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WO2024261312A2 - Sialyltransférases pour la production d'oligosaccharides sialylés - Google Patents

Sialyltransférases pour la production d'oligosaccharides sialylés Download PDF

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Publication number
WO2024261312A2
WO2024261312A2 PCT/EP2024/067548 EP2024067548W WO2024261312A2 WO 2024261312 A2 WO2024261312 A2 WO 2024261312A2 EP 2024067548 W EP2024067548 W EP 2024067548W WO 2024261312 A2 WO2024261312 A2 WO 2024261312A2
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Prior art keywords
cell
amino acid
oligosaccharide
acceptor
monosaccharide
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WO2024261312A3 (fr
Inventor
Joeri Beauprez
Thomas DECOENE
Annelies VERCAUTEREN
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Inbiose NV
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Inbiose NV
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    • 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
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • 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.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • 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
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/99—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)

Definitions

  • the present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation.
  • the present invention relates to newly identified sialyltransferases having alpha-2, 3- sialyltransferase activity on an acceptor which is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • the invention also describes methods for the production of a 3'sialylated oligosaccharide using any one of said newly identified sialyltransferases as well as the purification of said 3'sialylated oligosaccharide.
  • the present invention also provides a cell for production of said 3'sialylated oligosaccharide and the use of said cell in a cultivation or incubation.
  • HMOs human milk oligosaccharides
  • sialylated HMOs were observed to support several beneficial effects as described in the art.
  • sialylated oligosaccharides in human milk 3'sialyllactose, 5'sialyllactose, sialyllacto-N-tetraose a, sialyl lacto-N-tetraose b, sialyllacto-N-tetraose c and disialyllacto-N-tetraose are the most prevalent members.
  • Sialylated oligosaccharides are found to be a complex structure and their chemical or (chemo-)enzymatic syntheses has been proven challenging: there are extensive difficulties, e.g. control of stereochemistry, formation of specific linkages, availability of feedstocks, etc. As a consequence, alternative production methods have been developed, amongst which efforts in metabolic engineering of microorganisms to produce sialylated oligosaccharides have been made.
  • sialyltransferases have been identified and characterized to date, from bacterial species e. g. from Neisseria, Campylobacter, Pasteurella, Helicobacter and Photobacterium, as well as from mammals and viruses.
  • Sialyltransferases have been generally classified into six glycosyltransferase (GT) families, based on protein sequence similarities.
  • GT glycosyltransferase
  • Sialyltransferases are distinguished due to the glycosidic linkages that they form, e. g. into a-2,3-, a-2,6- and a-2,8-sialyltransferases.
  • sialyltransferases transfer the sialic acid residue from cytidine 5'-monophosphate sialic acid (e. g. CMP-NeuNAc) to a variety of acceptor molecules, usually a galactose (Gal) moiety, an N-acetylgalactosamine (GalNAc) moiety or an N- acetylglucosamine (GIcNAc) moiety or another sialic acid (Sia) moiety.
  • Gal galactose
  • GalNAc N-acetylgalactosamine
  • GIcNAc N- acetylglucosamine
  • a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, can be produced, preferably in an efficient, time and cost-effective way and which yields high amounts of the desired oligosaccharide.
  • this and other objects are achieved by providing newly identified alpha-2, 3- sialyltransferases as described herein, each of which can be used in a method for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • Such method comprising contacting a sialyltransferase with a mixture comprising a donor comprising a sialic acid residue, and an acceptor, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from the donor to the acceptor, thereby producing said 3'sialylated oligosaccharide, wherein said acceptor is a saccharide comprising at least one N- acetylglucosamine monosaccharide and a galactose monosaccharide, chosen from the list consisting of an oligosaccharide or a disaccharide.
  • any one of said newly identified alpha-2, 3-sialyltransferases can be used in a cell for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • the invention provides newly identified alpha-2, 3-sialyltransferases having alpha-2, 3- sialyltransferase activity on a galactose (Gal) residue, preferably a terminal Gal residue, of an acceptor wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide chosen from the list consisting of an oligosaccharide or disaccharide, and wherein any one or more of said alpha-2, 3-sialyltransferase comprise an amino acid sequence that is at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30, 31, 25, 18, 26 or 22, or that is at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues
  • the invention also provides methods and a cell for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • the present invention also provides methods for the purification of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • the present invention provides a cell which is metabolically engineered with any one of said newly identified alpha-2, 3-sialyltransferases as described herein and which comprises a pathway for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • the present invention also provides for newly identified alpha-2, 3-sialyltransferases for use in the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • the expressions “capable of... ⁇ verb>” and “capable to... ⁇ verb>” are preferably replaced with the active voice of said verb and vice versa.
  • the expression “capable of expressing” is preferably replaced with “expresses” and vice versa, i.e. "expresses” is preferably replaced with "capable of expressing”.
  • the verbs "to comprise”, “to have” and “to contain” and their conjugations are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • the verb "to comprise” may be replaced by “to consist of” or “to consist essentially of” and vice versa.
  • the verb "to consist of” may be replaced by "to consist essentially of” meaning that a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • 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”.
  • polynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotide(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triplestranded regions, or a mixture of single- and double-stranded regions.
  • 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.
  • the term "polynucleotide(s)” also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" according to the present invention.
  • 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”.
  • polynucleotides DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases.
  • polynucleotides are to be understood to be covered by the term “polynucleotides”.
  • 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. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to the skilled person.
  • 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
  • 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.
  • recombinant or “transgenic” or “metabolically engineered” or “genetically engineered” as used herein with reference to a cell or host cell are used interchangeably and indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e. a sequence "foreign to said cell” or a sequence "foreign to said location or environment in said cell”).
  • Such cells are described to be transformed with at least one heterologous or exogenous gene or are described to be transformed by the introduction of at least one heterologous or exogenous gene.
  • Metabolically engineered or recombinant or transgenic or genetically engineered cells can contain genes that are not found within the native (non-recombinant) form of the cell.
  • Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means.
  • the terms also encompass cells that contain a nucleic acid endogenous to the cell that has been modified or its expression or activity has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, replacement of a promoter; site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis and related techniques as known to a person skilled in the art. Accordingly, a "recombinant polypeptide" is one which has been produced by a recombinant cell.
  • the terms also encompass cells that have been modified by removing a nucleic acid endogenous to the cell by means of common well-known technologies for a skilled person (like e.g. knocking-out genes).
  • 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.
  • mutant or engineered cell or microorganism refers to a cell or microorganism which is genetically engineered.
  • 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.
  • modified expression of a gene relates to a change in expression compared to the wild-type expression of said gene in any phase of the production process of the desired 3'sialylated oligosaccharide. Said modified expression is either a lower or higher expression compared to the wild-type, wherein the term “higher expression” is also defined as “overexpression” of said gene in the case of an endogenous gene or “expression” in the case of a heterologous gene that is not present in the wild-type strain.
  • Lower expression is obtained by means of common well-known technologies for a skilled person (such as the usage of siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon mutagenesis, etc.) which are used to change the genes in such a way that they are less able (i.e. statistically significantly 'less able' compared to a functional wild-type gene) or completely unable (such as knocked-out genes) to produce functional final products.
  • 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 (for instance a sialyltransferase gene sequence) and optionally a transcription terminator is present, and leading to the expression of a functional active protein. Said expression is either constitutive or conditional or regulated or tuneable.
  • RNA polymerase e.g. the bacterial sigma factors like o 70 , ⁇ J 54 , or related o- factors and the yeast mitochondrial RNA polymerase specificity factor MTF1 that co-associate with the RNA polymerase core enzyme
  • transcription factors are CRP, Lacl, ArcA, Cra, IcIR in E. coli, or Aft2p, Crzlp, Skn7 in Saccharomyces cerevisiae, or DeoR, GntR, Fur in B. subtilis.
  • RNA polymerase is the catalytic machinery for the synthesis of RNA from a DNA template. RNA polymerase binds a specific DNA sequence to initiate transcription, for instance via a sigma factor in prokaryotic hosts or via MTFl in yeasts. Constitutive expression offers a constant level of expression with no need for induction or repression.
  • regulated expression is defined as a facultative or regulatory or tuneable expression of a gene that is only expressed upon a certain natural condition of the host (e.g. mating phase of budding yeast, stationary phase of bacteria), as a response to an inducer or repressor such as but not limited to glucose, allo-lactose, lactose, galactose, glycerol, arabinose, rhamnose, fucose, IPTG, methanol, ethanol, acetate, formate, aluminium, copper, zinc, nitrogen, phosphates, xylene, carbon or nitrogen depletion, or substrates or the produced product or chemical repression, as a response to an environmental change (e.g.
  • Regulated expression allows for control as to when a gene is expressed.
  • inducible expression by a natural inducer is defined as a facultative or regulatory expression of a gene that is only expressed upon a certain natural condition of the host (e.g. organism being in labour, or during lactation), as a response to an environmental change (e.g. including but not limited to hormone, heat, cold, pH shifts, light, oxidative or osmotic stress / signalling), or dependent on the position of the developmental stage or the cell cycle of said host cell including but not limited to apoptosis and autophagy.
  • 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
  • inducible expression upon chemical treatment is defined as a facultative or regulatory expression of a gene that is only expressed upon treatment with a chemical inducer or repressor, wherein said inducer and repressor comprise but are not limited to an alcohol (e.g. ethanol, methanol), a carbohydrate (e.g. glucose, galactose, glycerol, lactose, arabinose, rhamnose, fucose, allo-lactose), metal ions (e.g. aluminium, copper, zinc), nitrogen, phosphates, IPTG, acetate, formate, xylene.
  • an alcohol e.g. ethanol, methanol
  • carbohydrate e.g. glucose, galactose, glycerol, lactose, arabinose, rhamnose, fucose, allo-lactose
  • metal ions e.g. aluminium, copper, zinc
  • 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.
  • the control sequences that are suitable for prokaryotes for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • DNA for a presequence or secretory leader may be operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • Said control sequences can furthermore be controlled with external chemicals, such as, but not limited to, IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose via an inducible promoter or via a genetic circuit that either induces or represses the transcription or translation of said polynucleotide to a polypeptide.
  • 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.
  • wild-type refers to the commonly known genetic or phenotypical situation as it occurs in nature.
  • 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 expression of a protein refers to i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein or iii) expression and/or overexpression of a variant protein that has a higher activity compared to the wild-type (i.e. native in the expression host) protein.
  • non-native indicates that the 3'sialylated oligosaccharide is i) not naturally produced or ii) when naturally produced not in the same amounts by the cell; and that the cell has been genetically engineered to be able to produce said 3'sialylated oligosaccharide or to have a higher production of the 3'sialylated oligosaccharide.
  • 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 as used in the present invention.
  • Variants can be produced by amino acid substitution, deletion, addition, or combinations thereof. For instance, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (e.g. conservative mutations) will not have a major effect on the biological activity of the resulting molecule.
  • Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Whether a change in the amino acid sequence of a polypeptide of the invention results in a functional homolog can be readily determined by assessing the ability of the variant polypeptide to produce a response in cells in a fashion similar to the wild-type polypeptide.
  • “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 50.0%, 60.0%, 70.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0% 91.0% 92.0% 93.0% 94.0% 95.0% 95.5% 96.0% 96.5% 97.0% 97.5% 98.0% 98.5% 99.0% 99.5%, 100%, preferably at least 80.0%, more preferably at least 85.0%, even more preferably at least 87.0%, even more preferably at least 90.0%, even more preferably at least 95.0%, most preferably at least 97.0%, of the full-length
  • a fragment of a polynucleotide SEQ ID NO preferably means a nucleotide sequence which comprises or consists of said polynucleotide SEQ ID NO, wherein an amount of consecutive nucleotides is missing and wherein said amount is no more than 50.0%, 40.0%, 30.0% of the full-length of said polynucleotide SEQ ID NO, preferably no more than 20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, more preferably no more than 15.0%, even more preferably no more than 10.0%, even more preferably no more than 5.0%, most preferably no more than 2.5%, of the full-length of said polynucleotide SEQ ID NO and wherein said fragment retains a usable, functional
  • “Fragment”, with respect to a polypeptide refers to a subsequence of the polypeptide that performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide.
  • a “subsequence of the polypeptide” or “a stretch of amino acid residues” as described herein refers to a sequence of contiguous amino acid residues derived from the polypeptide.
  • a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription.
  • Fragments can vary in size from as few as 3 amino acid residues to the full length of the intact polypeptide, for example at least about 10 amino acid residues in length, for example at least about 20 amino acid residues in length, for example at least about 30 amino acid residues in length, for example at least about 150 amino acid residues in length, for example at least about 200 amino acid residues in length.
  • 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 50.0%, 60.0%, 70.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0% 85.0% 86.0% 87.0% 88.0% 89.0% 90.0% 91.0% 92.0% 93.0% 94.0% 95.0% 95.5% 96.0% 96.5% 97.0% 97.5% 98.0% 98.5% 99.0% 99.5°% 100% preferably at least 80.0% more preferably at least 85.0%, even more preferably at least 87.0%, even more preferably at least 90.0%, even more preferably at least 95.0%, most preferably at least 97.0% of the full-length of said polypeptide SEQ
  • 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 (or UniProt ID) and that performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent, as does
  • polypeptide SEQ ID NO SEQ ID NO
  • polypeptide UniProt ID 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. By conservative substitutions is intended substitutions of one hydrophobic amino acid for another or substitution of one polar amino acid for another or substitution of one acidic amino acid for another or substitution of one basic amino acid for another etc.
  • 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) el002514). 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.
  • 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.
  • manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated.
  • a domain can be characterized, for example, by a Pfam (El-Gebal i et al., Nucleic Acids Res. 47 (209) D427- D432), an IPR (InterPro domain) (http://ebi.ac.uk/interpro) (Mitchell et al., Nucleic Acids Res. 47 (2019) D351-D360), a protein fingerprint domain (PRINTS) (Attwood et al., Nucleic Acids Res. 31 (2003) 400-402), a SUBFAM domain (Gough et al., J. Mol. Biol. 313 (2001) 903-919), a TIGRFAM domain (Selengut et al., Nucleic Acids Res.
  • Protein or polypeptide sequence information and functional information can be provided by a comprehensive resource for protein sequence and annotation data like e.g. the Universal Protein Resource (UniProt) (www.uniprot.or ) (Nucleic Acids Res. 2021, 49 (DI), D480-D489).
  • UniProt comprises the expertly and richly curated protein database called the UniProt Knowledgebase (UniProtKB), together with the UniProt Reference Clusters (UniRef) and the UniProt Archive (UniParc).
  • the UniProt identifiers (UniProt ID) are unique for each protein present in the database.
  • sequence of a polypeptide is represented by a SEQ ID NO or a UniProt ID.
  • 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.
  • InterPro provides functional analysis of proteins by classifying them into families and predicting domains and important sites. To classify proteins in this way, InterPro uses predictive models, known as signatures, provided by several different databases (referred to as member databases) that make up the InterPro consortium. Protein signatures from these member databases are combined into a single searchable resource, capitalizing on their individual strengths to produce a powerful integrated database and diagnostic tool.
  • member databases predictive models, known as signatures, provided by several different databases (referred to as member databases) that make up the InterPro consortium. Protein signatures from these member databases are combined into a single searchable resource, capitalizing on their individual strengths to produce a powerful integrated database and diagnostic tool.
  • 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 50%, 60%, 70%, 80%, 90% 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. In a most preferred embodiment, a partial sequence of a reference polypeptide sequence means a stretch of at least 200 amino acid residues up to the total number of amino acid residues of a reference polypeptide sequence. Using the full-length of the reference sequence in a local sequence alignment results in a global percent identity score between the test and the reference sequence.
  • Percent identity can be determined using different algorithms like for example BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403- 410; Altschul et al., 1997, Nucleic Acids Res 25: 17, 3389-402), the Clustal Omega method (Sievers et al., 2011, Mol. Syst. Biol. 7:539), the MatGAT method (Campanella et al., 2003, BMC Bioinformatics, 4:29) or EMBOSS Needle.
  • a polypeptide comprising or consisting of an amino acid sequence having 60.0% or more sequence identity over a stretch of at least 150 amino acid residues of a reference polypeptide sequence is to be understood as that the amino acid sequence has 60.0%, 61.0%, 62.0% 63.0%, 64.0%, 65.0%, 66.0%, 67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72.0%, 73.0%, 74.0%, 75.0%, 76.0%, 77.0%, 78.0%, 79.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 9
  • a polypeptide comprising or consisting of an amino acid sequence having 60.0% or more sequence identity over a stretch of at least 200 amino acid residues of a reference polypeptide sequence is to be understood as that the amino acid sequence has 60.0%, 61.0%, 62.0% 63.0%, 64.0%, 65.0%, 66.0%, 67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72.0%, 73.0%, 74.0%, 75.0%, 76.0%, 77.0%, 78.0%, 79.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 9
  • a polypeptide comprising or consisting of an amino acid sequence having 50.0% or more sequence identity to the full-length sequence of a reference polypeptide sequence is to be understood as that the amino acid sequence has 50.0%, 51.0%, 52.0%, 53.0%, 54.0%, 55.0%, 56.0%, 57.0%, 58.0%, 59.0%, 60.0%, 61.0%, 62.0%, 63.0%, 64.0%, 65.0%, 66.0%, 67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72.0%, 73.0%, 74.0%, 75.0%, 76.0%, 77.0%, 78.0%, 79.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%,
  • a polypeptide comprising, consisting of or having an amino acid sequence having 50.0% or more sequence identity to the full-length amino acid sequence of a reference polypeptide, usually indicated with a SEQ. ID NO or UniProt ID, preferably has 50.0%, 55.0%, 60.0%, 65.0%, 70.0%, 75.0%, 80.0%, 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, more preferably has at least 50.0%, even more preferably has at least 55.0%, even more preferably has at least 60.0%, even more preferably has at least 65.0%, even more preferably has at least 70.0%, even more preferably has at least 80.0%, even more preferably has at least 85.0%, most preferably has at least 90.0%, sequence identity to the full length reference sequence.
  • a polypeptide comprising or consisting of an amino acid sequence having 65.0% or more sequence identity to the full-length sequence of a reference polypeptide sequence is to be understood as that the amino acid sequence has 65.0%, 66.0%, 67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72.0%, 73.0%, 74.0%, 75.0%, 76.0%, 77.0%, 78.0%, 79.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%
  • a polypeptide comprising, consisting of or having an amino acid sequence having 65.0% or more sequence identity to the full-length amino acid sequence of a reference polypeptide, usually indicated with a SEQ. ID NO or UniProt ID, preferably has 65.0%, 70.0%, 75.0%, 80.0%, 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, more preferably has at least 65.0%, even more preferably has at least 70.0%, even more preferably has at least 80.0%, even more preferably has at least 85.0%, most preferably has at least 90.0%, sequence identity to the full length reference sequence.
  • a polypeptide comprising or consisting of an amino acid sequence having 85.0% or more sequence identity over a stretch of at least 150 amino acid residues of a reference polypeptide sequence is to be understood as that the amino acid sequence has 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 100% sequence identity over a stretch of at least 150 amino acid residues of the reference polypeptide sequence.
  • a polypeptide comprising or consisting of an amino acid sequence having 85.0% or more sequence identity over a stretch of at least 200 amino acid residues of a reference polypeptide sequence is to be understood as that the amino acid sequence has 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 100% sequence identity over a stretch of at least 200 amino acid residues of the reference polypeptide sequence.
  • a polypeptide comprising or consisting of an amino acid sequence having 85.0% or more sequence identity to the full-length sequence of a reference polypeptide sequence is to be understood as that the amino acid sequence has 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 100% sequence identity to the full-length of the amino acid sequence of the reference polypeptide sequence.
  • a polypeptide comprising, consisting of or having an amino acid sequence having 80.0% or more sequence identity to the full-length amino acid sequence of a reference polypeptide, usually indicated with a SEQ ID NO or UniProt ID, preferably has 80.0%, 85.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0% or 99.0%, more preferably has at least 80.0%, even more preferably has at least 85.0%, most preferably has at least 90.0%, sequence identity to the full length reference sequence.
  • 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 refers 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
  • Neu4Ac is also known as 4-O-acetyl-5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid or 4-O-acetyl neuraminic acid and has C11H19NO9 as molecular formula.
  • Neu5Ac is also known as 5- acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, D-glycero-5-acetamido-3,5- dideoxy-D-galacto-non-2-ulo-pyranosonic acid, 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2- nonulopyranosonic acid, 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid, 5- (acetylamino)-3,5-dideoxy-D-glycero-D-galacto-non-2-nonulosonic acid or 5-(acetylamino)-3,5-dideoxy- D-glycero-D-galacto-non-2-ulopyranosonic acid and has C11H19
  • Neu4,5Ac2 is also known as N-acetyl-4-O-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 4-O-acetyl-N- acetylneuraminate, 4-acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonate, 4-acetate 5- (acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonate, 4-acetate-5-acetamido-3,5-dideoxy-D- glycero-D-galacto-nonulosonic acid or 4-acetate 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2- nonulosonic acid and has C13H21NO10 as molecular formula.
  • Neu5,7Ac2 is also known as 7-O-acetyl-N- acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, 7-O-acetyl-N-acetylneuraminate, 7-acetate 5- acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonate, 7-acetate-5-(acetylamino)-3,5-dideoxy-D- glycero-D-galacto-2-nonulosonate, 7-acetate-5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonic acid or 7-acetate 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid and has C13H21NO10 as molecular formula.
  • Neu5,8Ac2 is also known as 5-N-acetyl-8-O-acetyl neuraminic acid and has C13H21NO10 as molecular formula.
  • Neu5,9Ac2 is also known as N-acetyl-9-O-acetylneuraminic acid, 9-anana, 9-O-acetylsialic acid, 9-O-acetyl-N-acetylneuraminic acid, 5-N-acetyl-9-O-acetyl neuraminic acid, N,9-O-diacetylneuraminate or N,9-O-diacetylneuraminate and has C13H21NO10 as molecular formula.
  • Neu4,5,9Ac3 is also known as 5-N-acetyl-4,9-di-O-acetylneuraminic acid.
  • Neu5,7,9Ac3 is also known as 5-N-acetyl-7,9-di-O-acetylneuraminic acid.
  • Neu5,8,9Ac3 is also known as 5-N-acetyl-8,9-di-O- acetylneuraminic acid.
  • Neu4,5,7,9Ac4 is also known as 5-N-acetyl-4,7,9-tri-O-acetylneuraminic acid.
  • Neu5,7,8,9Ac4 is also known as 5-N-acetyl-7,8,9-tri-O-acetylneurarriinic acid.
  • Neu4,5,7,8,9Ac5 is also known as 5-N-acetyl-4,7,8,9-tetra-O-acetylneuraminic acid.
  • Neu5Gc is also known as N-glycolyl- neuraminic acid, N-glycolylneuraminicacid, N-glycolylneuraminate, N-glycoloyl-neuraminate, N-glycoloyl- neuraminic acid, N-glycoloylneuraminic acid, 3,5-dideoxy-5-((hydroxyacetyl)amino)-D-glycero-D-galacto- 2-nonulosonic acid, 3,5-dideoxy-5-(glycoloylamino)-D-glycero-D-galacto-2-nonulopyranosonic acid, 3,5- dideoxy-5-(glycoloylamino)-D-glycero-D-galacto-non-2-ulopyranosonic acid, 3,5-dideoxy-5- [(hydroxyacetyl)amino]-D-glycero-D-galacto-non-2-ulopyranosonic acid, D
  • 2-keto-3- deoxymanno-octulonic acid is also known as KDO, Kdo, kdo, 2-keto-3-deoxy-D-mannooctanoic acid, 2- oxo-3-deoxy-D-mannooctonic acid, 3-deoxy-D-manno-2-octulosonic acid, 3-deoxy-D-manno-oct-2-ulo- pyranosonic acid, 3-deoxy-D-manno-oct-2-ulosonic acid, 3-deoxy-D-manno-octulosonic acid, 3-deoxy-D- manno-oct-2-ulopyranosonic acid, ketodeoxyoctonic acid, ketodeoxyoctulonic acid, (6R)-6- (hydroxymethyl)-l-carboxy-2-deoxy-D-lyxo-hexopyranose, keto-deoxy-octulonic acid and has C8H14O8 as molecular formula.
  • 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.
  • alpha-2, 3-sialyltransferase alpha 2,3 sialyltransferase, “3-sialyltransferase”, “a-2,3- sialyltransferase”, “a 2,3 sialyltransferase”, “3 sialyltransferase”, “3-ST”, “3ST” or “a23-ST” as used in the present invention, are used interchangeably and refer to a glycosyltransferase that catalyzes the transfer of sialic acid from the donor CMP-sialic acid, to the acceptor molecule in an alpha-2, 3-linkage.
  • monosaccharide refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed either an aldose a ketose, a deoxysugar, a deoxy-aminosugar, a uronic acid, an aldonic acid, a ketoaldonic acid, an aldaric acid or a sugar alcohol, and contains one or more hydroxyl groups per molecule.
  • Monosaccharides are saccharides containing only one simple sugar. With the term polyol is meant an alcohol containing multiple hydroxyl groups. For example, glycerol, sorbitol, or mannitol.
  • phosphorylated monosaccharide refers to a monosaccharide, which is phosphorylated.
  • phosphorylated monosaccharides include but are not limited to glucose-1- phosphate, glucose-6-phosphate, glucose-l,6-bisphosphate, galactose-l-phosphate, fructose-6- phosphate, fructose-l,6-bisphosphate, fructose-l-phosphate, glucosamine-l-phosphate, glucosamine-6- phosphate, N-acetylglucosamine-l-phosphate, mannose-l-phosphate, mannose-6-phosphate or fucose- 1-phosphate.
  • Some, but not all, of these phosphorylated monosaccharides are precursors or intermediates for the production of activated monosaccharide.
  • activated monosaccharide refers to activated forms of monosaccharides.
  • activated monosaccharides include but are not limited to UDP-N- acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), UDP- glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy--L-arabino-4-hexulose, UDP-2- acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2- acetamido-2,6-dideoxy-L-mannose), dTDP-N-
  • CMP-sialic acid refers to a nucleotide-activated form of sialic acid comprising but not limited to CMP-Neu5Ac, CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP- Neu5,9Ac 2 , CMP-Neu5,7 (8,9) Ac 2 , CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) and CMP-KDO.
  • disaccharide refers to a saccharide polymer containing two simple sugars, i.e. monosaccharides. Such disaccharides contain monosaccharides preferably selected from the list of monosaccharides as used herein above.
  • disaccharides comprise lactose (Gal-pi,4-Glc), lacto- N-biose (Gal-pi,3-GlcNAc), N-acetyllactosamine (Gal-pi,4-GlcNAc), LacDiNAc (GalNAc-pi,4-GlcNAc), N- acetylgalactosaminylglucose (GalNAc-pi,4-Glc), Neu5Ac-ot2,3-Gal, Neu5Ac-o.2,6-Gal, fucopyranosyl-(l- 4)-N-glycolylneuraminic acid (Fuc-(l-4)-Neu5Gc), sucrose (Glc-al,2-Fru), maltose (Glc-al,4-Glc) and melibiose (Gal-ocl,6-Glc).
  • oligosaccharide as used in the context of the present invention preferably refers to a saccharide containing 2 up to and including 20 monosaccharides, i.e. the degree of polymerization (DP) is 2-20, more preferably refers to a saccharide containing 3 up to and including 20 monosaccharides, i.e. the degree of polymerization (DP) is 3-20.
  • oligosaccharide preferably refers to a saccharide consisting of 2-20, more preferably 3-20, monosaccharide units which are linked to each other via glycosidic bonds in a linear or in a branched structure.
  • a beta-glycosidic bond links carbon-1 of galactose (Gal) with the carbon-4 of glucose (Glc).
  • Each monosaccharide can be in the cyclic form (e.g., pyranose or furanose form).
  • Linkages between the individual monosaccharide units may include alpha l->2, alpha l->3, alpha l->4, alpha l->6, alpha 2->l, alpha 2->3, alpha 2->4, alpha 2->6, beta l->2, beta l->3, beta l->4, beta l->6, beta 2->l, beta 2->3, beta 2->4, and beta 2->5.
  • 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.
  • oligosaccharides include but are not limited to Lewis-type antigen oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigen, enterobacterial common antigen (EGA), the glycan chain present in lipopolysaccharides (LPS), the oligosaccharide repeats present in capsular polysaccharides, peptidoglycan (PG), amino-sugars, antigens of the human ABO blood group system, an animal oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans, a plant oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans, sialylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, fucosylated oligosaccharide, N-acetylglucos
  • Charged oligosaccharides are oligosaccharide structures that contain one or more negatively charged monosaccharide subunits including sialic acid, glucuronate and galacturonate. Charged oligosaccharides are also referred to as acidic oligosaccharides. In contrast, neutral (non-charged) oligosaccharides are non- sialylated oligosaccharides, and thus do not contain an acidic monosaccharide subunit.
  • Neutral oligosaccharides comprise non-charged fucosylated oligosaccharides that contain one or more fucose subunits in their glycan structure as well as non-charged non-fucosylated oligosaccharides that lack any fucose subunit.
  • Other examples of charged oligosaccharides are sulphated chitosans and deacetylated chitosans.
  • oligosaccharide or “acidic oligosaccharide” are used interchangeably and refer to an oligosaccharide with a negative charge.
  • the negatively charged oligosaccharide is a sialylated oligosaccharide.
  • a 'sialylated oligosaccharide' is to be understood as a negatively charged sialic acid containing oligosaccharide, i.e., an oligosaccharide having a sialic acid residue. It has an acidic nature.
  • sialylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of said monosaccharide subunit is a sialic acid residue.
  • a sialylated oligosaccharide can contain more than one sialic acid residue, e.g., two, three or more.
  • Said more than one sialic acid residue can be two, three or more identical sialic acid residues. Said more than one sialic acid residue can also be two, three or more different sialic acid residues.
  • a sialylated oligosaccharide can contain one or more Neu5Ac residues and one or more KDO residues.
  • 3'-SL (3'-sialyllactose or 3'SL or Neu5Ac-a2,3-Gal-pi,4-Glc), 3'-sialyllactosamine, 6-SL (6'sialyllactose, 6'-sialyllactose or 6'SL or Neu5Ac-a2,6-Gal-pi,4-Glc), 3,6-disialyllactose (Neu5Ac-a2,3- (Neu5Ac-a2,6)-Gal-pi,4-Glc), 6,6'-disialyllactose (Neu5Ac-a2,6-Gal-pi,4-(Neu5Ac-a2,6)-Glc), 8,3- disialyllactose (Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-pi,4-Glc), 6'-sialyllactosamine,
  • a '3'sialylated oligosaccharide' is to be understood as a negatively charged sialic acid containing oligosaccharide comprising an oligosaccharide or disaccharide which is alpha-2, 3-glycosidically linked to a sialic acid residue. It has an acidic nature.
  • sialylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of said monosaccharide subunit is an alpha-2, 3-glycosydically linked sialic acid residue.
  • a 3'sialylated oligosaccharide can contain more than one sialic acid residue, e.g., two, three or more. Said more than one sialic acid residue can be two, three or more identical sialic acid residues. Said more than one sialic acid residue can also be two, three or more different sialic acid residues.
  • a 3'sialylated oligosaccharide can contain one or more Neu5Ac residues and one or more KDO residues.
  • 3'-SL 3'-sialyllactose or 3'SL or Neu5Ac-a2,3-Gal-pi,4-Glc
  • 3'-sialyllactosamine 3,6- disialyllactose (Neu5Ac-a2,3-(Neu5Ac-a2,6)-Gal-pi,4-Glc), 8,3-disialyllactose (Neu5Ac-a2,8-Neu5Ac-a2,3- Gal-pi,4-Glc)
  • SGG hexasaccharide (Neu5Aca-2,3Gai -l,3GalNac -l,3Gala-l,4Gai -l,4Gal)
  • sialylated tetrasaccharide (Neu5Aca-2,3Gaip-l,4GlcNac -l,4GlcNAc), pentasaccharide
  • 3'sialylated lactosamine comprising oligosaccharide or "3'sialylated LacNAc comprising oligosaccharide”
  • 3'sLacNAc comprising oligosaccharide are used interchangeably and refer to an oligosaccharide with a negative charge comprising one or more sialic acid groups and one or more lactosamine disaccharides.
  • Some examples are 3'-sialyllactosamine, LSTd, sialyl lewis x, 3'-KDO- lactosamine.
  • 3'sialylated lacto-N-biose comprising oligosaccharide or "3'sialylated LNB comprising oligosaccharide”
  • 3'sLNB comprising oligosaccharide are used interchangeably and refer to an oligosaccharide with a negative charge comprising one or more sialic acid groups and one or more lacto- N-biose disaccharides.
  • Some examples are LSTa, sialyl-Lewis a, 3'-sialyllacto-N-biose.
  • LNB and “Lacto-N-biose” are used interchangeably and refer to the disaccharide Gal-pi,3- GIcNAc.
  • LNT II LNT-II
  • LN3 lacto-N-triose II
  • lacto-N-triose II lacto-N-triose
  • lacto-N-triose lacto-N-triose
  • GlcNAc i-3Gaipi-4Glc as used in the present invention
  • LNT lacto-N-tetraose
  • lacto-W-tetraose lacto-W-tetraose
  • Gaipi-3GlcNAcpi-3Gaipi-4Glc as used in the present invention, are used interchangeably.
  • LNnT lacto-N-neotetraose
  • lacto-W-neotetraose lacto-W-neotetraose
  • Gaipi-4GlcNAcpi- 3Gaipi-4Glc as used in the present invention, are used interchangeably.
  • LSTa LS-tetrasaccharide a
  • sialyl-lacto-N-tetraose a sialyllacto-N-tetraose a
  • Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,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- pi,3-(Neu5Ac-a2,6)-GlcNAc-pi,3-Gal-pi,4-Glc as used in the present invention, are used interchangeably.
  • LSTc "LS-Tetrasaccharide c", "Sialyl-lacto-N-tetraose c", “sialyllacto-N-tetraose c”, “sialyllacto-N-neotetraose c", or "Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc" as used in the present invention, are used interchangeably.
  • LSTd LS-tetrasaccharide d
  • LS-tetrasaccharide d sialyl-lacto-N-tetraose d
  • sialyllacto-N-tetraose d sialyllacto-N-neotetraose d
  • Neu5Ac-a2,3-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc as used in the present invention, are used interchangeably.
  • DSLNT disialyllacto-N-tetraose
  • DSLNT disialyllacto-N-tetraose
  • D'LNnT and “disialyllacto-N-neotetraose analog” as used in the present invention, are used interchangeably and refer to Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,4-GlcNAc-pi,3-)Gal-pi,4-Glc.
  • sialylated tetraose type 1 refers to Neu5Ac-a2,3-Gal-pi,3- GlcNAc-pi,3-Gal.
  • sialylated tetraose type 2 refers to Neu5Ac-a2,3-Gal-pi,4- GlcNAc-pi,3-Gal.
  • sialyl-Lewis a refers to Fuc-al,4-(Neu5Ac-a2,3-Gal-pi,3-) GIcNAc.
  • a 'neutral oligosaccharide' or a 'non-charged oligosaccharide' as used herein and as generally understood in the state of the art is an oligosaccharide that has no negative charge originating from a carboxylic acid group.
  • Examples of such neutral oligosaccharide are 2'-fucosyllactose (2'FL), 3-fucosyl lactose (3FL), 2', 3- difucosyllactose (diFL), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto- N-fucopentaose I (LNFP I), lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose II (LNFP II), lacto- N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-fucopentaose VI, lacto-N- neofucopentaose V (LNnFP V), lacto-N-
  • amino-sugar refers to a sugar molecule in which a hydroxyl group has been replaced with an amine group.
  • an antigen of the human ABO blood group system is an oligosaccharide. Such antigens of the human ABO blood group system are not restricted to human n structures.
  • Mammalian milk oligosaccharides or MMOs comprise oligosaccharides present in milk found in any phase during lactation including colostrum milk from humans (i.e. human milk oligosaccharides or HMOs) and mammals including but not limited to cows (Bos Taurus), sheep (Ovisaries), goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus), horses (Equus ferus caballus), pigs (Sus scropha), dogs (Canis lupus familiaris), ezo brown bears (Ursus arctos yesoensis), polar bear (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephant (Lo
  • mammalian milk oligosaccharide refers to oligosaccharides such as but not limited to 3- fucosyllactose, 2'-fucosyllactose, 6-fucosyl lactose, 2',3-difucosyllactose, 2',2-difucosyllactose, 3,4- difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, 3,6-disialyllactose, 6,6'-disialyllactose, 8,3- disialyllactose, 3,6-disialyllacto-N-tetraose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto
  • Human milk oligosaccharides are also known as human identical milk oligosaccharides which are chemically identical to the human milk oligosaccharides found in human breast milk, but which are biotechnologically produced (e.g. using cell free systems or cells and organisms comprising a bacterium, a fungus, a yeast, a plant, animal, or protozoan cell, preferably metabolically engineered cells and organisms).
  • Human identical milk oligosaccharides are marketed under the name HiMO.
  • HMOs comprise fucosylated oligosaccharides, non-fucosylated neutral oligosaccharides and sialylated oligosaccharides (see e.g.
  • HMOs Human Milk Oligosaccharides
  • Examples of HMOs comprise 3- fucosyllactose, 2'-fucosyllactose, 2',3-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, LN3, lacto-N- tetraose, lacto-N-neotetraose, lacto-N-fucopentaose ll 7 lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N-tetraose c, sialyllacto-N-tetraose c, sialyllacto-N-tetra
  • mammary cell 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • cell genetically modified for the production of a sialylated oligosaccharide or “cell metabolically engineered for the production of a sialylated oligosaccharide” within the context of the present disclosure refers to a cell of a microorganism which is genetically manipulated to comprise at least one sialyltransferase combined with any one or more of i) a gene encoding a glycosyltransferase necessary for the synthesis of said sialylated oligosaccharide, ii) a biosynthetic pathway to produce a nucleotide donor suitable to be transferred by said glycosyltransferase to a carbohydrate precursor, and/or iii) a biosynthetic pathway to produce a precursor or a mechanism of internalization of a precursor from the culture medium into the cell where it is glycosylated to produce the sialylated oligosaccharide.
  • pathway for production of a sialylated oligosaccharide is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of a sialylated oligosaccharide as defined herein.
  • Said pathway for production of a sialylated oligosaccharide can comprise but is not limited to pathways involved in the synthesis of a nucleotide-activated sugar and the transfer of said nucleotide-activated sugar to an acceptor to create a sialylated oligosaccharide of the present invention.
  • An example of such pathway is a sialylation pathway.
  • pathway comprises 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 chosen from the list comprising an L-glutamine— D-fructose-6-phosphate aminotransferase, a phosphoglucosamine mutase, an N-acetylglucosamine-6-P deacetylase, an N- acylglucosamine 2-epimerase, a UDP-N-acetylglucosamine 2-epimerase, an N-acetylmannosamine-6- phosphate 2-epimerase, a UDP-GIcNAc 2-epimerase/kinase, a glucosamine 6-phosphate N- acetyltransferase, an N-acetylglucosamine-6-phosphate phosphatase, a phosphoacetylglucosamine mutase, an N-acetylglucosamine 1-phosphate uridyly
  • L-glutamine— D-fructose-6-phosphate aminotransferase D-fructose-6-phosphate aminotransferase
  • glutamine — fructose-6-phosphate transaminase (isomerizing) hexosephosphate aminotransferase
  • glucosamine-6-phosphate isomerase glutamine-forming
  • glutamine-fructose-6-phosphate transaminase (isomerizing) "D- fructose-6-phosphate amidotransferase
  • fructose-6-phosphate aminotransferase "glucosaminephosphate isomerase
  • glucosamine 6-phosphate synthase synthase
  • GlcN6P synthase GFA
  • glms glmS
  • glmS*54 are used interchangeably and refer to an enzyme that catalyses the conversion of D-fructose-6-phosphate into
  • phosphoglucosamine mutase and “glmM” are used interchangeably and refer to an enzyme that catalyses the conversion of glucosamine-6-phosphate to glucosamine-l-phosphate. Phosphoglucosamine mutase can also catalyse the formation of glucose-6-P from glucose-l-P, although at a 1400-fold lower rate.
  • Alternative names for this enzyme comprise N-acetylglucosamine 2-epimerase, N- acetyl-D-glucosamine 2-epimerase, GIcNAc 2-epimerase, N-acyl-D-glucosamine 2-epimerase and N- acetylglucosamine epimerase.
  • a glucosamine 6-phosphate N-acetyltransferase is an enzyme that catalyses the transfer of an acetyl group from acetyl-CoA to D-glucosamine-6-phosphate thereby generating a free CoA and N-acetyl-D- glucosamine 6-phosphate.
  • Alternative names comprise aminodeoxyglucosephosphate acetyltransferase, D-glucosamine-6-P N-acetyltransferase, glucosamine 6-phosphate acetylase, glucosamine 6-phosphate N-acetyltransferase, glucosamine-phosphate N-acetyltransferase, glucosamine-6-phosphate acetylase, N-acetylglucosamine-6-phosphate synthase, phosphoglucosamine acetylase, phosphoglucosamine N- acetylase phosphoglucosamine N-acetylase, phosphoglucosamine transacetylase, GNA and GNA1.
  • N-acetylglucosamine-6-phosphate phosphatase refers to an enzyme that dephosphorylates N-acetylglucosamine-6-phosphate (GlcNAc-6-P) hereby synthesizing N-acetylglucosamine (GIcNAc).
  • phosphoacetylglucosamine mutase "acetylglucosamine phosphomutase", “acetylaminodeoxyglucose phosphomutase”, “phospho-N-acetylglucosamine mutase” and “N-acetyl-D- glucosamine 1,6-phosphomutase” are used interchangeably and refer to an enzyme that catalyses the conversion of N-acetyl-glucosamine 1-phosphate into N-acetylglucosamine 6-phosphate.
  • N-acetylglucosamine 1-phosphate uridylyltransferase "N-acetylglucosamine-l-phosphate uridyltransferase”
  • UDP-N-acetylglucosamine diphosphorylase "UDP-N-acetylglucosamine pyrophosphorylase”
  • uridine diphosphoacetylglucosamine pyrophosphorylase "UTP:2-acetamido-2- deoxy-alpha-D-glucose-l-phosphate uridylyltransferase”
  • UDP-GIcNAc pyrophosphorylase "GlmU uridylyltransferase”
  • Acetylglucosamine 1-phosphate uridylyltransferase "UDP-acetylglucosamine pyrophosphorylase”
  • uridine diphosphate-N-acetylglucosamine pyrophosphorylase "
  • glucosamine-l-phosphate acetyltransferase refers to an enzyme that catalyses the transfer of the acetyl group from acetyl coenzyme A to glucosamine-l-phosphate (GlcN-1-P) to produce N- acetylglucosamine-l-phosphate (GlcNAc-1-P).
  • glycosmll refers to a bifunctional enzyme that has both N-acetylglucosamine-l-phosphate uridyltransferase and glucosamine-l-phosphate acetyltransferase activity and that catalyses two sequential reactions in the de novo biosynthetic pathway for UDP-GIcNAc.
  • the C-terminal domain catalyses the transfer of acetyl group from acetyl coenzyme A to GlcN-1-P to produce GlcNAc-1-P, which is converted into UDP-GIcNAc by the transfer of uridine 5-monophosphate, a reaction catalysed by the N- terminal domain.
  • Neuronac synthase N-acetylneuraminic acid synthase
  • N-acetylneuraminate synthase sialic acid synthase
  • NeAc synthase N-acetylneuraminate synthase
  • NANA condensing enzyme "N- acetylneuraminate lyase synthase”
  • N-acetylneuraminic acid condensing enzyme as used herein are used interchangeably and refer to an enzyme capable to synthesize sialic acid (Neu5Ac) from N- acetylmannosamine (ManNAc) in a reaction using phosphoenolpyruvate (PEP).
  • N-acetylneuraminate lyase N-acetylneuraminate lyase
  • Neu5Ac lyase N-acetylneuraminate pyruvate-lyase
  • N- acetylneuraminic acid aldolase N- acetylneuraminic acid aldolase
  • NALase N-acetylneuraminic acid aldolase
  • NALase amino acid aldolase
  • sialate lyase sialate lyase
  • sialic acid aldolase sialic acid lyase
  • nanA N-acetylneuraminate lyase
  • ManNAc N- acetylmannosamine
  • N-acylneuraminate-9-phosphate synthase N-acylneuraminate-9-phosphate synthetase
  • NANA synthase NANAS
  • NANS NmeNANAS
  • N-acetylneuraminate pyruvate-lyase pyruvate- phosphorylating
  • N-acylneuraminate-9-phosphatase refers to an enzyme capable to dephosphorylate N- acylneuraminate-9-phosphate to synthesise N-acylneuraminate.
  • CMP-sialic acid synthase N-acylneuraminate cytidylyltransferase
  • CMP-sialate synthase CMP-NeuAc synthase
  • NeuroA CMP-N-acetylneuraminic acid synthase
  • a 'fucosylation pathway' as used herein is a biochemical pathway comprising at least one of the enzymes and their respective genes chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase combined with a fucosyltransferase leading to a 1,2; a 1,3; a 1,4 and/or a 1,6 fucosylated compounds.
  • a 'galactosylation pathway' as used herein is a biochemical pathway comprising at least one of the enzymes and their respective genes chosen from the list comprising galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase combined with a galactosyltransferase leading to a galactosylated compound comprising a mono-, di-, or oligosaccharide having an alpha or beta bound galactose on any one or more of the 2, 3, 4 and 5 hydroxyl group of said mono-, di-, or oligosaccharide.
  • An 'N-acetylglucosaminylation pathway' as used herein is a biochemical pathway comprising at least one of the enzymes and their respective genes chosen from the list comprising L-glutamine— D-fructose-6- phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase combined with a glycosyltransferase leading to a GIcNAc-modified compound comprising a mono-, di-, or oligosaccharide having an alpha or beta bound N-acetylglucosamine (GIcNAc) on any one or more of the 3, 4 and 6 hydroxyl group of said mono-, di- or oligosaccharide.
  • An 'N-acetylgalactosaminylation pathway' as used herein is a biochemical pathway comprising at least one of the enzymes and their respective genes chosen from the list comprising L-glutamine— D-fructose- 5-phosphate aminotransferase, phosphoglucosamine mutase, /V-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, UDP-N-acetylglucosamine 4-epimerase, UDP-glucose 4-epimerase, N-acetylgalactosamine kinase and UDP-GalNAc pyrophosphorylase combined with a glycosyltransferase leading to a GalNAc-modified compound comprising a mono-, di- or oligosaccharide having an alpha or beta bound N-acetylgalactosamine on
  • a 'mannosylation pathway' as used herein is a biochemical pathway comprising at least one of the enzymes and their respective genes chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase and mannose-l-phosphate guanylyltransferase combined with a glycosyltransferase leading to a mannosylated compound comprising a mono-, di- or oligosaccharide having an alpha or beta bound mannose on said mono-, di- or oligosaccharide.
  • An 'N-acetylmannosaminylation pathway' as used herein is a biochemical pathway comprising at least one of the enzymes and their respective genes chosen from the list comprising L-glutamine— D-fructose- 5-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N- acetylglucosamine-6-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase, N- acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, UDP- GIcNAc 2-epimerase and ManNAc kinase combined with a glycosyltransferase leading to a ManNAc- modified compound comprising a mono-, di- or oligosaccharide having an alpha or
  • pyruvate dehydrogenase pyruvate oxidase
  • POX pyruvate oxidase
  • poxB pyruvate:ubiquinone-8 oxidoreductase
  • lactate dehydrogenase D-lactate dehydrogenase
  • IdhA hsll
  • htpH htpH
  • D-LDH htpH
  • fermentative lactate dehydrogenase and "D-specific 2-hydroxyacid dehydrogenase” are used interchangeably and refer to an enzyme that catalyses the conversion of lactate into pyruvate hereby generating NADH.
  • purified refers to material that is substantially or essentially free from components that interfere with the activity of the biological molecule.
  • purified saccharides, oligosaccharides, proteins or nucleic acids of the invention are at least about 50%, 55%, 50%, 55%, 70%, 75%, 80% or 85% pure, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, or 99.0% pure as measured by band intensity on a silver-stained gel or other method for determining purity.
  • Purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized. For di- and/or oligosaccharides, purity can be determined using methods such as but not limited to thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis or mass spectroscopy.
  • 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 one or more bioproduct(s).
  • Such treatment can be carried out in a conventional manner by centrifugation, flocculation, flocculation with optional ultrasonic treatment, gravity filtration, microfiltration, foam separation or vacuum filtration (e.g. through a ceramic filter which can include a CeliteTM filter aid).
  • culture refers to the culture medium wherein the cell is cultivated, or fermented, the cell itself, and a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, that is produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell.
  • 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 said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, is produced.
  • Said mixture can comprise one or more enzyme(s), one or more precursor(s) and one or more acceptor(s) as defined herein present in a buffered solution and incubated for a certain time at a certain temperature enabling production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine and a galactose monosaccharide, catalysed by said one or more enzyme(s) using said one or more precursor(s) and said one or more acceptor(s) in said mixture.
  • Said mixture can also comprise i) the cell obtained after cultivation or incubation, optionally said cell is subjected to cell lysis, ii) a buffered solution or the cultivation or incubation medium wherein the cell was cultivated or fermented, and iii) said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, that is produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracell ularly) of the cell.
  • Said incubation can also be the cultivation as defined herein.
  • reactors and incubators 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.
  • CPI cell productivity index
  • precursor refers to substances that are taken up or synthetized by the cell for the specific production of a sialylated oligosaccharide according to the present invention.
  • a precursor can be an acceptor as defined herein, but can also be another substance, metabolite, that is first modified within the cell as part of the biochemical synthesis route of a sialylated oligosaccharide, preferably a 3'sialylated oligosaccharide, said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide.
  • precursor as used herein is also to be understood as a chemical compound that participates in a chemical or enzymatic reaction to produce another compound like e.g. an intermediate or an acceptor as defined herein, as part in the metabolic pathway of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • precursor as used herein is also to be understood as a donor that is used by a glycosyltransferase to modify an acceptor as defined herein with a sugar moiety in a glycosidic bond, as part in the metabolic pathway of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • Such precursors comprise the acceptors as defined herein, and/or dihydroxyacetone, glucosamine, N-acetylglucosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyllactose, phosphorylated sugars or sugar phosphates like e.g.
  • glucose-1- phosphate galactose-l-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6- bisphosphate, mannose-6-phosphate, mannose-l-phosphate, glycerol-3-phosphate, glyceraldehyde-3- phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N-acetylglucosamine-l-phosphate, N-acetylneuraminic acid-9- phosphate and nucleotide-activated sugars like nucleotide diphospho-sugars and nucleotide monophospho-sugars as defined herein like e.g.
  • UDP-glucose UDP-galactose, UDP-N-acetylglucosamine, CMP-sialic acid, GDP-mannose, GDP-4-dehydro-6-deoxy-a-D-mannose, GDP-fucose.
  • the cell is transformed to comprise and to express at least one nucleic acid sequence encoding a protein selected from the group consisting of lactose transporter, N-acetylneuraminic acid transporter, fucose transporter, glucose transporter, galactose transporter, transporter for a nucleotide-activated sugar wherein said transporter internalizes a to the medium added precursor for the synthesis of the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, of present invention.
  • a protein selected from the group consisting of lactose transporter, N-acetylneuraminic acid transporter, fucose transporter, glucose transporter, galactose transporter, transporter for a nucleotide-activated sugar wherein said transporter internalizes a to the medium added precursor for the synthesis of the 3'sialylated
  • acceptor 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-biose, N-acetyllactosamine, lacto-N-triose, lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-pentaose (LNP), lacto-N-neopentaose, para lacto-N- pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N-hexaose (LNH), lacto-N-N-
  • the present invention provides a method for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • the method comprises contacting a sialyltransferase with a mixture comprising a donor comprising a sialic acid residue, and an acceptor, preferably in a medium, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from the donor to the acceptor, thereby producing said 3'sialylated oligosaccharide.
  • the acceptor used in the method is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide and the saccharide is chosen from the list consisting of an oligosaccharide or a disaccharide.
  • the sialyltransferases used in the present invention have alpha-2, 3-sialyltransferase activity on an acceptor, and comprise an amino acid sequence that is i) at least 60.0 % identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 or ii) at least 85.0 % identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequence as represented by SEQ ID NO: 17, 14, 16, 28 or 29.
  • a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide preferably is a disaccharide-containing 3’sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably is a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide, even more preferably is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-
  • the acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably said acceptor is LacNAc, LNB, a LacNAc comprising oligosaccharide or an LNB comprising oligosaccharide, more preferably an LNT or an LNnT.
  • the present invention provides a method for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide wherein the method comprises providing i) CMP-sialic acid, ii) an acceptor as defined herein, and iii) a sialyltransferase as defined herein.
  • the method further comprises contacting the sialyltransferase and CMP-sialic acid with the acceptor, under conditions where the sialyltransferase catalyses the transfer of a sialic acid residue from said CMP-sialic acid to the acceptor resulting in the production of said 3'sialylated oligosaccharide.
  • the method further comprises separating said produced 3’sialylated oligosaccharide from the medium.
  • the present invention provides a method for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide wherein the method comprises contacting a cell extract comprising a sialyltransferase as defined herein with a mixture comprising a donor comprising a sialic acid residue, and an acceptor as defined herein, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from the donor to the acceptor, thereby producing said 3'sialylated oligosaccharide.
  • said 3'sialylated oligosaccharide is separated.
  • the sialyltransferase has alpha-2, 3- sialyltransferase activity on the acceptor and comprises an amino acid sequence that is at least 80.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.
  • the 3'sialylated oligosaccharide is produced in a cell-free system.
  • the present invention provides a method for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said method comprises the steps of: i. providing a cell, preferably a single cell, expressing, preferably heterologously expressing, more preferably overexpressing, even more preferably heterologously overexpressing, a sialyltransferase as defined herein; ii. providing CMP-sialic acid, optionally said CMP-sialic acid is produced by said cell, and iii.
  • an acceptor being a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, optionally said acceptor is produced by said cell, and iv.
  • the present invention provides a method for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said method comprises the steps of: i. providing a cell, preferably a single cell, expressing, preferably heterologously expressing, more preferably overexpressing, even more preferably heterologously overexpressing, a sialyltransferase as defined herein, ii. providing CMP-sialic acid, optionally said CMP-sialic acid is produced by said cell, and iii.
  • an acceptor being a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, optionally said acceptor is produced by said cell, and iv.
  • the sialylated oligosaccharide is produced by a cell, preferably a single cell, wherein said cell expresses a sialyltransferase as defined herein.
  • the 3'sialylated oligosaccharide is produced by a cell, preferably a single cell, wherein said cell expresses a sialyltransferase as described herein.
  • the cell used in the present invention is preferably a metabolically engineered cell as described herein.
  • said cell is metabolically engineered for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • the sialyltransferase as described herein comprises an amino acid sequence that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues.
  • said sialyltransferase comprises an amino acid sequence that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22.
  • said sialyltransferase comprises an amino acid sequence that is at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full- length amino acid sequences as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or 27.
  • the sialyltransferase as described herein comprises an amino acid sequence that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues.
  • said sialyltransferase comprises an amino acid sequence that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 17, 10, 14, 16, 18, 28 or 29.
  • the sialyltransferase as described herein comprises an amino acid sequence comprising a fragment of any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 and having alpha-2, 3-sialyltransferase activity on the acceptor as defined herein.
  • said sialyltransferase comprises an amino acid sequence as represented by any one of the SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.
  • 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 sialic acid residue is at least one chosen from the list consisting of Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulonic acid (KDO).
  • the sialic acid residue is Neu5Ac.
  • the donor comprising a sialic acid residue is CMP-sialic acid.
  • the donor comprising a sialic acid residue is chosen from the list consisting of CMP-Neu5Ac, CMP-Neu4Ac, CMP- Neu5Ac9N 3 , CMP-Neu4,5Ac z , CMP-Neu5,7Ac z , CMP-Neu5,9Ac z , CMP-Neu5,7(8,9)Ac z , CMP-N- glycolylneuraminic acid (CMP-Neu5Gc) and CMP-KDO.
  • the donor comprising a sialic acid residue is CMP-Neu5Ac.
  • said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is a disaccharide-containing 3'sialylated oligosaccharide as defined herein, and said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.
  • the cultivation medium contains at least one carbon source selected from the group consisting of glucose, fructose, sucrose, and glycerol.
  • the cultivation or incubation medium contains at least one compound selected from the group consisting of lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP- GIcNAc), sialic acid and CMP-sialic acid.
  • the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is recovered from the medium, the cultivation medium or incubation medium and/or from the cell or separated from the cultivation or incubation as explained herein.
  • the method comprises the use of a cultivation or incubation medium comprising at least one precursor and/or acceptor for the production of the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as described herein and/or the method comprises adding to the cultivation or incubation medium at least one precursor and/or acceptor feed for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • said precursor is selected from the group comprising a monosaccharide like e.g. galactose, fucose, sialic acid, GIcNAc, GalNAc; a nucleotide-activated sugar like e.g. CMP-sialic acid, UDP-Gal, UDP-GIcNAc, GDP-fucose; a disaccharide like e.g. lactose, LNB or LacNAc; and an oligosaccharide like e.g. lacto-N-triose (LN3), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).
  • a monosaccharide like e.g. galactose, fucose, sialic acid, GIcNAc, GalNAc
  • a nucleotide-activated sugar like e.g. CMP-sialic acid, UDP
  • said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, more preferably a LacNAc comprising oligosaccharide, an LNB comprising oligosaccharide, even more preferably an LNT or an LNnT.
  • said precursor is chosen from the list comprising sialic acid, CMP-sialic acid and lactose.
  • said acceptor is LNT or LNnT.
  • the conditions permissive to produce said 3'sialylated oligosaccharide comprise adding to the cultivation or incubation medium at least one precursor and/or acceptor feed for the production of said 3'sialylated oligosaccharide.
  • the conditions permissive to produce said 3'sialylated oligosaccharide comprise the use of a cultivation or incubation medium wherein said cultivation or incubation medium lacks any precursor and/or acceptor for the production of said 3'sialylated oligosaccharide and is combined with a further addition to said cultivation or incubation medium of at least one precursor and/or acceptor feed for the production of said 3'sialylated oligosaccharide.
  • the cultivation or incubation is contained in a reactor or incubator, as defined herein.
  • the volume of said reactor or incubator ranges from microlitre (p.L) scale to 10.000 m3 (cubic meter). In a preferred embodiment, the volume of said reactor or incubator ranges from 250 mL (millilitre) to 10.000 m3 (cubic meter).
  • the method for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide comprises at least one of the following steps: i) use of a cultivation or incubation medium comprising at least one precursor and/or acceptor; ii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and/or acceptor feed wherein the total reactor or incubator volume ranges from 250 ml (millilitre) to 10.000 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, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor and/or acceptor feed; iii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor
  • said precursor is chosen from the list comprising lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP- GIcNAc), sialic acid and CMP-sialic acid.
  • said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, as defined herein.
  • the method for the production of 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide comprises at least one of the following steps: i) use of a cultivation or incubation medium comprising at least one precursor and/or acceptor; ii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and/or acceptor in one pulse or in a discontinuous (pulsed) manner wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m 3 (cubic meter), preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor and/or acceptor feed pulse(s); iii) adding to the cultivation or incubation medium in a
  • said precursor is chosen from the list comprising lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N- acetylglucosamine (UDP-GIcNAc), sialic acid and CMP-sialic acid.
  • said acceptor is a saccharide as defined herein.
  • said precursor is chosen from the list comprising lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N- acetylglucosamine (UDP-GIcNAc), sialic acid and CMP-sialic acid.
  • said precursor is chosen from the list comprising lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N- acetylglucosamine (UDP-GIcNAc), sialic acid and CMP-sialic acid.
  • the method for the production of a 3'sialylated oligosaccharide comprises at least one of the following steps: i) use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter); ii) adding to the cultivation or incubation medium in a reactor or incubator a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m 3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of
  • 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.
  • the lactose is added already in the first phase of exponential growth together with the carbon-based substrate.
  • the sialyltransferases or alpha-2, 3-sialyltransferases according to the invention preferably have alpha-2, 3-sialyltransferase activity on a galactose (Gal) residue, preferably a terminal Gal residue, of an acceptor as defined herein.
  • Gal galactose
  • the present invention provides a metabolically engineered cell for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said cell has been metabolically engineered to possess, preferably to express, more preferably to heterologously express, even more preferably to overexpress, most preferably to heterologously overexpress, a sialyltransferase which has alpha-2, 3-sialyltransferase activity, and comprises an amino acid sequence that is i) at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22, or ii) at least 80.0% identical over a stretch of at least 150 amino acid residues
  • the present invention provides a metabolically engineered cell for the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as described herein, wherein said cell has been metabolically engineered to possess, preferably to express, more preferably to heterologously express, even more preferably to overexpress, most preferably to heterologously overexpress, a sialyltransferase which has alpha-2, 3-sialyltransferase activity and comprises an amino acid sequence that is at least 80.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8,
  • the sialyltransferase comprises an amino acid sequence that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ. ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.
  • the sialyltransferase comprises an amino acid sequence as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.
  • the cell contains a nucleic acid molecule which comprises a polynucleotide sequence that encodes any one of the sialyltransferases as described herein.
  • a metabolically engineered cell comprising a pathway for production of said 3'sialylated oligosaccharide.
  • pathways comprise but are not limited to pathways involved in the synthesis of monosaccharide, phosphorylated monosaccharide, nucleotide-activated sugar, and/or glycosylation pathways like e.g., a fucosylation, sialylation, galactosylation, N- acetylglucosaminylation, N-acetylgalactosaminylation, mannosylation and/or N- acetylmannosaminylation pathway.
  • Said pathway for production of a sialylated oligosaccharide preferably comprises at least one sialyltransferase as described herein.
  • the cell comprises one or more pathway(s) for monosaccharide synthesis.
  • Said pathways for monosaccharide synthesis comprise enzymes like e.g. carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases, dehydrogenases, enzymes involved in the synthesis of one or more nucleoside triphosphate(s) like UTP, GTP, ATP and CTP, enzymes involved in the synthesis of any one or more nucleoside mono- or diphosphates like e.g. UMP and UDP, respectively, and enzymes involved in the synthesis of phosphoenolpyruvate (PEP).
  • enzymes like e.g. carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases,
  • the cell comprises one or more pathway(s) for phosphorylated monosaccharide synthesis.
  • Said pathways for phosphorylated monosaccharide synthesis comprise enzymes involved in the synthesis of one or more monosaccharide(s), one or more nucleoside mono-, di- and/or triphosphate(s) and enzymes involved in the synthesis of phosphoenolpyruvate (PEP) like e.g., but not limited to PEP synthase, carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases and dehydrogenases.
  • PEP phosphoenolpyruvate
  • the cell comprises one or more pathways for the synthesis of one or more nucleotide-activated sugars.
  • Said pathways for nucleotide-activated sugar synthesis comprise enzymes like e.g.
  • PEP synthase carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases, dehydrogenases, mannose-6-phosphate isomerase, phosphomannomutase, mannose-1- phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, L- fucokinase/GDP-fucose pyrophosphorylase, L-glutamine— D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epimerase, UDP-
  • Said cell may further comprise and express at least one further glycosyltransferase that is involved in the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • the cell is metabolically engineered to comprise a pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.
  • the cell is metabolically engineered to comprise a pathway for production said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, and to have modified expression or activity of a sialyltransferase of present invention.
  • the cell comprises a recombinant sialyltransferase capable of modifying said acceptor and/or wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein or another acceptor as defined herein with one or more sialic acid molecules that is/are synthesized by any one or more sialic acid synthases like e.g.
  • Neu5Ac synthases expressed in the cell into said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.
  • the metabolically engineered 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, 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.
  • 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.
  • external chemicals e.g. IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose
  • the expression modules can be integrated in the genome of said cell or can be presented to said cell on a vector.
  • Said vector can be present in the form of a plasmid, cosmid, phage, liposome, or virus, which is to be stably transformed/transfected into said metabolically engineered cell.
  • Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • These vectors may contain selection markers such as but not limited to antibiotic markers, auxotrophic markers, toxinantitoxin markers, RNA sense/antisense markers.
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • 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 said 3'sialyl ated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein; or said recombinant gene is linked to other pathways in said cell that are not involved in the synthesis of a sialylated oligosaccharide.
  • Said recombinant genes encode endogenous proteins with a modified expression or activity, preferably said endogenous proteins are overexpressed; or said recombinant genes encode heterologous proteins that are heterogeneously introduced and expressed in said modified cell, preferably overexpressed.
  • the endogenous proteins can have a modified expression in the cell which also expresses a heterologous protein.
  • each of said expression modules present in said metabolically engineered cell is constitutive or tuneable as described herein.
  • the cell is modified in the expression or activity of at least one of said sialyltransferases.
  • said sialyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous sialyltransferase is overexpressed; alternatively said sialyltransferase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous sialyltransferase can have a modified expression in the cell which also expresses a heterologous sialyltransferase.
  • the cell comprises a pathway for production of a sialylated oligosaccharide, preferably a 3'sialylated oligosaccharide, comprising at least one sialyltransferase according to present invention.
  • said pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine and a galactose monosaccharide as defined herein, further comprises at least one enzyme chosen from the list comprising L-glutamine— D-fructose- 6-phosphate aminotransferase, a phosphoglucosamine mutase, an N-acetylglucosamine-6-P deacetylase, an N-acylglucosamine 2-epimerase, a UDP-N-acetylglucosamine 2-epimerase, an N-acetylmannosamine- 6-phosphate 2-epimerase, a UDP-GIcNAc 2-epimerase/kinase, a glucosamine 6-phosphate N- acetyltransferase, an N-acetylglucosamine-6-phosphate
  • the cell comprises a pathway for production said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said cell expresses at least one enzyme chosen from the list comprising an N- acylglucosamine 2-epimerase like is known e.g. from several species including Bacteroides ovatus, E. coli, Homo sapiens, Rattus norvegicus, a Neu5Ac synthase, a CMP sialic acid synthase like is known e.g.
  • GIcNAc N-acetylglucosamine
  • Such cell producing GIcNAc can express a phosphatase converting GlcNAc-6-phosphate into GIcNAc, like any one or more of e.g. the E.
  • coli HAD-like phosphatase genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and Ybill, PsMupP from Pseudomonas putida, ScDOGl from 5.
  • the cell is modified to produce GIcNAc. More preferably, the cell is modified for enhanced GIcNAc production. Said modification can be any one or more chosen from the group comprising knockout of a glucosamine-5-phosphate deaminase, an N-acetylglucosamine-6-phosphate deacetylase and/or an N-acetyl-D-glucosamine kinase and over-expression of an L-glutamine— D- fructose-6-phosphate aminotransferase and/or a glucosamine 6-phosphate N-acetyltransferase.
  • the cell comprises a pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said cell expresses at least one enzyme chosen from the list comprising an UDP-N-acetylglucosamine 2-epimerase like is known e.g. from several species including Campylobacter jejuni, E.
  • UDP-N- acetylglucosamine can be added to the cell and/or can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing an UDP-GIcNAc can express enzymes converting, e.g.
  • GIcNAc which is to be added to the cell, to UDP-GIcNAc.
  • These enzymes may be any one or more enzymes chosen from the list comprising an N-acetyl-D-glucosamine kinase, an N- acetylglucosamine-5-phosphate deacetylase, a phosphoglucosamine mutase, and an N- acetylglucosamine-l-phosphate uridylyltransferase / glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli.
  • the cell is modified to produce UDP- GIcNAc.
  • the cell is modified for enhanced UDP-GIcNAc production.
  • Said modification can be any one or more chosen from the group comprising knock-out of an N-acetylglucosamine-6-phosphate deacetylase, over-expression of an L-glutamine— D-fructose-6-phosphate aminotransferase, over- expression of a phosphoglucosamine mutase, and over-expression of an N-acetylglucosamine-1- phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase.
  • the cell comprises a pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said cell expresses at least one enzyme chosen from the list comprising an N-acetylmannosamine-6-phosphate 2-epimerase like is known e.g. from several species including E.
  • coli Haemophilus influenzae, Enterobacter sp., Streptomyces sp., an N- acylneuraminate-9-phosphate synthetase, an N-acylneuraminate-9-phosphatase like is known e.g. from Candidatus Magnetomorum sp. HK-1 or Bacteroides thetaiotaomicron, a Neu5Ac synthase, a CMP sialic acid synthase like is known e.g. from Neisseria meningitidis, and a sialyltransferase according to present invention, wherein the enzymes are as defined herein.
  • GlcNAc-6P N-acetyl-D-glucosamine 6-phosphate
  • 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.
  • the cell is modified for enhanced GlcNAc-6P production.
  • Said modification can be any one or more chosen from the group comprising knockout of a glucosamine-6-phosphate deaminase, an N-acetylglucosamine-6-phosphate deacetylase and overexpression of an L-glutamine— D-fructose-6-phosphate aminotransferase and/or a glucosamine 6- phosphate N-acetyltransferase.
  • the cell comprises a pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, wherein said cell expresses at least one enzyme chosen from the list comprising a bifunctional UDP-GIcNAc 2-epimerase/kinase like is known e.g. from several species including Homo sapiens, Rattus norvegicus and Mus musculus, an N-acylneuraminate-9-phosphate synthetase, an N-acylneuraminate-9-phosphatase like is known e.g.
  • UDP-N-acetylglucosamine can be added to the cell and/or can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing an UDP-GIcNAc can express enzymes converting, e.g. GIcNAc, which is to be added to the cell, to UDP-GIcNAc.
  • These enzymes may be an N-acetyl-D-glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, and an N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli.
  • the cell is modified to produce UDP-GIcNAc. More preferably, the cell is modified for enhanced UDP-GIcNAc production.
  • Said modification can be any one or more chosen from the group comprising knock-out of an N-acetylglucosamine-6-phosphate deacetylase, over-expression of an L-glutamine— D-fructose-6-phosphate aminotransferase, over-expression of a phosphoglucosamine mutase, and over-expression of an N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-1- phosphate acetyltransferase.
  • the cell used herein is optionally genetically engineered to import a precursor and/or an acceptor in the cell, by the introduction and/or overexpression of a transporter able to import the respective precursor and/or acceptor in the cell.
  • 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.
  • the cell used herein is optionally genetically engineered to produce polyisoprenoid alcohols like e.g. phosphorylated dolichol that can act as lipid carrier.
  • polyisoprenoid alcohols like e.g. phosphorylated dolichol that can act as lipid carrier.
  • the cell used herein is optionally genetically engineered to import lactose in the cell, by the introduction and/or overexpression of a lactose permease.
  • Said lactose permease is for example encoded by the lacY gene or the Iacl2 gene.
  • the cell expresses a membrane protein that is a transporter protein involved in transport of compounds and/or a sialylated oligosaccharide as defined in present invention out of the cell.
  • a membrane protein that is a transporter protein involved in transport of compounds and/or a sialylated oligosaccharide as defined in present invention out of the cell.
  • said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is preferably produced intracellularly.
  • a fraction or substantially all of said produced 3'sialylated oligosaccharide remains intracellularly and/or is excreted outside the cell either passively or through active transport.
  • the cell is transformed to comprise at least one nucleic acid sequence encoding a protein selected from the group comprising a lactose transporter like e.g. the LacY or Iacl2 permease, a glucose transporter, a galactose transporter, a transporter for a nucleotide-activated sugar like for example a transporter for UDP-GIcNAc, a transporter protein involved in transport of said 3’sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide a and a galactose monosaccharide s defined herein, out of the cell.
  • a lactose transporter like e.g. the LacY or Iacl2 permease
  • a glucose transporter e.g. the LacY or Iacl2 permease
  • a glucose transporter e.g. the LacY or Iacl2 permease
  • the cell is capable to synthesize N-acetylmannosamine (ManNAc), N-acetylmannosamine-6-phosphate (ManNAc-6- phosphate) and/or phosphoenolpyruvate (PEP).
  • ManNAc N-acetylmannosamine
  • ManNAc-6- phosphate N-acetylmannosamine-6-phosphate
  • PEP phosphoenolpyruvate
  • the cell comprises a pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, comprising a pathway for production of ManNAc.
  • ManNAc can be provided by an enzyme expressed in the cell or by the mechanism of the cell.
  • Such cell producing ManNAc can express an N-acylglucosamine 2-epimerase like is known e.g. from several species including Bacteroides ovatus, E. coli, Homo sapiens, Rattus norvegicus that converts GIcNAc into ManNAc.
  • the cell producing ManNAc can express an UDP-N-acetylglucosamine 2-epimerase like is known e.g. from several species including Campylobacter jejuni, E. coli, Neisseria meningitidis, Bacillus subtilis, Citrobacter rodentium that converts UDP-GIcNAc into ManNAc.
  • GIcNAc and/or UDP-GIcNAc can be added to the cell and/or provided by an enzyme expressed in the cell or by the mechanism of the cell as described herein.
  • the cell is modified for enhanced ManNAc production.
  • Said modification can be any one or more chosen from the group comprising knock-out of N-acetylmannosamine kinase, over-expression of N-acetylneuraminate lyase.
  • the cell comprises a pathway for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, comprising a pathway for production of ManNAc-6-phosphate.
  • ManNAc-6-phosphate can be provided by an enzyme expressed in the cell or by the mechanism of the cell.
  • Such cell producing ManNAc-6-phosphate can express a bifunctional UDP-GIcNAc 2- epimerase/kinase like is known e.g. from several species including Homo sapiens, Rattus norvegicus and Mus musculus that converts UDP-GIcNAc into ManNAc-6-phosphate.
  • the cell producing ManNAc-6-phosphate can express an N-acetylmannosamine-6-phosphate 2-epimerase that converts GlcNAc-6-phosphate into ManNAc-6-phosphate.
  • UDP-GIcNAc and/or GlcNAc-6-phosphate can be added to the cell and/or provided by an enzyme expressed in the cell or by the mechanism of the cell as described herein.
  • the cell is modified for enhanced ManNAc-6- phosphate production.
  • Said modification can be any one or more chosen from the group comprising overexpression of N-acetylglucosamine-6-phosphate deacetylase, over-expression of N-acetyl-D-glucosamine kinase, over-expression of phosphoglucosamine mutase, over-expression of N-acetylglucosamine-1- phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase.
  • the cell is further capable to synthesize any one or more nucleotide-activated sugars.
  • the cell is capable to synthesize one or more nucleotide-activated sugars chosen from the list comprising UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP- Gal), GDP-mannose (GDP-Man), UDP-glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy— L- arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-
  • the cell is capable to synthesize at least the nucleotide-activated sugar CMP-Neu5Ac.
  • the cell uses at least one of the synthesized nucleotide-activated sugars in the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.
  • UDP-GIcNAc can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing an UDP-GIcNAc can express enzymes converting, e.g. GIcNAc, which is to be added to the cell, to UDP-GIcNAc.
  • These enzymes may be any one or more of the list comprising an N-acetyl-D- glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, and an N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli.
  • the cell is modified to produce UDP-GIcNAc. More preferably, the cell is modified for enhanced UDP-GIcNAc production.
  • Said modification can be any one or more chosen from the group comprising knock-out of an N-acetylglucosamine-6-phosphate deacetylase, over-expression of an L-glutamine— D-fructose-6- phosphate aminotransferase, over-expression of a phosphoglucosamine mutase, and over-expression of an N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase.
  • 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.
  • the cell is modified for enhanced CMP-Neu5Ac production.
  • Said modification can be any one or more chosen from the group comprising knock-out of an N-acetylglucosamine-6-phosphate deacetylase, knock-out of a glucosamine- 6-phosphate deaminase, over-expression of a CMP-sialic acid synthetase, and over-expression of an N- acetyl-D-glucosamine-2-epimerase encoding gene.
  • the cell used herein is optionally genetically engineered to express the de novo synthesis of GDP-fucose.
  • GDP-fucose can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing GDP-fucose can express an enzyme converting, e.g., fucose, which is to be added to the cell, to GDP-fucose.
  • This enzyme may be, e.g., a bifunctional fucose kinase/fucose-l-phosphate guanylyltransferase, like Fkp from Bacteroidesfragilis, or the combination of one separate fucose kinase together with one separate fucose-l-phosphate guanylyltransferase like they are known from several species including Homo sapiens, Sus scrofa and Rattus norvegicus.
  • the cell is modified to produce GDP-fucose. More preferably, the cell is modified for enhanced GDP-fucose production.
  • Said modification can be any one or more chosen from the group comprising knock-out of an UDP-glucose:undecaprenyl-phosphate glucose-l-phosphate transferase encoding gene, over-expression of a GDP-L-fucose synthase encoding gene, over-expression of a GDP-mannose 4,6-dehydratase encoding gene, over-expression of a mannose-l-phosphate guanylyltransferase encoding gene, over-expression of a phosphomannomutase encoding gene and over-expression of a mannose-6-phosphate isomerase encoding gene.
  • 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 chosen from the group comprising knock-out of a bifunctional 5'- nucleotidase/UDP-sugar hydrolase encoding gene, knock-out of a galactose-l-phosphate uridylyltransferase encoding gene and over-expression of a UDP-glucose-4-epimerase encoding gene.
  • the cell used herein is optionally genetically engineered to express the de novo synthesis of UDP-GalNAc.
  • UDP-GalNAc can be synthesized from UDP-GIcNAc by the action of a single-step reaction using a UDP-N-acetylglucosamine 4-epimerase like e.g. wbgU from Plesiomonas shigelloides, gne from Yersinia enterocolitica or wbpP from Pseudomonas aeruginosa serotype 06.
  • 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-GIcNAc via an epimerization reaction performed by a UDP-GIcNAc 2-epimerase (like e.g. cap5P from Staphylococcus aureus, RffE from E. coli, Cpsl9fK from S. pneumoniae, and RfbC from S. enterica).
  • a UDP-GIcNAc 2-epimerase like e.g. cap5P from Staphylococcus aureus, RffE from E. coli, Cpsl9fK from S. pneumoniae, and RfbC from S. enterica.
  • the cell is modified to produce UDP-ManNAc. More preferably, the cell is modified for enhanced UDP-ManNAc production.
  • the cell expresses at least one further glycosyltransferase chosen from the list comprising fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N- acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N- glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino- 4,6-dideoxy-N-acetyl-beta-L
  • the fucosyltransferase is chosen from the list comprising alpha-1, 2-fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-1,3/4- fucosyltransferase, alpha-1, 4-fucosyltransferase and alpha-1, 6-fucosyltransferase.
  • the further sialyltransferase is chosen from the list comprising alpha-2, 3-sialyltransferase, alpha-2, 5-sialyltransferase, and alpha-2, 8-sialyltransferase.
  • the galactosyltransferase is chosen from the list comprising beta-1, 3-galactosyltransferase, N- acetylglucosamine beta-1, 3-galactosyltransferase, beta-1, 4-galactosyltransferase, N-acetylglucosamine beta-1, 4-galactosyltransferase, alpha-1, 3-galactosyltransferase and alpha-1, 4-galactosyltransferase.
  • the glucosyltransferase is chosen from the list comprising alpha-glucosyltransferase, beta-1, 2- glucosyltransferase, beta-1, 3-glucosyltransferase and beta-1, 4-glucosyltransferase.
  • the mannosyltransferase is chosen from the list comprising alpha-1, 2-mannosyltransferase, alpha-1, 3- mannosyltransferase and alpha-1, 6-mannosyltransferase.
  • the N- acetylglucosaminyltransferase is chosen from the list comprising galactoside beta-1, 3-N- acetylglucosaminyltransferase and beta-1, 6-N-acetylglucosaminyltransferase.
  • the N- acetylgalactosaminyltransferase is chosen from the list comprising 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 comprises a fucosylation pathway comprising at least one enzyme chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, fucosyltransferase.
  • a fucosylation pathway comprising at least one enzyme chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase,
  • the cell comprises a galactosylation pathway comprising at least one enzyme chosen from the list comprising galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase, galactosyltransferase.
  • a galactosylation pathway comprising at least one enzyme chosen from the list comprising galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase, galactosyltransferase.
  • the cell comprises an N-acetylglucosaminylation pathway comprising at least one enzyme chosen from the list comprising L-glutamine— D-fructose-6-phosphate aminotransferase, N- acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-1- phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase, N- acetylglucosaminyltransferase.
  • 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 IdhA 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 beta-galactosidase, galactoside O-acetyltransferase, N- acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphate glucose-l-phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N- acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man
  • the cell is using a precursor for the synthesis of said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein
  • the precursor is fed to the cell from the cultivation or incubation medium.
  • the cell is producing a precursor for the synthesis of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • the method results in the production of 45 g/L or more, preferably 50 g/L or more, more preferably 60 g/L or more, of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • the method results in the production of 45 g/L or more, preferably 50 g/L or more, more preferably 60 g/L or more of a 3'sLacNAc comprising oligosaccharide or a 3'sLNB comprising oligosaccharide.
  • the cell produces 90 g/L or more of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, in the whole broth and/or supernatant.
  • said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, produced in the whole broth and/or supernatant has a purity of at least 80% measured on the total amount of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, and its precursor produced by the cell in the whole broth and/or supernatant, respectively.
  • the method results in the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, with a purity equal to or greater than 80% measured on the total amount of said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, and its precursor.
  • the method results in the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein with a purity equal to or greater than 85% measured on the total amount of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined hereinx, and its precursor.
  • the method results in the production of said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, with a purity equal to or greater than 90% measured on the total amount of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, and its precursor.
  • the method results in the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, with a purity equal to or greater than 91%, equal to or greater than 92%, equal to or greater than 93%, equal to or greater than 94%, equal to or greater than 95%, equal to or greater than 96%, equal to or greater than 97%, equal to or greater than 98%, equal to or greater than 99% measured on the total amount of said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, and its precursor.
  • the method results in the production of a mixture comprising said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, together with lactose and sialic acid, wherein said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, has a purity equal to or greater than 80% measured on the total amount of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, an acceptor as defined herein, and sialic acid in said mixture and wherein said mixture comprises less than 10% of said saccharide as defined here
  • said mixture comprises less than 9% of said saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein. In an even more preferred embodiment, said mixture comprises less than 8% of said saccharide comprising at least one N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.
  • said mixture comprises less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of said saccharide comprising at least one N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.
  • said mixture comprises less than 5% sialic acid.
  • said mixture comprises less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1% sialic acid.
  • the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is chosen from the list comprising a milk oligosaccharide, O-antigen, the oligosaccharide repeats present in capsular polysaccharides, an oligosaccharide present in lipopolysaccharides and aminosugars.
  • the milk oligosaccharide is a mammalian milk oligosaccharide.
  • the milk oligosaccharide is a human milk oligosaccharide.
  • the cell is capable to synthesize a mixture of oligosaccharides.
  • the cell is capable to synthesize a mixture of di- and/or oligosaccharides, alternatively, the cell is capable to synthesize a mixture of sialic acid, di- and/or oligosaccharides.
  • Another aspect of the invention provides for a method and a cell wherein said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, is produced in and/or by a cell which is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell.
  • the latter bacterium preferably belongs to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus or the phylum of Actinobacteria.
  • the latter bacterium belonging to the phylum Proteobacteria belongs preferably to the family Enterobacteriaceae, preferably to the species Escherichia coli.
  • the latter bacterium preferably relates to any strain belonging to the species Escherichia coli such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultivated Escherichia coli strains - designated as E. coli K12 strains - which are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine.
  • 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 5. fradiae.
  • the latter bacterium belonging to the phylum Proteobacteria preferably belonging to the family of the Vibrionaceae, with member Vibrio natriegens.
  • the latter yeast preferably belongs to the phylum of the Ascomycota or the phylum of the Basidiomycota or the phylum of the Deuteromycota or the phylum of the Zygomycetes.
  • the latter yeast belongs preferably to the genus Saccharomyces (with members like e.g. Saccharomyces cerevisiae, S. bayanus, S. boulardii), Zygosaccharomyces, Pichia (with members like e.g. Pichia pastoris, P. anomala, P.
  • 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 chosen from the list comprising an epithelial cell like e.g., a mammary epithelial cell, an embryonic kidney cell (e.g., HEK293 or HEK 293T cell), a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell like e.g.
  • an 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
  • the latter insect cell is preferably derived from Spodoptera frugiperda like e.g., Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae, Trichoplusia ni like e.g., BTI-TN-5B1-4 cells or Drosophila melanogaster Wke e.g., Drosophila S2 cells.
  • the latter protozoan cell preferably is a Leishmania tarentolae cell.
  • the cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably from the group consisting of yeast cells, bacterial cells, archaebacterial cells, algae cells, and fungal cells as described herein.
  • the cell as described herein comprises a nucleic acid molecule comprising a polynucleotide sequence encoding a sialyltransferase as described herein and operably linked to control sequences recognized by the cell, wherein said sequence is foreign to the cell, said sequence further i) being integrated in the genome of said cell and/or ii) presented to said cell on a vector.
  • the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the synthesis of a sialylated oligosaccharide.
  • a further aspect of the present invention provides for an isolated nucleic acid molecule encoding a sialyltransferase wherein said sialyltransferase is an alpha-2, 3-sialyltransferase as defined herein.
  • the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues.
  • the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 12, 8, 11, 15, 13, 24, 30, 31, 25, 18, 26 or 22.
  • the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence that is at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 1, 6, 9, 3, 7, 23 or 27.
  • the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence that is at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues.
  • the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to the full-length amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29.
  • the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.
  • Another further aspect of the present invention provides for an isolated nucleic acid molecule encoding a sialyltransferase wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity on the galactose (Gal) residue of said saccharide as defined herein, and comprises an amino acid sequence that is at least 80.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.
  • the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full- length amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.
  • the sialyltransferase encoded by said isolated nucleic acid molecule comprises an amino acid sequence as represented by any one of SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.
  • Another aspect of the present invention provides for a vector comprising an isolated nucleic acid molecule encoding a sialyltransferase as described herein.
  • a cell to be stably cultured in a medium, wherein said medium can be any type of growth medium comprising minimal medium, complex medium or growth medium enriched in certain compounds like, for example, but not limited to, vitamins, trace elements, amino acids.
  • the microorganism or cell as used herein is capable to grow on a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium or a mixture thereof as the main carbon source.
  • main is meant the most important carbon source for the microorganism or cell for the production of the sialylated oligosaccharide of interest, biomass formation, carbon dioxide and/or by-products formation (such as acids and/or alcohols, such as acetate, lactate, and/or ethanol), i.e. 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99% of all the required carbon is derived from the aboveindicated carbon source.
  • 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.
  • the methods as described herein preferably comprises a step of separating said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide of present invention from said cultivation or incubation, otherwise said recovering the 3'sialylated oligosaccharide, from the cultivation or incubation medium and/or the cell.
  • separating from said cultivation or incubation means harvesting, collecting, or retrieving said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide from the cell and/or the medium of its cultivation or incubation.
  • the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide can be separated in a conventional mannerfrom the aqueous culture medium, in which the cell was cultivated or incubated.
  • the cultivation or incubation medium and/or cell extract together and separately can then be further used for separating said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide can be clarified in a conventional manner.
  • said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide is clarified by centrifugation, flocculation, decantation and/or filtration.
  • Another step of separating said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide preferably involves removing substantially all the eventually remaining proteins, peptides, amino acids, RNA and DNA, and any endotoxins and glycolipids that could interfere with the subsequent separation step, from said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide preferably after it has been clarified.
  • remaining proteins and related impurities can be removed from said 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide in a conventional manner.
  • remaining proteins, salts, by-products, colour, endotoxins and other related impurities are removed from said 3'sialylated oligosaccharide, by ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, electrophoresis (e.g.
  • affinity chromatography using affinity ligands including e.g. DEAE-Sepharose, poly-L-lysine and polymyxin-B, endotoxin-selective adsorber matrices), ion exchange chromatography (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange, inside-out ligand attachment), hydrophobic interaction chromatography and/or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography or electrodialysis. With the exception of size exclusion chromatography, remaining proteins and related impurities are retained by a chromatography medium or a selected membrane.
  • the methods as described herein also provide for a further purification of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide of present invention.
  • a further purification of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide may be accomplished, for example, by use of (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange, temperature adjustment, pH adjustment or pH adjustment with an alkaline or acidic solution to remove any remaining DNA, protein, LPS, endotoxins, or other impurity. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used.
  • Another purification step is accomplished by crystallization, evaporation or precipitation of said 3'sialylated oligosaccharide.
  • Another purification step is to dry, e.g. spray dry, lyophilize, spray freeze dry, freeze spray dry, band dry, belt dry, vacuum band dry, vacuum belt dry, drum dry, roller dry, vacuum drum dry or vacuum roller dry the produced 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • the separation and purification of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is made in a process, comprising the following steps in any order: a) contacting the cultivation or incubation or a clarified version thereof with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da ensuring the retention of the produced 3'sialylated oligosaccharide, and allowing at least a part of the proteins, salts, byproducts, colour and other related impurities to pass, b) conducting a diafiltration process on the retentate from step a), using said membrane, with an aqueous solution of an inorganic electrolyte, followed by optional diafiltration with pure water to remove excess of the electrolyte, c) and collecting the retentate enriched in said 3'sialylated
  • the separation and purification of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is made in a process, comprising the following steps in any order: subjecting the cultivation or incubation or a clarified version thereof to two membrane filtration steps using different membranes, wherein one membrane has a molecular weight cut-off of between about 300 to about 500 Dalton, and the other membrane as a molecular weight cut-off of between about 600 to about 800 Dalton.
  • the separation and purification of said 3'sialylated oligosaccharide is made in a process, comprising treating the cultivation or incubation or a clarified version thereof with a strong cation exchange resin in H + -form in a step and with a weak anion exchange resin in free base form in another step, wherein said steps can be performed in any order.
  • the separation and purification of said oligosaccharide is made in the following way.
  • the cultivation or incubation comprising the produced 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, biomass, medium components and contaminants is applied to the following purification steps: i) separation of biomass from the cultivation or incubation, ii) cationic ion exchanger treatment for the removal of positively charged material, iii) anionic ion exchanger treatment for the removal of negatively charged material, iv) nanofiltration step and/or electrodialysis step, wherein a purified solution comprising the produced 3'sialylated at a purity of greater than or equal to 80% is provided.
  • the purified solution is dried by any one or more drying steps chosen from the list comprising spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying and vacuum roller drying.
  • the separation and purification of the 3'sialylated oligosaccharide as defined herein is made in a process, comprising the following steps in any order: enzymatic treatment of the cultivation or incubation; removal of the biomass from the cultivation or incubation; ultrafiltration; nanofiltration; and a column chromatography step.
  • a column chromatography step is a single column or a multiple column.
  • the column chromatography step is simulated moving bed chromatography.
  • Such simulated moving bed chromatography preferably comprises i) at least 4 columns, wherein at least one column comprises a weak or strong cation exchange resin; and/or ii) four zones I, II, III and IV with different flow rates; and/or iii) an eluent comprising water; and/or iv) an operating temperature of 15 degrees to 60 degrees centigrade.
  • the present invention provides the 3'sialylated oligosaccharide as defined herein which is dried to powder by any one or more drying steps chosen from the list comprising spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying and vacuum roller drying, wherein the dried powder contains ⁇ 15% -wt. of water, preferably ⁇ 10% -wt. of water, more preferably ⁇ 7% -wt. of water, most preferably ⁇ 5% -wt. of water.
  • Another aspect of the present invention provides the use of a sialyltransferase that has alpha-2, 3- sialyltransferase activity on the galactose (Gal) residue of said saccharide comprising at least one N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein and that comprises an amino acid sequence: that is at least 50.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190,
  • 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.
  • Another aspect of the present invention provides the use of a sialyltransferase that has alpha-2, 3- sialyltransferase activity on the galactose (Gal) residue of a saccharide comprising at least one N- acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, and that comprises an amino acid sequence: that is at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the full-length amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 17, 7, 10, 14, 16, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31, or
  • 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.
  • Another aspect of the present invention provides the use of a cell as described herein for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein.
  • a further aspect of the present invention provides i) use of a method as described herein for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, ii) use of an isolated nucleic acid molecule as described herein for production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, or iii) use of a vector as described herein for production of said 3'sialylated oligosaccharide as defined herein.
  • a further aspect of the present invention provides an alpha-2, 3-sialyltransferase for use in the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity on the galactose (Gal) residue of an acceptor, wherein said acceptor is a saccharide comprising at least one N-acetylgluco
  • a further aspect provides for an alpha-2, 3-sialyltransferase as described herein wherein said 3'sialylated oligosaccharide is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-Lewis x.
  • the invention also relates to the 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide obtained by the methods according to the invention.
  • Said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide may be used for the manufacture of a preparation, as food additive, prebiotic, symbiotic, for the supplementation of baby food, adult food, infant animal feed, adult animal feed, or as either therapeutically or pharmaceutically active compound or in cosmetic applications.
  • said preparation comprises at least one 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide as defined herein, that is obtainable, preferably obtained, by the methods as described herein.
  • 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 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide can easily and effectively be provided, without the need for complicated, time and cost consuming synthetic processes.
  • the monosaccharide or the monomeric building blocks e.g. the monosaccharide or glycan unit composition
  • the anomeric configuration of side chains e.g. the monosaccharide or glycan unit composition
  • 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 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide, is methylated with methyl iodide and strong base in DMSO, hydrolysis is performed, a reduction to partially methylated alditols is achieved, an acetylation to methylated alditol acetates is performed, and the analysis is carried out by GLC/MS (gas- liquid chromatography coupled with mass spectrometry).
  • a partial depolymerization is carried out using an acid or enzymes to determine the structures.
  • the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is subjected to enzymatic analysis, e.g. it is contacted with an enzyme that is specific for a particular type of linkage, e.g., beta-galactosidase, or alphaglucosidase, etc., and NMR may be used to analyse the products.
  • the separated and preferably also purified 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide as described herein is incorporated into a food (e.g., human food or feed), dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine.
  • a food e.g., human food or feed
  • the 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide is mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine.
  • 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 3'sialylated oligosaccharide being a prebiotic produced and/or purified by a process disclosed in this specification, to promote growth of one or more beneficial microorganisms.
  • prebiotic ingredients for dietary supplements include other prebiotic molecules (such as HMDs) and plant polysaccharides (such as inulin, pectin, b-glucan and xylooligosaccharide).
  • a "probiotic" product typically contains live microorganisms that replace or add to gastrointestinal microflora, to the benefit of the recipient.
  • 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 3'sialylated 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 3'sialylated oligosaccharide is incorporated into a human baby food (e.g., infant formula).
  • Infant formula is generally a manufactured food for feeding to infants as a complete or partial substitute for human breast milk.
  • 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 3'sialylated oligosaccharide, produced and/or purified by a process in this specification is included in infant formula to provide nutritional benefits similar to those provided by the oligosaccharides in human breast milk.
  • the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, is mixed with one or more ingredients of the infant formula.
  • infant formula ingredients include non-fat milk, carbohydrate sources (e.g., lactose), protein sources (e.g., whey protein concentrate and casein), fat sources (e.g., vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and/or sunflower oil; and fish oils), vitamins (such as vitamins A, Bb, Bi2, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate) and possibly human milk oligosaccharides (HMDs).
  • carbohydrate sources e.g., lactose
  • protein sources e.g., whey protein concentrate and casein
  • fat sources e.g., vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and/or sunflower oil; and fish oils
  • vitamins such as vitamins A, Bb, Bi2, C and D
  • minerals such as potassium citrate, calcium 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.
  • 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.
  • the one or more infant formula ingredients comprise lactose, whey protein concentrate and/or high oleic safflower oil.
  • the concentration of the 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide, in the infant formula is approximately the same concentration as the concentration of the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide generally present in human breast milk.
  • the 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, is incorporated into a feed preparation, wherein said feed is chosen from the list comprising pet food, animal milk replacer, veterinary product, veterinary feed supplement, nutrition supplement, post weaning feed, or creep feed.
  • the methods and the cell of the invention preferably provide at least one of the following further surprising advantages when using a sialyltransferase as described herein:
  • sucrose Ys g sialylated oligosaccharide / g sucrose, preferably g 3'sialylated oligosaccharide / g sucrose
  • a 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide- containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N- acetylglucosamine, more preferably a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide, the method comprising: contacting a sialyltransferase with a mixture comprising a donor comprising a sialic acid residue, and an acceptor in a medium, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from the donor to the acceptor, thereby producing said 3'sia
  • Method for the production of a 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide- containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N- acetylglucosamine , said method comprising the steps of: a) providing i. CMP-sialic acid, ii.
  • acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, and iii.
  • sialyltransferase wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity and comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22, at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 17, 10,
  • Method according to any one of previous embodiments comprising: contacting a cell extract comprising said sialyltransferase with a mixture comprising said donor comprising a sialic acid residue, and said acceptor, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from the donor to the acceptor, thereby producing said 3'sialylated oligosaccharide.
  • Method for the production of a 3'sialylated oligosaccharide comprising at least an N- acetylglucosamine monosaccharide and a galactose monosaccharide, said method comprising the steps of: i.
  • sialyltransferase wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity, and comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22, or at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to the amino acid sequence as represented by SEQ ID NO: 17, 10, 14, 16, 28 or 29, ii.
  • CMP-sialic acid optionally said CMP-sialic acid is produced by said cell, and iii. providing an acceptor being a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, optionally said saccharide is produced by said cell, and iv. cultivating and/or incubating said cell under conditions permissive to express said sialyltransferase, optionally permissive to produce said CMP-sialic acid and/or said oligosaccharide or disaccharide, v. preferably, separating said 3'sialylated oligosaccharide, from said cultivation or incubation.
  • said cell is a metabolically engineered cell, preferably wherein said cell is metabolically engineered for the production of said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide.
  • sialyltransferase comprises an amino acid sequence: that is at least at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27 , 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%
  • the cultivation medium contains at least one carbon source selected from the group consisting of glucose, fructose, sucrose, and glycerol.
  • the medium, cultivation medium or incubation medium contains at least one compound selected from the group consisting of lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GIcNAc), sialic acid and CMP-sialic acid.
  • Method according to any one of previous embodiments wherein said 3'sialylated oligosaccharide, is recovered from the medium, cultivation or incubation medium and/or from the cell.
  • the method comprising: i) use of a medium, cultivation medium or incubation medium comprising at least one precursor and/or acceptor for the production of said 3'sialylated oligosaccharide, and/or ii) adding to the medium at least one precursor and/or acceptor feed for the production of said 3'sialylated oligosaccharide, preferably said precursor is chosen from the list comprising lactose, galactose, lacto-N-tetraose, lacto-N-neotetraose (LNnT), LacNAc, LNB, UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP- GIcNAc), sia
  • the method comprising at least one of the following steps: i) use of a cultivation or incubation medium comprising at least one precursor and/or acceptor; ii) adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and/or acceptor feed wherein the total reactor or incubator volume ranges from 250 mL to 10.000 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, preferably not more than two-fold, more preferably less than 2-fold of the volume of the cultivation or incubation medium before the addition of said precursor and/or acceptor feed; iii) Adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and/or acceptor feed wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incuba
  • sialyltransferase has alpha- 2,3-sialyltransferase activity on the galactose (Gal) residue of said acceptor and/or wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably LacNAC, LNB, a LacNAc comprising oligosaccharide or an LNB comprising oligosaccharide, even more preferably an LNT or an LNnT.
  • said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, preferably is a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide, more preferably is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-
  • sialyltransferase comprises an amino acid sequence: that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%, at least 60.0%, at least 65
  • said fungus belongs to a genus chosen from the group comprising Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus, preferably said yeast belongs to a genus chosen from the group comprising Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces or Debaromyces, preferably said plant cell is an algal cell or is derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant, preferably said animal cell is derived from non-human mammals, birds, fish, invertebrates, reptiles, amphibians or insects or is a genetically engineered cell line derived from human cells excluding embryonic stem cells, more preferably said human and non-human mammalian cell is an epithelial cell, an embryonic kidney cell, a fibro
  • sialyltransferase has alpha-2, 3- sialyltransferase activity on the galactose (Gal) residue of said acceptor and/or wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably LacNAC, LNB, a LacNAc comprising oligosaccharide or an LNB comprising oligosaccharide, even more preferably an LNT or an LNnT.
  • said 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide is a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, preferably is a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide, more preferably is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-
  • sialyltransferase wherein said sialyltransferase has alpha-2, 3-sialyltransferase activity on the galactose (Gal) residue of an acceptor, wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, and wherein said sialyltransferase comprises an amino acid sequence that is: at least 60.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues, to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22, at least 85.0% identical over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid
  • sialyltransferase comprises an amino acid sequence: that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%, at least 55.0%,
  • a vector comprising the nucleic acid molecule of any one of embodiment 25 or 26. 8.
  • sialyltransferase that has alpha-2, 3-sialyltransferase activity on the galactose (Gal) residue of an acceptor, wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and a galactose monosaccharide, wherein said saccharide is an oligosaccharide or a disaccharide, and wherein said sialyltransferase comprises an amino acid sequence: that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31
  • a cell according to any one of embodiments 16 to 24 for production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sLacNAc comprising oligosaccharide or a 3'sLNB comprising oligosaccharide or a, most preferably chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sia
  • a method according to any one of embodiments 1 to 15 for production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sLacNAc comprising oligosaccharide or a 3'sLNB comprising oligosaccharide, most preferably 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl- Lewis a, sialyl-Lewis x.
  • an isolated nucleic acid molecule for production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sLacNAc comprising oligosaccharide or a 3'sLNB comprising oligosaccharide, most preferably 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl- Lewis a, sialyl-Lewis x
  • a vector according to embodiment 27 for production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sLacNAc comprising oligosaccharide or a 3'sLNB comprising oligosaccharide, most preferably 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-Lewis x.
  • An alpha-2, 3-sialyltransferase for use in the production of a 3'sialylated oligosaccharide comprising at least an N-acetylglucosamine monosaccharide and a galactose monosaccharide, preferably a disaccharide-containing 3'sialylated oligosaccharide wherein said disaccharide consists of a galactose and a N-acetylglucosamine, more preferably a 3'sialylated LacNAc comprising oligosaccharide or a 3'sialylated LNB comprising oligosaccharide wherein said sialyltransferase has alpha-2, 3- sialyltransferase activity on the galactose (Gal) residue of an acceptor, wherein said acceptor is a saccharide comprising at least one N-acetylglucosamine monosaccharide and
  • sialyltransferase comprises an amino acid sequence: that is at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 98.5%, or at least 99.0% identical to any one of the amino acid sequences as represented by SEQ ID NO: 2, 1, 6, 12, 8, 11, 15, 13, 9, 3, 7, 23, 27, 24, 30 ,31, 25, 18, 26 or 22 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues, that is at least 50.0%
  • 3'sialyltransferase according to embodiment 33 or 34 wherein said 3'sialylated oligosaccharide is chosen from the list consisting of 3'SLNB, 3'SLacNAc, LST a, LST d, DSLNT, DS'LNnT, sialylated tetraose type 1, sialylated tetraose type 2, sialyl-Lewis a, sialyl-Lewis x.
  • 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 or Twist Bioscience. Proteins described in present disclosure are summarized in Tables 1 and 2 and 4. Unless stated otherwise, 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.
  • GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. (1970) 48: 443-453) to find the global (i.e. spanning the full-length sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps.
  • 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 (i.e. spanning the full-length sequences) 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.
  • the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147 (1); 195-7).
  • Standards such as but not limited to sucrose, lactose, LacNAc, lacto-N-biose (LNB), fucosylated LacNAc (2'FLacNAc, 3-FLacNAc), sialylated LacNAc, (3'SLacNAc, 6'SLacNAc), fucosylated LNB (2'FLNB, 4'FLNB), lacto-/V-triose II (LN3), lacto-N-tetraose (LNT), lacto-/V-neo-tetraose (LNnT), LNFP-I, LNFP-II, LNFP-III, LNFP- V, LSTa, LSTc and LSTd were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with in-house made standards.
  • Neutral oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Evaporative Light Scattering Detector (ELSD) or a Refractive Index (Rl) detection.
  • ELSD Evaporative Light Scattering Detector
  • Rl Refractive Index
  • a volume of 0.7 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm; 130 A; 1.7 pm) column with an Acquity UPLC BEH Amide VanGuard column, 130 A, 2. lx 5 mm.
  • the column temperature was 50°C.
  • the mobile phase consisted of a % water and % 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
  • Sialylated oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Refractive Index (Rl) detection.
  • a volume of 0. 5 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm; 130 A; 1.7 pm). 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 Rl 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 pm) on 35°C.
  • a gradient was used wherein 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. For column equilibration, the initial condition of 2 % of eluent B was restored in 1 min and maintained for 12 min.
  • Both neutral and sialylated sugars at low concentrations were analyzed on a Dionex HPAEC system with pulsed amperometric detection (PAD).
  • a volume of 5 pL of sample was injected on a Dionex CarboPac PA200 column 4 x 250 mm with a Dionex CarboPac PA200 guard column 4 x 50 mm.
  • the column temperature was set to 30°C.
  • a gradient was used wherein eluent A was deionized water, wherein eluent B was 200 mM Sodium hydroxide and wherein 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.
  • 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 95-well plates or in shake flasks contained 2.00 g/L NH 4 CI, 5.00 g/L (NH 4 ) 2 SO 4 , 2.993 g/L KH 2 PO 4 , 7.315 g/L K 2 HPO 4 , 8.372 g/L MOPS, 0.5 g/L NaCI, 0.5 g/L MgSO 4 .7H 2 O, 30 g/L sucrose or 30 g/L glycerol, 1 ml/L vitamin solution, 100 pl/L molybdate solution, and 1 mL/L selenium solution.
  • Vitamin solution consisted of 3.6 g/L FeCI 2 .4H 2 O, 5.0 g/L CaCI 2 .2H 2 O, 1.3 g/L MnCI 2 .2H 2 O, 0.38 g/L CuCI 2 .2H 2 O, 0.5 g/L CoCI 2 .6H 2 O, 0.94 g/L ZnCI 2 , 0.0311 g/L HaBO 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 NaMoO 4 .2H 2 O.
  • the selenium solution contained 42 g/L SeO 2 .
  • the minimal medium for fermentations contained 6.75 g/L NH 4 CI, 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 NaCI, 0.5 g/L MgSO 4 .7H 2 O, 30 g/L sucrose or 30 g/L glycerol, 1 mL/L vitamin solution, 100 piL/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above. As specified in the respective examples, 0.30 g/L 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 pm Sartorius). When necessary, 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 antibiotic e.g. chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L).
  • a preculture for the bioreactor was started from an entire 1 mL cryovial of a certain strain, inoculated in 250 m L 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 H 2 S0 4 and 20% NH4OH.
  • the exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.
  • 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). Plasmids were maintained in the host E.
  • coli DH5alpha (F phi80d/ocZAM15, A IJacZYA-argF] U169, deoR, recAl, endAl, hsdR17 (rk _ , mk + ), phoA, supE44, lambda', thi-1, gyrA96, relAl) bought from Invitrogen.
  • Escherichia coli K12 MG1655 [A-, F', rph-1] was obtained from the Coli Genetic Stock Center (US), CGSC Strain#: 7740, in March 2007.
  • Gene disruptions, gene introductions and gene replacements were performed using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). This technique is based on antibiotic selection after homologous recombination performed by lambda Red recombinase. Subsequent catalysis of a flippase recombinase ensures removal of the antibiotic selection cassette in the final production strain.
  • Transformants carrying a Red helper plasmid pKD46 were grown in 10 mL LB media with ampicillin, (100 mg/L) and L-arabinose (10 mM) at 30°C to an ODgoonm of 0.6.
  • the cells were made electrocompetent by washing them with 50 mL of ice-cold water, a first time, and with ImL ice cold water, a second time. Then, the cells were resuspended in 50 pL of ice-cold water. Electroporation was done with 50 pL of cells and 10-100 ng of linear double-stranded-DNA product by using a Gene PulserTM (BioRad) (600 Q, 25 pFD, and 250 volts).
  • BioRad Gene PulserTM
  • cells were added to 1 mL LB media incubated 1 h at 37°C, and finally spread onto LB-agar containing 25 mg/L of chloramphenicol or 50 mg/L of kanamycin to select antibiotic resistant transformants.
  • the selected mutants were verified by PCR with primers upstream and downstream of the modified region and were grown in LB-agar at 42°C for the loss of the helper plasmid. The mutants were tested for ampicillin sensitivity.
  • the linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and their derivates as template.
  • the primers used had a part of the sequence complementary to the template and another part complementary to the side on the chromosomal DNA where the recombination must take place.
  • the region of homology was designed 50-nt upstream and 50-nt downstream of the start and stop codon of the gene of interest.
  • the transcriptional starting point (+1) had to be respected.
  • PCR products were PCR-purified, digested with Dpnl, re-purified from an agarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0).
  • pCP20 plasmid which is an ampicillin and chloramphenicol resistant plasmid that shows temperature- sensitive replication and thermal induction of FLP synthesis.
  • the ampicillin-resistant transformants were selected at 30°C, after which a few were colony purified in LB at 42°C and then tested for loss of all antibiotic resistance and of the FLP helper plasmid. The gene knock outs and knock ins are checked with control primers.
  • 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.
  • GNA1 from Saccharomyces cerevisiae
  • N-acylglucosamine 2-epimerase like e.g. AGE from Bacteroides ovatus (UniProt ID A7LVG6)
  • N-acetylneuraminate synthase like e.g.
  • sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a UDP-N- acetylglucosamine 2-epimerase like e.g. NeuC from C. jejuni (UniProt ID Q93MP8) and an N- acetylneuraminate synthase like e.g. NeuB from Neisseria meningitidis (UniProt ID E0NCD4) or NeuB from Campylobacter jejuni (UniProt ID Q93MP9).
  • a UDP-N- acetylglucosamine 2-epimerase like e.g. NeuC from C. jejuni (UniProt ID Q93MP8)
  • an N- acetylneuraminate synthase like e.g. NeuB from Neisseria meningitidis (UniProt ID E0NCD4) or NeuB from Campylobacter jejuni (UniProt ID Q93MP9).
  • 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), an N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase like e.g. glmU from E. coli (UniProt ID P0ACC7), a UDP-N-acetylglucosamine 2-epimerase like e.g. NeuC from C.
  • a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120)
  • sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a bifunctional UDP- GIcNAc 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. HK-1 (UniProt ID KPA15328.1) or NANP from Bacteroides thetaiotaomicron (UniProt ID Q8A712).
  • NANS from Pseudomonas sp. UW4
  • N-acylneuraminate-9-phosphatase like e.g. NANP from Candidatus Magnetomorum sp. HK-1 (UniProt ID KPA15328.1) or NANP from Bacteroides thetaiotaomicron (UniProt ID
  • 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), an N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-1- phosphate acetyltransferase like e.g. glmU from E. coli (UniProt ID P0ACC7), a bifunctional UDP-GIcNAc 2- epimerase/N-acetylmannosamine kinase like e.g. GNE from M.
  • a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120)
  • 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. HK-1 (UniProt ID KPA15328.1) or NANP from Bacteroides thetaiotaomicron (UniProt ID Q.8A712).
  • 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 WO 2018/122225, and/or genomic knock-outs of the E.
  • coli genes comprising any one or more of nanT, poxB, IdhA, adhE, aldB, pflA, pfIC, 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. the mutant glmS*54 from E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006) ) and an acetyl-CoA synthetase like e.g. acs from E. coli (UniProt ID P27550).
  • sialylated oligosaccharide production like e.g. LSTa (Neu5Ac-a2,3-Gal-pi,3-GlcNAc- pi,3-Gal-pi,4-Glc)
  • said sialic acid production strains were further modified to express an N- acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from Pasteurella multocida (SEQ ID NO: 21) and to express one or more copies of a beta-galactoside alpha-2, 3-sialyltransferase like e.g.
  • PmultST2 from Pasteurella multocida (SEQ ID NO: 20) or the polypeptides with SEQ ID NO: 1 - 11, 23, 24, 25, 27, 28, 29 or 30.
  • 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. coli LacZ, LacY and LacA genes and with a genomic knock-in of a constitutive transcriptional unit for a lactose permease like e.g. E. coli LacY (UniProt ID P02920).
  • All modified strains producing sialic acid, CMP-sialic acid and/or sialylated oligosaccharides could optionally be adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E. coli ⁇ N (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Z. mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP from B. adolescentis (UniProt ID A0ZZH6).
  • a sucrose transporter like e.g. CscB from E. coli ⁇ N (UniProt ID E0IXR1)
  • a fructose kinase like e.g. Frk originating from Z. mobilis
  • a sucrose phosphorylase like e.g. BaSP from
  • sialylated oligosaccharide production like e.g. LSTd (Neu5Ac-ct2,3-Gal-pi,4-GlcNAc- pi,3-Gal-pi,4-Glc)
  • said sialic acid production strains were further modified to express an N- acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from Pasteurella multocida (SEQ ID NO: 21) and to express one or more copies of a beta-galactoside alpha-2, 3-sialyltransferase like e.g.
  • PmultST3 from Pasteurella multocida (SEQ ID NO: 19) or a beta-galactoside alpha-2, 3-sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30, 31, 4 or 5.
  • 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.
  • 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. IgtA (UniProt ID Q9JXQ6) from N. meningitidis.
  • the mutant 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.
  • 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.
  • the mutant 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. IgtB (Uniprot ID Q.51116, sequence version 02, 01 Dec 2000) from Neisseria meningitidis.
  • 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. IgtB (Uniprot ID Q.51116, sequence version 02, 01 Dec 2000) from Neisseria meningitidis.
  • LN3, LNT and/or LNnT production can further be optimized in the modified E. coli strains with genomic knock-outs of the E. coli genes comprising any one or more of galT, ushA, IdhA and agp.
  • the modified LN3, LNT and/or LNnT producing strains can also be optionally modified for enhanced UDP-GIcNAc production with a genomic knock-in of a constitutive transcriptional unit for an L-glutamine— D-fructose-6-phosphate aminotransferase like e.g. the mutant glmS*54 from E. coli (differing from the wild-type E.
  • the modified E. 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.
  • 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 enhancer tag like e.g. a SUMO-tag, an MBP-tag, His, FLAG, Strep-11, Halo-tag, NusA, thioredoxin, GST and/or the Fh8-tag to enhance their solubility (Costa et al., Front. Microbiol. 2014, https://doi.org/10.3389/fmicb.2014.00063; Fox et al., Protein Sci. 2001, 10 (3), 622-630; Jia and Jeaon, Open BioL 2016, 6: 160196).
  • a solubility enhancer tag like e.g. a SUMO-tag, an MBP-tag, His, FLAG, Strep-11, Halo-tag, NusA, thioredoxin, GST and/or the Fh8-tag to
  • the modified E. 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 (Baneyx F., Palumbo J. L. (2003) Improving Heterologous Protein Folding via Molecular Chaperone and Foldase Co-Expression. In: Vaillancourt P.E. (eds) E. coli Gene Expression Protocols. Methods in Molecular BiologyTM, vol 205. Humana Press). All constitutive promoters, UTRs and terminator sequences originated from the libraries described by Cambray et al. (Nucleic Acids Res.
  • An E. coli K-12 MG1655 strain modified for production of sialic acid comprising genomic knock-ins of constitutive transcriptional units containing the UDP-N-acetylglucosamine 2-epimerase NeuC from C. jejuni (UniProt ID Q93MP8) and the N-acetylneuraminate synthase NeuB from Neisseria meningitidis (UniProt ID E0NCD4), and modified for growth on sucrose as described in Example 2 was further modified with a genomic knock-in of a constitutive transcriptional unit for the galactoside beta-1, 3-N- acetylglucosaminyltransferase LgtA from N.
  • meningitidis (UniProt ID Q9JXQ6) and for the N- acetylglucosamine beta-1, 3-galactosyltransferase like e.g., wbgO (Uniprot ID D3QY14) from E. coli 055:1-17 to allow production of lacto-N-tetraose (LNT, Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc).
  • lacto-N-tetraose LNT, Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc
  • multiple copies of the LgtA and/or wbgO genes could be added to the modified E. coli strain.
  • the novel strain was transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuAfrom P. multocida with SEQID NO: 21 and for i) an alpha- 2, 3-sialyltransferase with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 7, 23, 24, 25, 27, 28, 29 or 30 or ii) an alpha-2, 3-sialyltransferase with SEQ ID NO: 20 acting as a reference alpha-2, 3-sialyltransferase.
  • the novel strains were evaluated in a growth experiment for production of LSTa (Neu5Ac-a2,3-Gal-pi,3-GlcNAc- pi,3-Gal-pi,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.
  • the measured LSTa concentration measured of strains expressing an alpha-2, 3-sialyltransferase with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 7, 23, 24, 25, 27, 28, 29 or 30 was averaged over all biological replicates and then normalized to the averaged LSTa concentration of a reference strain expressing an alpha-2, 3-sialyltransferase with SEQ ID NO: 20.
  • the experiment showed all strains each expressing a different alpha-2, 3-sialyltransferase, were able to produce LSTa.
  • strains expressing the sialyltransferase with SEQ ID NO: 1, 2, 3, 6, 8, 9, 10, 11, 7, 23, 24, 25, J , 28, 29 or 30, showed a production of LSTa that was higher than the production of LSTa measured in the reference strain with SEQ ID NO: 20, and also higher than the production of LSTa by a strain with prior art SEQ ID NO: 4 or 5.
  • Table 2 Relative production of LSTa (%) in modified E.
  • Example 2 coli strains expressing an alpha-2, 3-sialyltransferase with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 7, 23, 24, 25, 27, 28, 29, or 30 and compared to an alpha-2, 3- sialyltransferase with SEQ ID NO: 20 serving as a reference, when evaluated in a growth experiment according to the culture conditions provided in Example 2, in which the culture medium contained 30 g/L sucrose and 20 g/L lactose.
  • Example 3 All modified E. coli strains as described in Example 3 were evaluated in another growth experiment for production of LNT, LSTa (Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal- 1,4-Glc) and 3'SL (Neu5Ac-a2,3-Gal- 01,4-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.
  • the measured LNT, LSTa and 3'SL concentration measured of strains, expressing an alpha-2, 3-sialyltransferase with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 7, 23, 24, 25, 27, 28, 29 or 30, was averaged over all biological replicates and then normalized to the averaged LNT, LSTa and 3'SL concentration, respectively, of the reference strain expressing an alpha-2, 3-sialyltransferase with SEQ ID NO: 20.
  • the experiment showed all strains, each expressing a different alpha-2, 3-sialyltransferase, were able to produce LNT, LSTa and 3'SL.
  • the sialyltransferase with SEQ ID NO:1, 2, 3, 6, 8, 9, 10, 11, 7, 23, 24, 25, 27, 28, 29 or 30 had a better 3-sialyltransferase binding activity on LNT than the reference sialyltransferase, with all strains showing a production of LSTa that was higher than the production of LSTa measured in the reference strain and having less of non-sialylated LNT compared to the strain expressing SEQ ID NO: 20, and also higher than the production of LSTa by a strain with prior art SEQ ID NO: 4 or 5.
  • An E. coli K-12 MG1655 strain modified to produce LNT as described in Example 2 is further transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P. multocida with SEQ ID NO: 21 and for i) an alpha-2, 3-sialyltransferase with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 7, 23, 24, 25, 27, 28, 29 or 30 or ii) an alpha-2, 3-sialyltransferase with SEQ ID NO: 20 acting as a reference alpha-2, 3-sialyltransferase.
  • the novel strains are evaluated in a growth experiment for production of LSTa (Neu5Ac-oc2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc) according to the culture conditions provided in Example 2, in which the strains are cultivated in minimal medium with glycerol as carbon source and sialic acid and lactose added to the medium.
  • LSTa Neuronucleic acid
  • An E. coli K-12 MG1655 strain modified to produce LNT as described in Example 2 is further modified for growth on sucrose as described in Example 2.
  • the strain is transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P. multocida with SEQ ID NO: 21 and for i) an alpha-2, 3-sialyltransferase with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 7, 23, 24, 25, 27, 28, 29, or 30 or ii) an alpha-2, 3-sialyltransferase with SEQ ID NO: 20 acting as a reference alpha-2, 3-sialyltransferase.
  • the novel strains are evaluated in a growth experiment for production of LSTa (Neu5Ac-o.2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc) according to the culture conditions provided in Example 2, in which the strains are cultivated in minimal medium with sucrose as carbon source and sialic acid and lactose added to the medium.
  • LSTa Neuronucleic acid
  • Example 7 Production of LSTa with modified E. coli hosts when evaluated in a fed-batch fermentation process with sucrose and lactose
  • the modified E. coli strains expressing the alpha-2, 3-sialyltransferase with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 7, 23, 24, 25, 27, 28, 29 or 30 and able to produce LSTa as described in Example 3 were selected for further evaluation in a fed-batch fermentation process.
  • Fed-batch fermentations at bioreactor scale were performed as described in Example 2.
  • Sucrose was used as a carbon source and lactose was added in the batch medium. During fed-batch, sucrose was added via an additional feed.
  • regular broth samples were taken at several time points during the fermentation process and sugars produced were measured using UPLC as described in Example 1.
  • Another example provides the evaluation of alpha-2, 3-sialyltransferase activity of the enzymes with SEQ
  • Said enzyme can be produced in a cell-free expression system such as but not limited to the PURExpress system (NEB), or in a host organism such as but not limited to Escherichia coli or Saccharomyces cerevisiae, after which the enzyme can be isolated and optionally further purified.
  • the enzyme extract or purified enzyme is added to a reaction mixture together with CMP-sialic acid and a buffering component such as Tris-HCI or HEPES and a substrate like e.g. lacto-N-tetraose (LNT) or lactose.
  • reaction mixture is then incubated at a certain temperature (for example 37°C) for a certain amount of time (for example 8 hours, 16 hours, 24 hours), during which the LNT or lactose will be converted by the enzyme using CMP-sialic acid to LSTa or 3'SL, respectively.
  • a certain temperature for example 37°C
  • time for example 8 hours, 16 hours, 24 hours
  • the oligosaccharides are then separated from the reaction mixture by methods known in the art. Further purification of LSTa or 3'SL can be performed if preferred.
  • the production of LSTa or 3'SL is measured via analytical methods as described in Example 1 and known by the person skilled in the art.
  • An E. coli K-12 MG1655 strain modified for production of sialic acid comprising genomic knock-ins of constitutive transcriptional units containing the UDP-N-acetylglucosamine 2-epimerase NeuC from C. jejuni (UniProt ID Q93MP8) and the N-acetylneuraminate synthase NeuB from Neisseria meningitidis (UniProt ID E0NCD4), and modified for growth on sucrose as described in Example 2 was further modified with a genomic knock-in of a constitutive transcriptional unit for the galactoside beta-1, 3-N- acetylglucosaminyltransferase LgtA from N.
  • LgtA and/or LgtB genes could be added to the modified E. coli strain.
  • the novel strain was transformed with an expression plasmid containing a constitutive transcriptional unit for the N- acylneuraminate cytidylyltransferase neuA from P. multocida with SEQ ID NO: 21 and for i) an alpha-2, 3- sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30 31, 04 or 05or ii) an alpha-2, 3-sialyltransferase with SEQ ID NO: 19 acting as a reference alpha-2, 3-sialyltransferase.
  • the novel strains were evaluated in a growth experiment for production of LSTd (Neu5Ac-a2,3-Gal-pi,4- GlcNAc-pi,3-Gal-pi,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.
  • the measured LSTd concentration of strains expressing an alpha-2, 3- sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30, 31, 4 or 5 was averaged over all biological replicates and then normalized to the averaged LSTd concentration of a reference strain expressing an alpha-2, 3-sialyltransferase with SEQ ID NO 19.
  • the experiment showed all strains each expressing a different alpha-2, 3-sialyltransferase, were able to produce LSTd.
  • strains expressing the sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30 or 31 showed a production of LSTd that was higher than the production of LSTd measured in the reference strain with SEQ ID NO: 19, and also higher than the production of LSTd by a strain with prior art SEQ ID NO: 4 or 5.
  • Example 9 All modified E. coli strains as described in Example 9 were evaluated in another growth experiment for production of LNnT, LSTd (Neu5Ac-a2,3-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc) and 3'SL (Neu5Ac-ot2,3-Gal- pi,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.
  • the measured LNnT, LSTd and 3'SL concentration measured of strains expressing an alpha-2, 3-sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30, 31, 4 or 5 was averaged over all biological replicates and then normalized to the averaged LNnT, LSTd and 3'SL concentration, respectively, of the reference strain expressing an alpha-2, 3-sialyltransferase with SEQ ID NO: 19.
  • the experiment showed that all strains, each expressing a different alpha-2, 3-sialyltransferase, were able to produce LNnT, LSTd and 3'SL.
  • the sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30 or 31 had a better 3-sialyltransferase binding activity on LNnT than the reference sialyltransferase, with strains showing a production of LSTd that was higher than the production of LSTd measured in the reference strain with SEQ ID NO: 19.
  • the reference sialyltransferase (SEQ ID NO: 19) had a better 3-sialyltransferase binding activity on lactose than the sialyltransferases with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30 or 31 with the reference strain expressing SEQ ID NO: 19 showing a production of 3'SL that was higher than the production of 3'SL measured in strains expressing SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30, 31.
  • Example 11 Production of LSTd with a modified E. coli host expressing siolyltronsferoses with a high specificity for LNnT
  • Modified E. coli strains as described in Example 10 and expressing an alpha-2, 3-sialyltransferase with SEQ ID NO: 6, 14, 15, 18, 22 or 30 show to specifically sialylate LNnT.
  • the sialyltransferase with SEQ ID NO: 6, 14, 15, 18, 22 or 30 only showed 15% or less side production of 3'SL compared to the reference sialyltransferase with SEQ ID NO: 19. This indicates the alpha-2, 3- sialyltransferase with SEQ ID NO: 6, 14, 15, 18, 22 or 30 have a high preference to sialylate LNnT as a substrate.
  • An E. coli K-12 MG1655 strain modified to produce LNnT as described in Example 2 is further transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P. multocida with SEQ ID NO: 21 and for i) an alpha-2, 3-sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30, 31, 4 or 5 or ii) an alpha- 2, 3-sialyltransferase with SEQ ID NO: 19 acting as a reference alpha-2, 3-sialyltransferase.
  • the novel strains are evaluated in a growth experiment for production of LSTd (Neu5Ac-ot2,3-Gal-pi,4-GlcNAc-pi,3-Gal- pi,4-Glc) according to the culture conditions provided in Example 2, in which the strains are cultivated in minimal medium with glycerol as carbon source and sialic acid and lactose added to the medium.
  • LSTd Neuronucleic acid
  • An E. coli K-12 MG1655 strain modified to produce LNnT as described in Example 2 is further modified for growth on sucrose as described in Example 2.
  • the strain is transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P.
  • the novel strains are evaluated in a growth experiment for production of LSTd (Neu5Ac-oc2,3-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc) according to the culture conditions provided in Example 2, in which the strains are cultivated in minimal medium with sucrose as carbon source and sialic acid and lactose added to the medium.
  • Example 14 Production of LSTd with modified E. coli hosts when evaluated in a fed-batch fermentation process with sucrose and lactose
  • the modified E. coli strains expressing the alpha-2, 3-sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 1 , 29, 30, 31, 4 or 5 and able to produce LSTd as described in Example 9 were selected for further evaluation in a fed-batch fermentation process.
  • Fed-batch fermentations at bioreactor scale were performed as described in Example 2.
  • Sucrose was used as a carbon source and lactose was added in the batch medium. During fed-batch, sucrose was added via an additional feed.
  • regular broth samples were taken at several time points during the fermentation process and sugars produced were measured using UPLC as described in Example 1. Ill
  • Another example provides the evaluation of alpha-2, 3-sialyltransferase activity of the enzymes with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30, 31, 4 or 5 of the present invention in an in vitro enzymatic assay.
  • Said enzyme can be produced in a cell-free expression system such as but not limited to the PURExpress system (NEB), or in a host organism such as but not limited to Escherichia coli or Saccharomyces cerevisiae, after which the enzyme can be isolated and optionally further purified.
  • the enzyme extract or purified enzyme is added to a reaction mixture together with CMP-sialic acid and a buffering component such as Tris-HCI or HEPES and a substrate like e.g. lacto-N-neotetraose (LNnT) or lactose.
  • Said reaction mixture is then incubated at a certain temperature (for example 37°C) for a certain amount of time (for example 8 hours, 16 hours, 24 hours), during which the LNnT or lactose will be converted by the enzyme using CMP-sialic acid to LSTd or 3'SL, respectively.
  • the oligosaccharides are then separated from the reaction mixture by methods known in the art. Further purification of LSTd or 3'SL can be performed if preferred. At the end of the reaction or after separation and/or purification, the production of LSTd or 3'SL is measured via analytical methods as described in Example 1 and known by the person skilled in the art.
  • Example 16 Evaluation of production of 3sLNB with a modified E. coli host
  • An E. coli K-12 MG1655 strain modified for production of sialic acid comprising genomic knock-ins ofconstitutive transcriptional units containing the mutant glmS*54 from E. coli (differing from the wildtype E.
  • coli glmS having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et aL (Biochimie 88, 419-29 (2006)), the glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), the N-acylglucosamine 2-epimerase AGE from Bacteroides ovatus (UniProt ID A7LVG6) and the N-acetylneuraminate synthase NeuB from Campylobacter jejuni (UniProt ID Q93MP9), and modified for growth on sucrose as described in Example 2 was further modified with a genomic knock-in of a constitutive transcriptional unit of wbgO (Uniprot ID D3QY14) from E.
  • wbgO Uniprot ID D3QY14
  • the novel strain was transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P.
  • the novel strains were evaluated in a growth experiment for production of 3sLNB according to the culture conditions provided in Example 2, in which the strains were cultivated in minimal medium with 30 g/L sucrose. The strains were grown in four biological replicates in a 96-well plate.
  • the 3sLNB concentration measured of strains expressing an alpha-2, 3-sialyltransferase with SEQ ID NO: 1, 2, 3, 5, 6, 8, 9, 10, 11, 23, 24, 25, 27, 28, 29 or 30 was averaged over all biological replicates and then normalized to the averaged 3sLNB concentration of a reference strain expressing an alpha-2, 3- sialyltransferase with SEQ ID NO: 20.
  • Table 7 Relative production of 3sLNB (%) in modified E. coli strains expressing an alpha-2, 3- sialyltransferase with SEQ ID NO: 1, 2, 3, 5, 6, 8, 9, 10, 11, 23, 24, 25, 27, 28, 29 or 30 and compared to an alpha-2, 3-sialyltransferase with SEQ ID NO: 20 serving as a reference, when evaluated in a growth experiment according to the culture conditions provided in Example 2, in which the culture medium contained 30 g/L sucrose.
  • Example 17 Evaluation of production of 3sLacNAc with a modified E. coli host
  • An E. coli K-12 MG1655 strain modified for production of sialic acid comprising genomic knock-ins of constitutive transcriptional units containing the mutant glmS*54 from E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al.
  • meningitidis (UniProt ID Q51116) to allow production of LacNAc.
  • multiple copies of IgtB genes could be added to the modified E. coli strain.
  • the novel strain was transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuAfrom P.
  • the novel strains were evaluated in a growth experiment for production of 3sLacNAc according to the culture conditions provided in Example 2, in which the strains were cultivated in minimal medium with 30 g/L sucrose. The strains were grown in four biological replicates in a 96-well plate.
  • the 3sLacNAc concentration measured of strains expressing an alpha-2, 3-sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 25, 26, 1 , 29, 30, 31 or 5 was averaged over all biological replicates and then normalized to the averaged 3sLacNAc concentration of a reference strain expressing an alpha-2, 3-sialyltransferase with SEQ ID NO: 19.
  • Table 8 Relative production of 3sLacNAc (%) in modified E. coli strains expressing an alpha-2, 3- sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 25, 26, 27, 29, 30, 31 or 5 and compared to an alpha-2, 3-sialyltransferase with SEQ ID NO: 19 serving as a reference, when evaluated in a growth experiment according to the culture conditions provided in Example 2, in which the culture medium contained 30 g/L sucrose.
  • S. cerevisiae BY4742 created by Brachmann et al. (Yeast (1998) 14:115-32) was used, available in the Euroscarf culture collection. All mutant strains were created by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11:355-360, 1995).
  • 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. the mutant glmS*54 from E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al.
  • a constitutive transcriptional unit for a glucosamine 6-phosphate N-acetyltransferase like e.g. GNA1 from S. cerevisiae was added as well.
  • the plasmid further comprised constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), and a beta-galactoside alpha-2, 3-sialyltransferase like e.g.
  • PmultST2 from Pasteurella multocida SEQ ID NO: 20
  • a beta-galactoside alpha-2, 3-sialyltransferase like e.g. PmultST3 from Pasteurella multocida (SEQ ID NO: 19) having a beta-galactoside alpha-2, 3-sialyltransferase activity or the polypeptide with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.
  • 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 a 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. IgtA from N. meningitidis (UniProt ID Q9JXQ6) to produce LN3.
  • LN3 derived oligosaccharides like lacto-A/-neotetraose (LNnT, Gal- pi,4-GlcNAc-pi,3-Gal-pi,4-Glc)
  • LNnT lacto-A/-neotetraose
  • the modified LN3 producing strain was further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB (Uniprot ID Q51116) from Neisseria 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 SUMOstar tag (e.g. obtained from pYSUMOstar, Life Sensors, Malvern, PA) to enhance their solubility.
  • a SUMOstar tag e.g. obtained from pYSUMOstar, Life Sensors, Malvern, PA
  • mutant yeast strains were modified with a genomic knock-in of a constitutive transcriptional unit encoding a chaperone protein like e.g. Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssbl, Ssel, Sse2, Ssal, Ssa2, Ssa3, Ssa4, Ssb2, EcmlO, Sscl, Ssql, Sszl, Lhsl, Hsp82, Hsc82, Hsp78, Hspl04, Tcpl, Cct4, Cct8, Cct2, Cct3, Cct5, Cct6 or Cct7 (Gong et aL, 2009, Mol. Syst.
  • a chaperone protein like e.g. Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssbl, Ssel, Sse2, Ssal, Ssa2, Ssa3, Ssa4, Ssb2,
  • Plasmids were maintained in the host E. coli DH5alpha (F‘, phi80d/acZdeltaM15, delta(/acZYA-argF)U169, deoR, recAl, endAl, hsdR17(rk', mk + ), phoA, supE44, lambda', thi-1, gyrA96, relAl) bought from Invitrogen.
  • Genes that needed to be expressed be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, IDT or Twist Bioscience. 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.
  • 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.
  • Genes were expressed using synthetic constitutive promoters, as described by e.g. Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012), Redden and Alper (Nat. Commun. 2015, 6, 7810), Liu et al. (Microb. Cell Fact. 2020, 19, 38), Xu et al. (Microb. Cell Fact.2021, 20, 148) and Lee et al. (ACS Synth. Biol. 2015, 4(9), 975-986).
  • Example 19 Production ofLSTa with a modified S. cerevisiae host
  • a S. cerevisiae strain is modified for production of CMP-sialic acid and LNT and for expression of an alpha- 2,3-sialyltransferase as described in Example 18 with a first yeast expression plasmid comprising constitutive transcriptional units for LAC12 from K. lactis (UniProt ID P07921), the mutant glmS*54 from E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006)), the phosphatase SurE from E.
  • PmultST2 from Pasteurella multocida (SEQ ID NO: 20) having beta-galactoside alpha-2, 3- sialyltransferase activity or ii) a polypeptide with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 7, 23, 24, 25, 27, 28, 29 or 30, and a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli (UniProt ID P09147), LgtA from N. meningitidis (UniProt ID Q9JXQ6) and LgtB from N. meningitidis (UniProt ID Q51116).
  • the novel strains are evaluated for production of LSTa when evaluated in a 3-days growth experiment according to the culture conditions provided in Example 18 using appropriate selective medium comprising lactose.
  • Example 20 Production ofLSTd with a modified S. cerevisiae host S. cerevisiae strain is modified for production of CMP-sialic acid and LNT and for expression of an alpha- 2, 3-sialyltransferase as described in Example 18 with a first yeast expression plasmid comprising constitutive transcriptional units for LAC12 from K. lactis (UniProt ID P07921), the mutant glmS*54 from E. coli (differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al.
  • a PmultST3 from Pasteurella multocida (SEQ ID NO: 19) having beta-galactoside alpha-2, 3-sialyltransferase activity or ii) a polypeptide with SEQ ID NO 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30, 31, 4 or 5 and a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli (UniProt ID P09147), LgtA from N. meningitidis (UniProt ID Q9JXQ6) and LgtB from N. meningitidis (UniProt ID Q51116).
  • the novel strains are evaluated for production of LSTd when evaluated in a 3-days growth experiment according to the culture conditions provided in Example 18 using appropriate selective medium comprising lactose.
  • Two media are used to cultivate B. subtilis: i.e. a rich Luria Broth (LB) and a minimal medium for shake flask cultures.
  • 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 30g/Lglucose (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 7.0with 1 M KOH. Depending on the experiment lactose is added as a precursor.
  • the trace element mix consisted of 0.735 g/L CaCI 2 .2H 2 O, 0.1 g/L MnCI 2 .2H 2 O, 0.033 g/L CuCI 2 .2H 2 O, 0.06 g/L CoCI 2 .6H 2 O, 0.17 g/L ZnCI 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 FeCI 3 .6H 2 O, 1 g/L Na-citrate (Hoch 1973 PMC1212887).
  • Complex medium e.g. LB
  • a medium was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 pm Sartorius).
  • the medium was made selective by adding an antibiotic (e.g. zeocin (20mg/L)).
  • 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. 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. coli (UniProt ID P31120), an N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase like e.g. glmU from E.
  • a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120)
  • an N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase like e.g. glmU 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-acetylglucosamine 2-epimerase like e.g. AGE from B.
  • 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.
  • the sialic acid production strains further need to express an N-acylneuraminate cytidylyltransferase like e.g. neuA from P. multocida with SEQ ID NO: 21, and a beta-galactoside alpha- 2,3-sialyltransferase like e.g. PmultST2 from Pasteurella multocida (SEQ ID NO: 20) or a beta-galactoside alpha-2, 3-sialyltransferase like e.g.
  • N-acylneuraminate cytidylyltransferase like e.g. neuA from P. multocida with SEQ ID NO: 21, and a beta-galactoside alpha- 2,3-sialyltransferase like e.g. PmultST2 from Pasteurella multocida (SEQ ID NO: 20) or a beta-galactoside alpha-2, 3-sialyl
  • PmultST3 from Pasteurella multocida having a betagalactoside alpha-2, 3-sialyltransferase activity or the polypeptide with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.
  • 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.
  • 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).
  • the engineered strain was derived from B. subtilis comprising knockouts of the B. subtilis nagB and gamA genes and genomic knock-ins of constitutive transcriptional units containing a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. IgtA from N. meningitidis (UniProt ID Q9JXQ6) and a lactose permease like e.g. LacY from E. coli (UniProt ID P02920).
  • a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. IgtA from N. meningitidis (UniProt ID Q9JXQ6) and a lactose permease like e.g. LacY from E. coli (UniProt ID P02920).
  • the LN3 producing strain was further transformed with constitutive transcriptional units for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. IgtB from N. meningitidis (UniProt ID Q51116).
  • Genes that needed to be expressed be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.
  • 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.
  • 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 l/3rd of the optical density measured at 600 nm.
  • Example 22 Production ofLSTa or LSTd with a modified B. subtilis host
  • a wild-type B. subtilis strain is first modified for production of LN3 with genomic knockouts of the B. subtilis genes nagB and gamA together with genomic knock-ins of constitutive transcriptional units for the lactose permease LacY from E. coli (UniProt ID P02920) and IgtA from N. meningitidis (UniProt ID Q9JXQ6).
  • the modified B. subtilis strain is modified for production of LNnT with a genomic knock-in of a constitutive transcriptional unit for IgtB from N. meningitidis (UniProt ID Q51116).
  • the modified strain is transformed with an expression plasmid comprising constitutive transcriptional units for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (SEQ ID NO: 21) and any one of the alpha-2, 3-sialyltransferase with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 7, 23, 24, 25, 27, 28, 29, or 30 for LSTa production or any one of the alpha-2, 3-sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30, 31, 4 or 5 for LSTd production, respectively.
  • the novel strain is evaluated for production of LSTa or LSTd when evaluated in a 3-days growth experiment according to the culture conditions provided in Example 21 using appropriate selective medium comprising lactose.
  • TY tryptone-yeast extract
  • VWR 0.5% sodium chloride
  • 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 , I 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 CaCI 2 , 10 g/L FeSO 4 .7H 2 O, 10 g/L MnSO 4 .H 2 O, 1 g/L ZnSO 4 .7H 2 O, 0.2 g/L CuSO 4 , 0.02 g/L NiCI 2 .6H 2 O, 0.2 g/L biotin (pH 7.0) and 0.03 g/L protocatechuic acid.
  • Complex medium e.g. TY
  • a medium was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 pm Sartorius).
  • the medium was made selective by adding an antibiotic (e.g. kanamycin, ampicillin).
  • Corynebacterium glutamicum ATCC 13032 was used as available at the American Type Culture Collection. Integrative plasmid vectors based on the Cre/loxP technique as described by Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 Apr, 67(2):225-33) and temperature-sensitive shuttle vectors as described by Okibe et al. (J. Microbiol. Meth. 85, 2011, 155-163) are constructed for gene deletions, mutations and insertions. Suitable promoters for (heterologous) gene expression can be derived from Yim et al. (Biotechnol. Bioeng., 2013 Nov, 110(ll):2959-69). 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 Idh, 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), an N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase like e.g. glmU from E.
  • C. glutamicum comprising knockouts of the C. glutamicum Idh, 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), an N-acetylglucosamine-l-
  • 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 sialic acid production strains further need to express an N- acylneuraminate cytidylyltransferase like e.g. neuA from P. multocida with SEQ ID NO: 21, and a betagalactoside alpha-2, 3-sialyltransferase like e.g.
  • PmultST2 from Pasteurella multocida SEQ ID NO: 20
  • a beta-galactoside alpha-2, 3-sialyltransferase like e.g. PmultST3 from Pasteurella multocida (SEQ ID NO: 19) having a beta-galactoside alpha-2, 3-sialyltransferase activity or the polypeptide with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 7, 18, 22, 23, 24, 25, 26, TJ , 28, 29, 30 or 31.
  • 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).
  • the engineered strain was derived from C. glutamicum comprising knockouts of the C. glutamicum Idh, cgl2645 and nagB genes and genomic knock-ins of constitutive transcriptional units containing a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. IgtA from N. meningitidis (UniProt ID Q9JXQ6) and a lactose permease like e.g. LacY from E. coli (UniProt ID P02920).
  • C. glutamicum comprising knockouts of the C. glutamicum Idh, cgl2645 and nagB genes and genomic knock-ins of constitutive transcriptional units containing a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. IgtA from N. meningitidis (UniProt ID Q9JXQ6) and
  • the LN3 producing strain was further transformed with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. IgtB from N. meningitidis (UniProt ID Q51116).
  • Genes that needed to be expressed be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.
  • 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.
  • the cell performance index or CPI was determined by dividing the oligosaccharide concentrations, e.g. sialyllactose 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 l/3rd of the optical density measured at 600 nm.
  • Example 24 Production of LSTa or LSTd with a modified C. glutamicum host
  • a wild-type C. glutamicum strain is first modified for production of LN3 with genomic knockouts of the Idh, cgl2645 and nagB genes together with genomic knock-ins of constitutive transcriptional units for the lactose permease LacYfrom E. coli (UniProt ID P02920) and IgtA from N. meningitidis (UniProt ID Q9JXQ6).
  • the modified C. glutamicum strain is modified for production of LNnT with a genomic knock- in of a constitutive transcriptional unit for IgtB from N. meningitidis (UniProt ID Q51116).
  • the modified strain is transformed with an expression plasmid comprising constitutive transcriptional units for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (SEQ ID NO: 21) and any one of the alpha-2, 3-sialyltransferase with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 7, 23, 24, 25, 27, 28, 29, or30 for LSTa production or any one of the alpha-2, 3-sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 22, 25, 26, 27, 29, 30, 31, 4 or 5 for LSTd production, respectively.
  • the novel strain is evaluated for production of LSTa or LSTd when evaluated in a 3-days growth experiment according to the culture conditions provided in Example 23 using appropriate selective medium comprising lactose.
  • Example 25 Materials and methods Chlamydomonas reinhardtii
  • TAP Tris-acetate-phosphate
  • the TAP medium uses a lOOOx stock Hutner's trace element mix.
  • Hutner's trace element mix consisted of 50 g/L Na 2 EDTA.H 2 O (Titriplex III), 22 g/L ZnSO 4 .7H 2 O, 11.4 g/L H 3 BO 3 , 5 g/L MnCI 2 .4H 2 O, 5 g/L FeSO 4 .7H 2 O, 1.6 g/L CoCI 2 .6H 2 O, 1.6 g/L CuSO 4 .5H 2 O and 1.1 g/L (NH 4 ) s MoO 3 .
  • the TAP medium contained 2.42 g/LTris (tris(hydroxymethyl)aminomethane), 25 mg/L salt stock solution, 0.108 g/L K 2 HPO 4 , 0.054 g/L KH 2 PO 4 and 1.0 mL/L glacial acetic acid.
  • the salt stock solution consisted of 15 g/L NH4CI, 4 g/L MgSO 4 .7H 2 O and 2 g/L CaCI 2 .2H 2 O.
  • precursors like e.g. galactose, glucose, fructose, fucose, GIcNAc could be added.
  • Medium was sterilized by autoclaving (121°C, 21 min).
  • TAP medium was used containing 1% agar (of purified high strength, 1000 g/cm2).
  • C. reinhardtii wild-type strains 21gr (CC-1690, wild-type, mt+), 6145C (CC-1691, wild-type, mt-), CC-125 (137c, wild-type, mt+), CC-124 (137c, wild-type, mt-) as available from Chlamydomonas Resource Center (https://www.chlamycollection.org), University of Minnesota, U.S.A.
  • Expression plasmids originated from pSllO3, as available from Chlamydomonas Resource Center. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. Suitable promoters for (heterologous) gene expression can be derived from e.g. Scranton et al. (Algal Res. 2016, 15: 135-142). Targeted gene modification (like gene knock-out or gene replacement) can be carried using the Crispr-Cas technology as described e.g. by Jiang et al. (Eukaryotic Cell 2014, 13(11): 1465-1469).
  • Transformation via electroporation was performed as described by Wang et al. (Biosci. Rep. 2019, 39: BSR2018210).
  • Cells were grown in liquid TAP medium under constant aeration and continuous light with a light intensity of 8000 Lx until the cell density reached 1.0-2.0 x 10 7 cells/mL. Then, the cells were inoculated into fresh liquid TAP medium in a concentration of 1.0 x 10 s cells/mL and grown under continuous light for 18-20 h until the cell density reached 4.0 x 10 s cells/mL.
  • cells were collected by centrifugation at 1250 g for 5 min at room temperature, washed and resuspended with pre-chilled liquid TAP medium containing 60 mM sorbitol (Sigma, U.S.A.), and iced for 10 min. Then, 250 pL of cell suspension (corresponding to 5.0 x 10 7 cells) were placed into a pre-chilled 0.4 cm electroporation cuvette with 100 ng plasmid DNA (400 ng/mL). Electroporation was performed with 6 pulses of 500 V each having a pulse length of 4 ms and pulse interval time of 100 ms using a BTX ECM830 electroporation apparatus (1575 Q, 50 pFD).
  • the cuvette was immediately placed on ice for 10 min. Finally, the cell suspension was transferred into a 50 mL conical centrifuge tube containing 10 mL of fresh liquid TAP medium with 60 mM sorbitol for overnight recovery at dim light by slowly shaking. After overnight recovery, cells were recollected and plated with starch embedding method onto selective 1.5% (w/v) agar- TAP plates containing ampicillin (100 mg/L) or chloramphenicol (100 mg/L). Plates were then incubated at 23 +-0.5°C under continuous illumination with a light intensity of 8000 Lx. Cells were analysed 5-7 days later.
  • C. reinhardtii cells were modified with transcriptional units comprising the gene encoding the galactokinase from Arabidopsis thaliana (KIN, UniProt ID Q9SEE5) and the gene encoding the UDP-sugar pyrophosphorylase (USP) from A. thaliana (UniProt ID Q9C5I1).
  • C. reinhardtii cells were modified with a constitutive transcriptional unit comprising a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. IgtA from N. meningitidis (UniProt ID Q9JXQ6).
  • the LN3 producing strain was further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. IgtB from N. meningitidis (UniProt ID Q51116).
  • C. reinhardtii cells were modified with constitutive transcriptional units for a UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase like e.g. GN E from Homo sapiens (UniProt ID Q9Y223) or a mutant form of the human GNE polypeptide comprising the R263L mutation, an N-acylneuraminate-9-phosphate synthetase like e.g. NANS from Homo sapiens (UniProt ID Q9NR45) and an N-acylneuraminate cytidylyltransferase like e.g.
  • C. reinhardtii cells are modified with a CMP-sialic acid transporter like e.g. CST from Mus musculus (UniProt ID Q61420), and a beta-galactoside alpha-2, 3-sialyltransferase like e.g. PmultST2 from Pasteurella multocida (SEQ ID NO: 20) or a beta-galactoside alpha-2, 3-sialyltransferase like e.g.
  • PmultST3 from Pasteurella multocida (SEQ ID NO: 19) having a beta-galactoside alpha-2, 3-sialyltransferase activity or the polypeptide with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 7, 18, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.
  • Genes that needed to be expressed be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9, Twist Biosciences or IDT.
  • 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.
  • cells could be cultivated in closed systems like e.g. vertical or horizontal tube photobioreactors, stirred tank photobioreactors or flat panel photobioreactors as described by Chen et al. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827).
  • closed systems like e.g. vertical or horizontal tube photobioreactors, stirred tank photobioreactors or flat panel photobioreactors as described by Chen et al. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827).
  • C. reinhardtii cells are engineered as described in Example 25 for production of UDP-Gal with genomic knock-ins of constitutive transcriptional units comprising the galactokinase from A. thaliana (KIN, UniProt ID Q9SEE5) and the UDP-sugar pyrophosphorylase (USP) from A. thaliana (UniProt ID Q9C5I1).
  • the engineered cells are modified for CMP-sialic acid synthesis with genomic knock-ins of constitutive transcriptional units comprising a mutant form of the UDP-/V-acetylglucosamine-2- epimerase/W-acetylmannosamine kinase GNE from Homo sapiens (UniProt ID Q9Y223) differing from the native polypeptide with a R263L mutation, the N-acylneuraminate-9-phosphate synthetase NANS from Homo sapiens (UniProt ID Q9NR45), the N-acylneuraminate cytidylyltransferase CMAS from Homosapiens (UniProt ID Q8NFW8) and the CMP-sialic acid transporter CST from Mus musculus (UniProt ID Q61420).
  • genomic knock-ins of constitutive transcriptional units comprising a mutant form of the UDP-/V-acetylglucosamine-2- epime
  • the engineered cells are modified with an expression plasmid comprising constitutive transcriptional units comprising any one of the alpha-2, 3-sialyltransferase with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 7, 19, 20, 23, 24, 25, 27, 28, 29 or 30 for LSTa production or any one of the alpha-2, 3- sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 19, 20, 22, 25, 26, 27, 29, 30, 31, 4 or 5 for LSTd production, respectively, the galactoside beta-1, 3-N-acetylglucosaminyltransferase IgtA from N.
  • Fresh adipose tissue is obtained from slaughterhouses (e.g. cattle, pigs, sheep, chicken, ducks, catfish, snake, frogs) or liposuction (e.g. in case of humans, after informed consent) and kept in phosphate buffer saline supplemented with antibiotics. 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.
  • FBS farnesoid bovine serum
  • Ahmad and Shakoori 2013, Stem Cell Regen. Med. 9(2): 29-36, which is incorporated herein by reference in its entirety for all purposes, describes certain variation(s) of the method(s) described herein in this example.
  • 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. 30(10): 2164-2174
  • the mesenchymal cells isolated from adipose tissue of different animals or from milk as described above can be differentiated into mammary-like epithelial and luminal cells in 2D and 3D culture systems. See, for example, Huynh et al. 1991. Exp Cell Res. 197(2): 191 -199; Gibson et al. 1991, In Vitro Cell Dev Biol Anim. 27(7): 585-594; Blatchford et al. 1999; Animal Cell Technology': Basic & Applied Aspects, Springer, Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res 11(3): 26-43; and Arevalo et al. 2015, Am J Physiol Cell Physiol. 310(5): C348 - C356; each of which is incorporated herein by reference in their entireties for all purposes.
  • 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.
  • serum is removed from the complete induction medium.
  • 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.
  • 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 WO 2021/067641, which is incorporated herein by reference in its entirety for all purposes.
  • remodelling systems such as CRISPR/Cas9
  • 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.
  • the cells 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.
  • 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.
  • Example 28 Production of LSTa or LSTd in a non-mammary adult stem cell
  • Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 17 are modified via CRISPR-CAS to express the GlcN6P synthase from Homo sapiens (UniProt ID Q06210), the glucosamine 6-phosphate N-acetyltransferase from Homo sapiens (UniProt ID Q96EK6), the phosphoacetylglucosamine mutase from Homo sapiens (UniProt ID 095394), the UDP-N- acetylhexosamine pyrophosphorylase from Homo sapiens (UniProt ID Q16222), the galactoside beta-1, 3- N-acetylglucosaminyltransferase LgtA from N.
  • meningitidis (UniProt ID Q9JXQ6), the N-acetylglucosamine beta-1, 4-galactosyltransferase IgtB from N. meningitidis (UniProt ID Q51116), the N-acylneuraminate cytidylyltransferases neuA from Mas musculus (UniProt ID Q99KK2) and any one of the alpha-2, 3- sialyltransferase with SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 19, 20, 22, 7, 23, 24, 25, 27, 28, 29, or 30 for LSTa production or any one of the alpha-2, 3-sialyltransferase with SEQ ID NO: 6, 12, 13, 14, 15, 10, 16, 17, 3, 8, 7, 18, 19, 20, 22, 25, 26, 1 , 29, 30, 31, 4 or 5 for LSTd production, respectively.
  • 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 for about 7 days. After cultivation as described in Example 1 , cells are subjected to UPLC to analyse for production of LSTa and/or LSTd.

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Abstract

La présente invention relève du domaine technique de la biologie synthétique, de l'ingénierie métabolique et de la culture cellulaire. La présente invention concerne des sialyltransférases récemment identifiées présentant une activité alpha-2,3-sialyltransférase sur un accepteur qui est un saccharide comprenant au moins un monosaccharide de N-acétylglucosamine et un monosaccharide de galactose. L'invention décrit également des procédés de production d'un oligosaccharide 3'-sialylé à l'aide de l'une quelconque des sialyltransférases récemment identifiées, ainsi que la purification dudit oligosaccharide 3'-sialylé. La présente invention propose également une cellule pour la production dudit oligosaccharide 3'-sialylé et l'utilisation de ladite cellule dans une culture ou une incubation.
PCT/EP2024/067548 2023-06-21 2024-06-21 Sialyltransférases pour la production d'oligosaccharides sialylés Pending WO2024261312A2 (fr)

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