-Sepharose, poly-L-lysine and polymyxin-B, endotoxin-selective adsorber matrices), ion exchange chromatography (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange, inside-out ligand attachment), hydrophobic interaction chromatography and / or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography or electrodialysis. With the exception of size exclusion chromatography, remaining proteins and related impurities are retained by a chromatography medium or a selected membrane.

[0266] In a further preferred embodiment, the methods as described herein also provide for a further purification of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide of present invention. A further purification of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide may be accomplished, for example, by use of (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange, temperature adjustment, pH adjustment or pH adjustment with an alkaline or acidic solution to remove any remaining DNA, protein, LPS, endotoxins, or other impurity. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used. Another purification step is accomplished by crystallization, evaporation or precipitation of said 3'sialylated oligosaccharide. Another purification step is to dry, e.g. spray dry, lyophilize, spray freeze dry, freeze spray dry, band dry, belt dry, vacuum band dry, vacuum belt dry, drum dry, roller dry, vacuum drum dry or vacuum roller dry the produced 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.

[0267] In an exemplary embodiment, the separation and purification of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is made in a process, comprising the following steps in any order: a) contacting the cultivation or incubation or a clarified version thereof with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da ensuring the retention of the produced 3'sialylated oligosaccharide, and allowing at least a part of the proteins, salts, byproducts, colour and other related impurities to pass, b) conducting a diafiltration process on the retentate from step a), using said membrane, with an aqueous solution of an inorganic electrolyte, followed by optional diafiltration with pure water to remove excess of the electrolyte, c) and collecting the retentate enriched in said 3'sialylated oligosaccharide, in the form of a salt from the cation of said electrolyte. In an alternative exemplary embodiment, the separation and purification of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is made in a process, comprising the following steps in any order: subjecting the cultivation or incubation or a clarified version thereof to two membrane filtration steps using different membranes, wherein one membrane has a molecular weight cut-off of between about 300 to about 500 Dalton, and the other membrane as a molecular weight cut-off of between about 600 to about 800 Dalton.

[0268] In an alternative exemplary embodiment, the separation and purification of said 3'sialylated oligosaccharide, is made in a process, comprising treating the cultivation or incubation or a clarified version thereof with a strong cation exchange resin in H+-form in a step and with a weak anion exchange resin in free base form in another step, wherein said steps can be performed in any order.

[0269] In an alternative exemplary embodiment, the separation and purification of said oligosaccharide, is made in the following way. The cultivation or incubation comprising the produced 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, biomass, medium components and contaminants is applied to the following purification steps: i) separation of biomass from the cultivation or incubation, ii) cationic ion exchanger treatment for the removal of positively charged material, iii) anionic ion exchanger treatment for the removal of negatively charged material, iv) nanofiltration step and / or electrodialysis step, wherein a purified solution comprising the produced 3'sialylated at a purity of greater than or equal to 80% is provided. Optionally the purified solution is dried by any one or more drying steps chosen from the list comprising spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying and vacuum roller drying.

[0270] In an alternative exemplary embodiment, the separation and purification of the 3'sialylated oligosaccharide as defined herein is made in a process, comprising the following steps in any order: enzymatic treatment of the cultivation or incubation; removal of the biomass from the cultivation or incubation; ultrafiltration; nanofiltration; and a column chromatography step. Preferably such column chromatography is a single column or a multiple column. Further preferably the column chromatography step is simulated moving bed chromatography. Such simulated moving bed chromatography preferably comprises i) at least 4 columns, wherein at least one column comprises a weak or strong cation exchange resin; and / or ii) four zones I, II, III and IV with different flow rates; and / or iii) an eluent comprising water; and / or iv) an operating temperature of 15 degrees to 60 degrees centigrade. In a specific embodiment, the present invention provides the 3'sialylated oligosaccharide as defined herein which is dried to powder by any one or more drying steps chosen from the list comprising spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying and vacuum roller drying, wherein the dried powder contains < 15% -wt. of water, preferably < 10% -wt. of water, more preferably < 7% -wt. of water, most preferably < 5% -wt. of water.

[0271] Another aspect of the present invention provides the use of a sialyltransferase that has alpha-2, 3- sialyltransferase activity on the galactose (Gal) residue of said saccharide comprising at least one N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein and that comprises an amino acid sequence: that is at least 50.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 250, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22, that is at least at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 95.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 1, 5, 9, 3, 7, 23 or 27, that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29, or

[0272] - as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27 , 28, 29, 30 or 31, for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.

[0273] Another aspect of the present invention provides the use of a sialyltransferase that has alpha-2, 3- sialyltransferase activity on the galactose (Gal) residue of a saccharide comprising at least one N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, and that comprises an amino acid sequence: that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31, or

[0274] - as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31, for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.

[0275] Another aspect of the present invention provides the use of a cell as described herein for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.

[0276] A further aspect of the present invention provides i) use of a method as described herein for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, ii) use of an isolated nucleic acid molecule as described herein for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, or iii) use of a vector as described herein for production of said 3'sialylated oligosaccharide as defined herein.

[0277] A further aspect of the present invention provides an alpha-2, 3-sialyltransferase for use in the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity on the galactose (Gal) residue of an acceptor, wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, and wherein said sialyltransferase comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22, at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29. that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22, that is at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or 27, that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29, or

[0278] - as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

[0279] A further aspect provides for an alpha-2, 3-sialyltransferase as described herein wherein said 3'sialylated oligosaccharide is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-Lewis x.

[0280] Furthermore, the invention also relates to the 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide obtained by the methods according to the invention. Said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide may be used for the manufacture of a preparation, as food additive, prebiotic, symbiotic, for the supplementation of baby food, adult food, infant animal feed, adult animal feed, or as either therapeutically or pharmaceutically active compound or in cosmetic applications. In a preferred embodiment, said preparation comprises at least one 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, that is obtainable, preferably obtained, by the methods as described herein. In another preferred embodiment, a preparation is provided that further comprises at least one probiotic microorganism. In another preferred embodiment of present invention, said preparation is a nutritional composition. In a more preferred embodiment, said preparation is a medicinal formulation, a dietary supplement, a dairy drink or an infant formula.

[0281] With the novel methods, the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide can easily and effectively be provided, without the need for complicated, time and cost consuming synthetic processes.

[0282] For identification of the 3’sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide of present invention produced as described herein, the monosaccharide or the monomeric building blocks (e.g. the monosaccharide or glycan unit composition), the anomeric configuration of side chains, the presence and location of substituent groups, degree of polymerization / molecular weight and the linkage pattern can be identified by standard methods known in the art, such as, e.g. methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas chromatographymass spectrometry), MALDI-MS (Matrix-assisted laser desorption / ionization-mass spectrometry), ESI-MS (Electrospray ionization-mass spectrometry), HPLC (High-Performance Liquid chromatography with ultraviolet or refractive index detection), HPAEC-PAD (High-Performance Anion-Exchange chromatography with Pulsed Amperometric Detection), CE (capillary electrophoresis), IR (infrared) / Raman spectroscopy, and NMR (Nuclear magnetic resonance) spectroscopy techniques. The crystal structure can be solved using, e.g., solid-state NMR, FT-IR (Fourier transform infrared spectroscopy), and WAXS (wide-angle X-ray scattering). The degree of polymerization (DP), the DP distribution, and polydispersity can be determined by, e.g., viscosimetry and SEC (SEC-HPLC, high performance size-exclusion chromatography). To identify the monomeric components of the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide methods such as e.g. acid-catalysed hydrolysis, HPLC (high performance liquid chromatography) or GLC (gas-liquid chromatography) (after conversion to alditol acetates) may be used. To determine the glycosidic linkages, the 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide, is methylated with methyl iodide and strong base in DMSO, hydrolysis is performed, a reduction to partially methylated alditols is achieved, an acetylation to methylated alditol acetates is performed, and the analysis is carried out by GLC / MS (gas- liquid chromatography coupled with mass spectrometry). To determine the glycan sequence, a partial depolymerization is carried out using an acid or enzymes to determine the structures. To identify the anomeric configuration, the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is subjected to enzymatic analysis, e.g. it is contacted with an enzyme that is specific for a particular type of linkage, e.g., beta-galactosidase, or alphaglucosidase, etc., and NMR may be used to analyse the products.

[0283] The separated and preferably also purified 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as described herein is incorporated into a food (e.g., human food or feed), dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine. In some embodiments, the 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide is mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine.

[0284] In some embodiments, the dietary supplement comprises at least one prebiotic ingredient and / or at least one probiotic ingredient.

[0285] A "prebiotic" is a substance that promotes growth of microorganisms beneficial to the host, particularly microorganisms in the gastrointestinal tract. In some embodiments, a dietary supplement provides multiple prebiotics, including the 3'sialylated oligosaccharide being a prebiotic produced and / or purified by a process disclosed in this specification, to promote growth of one or more beneficial microorganisms. Examples of prebiotic ingredients for dietary supplements include other prebiotic molecules (such as HMDs) and plant polysaccharides (such as inulin, pectin, b-glucan and xylooligosaccharide). A "probiotic" product typically contains live microorganisms that replace or add to gastrointestinal microflora, to the benefit of the recipient. Examples of such microorganisms include Lactobacillus species (for example, L. acidophilus and L. bulgaricus), Bifidobacterium species (for example, B. animalis, B. longum and B. infantis (e.g., Bi-26)), and Saccharomyces boulardii. In some embodiments, a 3'sialylated oligosaccharide produced and / or purified by a process of this specification is orally administered in combination with such microorganism.

[0286] Examples of further ingredients for dietary supplements include oligosaccharides (such as 2'- fucosyllactose, 3-fucosyllactose, 6'-sialyllactose), disaccharides (such as lactose), monosaccharides (such as glucose, galactose, L-fucose, sialic acid, glucosamine and N-acetylglucosamine), thickeners (such as gum arabic), acidity regulators (such as trisodium citrate), water, skimmed milk, and flavourings.

[0287] In some embodiments, the 3'sialylated oligosaccharide, is incorporated into a human baby food (e.g., infant formula). Infant formula is generally a manufactured food for feeding to infants as a complete or partial substitute for human breast milk. In some embodiments, infant formula is sold as a powder and prepared for bottle- or cup-feeding to an infant by mixing with water. The composition of infant formula is typically designed to be roughly mimic human breast milk. In some embodiments, a 3'sialylated oligosaccharide, produced and / or purified by a process in this specification is included in infant formula to provide nutritional benefits similar to those provided by the oligosaccharides in human breast milk. In some embodiments, the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, is mixed with one or more ingredients of the infant formula. Examples of infant formula ingredients include non-fat milk, carbohydrate sources (e.g., lactose), protein sources (e.g., whey protein concentrate and casein), fat sources (e.g., vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and / or sunflower oil; and fish oils), vitamins (such as vitamins A, Bb, Bi2, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate) and possibly human milk oligosaccharides (HMDs). Such HMOs may include, for example, DiFL, lacto-N-triose II, LNT, LNnT, lacto-N-fucopentaose I, lacto-N-neofucopentaose, lacto-N-fucopentaose II, lacto-N- fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, 6'-galactosyllactose, 3'- galactosyllactose, lacto-N-hexaose and lacto- N-neohexaose.

[0288] In some embodiments, the one or more infant formula ingredients comprise non-fat milk, a carbohydrate source, a protein source, a fat source, and / or a vitamin and mineral.

[0289] In some embodiments, the one or more infant formula ingredients comprise lactose, whey protein concentrate and / or high oleic safflower oil.

[0290] In some embodiments, the concentration of the 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide, in the infant formula is approximately the same concentration as the concentration of the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide generally present in human breast milk.

[0291] In some embodiments, the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, is incorporated into a feed preparation, wherein said feed is chosen from the list comprising pet food, animal milk replacer, veterinary product, veterinary feed supplement, nutrition supplement, post weaning feed, or creep feed.

[0292] As will be shown in the examples herein, the methods and the cell of the invention preferably provide at least one of the following further surprising advantages when using a sialyltransferase as described herein:

[0293] Higher titres of the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide (g / L),

[0294] Higher purity of the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide (g / L),

[0295] A purity of the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, (g / L) equal to or greater than 80%, preferably equal to or greater than 85%, more preferably equal to or greater than 90%, even more preferably equal to or greater than 91%, even more preferably equal to or greater than 92%, even more preferably equal to or greater than 93%, even more preferably equal to or greater than 94%, even more preferably equal to or greater than 95%, even more preferably equal to or greater than 96%, even more preferably equal to or greater than 97%, even more preferably equal to or greater than 98%, even more preferably equal to or greater than 99%,

[0296] Higher lactose conversion, leading to lower lactose concentration at end of fermentation (g / L lactose),

[0297] Lower sialic acid formation (g / L),

[0298] Higher production rate r (g sialylated oligosaccharide / L / h, preferably g 3'sialylated oligosaccharide / L / h),

[0299] Higher cell performance index CPI (g sialylated oligosaccharide / g X, preferably g 3'sialylated oligosaccharide / g X),

[0300] Higher specific productivity Qp (g sialylated oligosaccharide / g X / h, preferably g 3'sialylated oligosaccharide / g X / h),

[0301] Higher yield on the carbon source used Y (g sialylated oligosaccharide / g carbon source used, preferably g 3'sialylated oligosaccharide / g carbon source used),

[0302] Higher yield on sucrose Ys (g sialylated oligosaccharide / g sucrose, preferably g 3'sialylated oligosaccharide / g sucrose),

[0303] Higher uptake / conversion rate of the carbon source used Q. (g carbon source / g X / h),

[0304] Higher sucrose uptake / conversion rate Qs (g sucrose / g X / h),

[0305] Higher lactose conversion / consumption rate rs (g lactose / h),

[0306] Higher secretion, excretion or extracellular transport of the 3'sialylated oligosaccharide, and / or Higher growth speed of the production host, when compared to a method or a cell using an identical setup or enzymatic or genetic background but lacking the use of a sialyltransferase as described herein.

[0307] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described above and below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, purification steps are performed according to the manufacturer's specifications.

[0308] Further advantages follow from the specific embodiments and the examples. It goes without saying that the abovementioned features and the features which are still to be explained below can be used not only in the respectively specified combinations, but also in other combinations or on their own, without departing from the scope of the present invention.

[0309] Moreover, the present invention relates to the following specific embodiments:

[0310] 1. Method for the production of a 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide- containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N- acetylglucosamine, more preferably a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide, the method comprising: contacting a sialyltransferase with a mixture comprising a donor comprising a sialic acid residue, and an acceptor in a medium, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from the donor to the acceptor, thereby producing said 3'sialylated oligosaccharide, wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, chosen from the list consisting of an oligosaccharide or a disaccharide and wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity , and comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30, 31, 25, 18, 26 or 22, at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29.

[0311] 2. Method for the production of a 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide- containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N- acetylglucosamine , said method comprising the steps of: a) providing i. CMP-sialic acid, ii. an acceptor, wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, and iii. a sialyltransferase, wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity and comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22, at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 17, 10,

[0312] 14, 16, 28 or 29, b) contacting said sialyltransferase and CMP-sialic acid with said acceptor, under conditions where the sialyltransferase catalyses the transfer of a sialic acid residue from said CMP-sialic acid to the acceptor resulting in the production of said 3'sialylated oligosaccharide, c) preferably, separating said produced 3'sialylated oligosaccharide.

[0313] 3. Method according to any one of previous embodiments, the method comprising: contacting a cell extract comprising said sialyltransferase with a mixture comprising said donor comprising a sialic acid residue, and said acceptor, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from the donor to the acceptor, thereby producing said 3'sialylated oligosaccharide.

[0314] 4. Method according to any one of previous embodiments, wherein said 3'sialylated oligosaccharide is produced in a cell-free system.

[0315] 5. Method for the production of a 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide, said method comprising the steps of: i. providing a cell, preferably a single cell, expressing, preferably heterologously expressing, more preferably overexpressing, even more preferably heterologously overexpressing, a sialyltransferase wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity, and comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22, or at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29, ii. providing CMP-sialic acid, optionally said CMP-sialic acid is produced by said cell, and iii. providing an acceptor being a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, optionally said saccharide is produced by said cell, and iv. cultivating and / or incubating said cell under conditions permissive to express said sialyltransferase, optionally permissive to produce said CMP-sialic acid and / or said oligosaccharide or disaccharide, v. preferably, separating said 3'sialylated oligosaccharide, from said cultivation or incubation.

[0316] 6. Method according to embodiment 5, wherein said cell is a metabolically engineered cell, preferably wherein said cell is metabolically engineered for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.

[0317] 7. Method according to any one of previous embodiments, wherein said sialyltransferase comprises an amino acid sequence: that is at least at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27 , 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22, that is at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or 27, that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29, or

[0318] - as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

[0319] 8. Method according to any one of embodiment 5 to 7, wherein the cultivation medium contains at least one carbon source selected from the group consisting of glucose, fructose, sucrose, and glycerol.

[0320] 9. Method according to any one of previous embodiments, wherein the medium, cultivation medium or incubation medium contains at least one compound selected from the group consisting of lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GIcNAc), sialic acid and CMP-sialic acid.

[0321] 10. Method according to any one of previous embodiments, wherein said 3'sialylated oligosaccharide, is recovered from the medium, cultivation or incubation medium and / or from the cell. Method according to any one of previous embodiments, the method comprising: i) use of a medium, cultivation medium or incubation medium comprising at least one precursor and / or acceptor for the production of said 3'sialylated oligosaccharide, and / or ii) adding to the medium at least one precursor and / or acceptor feed for the production of said 3'sialylated oligosaccharide, preferably said precursor is chosen from the list comprising lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP- GIcNAc), sialic acid and CMP-sialic acid, preferably said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, more preferably a LacNAc comprising oligosaccharide, an LNB comprising oligosaccharide, even more preferably an LNT or an LNnT. Method according to any one of previous embodiments, the method comprising at least one of the following steps: i) use of a cultivation or incubation medium comprising at least one precursor and / or acceptor; ii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and / or acceptor feed wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the cultivation or incubation medium before the addition of said precursor and / or acceptor feed; iii) Adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and / or acceptor feed wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor and / or acceptor feed and wherein preferably, the pH of said precursor and / or acceptor feed is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor and / or acceptor feed is kept between 20°C and 80°C; iv) Adding at least one precursor and / or acceptor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor and / or acceptor feeding solution; v) Adding at least one precursor and / or acceptor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor and / or acceptor feeding solution and wherein the concentration of said precursor and / or acceptor feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably, 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably, 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of said precursor and / or acceptor feeding solution is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor and / or acceptor feeding solution is kept between 20°C and 80°C; said method resulting in said 3'sialylated oligosaccharide, , with a concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the cultivation or incubation, wherein preferably said precursor is chosen from the list comprising lactose, galactose, lacto-N- tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N- acetylglucosamine (UDP-GIcNAc), sialic acid and CMP-sialic acid, and wherein preferably said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide. Method according to any one of embodiments 1 to 11, the method comprising at least one of the following steps: i) use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter); ii) use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter); iii) adding to the cultivation or incubation medium in a reactor or incubator a precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than twofold, more preferably less than 2-fold of the volume of the cultivation or incubation medium before the addition of said precursor feed; iv) adding to the cultivation or incubation medium in a reactor or incubator an acceptor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mLto 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the cultivation or incubation medium before the addition of said acceptor feed; v) adding to the cultivation or incubation medium a precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the cultivation or incubation medium before the addition of said precursor feed and wherein preferably, the pH of said precursor feed is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor feed is kept between 20°C and 80°C; vi) adding to the cultivation or incubation medium an acceptor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the cultivation or incubation medium before the addition of said acceptor feed and wherein preferably, the pH of said acceptor feed is set between 2.0 and 10.0 and wherein preferably, the temperature of said acceptor feed is kept between 20°C and 80°C; vii) adding a precursor and / or acceptor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor and / or acceptor feeding solution; viii) adding a precursor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor feeding solution and wherein the concentration of said precursor feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably, 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably, 550 g / L, most preferably 600 g / L; and wherein preferably the pH of said precursor feeding solution is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor feeding solution is kept between 20°C and 80°C; ix) Adding an acceptor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of an acceptor feeding solution and wherein the concentration of said acceptor feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably, 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably, 550 g / L, most preferably 600 g / L; and wherein preferably the pH of said acceptor feeding solution is set between 2.0 and 10.0 and wherein preferably, the temperature of said acceptor feeding solution is kept between 20°C and 80°C; said method resulting in said 3'sialylated oligosaccharide, with a concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the cultivation or incubation, wherein preferably said precursor is chosen from the list comprising lactose, galactose, lacto-N- tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N- acetylglucosamine (UDP-GIcNAc), sialic acid and CMP-sialic acid, and wherein preferably said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide.

[0322] 14. Method according to any one of the previous embodiments, wherein said sialyltransferase has alpha- 2,3-sialyltransferase activity on the galactose (Gal) residue of said acceptor and / or wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably LacNAC, LNB, a LacNAc comprising oligosaccharide or an LNB comprising oligosaccharide, even more preferably an LNT or an LNnT.

[0323] 15. Method according to any one of previous embodiments, wherein said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, preferably is a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide, more preferably is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-Lewis x, preferably said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide is a milk oligosaccharide.

[0324] 16. A metabolically engineered cell for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said cell has been metabolically engineered to possess, to express or to overexpress, a sialyltransferase wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity and comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22, or at least 80.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29. Cell according to embodiment 16, wherein said sialyltransferase comprises an amino acid sequence: that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22, that is a at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or 27, that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29, or

[0325] - as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31. Cell according to any one of embodiment 16 or 17, wherein said cell contains a nucleic acid molecule which comprises a polynucleotide sequence that encodes said sialyltransferase. Cell according to any one of embodiments 16 to 18, wherein said cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell, preferably said bacterium is an Escherichia coli strain, more preferably an Escherichia coli strain which is a K-12 strain, even more preferably the Escherichia coli K-12 strain is E. coli MG1655, preferably said fungus belongs to a genus chosen from the group comprising Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus, preferably said yeast belongs to a genus chosen from the group comprising Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces or Debaromyces, preferably said plant cell is an algal cell or is derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant, preferably said animal cell is derived from non-human mammals, birds, fish, invertebrates, reptiles, amphibians or insects or is a genetically engineered cell line derived from human cells excluding embryonic stem cells, more preferably said human and non-human mammalian cell is an epithelial cell, an embryonic kidney cell, a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell, an NIH-3T3 cell, a non-mammary adult stem cell or derivatives thereof, more preferably said insect cell is derived from Spodoptera frugiperda, Bombyx mori, Mamestra brassicae, Trichoplusia ni or Drosophila melanogaster, preferably said protozoan cell is a Leishmania tarentolae cell. Cell according to any one of embodiments 16 to 19, wherein said cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably from the group consisting of yeast cells, bacterial cells, archaebacterial cells, algae cells, and fungal cells. Cell according to any one of embodiments 16 to 20, wherein said cell comprises a nucleic acid molecule comprising a polynucleotide sequence encoding said sialyltransferase and operably linked to control sequences recognized by the cell, wherein said sequence is foreign to the cell, said sequence further i) being integrated in the genome of said cell and / or ii) presented to said cell on a vector. Cell according to any one of embodiments 16 to 21, wherein said sialyltransferase has alpha-2, 3- sialyltransferase activity on the galactose (Gal) residue of said acceptor and / or wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably LacNAC, LNB, a LacNAc comprising oligosaccharide or an LNB comprising oligosaccharide, even more preferably an LNT or an LNnT. Cell according to any one of embodiments 16 to 22, wherein said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, preferably is a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide, more preferably is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-Lewis x.

[0326] 24. Cell according to any one of embodiments 16 to 23, wherein said cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and / or required for the synthesis of said 3'sialylated oligosaccharide.

[0327] 25. An isolated nucleic acid molecule encoding a sialyltransferase wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity on the galactose (Gal) residue of an acceptor, wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, and wherein said sialyltransferase comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22, at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29.

[0328] 26. An isolated nucleic acid molecule according to embodiment 25, wherein said sialyltransferase comprises an amino acid sequence: that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22, that is at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or 27, that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29, or

[0329] - as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31. J. A vector comprising the nucleic acid molecule of any one of embodiment 25 or 26. 8. Use of a sialyltransferase that has alpha-2, 3-sialyltransferase activity on the galactose (Gal) residue of an acceptor, wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, and wherein said sialyltransferase comprises an amino acid sequence: that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by EQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22, that is at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or 27, that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29, or

[0330] - as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31, for production of said 3'sialylated oligosaccharide.

[0331] 29. Use of a cell according to any one of embodiments 16 to 24 for production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sLacNAc comprising oligosaccharide or a 3'sLNB comprising oligosaccharide or a, most preferably chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-Lewis x.

[0332] 30. Use of a method according to any one of embodiments 1 to 15 for production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sLacNAc comprising oligosaccharide or a 3'sLNB comprising oligosaccharide, most preferably 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl- Lewis a, sialyl-Lewis x.

[0333] 31. Use of an isolated nucleic acid molecule according to any one of embodiment 25 or 26 for production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sLacNAc comprising oligosaccharide or a 3'sLNB comprising oligosaccharide, most preferably 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl- Lewis a, sialyl-Lewis x.

[0334] 32. Use of a vector according to embodiment 27 for production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sLacNAc comprising oligosaccharide or a 3'sLNB comprising oligosaccharide, most preferably 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-Lewis x.

[0335] 33. An alpha-2, 3-sialyltransferase for use in the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide wherein said sialyltransferase has alpha-2, 3- sialyltransferase activity on the galactose (Gal) residue of an acceptor, wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, and wherein said sialyltransferase comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22, at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29. An alpha-2, 3-sialyltransferase according to embodiment 33, wherein said sialyltransferase comprises an amino acid sequence: that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22, that is at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or 27, that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29, or - as ...

Claims

Claims1. Method for the production of a 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide- containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N- acetylglucosamine, more preferably a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide, the method comprising: contacting a sialyltransferase with a mixture comprising a donor comprising a sialic acid residue, and an acceptor, in a medium under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from the donor to the acceptor, thereby producing said 3'sialylated oligosaccharide, wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, chosen from the list consisting of an oligosaccharide or a disaccharide and wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity, and comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30, 31, 25, 18, 26 or 22, or at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29.

2. Method according to claim 1 wherein in said method the donor comprising a sialic acid residue is CMP-sialic acid, and said sialyltransferase and CMP-sialic acid are contacted with said acceptor under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from said CMP- sialic acid to the acceptor resulting in the production of said 3'sialylated oligosaccharide, preferably, said produced 3'sialylated oligosaccharide is separated from the medium.

3. Method according to any one of previous claims, the method comprising: contacting a cell extract comprising said sialyltransferase with a mixture comprising said donor comprising a sialic acid residue, and said acceptor, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from the donor to the acceptor, thereby producing said 3'sialylated oligosaccharide.

4. Method according to any one of previous claims, wherein said 3'sialylated oligosaccharide is produced in a cell-free system.

5. Method according to claim 1, wherein said method comprises the steps of: i. providing a cell, preferably a single cell, expressing, preferably heterologously expressing, more preferably overexpressing, even more preferably heterologously overexpressing, said sialyltransferase ii. providing the donor comprising a sialic acid residue, wherein said donor is CMP-sialic acid,optionally said CMP-sialic acid is produced by said cell, and iii. providing said acceptor, optionally said acceptor is produced by said cell, and iv. cultivating and / or incubating said cell under conditions permissive to express said sialyltransferase, optionally permissive to produce said CMP-sialic acid and / or said acceptor, v. preferably, separating said 3'sialylated oligosaccharide from said cultivation or incubation.

6. Method according to claim 5, wherein said cell is a metabolically engineered cell, preferably wherein said cell is metabolically engineered for the production of said 3'sialylated oligosaccharide.

7. Method according to any one of previous claims, wherein said sialyltransferase comprises an amino acid sequence: that is at least at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22, that is at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or 27, that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29, or- as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, TJ , 28, 29, 30 or 31.

8. Method according to any one of claim 5 to 7, wherein the cultivation medium contains at least one carbon source selected from the group consisting of glucose, fructose, sucrose, and glycerol.

9. Method according to any one of previous claims, wherein the medium contains at least one compound selected from the group consisting of lactose, galactose, lacto-N-tetraose, lacto-N- neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP- GIcNAc), sialic acid and CMP-sialic acid.

10. Method according to any one of previous claims, wherein said 3'sialylated oligosaccharide is recovered from the medium, cultivation or incubation medium and / or from the cell.

11. Method according to any one of previous claims, the method comprising: i) use of a medium, cultivation medium or incubation medium comprising at least one precursor and / or acceptor for the production of said 3'sialylated oligosaccharide, and / or ii) adding to the medium at least one precursor and / or acceptor feed for the production of said 3'sialylated oligosaccharide, preferably said precursor is chosen from the list comprising lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP- GIcNAc), sialic acid and CMP-sialic acid, preferably said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, more preferably a LacNAc comprising oligosaccharide, an LNB comprising oligosaccharide, even more preferably an LNT or an LNnT.

12. Method according to any one of previous claims, the method comprising at least one of the following steps: i) use of a cultivation or incubation medium comprising at least one precursor and / or acceptor; ii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and / or acceptor feed wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the cultivation or incubation medium before the addition of said precursor and / or acceptor feed; iii) Adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and / or acceptor feed wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m3(cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor and / or acceptor feed and wherein preferably, the pH of said precursor and / or acceptor feed is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor and / or acceptor feed is kept between 20°C and 80°C;iv) Adding at least one precursor and / or acceptor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor and / or acceptor feeding solution; v) Adding at least one precursor and / or acceptor feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor and / or acceptor feeding solution and wherein preferably the concentration of said precursor and / or acceptor feeding solution is 50 g / L, preferably 75 g / L, more preferably 100 g / L, more preferably 125 g / L, more preferably 150 g / L, more preferably 175 g / L, more preferably 200 g / L, more preferably 225 g / L, more preferably 250 g / L, more preferably 275 g / L, more preferably 300 g / L, more preferably 325 g / L, more preferably 350 g / L, more preferably 375 g / L, more preferably, 400 g / L, more preferably 450 g / L, more preferably 500 g / L, even more preferably, 550 g / L, most preferably 600 g / L; and wherein preferably, the pH of said precursor and / or acceptor feeding solution is set between 2.0 and 10.0 and wherein preferably, the temperature of said precursor and / or acceptor feeding solution is kept between 20°C and 80°C; said method resulting in said 3’sialylated oligosaccharide with a concentration of at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L in the final volume of the cultivation or incubation, wherein preferably said precursor is chosen from the list comprising lactose, galactose, lacto-N- tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N- acetylglucosamine (UDP-GIcNAc), sialic acid and CMP-sialic acid, and wherein preferably said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide.

13. Method according to any one of the previous claims, wherein said sialyltransferase has alpha-2, 3- sialyltransferase activity on the galactose (Gal) residue of said acceptor and / or wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably LacNAC, LNB, a LacNAc comprising oligosaccharide or an LNB comprising oligosaccharide, even more preferably an LNT or an LNnT.

14. Method according to any one of previous claims, wherein said 3'sialylated oligosaccharide is a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, preferably is a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide, more preferably is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-Lewis x, preferably said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide is a milk oligosaccharide.

15. A metabolically engineered cell for the production of a 3'sialylated oligosaccharide comprising at leastan N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said cell has been metabolically engineered to express or to overexpress a sialyltransferase wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity and comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22, or at least 80.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29.

16. Cell according to claim 15, wherein said sialyltransferase comprises an amino acid sequence: that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22, that is a at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or U, that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29, or- as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

17. Cell according to any one of claim 15 or 16, wherein said cell contains a nucleic acid molecule which comprises a polynucleotide sequence that encodes said sialyltransferase.

18. Cell according to any one of claims 15 to 17, wherein said cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably from the group consisting of yeast cells, bacterial cells, archaebacterial cells, algae cells, and fungal cells.

19. Cell according to any one of claims 15 to 18, wherein said cell comprises a nucleic acid molecule comprising a polynucleotide sequence encoding said sialyltransferase and operably linked to control sequences recognized by the cell, wherein said sequence is foreign to the cell, said sequence further i) being integrated in the genome of said cell and / or ii) presented to said cell on a vector.

20. Cell according to any one of claims 15 to 19, wherein said sialyltransferase has alpha-2, 3- sialyltransferase activity on the galactose (Gal) residue of said acceptor and / or wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably LacNAC, LNB, a LacNAc comprising oligosaccharide or an LNB comprising oligosaccharide, even more preferably an LNT or an LNnT.

21. Cell according to any one of claims 15 to 20, wherein said 3'sialylated oligosaccharide is a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, preferably is a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide, more preferably is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-Lewis x.

22. Cell according to any one of claims 15 to 21, wherein said cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and / or required for the synthesis of said 3'sialylated oligosaccharide.

23. An alpha-2, 3-sialyltransferase for use in the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide wherein said sialyltransferase has alpha-2, 3- sialyltransferase activity on the galactose (Gal) residue of an acceptor, wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, and wherein said sialyltransferase comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22,at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29.

24. An alpha-2, 3-sialyltransferase according to claim 23, wherein said sialyltransferase comprises an amino acid sequence: that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22, that is at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or 27, that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29, or- as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

25. An alpha-2, 3-sialyltransferase according to claim 23 or 24 wherein said 3'sialylated oligosaccharide is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-Lewis x.

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