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WO2025224348A1 - Production d'un mélange d'oligosaccharides de lait - Google Patents

Production d'un mélange d'oligosaccharides de lait

Info

Publication number
WO2025224348A1
WO2025224348A1 PCT/EP2025/061445 EP2025061445W WO2025224348A1 WO 2025224348 A1 WO2025224348 A1 WO 2025224348A1 EP 2025061445 W EP2025061445 W EP 2025061445W WO 2025224348 A1 WO2025224348 A1 WO 2025224348A1
Authority
WO
WIPO (PCT)
Prior art keywords
gal
lacto
sialylated
glcnac
neu5ac
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/061445
Other languages
English (en)
Inventor
Sofie AESAERT
Joeri Beauprez
Thomas DECOENE
Annelies VERCAUTEREN
Tom Verhaeghe
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Inbiose NV
Original Assignee
Inbiose NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/EP2024/061529 external-priority patent/WO2024223815A1/fr
Application filed by Inbiose NV filed Critical Inbiose NV
Publication of WO2025224348A1 publication Critical patent/WO2025224348A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01056—N-acetylneuraminate synthase (2.5.1.56)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07043—N-Acylneuraminate cytidylyltransferase (2.7.7.43)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01183—UDP-N-acetylglucosamine 2-epimerase (hydrolysing) (3.2.1.183)

Definitions

  • the present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation.
  • the present invention relates to methods for the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide as well as the purification of said milk oligosaccharide mixture.
  • the present invention also provides a cell for production of said milk oligosaccharide mixture and the use of said cell in a cultivation or incubation.
  • Non-sialylated milk oligosaccharides are oligosaccharides that have no sialic acid residue.
  • Non-sialylated milk oligosaccharides comprise both non-charged (neutral) milk oligosaccharides and negatively charged milk oligosaccharides.
  • Examples of non-sialylated milk oligosaccharides comprise fucosylated oligosaccharides comprising at least one fucose residue like e.g.
  • LNFP I lacto-N-neofucopentaose I
  • non-sialylated milk oligosaccharides comprise non-fucosylated oligosaccharides comprising a galactose (Gal) residue, an N-acetylglucosamine (GlcNAc) residue, an N-acetylgalactosamine (GalNAc) residue, an N-acetyllactosamine (LacNAc, Gal- ⁇ 1,4-GlcNAc) epitope and/or a lacto-N-biose (LNB, Gal- ⁇ 1,3-GlcNAc) epitope, like e.g.
  • lacto-N-triose II LN3
  • lacto-N-tetraose LNT
  • lacto-N-neotetraose LNnT
  • 6'-galactosyllactose 3'-galactosyllactose
  • lacto-N-hexaose lacto-N-neohexaose
  • para-lacto-N- hexaose para-lacto-N-neohexaose as important members.
  • Cell-based production of oligosaccharides and of oligosaccharide mixtures also makes use of glycosyltransferases and although preferred over chemo-enzymatic synthesis, cell-based methods also need tight control of spatiotemporal synthesis of nucleotide-sugar donors and/or availability of adequate levels of nucleotide-sugar donors in proximity of complementary glycosyltransferases.
  • this and other objects are achieved by providing methods and a cell for production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide as described herein.
  • the present invention also provides methods for the purification of said mixture.
  • the present invention provides a cell which is metabolically engineered as described herein.
  • This invention also provides a purified mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide by the above-referenced process.
  • the expression “capable of expressing” is preferably replaced with “expresses” and vice versa, i.e., “expresses” is preferably replaced with “capable of expressing”.
  • the verb "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.
  • the verb “to comprise” may be replaced by “to consist” or “to consist essentially of” and vice versa.
  • the verb “to consist” 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.
  • 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.
  • 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”.
  • 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”.
  • 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.
  • 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 triple- stranded regions, or a mixture of single- and double-stranded regions.
  • polynucleotide 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.
  • 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.
  • 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.
  • 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.
  • polynucleotide(s) also embraces short polynucleotides often referred to as oligonucleotide(s).
  • 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.
  • 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.
  • 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
  • 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.
  • polynucleotide encoding a polypeptide 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Recombinant or metabolically engineered or genetically engineered or transgenic 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).
  • 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.
  • 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.
  • 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.
  • exogenous 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.
  • exogenous refers to any polynucleotide, polypeptide or protein sequence which originates from outside the cell under study and not a natural part of the cell or which is not occurring at its natural location in the cell chromosome or plasmid.
  • 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.
  • 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.
  • 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.
  • a promoter operably linked to a gene to which it is not operably linked to in its natural state i.e.
  • heterologous promoter in the genome of a non- genetically engineered organism
  • 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 mixture of at least two milk oligosaccharides as described herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • RNA polymerase e.g., the bacterial sigma factors like s70, s54, or related s- factors and the yeast mitochondrial RNA polymerase specificity factor MTF1 that co-associate with the RNA polymerase core enzyme
  • transcription factors are CRP, LacI, ArcA, Cra, IclR in E. coli, or, Aft2p, Crz1p, Skn7 in Saccharomyces cerevisiae, or, DeoR, GntR, Fur in B. subtilis.
  • 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 MTF1 in yeasts. Constitutive expression offers a constant level of expression with no need for induction or repression.
  • regulated expression is defined as expression that is regulated by transcription factors other than the subunits of RNA polymerase (e.g. bacterial sigma factors) under certain growth conditions. Examples of such transcription factors are described above.
  • 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.
  • control sequences can be, but are not limited to, promoter sequences, ribosome binding sequences, Shine Dalgarno sequences, Kozak sequences, transcription terminator sequences.
  • control sequences that are suitable for prokaryotes 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.
  • 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.
  • "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.
  • wildtype refers to the commonly known genetic or phenotypical situation as it occurs
  • modified expression of a protein 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.
  • modified activity of a protein relates to a non-native activity of the protein in any phase of the production process of the desired mixture of at least two milk oligosaccharides as described herein.
  • 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.
  • non-native indicates that said mixture 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 mixture or to have a higher production of the mixture.
  • 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.
  • 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 non- limiting 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.
  • the term “non-mammary cell(s)” may generally include any mammalian cell of non- mammary lineage.
  • 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.
  • molecular biology and genome editing techniques can be engineered to eliminate, silence, or attenuate myriad genes simultaneously.
  • Variant(s) 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.
  • the present invention contemplates making functional variants by modifying the structure of an enzyme, like e.g. a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, N- acetylneuraminate synthase and/or N-acylneuraminate cytidylyltransferase, as used in the present invention.
  • Variants can be produced by amino acid substitution, deletion, addition, or combinations thereof.
  • a variant can be produced as a fusion protein comprising at least one portion of an enzyme, like e.g. a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminate synthase and/or N-acylneuraminate cytidylyltransferase, of present invention fused to at least one portion comprising a peptide tag.
  • Said peptide tag may be used to assist protein folding of said enzyme, assist post expression purification, protect the enzyme from the action of degradative enzymes, and/or assist the enzyme in passing through the cell membrane.
  • peptide tag comprises, e.g., a SUMO tag, an MBP tag, a His tag, a FLAG tag, a Strep-II tag, a Halo-tag, a NusA tag, thioredoxin, GST and/or a Fh8-tag.
  • the fusion protein may be designed to include at least one cleavable peptide linker so that the enzyme of interest can be subsequently recovered from the fusion protein.
  • the fusion protein may be designed to include a plurality of inclusion body tags, cleavable peptide linkers, and regions encoding the enzyme of interest.
  • “Fragment” 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.
  • 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.
  • 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.
  • a usable, functional characteristic e.g. activity
  • 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 45.0 %, 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 80.0
  • 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
  • “Fragment”, with respect to a polypeptide, refers to a subsequence of the polypeptide which 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.
  • 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 100 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.
  • a fragment of a polypeptide SEQ ID NO 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.
  • a fragment of a polypeptide SEQ ID NO 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 45.0 %, 50.0 %, 55.0 %, 60.0 %, 65.0 %, 70.0 %, 75.0 %, 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 %,
  • a fragment of a polypeptide SEQ ID NO 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
  • polypeptide SEQ ID NO SEQ ID NO
  • polypeptide UniProt ID 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.
  • conservative substitutions 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.
  • 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.
  • 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) e1002514). Homologs can be identified by analysis of nucleotide and polypeptide sequence alignments.
  • 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.
  • 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.
  • 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.
  • 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.org) (Nucleic Acids Res.2021, 49(D1), 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.
  • the sequence of a polypeptide is represented by a SEQ ID NO or an UniProt ID.
  • 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.
  • the content of each database is fixed at each release and is not to be 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.
  • 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.
  • sequence comparison one sequence acts as a reference sequence, to which test sequences are compared.
  • 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 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 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85%, 87.5 %, 90 %, 91 %, 92 %, 93 %, 94 % or 95 % of the full-length reference sequence.
  • 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.
  • a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence having 80 % 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 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 %
  • a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence having 80 % 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 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 %,
  • a polypeptide comprising, consisting of or consisting essentially of an amino acid sequence having 80 % 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 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
  • a polypeptide comprising, consisting of or having an amino acid sequence having 80 % 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 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
  • a polynucleotide sequence comprising, consisting of or having a nucleotide sequence having 80 % or more sequence identity to the full-length nucleotide sequence of a reference polynucleotide sequence, usually indicated with a SEQ ID NO, 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 85.0%, even more preferably has at least 87.50%, even more preferably has at least 90.0% sequence identity to the full-length reference polynucleotide sequence.
  • 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.
  • 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).
  • KDO 2-keto-3-deoxymanno-octulonic acid
  • free sialic acid refers to unbound sialic acid, i.e. a sialic acid residue that is not bound to another molecule or that is not present as part of a disaccharide, an oligosaccharide, a polysaccharide or a glycan.
  • sialic acid residue refers to a sialic acid molecule that is bound to another molecule or that is present as part of a disaccharide, an oligosaccharide, a polysaccharide or a glycan.
  • glycosyltransferase 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).
  • 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
  • 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.
  • monosaccharide refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed either an aldose or ketose, and contains one or more hydroxyl groups per molecule. Monosaccharides are saccharides containing only one simple sugar.
  • phosphorylated monosaccharide refers to a monosaccharide that is phosphorylated.
  • Examples of phosphorylated monosaccharides include but are not limited to glucose-1- phosphate, glucose-6-phosphate, glucose-1,6-bisphosphate, galactose-1-phosphate, fructose-6- phosphate, fructose-1,6-bisphosphate, fructose-1-phosphate, glucosamine-1-phosphate, glucosamine-6- phosphate, N-acetylglucosamine-1-phosphate, mannose-1-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.
  • activated monosaccharide refers to activated forms of monosaccharides.
  • activated monosaccharides include but are not limited to UDP-N- acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), 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
  • CMP-sialic acid refers to a nucleotide-activated form of sialic acid comprising but not limited to CMP-Neu5Ac, CMP-Neu4Ac, CMP-Neu5Ac9N 3 , CMP-Neu4,5Ac 2 , CMP-Neu5,7Ac 2 , CMP- Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) and CMP-KDO.
  • disaccharide refers to a saccharide polymer containing two simple sugars, i.e. monosaccharides.
  • examples of disaccharides comprise lactose (Gal- ⁇ 1,4-Glc), lacto-N-biose (Gal- ⁇ 1,3- GlcNAc), N-acetyllactosamine (Gal- ⁇ 1,4-GlcNAc), LacDiNAc (GalNAc- ⁇ 1,4-GlcNAc), N- acetylgalactosaminylglucose (GalNAc- ⁇ 1,4-Glc), Neu5Ac- ⁇ 2,3-Gal, Neu5Ac- ⁇ 2,6-Gal, fucopyranosyl- (1- 4)-N-glycolylneuraminic acid (Fuc-(1-4)-Neu5Gc), sucrose (Glc- ⁇ 1,2-Fru), maltose (Glc- ⁇ 1,4-G
  • Oleaccharide refers to a saccharide polymer containing a small number, typically three to twenty, preferably three to ten, of simple sugars, i.e., monosaccharides.
  • the oligosaccharide as used in the present invention can be a linear structure or can include branches.
  • the linkage e.g., glycosidic linkage, galactosidic linkage, glucosidic linkage, etc.
  • linkage between two sugar units can be expressed, for example, as 1,4, 1->4, or (1-4), used interchangeably herein.
  • Gal-b1,4-Glc For example, the terms “Gal-b1,4-Glc”, “Gal- ⁇ 1,4-Glc”, “b-Gal-(1->4)-Glc”, “ ⁇ -Gal- (1->4)-Glc”, “Galbeta1-4-Glc”, “Gal-b(1-4)-Glc” and “Gal- ⁇ (1-4)-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 1->2, alpha 1->3, alpha 1->4, alpha 1->6, alpha 2->1, alpha 2->3, alpha 2->4, alpha 2->6, beta 1->2, beta 1->3, beta 1->4, beta 1->6, beta 2->1, beta 2->3, beta 2->4, and beta 2->6.
  • An oligosaccharide can contain both alpha- and beta-glycosidic bonds or can contain only alpha- glycosidic or only beta-glycosidic bonds.
  • polysaccharide refers to a compound consisting of a large number, typically more than twenty, of monosaccharides linked glycosidically.
  • sialylated milk oligosaccharide is to be understood as a negatively charged sialic acid containing milk oligosaccharide, i.e., a milk oligosaccharide having a sialic acid residue as defined herein. It has an acidic nature.
  • sialylated milk 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.
  • Said sialylated milk oligosaccharide may further comprise a monosaccharide subunit selected from the list comprising, con fucose, galactose, glucose, GlcNAc, GalNAc, mannose, ManNAc, xylose, rhamnose, arabinose, fructose.
  • a sialylated milk 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.
  • a sialylated milk oligosaccharide can contain one or more Neu5Ac residues and one or more KDO residues. Said one or more sialic acid residues can be present in said sialylated milk oligosaccharide in an alpha-2,3 glycosidic linkage, in an alpha-2,6 glycosidic linkage and/or in an alpha-2,8 glycosidic linkage.
  • 3-SL (3 ⁇ -sialyllactose or 3’SL or Neu5Ac- ⁇ 2,3-Gal- ⁇ 1,4-Glc), 3'-sialyllactosamine, 6-SL (6’sialyllactose, 6 ⁇ -sialyllactose or 6’SL or Neu5Ac- ⁇ 2,6-Gal- ⁇ 1,4-Glc), 3,6-disialyllactose (Neu5Ac- ⁇ 2,3-(Neu5Ac- ⁇ 2,6)-Gal- ⁇ 1,4-Glc), 6,6’-disialyllactose (Neu5Ac- ⁇ 2,6-Gal- ⁇ 1,4-(Neu5Ac- ⁇ 2,6)-Glc), 8,3-disialyllactose (Neu5Ac- ⁇ 2,8-Neu5Ac- ⁇ 2,3-Gal- ⁇ 1,4- Glc), 6'-sialyllactosamine, oligo
  • a non-sialylated milk oligosaccharide as used herein refers to a milk oligosaccharide that has no sialic acid residue as defined herein.
  • a non-sialylated milk oligosaccharide can be a negatively charged milk oligosaccharide or can be a neutral milk oligosaccharide as described herein.
  • Examples of non-sialylated negatively charged milk oligosaccharides comprise sulphated milk oligosaccharides like e.g.
  • non-sialylated neutral milk oligosaccharide comprises neutral fucosylated milk oligosaccharides and neutral non-fucosylated milk oligosaccharides as described herein.
  • neutral oligosaccharide and ‘non-charged’ milk oligosaccharide as used herein are used interchangeably and refer, as generally understood in the state of the art, to a milk oligosaccharide that has no negative charge originating from a carboxylic acid group.
  • Examples of such neutral milk oligosaccharide are 2'-fucosyllactose (2’FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-triose II (LN3, GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc), lacto-N-tetraose (LNT, Gal ⁇ 1-3GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc), lacto-N-neotetraose (LNnT, Gal ⁇ 1-4GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc), lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaos
  • a ‘fucosylated milk oligosaccharide’ as used herein and as generally understood in the state of the art is a milk oligosaccharide that is carrying a fucose-residue.
  • Such fucosylated milk 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 fucose.
  • a fucosylated milk oligosaccharide can contain more than one fucose residue, e.g., two, three or more.
  • a fucosylated milk oligosaccharide can be a neutral milk oligosaccharide or can comprise one or more sialic acid residues. Fucose can be linked to other monosaccharide subunits comprising glucose, galactose, GlcNAc via alpha-glycosidic bonds comprising alpha-1,2 alpha-1,3, alpha-1,4, alpha-1,6 linkages.
  • Examples comprise 2'-fucosyllactose (2’FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL), Lacto-N- fucopentaose I (LNFP I), Gal-a1,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal-LNFP I), GalNAc-a1,3- (Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc-LNFP I), Lacto-N-fucopentaose II (LNFP II), Lacto- N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lac
  • neutral fucosylated milk oligosaccharides comprise 2'-fucosyllactose (2’FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL), Lacto-N-fucopentaose I (LNFP I), Gal-a1,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal-LNFP I), GalNAc-a1,3-(Fuc-a1,2-)Gal-b1,3- GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc-LNFP I), Lacto-N-fucopentaose II (LNFP II), Lacto-N-fucopentaose III (LNFP III), lacto-N-fu
  • a ‘neutral non-fucosylated milk oligosaccharide’ as used herein refers to a milk oligosaccharide that has no negative charge and that does not comprise a sialic acid residue nor a fucose residue.
  • Examples of neutral non-fucosylated milk oligosaccharides comprise lacto-N-triose II (LN3, GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc), lacto-N-tetraose (LNT, Gal ⁇ 1-3GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc), lacto-N-neotetraose (LNnT, Gal ⁇ 1-4GlcNAc ⁇ 1- 3Gal ⁇ 1-4Glc), 6'-galactosyllactose, 3'-galactosyllactose, GlcNAc-b1,6-(GlcNAc-b1,3-)Gal-b1,4-Glc, lacto-N- penta
  • a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide is used interchangeably and refer to a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide as described herein and at least one non-sialylated milk oligosaccharide as described herein.
  • Milk oligosaccharides or MOs 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 (i.e.
  • mammalian milk oligosaccharides or MMOs including but not limited to cows (Bos Taurus), sheep (Ovis aries), 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
  • MMOs refer to oligosaccharides such as but not limited to 3-fucosyllactose, 2 ⁇ -fucosyllactose, 6-fucosyllactose, 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, lac
  • HMOs refer to oligosaccharides such as but not limited to 3-fucosyllactose, 2 ⁇ -fucosyllactose, 6-fucosyllactose, 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
  • LNT II LNT-II
  • LN3 lacto-N-triose II
  • lacto-N-triose II lacto-N-triose
  • lacto-N-triose lacto-N-triose
  • GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc as used in the present invention
  • LNnT lacto-N-neotetraose
  • lacto-N-neotetraose lacto-N-neotetraose
  • Gal ⁇ 1-4GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc as used in the present invention, are used interchangeably.
  • LSTa LS-Tetrasaccharide a
  • Sialyl-lacto-N-tetraose a sialyllacto-N-tetraose a
  • Neu5Ac- ⁇ 2,3-Gal- ⁇ 1,3-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc as used in the present invention, are used interchangeably.
  • LSTb LS-Tetrasaccharide b
  • Sialyl-lacto-N-tetraose b Sialyllacto-N- tetraose b
  • Gal- ⁇ 1,3-(Neu5Ac- ⁇ 2,6)-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc as used in the present invention, are used interchangeably.
  • LSTc LS-Tetrasaccharide c
  • Sialyl-lacto-N-tetraose c sialyl-lacto-N-tetraose c
  • sialyllacto- N-tetraose c sialyllacto-N-neotetraose c
  • Neu5Ac- ⁇ 2,6-Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc as used in the present invention
  • LSTd LS-Tetrasaccharide d
  • Sialyl- lacto-N-tetraose d sialyl- lacto-N-tetraose d
  • sialyllacto-N-tetraose d sialyllacto-N-neotetraose d
  • Neu5Ac- ⁇ 2,3-Gal- ⁇ 1,4- GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc as used in the present invention
  • DSLNnT and “Disialyllacto-N-neotetraose” are used interchangeably and refer to Neu5Ac- ⁇ 2,6-Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-[Neu5Ac- ⁇ 2,6]-Gal- ⁇ 1,4-Glc.
  • DSLNT and “Disialyllacto-N- tetraose” are used interchangeably and refer to Neu5Ac- ⁇ 2,6-(Neu5Ac- ⁇ 2,3-Gal- ⁇ 1,3)-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc.
  • membrane transporter proteins refers to proteins that are part of or interact with the cell membrane and control the flow of molecules and information across the cell. The membrane proteins are thus involved in transport, be it import into or export out of the cell.
  • membrane transporter proteins can be but are not limited to porters, P-P-bond-hydrolysis-driven transporters, ⁇ - Barrel Porins, auxiliary transport proteins and phosphotransfer-driven group translocators (Forrest et al., Biochim. Biophys. Acta 1807 (2011) 167-188; Lengeler, J. Mol. Microbiol. Biotechnol. 25 (2015) 79-93; Moraes and Reithmeier, Biochim. Biophys.
  • pathway for production of at least one sialylated milk oligosaccharide is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of at least one sialylated milk oligosaccharide as defined herein.
  • Said pathway for production of at least one sialylated milk 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 at least one sialylated milk 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.
  • a ‘sialylation pathway’ is a biochemical pathway consisting of at least one of the enzymes and their respective genes selected from the list comprising, consisting of or consisting essentially of an L- glutamine—D-fructose-6-phosphate aminotransferase, a phosphoglucosamine mutase, an N- acetylglucosamine-6-P deacetylase, an N-acylglucosamine 2-epimerase, a hydrolyzing UDP-N- acetylglucosamine 2-epimerase, an N-acetylmannosamine-6-phosphate 2-epimerase, a UDP-GlcNAc 2- epimerase/kinase, a glucosamine 6-phosphate N-acetyltransferase, an N-acetylglucosamine-6-phosphate phosphatase, a phosphoacetylglucosamine mutase, an N-acetylgluco
  • nnaA and the neuC polypeptide are examples of hydrolyzing UDP-N- acetyl-D-glucosamine-2-epimerases.
  • N-acetylneuraminate synthase “N-acetylneuraminic acid synthase”, “N-acetylneuraminic acid synthetase”, “sialic acid synthase”, “sialic acid synthetase” and “neuB” are used interchangeably and refer to an enzyme that catalyses the conversion of N-acetylmannosamine (ManNAc) to N- acetylneuraminate (or sialic acid, Neu5Ac).
  • ManNAc N-acetylmannosamine
  • Neu5Ac sialic acid, Neu5Ac
  • N-acylneuraminate cytidylyltransferase CMP-N-acetylneuraminic acid synthase
  • CMP-NeuNAc synthase CMP-NeuNAc synthetase
  • CMP-sialic acid synthase CMP-sialic acid synthetase
  • neutralA a biochemical pathway that results in the cytoplasmic environment of the cell to be reducing, wherein said reducing environment negatively influences proper protein folding and blocks disulfide bond formation.
  • Examples of a reductive pathway comprise but are not limited to the reductive acetyl-CoA-pathway, the reductive pyrimidine catabolic pathway, the reductive citric acid cycle, the thiol-redox pathway and the reductive glycine pathway.
  • a reducing cytoplasm means e.g. that the NADP+:NADPH ratio in said cytoplasmic environment is low and/or that the glutathione (GSH) levels are high (e.g., 10 mM). Reducing environments may affect the oxidation state of a molecule, thereby altering its solubility.
  • glutathione reductase glutathione reductase (NADPH)
  • Glutathione S-reductase glutathione S-reductase
  • GSH reductase glutathione S-reductase
  • GSG reductase oxidized-glutathione oxidoreductase
  • NADPH-glutathione reductase glutathione reductase
  • NADPH-GSSG reductase glutathione disulfide + H+ + NADPH.
  • thioredoxin reductase thioredoxin-disulfide reductase
  • NADPH oxidized thioredoxin oxidoreductase
  • NADPH thioredoxin reductase
  • NADP thioredoxin reductase
  • trxB thioredoxin reductase (NADPH)
  • TRXR TRXR
  • disulfide bond isomerase protein disulfide-isomerase
  • S-S rearrangase S-S rearrangase
  • PDI PDI-disulfide bond isomerase
  • disulfide-isomerase protein disulfide-isomerase
  • S-S rearrangase S-S rearrangase
  • PDI PDI
  • disulfide bond isomerase protein disulfide-isomerase
  • PDI protein disulfide-isomerase
  • restrictionone refers to an enzyme that assist in protein folding. Examples are PDI, SecB, ERp57, heat shock proteins or Hsps, such as e.g., Hsp10, Hsp60, Hsp70, Hsp90.
  • pyruvate dehydrogenase pyruvate oxidase
  • POX pyruvate oxidase
  • poxB pyruvate:ubiquinone-8 oxidoreductase
  • lactate dehydrogenase D-lactate dehydrogenase
  • ldhA hslI
  • htpH htpH
  • D-LDH htpH
  • fermentative lactate dehydrogenase D-specific 2-hydroxyacid dehydrogenase
  • D-specific 2-hydroxyacid dehydrogenase D-specific 2-hydroxyacid dehydrogenase
  • lactate dehydrogenase means to introduce the activity of transport of a solute over the cytoplasm membrane and/or the cell wall. Said transport may be enabled by introducing and/or increasing the expression of a membrane transporter protein as described in the present invention.
  • enhanced efflux means to improve the activity of transport of a solute over the cytoplasm membrane and/or the cell wall. Transport of a solute over the cytoplasm membrane and/or cell wall may be enhanced by introducing and/or increasing the expression of a membrane transporter protein as described in the present invention. “Expression” of a membrane transporter protein is defined as “overexpression” of the gene encoding said membrane transporter protein in the case said gene is an endogenous gene or “expression” in the case the gene encoding said membrane transporter protein is a heterologous gene that is not present in the wild-type strain or cell.
  • purified refers to material that is substantially or essentially free from components that interfere with the activity of the biological molecule.
  • purified refers to material that is substantially or essentially free from components that normally accompany the material as found in its native state.
  • purified saccharides, oligosaccharides, proteins or nucleic acids of the invention are at least about 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 % or 85 % pure, usually at least about 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 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 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.
  • 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.
  • 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 mixture of at least two milk oligosaccharides as described herein.
  • 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 CeliteTM filter aid).
  • the term “cultivation” refers to the culture medium wherein the cell is cultivated, or fermented, the cell itself, and a mixture of at least two milk oligosaccharides as described herein that is produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell.
  • culture medium and “cultivation medium” as used herein are used interchangeably and refer to the medium wherein the cell is cultivated.
  • incubation refers to a mixture wherein a mixture of at least two milk oligosaccharides as described herein is produced.
  • Said incubation 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 a mixture of at least two milk oligosaccharides as described herein, catalysed by said one or more enzyme(s) using said one or more precursor(s) and said one or more acceptor(s) in said incubation.
  • Said incubation 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) a mixture of at least two milk oligosaccharides as described herein that is produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell.
  • Said incubation can also be the cultivation as defined herein.
  • the terms “reactor” and “incubator” refer to the recipient filled with the cultivation or incubation.
  • 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.
  • cell productivity index (CPI) refers to the mass of the sialylated milk oligosaccharide produced by the cells divided by the mass of the cells produced in the culture.
  • cell productivity index (CPI) refers to the mass of the non-sialylated milk oligosaccharide produced by the cells divided by the mass of the cells produced in the culture.
  • precursor refers to substances which are taken up or synthetized by the cell for the specific production of a sialylated milk oligosaccharide and/or a non-sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides according to the present invention.
  • a precursor can be an acceptor as defined herein, but can also be another substance, metabolite, which is first modified within the cell as part of the biochemical synthesis route of a sialylated milk oligosaccharide and/or a non-sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides.
  • 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 a sialylated milk oligosaccharide and/or a non- sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides.
  • 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 a sialylated milk oligosaccharide and/or a non-sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides.
  • Such precursors comprise the acceptors as defined herein, and/or a sialic acid residue, sialic acid, dihydroxyacetone, glucose, galactose, glucosamine, N-acetylglucosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyllactose, phosphorylated sugars or sugar phosphates like e.g.
  • glucose-1-phosphate galactose-1-phosphate, glucose-6- phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, mannose-6-phosphate, mannose-1- phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N- acetylglucosamine-1-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-Glc
  • UDP-galactose UDP-Gal
  • UDP-N-acetylglucosamine UDP-GlcNAc
  • UDP-N- acetylgalactosamine UDP-GalNAc
  • CMP-sialic acid CMP-Neu5Ac, GDP-mannose, GDP-4-dehydro-6- deoxy- ⁇ -D-mannose, GDP-fucose.
  • the cell is transformed to comprise and to express at least one nucleic acid sequence encoding a protein selected from the list 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 6’sialylated oligosaccharide of present invention.
  • the term “acceptor” as used herein refers to a mono-, di- or oligosaccharide, which can be modified by a glycosyltransferase.
  • acceptors comprise glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, lacto-N-triose (LN3), 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-hept
  • two important nucleotide-sugar donors in the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide are CMP-sialic acid, like e.g. CMP-Neu5Ac, and UDP- GlcNAc.
  • CMP-sialic acid is used in the production of the sialylated milk oligosaccharide, providing the at least one sialic acid residue that is present in the sialylated milk oligosaccharide.
  • UDP-GlcNAc can be used both in the production of the sialylated milk oligosaccharide and in the production of the non-sialylated milk oligosaccharide.
  • UDP-GlcNAc may, in the pathway towards the production of a sialylated milk oligosaccharide, act as a precursor in the synthesis of CMP-sialic acid: a hydrolyzing UDP-N-acetyl-D- glucosamine-2-epimerase first converts UDP-GlcNAc into N-acetylmannosamine (ManNAc), followed by synthesis of CMP-sialic acid out of ManNAc via consecutive action of an N-acetylneuraminate synthase and an N-acylneuraminate cytidylyltransferase enzyme.
  • ManNAc N-acetylmannosamine
  • UDP-GlcNAc may, in the pathway towards the production of a sialylated milk oligosaccharide, be directly used by an appropriate glycosyltransferase as a donor for addition of a GlcNAc residue in the growing oligosaccharide chain of a sialylated milk oligosaccharide.
  • UDP-GlcNAc can be used as nucleotide- sugar donor for appropriate glycosyltransferases in the pathway towards the production of a non- sialylated milk oligosaccharide, by providing a GlcNAc residue that can be added to a growing oligosaccharide chain of a non-sialylated milk oligosaccharide.
  • UDP-GlcNAc is a central molecule both in the production of the at least one sialylated milk oligosaccharide and the at least one non-sialylated milk oligosaccharide in the mixture of at least two milk oligosaccharides as described herein.
  • UDP-GlcNAc for production of CMP-sialic acid and/or as donor for a GlcNAc residue impacts the production and thus the concentration of the at least one sialylated milk oligosaccharide in said mixture of at least two milk oligosaccharides. Also, usage of UDP-GlcNAc impacts the production of the at least one non-sialylated oligosaccharide in said mixture.
  • UDP-GlcNAc in the pathway towards cellular CMP-sialic acid synthesis and to have more UDP-GlcNAc available as donor for GlcNAc of an appropriate glycosyltransferase, one could synthesize ManNAc via a pathway generating unconjugated (i.e. free) GlcNAc wherein said GlcNAc is converted into ManNAc via an N-acylglucosamine 2-epimerase.
  • synthesis of free GlcNAc in the context of a cellular system for production of a human milk oligosaccharide mixture is not preferred due to the unwanted side-production of unconjugated, i.e.
  • N-acetyllactosamine LacNAc, Gal- ⁇ 1,4- GlcNAc
  • LNB lacto-N-biose
  • LNB lacto-N-biose
  • Gal- ⁇ 1,3-GlcNAc lacto-N-biose
  • the use of an N-acylglucosamine 2-epimerase is not desired when designing a cellular production system for the production of a mixture of at least two human milk oligosaccharides comprising at least one sialylated human milk oligosaccharide and at least one non-sialylated human milk oligosaccharide.
  • Said side-production of LacNAc and/or LNB is less and/or not problematic when producing a mixture of at least two mammalian non-human milk oligosaccharides comprising at least one sialylated mammalian non-human milk oligosaccharide and at least one non- sialylated mammalian non-human milk oligosaccharide.
  • UDP-GlcNAc oligosaccharide Increasing the availability of the UDP-GlcNAc pool in the cell by enhancing UDP-GlcNAc synthesis is also not an appropriate solution, as this will not result in cellular production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide either, but will result in the production of a single milk oligosaccharide and not in a milk oligosaccharide mixture comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk oligosaccharide like is e.g. described in WO 2014/153253.
  • the present invention provides a cell metabolically engineered for the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide, said cell comprising: - a pathway for production of said at least one sialylated milk oligosaccharide, wherein said pathway comprises production of UDP-N-acetylglucosamine (UDP-GlcNAc), conversion of said UDP-GlcNAc into N-acetylmannosamine (ManNAc) by action of a hydrolyzing UDP-N-acetyl-D- glucosamine-2-epimerase, and conversion of said ManNAc into CMP-sialic acid by consecutive action of an N-acetylneuraminate synthase and an N-acylneuraminate cytidylyltransferase, and
  • the present invention provides a method for the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk oligosaccharide by a cell, the method comprising cultivating and/or incubating a cell as described herein in cultivation and/or incubation medium under conditions permissive to produce said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide.
  • the method further comprises separation and/or purification of said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide from said cultivation and/or incubation.
  • the cell of present invention comprises a pathway for production of the at least one sialylated milk oligosaccharide that is present in said mixture of at least two milk oligosaccharides as described herein.
  • said pathway for production of said at least one sialylated milk oligosaccharide is a sialylation pathway.
  • the cell is genetically engineered to comprise a sialylation pathway.
  • the cell comprises a sialylation pathway wherein said sialylation pathway has been genetically engineered.
  • the cell is genetically engineered for production of a sialic acid residue as described herein.
  • 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 the at least one sialylated milk oligosaccharide that is present in said mixture of at least two milk oligosaccharides as described herein preferably comprises at least one sialyltransferase.
  • Said cell may further comprise and express at least one further glycosyltransferase that is involved in the production of said at least one sialylated milk oligosaccharide.
  • the cell is genetically engineered to comprise a pathway for production of the at least one sialylated milk oligosaccharide that is present in said mixture of at least two milk oligosaccharides as described herein, and to have modified expression or activity of a sialyltransferase.
  • the cell as described herein comprises a sialylation pathway comprising at least one enzyme selected from the list comprising, consisting of or consisting essentially of L-glutamine—D-fructose-6- phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N- acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6- phosphate 2-epimerase, UDP-GlcNAc 2-epimerase/kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N- acetylglucosamine 1-phosphate uridylyltransferase, glucos
  • the cell of present invention comprises a pathway for production of the at least one sialylated milk oligosaccharide that is present in said mixture of at least two milk oligosaccharides as described herein, wherein said pathway comprises production of UDP-N- acetylglucosamine (UDP-GlcNAc), conversion of said UDP-GlcNAc into N-acetylmannosamine (ManNAc) by action of a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, and conversion of said ManNAc into CMP-sialic acid by consecutive action of an N-acetylneuraminate synthase and an N-acylneuraminate cytidylyltransferase.
  • UDP-N- acetylglucosamine UDP-N- acetylglucosamine
  • ManNAc N-acetylmannosamine
  • the cell used herein is optionally genetically engineered to express the de novo synthesis of UDP-GlcNAc.
  • UDP-GlcNAc can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing an UDP-GlcNAc can express enzymes converting, e.g. GlcNAc, which is to be added to the cell, to UDP-GlcNAc.
  • These enzymes may be any one or more of the list comprising, consisting of or consisting essentially of an N-acetyl-D-glucosamine kinase, an N- acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, and an N- acetylglucosamine-1-phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli.
  • the cell is modified to produce UDP- GlcNAc. More preferably, the cell is modified for enhanced UDP-GlcNAc production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of 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-1-phosphate acetyltransferase.
  • the cell of present invention comprises a pathway for production of the at least one non-sialylated milk oligosaccharide that is present in said mixture of at least two milk oligosaccharides as described herein.
  • said pathway for production of said at least one non-sialylated milk oligosaccharide is selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N- acetylmannosaminylation pathway.
  • the amount of the at least one sialylated milk oligosaccharide that is produced by the cell of present invention in said mixture of at least two milk oligosaccharides is determined by: - choice of the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminate synthase and/or N-acylneuraminate cytidylyltransferase, - swapping the native promoter of any one of the genes encoding the hydrolyzing UDP-N-acetyl- D-glucosamine-2-epimerase, N-acetylneuraminate synthase or N-acylneuraminate cytidylyltransferase with a promoter of interest, - swapping the native 5’untranslated region (5’UTR) of any one of the genes encoding the hydrolyzing UDP-N-acetyl-D-glucosamine
  • the amount of the at least one sialylated milk oligosaccharide that is produced by the cell of present invention in said mixture of at least two milk oligosaccharides is determined by the activity and/or expression levels of any one of the hydrolyzing UDP-N-acetyl-D- glucosamine-2-epimerase, N-acetylneuraminate synthase and/or N-acylneuraminate cytidylyltransferase that is/are expressed in said pathway for production of said at least one sialylated milk oligosaccharide in said cell, since said hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminate synthase and/or N-acylneuraminate cytidylyltransferase are involved in conversion of UDP-GlcNAc into CMP-sialic acid.
  • a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase is able to convert and/or converts UDP-GlcNAc into ManNAc; an N-acetylneuraminate synthase is able to convert and/or converts ManNAc into sialic acid; an N-acylneuraminate cytidylyltransferase is able to convert and/or converts sialic acid into CMP-sialic acid.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, N- acetylneuraminate synthase and/or N-acylneuraminate cytidylyltransferase thus orchestrate (1) the use of the UDP-GlcNAc pool that is available in the cell towards synthesis of CMP-sialic acid and (2) the production and/or availability of CMP-sialic acid to be used in the production of said at least one sialylated milk oligosaccharide.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminate synthase and/or N-acylneuraminate cytidylyltransferase also affects the UDP-GlcNAc pool that is available for compatible glycosyltransferases, like e.g. N-acetylglucosaminyltransferases, that transfer GlcNAc from said UDP-GlcNAc to a growing oligosaccharide chain of a sialylated milk oligosaccharide and/or a non- sialylated milk oligosaccharide.
  • compatible glycosyltransferases like e.g. N-acetylglucosaminyltransferases
  • the activity and/or expression levels of any one of hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminate synthase and N-acylneuraminate cytidylyltransferase affects production of said at least one sialylated milk oligosaccharide and possibly production of said at least one non-sialylated milk oligosaccharide.
  • the amount of the at least one sialylated milk oligosaccharide that is produced by the cell of present invention in said mixture of at least two milk oligosaccharides can be increased or decreased based on the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminate synthase and/or N- acylneuraminate cytidylyltransferase that is selected to be expressed in the cell wherein the selected hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminate synthase and/or N- acylneuraminate cytidylyltransferase can have a higher or lower enzymatic activity compared to the enzymatic activity of the native hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, N- acetylneuraminate
  • the cell of present invention may also have no native genes encoding a hydrolyzing UDP-N-acetyl- D-glucosamine-2-epimerase, N-acetylneuraminate synthase and/or N-acylneuraminate cytidylyltransferase and may be modified to express a recombinant gene encoding a hydrolyzing UDP-N- acetyl-D-glucosamine-2-epimerase, N-acetylneuraminate synthase and/or N-acylneuraminate cytidylyltransferase.
  • the amount of the at least one sialylated milk oligosaccharide that is produced by the cell of present invention in said mixture of at least two milk oligosaccharides can be increased or decreased by amending the expression levels of the genes encoding the selected hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminate synthase and/or N-acylneuraminate cytidylyltransferase.
  • the term “amending” as used herein is to be understood as “increasing” or “decreasing”.
  • Expression levels of a gene can be amended by swapping the native promoter of the gene with a promoter of interest, wherein said promoter of interest has a different, increased or decreased activity to control expression of the gene compared to the native promoter of said gene.
  • said promoter of interest is selected from the list consisting of SEQ ID NO 15, 16 and 17.
  • the expression levels of a gene can be amended by swapping the native 5’untranslated region (5’UTR) of the gene with a 5’UTR of interest, wherein said 5’UTR of interest has a different, increased or decreased activity to control expression of the gene compared to the native 5’UTR of said gene.
  • said 5’UTR of interest is selected from the list consisting of SEQ ID NO 18 and 19.
  • the expression levels of a gene can be amended by modifying the copy number of a gene.
  • the term “modifying” as used herein is to be understood as “increasing” or “decreasing”.
  • the expression levels of a gene can be amended by expressing the gene from a different locus on the chromosome compared to its native location on the chromosome.
  • the expression levels of a gene can be amended by introduction of a transcriptional unit of said gene on the chromosome of the cell and/or on a vector that is used to transform said cell.
  • the expression levels of a gene can be amended by the choice of vector that is used to express the gene from.
  • Types of vectors that can be used herein comprise but are not limited to high-copy plasmids, low-copy plasmids, episomal vectors, cosmids.
  • the cell expresses a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminate synthase and N- acylneuraminate cytidylyltransferase as described herein.
  • the cell is modified in the expression or activity of a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase, an N- acetylneuraminate synthase and/or N-acylneuraminate cytidylyltransferase as described herein.
  • said hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous hydrolyzing UDP-N-acetyl-D- glucosamine-2-epimerase is overexpressed; alternatively said hydrolyzing UDP-N-acetyl-D-glucosamine- 2-epimerase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase can have a modified expression in the cell which also expresses a heterologous hydrolyzing UDP-N-acetyl-D- glucosamine-2-epimerase.
  • said N-acetylneuraminate synthase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous N-acetylneuraminate synthase is overexpressed; alternatively said N-acetylneuraminate synthase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous N-acetylneuraminate synthase can have a modified expression in the cell which also expresses a heterologous N- acetylneuraminate synthase.
  • said N-acylneuraminate cytidylyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous N-acylneuraminate cytidylyltransferase is overexpressed; alternatively said N-acylneuraminate cytidylyltransferase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous N- acylneuraminate cytidylyltransferase can have a modified expression in the cell which also expresses a heterologous N-acylneuraminate cytidylyltransferase.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase has hydrolyzing UDP-N-acetyl-D-glucosamine-2- epimerase activity and comprises an amino acid sequence comprising a conserved motif [ACILM][GST]N[ST][ST]XXXX[DE][ACGILMST][ACDEGIMPQSTV] with SEQ ID NO 08, wherein X can be any amino acid residue.
  • the X present in said conserved motif indicates that any single amino acid is possible.
  • the amino acid can be one of the 20 common amino acids encoded in the genetic code of life (i.e.
  • a common amino acid like e.g. L-ornithine
  • each X is an amino acid that results from a new, independent selection made out of the list of possible amino acid residues.
  • each X present in the conserved motif with SEQ ID NO 08 refers to the same amino acid or to a different amino acid.
  • the hydrolyzing UDP-N-acetyl- D-glucosamine-2-epimerase has hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase activity and comprises, consists of or consists essentially of an amino acid sequence that is at least 80 % identical over a stretch of at least 150 amino acid residues to any one of the amino acid sequences as represented by SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 09.
  • the hydrolyzing UDP-N- acetyl-D-glucosamine-2-epimerase has hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase activity and comprises, consists of or consists essentially of an amino acid sequence that is at least 80 % identical over a stretch of at least 200 amino acid residues to any one of the amino acid sequences as represented by SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 09.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase has hydrolyzing UDP-N-acetyl-D-glucosamine-2- epimerase activity and comprises an amino acid sequence comprising an IPR domain selected from the list comprising IPR003331, IPR020004 and IPR029767 as defined by InterPro 90.0 as released on 4th August 2022.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase has hydrolyzing UDP-N-acetyl-D-glucosamine-2- epimerase activity and comprises an amino acid sequence comprising a PF02350 motif as defined by PFAM 32.0 as released in September 2018.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase has hydrolyzing UDP-N-acetyl-D-glucosamine-2- epimerase activity and comprises an amino acid sequence comprising a cd03786 motif as defined by CDD v3.17 as released in September 2016.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase has hydrolyzing UDP-N-acetyl-D-glucosamine-2- epimerase activity and comprises an amino acid sequence comprising a panther domain selected from the list comprising PTHR43174 and PTHR43174:SF3 as defined by PANTHER 17.0 as released on 23 rd February 2022.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase has hydrolyzing UDP-N-acetyl-D-glucosamine-2- epimerase activity and comprises an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, or at least 99 % identical to any one of the amino acid sequences as represented by SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 09 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.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase has hydrolyzing UDP-N-acetyl-D-glucosamine-2- epimerase activity and comprises an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, or at least 99 % identical to any one of the full-length amino acid sequences as represented by SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 09.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence as represented by any one of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 09 and comprising UDP-N-acetyl-D-glucosamine-2- epimerase activity.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase is a nnaA polypeptide or a neuC polypeptide.
  • the cell is modified with one or more expression modules.
  • 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 (UTR) including 5’UTR and 3’UTR sequences, 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).
  • the expression of each of said expression modules can be constitutive or is created by a natural or chemical inducer.
  • 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.
  • a certain natural condition of the host e.g. organism being in labour, or during lactation
  • an environmental change e.g. including but not limited to hormone, heat, cold, pH shifts, light, oxidative or osmotic stress / signalling
  • 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.
  • 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, toxin- antitoxin markers, RNA sense/antisense markers.
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • 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.
  • 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.
  • 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 at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides as described herein; or said recombinant gene is linked to other pathways in said cell that are not involved in the synthesis of at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides as described herein.
  • 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.
  • the expression of each of said expression modules present in said metabolically engineered cell is constitutive or tuneable as described herein.
  • the cell comprises one or more pathway(s) for monosaccharide synthesis. More preferably, the cell is genetically engineered to comprise 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).
  • enzymes like e.g. carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases
  • the cell comprises one or more pathway(s) for phosphorylated monosaccharide synthesis. More preferably, the cell is genetically engineered to comprise 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.
  • PEP phosphoenolpyruvate
  • the cell comprises one or more pathways for the synthesis of one or more nucleotide-activated sugars. More preferably, the cell is genetically engineered to comprise one or more pathway(s) 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
  • the cell expresses at least one enzyme selected from the list comprising, consisting of or consisting essentially of 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.
  • an enzyme selected from the list comprising, consisting of or consisting essentially of 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,
  • GlcNAc N-acetylglucosamine
  • Such cell producing GlcNAc can express a phosphatase converting GlcNAc-6-phosphate into GlcNAc, like any one or more of e.g. the E.
  • coli HAD-like phosphatase genes comprising, consisting of or consisting essentially of 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 YbiU, PsMupP from Pseudomonas putida, ScDOG1 from S.
  • the cell is modified to produce GlcNAc. More preferably, the cell is modified for enhanced GlcNAc production. Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knockout of a glucosamine-6-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.
  • the cell expresses at least one enzyme selected from the list comprising, consisting of or consisting essentially of 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.
  • 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.
  • 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 selected from the list comprising, consisting of or consisting essentially of knockout of a glucosamine-6-phosphate deaminase, an N-acetylglucosamine-6-phosphate deacetylase and over-expression of an L-glutamine—D- fructose-6-phosphate aminotransferase and/or a glucosamine 6-phosphate N-acetyltransferase.
  • the cell expresses at least one enzyme selected from the list comprising, consisting of or consisting essentially of a bifunctional UDP-GlcNAc 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.
  • UDP-GlcNAc is provided by an enzyme expressed in the cell or by the metabolism of the cell. Additionally, UDP-GlcNAc can be added to the cell. Such cell producing an UDP-GlcNAc can express enzymes converting, e.g. GlcNAc, which is to be added to the cell, to UDP-GlcNAc.
  • These enzymes may be an N-acetyl-D-glucosamine kinase, an N-acetylglucosamine-6- phosphate deacetylase, a phosphoglucosamine mutase, and an N-acetylglucosamine-1-phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli.
  • the cell is modified to produce UDP-GlcNAc. More preferably, the cell is modified for enhanced UDP-GlcNAc production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of 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-1-phosphate acetyltransferase.
  • the cell expresses at least one enzyme selected from the list comprising, consisting of or consisting essentially of a d-arabinose 5-phosphate isomerase, a KDO-8P synthase, a KDO 8-phosphate phosphatase, a CMP-KDO synthetase from different species like e.g. Escherichia coli, Pseudomonas aeruginosa, Agrobacterium sp. and a sialyltransferase.
  • the cell is capable to make CMP-KDO. More preferably, the cell is modified to produce CMP-KDO.
  • the cell is modified for enhanced CMP-KDO production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of over-expression of a d-arabinose 5-phosphate isomerase, a KDO- 8P synthase, a KDO 8-phosphate phosphatase and/or a CMP-KDO synthetase encoding gene.
  • the cell further comprises a pathway selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N- acetylmannosaminylation pathway.
  • the cell is genetically engineered to comprise at least one pathway selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N- acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N- acetylmannosaminylation pathway.
  • the cell comprises at least one pathway selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N- acetylmannosaminylation pathway wherein at least one of said pathway(s) has/have been genetically engineered.
  • the cell as described herein comprises a fucosylation pathway comprising at least one enzyme selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-1- phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-1-phosphate guanylyltransferase, fucosyltransferase.
  • a fucosylation pathway comprising at least one enzyme selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-1- phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kina
  • the cell as described herein comprises a galactosylation pathway comprising at least one enzyme selected from the list comprising, consisting of or consisting essentially of galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridylyltransferase, phosphoglucomutase, galactosyltransferase.
  • the cell as described herein comprises an N-acetylglucosaminylation pathway comprising at least one enzyme selected from the list comprising, consisting of or consisting essentially of L-glutamine—D-fructose-6-phosphate aminotransferase, N- acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-1- phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase, N- acetylglucosaminyltransferase.
  • the cell as described herein comprises an N-acetylgalactosaminylation pathway comprising at least one enzyme selected from the list comprising, consisting of or consisting essentially of L-glutamine—D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-1- phosphate acetyltransferase, bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase, UDP-N-acetylglucosamine 4-epimerase, UDP-glucose 4-epimerase, N-acetylgalactosamine kinase, UDP-N-acetylgalactosamine pyrophosphorylase and
  • the cell as described herein comprises an mannosylation pathway comprising at least one enzyme selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-1- phosphate guanylyltransferase and mannosyltransferase.
  • the cell as described herein comprises an N-acetylmannosaminylation pathway comprising at least one enzyme selected from the list comprising, consisting of or consisting essentially of L-glutamine—D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, glucosamine-1-phosphate acetyltransferase, bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1- phosphate acetyltransfer
  • the cell is capable to produce and/or produces N-acetylmannosamine (ManNAc).
  • ManNAc can be provided by an enzyme expressed in the cell or by the mechanism of the cell.
  • the cell expresses a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase that converts UDP-GlcNAc into ManNAc.
  • the cell producing ManNAc can further express an N- acylglucosamine 2-epimerase like is known e.g. from several species including Bacteroides ovatus, E.
  • the cell comprises a pathway for production of ManNAc.
  • the cell is modified for enhanced ManNAc production. Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knock-out of N-acetylmannosamine kinase, over-expression of N- acetylneuraminate lyase, overexpression of a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase.
  • the cell is capable to produce and/or produces N-acetylmannosamine-6-phosphate (ManNAc- 6-phosphate).
  • the cell comprises 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-GlcNAc 2-epimerase/kinase like is known e.g.
  • 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-GlcNAc 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.
  • the cell is modified for enhanced ManNAc-6-phosphate production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of over-expression 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-1-phosphate acetyltransferase.
  • the cell as described herein is capable to produce and/or produces any one or more nucleotide-activated sugars.
  • the cell as described herein comprises a pathway for the synthesis of one or more nucleotide- activated sugars.
  • the cell as described herein is genetically engineered for production of any one or more nucleotide-activated sugars.
  • the nucleotide-activated sugar is selected from the list comprising, consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N- acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), 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,
  • the cell is capable to synthesize at least the nucleotide-activated sugar CMP-Neu5Ac. In another more preferred embodiment, the cell is capable to synthesize at least the nucleotide-activated sugar CMP-KDO.
  • the cell uses at least one of the synthesized nucleotide-activated sugars in the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide as described herein.
  • 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.
  • 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 selected from the list comprising, consisting of or consisting essentially of 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.
  • the cell used herein is optionally genetically engineered to express the de novo synthesis of CMP-KDO.
  • CMP-KDO can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing CMP-KDO can express an enzyme converting, e.g., KDO to CMP-KDO.
  • This enzyme may be a CMP-KDO synthetase, like the 3-deoxy-manno-octulosonate cytidylyltransferase kdsB from several species including Escherichia coli, Arabidopsis thaliana, Pseudomonas aeruginosa, Xanthomonas campestris.
  • the cell is modified to produce CMP-KDO. More preferably, the cell is modified for enhanced CMP-KDO production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of over-expression of a d-arabinose 5-phosphate isomerase, a KDO-8P synthase, a KDO 8-phosphate phosphatase and/or a CMP-KDO synthetase encoding gene.
  • 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-1-phosphate guanylyltransferase, like Fkp from Bacteroides fragilis, or the combination of one separate fucose kinase together with one separate fucose-1-phosphate guanylyltransferase like they are known from several species including Homo sapiens, Sus scrofa and Rattus norvegicus.
  • the cell is modified to produce GDP-fucose.
  • the cell is modified for enhanced GDP-fucose production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knock-out of an UDP-glucose:undecaprenyl-phosphate glucose-1-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-1-phosphate guanylyltransferase encoding gene, over-expression of a phosphomannomutase encoding gene and over- expression of a mannose-6-phosphate isomerase encoding gene.
  • 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.
  • 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 selected from the list comprising, consisting of or consisting essentially of knock-out of a bifunctional 5’-nucleotidase/UDP-sugar hydrolase encoding gene, knock-out of a galactose-1-phosphate uridylyltransferase encoding gene and over-expression of a UDP-glucose-4- epimerase encoding gene.
  • the cell used herein is optionally genetically engineered to express the de novo synthesis of UDP-GalNAc.
  • UDP-GalNAc can be synthesized from UDP-GlcNAc 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 O6.
  • the cell is modified to produce UDP-GalNAc. More preferably, the cell is modified for enhanced UDP-GalNAc production.
  • 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-GlcNAc via an epimerization reaction performed by a UDP-GlcNAc 2-epimerase (like e.g. cap5P from Staphylococcus aureus, RffE from E. coli, Cps19fK from S. pneumoniae, and RfbC from S. enterica).
  • a UDP-GlcNAc 2-epimerase like e.g. cap5P from Staphylococcus aureus, RffE from E. coli, Cps19fK from S. pneumoniae, and RfbC from S. enterica.
  • the cell is modified to produce UDP-ManNAc. More preferably, the cell is modified for enhanced UDP-ManNAc production.
  • the cell as described herein possesses, preferably expresses, more preferably overexpresses one or more genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6- dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-1-phosphate guanylyltransferase, L-glutamine—D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N- acetylglucosamine 2-epimerase, N-acet
  • the cell as described herein possesses, preferably expresses, more preferably overexpresses, one or more glycosyltransferase(s) selected from the list comprising, consisting of or consisting essentially of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N- acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N- acetyltransferase(s) selected from the list comprising, consisting
  • the cell as described herein is genetically engineered to express and/or to over-express one or more glycosyltransferase(s) selected from the list comprising, consisting of or consisting essentially of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N- acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N- glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyl
  • the fucosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1,2-fucosyltransferase, alpha-1,3-fucosyltransferase, alpha-1,3/4- fucosyltransferase, alpha-1,4-fucosyltransferase and alpha-1,6-fucosyltransferase.
  • the sialyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase, and alpha-2,8- sialyltransferase.
  • the galactosyltransferase is selected from the list comprising, consisting of or consisting essentially of 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.
  • the glucosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-glucosyltransferase, beta-1,2-glucosyltransferase, beta-1,3- glucosyltransferase and beta-1,4-glucosyltransferase.
  • the mannosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1,2-mannosyltransferase, alpha-1,3-mannosyltransferase and alpha-1,6-mannosyltransferase.
  • the N-acetylglucosaminyltransferase is selected from the list comprising, consisting of or consisting essentially of galactoside beta-1,3-N- acetylglucosaminyltransferase and beta-1,6-N-acetylglucosaminyltransferase.
  • the N-acetylgalactosaminyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1,3-N-acetylgalactosaminyltransferase.
  • the cell is modified in the expression or activity of at least one of said glycosyltransferases.
  • 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.
  • the cell as described herein is capable to produce and/or produces phosphoenolpyruvate (PEP).
  • PEP phosphoenolpyruvate
  • the cell comprises a pathway for production of PEP.
  • the cell as described herein is modified for enhanced production and/or supply of PEP compared to a non-modified progenitor.
  • one or more PEP-dependent, sugar-transporting phosphotransferase system(s) is/are disrupted such as but not limited to: 1) the N-acetyl-D-glucosamine Npi-phosphotransferase (EC 2.7.1.193), which is for instance encoded by the nagE gene (or the cluster nagABCD) in E.
  • ManXYZ which encodes the Enzyme ll Man complex (mannose PTS permease, protein-Npi- phosphohistidine-D-mannose phosphotransferase) that imports exogenous hexoses (mannose, glucose, glucosamine, fructose, 2- deoxyglucose, mannosamine, N-acetylglucosamine, etc.) and releases the phosphate esters into the cell cytoplasm, 3) the glucose-specific PTS transporter (for instance encoded by PtsG/Crr) which takes up glucose and forms glucose-6-phosphate in the cytoplasm, 4) the sucrose-specific PTS transporter which takes up sucrose and forms sucrose-6-phosphate in the cytoplasm, 5) the fructose-specific PTS transporter (for instance encoded by the genes fruA and fruB and the kinase fruK which takes up fructose and forms in a first step fructose-1
  • the full PTS system is disrupted by disrupting the PtsIH/Crr gene cluster.
  • the cell is further modified to compensate for the deletion of a PTS system of a carbon source by the introduction and/or overexpression of the corresponding permease.
  • permeases or ABC transporters that comprise but are not limited to transporters that specifically import lactose such as e.g. the transporter encoded by the LacY gene from E. coli, sucrose such as e.g. the transporter encoded by the cscB gene from E. coli, glucose such as e.g.
  • the transporter encoded by the galP gene from E. coli fructose such as e.g. the transporter encoded by the fruI gene from Streptococcus mutans, or the Sorbitol/mannitol ABC transporter such as the transporter encoded by the cluster SmoEFGK of Rhodobacter sphaeroides, the trehalose/sucrose/maltose transporter such as the transporter encoded by the gene cluster ThuEFGK of Sinorhizobium meliloti and the N- acetylglucosamine/galactose/glucose transporter such as the transporter encoded by NagP of Shewanella oneidensis.
  • fructose such as e.g. the transporter encoded by the fruI gene from Streptococcus mutans
  • Sorbitol/mannitol ABC transporter such as the transporter encoded by the cluster SmoEFGK of Rhodobacter
  • Examples of combinations of PTS deletions with overexpression of alternative transporters are: 1) the deletion of the glucose PTS system, e.g. ptsG gene, combined with the introduction and/or overexpression of a glucose permease (e.g. galP of glcP), 2) the deletion of the fructose PTS system, e.g. one or more of the fruB, fruA, fruK genes, combined with the introduction and/or overexpression of fructose permease, e.g. fruI, 3) the deletion of the lactose PTS system, combined with the introduction and/or overexpression of lactose permease, e.g.
  • the deletion of the sucrose PTS system combined with the introduction and/or overexpression of a sucrose permease, e.g. cscB.
  • the cell is modified to compensate for the deletion of a PTS system of a carbon source by the introduction of carbohydrate kinases, such as glucokinase (EC 2.7.1.1, EC 2.7.1.2, EC 2.7.1.63), galactokinase (EC 2.7.1.6), and/or fructokinase (EC 2.7.1.3, EC 2.7.1.4).
  • carbohydrate kinases such as glucokinase (EC 2.7.1.1, EC 2.7.1.2, EC 2.7.1.63), galactokinase (EC 2.7.1.6), and/or fructokinase (EC 2.7.1.3, EC 2.7.1.4).
  • the cell is modified by the introduction of or modification in any one or more of the list comprising, consisting of or consisting essentially of phosphoenolpyruvate synthase activity (EC: 2.7.9.2 encoded for instance in E. coli by ppsA), phosphoenolpyruvate carboxykinase activity (EC 4.1.1.32 or EC 4.1.1.49 encoded for instance in Corynebacterium glutamicum by PCK or in E. coli by pckA, resp.), phosphoenolpyruvate carboxylase activity (EC 4.1.1.31 encoded for instance in E.
  • coli by ppc oxaloacetate decarboxylase activity
  • EC 4.1.1.112 encoded for instance in E. coli by eda oxaloacetate decarboxylase activity
  • EC 2.7.1.40 encoded for instance in E. coli by pykA and pykF pyruvate carboxylase activity
  • malate dehydrogenase activity EC 1.1.1.38 or EC 1.1.1.40 encoded for instance in E. coli by maeA or maeB, resp.
  • the cell is modified by a reduced activity of phosphoenolpyruvate carboxylase activity, and/or pyruvate kinase activity, preferably a deletion of the genes encoding for phosphoenolpyruvate carboxylase, the pyruvate carboxylase activity and/or pyruvate kinase.
  • the cell as described herein comprises a modification for reduced production of acetate compared to a non-modified progenitor.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of overexpression of an acetyl-coenzyme A synthetase, a full or partial knock-out or rendered less functional pyruvate dehydrogenase and a full or partial knock- out or rendered less functional lactate dehydrogenase.
  • the cell is modified in the expression or activity of at least one acetyl-coenzyme A synthetase like e.g. acs from E. coli, S. cerevisiae, H. sapiens, M. musculus.
  • said acetyl-coenzyme A synthetase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous acetyl-coenzyme A synthetase is overexpressed; alternatively, said acetyl-coenzyme A synthetase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous acetyl-coenzyme A synthetase can have a modified expression in the cell which also expresses a heterologous acetyl-coenzyme A synthetase.
  • 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.
  • 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.
  • the cell has a full knock-out in the poxB encoding gene resulting in a cell lacking pyruvate dehydrogenase activity.
  • 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.
  • 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.
  • the cell has a full knock-out in the ldhA encoding gene resulting in a cell lacking lactate dehydrogenase activity.
  • 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, consisting of or consisting essentially of 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-1- phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N- acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man,
  • one or more gene(s) involved in one or more reductive pathway(s) in the cell of present invention is/are rendered less functional compared to a non-modified progenitor or is/are knocked-out.
  • a reductive pathway comprise but are not limited to the reductive acetyl-CoA-pathway, the reductive pyrimidine catabolic pathway, the reductive citric acid cycle, the thiol-redox pathway and the reductive glycine pathway.
  • one or more gene(s) involved in one or more reductive pathway(s) is/are rendered less functional by insertion, deletion and/or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) selected from the list comprising, consisting of or consisting essentially of promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said one or more gene(s).
  • said one or more gene(s) may be selected from the list comprising, consisting of or consisting essentially of gene(s) encoding formate dehydrogenase, formate–tetrahydrofolate ligase, methenyltetrahydrofolate cyclohydrolase, glycine dehydrogenase/decarboxylating, glycine cleavage system protein, glutamate dehydrogenase, glycine reductase complex, CO2 reductase, folate synthetase, folate cyclohydrolase, folate dehydrogenase, folate reductase, methyltransferase, pyruvate synthase, phosphotransacetylase, acetate kinase, ATPase, acetyl-CoA carbonylase/synthase, methylenetetrahydrofolate reductase, methylenetetrahydrolate
  • said one or more genes involved in one or more reductive pathway(s) is/are selected from the list comprising, consisting of or consisting essentially of a glutathione reductase and a thioredoxin reductase.
  • the cell as described herein possesses, preferably expresses, more preferably overexpresses, at least one gene selected from the list comprising, consisting of or consisting essentially of genes encoding a disulfide bond isomerase, a thiol oxidase and a chaperone.
  • 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 production of said at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said milk oligosaccharide mixture as described herein.
  • the cell is capable to produce, preferably produces, said at least one sialylated milk oligosaccharide and/or said at least one non-sialylated milk oligosaccharide from one or more precursor(s).
  • said precursor is lactose.
  • the precursor is fed to the cell from the cultivation or incubation medium.
  • the cell is capable to produce, preferably produces, one or more precursor(s) for the synthesis of said at least one sialylated milk oligosaccharide and/or said at least one non-sialylated milk oligosaccharide of the milk oligosaccharide mixture as described herein. More preferably, the cell is capable to produce, preferably produces, all of said one or more precursor(s) for the synthesis of said at least one sialylated milk oligosaccharide and/or said at least one non-sialylated milk oligosaccharide of the milk oligosaccharide mixture as described herein.
  • the cell is genetically engineered for the production of at least one of said one or more precursor(s) for the synthesis of said at least one sialylated milk oligosaccharide and/or said at least one non-sialylated milk oligosaccharide of the milk oligosaccharide mixture as described herein. More preferably, the cell is genetically engineered for the production of all of said one or more precursor(s) for the synthesis of said at least one sialylated milk oligosaccharide and/or said at least one non-sialylated milk oligosaccharide of the milk oligosaccharide mixture as described herein.
  • At least one of said one or more precursor(s) is internalized in said cell via one or more membrane protein(s).
  • said one or more precursor(s) is selected from the list comprising, consisting of or consisting essentially of sialic acid, a sialic acid residue, 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; KDO, CMP-sialic acid, CMP-Neu5Ac, glucose, galactose, GlcNAc, GalNAc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc.
  • the cell as described herein is optionally genetically engineered to import a precursor and/or an acceptor in said cell, by the introduction and/or overexpression of a transporter able to import the respective precursor and/or acceptor in the cell.
  • a 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.
  • MFS major facilitator superfamily
  • ABS ATP-binding cassette
  • the cell as described herein is optionally genetically engineered to produce polyisoprenoid alcohols like e.g., phosphorylated dolichol that can act as lipid carrier.
  • the cell as described herein is an E. coli or yeast with a lactose permease positive phenotype.
  • said lactose permease is coded by the gene LacY or LAC12, respectively.
  • the cell as described herein is optionally genetically engineered to import lactose in the cell, by the introduction and/or overexpression of a lactose permease, like e.g., encoded by the LacY gene or the LAC12 gene.
  • the cell as described herein expresses a membrane protein that is a transporter protein involved in transport of compounds, like e.g., at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides as defined in present invention out of the cell.
  • a membrane protein that is a transporter protein involved in transport of compounds, like e.g., at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides as defined in present invention out of the cell.
  • a membrane protein that is a transporter protein involved in transport of compounds, like e.g., at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said mixture of at
  • the cell as described herein expresses a membrane transporter protein or a polypeptide having transport activity hereby transporting compounds across the outer membrane of the cell wall.
  • the cell as described herein expresses more than one membrane transporter protein or polypeptide having transport activity hereby transporting compounds across the outer membrane of the cell wall.
  • the cell is modified in the expression or activity of said membrane transporter protein or polypeptide having transport activity.
  • Said membrane transporter protein or polypeptide having transport activity is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous membrane transporter protein or polypeptide having transport activity is overexpressed; alternatively said membrane transporter protein or polypeptide having transport activity is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous membrane transporter protein or polypeptide having transport activity can have a modified expression in the cell which also expresses a heterologous membrane transporter protein or polypeptide having transport activity.
  • the membrane transporter protein or polypeptide having transport activity is selected from the list comprising, consisting of or consisting essentially of porters, P-P-bond- hydrolysis-driven transporters, ⁇ -barrel porins, auxiliary transport proteins and phosphotransfer-driven group translocators.
  • the porters comprise MFS transporters, sugar efflux transporters and siderophore exporters.
  • the P-P-bond-hydrolysis-driven transporters comprise ABC transporters and siderophore exporters.
  • the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides.
  • the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of one or more precursor(s) to be used in said production of at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides.
  • the membrane transporter protein or polypeptide having transport activity provides improved production of at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides.
  • the membrane transporter protein or polypeptide having transport activity provides enabled efflux of at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides. In an alternative and/or additional preferred embodiment, the membrane transporter protein or polypeptide having transport activity provides enhanced efflux of at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides.
  • the cell as described herein is transformed to comprise at least one nucleic acid sequence encoding a protein selected from the list comprising, consisting of or consisting essentially of a lactose transporter like e.g. the LacY or lac12 permease, a glucose transporter, a galactose transporter, a transporter for a nucleotide-activated sugar like for example a transporter for UDP-GlcNAc, a transporter protein involved in transport of at least one sialylated milk oligosaccharide and/or at least one non- sialylated milk oligosaccharide of said mixture of at least two milk oligosaccharides out of the cell.
  • a lactose transporter like e.g. the LacY or lac12 permease, a glucose transporter, a galactose transporter, a transporter for a nucleotide-activated sugar like for example a transporter for UDP-GlcNAc,
  • the cell as described herein expresses a membrane transporter protein belonging to the family of MFS transporters like e.g., an MdfA polypeptide of the multidrug transporter MdfA family from species comprising, consisting of or consisting essentially of E. coli (UniProt ID P0AEY8), Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), Citrobacter youngae (UniProt ID D4BC23) and Yokenella regensburgei (UniProt ID G9Z5F4).
  • a membrane transporter protein belonging to the family of MFS transporters like e.g., an MdfA polypeptide of the multidrug transporter MdfA family from species comprising, consisting of or consisting essentially of E. coli (UniProt ID P0AEY8), Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), Citro
  • the cell as described herein expresses a membrane transporter protein belonging to the family of sugar efflux transporters like e.g., a SetA polypeptide of the SetA family from species comprising, consisting of or consisting essentially of E. coli (UniProt ID P31675, sequence version 03 (11 Oct 2004)) and Citrobacter koseri (UniProt ID A0A078LM16).
  • the cell as described herein expresses a membrane transporter protein belonging to the family of siderophore exporters like e.g., the E. coli entS (UniProt ID P24077, sequence version 02 (01 Nov 1997)), the K.
  • the cell as described herein expresses a membrane transporter protein belonging to the family of ABC transporters like e.g., oppF from E. coli (UniProt ID P77737), lmrA from Lactococcus lactis subsp. lactis bv. diacetylactis (UniProt ID A0A1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4).
  • a membrane transporter protein belonging to the family of ABC transporters like e.g., oppF from E. coli (UniProt ID P77737), lmrA from Lactococcus lactis subsp. lactis bv. diacetylactis (UniProt ID A0A1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4).
  • the cell expresses more than one membrane transporter protein selected from the list comprising, consisting of or consisting essentially of a lactose transporter like e.g. the LacY or lac12 permease, a fucose transporter, a glucose transporter, a galactose transporter, a transporter for a nucleotide-activated sugar like for example a transporter for UDP-GlcNAc, UDP-Gal and/or GDP-Fuc, the MdfA protein from E.
  • a lactose transporter like e.g. the LacY or lac12 permease, a fucose transporter, a glucose transporter, a galactose transporter, a transporter for a nucleotide-activated sugar like for example a transporter for UDP-GlcNAc, UDP-Gal and/or GDP-Fuc, the MdfA protein from E.
  • a lactose transporter like e.
  • the cell is transformed to comprise at least one nucleic acid sequence encoding a membrane transporter protein selected from the list comprising, consisting of or consisting essentially of a siderophore exporter, a major facilitator superfamily (MFS) transporter, an ATP-binding cassette (ABC) transporter or a sugar efflux transporter.
  • a membrane transporter protein selected from the list comprising, consisting of or consisting essentially of a siderophore exporter, a major facilitator superfamily (MFS) transporter, an ATP-binding cassette (ABC) transporter or a sugar efflux transporter.
  • the at least one sialylated milk oligosaccharide present in said mixture of at least two milk oligosaccharides of present invention is a sialylated milk oligosaccharide having at least one sialic acid residue selected 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).
  • KDO 2-keto-3- deoxymanno-octulonic acid
  • the at least one sialylated milk oligosaccharide present in said mixture of at least two milk oligosaccharides of present invention is selected from the list comprising, consisting of or consisting essentially of a sialylated mammalian milk oligosaccharide (MMO), a sialylated human milk oligosaccharide (HMO), N- acetyllactosamine containing sialylated milk oligosaccharide, lacto-N-biose containing sialylated milk oligosaccharide, 3’sialyllactose (3’SL), 6’sialyllactose (6’SL), 3'-sialyllactosamine, 6’-sialyllactosamine, oligosaccharide comprising 6’-sialyllactosamine, oligosaccharide comprising 6’-sialyllactosamine, oligosaccharide
  • the at least one non-sialylated milk oligosaccharide present in said mixture of at least two milk oligosaccharides of present invention is selected from the list comprising, consisting of or consisting essentially of non-sialylated neutral milk oligosaccharide, non-sialylated neutral MMO, non-sialylated neutral HMO, non-sialylated negatively charged oligosaccharide, non-sialylated negatively charged MMO, non-sialylated negatively charged HMO, sulphated milk oligosaccharides, 2'-fucosyllactose (2’FL), 3- fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N- triose II (LN)
  • the mixture of at least two milk oligosaccharides of present invention comprises at least one sialylated milk oligosaccharide selected from the list comprising and 3’SL, 6’SL, LSTa, LSTb, LSTc, LSTd, DSLNT and DSLNnT and at least one non-sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N- fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N- difuco
  • the mixture of at least two milk oligosaccharides of present invention comprises (1) 3’SL and/or 6’SL and (2) at least one non- sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, monofucosyllacto-N-hexaose-III, difu
  • the mixture of at least two milk oligosaccharides of present invention comprises (1) 3’SL and/or LSTa and (2) at least one non- sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lac
  • said mixture further comprises DSLNT. In another more preferred embodiment, said mixture further comprises DSLNnT. In another more preferred embodiment, said mixture further comprises LSTc. In another more preferred embodiment, said mixture further comprises LSTb. In another more preferred embodiment, said mixture further comprises LSTd.
  • the mixture of at least two milk oligosaccharides of present invention comprises (1) 3’SL and/or LSTd and (2) at least one non- sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, monofucosyllacto-N-hexaose-III, difu
  • the mixture of at least two milk oligosaccharides of present invention comprises (1) LSTa and/or LSTd and (2) at least one non- sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difuco
  • the mixture of at least two milk oligosaccharides of present invention comprises (1) 6’SL and/or LSTb and (2) at least one non- sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V
  • said mixture further comprises 3’SL. In another more preferred embodiment, said mixture further comprises LSTa. In another more preferred embodiment, said mixture further comprises LSTc. In another more preferred embodiment, said mixture further comprises LSTd. In another more preferred embodiment, said mixture further comprises DSLNT. In another more preferred embodiment, said mixture further comprises DSLNnT.
  • the mixture of at least two milk oligosaccharides of present invention comprises (1) 6’SL and/or LSTc and (2) at least one non- sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, monofucosyllacto-N-hexaose-III, difu
  • said mixture further comprises 3’SL. In another more preferred embodiment, said mixture further comprises LSTa. In another more preferred embodiment, said mixture further comprises LSTb. In another more preferred embodiment, said mixture further comprises LSTd. In another more preferred embodiment, said mixture further comprises DSLNT. In another more preferred embodiment, said mixture further comprises DSLNnT.
  • the mixture of at least two milk oligosaccharides of present invention comprises 3’SL, 6’SL, LSTa, LSTc, LSTd, 2’FL, 3-FL, DiFL, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNnFP-I, LNDFH-I, LNDFH-II and LNnDFH.
  • the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNT, 3’SL and 6’SL.
  • the mixture of at least two milk oligosaccharides of present invention comprises twelve or more oligosaccharides selected from the list consisting of 3’SL, 6’SL, LSTa, LSTc, LSTd, 2’FL, 3-FL, DiFL, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP- VI, LNnFP-I, LNDFH-I, LNDFH-II and LNnDFH.
  • the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNT, 3’SL and 6’SL. In another preferred embodiment of the method and/or cell of present invention, the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNnT, 3’SL and 6’SL. In another preferred embodiment of the method and/or cell of present invention, the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNT, LNnT, 3’SL and 6’SL.
  • the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNT, 3’SL, 6’SL and sialic acid. In another preferred embodiment of the method and/or cell of present invention, the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNnT, 3’SL, 6’SL and sialic acid. In another preferred embodiment of the method and/or cell of present invention, the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNT, LNnT, 3’SL, 6’SL and sialic acid.
  • the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNT, 3’SL, 6’SL and l-fucose. In another preferred embodiment of the method and/or cell of present invention, the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNnT, 3’SL, 6’SL and l-fucose. In another preferred embodiment of the method and/or cell of present invention, the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNT, LNnT, 3’SL, 6’SL and l-fucose.
  • the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNT, 3’SL, 6’SL, sialic acid and l-fucose.
  • the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNnT, 3’SL, 6’SL, sialic acid and l- fucose.
  • the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNT, LNnT, 3’SL, 6’SL, sialic acid and l-fucose.
  • the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, DiFL, LNT, 3’SL and 6’SL.
  • the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, LNT, LNnT, 3’SL and 6’SL.
  • the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, DiFL, LNT, LNnT, 3’SL and 6’SL. In another preferred embodiment of the method and/or cell of present invention, the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3FL, DiFL, LNT, LNnT, 3’SL and 6’SL. In another preferred embodiment of the method and/or cell of present invention, the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, DiFL, LNT, LNnT, 3’SL and 6’SL.
  • the mixture of at least two milk oligosaccharides of present invention comprises 2’FL, 3-FL, LNT, LNnT, 3’SL and 6’SL.
  • the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide as described herein comprises two or more sialylated milk oligosaccharides.
  • the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide as described herein comprises less sialylated milk oligosaccharide than non-sialylated milk oligosaccharides.
  • the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide as described herein comprises less non-sialylated milk oligosaccharide than sialylated milk oligosaccharides.
  • the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide as described herein does not comprise free sialic acid, wherein said free sialic acid is selected 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).
  • KDO 2-keto-3-deoxymanno-octulonic acid
  • the mixture of at least two milk oligosaccharides of present invention further comprises free sialic acid selected 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).
  • free sialic acid selected from the list consisting of Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulonic
  • the mixture of at least two milk oligosaccharides of present invention further comprises a monosaccharide. In another and/or additional preferred embodiment of the method and/or cell of present invention, the mixture of at least two milk oligosaccharides of present invention further comprises a disaccharide. In another and/or additional preferred embodiment of the method and/or cell of present invention, the mixture of at least two milk oligosaccharides of present invention further comprises lactose.
  • the mixture of at least two milk oligosaccharides of present invention comprises at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide and sialic acid.
  • the mixture of at least two milk oligosaccharides of present invention comprises at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, lactose and sialic acid.
  • the mixture of at least two milk oligosaccharides of present invention comprises at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide and lactose.
  • the mixture of at least two milk oligosaccharides of present invention comprises at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, lactose and free sialic acid wherein said mixture comprises less than 10 % lactose and/or less than 5 % free sialic acid.
  • said mixture comprises less than 9 % lactose. In an even more preferred embodiment, said mixture comprises less than 8 % lactose. 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 % lactose. In an additional and/or alternative more preferred embodiment, said mixture comprises less than 5 % free 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 % free sialic acid.
  • the mixture of at least two milk oligosaccharides of present invention comprises at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide and l-fucose.
  • the mixture of at least two milk oligosaccharides of present invention comprises at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, lactose and l-fucose.
  • the mixture of at least two milk oligosaccharides of present invention comprises at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, free sialic acid and l-fucose.
  • the mixture of at least two milk oligosaccharides of present invention comprises at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, lactose, free sialic acid and l-fucose.
  • the cell is selected from the list consisting of prokaryotic cells and eukaryotic cells, optionally, said cell is selected from the list consisting of yeast cells, bacterial cells, archaebacterial cells, algae cells, fungal cells, plant cells, animal cells, insect cells, protozoan cells.
  • the cell 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.
  • 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.
  • 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 S. 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.
  • 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.
  • 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.
  • 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.
  • Both human and non-human mammalian cells are preferably selected from the list comprising, consisting of or consisting essentially of 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 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.
  • 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 like e.g., Drosophila S2 cells.
  • the latter protozoan cell preferably is a Leishmania tarentolae cell.
  • a cell to be stably cultured in a cultivation or incubation medium wherein said cultivation or incubation medium can be any type of growth medium comprising, consisting of or consisting essentially of minimal medium, complex medium or growth medium enriched in certain compounds like, for example, but not limited to, vitamins, trace elements, amino acids.
  • the 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.
  • main is meant the most important carbon source for the cell for the production of the oligosaccharide mixture 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 above-indicated carbon source.
  • 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.
  • a precursor as defined herein cannot be used as a carbon source for the production of the oligosaccharide mixture of present invention.
  • the cultivation or incubation medium contains at least one carbon source selected from the list comprising, consisting of or consisting essentially of a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract.
  • said carbon source is selected from the list comprising, consisting of or consisting essentially of glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose, maltooligosaccharides, 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.
  • the cultivation or incubation medium contains at least one compound selected from the list comprising, consisting of or consisting essentially of lactose, GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc (lacto-N-triose, LN3), Gal- ⁇ 1,3-GlcNAc- ⁇ 1,3- Gal- ⁇ 1,4-Glc (lacto-N-tetraose, LNT), Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc (lacto-N-neotetraose, LNnT), galactose, glucose, sialic acid, Neu5Ac, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, GlcNAc, GalNAc, UDP- GlcNAc, UDP-GalNAc, and UDP-galactose (UDP-Gal).
  • lactose GlcNAc- ⁇ 1,3
  • 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.
  • 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.
  • the conditions permissive to produce a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide as described herein comprise the use of a cultivation or incubation medium comprising at least one precursor and/or acceptor for the production of said at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide in said milk oligosaccharide mixture as described herein.
  • the cultivation or incubation medium contains at least one precursor and/or acceptor, wherein said precursor is selected from the list comprising, consisting of or consisting essentially of a monosaccharide like e.g. galactose, glucose, fucose, sialic acid, GlcNAc, GalNAc; a nucleotide-activated sugar like e.g. CMP-sialic acid, CMP- Neu5Ac, CMP-KDO, UDP-Gal, UDP-GlcNAc, GDP-fucose; a disaccharide like e.g. lactose; and an oligosaccharide like e.g.
  • a monosaccharide like e.g. galactose, glucose, fucose, sialic acid, GlcNAc, GalNAc
  • a nucleotide-activated sugar like e.g. CMP-sialic acid, CMP- Neu5Ac, C
  • lacto-N-triose LN3
  • lacto-N-tetraose LNT
  • lacto-N-neotetraose LNnT
  • acceptor is selected from the list comprising, consisting of or consisting essentially of a disaccharide like e.g. lactose; an oligosaccharide like e.g. LN3, LNT, LNnT.
  • said precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, a sialic acid residue, 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; Neu4,5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc; KDO, CMP-sialic acid, CMP-Neu5Ac, glucose, galactose, GlcNAc, GalNAc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc.
  • said acceptor is selected from the list comprising, consisting of or consisting essentially of lactose, LN3, LNT and LNnT.
  • the conditions permissive to produce said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide as described herein comprise the use of a cultivation or incubation medium and adding to said cultivation or incubation medium at least one precursor and/or acceptor feed for the production of said at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said milk oligosaccharide mixture.
  • the conditions permissive to produce said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide as described herein, 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 at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said milk oligosaccharide mixture, 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 at least one sialylated milk oligosaccharide and/or at least one non-sialylated milk oligosaccharide of said milk oligosaccharide mixture.
  • said precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, a sialic acid residue, 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; Neu4,5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc; KDO, CMP-sialic acid, CMP-Neu5Ac, glucose, galactose, GlcNAc, GalNAc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc.
  • said acceptor is selected from the list comprising, consisting of or consisting essentially of lactose, LN3, LNT and LNnT.
  • the cultivation or incubation is contained in a reactor or incubator, as defined herein.
  • the volume of said reactor or incubator ranges from microlitre ( ⁇ 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).
  • said precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, CMP-sialic acid, CMP-Neu5Ac, glucose, galactose, GlcNAc, GalNAc, UDP-GlcNAc, UDP-GalNAc and UDP-Gal.
  • said acceptor is selected from the list comprising, consisting of or consisting essentially of lactose, LN3, LNT and LNnT.
  • said precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, CMP-sialic acid, CMP-Neu5Ac, glucose, galactose, GlcNAc, GalNAc, UDP-GlcNAc, UDP-GalNAc and UDP- Gal.
  • said acceptor is selected from the list comprising, consisting of or consisting essentially of lactose, LN3, LNT and LNnT.
  • said precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, CMP-sialic acid, CMP-Neu5Ac, glucose, galactose, GlcNAc, GalNAc, UDP-GlcNAc, UDP-GalNAc and UDP- Gal.
  • said acceptor is selected from the list comprising, consisting of or consisting essentially of lactose, LN3, LNT and LNnT.
  • 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.
  • 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.
  • the cells are cultivated or incubated for at least about 60, 80, 100, or about 120 hours or in a continuous manner.
  • 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.
  • 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.
  • a carbon source preferably glucose or sucrose
  • the lactose is added already in the first phase of exponential growth together with the carbon-based substrate.
  • the methods of present invention result in the production of 0.1 g/L or more, preferably 0.5 g/L or more, more preferably 1 g/L or more, more preferably 5 g/L or more, even more preferably 10 g/L or more, even more preferably 20 g/L or more, even more preferably 30 g/L or more, even preferably 40 g/L or more, most preferably 50 g/L or more of total milk oligosaccharides.
  • the cell of present invention produces 0.1 g/L or more, preferably 0.5 g/L or more, more preferably 1 g/L or more, more preferably 5 g/L or more, even more preferably 10 g/L or more, even more preferably 20 g/L or more, even more preferably 30 g/L or more, even preferably 40 g/L or more, most preferably 50 g/L or more of total milk oligosaccharides.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide and free sialic acid.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, lactose and free sialic acid.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide and lactose.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, lactose and free sialic acid wherein said mixture comprises less than 10 % lactose and/or less than 5 % free sialic acid.
  • said mixture comprises less than 9 % lactose. In an even more preferred embodiment, said mixture comprises less than 8 % lactose. 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 % lactose. In an additional and/or alternative more preferred embodiment, said mixture comprises less than 5 % of free sialic acid.
  • said mixture comprises less than 4 %, less than 3 %, less than 2 %, less than 1 %, less than 0.5 %, less than less than 0.2 %, and/or less than 0.1 % of free sialic acid, wherein free said sialic acid is selected 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).
  • free said sialic acid is selected from the list consisting of Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu
  • said mixture does not comprise free sialic acid as defined herein.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides of present invention comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide and l-fucose.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides of present invention comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, lactose and l-fucose.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides of present invention comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, free sialic acid and l-fucose.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides of present invention comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, lactose, free sialic acid and l-fucose.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide selected from the list comprising and 3’SL, 6’SL, LSTa, LSTb, LSTc, LSTd, DSLNT and DSLNnT and at least one non-sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexao
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides comprising (1) 3’SL and/or 6’SL and (2) at least one non-sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, monofucosyllacto-N-hexaose-III, difu
  • said mixture further comprises LSTa. In another more preferred embodiment, said mixture further comprises LSTb. In another more preferred embodiment, said mixture further comprises LSTc. In another more preferred embodiment, said mixture further comprises LSTd.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides comprising (1) 3’SL and/or LSTa and (2) at least one non-sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lac
  • said mixture further comprises DSLNT. In another more preferred embodiment, said mixture further comprises DSLNnT. In another more preferred embodiment, said mixture further comprises LSTc. In another more preferred embodiment, said mixture further comprises LSTb. In another more preferred embodiment, said mixture further comprises LSTd.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides comprising (1) 3’SL and/or LSTd and (2) at least one non-sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, monofucosyllacto-N-hexaose-III, difu
  • said mixture further comprises DSLNT.
  • said mixture further comprises DSLNnT.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides comprising (1) LSTa and/or LSTd and (2) at least one non-sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difuco
  • said mixture further comprises DSLNT. In another more preferred embodiment, said mixture further comprises DSLNnT. In another more preferred embodiment, said mixture further comprises LSTb. In another more preferred embodiment, said mixture further comprises LSTc.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides comprising (1) 6’SL and/or LSTb and (2) at least one non-sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V
  • said mixture further comprises 3’SL. In another more preferred embodiment, said mixture further comprises LSTa. In another more preferred embodiment, said mixture further comprises LSTc. In another more preferred embodiment, said mixture further comprises LSTd. In another more preferred embodiment, said mixture further comprises DSLNT. In another more preferred embodiment, said mixture further comprises DSLNnT.
  • the methods of present invention result in the production of a mixture of at least two milk oligosaccharides comprising (1) 6’SL and/or LSTc and (2) at least one non-sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, monofucosyllacto-N-hexaose-III, difu
  • said mixture further comprises 3’SL. In another more preferred embodiment, said mixture further comprises LSTa. In another more preferred embodiment, said mixture further comprises LSTb. In another more preferred embodiment, said mixture further comprises LSTd. In another more preferred embodiment, said mixture further comprises DSLNT. In another more preferred embodiment, said mixture further comprises DSLNnT.
  • the methods of present invention result in the production of an oligosaccharide mixture comprising 3’SL, 6’SL, LSTa, LSTc, LSTd, 2’FL, 3-FL, DiFL, LNT, LNnT, LNFP-I, LNFP- II, LNFP-III, LNFP-V, LNFP-VI, LNnFP-I, LNDFH-I, LNDFH-II and LNnDFH.
  • the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, 3’SL and 6’SL.
  • the methods of present invention result in the production of an oligosaccharide mixture comprising twelve or more oligosaccharides selected from the list consisting of 3’SL, 6’SL, LSTa, LSTc, LSTd, 2’FL, 3-FL, DiFL, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNnFP-I, LNDFH-I, LNDFH-II and LNnDFH.
  • the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, 3’SL and 6’SL.
  • the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3- FL, LNnT, 3’SL and 6’SL. In another preferred embodiment, the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, LNnT, 3’SL and 6’SL. In another preferred embodiment, the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, 3’SL, 6’SL and sialic acid.
  • the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3- FL, LNnT, 3’SL, 6’SL and sialic acid. In another preferred embodiment, the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, LNnT, 3’SL, 6’SL and sialic acid. In another preferred embodiment, the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, 3’SL, 6’SL and l-fucose.
  • the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3-FL, LNnT, 3’SL, 6’SL and l-fucose. In another preferred embodiment, the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, LNnT, 3’SL, 6’SL and l-fucose. In another preferred embodiment, the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, 3’SL, 6’SL, sialic acid and l-fucose.
  • the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3-FL, LNnT, 3’SL, 6’SL, sialic acid and l-fucose. In another preferred embodiment, the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, LNnT, 3’SL, 6’SL, sialic acid and l-fucose. In another preferred embodiment, the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, DiFL, LNT, 3’SL and 6’SL.
  • the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, LNT, LNnT, 3’SL and 6’SL. In another preferred embodiment, the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, DiFL, LNT, LNnT, 3’SL and 6’SL. In another preferred embodiment, the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3FL, DiFL, LNT, LNnT, 3’SL and 6’SL.
  • the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, DiFL, LNT, LNnT, 3’SL and 6’SL. In another preferred embodiment, the methods of present invention result in the production of an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, LNnT, 3’SL and 6’SL.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide and sialic acid.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide, at least one non- sialylated milk oligosaccharide, lactose and sialic acid.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide and lactose.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, lactose and sialic acid wherein said mixture comprises less than 10 % lactose and/or less than 5 % free sialic acid. In a more preferred embodiment, said mixture comprises less than 9 % lactose. In an even more preferred embodiment, said mixture comprises less than 8 % lactose.
  • said mixture comprises less than 7 %, less than 6 %, less than 5 %, less than 4 %, less than 3 %, less than 2 %, less than 1 % lactose. In an additional and/or alternative more preferred embodiment, said mixture comprises less than 5 % free 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 % free sialic acid.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides of present invention comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide and l-fucose.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides of present invention comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, lactose and l-fucose.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides of present invention comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, free sialic acid and l-fucose.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides of present invention comprising at least one sialylated milk oligosaccharide, at least one non-sialylated milk oligosaccharide, lactose, free sialic acid and l-fucose.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide selected from the list comprising and 3’SL, 6’SL, LSTa, LSTb, LSTc, LSTd, DSLNT and DSLNnT and at least one non-sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difu
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides comprising (1) 3’SL and/or 6’SL and (2) at least one non- sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, monofucosyllacto-N-hexaose-III, d
  • said mixture further comprises LSTa. In another more preferred embodiment, said mixture further comprises LSTb. In another more preferred embodiment, said mixture further comprises LSTc. In another more preferred embodiment, said mixture further comprises LSTd.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides comprising (1) 3’SL and/or LSTa and (2) at least one non- sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V
  • said mixture further comprises DSLNT. In another more preferred embodiment, said mixture further comprises DSLNnT. In another more preferred embodiment, said mixture further comprises LSTc. In another more preferred embodiment, said mixture further comprises LSTb. In another more preferred embodiment, said mixture further comprises LSTd.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides comprising (1) 3’SL and/or LSTd and (2) at least one non- sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, monofucosyllacto-N-hexaose-III, d
  • said mixture further comprises DSLNT.
  • said mixture further comprises DSLNnT.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides comprising (1) LSTa and/or LSTd and (2) at least one non- sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- dif
  • said mixture further comprises DSLNT. In another more preferred embodiment, said mixture further comprises DSLNnT. In another more preferred embodiment, said mixture further comprises LSTb. In another more preferred embodiment, said mixture further comprises LSTc.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides comprising (1) 6’SL and/or LSTb and (2) at least one non- sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaos
  • said mixture further comprises 3’SL. In another more preferred embodiment, said mixture further comprises LSTa. In another more preferred embodiment, said mixture further comprises LSTc. In another more preferred embodiment, said mixture further comprises LSTd. In another more preferred embodiment, said mixture further comprises DSLNT. In another more preferred embodiment, said mixture further comprises DSLNnT.
  • the cell of present invention is capable of producing and/or produces a mixture of at least two milk oligosaccharides comprising (1) 6’SL and/or LSTc and (2) at least one non- sialylated milk oligosaccharide selected from the list comprising 2’FL, 3-FL, DiFL, LN3, LNT, LNnT, lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, monofucosyllacto-N-hexaose-III, d
  • said mixture further comprises 3’SL. In another more preferred embodiment, said mixture further comprises LSTa. In another more preferred embodiment, said mixture further comprises LSTb. In another more preferred embodiment, said mixture further comprises LSTd. In another more preferred embodiment, said mixture further comprises DSLNT. In another more preferred embodiment, said mixture further comprises DSLNnT.
  • the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 3’SL, 6’SL, LSTa, LSTc, LSTd, 2’FL, 3-FL, DiFL, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNnFP-I, LNDFH-I, LNDFH-II and LNnDFH.
  • the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, 3’SL and 6’SL.
  • the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising twelve or more oligosaccharides selected from the list consisting of 3’SL, 6’SL, LSTa, LSTc, LSTd, 2’FL, 3-FL, DiFL, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNnFP-I, LNDFH-I, LNDFH-II and LNnDFH.
  • the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, 3’SL and 6’SL.
  • the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNnT, 3’SL and 6’SL. In another preferred embodiment, the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, LNnT, 3’SL and 6’SL. In another preferred embodiment, the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, 3’SL, 6’SL and sialic acid.
  • the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNnT, 3’SL, 6’SL and sialic acid. In another preferred embodiment, the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, LNnT, 3’SL, 6’SL and sialic acid. In another preferred embodiment, the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, 3’SL, 6’SL and l-fucose.
  • the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNnT, 3’SL, 6’SL and l-fucose. In another preferred embodiment, the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, LNnT, 3’SL, 6’SL and l-fucose. In another preferred embodiment, the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, 3’SL, 6’SL, sialic acid and l-fucose.
  • the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNnT, 3’SL, 6’SL, sialic acid and l-fucose. In another preferred embodiment, the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, LNnT, 3’SL, 6’SL, sialic acid and l-fucose. In another preferred embodiment, the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, DiFL, LNT, 3’SL and 6’SL.
  • the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, LNT, LNnT, 3’SL and 6’SL. In another preferred embodiment, the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, DiFL, LNT, LNnT, 3’SL and 6’SL. In another preferred embodiment, the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3FL, DiFL, LNT, LNnT, 3’SL and 6’SL.
  • the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, DiFL, LNT, LNnT, 3’SL and 6’SL. In another preferred embodiment, the cell of present invention is capable of producing and/or produces an oligosaccharide mixture comprising 2’FL, 3-FL, LNT, LNnT, 3’SL and 6’SL.
  • the methods as described herein preferably comprises a step of separating the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide of present invention from the cultivation or incubation, thereby recovering said milk oligosaccharide mixture from the cultivation or incubation medium and/or the cell.
  • separating from said cultivation or incubation means harvesting, collecting, or retrieving said milk oligosaccharide mixture from the cell and/or the medium of its cultivation or incubation.
  • the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide can be separated in a conventional manner from the aqueous culture medium, in which the cell was cultivated or incubated.
  • milk oligosaccharide mixture is still present in the cells producing the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide
  • conventional manners to free or to extract said milk oligosaccharide mixture 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 mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide.
  • This preferably involves clarifying said milk oligosaccharide mixture to remove suspended particulates and contaminants, particularly cells, cell components, insoluble metabolites and debris produced by culturing or incubating the genetically engineered cell.
  • said milk oligosaccharide mixture can be clarified in a conventional manner.
  • said milk oligosaccharide mixture is clarified by centrifugation, flocculation, decantation and/or filtration.
  • Another step of separating said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide 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 milk oligosaccharide mixture, preferably after it has been clarified.
  • remaining proteins and related impurities can be removed from said milk oligosaccharide mixture in a conventional manner.
  • remaining proteins, salts, by-products, colour, endotoxins and other related impurities are removed from said milk oligosaccharide mixture 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.
  • the methods as described herein also provide for a further purification of the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide of present invention.
  • a further purification of said milk oligosaccharide mixture may be accomplished, for example, by use of (activated) charcoal or carbon, nanofiltration, ultrafiltration, ion exchange, 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 milk oligosaccharide mixture.
  • 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 milk oligosaccharide mixture.
  • the separation and purification of the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide 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 t he produced milk oligosaccharide mixture and allowing at least a part of the proteins, salts, by- products, 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 milk oligosaccharide
  • the separation and purification of said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk oligosaccharide 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 has a molecular weight cut-off of between about 600 to about 800 Dalton.
  • the separation and purification of said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk 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.
  • the separation and purification of said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk oligosaccharide is made in the following way.
  • the cultivation or incubation comprising the produced milk oligosaccharide mixture, biomass, medium components and contaminants is applied to the following purification steps: i) separation of biomass from the cultivation or incubation, i i) 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 milk oligosaccharide mixture at a purity of greater than or equal to 80 % is provided.
  • the purified solution is dried by any one or more drying steps selected from the list comprising, consisting of or consisting essentially of 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.
  • the separation and purification of said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk oligosaccharide is performed as described in WO2022/034078.
  • the separation and purification of the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk oligosaccharide 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.
  • enzymatic treatment of the cultivation or incubation removal of the biomass from the cultivation or incubation
  • ultrafiltration nanofiltration
  • nanofiltration nanofiltration
  • a 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.
  • the present invention provides the produced mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide which is dried to powder by any one or more drying steps selected from the list comprising, consisting of or consisting essentially of 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.
  • Another aspect of the present invention provides the use of a cell of the invention for the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide as described herein.
  • Another aspect of the present invention provides the use of a method of the invention for the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk oligosaccharide as described herein.
  • the invention also relates to the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide obtained by the methods according to the invention.
  • Said milk oligosaccharide mixture 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.
  • said preparation comprises a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide that is obtainable, preferably obtained, by the methods as described herein.
  • a preparation is provided that further comprises at least one probiotic microorganism.
  • said preparation is a nutritional composition.
  • said preparation is a medicinal formulation, a dietary supplement, a dairy drink or an infant formula.
  • the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide can easily and effectively be provided, without the need for complicated, time and cost consuming synthetic processes.
  • 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.
  • 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).
  • SEC-HPLC high performance size-exclusion chromatography
  • the milk oligosaccharides in said milk oligosaccharide mixture of present invention are 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).
  • a partial depolymerization is carried out using an acid or enzymes to determine the structures.
  • the milk oligosaccharides in said milk oligosaccharide mixture of present invention are 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 alpha-glucosidase, etc., and NMR may be used to analyse the products.
  • the separated and preferably also purified mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide as described herein is incorporated into a food (e.g., human food or feed), dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine.
  • a food e.g., human food or feed
  • the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide is mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine.
  • the dietary supplement comprises at least one prebiotic ingredient and/or at least one probiotic ingredient.
  • a “prebiotic” is a substance that promotes growth of microorganisms beneficial to the host, particularly microorganisms in the gastrointestinal tract.
  • a dietary supplement provides multiple prebiotics, including the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide being a prebiotic produced and/or purified by a process disclosed in this specification, to promote growth of one or more beneficial microorganisms.
  • prebiotic ingredients for dietary supplements include other prebiotic molecules (such as HMOs) 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.
  • a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide produced and/or purified by a process of this specification is orally administered in combination with such microorganism.
  • 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
  • the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk 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.
  • 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.
  • a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk 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.
  • the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide is mixed with one or more ingredients of the infant formula.
  • 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, B3, B6, B12, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate) and possibly human milk oligosaccharides (HMOs).
  • 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, B3, B6, B12, C and D
  • minerals such as potassium cit
  • 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.
  • DiFL lacto-N-triose II, LNT, LNnT
  • lacto-N-fucopentaose I lacto-N-
  • 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. In some embodiments, the one or more infant formula ingredients comprise lactose, whey protein concentrate and/or high oleic safflower oil. In some embodiments, the concentration of the milk oligosaccharides in the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide added in the infant formula is approximately the same concentration as the concentration of the milk oligosaccharides generally present in human breast milk.
  • the milk oligosaccharides produced by the methods and/or cells disclosed herein can be described by their ratios in a mixture of milk oligosaccharides.
  • the "ratio" as described herein is understood as the ratio between two amounts of milk oligosaccharides, such as, but not limited to, the amount of one milk oligosaccharide divided by the amount of the other milk oligosaccharide, or the amount of one milk oligosaccharide divided by the total amount of milk oligosaccharides.
  • ratio and “relative abundance” as used herein are used interchangeably and can be expressed in weight percentage (weight %), mass percentage (mass %) or in mole percentage (mol %).
  • the relative abundance of a milk oligosaccharide or of a fraction of milk oligosaccharides is expressed herein as a weight percentage (weight %).
  • the relative abundance of the sialylated milk oligosaccharides in the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide is less than 49.9 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of the sialylated milk oligosaccharides in the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide is at least 5 %, preferably at least 7 %, more preferably at least 10 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of the sialylated milk oligosaccharides in the milk oligosaccharide mixture of present invention is less than 20 %, preferably less than 15 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of said sialylated milk oligosaccharides in said milk oligosaccharide mixture is preferably 5 to 20 %, preferably 5 to 15 %, more preferably 10 to 15 %, even more preferably 12 to 14 % on the total amount of milk oligosaccharides present in said mixture, most preferably reflecting the relative abundance of sialylated milk oligosaccharides in the oligosaccharide fraction of human breast milk and/or colostrum.
  • the relative abundance of fucosylated milk oligosaccharides in the fraction of non-sialylated milk oligosaccharides present in said milk oligosaccharide mixture of present invention is at least 10 %, preferably at least 20%, more preferably at least 30%, most preferably at least 35% on the total amount of non-sialylated milk oligosaccharides present in said milk oligosaccharide mixture.
  • the relative abundance of fucosylated milk oligosaccharides in the fraction of non-sialylated milk oligosaccharides present in said milk oligosaccharide mixture is 10-60%, preferably 20-60%, more preferably 30-60%, even more preferably 30-50%, even more preferably 35-50% on the total amount of non-sialylated milk oligosaccharides present in said milk oligosaccharide mixture, most preferably reflecting the relative abundance of fucosylated oligosaccharides in the non-sialylated oligosaccharide fraction in human breast milk and/or colostrum.
  • the relative abundance of the fucosylated milk oligosaccharides in the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide is at least 10 %, preferably at least 20 %, more preferably at least 30 %, more preferably at least 35 %, even more preferably at least 40%, most preferably at least 50 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of the fucosylated milk oligosaccharides in the milk oligosaccharide mixture of present invention is less than 90 %, preferably less than 85 %, more preferably less than 80 %, even more preferably less than 70 %, even more preferably less than 60 %, even more preferably less than 55%, most preferably less than 50 %, on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of said fucosylated milk oligosaccharides in said milk oligosaccharide mixture is preferably 10 to 90 %, preferably 20 to 80 %, more preferably 30 to 60 %, even more preferably 35 to 50 % on the total amount of milk oligosaccharides present in said mixture, most preferably reflecting the relative abundance of fucosylated milk oligosaccharides in the oligosaccharide fraction of human breast milk and/or colostrum.
  • the relative abundance of each milk oligosaccharide in the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide of present invention is at least 3 %, preferably at least 5 %, more preferably at least 10 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of the sialylated milk oligosaccharides in the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide is at least 30 %, preferably at least 40 %, more preferably at least 50 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of the sialylated milk oligosaccharides in the milk oligosaccharide mixture of present invention is less than 75 %, preferably less than 60 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of said sialylated milk oligosaccharides in said milk oligosaccharide mixture is preferably 30 to 75 %, preferably 30 to 60 %, more preferably 50 % on the total amount of milk oligosaccharides present in said mixture, most preferably reflecting the relative abundance of sialylated milk oligosaccharides in the oligosaccharide fraction of mammalian milk.
  • the relative abundance of the sialylated milk oligosaccharides in the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide is at least 50 %, preferably at least 55 %, more preferably at least 60 %, even more preferably at least 62 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of the sialylated milk oligosaccharides in the milk oligosaccharide mixture of present invention is less than 75 %, preferably less than 65 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of said sialylated milk oligosaccharides in said milk oligosaccharide mixture is preferably 50 to 75 %, preferably 60 to 75 %, more preferably 60 to 65 %, most preferably 62 % on the total amount of milk oligosaccharides present in said mixture, most preferably reflecting the relative abundance of sialylated milk oligosaccharides in the oligosaccharide fraction of mammalian milk.
  • the relative abundance of the sialylated milk oligosaccharides in the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide is at least 30 %, preferably at least 35 %, more preferably at least 40 %, even more preferably at least 45 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of the sialylated milk oligosaccharides in the milk oligosaccharide mixture of present invention is less than 55 %, preferably less than 50 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of said sialylated milk oligosaccharides in said milk oligosaccharide mixture is preferably 30 to 55 %, preferably 35 to 50 %, more preferably 40 to 45 %, most preferably 45 % on the total amount of milk oligosaccharides present in said mixture, most preferably reflecting the relative abundance of sialylated milk oligosaccharides in the oligosaccharide fraction of mammalian milk.
  • the mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide is incorporated into a feed preparation, wherein said feed is selected from the list comprising, consisting of or consisting essentially of pet food, animal milk replacer, veterinary product, veterinary feed supplement, nutrition supplement, post weaning feed, or creep feed.
  • the methods and the cell of the invention preferably provide at least one of the following further surprising advantages as described herein: - Modified titres of the sialylated milk oligosaccharide (g/L) in the mixture of at least two milk oligosaccharides as described herein, - Modified production rate r for the sialylated milk oligosaccharide in the mixture of at least two milk oligosaccharides as described herein (g sialylated milk oligosaccharide / L/h), - Modified cell performance index CPI for the sialylated milk oligosaccharide in the mixture of at least two milk oligosaccharides as described herein (g sialylated milk oligosaccharide / g X), - Modified specific productivity Qp (g sialylated milk oligosaccharide /g X /h), - Modified yield on the carbon source used Y (g
  • a cell metabolically engineered for the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide, said cell comprising: - a pathway for production of said at least one sialylated milk oligosaccharide, wherein said pathway comprises production of UDP-N-acetylglucosamine (UDP-GlcNAc), conversion of said UDP-GlcNAc into N-acetylmannosamine (ManNAc) by action of a hydrolyzing UDP-N-acetyl-D- glucosamine-2-epimerase, and conversion of said ManNAc into CMP-sialic acid by consecutive action of an N-acetylneuraminate synthase and an N-acylneuraminate cytidylyltransferas
  • said mixture further comprises: - a sialic acid residue selected 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), - a monosaccharide, and/or - a disaccharide. 7.
  • KDO 2-keto-3-deoxymanno-octulonic acid
  • said mixture comprises: - less sialylated milk oligosaccharide than non-sialylated milk oligosaccharides, or - less non-sialylated milk oligosaccharide than sialylated milk oligosaccharides.
  • the relative abundance of the sialylated milk oligosaccharides in said mixture is: - less than 49.9 % on the total amount of milk oligosaccharides present in said mixture, and/or - 5 to 20 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of the sialylated milk oligosaccharides in said mixture is: - 30 to 75 % on the total amount of milk oligosaccharides present in said mixture, preferably 30 to 60 % on the total amount of milk oligosaccharide present in said mixture, more preferably 50 % on the total amount of milk oligosaccharides present in said mixture, - 50 to 75 % on the total amount of milk oligosaccharides present in said mixture, preferably 60 to 75 % on the total amount of milk oligosaccharides present in said mixture, more preferably 60 to 65 % on the total amount of milk oligosaccharides present in said mixture, most preferably 62 % on the total amount of milk oligosaccharides present in said mixture, or - 30 to 55 % on the total amount of milk oligosaccharides present in said mixture, preferably 35 to 50 % on the total amount of milk oligosaccharides present in said mixture, preferably 35 to 50
  • said pathway for production of said at least one non-sialylated milk oligosaccharide is selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N- acetylmannosaminylation pathway. 15.
  • said cell further comprises a pathway selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N- acetylmannosaminylation pathway, - is genetically engineered to comprise at least one pathway selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N- acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway, and/or - comprises at least one pathway selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannos
  • glycosyltransferase is selected from the list comprising, consisting of or consisting essentially of 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- alt
  • nucleotide-activated sugar is selected from the list comprising, consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GlcNAc), UDP- N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), 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-
  • said precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, a sialic acid residue, 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; Neu4,5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc; KDO, CMP-sialic acid, CMP-Neu5Ac, glucose, galactose, GlcNAc, GalNAc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc.
  • said at least one sialylated milk oligosaccharide is: - a sialylated milk oligosaccharide having at least one sialic acid residue selected from the list consisting of Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; N eu5,7,9Ac3; 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), and - selected from the list comprising, consisting of or consisting essentially of a sialylated mammalian milk oligosaccharide (MMO), a sialylated human milk oligosaccharide (HMO), N-acetyllactos
  • MMO sialylated mamm
  • non-sialylated oligosaccharide is selected from the list comprising, consisting of or consisting essentially of non- sialylated neutral milk oligosaccharide, non-sialylated neutral MMO, non-sialylated neutral HMO, non-sialylated negatively charged oligosaccharide, non-sialylated negatively charged MMO, non- sialylated negatively charged HMO, sulphated milk oligosaccharides, 2'-fucosyllactose (2’FL), 3- fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto- N-triose II (LN3, GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc), lacto-N-tetraose
  • Cell according to any one of previous embodiments wherein said cell is selected from the list consisting of prokaryotic cells and eukaryotic cells.
  • Cell according to any one of previous embodiments wherein said cell is: - selected from the list consisting of yeast cells, bacterial cells, archaebacterial cells, algae cells, fungal cells, plant cells, animal cells, insect cells, protozoan cells, and/or - an E. coli or yeast with a lactose permease positive phenotype, preferably wherein said lactose permease is coded by the gene LacY or LAC12, respectively.
  • said cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell
  • said bacterium belongs to a phylum selected from the list comprising Proteobacteria, Firmicutes, Cyanobacteria, Deinococcus-Thermus and Actinobacteria; more preferably, said bacterium belongs to a family selected from the list comprising Enterobacteriaceae, Bacillaceae, Lactobacillaceae, Corynebacteriaceae and Vibrionaceae; even more preferably, said bacterium is s elected from the list comprising an Escherichia coli strain, a Bacillus subtilis strain, a Vibrio natriegens strain; even more preferably said Escherichia coli strain is a K-12 strain, most preferably said Escherichia coli K-12 strain is E.
  • said fungus belongs to a genus selected from the list comprising Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus, - preferably, said yeast belongs to a genus selected from the list comprising Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces, D ebaromyces, Candida, Schizosaccharomyces, Schwanniomyces or Torulaspora; more preferably, said yeast is selected from the list consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwannio
  • Method for the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide by a cell comprising: i. cultivating and/or incubating a cell of any one of previous embodiments, in cultivation and/or incubation medium under conditions permissive to produce said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk oligosaccharide, ii.
  • Method according to embodiment 32 wherein the method comprises: - Using a cultivation or incubation medium comprising one or more precursor(s) that is/are used for production of said at least one sialylated milk oligosaccharide and/or said at least one non-sialylated milk oligosaccharide, and/or - Adding to the cultivation or incubation medium one or more precursor(s) that is/are used for production of said at least one sialylated milk oligosaccharide and/or said at least one non- sialylated milk oligosaccharide.
  • Method according to any one of embodiment 32 or 33 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 m 3 (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, not more than two- fold, and/or 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 m 3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incuba,
  • Method according to any one of embodiments 32 to 34 comprising at least one of the following steps: i) Use of a cultivation or incubation medium comprising at least 50, at least 75, at least 100, at least 120 and/or 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 m 3 (cubic meter); ii) Use of a cultivation or incubation medium comprising at least 50, at least 75, at least 100, at least 120 and/or 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 m 3 (cubic meter); iii) Adding to the cultivation or incubation medium in a reactor or incubator a precursor feed comprising at least 50, at least 75, at least 100, at least 120 and/or 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 m 3 (
  • - said precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, a sialic acid residue, Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; N eu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc; KDO, CMP-sialic acid, CMP-Neu5Ac, glucose, galactose, GlcNAc, GalNAc, UDP-Gal, UDP- GlcNAc, and UDP-GalNAc, and/or - said acceptor is selected from the list comprising, consisting of or consisting essentially of lactose, LN3, LNT and LNnT.
  • Method according to embodiment 38 wherein said: - separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography, and/or - purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration, ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying or lyophilization. 40.
  • a cell according to any one of embodiments 1 to 31 for the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide. More specifically, the present invention relates to the following preferred specific embodiments: 1.
  • said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk oligosaccharide further comprises: - free sialic acid selected 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), - a monosaccharide, and/or - a disaccharide.
  • KDO 2-keto-3-deoxymanno-octulonic acid
  • said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk oligosaccharide comprises: - less sialylated milk oligosaccharide than non-sialylated milk oligosaccharides, or - less non-sialylated milk oligosaccharide than sialylated milk oligosaccharides.
  • the relative abundance of the sialylated milk oligosaccharides in said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide is: - less than 49.9 % on the total amount of milk oligosaccharides present in said mixture, and/or - 5 to 20 % on the total amount of milk oligosaccharides present in said mixture.
  • the relative abundance of the sialylated milk oligosaccharides in said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide is: - 30 to 75 %, 30 to 60 %, or 50 % on the total amount of milk oligosaccharides present in said mixture, - 50 to 75 %, 60 to 75 %, 60 to 65 %, or 62 % on the total amount of milk oligosaccharides present in said mixture, or - 30 to 55 %, 35 to 50 %, 40 to 45 %, or 45 % on the total amount of milk oligosaccharides present in said mixture.
  • said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk oligosaccharide - does not comprise free sialic acid, or - comprises less than 1 %, less than 0.5 %, less than 0.2 %, and/or less than 0.1 % of free sialic acid, wherein said free sialic acid is selected 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).
  • KDO 2-keto-3-deoxymanno-octulonic acid
  • said pathway for production of said at least one sialylated milk oligosaccharide is a sialylation pathway.
  • said cell - is genetically engineered to comprise a sialylation pathway, - comprises a sialylation pathway wherein said sialylation pathway has been genetically engineered, and/or - is genetically engineered for production of a sialic acid residue.
  • said pathway for production of said at least one non-sialylated milk oligosaccharide is selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N- acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway.
  • said cell further comprises a pathway selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N- acetylmannosaminylation pathway
  • - is genetically engineered to comprise at least one pathway selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N- acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway
  • - comprises at least one pathway selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosy
  • said one or more gene(s) in one or more reductive pathway(s) is/are rendered less functional by insertion, deletion and/or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) selected from the list comprising promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said one or more gene(s).
  • Cell according to any one of previous preferred embodiments wherein said cell: - possesses, expresses and/or overexpresses one or more glycosyltransferase(s), and/or - is genetically engineered to express and/or to over-express one or more glycosyltransferase(s).
  • glycosyltransferase(s) is selected from the list comprising, consisting of or consisting essentially of 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-
  • Cell according to any one of previous preferred embodiments, wherein said cell: - is capable to produce and/or produces a nucleotide-activated sugar, - is genetically engineered for production of a nucleotide-activated sugar, and/or - comprises a pathway for the synthesis of a nucleotide-activated sugar.
  • nucleotide-activated sugar is selected from the list comprising, consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP- glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), 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-R
  • said precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, a sialic acid residue, Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; N eu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc; KDO, CMP-sialic acid, CMP-Neu5Ac, glucose, galactose, GlcNAc, GalNAc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc.
  • sialylated milk oligosaccharide is: - a sialylated milk oligosaccharide having at least one sialic acid residue selected 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), and - selected from the list comprising, consisting of or consisting essentially of a sialylated mammalian milk oligosaccharide (MMO), a sialylated human milk oligosaccharide (HMO), N-acetyllactos
  • MMO sialylated mammalian milk oligos
  • non- sialylated oligosaccharide is selected from the list comprising, consisting of or consisting essentially of non-sialylated neutral milk oligosaccharide, non-sialylated neutral MMO, non-sialylated neutral HMO, non-sialylated negatively charged oligosaccharide, non-sialylated negatively charged MMO, non-sialylated negatively charged HMO, sulphated milk oligosaccharides, 2'-fucosyllactose (2’FL), 3- fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto- N-triose II (LN3, GlcNAc ⁇ 1-3Gal ⁇ 1-4Glc), lacto-N-tetraose
  • Method for the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide by a cell comprising: i . cultivating and/or incubating a cell of any one of previous preferred embodiments, in cultivation and/or incubation medium under conditions permissive to produce said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non- sialylated milk oligosaccharide, i i.
  • Method according to preferred embodiment 30, wherein the method comprises: - Using a cultivation or incubation medium comprising one or more precursor(s) that is/are used for production of said at least one sialylated milk oligosaccharide and/or said at least one non-sialylated milk oligosaccharide in said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide, and/or - Adding to the cultivation or incubation medium one or more precursor(s) that is/are used for production of said at least one sialylated milk oligosaccharide and/or said at least one non- sialylated milk oligosaccharide in said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligos
  • Method according to any one of preferred embodiments 30 to 32 comprising at least one of the following steps: i) Use of a cultivation or incubation medium comprising at least 50, at least 75, at least 100, at least 120 and/or at least 150 grams of precursor, used for production of said at least one sialylated milk oligosaccharide and/or said at least one non-sialylated milk oligosaccharide in said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide, per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter), ii) Use of a cultivation or incubation medium comprising at least 50, at least 75, at least 100, at least 120 and/or at least 150 grams of acceptor, used for production of said at least one sialylated milk oligo
  • - precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, a sialic acid residue, Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; N eu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc; KDO, CMP-sialic acid, CMP-Neu5Ac, glucose, galactose, GlcNAc, GalNAc, UDP-Gal, UDP- GlcNAc, and UDP-GalNAc, and/or - acceptor is selected from the list comprising, consisting of or consisting essentially of lactose, LN3, LNT and LNnT.
  • Method according to any one of preferred embodiments 30 to 34 wherein: - said method results in the production of 0.1 g/L or more, 0.5 g/L or more, 1 g/L or more, 5 g/L or more, 10 g/L or more, 20 g/L or more, 30 g/L or more, 40 g/L or more, and/or 50 g/L or more of total milk oligosaccharides, - said cell produces 0.1 g/L or more, 0.5 g/L or more, 1 g/L or more, 5 g/L or more, 10 g/L or more, 20 g/L or more, 30 g/L or more, 40 g/L or more, and/or 50 g/L or more of total milk oligosaccharides, - said mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligos
  • Method according to preferred embodiment 36 wherein said: - separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography, and/or - purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration, ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying or lyophilization. 38.
  • a cell according to any one of preferred embodiments 1 to 29 for the production of a mixture of at least two milk oligosaccharides comprising at least one sialylated milk oligosaccharide and at least one non-sialylated milk oligosaccharide.
  • the invention will be described in more detail in the examples. The following examples will serve as further illustration and clarification of the present invention and are not intended to be limiting. Examples Example 1. Calculation of percentage identity between nucleotide or polypeptide sequences Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA.
  • GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol.
  • the BLAST algorithm (Altschul et al., J. Mol. Biol. (1990) 215: 403-10) calculates the global percentage sequence identity (i.e., over the full-length sequence) and performs a statistical analysis of the similarity between the two sequences.
  • the software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologs may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage.
  • Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics (2003) 4:29). Minor manual editing may be performed to optimize alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologs, specific domains may also be used, to determine the so-called local sequence identity.
  • the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1); 195-7).
  • the Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium).
  • the minimal medium used in the cultivation experiments in 96-well plates or in shake flasks contained 2.00 g/L NH 4 Cl, 5.00 g/L (NH 4 ) 2 SO 4 , 2.993 g/L KH2PO4, 7.315 g/L K2HPO4, 8.372 g/L MOPS, 0.5 g/L NaCl, 0.5 g/L MgSO4.7H2O, 30 g/L sucrose or 30 g/L glycerol, 1 ml/L vitamin solution, 100 ⁇ l/L molybdate solution, and 1 mL/L selenium solution.
  • Vitamin solution consisted of 3.6 g/L FeCl2.4H2O, 5.0 g/L CaCl2.2H2O, 1.3 g/L MnCl2.2H2O, 0.38 g/L CuCl2.2H2O, 0.5 g/L CoCl2.6H2O, 0.94 g/L ZnCl 2 , 0.0311 g/L H 3 BO 4 , 0.4 g/L Na 2 EDTA.2H 2 O and 1.01 g/L thiamine.HCl.
  • the molybdate solution contained 0.967 g/L NaMoO4.2H2O.
  • the selenium solution contained 42 g/L Seo2.
  • the minimal medium for fermentations contained 6.75 g/L NH 4 Cl, 1.25 g/L (NH 4 ) 2 SO 4 , 2.93 g/L KH 2 PO 4 and 7.31 g/L KH 2 PO 4 , 0.5 g/L NaCl, 0.5 g/L MgSO4.7H2O, 30 g/L sucrose or 30 g/L glycerol, 1 mL/L vitamin solution, 100 ⁇ L/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above.
  • sialic acid and/or 20 g/L lactose were additionally added to the medium.
  • Complex medium was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 ⁇ m Sartorius).
  • the medium was made selective by adding an antibiotic: e.g., chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L).
  • an inducer for inducible gene expression like e.g., IPTG or arabinose was added.
  • a preculture for the bioreactor was started from an entire 1 mL cryovial of a certain strain, inoculated in 250 mL or 500 mL minimal medium in a 1 L or 2.5 L shake flask and incubated for 24 h at 37°C on an orbital shaker at 200 rpm.
  • a 5 L bioreactor was then inoculated (250 mL inoculum in 2 L batch medium); the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsoder, Germany). Culturing condition were set to 37 °C, and maximal stirring; pressure gas flow rates were dependent on the strain and bioreactor.
  • the pH was controlled at 6.8 using 0.5 M H2S04 and 20% NH4OH.
  • Plasmids pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains an FRT-flanked chloramphenicol resistance (cat) gene), pKD4 (contains an FRT-flanked kanamycin resistance (kan) gene), and pCP20 (expresses FLP recombinase activity) plasmids were obtained from Prof. R. Cunin (Vrije Universiteit Brussel, Belgium in 2007). The pET28b(+) vector was obtained from Millipore and adapted for Golden Gate cloning.
  • Plasmids were maintained in the host E. coli DH5alpha (F-, phi80dlacZ ⁇ M15, ⁇ (lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(rk-, mk + ), phoA, supE44, lambda-, thi-1, gyrA96, relA1) bought from Invitrogen.
  • Strains and mutations Escherichia coli K12 MG1655 [ ⁇ -, F-, rph-1] was obtained from the Coli Genetic Stock Center (US), CGSC Strain#: 7740, in March 2007.
  • the mutant strain was derived from E. coli K12 MG1655 comprising genomic knock-ins of constitutive transcriptional units containing a hydrolyzing UDP-N- acetylglucosamine 2-epimerase selected from the list consisting of SEQ ID NO 01, 02, 03, 04, 05, 06, 07 and 09 and an N-acetylneuraminate synthase like e.g., NeuB from N. meningitidis (UniProt ID E0NCD4) or NeuB from C.
  • UDP-N- acetylglucosamine 2-epimerase selected from the list consisting of SEQ ID NO 01, 02, 03, 04, 05, 06, 07 and 09
  • an N-acetylneuraminate synthase like e.g., NeuB from N. meningitidis (UniProt ID E0NCD4) or NeuB from C.
  • sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, Sequence version 03 (23 Jan 2007)), an N- acetylglucosamine-1-phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase like e.g. glmU from E.
  • a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, Sequence version 03 (23 Jan 2007)
  • an N- acetylglucosamine-1-phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase like e.g. glmU from E.
  • coli (UniProt ID P0ACC7), a hydrolyzing UDP-N-acetylglucosamine 2-epimerase selected from the list consisting of SEQ ID NO 01, 02, 03, 04, 05, 06, 07 and 09 and an N-acetylneuraminate synthase like e.g. NeuB from N. meningitidis (UniProt ID E0NCD4) or NeuB from C. jejuni (UniProt ID Q93MP9).
  • the mutant strain was derived from E.
  • coli K12 MG1655 comprising genomic knock-ins of constitutive transcriptional units containing one or more copies of a glucosamine 6- phosphate N-acetyltransferase like e.g. GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), an N- acylglucosamine 2-epimerase like e.g. AGE from Bacteroides ovatus (UniProt ID A7LVG6) and an N- acetylneuraminate synthase like e.g. NeuB from Neisseria meningitidis (UniProt ID E0NCD4) or NeuB Campylobacter jejuni (UniProt ID Q93MP9).
  • a glucosamine 6- phosphate N-acetyltransferase like e.g. GNA1 from Saccharomyces cerevisiae (UniProt ID P43577)
  • sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a bifunctional UDP- GlcNAc 2-epimerase/N-acetylmannosamine kinase like e.g. GNE from Mus musculus (strain C57BL/6J) (UniProt ID Q91WG8), an N-acylneuraminate-9-phosphate synthetase like e.g. NANS from Pseudomonas sp. UW4 (UniProt ID K9NPH9) and an N-acylneuraminate-9-phosphatase like e.g. NANP from Candidatus Magnetomorum sp.
  • a bifunctional UDP- GlcNAc 2-epimerase/N-acetylmannosamine kinase like e.g. GNE from Mus musculus (strain C57BL/6J) (UniProt ID Q91WG8)
  • sialic acid production can be obtained by genomic knock- ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03 (23 Jan 2007)), an N-acetylglucosamine-1-phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase like e.g. glmU from E.
  • a phosphoglucosamine mutase like e.g. glmM from E. coli
  • an N-acetylglucosamine-1-phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase like e.g. glmU from E.
  • coli (UniProt ID P0ACC7), a bifunctional UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase like e.g. GNE from M. musculus (strain C57BL/6J) (UniProt ID Q91WG8), an N-acylneuraminate-9-phosphate synthetase like e.g. NANS from Pseudomonas sp. UW4 (UniProt ID K9NPH9) and an N-acylneuraminate-9-phosphatase like e.g. NANP from C. Magnetomorum sp. HK-1 (UniProt ID KPA15328.1) or NANP from B.
  • UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase like e.g. GNE from M. musculus (strain C57BL/6J) (UniProt ID Q91WG8)
  • Sialic acid production can further be optimized in the modified E. coli strain with genomic knock-outs of the E. coli genes comprising any one or more of nagA, nagB, nagC, nagD, nagE, nanA, nanE, nanK, manX, manY and manZ as described in WO18122225, and/or genomic knock-outs of the E.
  • coli genes comprising any one or more of nanT, poxB, ldhA, adhE, aldB, pflA, pflC, ybiY, ackA and/or pta and with genomic knock- ins of constitutive transcriptional units comprising one or more copies of an L-glutamine—D-fructose-6- phosphate aminotransferase like e.g. glmS from E. coli (UniProt ID P17169, Sequence version 04 (23 Jan 2007)) and an acetyl-CoA synthetase like e.g. acs from E. coli (UniProt ID P27550, Sequence version 02 (01 Oct 1993)).
  • sialic acid production strains were further modified to express an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from Pasteurella multocida (UniProt ID A0A849CI62) and/or the NeuA enzyme from Campylobacter jejuni (UniProt ID Q93MP7) and to express an alpha-2,3-sialyltransferase like e.g.
  • PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3), SEQ ID NO 11 or SEQ ID NO 14, an alpha-2,6-sialyltransferase like e.g. PdbST6 from Photobacterium damselae (UniProt ID O66375), SEQ ID NO 12 or SEQ ID NO 13.
  • Constitutive transcriptional units of the N-acylneuraminate cytidylyltransferase and the sialyltransferase(s) can be delivered to the modified strain either via genomic knock-in or via expression plasmids.
  • the strains were additionally modified with genomic knock-outs of the E.
  • sialic acid and/or sialylated milk oligosaccharide production can further be optimized in the mutant E. coli strains with genomic knock-ins of constitutive transcriptional units comprising a membrane transporter protein like e.g. a sialic acid transporter like e.g. nanT from E. coli K- 12 MG1655 (UniProt ID P41036, sequence version 02 (01 Nov 1995)), nanT from E.
  • a membrane transporter protein like e.g. a sialic acid transporter like e.g. nanT from E. coli K- 12 MG1655 (UniProt ID P41036, sequence version 02 (01 Nov 1995)
  • nanT from E.
  • coli O6:H1 (UniProt ID Q8FD59), nanT from E. albertii (UniProt ID B1EFH1) or a porter like e.g. EntS from E. coli (UniProt ID P24077, sequence version 02 (01 Nov 1997)), EntS from Kluyvera ascorbata (UniProt ID A0A378GQ13) or EntS from Salmonella enterica subsp.
  • arizonae (UniProt ID A0A6Y2K4E8), MdfA from Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), MdfA from Citrobacter youngae (UniProt ID D4BC23), MdfA from E. coli (UniProt ID P0AEY8), MdfA from Yokenella regensburgei (UniProt ID G9Z5F4), iceT from E. coli (UniProt ID A0A024L207), iceT from Citrobacter youngae (UniProt ID D4B8A6), SetA from E.
  • the modified strain was derived from E. coli K12 MG1655 and modified with a knock-out of the E. coli lacZ, lacY, lacA and nagB genes and with genomic knock-ins of constitutive transcriptional units for a lactose permease like e.g. the E. coli LacY (UniProt ID P02920) and a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g. lgtA (UniProt ID Q9JXQ6) from N. meningitidis.
  • a lactose permease like e.g. the E. coli LacY (UniProt ID P02920) and a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g. lgtA (UniProt ID Q9JXQ6) from N. meningitidis.
  • the modified LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N-acetylglucosamine beta-1,4-galactosyltransferase like e.g. LgtB (Uniprot ID Q51116, Sequence version 02 (01 Dec 2000)) from N. meningitidis.
  • LgtB Uniprot ID Q51116, Sequence version 02 (01 Dec 2000)
  • the modified LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N- acetylglucosamine beta-1,3-galactosyltransferase like e.g. WbgO (Uniprot ID D3QY14) from E. coli O55:H7.
  • LN3 and/or LNnT production can further be optimized in the modified E.
  • the modified LN3 and/or LNnT producing strains can also be optionally modified for enhanced UDP-GlcNAc production with a genomic knock-in of a constitutive transcriptional unit for an L-glutamine—D-fructose-6-phosphate aminotransferase like e.g., glmS from E. coli (UniProt ID P17169, Sequence version 04, 23 Jan 2007).
  • coli strains can also optionally be adapted with a genomic knock-in of a constitutive transcriptional unit for a UDP-glucose-4-epimerase like e.g., galE from E. coli (UniProt ID P09147), a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, Sequence version 03, 23 Jan 2007) and an N-acetylglucosamine-1-phosphate uridylyltransferase / glucosamine-1-phosphate acetyltransferase like e.g. glmU from E. coli (UniProt ID P0ACC7).
  • a genomic knock-in of a constitutive transcriptional unit for a UDP-glucose-4-epimerase like e.g., galE from E. coli (UniProt ID P09147)
  • the mutant strain was derived from E. coli K12 MG1655 comprising a knock-out of the E. coli wcaJ gene and genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E. coli W (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6).
  • a sucrose transporter like e.g. CscB from E. coli W (UniProt ID E0IXR1)
  • a fructose kinase like e.g. Frk originating from Zymomonas mobilis
  • a sucrose phosphorylase like e.g
  • GDP-fucose production can further be optimized in the mutant E. coli strain by genomic knock-outs of any one or more of the E. coli genes comprising glgC, agp, pfkA, pfkB, pgi, arcA, iclR, pgi and lon as described in WO2016075243 and WO2012007481.
  • GDP-fucose production can additionally be optimized comprising genomic knock-ins of constitutive transcriptional units for a mannose-6-phosphate isomerase like e.g. manA from E. coli (UniProt ID P00946), a phosphomannomutase like e.g. manB from E.
  • GDP-fucose production can also be obtained by genomic knock-outs of the E.
  • the mutant GDP-fucose production strain was additionally modified with an expression plasmid comprising a constitutive transcriptional unit for an alpha-1,2-fucosyltransferase like e.g. HpFutC from H. pylori (UniProt ID Q9X435) and/or an alpha-1,3-fucosyltransferase like e.g. HpFucT from H. pylori (UniProt ID O30511).
  • an expression plasmid comprising a constitutive transcriptional unit for an alpha-1,2-fucosyltransferase like e.g. HpFutC from H. pylori (UniProt ID Q9X435) and/or an alpha-1,3-fucosyltransferase like e.g. HpFucT from H. pylori (UniProt ID O30511).
  • any one or more of the glycosyltransferases, the proteins involved in nucleotide-activated sugar synthesis and/or the membrane transporter proteins were N- and/or C- terminally fused to a solubility enhancer tag like e.g. a SUMO-tag, an MBP-tag, His, FLAG, Strep-II, Halo- tag, NusA, thioredoxin, GST and/or the Fh8-tag to enhance their solubility.
  • a solubility enhancer tag like e.g. a SUMO-tag, an MBP-tag, His, FLAG, Strep-II, Halo- tag, NusA, thioredoxin, GST and/or the Fh8-tag to enhance their solubility.
  • a solubility enhancer tag like e.g. a SUMO-tag, an MBP-tag, His, FLAG, Strep-II, Halo- tag, NusA, thiore
  • coli strains were modified with a genomic knock-ins of a constitutive transcriptional unit encoding a chaperone protein like e.g., DnaK, DnaJ, GrpE or the GroEL/ES chaperonin system.
  • a chaperone protein like e.g., DnaK, DnaJ, GrpE or the GroEL/ES chaperonin system.
  • the mutant E. coli strains are modified to create a glycominimized E.
  • coli strain comprising genomic knock-out of any one or more of non-essential glycosyltransferase genes comprising pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcaI, wcaJ, wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA and yaiP.
  • non-essential glycosyltransferase genes comprising pgaC, pgaD,
  • wild-type E. coli K12 MG1655 cells or mutant E. coli K12 MG1655 cells modified for synthesis of a milk oligosaccharide were modified for protein production by mutation in or genomic knock-out of one or more cytoplasmic reductive pathway genes like e.g., glutathione reductase (gor) and/or thioredoxin reductase (trxB) rendering said genes less functional or eliminated compared to non-modified cells.
  • cytoplasmic reductive pathway genes like e.g., glutathione reductase (gor) and/or thioredoxin reductase (trxB) rendering said genes less functional or eliminated compared to non-modified cells.
  • the mutant cells were further modified with genomic knock-ins of constitutive transcriptional units containing one or more copies of a disulfide bond isomerase, a thiol oxidase and/or a chaperone like e.g., dsbC from E. coli (UniProt ID P0AEG6), PDI from S. cerevisiae (UniProt ID P17967, Sequence version 02 (02 Jun 2021)), QSOX from Homo sapiens (UniProt ID O00391, Sequence version 03 (02 Jun 2021)).
  • the mutant cells were further modified with genomic knock-outs of one or more gene(s) encoding a protease like e.g.
  • nucleotide-sugar degrading enzyme like e.g. ushA.
  • All constitutive promoters, UTRs and terminator sequences originated from the libraries described by Cambray et al. (Nucleic Acids Res.2013, 41(9), 5139-5148), Dunn et al. (Nucleic Acids Res.1980, 8, 2119- 2132), Edens et al. (Nucleic Acids Res.1975, 2, 1811-1820), Kim and Lee (FEBS Letters 1997, 407, 353-356) and Mutalik et al. (Nat.
  • Saccharomyces cerevisiae Media and cultivation Strains were grown on Synthetic Defined yeast medium with Complete Supplement Mixture (SD CSM) or CSM drop-out (SD CSM-Ura, SD CSM-Trp, SD CSM-His) containing 6.7 g/L Yeast Nitrogen Base without amino acids (YNB w/o AA, Difco), 20 g/L agar (Difco) (solid cultures), 22 g/L glucose monohydrate or 20 g/L lactose and 0.79 g/L CSM or 0.77 g/L CSM-Ura, 0.77 g/L CSM-Trp, or 0.77 g/L CSM-His (MP Biomedicals).
  • SD CSM Complete Supplement Mixture
  • SD CSM-Ura CSM drop-out
  • SD CSM-His containing 6.7 g/L Yeast Nitrogen Base without amino acids
  • 20 g/L agar Difco
  • solid cultures 22
  • yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were grown for 2-3 days at 30 °C. Starting from a single colony, a preculture was grown over night in 5 mL at 30 °C, shaking at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL media. These shake flasks were incubated at 30 °C with an orbital shaking of 200 rpm. Strains and plasmids S. cerevisiae BY4742 created by Brachmann et al. (Yeast (1998) 14:115-32) was used, available in the Euroscarf culture collection.
  • a yeast expression plasmid was derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the TRP1 selection marker and constitutive transcriptional units for an L-glutamine—D-fructose-6-phosphate aminotransferase like e.g. glmS from E. coli (UniProt ID P17169, sequence version 04 (23 Jan 2007)), a phosphoglucosamine mutase like e.g. glmM from E.
  • the plasmid further comprised constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), and an alpha-2,3-sialyltransferase like e.g.
  • PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3), SEQ ID NO 11 or SEQ ID NO 14, and/or an alpha-2,6-sialyltransferase like e.g. PdbST6 from Photobacterium damselae (UniProt ID O66375), SEQ ID NO 12 or SEQ ID NO 13.
  • a yeast expression plasmid like p2a_2 ⁇ _Fuc (Chan 2013, Plasmid 70, 2-17) can be used for expression of foreign genes in S. cerevisiae. This plasmid contains an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E.
  • This plasmid is further modified with constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), a GDP-mannose 4,6-dehydratase like e.g. gmd from E. coli (UniProt ID P0AC88) and a GDP-L-fucose synthase like e.g. fcl from E. coli (UniProt ID P32055).
  • a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921)
  • a GDP-mannose 4,6-dehydratase like e.g. gmd from E. coli (UniProt ID P0AC88)
  • a GDP-L-fucose synthase like e.g. fcl from E. coli (UniProt ID P32055).
  • the yeast expression plasmid p2a_2 ⁇ _Fuc2 can be used as an alternative expression plasmid of the p2a_2 ⁇ _Fuc plasmid comprising next to the ampicillin resistance gene, the bacterial ori, the 2 ⁇ yeast ori and the Ura3 selection marker constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), a fucose permease like e.g. fucP from E. coli (UniProt ID P11551) and a bifunctional enzyme with fucose kinase/fucose-1-phosphate guanylyltransferase activity like e.g.
  • a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921)
  • a fucose permease like e.g. fucP from E. coli (Un
  • the p2a_2 ⁇ _Fuc and its variant the p2a_2 ⁇ _Fuc2 additionally contained a constitutive transcriptional unit for an alpha-1,2- fucosyltransferase like e.g. HpFutC from H. pylori (UniProt ID Q9X435) and/or an alpha-1,3- fucosyltransferase like e.g. HpFucT from H. pylori (UniProt ID O30511).
  • a constitutive transcriptional unit for an alpha-1,2- fucosyltransferase like e.g. HpFutC from H. pylori (UniProt ID Q9X435) and/or an alpha-1,3- fucosyltransferase like e.g. HpFucT from H. pylori (UniProt ID O30511).
  • a yeast expression plasmid can be derived from the pRS420- plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the HIS3 selection marker and a constitutive transcriptional unit for an UDP-glucose-4-epimerase like e.g. galE from E. coli (UniProt ID P09147).
  • This plasmid can be further modified with constitutive transcriptional units for a lactose permease like e.g. LAC12 from K.
  • lactis (UniProt ID P07921) and a galactoside beta-1,3-N- acetylglucosaminyltransferase activity like e.g. lgtA from N. meningitidis (UniProt ID Q9JXQ6) to produce LN3.
  • the mutant LN3 producing strains were further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,3- galactosyltransferase like e.g. WbgO (Uniprot ID D3QY14) from E. coli O55:H7.
  • LN3 derived oligosaccharides like lacto-N-neotetraose (LNnT, Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc)
  • LNnT lacto-N-neotetraose
  • the mutant LN3 producing strain were further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,4-galactosyltransferase like e.g. LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis.
  • any one or more of the glycosyltransferases and/or the proteins involved in nucleotide-activated sugar synthesis were N- and/or C-terminally fused to a solubility tag like e.g., a SUMOstar tag (e.g. obtained from pYSUMOstar, Life Sensors, Malvern, PA) or an MBP-tag to enhance their solubility.
  • a solubility tag like e.g., a SUMOstar tag (e.g. obtained from pYSUMOstar, Life Sensors, Malvern, PA) or an MBP-tag to enhance their solubility.
  • the mutant yeast strains were modified with a genomic knock-in of a constitutive transcriptional unit encoding a chaperone protein like e.g.
  • coli DH5alpha (F-, phi80dlacZdeltaM15, delta(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk-, mk + ), phoA, supE44, lambda-, thi-1, gyrA96, relA1) bought from Invitrogen.
  • the LB medium consisted of 1% tryptone peptone (Difco), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR).
  • Luria Broth agar (LBA) plates consisted of the LB media, with 12 g/L agar (Difco) added.
  • the minimal medium contained 2.00 g/L (NH 4 ) 2 SO 4 , 7.5 g/L KH 2 PO 4 , 17.5 g/L K 2 HPO 4 , 1.25 g/L Na-citrate, 0.25 g/L MgSO 4 .7H 2 O, 0.05 g/L tryptophan, from 10 up to 30 g/L glucose (or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose), 10 mL/L trace element mix and 10 mL/L Fe-citrate solution.
  • the medium was set to a pH of 7.0 with 1 M KOH. Depending on the experiment lactose is added as a precursor.
  • the trace element mix consisted of 0.735 g/L CaCl 2 .2H 2 O, 0.1 g/L MnCl 2 .2H 2 O, 0.033 g/L CuCl 2 .2H 2 O, 0.06 g/L CoCl 2 .6H 2 O, 0.17 g/L ZnCl 2 , 0.0311 g/L H 3 BO 4 , 0.4 g/L Na 2 EDTA.2H 2 O and 0.06 g/L Na 2 MoO 4 .
  • the Fe-citrate solution contained 0.135 g/L FeCl3.6H2O, 1 g/L Na-citrate (Hoch 1973 PMC1212887).
  • Complex medium e.g. LB
  • LB was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 ⁇ m Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g. zeocin (20mg/L)).
  • a preculture of 96-well microtiter plate experiments was started from a cryovial or a single colony from an LB plate, in 150 ⁇ L LB and was incubated overnight at 37 °C on an orbital shaker at 800 rpm.
  • a dilution of the cultures was made to measure the optical density at 600 nm.
  • the cell performance index or CPI was determined by dividing the oligosaccharide concentrations by the biomass, in relative percentages compared to a reference strain. The biomass is empirically determined to be approximately 1/3rd of the optical density measured at 600 nm.
  • Strains, plasmids and mutations Bacillus subtilis 168 is used as available at the Bacillus Genetic Stock Center (Ohio, USA). Plasmids for gene deletion via Cre/lox are constructed as described by Yan et al. (Appl & Environm microbial, Sept 2008, p5556-5562).
  • Gene disruption is done via homologous recombination with linear DNA and transformation via the electroporation as described by Xue et al. (J. microb. Meth. 34 (1999) 183-191).
  • the method of gene knockouts is described by Liu et al. (Metab. Engine. 24 (2014) 61-69). This method uses 1000 bp homologies up- and downstream of the target gene.
  • Integrative vectors as described by Popp et al. (Sci. Rep., 2017, 7, 15158) are used as expression vector and could be further used for genomic integrations if necessary.
  • a suitable promoter for expression can be derived from the part repository (iGem): sequence id: BBa_K143012, BBa_K823000, BBa_K823002 or BBa_K823003. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.
  • the engineered strain was derived from B. subtilis comprising knockouts of the B. subtilis nagA, nagB and gamA genes and genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g. glmM from E.
  • Sialic acid production can also be obtained in modified B. subtilis comprising knockouts of the B. subtilis nagA, nagB and gamA genes and genomic knock-ins of constitutive transcriptional units containing an N-acylglucosamine 2-epimerase like e.g., AGE from B. ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase like e.g., NeuB from N. meningitidis (UniProt ID E0NCD4).
  • an N-acylglucosamine 2-epimerase like e.g., AGE from B. ovatus (UniProt ID A7LVG6)
  • an N-acetylneuraminate synthase like e.g., NeuB from N. meningitidis (UniProt ID E0NCD4).
  • the modified strain can further be modified with a genomic knock-in of one or more constitutive transcriptional units containing a glutamine--fructose-6-P-aminotransferase like e.g., the native glutamine--fructose-6-P-aminotransferase glmS (UniProt ID P0CI73).
  • the strains were also modified for expression of a phosphatase like e.g., SurE from E. coli (UniProt ID P0A840).
  • the sialic acid production strains further need to express an N-acylneuraminate cytidylyltransferase like e.g. NeuA enzyme from P. multocida (UniProt ID A0A849CI62), and an alpha-2,3-sialyltransferase like e.g. PmultST3 from P. multocida (UniProt ID Q9CLP3), SEQ ID NO 11 or SEQ ID NO 14, and/or an alpha-2,6- sialyltransferase like e.g. PdbST6 from P.
  • an N-acylneuraminate cytidylyltransferase like e.g. NeuA enzyme from P. multocida (UniProt ID A0A849CI62)
  • an alpha-2,3-sialyltransferase like e.g. PmultST3 from P. multocida (UniProt ID Q
  • Constitutive transcriptional units of the N-acylneuraminate cytidylyltransferase and the sialyltransferases can be delivered to the engineered strain either via genomic knock-in or via expression plasmids. If the engineered strains producing sialic acid and CMP-sialic acid were intended to make sialylated lactose structures, the strains were additionally modified with a genomic knock-in of a constitutive transcriptional unit for a lactose permease like e.g. the E. coli LacY (UniProt ID P02920). In an example for the production of LN3, the B.
  • subtilis strain is modified with a genomic knock-in of constitutive transcriptional units comprising a lactose importer (such as e.g. the E. coli lacY with UniProt ID P02920) and a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g. LgtA from N. meningitidis (UniProt ID Q9JXQ6).
  • the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,3-galactosyltransferase like e.g. WbgO from E.
  • the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,4-galactosyltransferase like e.g. LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000).
  • the N-acetylglucosamine beta-1,3-galactosyltransferase and the N-acetylglucosamine beta-1,4-galactosyltransferase can be delivered to the strain either via genomic knock-in or from an expression plasmid.
  • the B. subtilis strains are modified with a constitutive transcriptional unit for an alpha-1,2-fucosyltransferase like e.g. HpFutC from H. pylori (UniProt ID Q9X435) and/or an alpha-1,3-fucosyltransferase like e.g. HpFucT from H. pylori (UniProt ID O30511).
  • D. Corynebacterium glutamicum Media and cultivation Two different media are used to cultivate C. glutamicum: i.e., a rich tryptone-yeast extract (TY) medium and a minimal medium.
  • the TY medium consisted of 1.6% tryptone (Difco), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR).
  • TY agar (TYA) plates consisted of the TY media, with 12 g/L agar (Difco) added.
  • the minimal medium for the shake flask experiments contained 20 g/L (NH 4 ) 2 SO 4 , 5 g/L urea, 1 g/L KH 2 PO 4 , 1 g/L K 2 HPO 4 , 0.25 g/L MgSO 4 .7H 2 O, 42 g/L MOPS, from 10 up to 30 g/L glucose (or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose) and 1 mL/L trace element mix. Depending on the experiment lactose is added as a precursor.
  • the trace element mix consisted of 10 g/L CaCl2, 10 g/L FeSO4.7H2O, 10 g/L MnSO4.H2O, 1 g/L ZnSO4.7H2O, 0.2 g/L CuSO4, 0.02 g/L NiCl2.6H2O, 0.2 g/L biotin (pH 7.0) and 0.03 g/L protocatechuic acid.
  • Complex medium e.g., TY, was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 ⁇ m Sartorius). When necessary, the medium was made selective by adding an antibiotic (e.g., kanamycin, ampicillin).
  • a preculture of 96-well microtiter plate experiments was started from a cryovial or a single colony from a TY plate, in 150 ⁇ L TY and was incubated overnight at 37 °C on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, with 400 ⁇ L minimal medium by diluting 400x. Each strain was grown in multiple wells of the 96-well plate as biological replicates. These final 96-well culture plates were then incubated at 37 °C on an orbital shaker at 800 rpm for 72h, or shorter, or longer.
  • the cell performance index or CPI was determined by dividing the oligosaccharide concentrations, e.g., 3’SL concentrations, measured in the whole broth by the biomass, in relative percentages compared to the reference strain.
  • the biomass is empirically determined to be approximately 1/3rd of the optical density measured at 600 nm.
  • Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.
  • the engineered strain was derived from C. glutamicum comprising knockouts of the C. glutamicum ldh, cgl2645 and nagB genes and genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03 (23 Jan 2007)), an N-acetylglucosamine-1-phosphate uridylyltransferase/glucosamine-1-phosphate acetyltransferase like e.g.
  • glmU from E. coli (UniProt ID P0ACC7), a hydrolyzing UDP-N-acetylglucosamine 2-epimerase selected from the list consisting of SEQ ID NO 01, 02, 03, 04, 05, 06, 07 and 09, and an N-acetylneuraminate synthase like e.g. neuB from N. meningitidis (UniProt ID E0NCD4).
  • the modified strain can further be modified with a genomic knock-in of one or more constitutive transcriptional units containing a glutamine--fructose-6-P-aminotransferase like e.g., the native glutamine--fructose-6-P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 02 (23 Jan 2007)).
  • a glutamine--fructose-6-P-aminotransferase like e.g., the native glutamine--fructose-6-P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 02 (23 Jan 2007)).
  • the sialic acid production strains further need to express an N-acylneuraminate cytidylyltransferase like e.g. NeuA enzyme from P.
  • multocida (UniProt ID A0A849CI62), and an alpha-2,3-sialyltransferase like e.g. PmultST3 from P. multocida (UniProt ID Q9CLP3), SEQ ID NO 11 or SEQ ID NO 14, and/or an alpha-2,6-sialyltransferase like e.g. PdbST6 from P. damselae (UniProt ID O66375), SEQ ID NO 12 or SEQ ID NO 13.
  • PmultST3 from P. multocida
  • SEQ ID NO 11 SEQ ID NO 14
  • alpha-2,6-sialyltransferase like e.g. PdbST6 from P. damselae (UniProt ID O66375), SEQ ID NO 12 or SEQ ID NO 13.
  • Constitutive transcriptional units of the N- acylneuraminate cytidylyltransferase and the sialyltransferases can be delivered to the engineered strain either via genomic knock-in or via expression plasmids. If the engineered strains producing sialic acid and CMP-sialic acid were intended to make sialylated lactose structures, the strains were additionally modified with a genomic knock-in of a constitutive transcriptional unit for a lactose permease like e.g., the E. coli LacY (UniProt ID P02920). In an example for the production of lactose-based oligosaccharides, C.
  • glutamicum mutant strains are created to contain a gene coding for a lactose importer (such as e.g. the E. coli lacY with UniProt ID P02920).
  • a lactose importer such as e.g. the E. coli lacY with UniProt ID P02920
  • the C. glutamicum strain is modified with a genomic knock-in of constitutive expression units comprising a lactose importer (such as e.g. the E. coli lacY with UniProt ID P02920) and a galactoside beta-1,3-N-acetylglucosaminyltransferase like e.g. LgtA from N. meningitidis (UniProt ID Q9JXQ6).
  • the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,3-galactosyltransferase like e.g. WbgO from E. coli O55:H7 (UniProt ID D3QY14).
  • the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1,4-galactosyltransferase like e.g. LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000).
  • the N-acetylglucosamine beta-1,3-galactosyltransferase and the N-acetylglucosamine beta-1,4-galactosyltransferase can be delivered to the strain either via genomic knock-in or from an expression plasmid.
  • the mutant C. glutamicum strains are further modified with a constitutive transcriptional unit for an alpha-1,2-fucosyltransferase like e.g. HpFutC from H. pylori (UniProt ID Q9X435) and/or an alpha-1,3-fucosyltransferase like e.g.
  • HpFucT from H. pylori (UniProt ID O30511).
  • slaughterhouses e.g. cattle, pigs, sheep, chicken, ducks, catfish, snake, frogs
  • liposuction e.g., in case of humans, after informed consent
  • Enzymatic digestion of the adipose tissue is performed followed by centrifugation to isolate mesenchymal stem cells.
  • the isolated mesenchymal stem cells are transferred to cell culture flasks and grown under standard growth conditions, e.g., 37°C, 5% CO2.
  • the initial culture medium includes DMEM-F12, RPMI, and Alpha-MEM medium (supplemented with 15% foetal bovine serum), and 1% antibiotics.
  • the culture medium is subsequently replaced with 10% FBS (foetal bovine serum)-supplemented media after the first passage.
  • FBS foetal bovine serum
  • Isolation of mesenchymal stem cells from milk This example illustrates isolation of mesenchymal stem cells from milk collected under aseptic conditions from human or any other mammal(s) such as described herein.
  • An equal volume of phosphate buffer saline is added to diluted milk, followed by centrifugation for 20 min.
  • the cell pellet is washed thrice with phosphate buffer saline and cells are seeded in cell culture flasks in DMEM-F12, RPMI, and Alpha-MEM medium supplemented with 10% foetal bovine serum and 1% antibiotics under standard culture conditions.
  • Hassiotou et al. 2012, Stem Cells.
  • the isolated cells were initially seeded in culture plates in growth media supplemented with 10 ng/mL epithelial growth factor and 5 pg/mL insulin.
  • growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/mL penicillin, 100 ug/mL streptomycin), and 5 pg/mL insulin for 48h.
  • penicillin-streptomycin 100 U/mL penicillin, 100 ug/mL streptomycin
  • 5 pg/mL insulin for 48h.
  • the cells were fed with complete growth medium containing 5 pg/mL insulin, 1 pg/mL hydrocortisone, 0.65 ng/mL triiodothyronine, 100 nM dexamethasone, and 1 pg/mL prolactin.
  • the isolated cells were trypsinized and cultured in Matrigel, hyaluronic acid, or ultra- low attachment surface culture plates for six days and induced to differentiate and lactate by adding growth media supplemented with 10 ng/mL epithelial growth factor and 5 pg/mL insulin.
  • growth media supplemented with 10 ng/mL epithelial growth factor and 5 pg/mL insulin.
  • cells were fed with growth medium supplemented with 2% foetal bovine serum, 1% penicillin-streptomycin (100 U/mL penicillin, 100 ug/mL streptomycin), and 5 pg/mL insulin for 48h.
  • the cells were fed with complete growth medium containing 5 pg/mL insulin, 1 pg/mL hydrocortisone, 0.65 ng/mL triiodothyronine, 100 nM dexamethasone, and 1 pg/mL prolactin. After 24h, serum is removed from the complete induction medium. Method of making mammary-like cells In a next step, the cells are brought to induced pluripotency by reprogramming with viral vectors encoding for Oct4, Sox2, Klf4, and c-Myc.
  • the resultant reprogrammed cells are then cultured in Mammocult media (available from Stem Cell Technologies), or mammary cell enrichment media (DMEM, 3% FBS, estrogen, progesterone, heparin, hydrocortisone, insulin, EGF) to make them mammary-like, from which expression of select milk components can be induced.
  • Mammocult media available from Stem Cell Technologies
  • DMEM mammary cell enrichment media
  • epigenetic remodelling is performed using remodelling systems such as CRISPR/Cas9, to activate select genes of interest, such as casein, a- lactalbumin to be constitutively on, to allow for the expression of their respective proteins, and/or to down-regulate and/or knock-out select endogenous genes as described e.g., in WO21067641, which is incorporated herein by reference in its entirety for all purposes.
  • Cultivation Completed growth media includes high glucose DMEM/F12, 10% FBS, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/mL EGF, and 5 pg/mL hydrocortisone.
  • Completed lactation media includes high glucose DMEM/F12, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/mL EGF, 5 pg/mL hydrocortisone, and 1 pg/mL prolactin (5ug/mL in Hyunh 1991).
  • Cells are seeded at a density of 20,000 cells/cm2 onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media. Upon exposure to the lactation media, the cells start to differentiate and stop growing.
  • the cells start secreting lactation product(s) such as milk lipids, lactose, casein and whey into the media.
  • lactation product(s) such as milk lipids, lactose, casein and whey into the media.
  • a desired concentration of the lactation media can be achieved by concentration or dilution by ultrafiltration.
  • a desired salt balance of the lactation media can be achieved by dialysis, for example, to remove unwanted metabolic products from the media.
  • Hormones and other growth factors used can be selectively extracted by resin purification, for example the use of nickel resins to remove His-tagged growth factors, to further reduce the levels of contaminants in the lactated product.
  • Optical density Cell density of the cultures was frequently monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or with a Spark 10M microplate reader, Tecan, Switzerland). The maximum growth speed (mumax) was calculated based on the observed optical densities at 600nm using the R package grofit.
  • G. Heterologous and homologous expression Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: IDT, Twist Bioscience, DNA2.0 or Gen9. Proteins, promoter and 5’UTR sequences described in present disclosure are summarized in Table 1.
  • the UniProt IDs of the proteins described correspond to their sequence version 01 as present in the UniProt Database version release 2021_03 of 09 June 2021. Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier. Table 1.
  • a volume of 0.7 ⁇ L sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 ⁇ ;1.7 ⁇ m) column with an Acquity UPLC BEH Amide VanGuard column, 130 ⁇ , 2.1x 5 mm.
  • the column temperature was 50 °C.
  • the mobile phase consisted of a 1 ⁇ 4 water and 3 ⁇ 4 acetonitrile solution to which 0.2 % triethylamine was added.
  • the method was isocratic with a flow of 0.130 mL/min.
  • the ELS detector had a drift tube temperature of 50 °C and the N2 gas pressure was 50 psi, the gain 200 and the data rate 10 pps.
  • the temperature of the RI detector was set at 35 °C.
  • Sialylated oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Refractive Index (RI) detection.
  • RI Refractive Index
  • a volume of 0.5 ⁇ L sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 ⁇ ;1.7 ⁇ m).
  • the column temperature was 50 °C.
  • the mobile phase consisted of a mixture of 70 % acetonitrile, 26 % ammonium acetate buffer (150 mM) and 4 % methanol to which 0.05 % pyrrolidine was added.
  • the method was isocratic with a flow of 0.150 mL/min.
  • the temperature of the RI detector was set at 35 °C. Both neutral and sialylated sugars were analyzed on a Waters Acquity H-class UPLC with Refractive Index (RI) detection. A volume of 0.5 ⁇ L sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 ⁇ ;1.7 ⁇ m). The column temperature was 50°C. The mobile phase consisted of a mixture of 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which 0.1% triethylamine was added. The method was isocratic with a flow of 0.260 mL/min. The temperature of the RI detector was set at 35°C.
  • a Waters Xevo TQ-MS with Electron Spray Ionisation (ESI) was used with a desolvation temperature of 450 °C, a nitrogen desolvation gas flow of 650 L/h and a cone voltage of 20 V.
  • the MS was operated in selected ion monitoring (SIM) in negative mode for all oligosaccharides. Separation was performed on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1 x 100 mm; 3 ⁇ m) on 35 °C.
  • eluent A was ultrapure water with 0.1 % formic acid and wherein eluent B was acetonitrile with 0.1 % formic acid.
  • the oligosaccharides were separated in 55 min using the following gradient: an initial increase from 2 to 12 % of eluent B over 21 min, a second increase from 12 to 40 % of eluent B over 11 min and a third increase from 40 to 100 % of eluent B over 5 min.
  • As a washing step 100 % of eluent B was used for 5 min.
  • the initial condition of 2 % of eluent B was restored in 1 min and maintained for 12 min.
  • samples were diluted two times in acetonitrile and subsequently mixed with 2,5-dihydroxybenzoic acid (DHB, Merck Life Science B.V.) matrix and Girard’s Reagent T (GT, Merck Life Science B.V.).
  • Samples were analysed using a 4800 Plus MALDI TOF/TOF Analyser (Applied Biosystems, Germany) with an Nd:YAG laser (200 Hz, 355 nm) controlled by the 4000 Series Explorer software version 3.5.3 (Applied Biosystems, Germany).
  • the instrument was operated in positive ion mode with delayed extraction and an acceleration voltage of 20 kV with a grid of 15.6 kV.
  • eluent A was deionized water
  • eluent B was 200 mM Sodium hydroxide
  • eluent C was 500 mM Sodium acetate.
  • the oligosaccharides were separated in 60 min while maintaining a constant ratio of 25 % of eluent B using the following gradient: an initial isocratic step maintained for 10 min of 75 % of eluent A, an initial increase from 0 to 4 % of eluent C over 8 min, a second isocratic step maintained for 6 min of 71 % of eluent A and 4 % of eluent C, a second increase from 4 to 12 % of eluent C over 2.6 min, a third isocratic step maintained for 3.4 min of 63 % of eluent A and 12 % of eluent C and a third increase from 12 to 48 % of eluent C over 5 min.
  • Total DNA is measured by means of a Threshold assay (Molecular Devices), based on an immunoassay allowing to measure as low as 2 pg of DNA in a sample in solution. Double stranded DNA is measured by means of SpectraMax® QuantTM AccuBlueTM Pico dsDNA Assay Kit (Molecular Devices) having a linear range between 5 pg and 3 ng of dsDNA.
  • Threshold assay based on an immunoassay allowing to measure as low as 2 pg of DNA in a sample in solution.
  • Double stranded DNA is measured by means of SpectraMax® QuantTM AccuBlueTM Pico dsDNA Assay Kit (Molecular Devices) having a linear range between 5 pg and 3 ng of dsDNA.
  • Example 3 Evaluation of production of a mixture comprising LSTc and LNnT with a modified E. coli host An E.
  • coli K-12 MG1655 strain modified for uptake of sucrose as described in Example 2 is further modified for production of sialic acid with genomic knock-ins of constitutive transcriptional units containing a hydrolyzing UDP-N-acetylglucosamine 2-epimerase selected from the list consisting of SEQ ID NO 01, 02, 03, 04, 05, 06, 07 and 09 and the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4).
  • the mutant strains are further modified by knocking in a constitutive transcriptional unit for the beta-1,3-N-acetylglucosaminyltransferase lgtA from N.
  • mutant strains are transformed with an expression plasmid comprising constitutive transcriptional units for the N- acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2,6- sialyltransferase from B. psittacipulmonis with SEQ ID NO 13.
  • the novel strains are evaluated in a 96-well plate according to the culture conditions provided in Example 2 in which the strain is cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. After 72h of incubation, the culture broth is harvested and analysed for production of a mixture comprising the sialylated milk oligosaccharide LSTc (Neu5Ac- ⁇ 2,6-Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc) and the non-sialylated milk oligosaccharides LN3 and LNnT as described in Example 2.
  • LSTc sialylated milk oligosaccharide
  • the strain was further modified with genomic knock-ins of constitutive transcriptional units containing nucleotide sequences encoding the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4) and a hydrolyzing UDP-N-acetylglucosamine-2-epimerase selected from the list consisting of SEQ ID NO 01, 02, 05 and 09.
  • the strains were modified with a genomic knock-in of constitutive transcriptional units for the phosphomannomutase manB from E.
  • strains A, B, C and D were further modified with a plasmid containing a constitutive transcriptional unit containing nucleotide sequences encoding neuA from C. jejuni (UniProt ID Q93MP7) and a constitutive transcriptional unit containing nucleotide sequences encoding the alpha-2,6-sialyltransferase from Basilea psittacipulmonis with SEQ ID NO 13.
  • the novel strains were modified for growth on sucrose as in Example 2, resulting in strains E, F, G and H (See Table 2).
  • the novel strains E-H were evaluated in a growth experiment for production of an oligosaccharide mixture comprising 6’SL, LN3, LNT, LNnT, pLNnH (para-lacto-N-neohexaose, Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc), LSTc and/or Neu5Ac-a2,6-Gal- ⁇ 1,3-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc according to the culture conditions provided in Example 2 in which the strains were cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose.
  • the strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed on UPLC. Results are shown in Table 2. The experiment demonstrated that the strains produced an oligosaccharide mixture comprising 0.5 to 71.3 weight % sialylated oligosaccharides and 99.5 to 28.7 weight % non-sialylated milk oligosaccharides, calculated relatively to the total amount of milk oligosaccharides produced.
  • the fraction of sialylated milk oligosaccharides comprised 6’SL, LSTc and Neu5Ac-a2,6-Gal- ⁇ 1,3-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc.
  • the fraction of non-sialylated oligosaccharide comprised LN3, LNnT, LNT and pLNnH. Table 2. Production of a mixture comprising sialylated oligosaccharides (weight %) and non-sialylated oligosaccharides (weight %) in mutant E.
  • Example 5 Evaluation of production of a mixture of sialylated and non-sialylated milk oligosaccharides with a modified E. coli host An E.
  • coli K-12 MG1655 strain modified for production of LNT and LNnT as described in Example 4 was further modified for production of sialic acid with genomic knock-ins of constitutive transcriptional units containing nucleotide sequences encoding the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4) and a hydrolyzing UDP-N-acetylglucosamine-2-epimerase selected from the list consisting of SEQ ID NO 01, 02 and 09.
  • the novel strains 10-18 were evaluated in a growth experiment for production of an oligosaccharide mixture comprising 6’SL, LN3, LNT, LNnT, pLNnH (para- lacto-N-neohexaose, Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc), LSTc and/or Neu5Ac- a2,6-Gal- ⁇ 1,3-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc according to the culture conditions provided in Example 2 in which the strains were cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose.
  • each strain with a particular transcriptional unit of a hydrolyzing UDP-N- acetylglucosamine-2-epimerase tested produced a mixture comprising 0.5 to 68.7 weight % sialylated milk oligosaccharides and 99.5 to 31.3 weigh% non-sialylated milk oligosaccharides, calculated relatively to the total amount of oligosaccharides produced.
  • the fraction of sialylated oligosaccharides comprised 6’SL, LSTc and Neu5Ac-a2,6-Gal- ⁇ 1,3-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc.
  • the fraction of non-sialylated oligosaccharides comprised LN3, LNnT, LNT and pLNnH.
  • the mutant E. coli strains A, B, C and D as described in Example 4 were further modified to allow production of sialylated milk oligosaccharides with a plasmid containing a constitutive transcriptional unit containing nucleotide sequences encoding neuA from C. jejuni (UniProt ID Q93MP7) and a constitutive transcriptional unit containing nucleotide sequences encoding the alpha-2,3-sialyltransferase SagalST from Streptococcus agalactiae with SEQ ID NO 14.
  • the novel strains were modified for growth on sucrose as in Example 2, resulting in strains I, J, K and L (See Table 4).
  • the novel strains I- L were evaluated in a growth experiment for production of an oligosaccharide mixture comprising 3’SL, LN3, LNT, LNnT, pLNnH, LSTd and/or LSTa according to the culture conditions provided in Example 2 in which the strains were cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. The strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed on UPLC.
  • each strain with a particular transcriptional unit of a hydrolyzing UDP-N-acetylglucosamine-2-epimerase tested produced an oligosaccharide mixture comprising 20 to 57.4 weight % sialylated milk oligosaccharides and 80 to 42.6 weight % non-sialylated milk oligosaccharides, calculated relatively to the total amount of oligosaccharides produced.
  • the fraction of sialylated milk oligosaccharides comprised 3’SL and LSTd.
  • the fraction of non-sialylated milk oligosaccharides comprised LN3, LNnT, LNT and pLNnH.
  • Example 7 Evaluation of production of a mixture of sialylated and non-sialylated milk oligosaccharides with a modified E. coli host The mutant E.
  • coli strains 1-9 as described in Example 5 were further modified to allow production of sialylated oligosaccharides with a plasmid containing a constitutive transcriptional unit containing nucleotide sequences encoding neuA from C. jejuni (UniProt ID Q93MP7) and a constitutive transcriptional unit containing nucleotide sequences encoding the alpha-2,3-sialyltransferase SagalST from Streptococcus agalactiae with SEQ ID NO 14.
  • the novel strains were modified for growth on sucrose as in Example 2, resulting in strains 19-27 (See Table 5).
  • the novel strains 19-27 were evaluated in a growth experiment for production of an oligosaccharide mixture comprising 3’SL, LN3, LNT, LNnT, pLNnH, LSTd and/or LSTa according to the culture conditions provided in Example 2 in which the strains were cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. The strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed on UPLC.
  • each strain with a particular transcriptional unit of a hydrolyzing UDP-N-acetylglucosamine-2-epimerase tested produced an oligosaccharide mixture comprising 1.2 to 60.9 weight % sialylated oligosaccharides and 98.8 to 39.1 weight % non-sialylated oligosaccharides, calculated relatively to the total amount of oligosaccharides produced.
  • the fraction of sialylated oligosaccharides comprised 3’SL and LSTd.
  • the fraction of non- sialylated oligosaccharide comprised LN3, LNnT, LNT and pLNnH.
  • Example 8 Evaluation of production of a mixture of sialylated and non-sialylated milk oligosaccharides with a modified E.
  • the mutant E. coli strains A, B, C and D as described in Example 4 were further modified to allow production of sialylated milk oligosaccharides with a plasmid containing a constitutive transcriptional unit containing nucleotide sequences encoding neuA from C. jejuni (UniProt ID Q93MP7) and a constitutive transcriptional unit containing nucleotide sequences encoding the alpha-2,6-sialyltransferase from B. psittacipulmonis with SEQ ID NO 13 and the alpha-2,3-sialyltransferase SagalST from Streptococcus agalactiae with SEQ ID NO 14.
  • the novel strains were modified for growth on sucrose as in Example 2, resulting in strains M, N, O and P (See Table 6).
  • the novel strains M-P were evaluated in a growth experiment for production of an oligosaccharide mixture comprising 3’SL, 6’SL, LN3, LNT, LNnT, pLNnH, LSTc, LSTd, LSTa and/or Neu5Ac-a2,6-Gal- ⁇ 1,3-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc according to the culture conditions provided in Example 2 in which the strains were cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose.
  • each strain was grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed on UPLC. The experiment demonstrated that each strain with a particular transcriptional unit of a hydrolyzing UDP-N- acetylglucosamine-2-epimerase tested produced an oligosaccharide mixture comprising 20.3 to 95 weight % sialylated milk oligosaccharides and 79.7 to 5 weight % non-sialylated milk oligosaccharides, calculated relatively to the total amount of oligosaccharides produced.
  • the fraction of sialylated milk oligosaccharides comprised 3SL, 6SL, LSTc, LSTd and Neu5Ac-a2,6-Gal- ⁇ 1,3-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc.
  • the fraction of non-sialylated milk oligosaccharides comprised LN3, LNnT, LNT and pLNnH.
  • the data clearly showed that the production of sialylated milk oligosaccharides and non-sialylated milk oligosaccharides could be tuned based on the hydrolyzing UDP-N-acetylglucosamine-2-epimerase selected (see Table 6). Table 6.
  • expression of said hydrolyzing UDP-N-acetylglucosamine- 2-epimerases was varied by the use of a promoter of interest selected from the list consisting of SEQ ID NO 15, 16 and 17, and by the use of a 5’UTR of interest selected from the list consisting of SEQ ID NO 18 and 19.
  • the strains were further modified as described in Example 4 to allow production of GDP-fucose.
  • the mutant E. coli strains were further modified to allow production of sialylated oligosaccharides with a plasmid containing a constitutive transcriptional unit containing nucleotide sequences encoding neuA from C.
  • the novel strains 28-36 were evaluated in a growth experiment for production of an oligosaccharide mixture comprising 3’SL, 6’SL, LN3, LNT, LNnT, pLNnH, LSTc, LSTd, LSTa and/or Neu5Ac-a2,6-Gal- ⁇ 1,3-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc according to the culture conditions provided in Example 2 in which the strains were cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. The strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed on UPLC.
  • each strain with a particular transcriptional unit of a hydrolyzing UDP-N-acetylglucosamine-2-epimerase tested produced an oligosaccharide mixture comprising 3.4 to 95 weight % sialylated milk oligosaccharides and 96.6 to 5 weight % non-sialylated milk oligosaccharides, calculated relatively to the total amount of oligosaccharides produced.
  • the fraction of sialylated milk oligosaccharides comprised 3SL, 6SL, LSTc, LSTd and Neu5Ac-a2,6-Gal- ⁇ 1,3-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc.
  • the fraction of non-sialylated milk oligosaccharides comprised LN3, LNnT, LNT and pLNnH.
  • Table 7 Production of a mixture comprising sialylated oligosaccharides (weight %) and non-sialylated oligosaccharides (weight %) in mutant E.
  • Example 10 coli strains 28-36 expressing an alpha-2,6-sialyltransferase with SEQ ID NO 13 and an alpha-2,3-sialyltransferase with SEQ ID NO 14 and a hydrolyzing UDP-N- acetylglucosamine-2-epimerase (with a SEQ ID NO 01, 02 or 09) with varying expression levels by expression under control of a selected promoter sequence (SEQ ID NO 15, 16 or 17) and 5’UTR sequence (SEQ ID NO 18 or 19) when evaluated in a growth experiment according to the culture conditions provided in Example 2, in which the cultivation medium contained 30 g/L sucrose and 20 g/L lactose.
  • Example 10 Example 10
  • each strain with a particular transcriptional unit of a hydrolyzing UDP-N-acetylglucosamine-2-epimerase tested produced an oligosaccharide mixture comprising of 5.5 to 25 weight % sialylated milk oligosaccharides and 94.5 to 75 weight % non-sialylated milk oligosaccharides.
  • the fraction of sialylated oligosaccharides comprised 3’SL and LSTd.
  • the fraction of non-sialylated milk oligosaccharides comprised LN3, LNnT, LNT and pLNnH.
  • Example 11 Evaluation of production of a mixture of sialylated and non-sialylated oligosaccharides with a modified E. coli host on 5L bioreactor An E.
  • coli K-12 MG1655 strain modified for production of LNT and LNnT as described in Example 4 is further modified to allow production of sialic acid with genomic knock-ins of constitutive transcriptional units containing nucleotide sequences encoding the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4) and a hydrolyzing UDP-N-acetylglucosamine-2-epimerase selected from the list consisting of SEQ ID NO 01, 02, 03, 04, 05, 0607 and 09.
  • the genes encoding said hydrolyzing UDP- N-acetylglucosamine-2-epimerase enzymes are cloned under control of promoter with SEQ ID NO 15 and of 5’UTR with SEQ ID NO 18. All mutant strains are further modified as described in Example 4 to allow production of GDP-fucose. To allow production of fucosylated and sialylated milk oligosaccharides the strains are further modified with a plasmid containing a constitutive transcriptional unit containing nucleotide sequences encoding neuA from C.
  • the novel strains are evaluated in a growth experiment for production of an oligosaccharide mixture comprising 2’FL, 3’SL, DiFL, LNFP-I, LSTa, LSTd, LNT, LNnT, LN3 and pLNnH according to the culture conditions provided in Example 2 in which the strains are cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. The strains are grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested, and the sugars are analysed on UPLC. Example 12. Evaluation of production of a mixture of fucosylated, sialylated and neutral milk oligosaccharides with a modified E.
  • mutant E. coli strains A and D expressing a hydrolyzing UDP-N-acetylglucosamine-2-epimerase with either SEQ ID NO 01 or 09, respectively, as described in Example 4, were further modified with a genomic knock-in of a constitutive transcriptional unit containing nucleotide sequences encoding neuA from P. multocida (UniProt ID A0A849CI62) and a constitutive transcriptional unit containing nucleotide sequences encoding the alpha- 2,6-sialyltransferase from B.
  • strains S and T were further modified with a plasmid expressing a constitutive transcriptional unit containing nucleotide sequences encoding the alpha-1,2-fucosyltransferase from Helicobacter sp.
  • MIT 01-6242 UniProt ID A0A1B1U4V1
  • a constitutive transcriptional unit containing nucleotide sequences encoding the alpha-1,3- fucosyltransferase from Porphyromonas catoniae (UniProt ID Z4WWI2) resulting in strains U and V.
  • the novel strains U and V were modified for growth on sucrose as in Example 2, resulting in strains W and X (See Table 9).
  • the novel strains W and X were evaluated in a growth experiment for production of an oligosaccharide mixture comprising Fuc- ⁇ 1,2-Gal- ⁇ 1,3-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-(Fuc- ⁇ 1,3)-Glc, Fuc- ⁇ 1,2-Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-(Fuc- ⁇ 1,3)-Glc, LNnDFH-I, Fuc- ⁇ 1,4-(Fuc- ⁇ 1,2-Gal- ⁇ 1,3)-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-(Fuc- ⁇ 1,3)-Glc, LNDFH-I, LNFP-I, LNnFP-I, 2’FL, LNFP-VI, LNnDFH-II
  • the strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed on UPLC. Results are shown in Table 9. The experiment demonstrated that the strains produced an oligosaccharide mixture comprising 83.9 to 84.7 weight % fucosylated oligosaccharides, 10.1 to 10.3 weight % sialylated oligosaccharides and 5.2 to 5.9 weight % neutral milk oligosaccharides, calculated relatively to the total amount of milk oligosaccharides produced.
  • the fraction of fucosylated milk oligosaccharides comprised Fuc- ⁇ 1,2-Gal- ⁇ 1,3-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-(Fuc- ⁇ 1,3)-Glc, Fuc- ⁇ 1,2-Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-(Fuc- ⁇ 1,3)-Glc, LNnDFH-I, Fuc- ⁇ 1,4-(Fuc- ⁇ 1,2-Gal- ⁇ 1,3)-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-(Fuc- ⁇ 1,3)-Glc, LNDFH-I, LNFP-I, LNnFP-I, 2’FL, LNFP-VI, LNnDFH-II, LNFP-III, LNDFH-II, 3-FL and DiFL.
  • the fraction of sialylated milk oligosaccharides comprised 6’SL and LSTc.
  • the fraction of neutral oligosaccharides comprised LN3, LNnT and LNT.
  • Table 9 Production of a mixture comprising fucosylated oligosaccharides, sialylated oligosaccharides and neutral oligosaccharides (weight %) in mutant E. coli strains W and X expressing a hydrolyzing UDP- N-acetylglucosamine-2-epimerase (with a SEQ ID NO 01 or 09) when evaluated in a growth experiment according to the culture conditions provided in Example 2, in which the cultivation medium contained 30 g/L sucrose and 20 g/L lactose.
  • Example 13 Example 13
  • strain W with a transcriptional unit of the hydrolyzing UDP-N-acetylglucosamine-2-epimerase with SEQ ID NO 01 tested produced an oligosaccharide mixture comprising of 67.9 weight % fucosylated milk oligosaccharides, 12.8 weight % sialylated milk oligosaccharides and 19.1 weight % neutral milk oligosaccharides.
  • the fraction of fucosylated oligosaccharides comprised Fuc- ⁇ 1,2-Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-(Fuc- ⁇ 1,3)-Glc, LNDFH- I, LNFP-I, LNnFP-I, 2’FL, LNFP-VI, LNFP-III, 3-FL and DiFL
  • the fraction sialylated oligosaccharides comprised 6’SL, LSTc, Neu5Ac- ⁇ 2,6-(Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3)-Gal- ⁇ 1,4-Glc and Neu5Ac- ⁇ 2,6-Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3- Gal- ⁇ 1,4-GlcNAc- ⁇ 1,3-Gal- ⁇ 1,4-Glc.
  • the fraction of neutral milk oligosaccharides comprised LN3, LNnT and LNT.

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Abstract

La présente invention se rapporte au domaine technique de la biologie synthétique, de l'ingénierie métabolique et de la culture cellulaire. La présente invention concerne des procédés de production d'un mélange d'au moins deux oligosaccharides de lait comprenant au moins un oligosaccharide de lait sialylé et au moins un oligosaccharide de lait non sialylé ainsi que la purification dudit mélange d'oligosaccharides de lait. La présente invention concerne également une cellule pour la production dudit mélange d'oligosaccharide de lait et l'utilisation de ladite cellule dans une culture ou une incubation.
PCT/EP2025/061445 2024-04-26 2025-04-25 Production d'un mélange d'oligosaccharides de lait Pending WO2025224348A1 (fr)

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PCT/EP2024/061529 WO2024223815A1 (fr) 2023-04-26 2024-04-26 Production d'un oligosaccharide chargé négativement par une cellule
EPPCT/EP2024/061529 2024-04-26
EP24209968.7 2024-10-30
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EP24209968 2024-10-30
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