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WO2024223815A1 - Production d'un oligosaccharide chargé négativement par une cellule - Google Patents

Production d'un oligosaccharide chargé négativement par une cellule Download PDF

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Publication number
WO2024223815A1
WO2024223815A1 PCT/EP2024/061529 EP2024061529W WO2024223815A1 WO 2024223815 A1 WO2024223815 A1 WO 2024223815A1 EP 2024061529 W EP2024061529 W EP 2024061529W WO 2024223815 A1 WO2024223815 A1 WO 2024223815A1
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Prior art keywords
gal
neu5ac
oligosaccharide
glcnac
cell
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Inventor
Joeri Beauprez
Thomas DECOENE
Annelies VERCAUTEREN
Tom Verhaeghe
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Inbiose NV
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Inbiose NV
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Priority to CN202480027924.2A priority Critical patent/CN121079401A/zh
Publication of WO2024223815A1 publication Critical patent/WO2024223815A1/fr
Priority to PCT/EP2025/061445 priority patent/WO2025224348A1/fr
Anticipated expiration legal-status Critical
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01183—UDP-N-acetylglucosamine 2-epimerase (hydrolysing) (3.2.1.183)

Definitions

  • the present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation.
  • the invention provides a cell for production of a negatively charged, preferably sialylated, oligosaccharide wherein said cell is genetically engineered to possess or express, preferably to overexpress a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase.
  • the invention further provides use of said cell in a cultivation or incubation.
  • the invention also describes methods for the production of a negatively charged, preferably sialylated, oligosaccharide using said cell as well as the purification of said negatively charged, preferably sialylated, oligosaccharide.
  • Oligosaccharides are very diverse in chemical structure and are composed of miscellaneous monosaccharides, such as e.g., glucose, galactose, N-acetylglucosamine, xylose, rhamnose, fucose, mannose, N-acetylneuraminic acid, N-acetylgalactosamine, galactosamine, glucosamine, glucuronic acid, galacturonic acid. Oligosaccharides are widely distributed in all living organisms and play important roles in a variety of physiological and pathological processes, such as cell metastasis, signal transduction, intercellular adhesion, inflammation, and immune response.
  • miscellaneous monosaccharides such as e.g., glucose, galactose, N-acetylglucosamine, xylose, rhamnose, fucose, mannose, N-acetylneuraminic acid, N-acetylgalactosamine, galact
  • oligosaccharides comprises mammalian milk oligosaccharides (MMOs) and human milk oligosaccharides (HMOs) found in mammalian and human milk, respectively.
  • MMOs mammalian milk oligosaccharides
  • HMOs human milk oligosaccharides
  • milk oligosaccharides serve as a substrate for beneficial bacteria like Bifidobacteria or Lactobacilli.
  • Milk oligosaccharides are further known to act as decoys to reduce the risk of infections by bacterial and viral pathogens which adhere to human cells by binding to these cells' surface glycoproteins. Additionally, various milk oligosaccharides possess an anti-inflammatory effect and act as immunomodulators (e.g., reducing the risk of developing food allergies). Many of these milk oligosaccharides are negatively charged oligosaccharides. Among negatively charged HMOs, sialylated HMOs (SHMOs) were observed to support several beneficial effects as described in the art.
  • SHMOs sialylated HMOs
  • sialylated oligosaccharides in human milk 3'sialyllactose, 6'sialyllactose, sialyllacto-N-tetraose a, sialyl lacto-N-tetraose b, sialyllacto-N-tetraose c and disialyllacto-N-tetraose are the most prevalent members.
  • Negatively charged 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. Therefore, alternative production methods have been developed, amongst which efforts in metabolic engineering of microorganisms to produce negatively charged oligosaccharides have been made.
  • the present invention provides methods and a cell for the production of a negatively charged, preferably sialylated, oligosaccharide.
  • the present invention also provides methods for the purification of said negatively charged, preferably sialylated, oligosaccharide.
  • the present invention provides a cell which is genetically engineered as described herein to possess or express, preferably to over-express, a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase wherein said hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising a conserved motif with SEQ.
  • This invention also provides a purified negatively charged, preferably sialylated, oligosaccharide by the above-referenced process. Further benefits of the teachings of this invention will be apparent to one skilled in the art from reading this invention.
  • the features “synthesize”, “synthesized” and “synthesis” are interchangeably used with the features “produce”, “produced” and “production”, respectively.
  • 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 verb "to comprise”, “to have” and “to contain” and their conjugations are used in their nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • the verb "to comprise” may be replaced by “to consist” or “to consist essentially of” and vice versa.
  • the verb “to consist” may be replaced by "to consist essentially of” meaning that a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • the articles “a” and “an” are preferably replaced by "at least one", more preferably “at least two”, even more preferably by “at least three", even more preferably by “at least four", even more preferably by "at least five", even more preferably by “at least six", most preferably by "at least two”.
  • the word “about” or “approximately” when used in association with a numerical value (e.g., “about 10") or with a range (e.g., "about x to approximately y”) preferably means that the value or range is interpreted as being as accurate as the method used to measure it.
  • polynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • 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 as well known to the skilled person. The same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Furthermore, a given polypeptide may contain many types of modifications.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid sidechains, and the amino or carboxyl termini.
  • Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention.
  • the term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions that also may contain coding and/or non-coding sequences.
  • isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • a “synthetic" sequence as the term is used herein, means any sequence that has been generated synthetically and not directly isolated from a natural source.
  • Synthesized as the term is used herein, means any synthetically generated sequence and not directly isolated from a natural source.
  • Recombinant means genetically engineered DNA prepared by transplanting or splicing genes from one species into the cells of a host organism of a different species. Such DNA becomes part of the host's genetic makeup and is replicated.
  • recombinant or “transgenic” or “metabolically engineered” or “genetically engineered” as used herein with reference to a cell or host cell are used interchangeably and indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence "foreign to said cell” or a sequence "foreign to said location or environment in said cell”).
  • Such cells are described to be transformed with at least one heterologous or exogenous gene or are described to be transformed by the introduction of at least one heterologous or exogenous gene.
  • Recombinant or metabolically engineered or genetically engineered or transgenic cells can contain genes that are not found within the native (non-recombinant) form of the cell.
  • Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means.
  • the terms also encompass cells that contain a nucleic acid endogenous to the cell that has been modified or its expression or activity has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, replacement of a promoter; site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis and related techniques as known to a person skilled in the art.
  • a "recombinant polypeptide” is one which has been produced by a recombinant cell.
  • the terms also encompass cells that have been modified by removing a nucleic acid endogenous to the cell by means of common well-known technologies for a skilled person (like e.g. knocking-out genes).
  • heterologous sequence or a “heterologous nucleic acid”, as used herein, is one that originates from a source foreign to the particular cell (e.g., from a different species), or, if from the same source, is modified from its original form or place in the genome.
  • a heterologous nucleic acid operably linked to a promoter is from a source different from that from which the promoter was derived, or, if from the same source, is modified from its original form or place in the genome.
  • the heterologous sequence may be stably introduced, e.g., by transfection, transformation, conjugation or transduction, into the genome of the host cell, wherein techniques may be applied which will depend on the cell and the sequence that is to be introduced.
  • mutant or "engineered” cell as used within the context of the present invention refers to a cell which is genetically engineered.
  • exogenous within the context of the present disclosure refers to any polynucleotide, polypeptide or protein sequence that is a natural part of a cell and is occurring at its natural location in the cell chromosome and of which the control of expression has not been altered compared to the natural control mechanism acting on its expression.
  • exogenous refers to any polynucleotide, polypeptide or protein sequence which originates from outside the cell under study and not a natural part of the cell or which is not occurring at its natural location in the cell chromosome or plasmid.
  • heterologous when used in reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme refers to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is from a source or derived from a source other than the host organism.
  • 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.
  • heterologous means that the regulatory sequence or auxiliary sequence is not naturally associated with the gene with which the regulatory or auxiliary nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome.
  • a promoter operably linked to a gene to which it is not operably linked to in its natural state i.e.
  • heterologous promoter in the genome of a non-genetically engineered organism is referred to herein as a "heterologous promoter," even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked.
  • 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 negatively charged, preferably 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 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 s 70 , s 54 , or related s- factors and the yeast mitochondrial RNA polymerase specificity factor MTFl 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 expression that is regulated by transcription factors other than the subunits of RNA polymerase (e.g. bacterial sigma factors) under certain growth conditions. Examples of such transcription factors are described above. Commonly expression regulation is obtained by means of an inducer, such as but not limited to IPTG, arabinose, rhamnose, fucose, allo-lactose or pH shifts, or temperature shifts or carbon depletion or substrates or the produced product.
  • inducer such as but not limited to IPTG, arabinose, rhamnose, fucose, allo-lactose or pH shifts, or temperature shifts or carbon depletion or substrates or the produced product.
  • 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.
  • wildtype 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.
  • modified activity of a protein relates to a non-native activity of the protein in any phase of the production process of the desired negatively charged, preferably sialylated, oligosaccharide.
  • non-native as used herein with reference to the activity of a protein indicates that the protein has been modified to have an abolished, impaired, reduced, delayed, higher, accelerated or improved activity compared to the native activity of said protein.
  • a modified activity of a protein is obtained by modified expression of said protein or is obtained by expression of a modified, i.e., mutant form of the protein.
  • a mutant form of the protein can be obtained by expression of a mutant form of the gene encoding the protein, e.g., comprising a deletion, an insertion and/or a mutation of one or more nucleotides compared to the native gene sequence.
  • a mutant form of a gene can be obtained by techniques well-known to a person skilled in the art, such as but not limited to site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis.
  • non-native indicates that the negatively charged, preferably 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 negatively charged, preferably sialylated, oligosaccharide or to have a higher production of the negatively charged, preferably 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.
  • a variant can be produced as a fusion protein comprising at least one portion of an enzyme of present invention fused to at least one portion comprising a peptide tag.
  • Said peptide tag may be used to assist protein folding of said enzyme, assist post expression purification, protect the enzyme from the action of degradative enzymes, and/or assist the enzyme in passing through the cell membrane.
  • peptide tag comprises, e.g., a SUMO tag, an MBP tag, a His tag, a FLAG tag, a Strep-ll tag, a Halo-tag, a NusA tag, thioredoxin, GST and/or a Fh8-tag.
  • the fusion protein may be designed to include at least one cleavable peptide linker so that the enzyme of interest can be subsequently recovered from the fusion protein.
  • the fusion protein may be designed to include a plurality of inclusion body tags, cleavable peptide linkers, and regions encoding the enzyme of interest.
  • “Fragment” refers to a clone or any part of a polynucleotide molecule, particularly a part of a polynucleotide that retains a usable, functional characteristic of the full-length polynucleotide molecule.
  • Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation.
  • polynucleotide fragment refers to any subsequence of a polynucleotide SEQ ID NO, typically, comprising or consisting of at least about 9, 10, 11, 12 consecutive nucleotides from said polynucleotide SEQ ID NO, for example at least about 30 nucleotides or at least about 50 nucleotides of any of the polynucleotide sequences provided herein.
  • Exemplary fragments can additionally or alternatively include fragments that comprise, consist essentially of, or consist of a region that encodes a conserved family domain of a polypeptide.
  • Exemplary fragments can additionally or alternatively include fragments that comprise a conserved domain of a polypeptide.
  • a fragment of a polynucleotide SEQ ID NO preferably means a nucleotide sequence which comprises or consists of said polynucleotide SEQ ID NO wherein no more than about 200, 150, 100, 50 or 25 consecutive nucleotides are missing, preferably no more than about 50 consecutive nucleotides are missing, and which retains a usable, functional characteristic (e.g. activity) of the full-length polynucleotide molecule which can be assessed by the skilled person through routine experimentation.
  • a usable, functional characteristic e.g. activity
  • a fragment of a polynucleotide SEQ ID NO preferably means a nucleotide sequence which comprises or consists of an amount of consecutive nucleotides from said polynucleotide SEQ ID NO and wherein said amount of consecutive nucleotides is at least 50 %, 60 %, 70 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95.5%, 96 %, 96.5 %, 97 %, 97.5 %, 98 %, 98.5 %, 99 %, 99.5 %, 100 %, preferably at least 80 %, more preferably at least 85 %, even more preferably at least 87 %, even more preferably at least 90 %, even more preferably at least 95 %,
  • 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 %, 40 %, 30 % of the full-length of said polynucleotide SEQ ID NO, preferably no more than 20 %, 15 %, 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4.5 %, 4 %, 3.5 %, 3 %, 2.5 %, 2 %, 1.5 %, 1 %, 0.5 %, more preferably no more than 15 %, even more preferably no more than 10 %, even more preferably no more than 5 %, most preferably no more than 2.5 %, of the full-length of said poly
  • “Fragment”, with respect to a polypeptide refers to a subsequence of the polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide.
  • a “subsequence of the polypeptide” or “a stretch of amino acid residues” as described herein refers to a sequence of contiguous amino acid residues derived from the polypeptide.
  • a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription.
  • Fragments can vary in size from as few as 3 amino acid residues to the full length of the intact polypeptide, for example at least about 10 amino acid residues in length, for example at least about 20 amino acid residues in length, for example at least about 30 amino acid residues in length, for example at least about 100 amino acid residues in length, for example at least about 150 amino acid residues in length, for example at least about 200 amino acid residues in length.
  • a fragment of a polypeptide SEQ ID NO preferably means a polypeptide sequence which comprises or consists of said polypeptide SEQ ID NO (or UniProt ID) wherein no more than about 200, 150, 125, 100, 80, 60, 50, 40, 30, 20 or 15 consecutive amino acid residues are missing, preferably no more than about 100 consecutive amino acid residues are missing, more preferably no more than about 50 consecutive amino acid residues are missing, even more preferably no more than about 40 consecutive amino acid residues are missing, and performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent, as does the intact polypeptide which can be routinely assessed by the skilled person.
  • a fragment of a polypeptide SEQ ID NO preferably means a polypeptide sequence which comprises or consists of an amount of consecutive amino acid residues from said polypeptide SEQ ID NO (or UniProt ID) and wherein said amount of consecutive amino acid residues is at least 50 %, 60 %, 70 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 95.5 %, 96 %, 96.5 %, 97 %, 97.5 %, 98 %, 98.5 %, 99 %, 99.5 %, 100 %, preferably at least 80 %, more preferably at least 85 %, even more preferably at least 87 %, even more preferably at least 90 %, even more preferably at least 90 %, even more preferably
  • 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 %, 40 %, 30 % of the full-length of said polypeptide SEQ ID NO (or UniProt ID), preferably no more than 20 %, 15 %, 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4.5 %, 4 %, 3.5 %, 3 %, 2.5 %, 2 %, 1.5 %, 1 %, 0.5 %, more preferably no more than 15 %, even more preferably no more than 10 %, even more preferably no more than 5 %, most preferably no more than 2.5 %, of the full-length of said polypeptide SEQ ID NO (or UniProt ID) and which performs at least one biological function of
  • 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. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of the nucleotides or polypeptides of interest. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI- BLAST analysis of non-redundant databases using the amino acid sequence of a reference polypeptide sequence. The amino acid sequence is, in some instances, deduced from the nucleotide sequence. Typically, those polypeptides in the database that have greater than 40 % sequence identity to a polypeptide of interest are candidates for further evaluation for suitability as a homologous polypeptide.
  • Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another or substitution of one acidic amino acid for another or substitution of one basic amino acid for another etc.
  • conservative substitutions is intended combinations such as glycine by alanine and vice versa; valine, isoleucine and leucine by methionine and vice versa; aspartate by glutamate and vice versa; asparagine by glutamine and vice versa; serine by threonine and vice versa; lysine by arginine and vice versa; cysteine by methionine and vice versa; and phenylalanine and tyrosine by tryptophan and vice versa. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated.
  • a domain can be characterized, for example, by a Pfam (El-Gebali et al., Nucleic Acids Res. 47 (2019) 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.org) (Nucleic Acids Res. 2021, 49(D1), D480-D489).
  • UniProt comprises the expertly and richly curated protein database called the UniProt Knowledgebase (UniProtKB), together with the UniProt Reference Clusters (UniRef) and the UniProt Archive (UniParc).
  • the UniProt identifiers (UniProt ID) are unique for each protein present in the database.
  • the sequence of a polypeptide is represented by a SEQ. ID NO or an UniProt ID. Unless stated otherwise, the UniProt IDs of the proteins described correspond to their sequence version 01 as present in the UniProt Database (www.uniprot.org) version release 2021_03 and consulted on 09 June 2021.
  • 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.
  • 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.
  • 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 80 % or more sequence identity over a stretch of at least 150 amino acid residues of a reference polypeptide sequence is to be understood as that the amino acid sequence 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 91.5 %, 92 %, 92.5 %, 93 %, 93.5 %, 94 %, 94.5 %, 95 %, 95.5 %, 96 %, 96.5 %, 97 %,
  • a polypeptide comprising or consisting of an amino acid sequence having 80 % or more sequence identity over a stretch of at least 200 amino acid residues of a reference polypeptide sequence is to be understood as that the amino acid sequence 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 91.5 %, 92 %, 92.5 %, 93 %, 93.5 %, 94 %, 94.5 %, 95 %, 95.5 %, 96 %, 96.5 %, 97 %,
  • a polypeptide comprising or consisting of an amino acid sequence having 80 % or more sequence identity to the full-length sequence of a reference polypeptide sequence is to be understood as that the amino acid sequence has 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %,
  • a polypeptide comprising, consisting of or having an amino acid sequence having 80 % or more sequence identity to the full-length amino acid sequence of a reference polypeptide, usually indicated with a SEQ ID NO or UniProt ID, preferably has 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 %, more preferably has at least 85 %, even more preferably has at least 90 %, sequence identity to the full length reference sequence.
  • a polynucleotide sequence comprising, consisting of or having a nucleotide sequence having 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 %, more preferably has at least 85 %, even more preferably has at least 90 % 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,9-0-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-acetylneuraminic 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-glycolylneuraminic acid, 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,
  • 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, UDP-4-amin
  • 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” or “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.
  • alpha-2, 6-sialyltransferase alpha 2,6 sialyltransferase, “6-sialyltransferase, "a-2,6- sialyltransferase”, “a 2,6 sialyltransferase”, “6 sialyltransferase”, “6-ST” or “6ST” or “a26-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, 6-linkage.
  • alpha-2, 8-sialyltransferase alpha 2,8 sialyltransferase, “8-sialyltransferase, "a-2,8- sialyltransferase”, “a 2,8 sialyltransferase”, “8 sialyltransferase”, “8-ST” or “8ST” or “a28-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, 8-linkage.
  • monosaccharide refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed as 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.
  • 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-acet
  • 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- Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) and CMP-KDO.
  • disaccharide refers to a saccharide polymer containing two simple sugars, i.e., monosaccharides.
  • examples of disaccharides comprise lactose (Gal-pi,4-Glc), lacto-N-biose (Gal-pi,3- GIcNAc), N-acetyllactosamine (Gal-pi,4-GlcNAc), LacDiNAc (GalNAc-pi,4-GlcNAc), N- acetylgalactosaminylglucose (GalNAc-pi,4-Glc), Neu5Ac-a2,3-Gal, Neu5Ac-a2,6-Gal, fucopyranosyl- (1- 4)-N-glycolylneuraminic acid (Fuc-(l-4)-Neu5Gc), sucrose (Glc-al,2-Fru), maltose (Gl)
  • Oleaccharide refers to a saccharide polymer containing a small number, typically three to twenty, preferably three to ten, of simple sugars, i.e., monosaccharides.
  • the oligosaccharide as used in the present invention can be a linear structure or can include branches.
  • the linkage e.g., glycosidic linkage, galactosidic linkage, glucosidic linkage, etc.
  • linkage between two sugar units can be expressed, for example, as 1,4, l->4, or (1-4), used interchangeably herein.
  • Gal-bl,4-Glc For example, the terms "Gal-bl,4-Glc”, “Gal-pi,4-Glc”, “b-Gal-(l->4)-Glc”, “P-Gal- (l->4)-Glc”, “Galbetal-4-Glc”, “Gal-b(l-4)-Glc” and “Gal-P(l-4)-Glc” have the same meaning, i.e. a beta- glycosidic bond links carbon-1 of galactose (Gal) with the carbon-4 of glucose (Glc).
  • Each monosaccharide can be in the cyclic form (e.g., pyranose or furanose form).
  • Linkages between the individual monosaccharide units may include alpha l->2, alpha l->3, alpha l->4, alpha l->6, alpha 2->l, alpha 2->3, alpha 2->4, alpha 2->6, beta l->2, beta l->3, beta l->4, beta l->6, beta 2->l, beta 2->3, beta 2->4, and beta 2->6.
  • An oligosaccharide can contain both alpha- and beta-glycosidic bonds or can contain only alpha- glycosidic or only beta-glycosidic bonds.
  • polysaccharide refers to a compound consisting of a large number, typically more than twenty, of monosaccharides linked glycosidically.
  • oligosaccharides include but are not limited to Lewis-type antigen oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigen, enterobacterial common antigen (ECA), 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
  • 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 one or more sialic acid residue(s). It has an acidic nature.
  • Such sialylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of said monosaccharide subunit is a sialic acid residue as described herein.
  • 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.
  • Some examples are 3-SL (3'-sialyllactose or 3'SL or Neu5Ac-a2,3-Gal-pi,4-Glc), 3'-sialyllactosamine, 6-SL (6'sialyllactose, 6'-sialyllactose or 6'SL or Neu5Ac-a2,6-Gal-pi,4-Glc), 3,6-disialyllactose (Neu5Ac-a2,3- (Neu5Ac-a2,6)-Gal-pi,4-Glc), 6,6'-disialyllactose (Neu5Ac-a2,6-Gal-pi,4-(Neu5Ac-a2,6)-Glc), 8,3- disialyllactose (Neu5Ac-a
  • 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.
  • 6' sialyllactose 6,' -sialyllactose, "alpha-2, 6-sialyllactose”, “alpha 2,6 sialyllactose”, “a-2,6-sialyllactose”, “a 2,6 sialyllactose”, “6SL” or “6'SL” as used in the present invention, are used interchangeably and refer to the product obtained by the catalysis of the alpha-2, 6-fucosyltransferase transferring the sialic acid group from CMP-Neu5Ac to lactose in an alpha-2, 6-linkage.
  • 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- i,4-GlcNAc- i,3-Gal- i,4-Glc" as used in the present invention, are used interchangeably.
  • LSTd LS-Tetrasaccharide d
  • Sialyl-lacto-N-tetraose 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
  • DSLNnT and “Disialyllacto-N- neotetraose” are used interchangeably and refer to Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-[Neu5Ac-a2,6]- Gal-pi,4-Glc.
  • DSLNT and “Disialyllacto-N-tetraose” are used interchangeably and refer to Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,3)-GlcNAc-pi,3-Gal-pi,4-Glc.
  • 'neutral oligosaccharide' and 'non-charged' oligosaccharide as used herein are used interchangeably and refer, as generally understood in the state of the art, to an oligosaccharide that has no negative charge originating from a carboxylic acid group.
  • Examples of such neutral oligosaccharide are 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 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 ll) 7 lacto- N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-fucopentaose VI, lacto-
  • LNT II "LNT-II", “LN3", "lacto-N-triose II", “lacto-/V-triose II”, “lacto-N-triose”, “lacto-/V-triose” or “GlcNAcpi-3Gaipi-4Glc” as used in the present invention, are used interchangeably.
  • LNT lacto-N-tetraose
  • lacto-/V-tetraose or "Gaipi-3GlcNAcpi-3Gaipi-4Glc” as used in the present invention, are used interchangeably.
  • LNnT lacto-N-neotetraose
  • lacto-/V-neotetraose lacto-/V-neotetraose
  • neo-LNT lacto-/V-neotetraose
  • Gaipi-4GlcNAcpi-3Gaipi-4Glc as used in the present invention
  • LNB lacto-N-neotetraose
  • lacto-/V-neotetraose lacto-/V-neotetraose
  • neo-LNT lacto-/V-neotetraose
  • Gaipi-4GlcNAcpi-3Gaipi-4Glc as used in the present invention
  • a 'fucosylated oligosaccharide' as used herein and as generally understood in the state of the art is an oligosaccharide that is carrying a fucose-residue.
  • Such fucosylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of said monosaccharide subunit is a fucose.
  • a fucosylated oligosaccharide can contain more than one fucose residue, e.g., two, three or more.
  • a fucosylated oligosaccharide can be a neutral oligosaccharide or a charged oligosaccharide e.g., also comprising sialic acid structures. Fucose can be linked to other monosaccharide subunits comprising glucose, galactose, GIcNAc via alpha-glycosidic bonds comprising alpha-1,2 alpha-1,3, alpha-1,4, alpha-1,6 linkages.
  • O-antigen refers to the repetitive glycan component of the surface lipopolysaccharide (LPS) of Gram-negative bacteria.
  • lipopolysaccharide or “LPS” refers to glycolipids found in the outer membrane of Gram-negative bacteria which are composed of a lipid A, a core oligosaccharide and the O-antigen.
  • capsule polysaccharides refers to long-chain polysaccharides with oligosaccharide repeat structures that are present in bacterial capsules, the latter being a polysaccharide layer that lies outside the cell envelope.
  • peptidoglycan or “murein” refers to an essential structural element in the cell wall of most bacteria, being composed of sugars and amino acids, wherein the sugar components consist of alternating residues of beta-1,4 linked GIcNAc and N-acetylmuramic acid.
  • 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 structures.
  • Said structures involve the A determinant GalNAc-alphal,3(Fuc-alphal,2)- Gal-, the B determinant Gal-alphal,3(Fuc-alphal,2)-Gal- and the H determinant Fuc-alphal,2-Gal- that are present on disaccharide core structures comprising Gal-betal,3-GlcNAc, Gal-betal,4-GlcNAc, Gal- betal,3-GalNAc and Gal-betal,4-Glc.
  • Mammalian milk oligosaccharides 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 (Ovis aries), goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus), horses (Eguus 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 (Loxodon
  • mammalian milk oligosaccharide or “MMO” refers to oligosaccharides such as but not limited to 3-fucosyllactose, 2'-fucosyllactose, 6-fucosyllactose, 2', 3- difucosyllactose, 2',2-difucosyllactose, 3,4-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, 3,6- disialyllactose, 6,6'-disialyllactose, 8,3-disialyllactose, 3,6-disialyllacto-N-tetraose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaos
  • human milk oligosaccharide refers to oligosaccharides found in human breast milk, including preterm human milk, colostrum and term human milk. HMOs comprise fucosylated oligosaccharides, non-fucosylated neutral oligosaccharides and sialylated oligosaccharides (see e.g., Chen X., Chapter Four: Human Milk Oligosaccharides (HMOS): Structure, Function, and Enzyme-Catalyzed Synthesis in Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)).
  • HMOS Human Milk Oligosaccharides
  • HMOs comprise 3- fucosyllactose, 2'-fucosyllactose, 2',3-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, LN3, lacto-N- tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, difucosyllacto-N-tetraose, lacto-N-hexao
  • mammary cell(s) generally refers to mammalian mammary epithelial cell(s), mammalian mammary-epithelial luminal cell(s), or mammalian epithelial alveolar cell(s), or any combination thereof.
  • mammary-like cell(s) generally refers to mammalian cell(s) having a phenotype/genotype similar (or substantially similar) to natural mammalian mammary cell(s) but is/are derived from mammalian non-mammary cell source(s).
  • Such mammalian mammary-like cell (s) may be engineered to remove at least one undesired genetic component and/or to include at least one predetermined genetic construct that is typical of a mammalian mammary cell.
  • Non-limiting examples of mammalian mammary-like cell(s) may include mammalian mammary epithelial-like cell(s), mammalian mammary epithelial luminal-like cell(s), mammalian non-mammary cell(s) that exhibits one or more characteristics of a cell of a mammalian mammary cell lineage, or any combination thereof.
  • 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.
  • a mammalian cell e.g., derived from a mammary cell lineage or a non-mammary cell lineage
  • 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.
  • a 'sialylation pathway' is a biochemical pathway consisting of at least one of the enzymes and their respective genes selected 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
  • Said pathway for production of a negatively charged, preferably sialylated, oligosaccharide may comprise a pathway for synthesis and/or import of a co-factor and/or a precursor used in said pathway for production of said negatively charged, preferably sialylated, oligosaccharide.
  • reductive pathway is a biochemical pathway that results in the cytoplasmic environment of the cell to be reducing, wherein said reducing environment negatively influences proper protein folding and blocks disulfide bond formation.
  • a reductive pathway comprise but are not limited to the reductive acetyl-CoA-pathway, the reductive pyrimidine catabolic pathway, the reductive citric acid cycle, the thiol-redox pathway and the reductive glycine pathway.
  • a reducing cytoplasm means e.g. that the NADP+:NADPH ratio in said cytoplasmic environment is low and/or that the glutathione (GSH) levels are high (e.g., 10 mM).
  • Glutathione reductase glutathione reductase (NADPH)
  • Glutathione S-reductase glutathione S-reductase
  • GSH reductase glutathione S-reductase
  • GSH reductase glutathione S-reductase
  • GSH reductase glutathione S-reductase
  • GSH reductase glutathione S-reductase
  • GSH reductase glutathione S-reductase
  • GSH reductase glutathione S-reductase
  • GSH reductase glutathione S-reductase
  • GSH reductase glutathione S-reductase
  • GSH reductase glutathione S-reductase
  • GSH reductase glutathione S-reductase
  • thioredoxin reductase thioredoxin-disulfide reductase
  • NADPH oxidized thioredoxin oxidoreductase
  • NADPH— thioredoxin reductase NADP— thioredoxin reductase
  • thioredoxin reductase NADPH
  • trxB thioredoxin reductase (NADPH)
  • disulfide bond isomerase protein disulfide-isomerase
  • S-S rearrangase S-S rearrangase
  • PDI PDI
  • disulfide oxidoreductase disulfide oxidoreductase 2
  • thiokdisulfide oxidoreductase dsbC
  • xprA xprA
  • the term “chaperone” refers to an enzyme that assist in protein folding. Examples are PDI, SecB, ERp57, heat shock proteins or Hsps, such as e.g., HsplO, Hsp60, Hsp70, Hsp90.
  • Hsps heat shock proteins
  • 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.
  • membrane transporter proteins refers to proteins that are part of or interact with the cell membrane and control the flow of molecules and information across the cell. The membrane proteins are thus involved in transport, be it import into or export out of the cell.
  • membrane transporter proteins can be but are not limited to porters, P-P-bond-hydrolysis-driven transporters, - Barrel Porins, auxiliary transport proteins and phosphotransfer-driven group translocators (Forrest et al., Biochim. Biophys. Acta 1807 (2011) 167-188; Lengeler, J. Mol. Microbiol. Biotechnol. 25 (2015) 79-93; Moraes and Reithmeier, Biochim. Biophys. Acta 1818 (2012), 2687-2706; Saier et al., Nucleic Acids Res. 44 (2016) D372-D379).
  • Siderophore as used herein is referring to the secondary metabolite of various microorganisms which are mainly ferric ion specific chelators. These molecules have been classified as catecholate, hydroxamate, carboxylate and mixed types. Siderophores are in general synthesized by a nonribosomal peptide synthetase (NRPS) dependent pathway or an NRPS independent pathway (NIS). The most important precursor in NRPS-dependent siderophore biosynthetic pathway is chorismate.
  • NRPS nonribosomal peptide synthetase
  • NPS NRPS independent pathway
  • 3-DHBA could be formed from chorismate by a three-step reaction catalysed by isochorismate synthase, isochorismatase, and 1, 3-dihydroxybenzoate-2, 3-dehydrogenase.
  • Siderophores can also be formed from salicylate which is formed from isochorismate by isochorismate pyruvate lyase.
  • ornithine is used as precursor for siderophores, biosynthesis depends on the hydroxylation of ornithine catalysed by L- ornithine N5-monooxygenase. In the NIS pathway, an important step in siderophore biosynthesis is N(6)- hydroxylysine synthase.
  • a transporter is needed to export the siderophore outside the cell.
  • MFS major facilitator superfamily
  • MOP Multidrug/Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily
  • RPD resistance, nodulation and cell division superfamily
  • ABC ABC superfamily.
  • the genes involved in siderophore export are clustered together with the siderophore biosynthesis genes.
  • siderophore exporter refers to such transporters needed to export the siderophore outside of the cell.
  • the ATP-binding cassette (ABC) superfamily contains both uptake and efflux transport systems, and the members of these two porter groups generally cluster loosely together. ATP hydrolysis without protein phosphorylation energizes transport. There are dozens of families within the ABC superfamily, and family generally correlates with substrate specificity. Members are classified according to class 3.A.1 as defined by the Transporter Classification Database operated by the Saier Lab Bioinformatics Group available via www.tcdb.org and providing a functional and phylogenetic classification of membrane transporter proteins.
  • MFS The major facilitator superfamily
  • TMSs transmembrane a- helical spanners
  • SET or “Sugar Efflux Transporter” as used herein refers to membrane proteins of the SET family which are proteins with InterPRO domain IPR004750 and/or are proteins that belong to the eggNOGv4.5 family ENOG410XTE9. Identification of the InterPro domain can be done by using the online tool on https://www.ebi.ac.uk/interpro/ or a standalone version of InterProScan (https://www.ebi.ac.uk/interpro/download.html) using the default values. Identification of the orthology family in eggNOGv4.5 can be done using the online version or a standalone version of eggNOG-mappervl (http://eggnogdb.embl.de/#/app/home).
  • the term “enabled efflux” means to introduce the activity of transport of a solute over the cytoplasm membrane and/or the cell wall. Said transport may be enabled by introducing and/or increasing the expression of a membrane transporter protein as described in the present invention.
  • the term “enhanced efflux” means to improve the activity of transport of a solute over the cytoplasm membrane and/or the cell wall. Transport of a solute over the cytoplasm membrane and/or cell wall may be enhanced by introducing and/or increasing the expression of a membrane transporter protein as described in the present invention.
  • “Expression” of a membrane transporter protein is defined as “overexpression” of the gene encoding said membrane transporter protein in the case said gene is an endogenous gene or “expression” in the case the gene encoding said membrane transporter protein is a heterologous gene that is not present in the wild-type strain or cell.
  • purified refers to material that is substantially or essentially free from components that interfere with the activity of the biological molecule.
  • purified refers to material that is substantially or essentially free from components that normally accompany the material as found in its native state.
  • purified saccharides, oligosaccharides, proteins or nucleic acids of the invention are at least about 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 % or 85 % pure, usually at least about 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99.0 % pure as measured by band intensity on a silver-stained gel or other method for determining purity.
  • Purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization upon staining.
  • 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 negatively charged, preferably sialylated, oligosaccharide.
  • aqueous medium like e.g., a cultivation or an incubation
  • 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 negatively charged, preferably sialylated, oligosaccharide 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.
  • the term "incubation” refers to a mixture wherein a negatively charged, preferably sialylated, oligosaccharide 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 a negatively charged, preferably sialylated, oligosaccharide, 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) a negatively charged, preferably sialylated, oligosaccharide that is produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell.
  • Said incubation can also be the cultivation as defined herein.
  • 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 which are taken up or synthetized by the cell for the specific production of a negatively charged, preferably sialylated, oligosaccharide according to the present invention.
  • a precursor can be an acceptor as defined herein, but can also be another substance, metabolite, co-factor, which is first modified within the cell as part of the biochemical synthesis route of a negatively charged, preferably sialylated, oligosaccharide.
  • precursor as used herein is also to be understood as a chemical compound that participates in an incubation, a chemical or an enzymatic reaction to produce another compound like e.g., an intermediate or an acceptor as defined herein, as part in the metabolic pathway of a negatively charged, preferably sialylated, oligosaccharide.
  • precursor as used herein is also to be understood as a donor that is used by a glycosyltransferase to modify an acceptor as defined herein with a sugar moiety in a glycosidic bond, as part in the metabolic pathway of a negatively charged, preferably sialylated, oligosaccharide.
  • 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-l-phosphate galactose-1- phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-l,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 used to produce the negatively charged, preferably sialylated, oligosaccharide is transformed to comprise and to express at least one nucleic acid sequence encoding a protein selected from the list consisting of lactose transporter, N-acetylneuraminic acid transporter, fucose transporter, glucose transporter, galactose transporter, transporter for a nucleotide-activated sugar wherein said transporter internalizes a to the medium added precursor for the synthesis of the negatively charged, preferably sialylated, oligosaccharide of present invention.
  • a protein selected from the list consisting of lactose transporter, N-acetylneuraminic acid transporter, fucose transporter, glucose transporter, galactose transporter, transporter for a nucleotide-activated sugar wherein said transporter internalizes a to the medium added precursor for the synthesis of the negatively charged, preferably sialylated, oligosaccharide
  • 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, N-acetylglucosamine, maltose, sucrose, lactose, lactulose, lactobionic acid (LBA), N- acetyllactosamine, lacto-N-biose, 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-novopentao
  • the present invention provides a cell that comprises a pathway for production of a negatively charged, preferably sialylated, oligosaccharide, the cell being genetically engineered to possess or express, preferably to over-express, a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase wherein said hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising a conserved motif with SEQ. ID NO 12 and having hydrolyzing UDP-N-acetyl-D-glucosamine-2- epimerase activity.
  • the present invention provides a method for the production of a negatively charged, preferably sialylated, oligosaccharide, wherein the method comprises cultivating and/or incubating a cell as described herein, in cultivation and/or incubation medium under conditions permissive to produce a negatively charged, preferably sialylated, oligosaccharide.
  • 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/precursor/acceptor concentration.
  • the permissive conditions may include a temperature-range of 30 +/- 20 degrees centigrade, a pH-range of 7 +/- 3.
  • the negatively charged, preferably sialylated, oligosaccharide is separated from said cultivation and/or incubation.
  • the oligosaccharide is purified from said cultivation and/or incubation.
  • the cell comprises a pathway for production of a negatively charged, preferably sialylated, oligosaccharide.
  • pathway for production of a negatively charged, preferably sialylated, oligosaccharide is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of a negatively charged, preferably sialylated, oligosaccharide as defined herein.
  • Said pathway for production of a negatively charged, preferably 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 negatively charged, preferably sialylated, oligosaccharide of the present invention.
  • Examples of such pathways comprise but are not limited to a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway and an N-acetylmannosaminylation pathway.
  • the cell is genetically engineered to possess or express a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase as described herein. In a preferred embodiment, the cell is genetically engineered to over-express a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase as described herein.
  • the genetically engineered cell is modified with gene expression modules wherein the expression from any one of said expression modules is constitutive or is tuneable.
  • 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.
  • the cell is modified with one or more expression modules.
  • the expression modules can be integrated in the genome of said cell or can be presented to said cell on a vector.
  • Said vector can be present in the form of a plasmid, cosmid, phage, liposome, or virus, which is to be stably transformed/transfected into said metabolically engineered cell.
  • Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • These vectors may contain selection markers such as but not limited to antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA sense/antisense markers.
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard.
  • the appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., see above.
  • cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
  • Introduction of a polynucleotide into the cell can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986), and Sambrook et al., 1989, supra.
  • an expression module comprises polynucleotides for expression of at least one recombinant gene.
  • Said recombinant gene is involved in the pathway for production of a negatively charged, preferably sialylated, oligosaccharide; or said recombinant gene is linked to other pathways in said cell that are not involved in the synthesis of a negatively charged, preferably 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 is constitutive or tuneable as defined herein.
  • the cell is modified in the expression or activity of a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase as described herein.
  • said hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous hydrolyzing UDP-N-acetyl-D- glucosamine-2-epimerase is overexpressed; alternatively said hydrolyzing UDP-N-acetyl-D-glucosamine- 2-epimerase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase can have a modified expression in the cell which also expresses a heterologous hydrolyzing UDP-N-acetyl-D- glucosamine-2-epimerase.
  • the amino acid can be one of the 20 common amino acids encoded in the genetic code of life (i.e. A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V) or can be a modified amino acid (e.g. due to decomposition of a common amino acid like e.g. L-ornithine, or modified by e.g. hydroxylation, carboxylation, phosphorylation, methylation, acetylation, glycosylation, ADP-ribosylation or ubiquitination of a common amino acid).
  • a common amino acid like e.g. L-ornithine, or modified by e.g. hydroxylation, carboxylation, phosphorylation, methylation, acetylation, glycosylation, ADP-ribosylation or ubiquitination of a common amino acid.
  • each X is an amino acid that results from a new, independent selection made out of the list of possible amino acid residues.
  • each X present in the conserved motif with SEQ ID NO 12 refers to the same amino acid or to a different amino acid.
  • the hydrolyzing UDP-N-acetyl- D-glucosamine-2-epimerase comprises, consists of or consists essentially of an amino acid sequence that is at least 80 % identical over a stretch of at least 150 amino acid residues to any one of the amino acid sequences as represented by SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 08.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase comprises, consists of or consists essentially of an amino acid sequence that is at least 80 % identical over a stretch of at least 200 amino acid residues to any one of the amino acid sequences as represented by SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 08.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising an IPR domain selected from the list comprising IPR003331, IPR020004 and IPR029767 as defined by InterPro 90.0 as released on 4 th August 2022.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising a PF02350 motif as defined by PFAM 32.0 as released in September 2018.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising a cd03786 motif as defined by CDD v3.17 as released in September 2016.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising a panther domain selected from the list comprising PTHR43174 and PTHR43174:SF3 as defined by PANTHER 17.0 as released on 23 rd February 2022.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, or at least 99 % identical to any one of the amino acid sequences as represented by SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 08 over a stretch of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290 or at least 300 amino acid residues.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence that is at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, or at least 99 % identical to any one of the full-length amino acid sequences as represented by SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 08.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence as represented by any one of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 08 and comprising UDP-N-acetyl-D-glucosamine-2- epimerase activity.
  • the hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase is a nnaA polypeptide.
  • the cell contains a nucleic acid molecule which comprises a polynucleotide sequence which encodes a hydrolyzing UDP-N-acetyl-D- glucosamine-2-epimerase as described herein.
  • the nucleic acid molecule is operably linked to control sequences recognized by the cell, said nucleic acid molecule further i) being integrated in the genome of said cell and/or ii) presented to said cell on a vector.
  • the nucleic acid molecule is foreign to said cell.
  • the cell is genetically engineered for production of a negatively charged, preferably sialylated, oligosaccharide, wherein the cell comprises a pathway for production of said oligosaccharide.
  • the cell is genetically engineered for production of two or more negatively charged, preferably sialylated, oligosaccharides.
  • the cell is genetically engineered for an enhanced production of a negatively charged, preferably sialylated, oligosaccharide, an enhanced uptake of one or more precursor(s) and/or acceptor(s) that is/are used in the synthesis of a negatively charged, preferably sialylated, oligosaccharide, a better and/or enhanced efflux of the negatively charged, preferably sialylated, oligosaccharide, a decreased production of by-products like e.g. acids, an increased availability of co-factors like e.g.
  • ATP ATP, NADP, NADPH, and/or better and/or enhanced metabolic flux through any one of the sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N- acetylgalactosaminylation, mannosylation, and/or N-acetylmannosaminylation pathway present in the cell.
  • the pathway for production of said negatively charged, preferably sialylated, oligosaccharide is a sialylation pathway.
  • the cell is genetically engineered to comprise said sialylation pathway.
  • the cell comprises said sialylation pathway wherein said sialylation pathway has been genetically engineered.
  • the cell has been metabolically engineered to comprise a sialylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6- phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N- acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6- phosphate 2-epimerase, UDP-GIcNAc 2-epimerase/kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N- acetylglucosamine 1-phosphate uridylyltransfer
  • the cell further comprises a pathway selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway.
  • the cell is genetically engineered to comprise at least one pathway selected from the list comprising, consisting of or consisting essentially of said fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway.
  • the cell comprises at least one pathway selected from the list comprising, consisting of or consisting essentially of said fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway wherein at least one of said fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N- acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway has/have been genetically engineered.
  • the cell comprises a fucosylation pathway.
  • the cell is metabolically engineered to comprise a fucosylation pathway.
  • the cell has been metabolically engineered to comprise a fucosylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-1- phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase and fucosyltransferase has/have a modified and/or enhanced expression.
  • the cell comprises a galactosylation pathway.
  • the cell is metabolically engineered to comprise a galactosylation pathway.
  • the cell has been metabolically engineered to comprise a galactosylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of galactose-l-epimerase, galactokinase, glucokinase, galactose-1- phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase and galactosyltransferase has/have a modified and/or enhanced expression.
  • the cell comprises an 'N- acetylglucosaminylation' pathway.
  • the cell is metabolically engineered to comprise an N-acetylglucosaminylation pathway.
  • the cell has been metabolically engineered to comprise an N-acetylglucosaminylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N- acetylglucosamine-l-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, and a glycosyltransferase transferring GIcNAc has/have a modified and/or enhanced expression.
  • the cell comprises an 'N- acetylgalactosaminylation' pathway.
  • the cell is metabolically engineered to comprise an N-acetylgalactosaminylation pathway.
  • the cell has been metabolically engineered to comprise an N-acetylgalactosaminylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, UDP-N-acetylglucosamine 4-epimerase, UDP-glucose 4-epimerase, N-acetylgalactosamine kinase and/or UDP-N-acetylgalact
  • the cell comprises a 'mannosylation' pathway.
  • the cell is metabolically engineered to comprise a mannosylation pathway.
  • the cell has been metabolically engineered to comprise a mannosylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase and/or mannose-l-phosphate guanylyltransferase and mannosyltransferase has/have a modified and/or enhanced expression.
  • the cell comprises an 'N- acetylmannosaminylation' pathway.
  • the cell is metabolically engineered to comprise an N-acetylmannosaminylation pathway.
  • the cell has been metabolically engineered to comprise an N-acetylmannosaminylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N- acetylglucosamine-6-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase, N- acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine
  • 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).
  • PEP phosphoenolpyruvate
  • 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.
  • 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-acetylglucosamine epimerase, UDP-
  • the cell possesses, preferably expresses, more preferably overexpresses, one or more glycosyltransferase(s) selected from the list comprising, consisting of or consisting essentially of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N- acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N- glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases
  • the fucosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1, 2-fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-1, 4-fucosyltransferase and alpha-1, 6-fucosyltransferase.
  • the further sialyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase, and alpha-2, 8-sialyltransferase.
  • the galactosyltransferase is selected from the list comprising, consisting of or consisting essentially of beta-1, 3-galactosyltransferase, N-acetylglucosamine beta-1, 3-galactosyltransferase, beta- 1,4-galactosyltransferase, N-acetylglucosamine beta-1, 4-galactosyltransferase, alpha-1, 3- galactosyltransferase and alpha-1, 4-galactosyltransferase.
  • the glucosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-glucosyltransferase, beta-1, 2-glucosyltransferase, beta-1, 3-glucosyltransferase and beta-1, 4-glucosyltransferase.
  • the mannosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1, 2-mannosyltransferase, alpha-1, 3-mannosyltransferase and alpha-1, 6- mannosyltransferase.
  • the N-acetylglucosaminyltransferase is selected from the list comprising, consisting of or consisting essentially of galactoside beta-1, 3-N-acetylglucosaminyltransferase and beta-1, 6-N- acetylglucosaminyltransferase.
  • the N-acetylgalactosaminyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1, 3-N-acetylgalactosaminyltransferase.
  • the cell is modified in the expression or activity of at least one of said glycosyltransferases.
  • said glycosyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous glycosyltransferase is overexpressed; alternatively said glycosyltransferase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous glycosyltransferase can have a modified expression in the cell which also expresses a heterologous glycosyltransferase.
  • the cell is capable to produce, preferably produces, one or more nucleotide-activated sugars, preferably said cell is genetically engineered for production of one or more of said nucleotide-activated sugar(s).
  • said one or more nucleotide-activated sugar(s) is/are selected from the list comprising, consisting of or consisting essentially of 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), GDP-fucose (GDP-Fuc), UDP-glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy- -L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L
  • the cell comprises a pathway for the synthesis of a nucleotide-activated sugar selected from the list comprising, consisting of or consisting essentially of 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), GDP-fucose, (GDP-Fuc), UDP-glucuronate, UDP-galacturonate, UDP-2- acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-
  • 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, consisting of or consisting essentially of an N-acetyl-D-glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, N-acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, and a bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli.
  • the cell is modified to produce UDP-GIcNAc.
  • the cell used herein is optionally genetically modified 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 selected from the list comprising, consisting of or consisting essentially of knock-out of an N-acetylglucosamine-6-phosphate deacetylase, knock-out of a glucosamine-6-phosphate deaminase, over-expression of a CMP-sialic acid synthetase, and over-expression of an N-acetyl-D-glucosamine-2-epimerase encoding gene.
  • the cell used herein is optionally genetically modified 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 selected from the list comprising, consisting of or consisting essentially of 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 overexpression of a mannose-6-phosphate isomerase encoding gene.
  • the cell used herein is optionally genetically modified to express the de novo synthesis of UDP-Gal.
  • UDP-Gal can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing UDP-Gal can express an enzyme converting, e.g. UDP-glucose, to UDP-Gal.
  • This enzyme may be, e.g., the UDP-glucose-4-epimerase GalE like as known from several species including Homo sapiens, Escherichia coli, and Rattus norvegicus.
  • the cell is modified to produce UDP- Gal. More preferably, the cell is modified for enhanced UDP-Gal production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knock-out of a bifunctional 5'-nucleotidase/UDP-sugar hydrolase encoding gene, knock-out of a galactose-l-phosphate uridylyltransferase encoding gene and over-expression of an UDP-glucose-4-epimerase encoding gene.
  • the cell used herein is optionally genetically modified 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 an 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 modified to express the de novo synthesis of UDP-ManNAc.
  • UDP-ManNAc can be synthesized directly from UDP-GIcNAc via an epimerization reaction performed by an UDP-GIcNAc 2-epimerase (like e.g. cap5P from Staphylococcus aureus, RffE from E. coli, Cpsl9fK from S. pneumoniae, and RfbC from S. enterica).
  • an 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 possesses, preferably expresses, one or more genes selected from the list comprising, consisting of or consisting essentially of 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, L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N- acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6- phosphate 2-ep
  • the cell overexpresses one or more genes selected from the list comprising, consisting of or consisting essentially of 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, L- glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N- acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2- epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GIcNAc 2-epimerase/
  • the cell is capable to produce, preferably produces, N-acetylmannosamine (ManNAc), a sialic acid residue and/or N- acetylglucosamine (GIcNAc), wherein said sialic acid residue is selected from the list comprising 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).
  • ManNAc N-acetylmannosamine
  • GIcNAc N- acetylglucosamine
  • the cell comprises a pathway for production of ManNAc, a sialic acid residue and/or GIcNAc.
  • the cell is genetically engineered for production of ManNAc, a sialic acid residue and/or GIcNAc.
  • the negatively charged oligosaccharide produced by a cell of present invention is a sialylated oligosaccharide having at least one sialic acid residue selected from the list comprising, consisting of or consisting essentially of Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulonic acid (KDO).
  • KDO 2-keto-3-deoxymanno-octulonic acid
  • the negatively charged oligosaccharide is an oligosaccharide selected from the list comprising, consisting of or consisting essentially of a negatively charged, preferably sialylated, milk oligosaccharide, preferably a negatively charged, more preferably sialylated, mammalian milk oligosaccharide (MMO), more preferably a negatively charged, more preferably sialylated, human milk oligosaccharide (HMO); O-antigen; the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an amino-sugar; Lewis-type antigen oligosaccharide; a negatively charged, preferably sialylated, animal oligosaccharide; a negatively charged, preferably sialylated, plant oligosaccharide; N- acetyllactosamine containing negatively charged, preferably sialylated, oligosaccharide and lac
  • the negatively charged oligosaccharide is a mammalian milk oligosaccharide (MMO) as described herein.
  • the negatively charged oligosaccharide is a human milk oligosaccharide (HMO) as described herein.
  • the negatively charged oligosaccharide is an animal oligosaccharide selected from the list consisting of N- glycans and O-glycans.
  • the negatively charged oligosaccharide is a plant oligosaccharide selected from the list consisting of N-glycans and O-glycans.
  • N-glycans and O-glycans refer to the oligosaccharide structures as known by the person skilled in the art wherein said structures are not attached to a protein or a peptide.
  • the negatively charged oligosaccharide is selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), 3'-sialyllactosamine, 3,6-disialyllactose, 8,3- disialyllactose, sialylated lacto-N-triose, sialyllacto-N-tetraose a (LSTa), KDOa-2,3Gaip-l,3GlcNAcP- l,3Gaip-l,4Glc, sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyll
  • the oligosaccharide in the context of the present invention is preferably in free form, i.e., the oligosaccharide does not contain any protective group.
  • the cell is capable to produce, preferably produces, said negatively charged, preferably sialylated, oligosaccharide from one or more precursor(s) as defined herein.
  • the precursor is lactose.
  • said one or more precursor(s) is/are fed to the cell from the culture medium or the incubation.
  • the cell is capable to produce, preferably produces, at least one of said one or more precursor(s).
  • the cell is capable to produce, preferably produces, all of said one or more precursor(s).
  • the cell is capable to produce, preferably produces, lactose.
  • the cell is genetically engineered for the production of at least one of said one or more precursor(s). In an even more preferred embodiment, the cell is genetically engineered for the production of all of said one or more precursor(s). In another more preferred embodiment, the cell is genetically engineered for the production of lactose. In another more preferred embodiment, at least one of said one or more precursor(s) is internalized in said cell via one or more membrane protein(s), preferably said membrane protein(s) is/are membrane transporter protein(s) as described herein.
  • the precursor(s) that is/are used by the cell for the production of said negatively charged, preferably sialylated, oligosaccharide is/are completely converted into said negatively charged, preferably sialylated, oligosaccharide.
  • the cell is capable to produce, preferably produces, phosphoenolpyruvate (PEP).
  • PEP phosphoenolpyruvate
  • the cell comprises a pathway for production of PEP.
  • the cell is modified for enhanced synthesis and/or supply of phosphoenolpyruvate (PEP) compared to a nonmodified progenitor.
  • PEP phosphoenolpyruvate
  • one or more PEP-dependent, sugar-transporting phosphotransferase system(s) is/are disrupted such as but not limited to: 1) the N-acetyl-D-glucosamine Npi-phosphotransferase (EC 2.7.1.193), which is for instance encoded by the nagE gene (or the cluster nagABCD) in E.
  • ManXYZ which encodes the Enzyme II Man complex (mannose PTS permease, protein-Npi- phosphohistidine-D-mannose phosphotransferase) that imports exogenous hexoses (mannose, glucose, glucosamine, fructose, 2- deoxyglucose, mannosamine, N-acetylglucosamine, etc.) and releases the phosphate esters into the cell cytoplasm, 3) the glucose-specific PTS transporter (for instance encoded by PtsG/Crr) which takes up glucose and forms glucose-6-phosphate in the cytoplasm, 4) the sucrose-specific PTS transporter which takes up sucrose and forms sucrose-6-phosphate in the cytoplasm, 5) the fructose-specific PTS transporter (for instance encoded by the genes fruA and fruB and the kinase fruK which takes up fructose and forms in a first step fructose-l
  • the full PTS system is disrupted by disrupting the PtsIH/Crr gene cluster.
  • the cell is further modified to compensate for the deletion of a PTS system of a carbon source by the introduction and/or overexpression of the corresponding permease.
  • permeases or ABC transporters that comprise but are not limited to transporters that specifically import lactose such as e.g. the transporter encoded by the LacY gene from E. coli, sucrose such as e.g. the transporter encoded by the cscB gene from E. coli, glucose such as e.g. the transporter encoded by the galP gene from E. coli, fructose such as e.g.
  • the transporter encoded by the frul gene from Streptococcus mutans, or the Sorbitol/mannitol ABC transporter such as the transporter encoded by the cluster SmoEFGK of Rhodobacter sphaeroides, the trehalose/sucrose/maltose transporter such as the transporter encoded by the gene cluster ThuEFGK of Sinorhizobium meliloti and the N- acetylglucosamine/galactose/glucose transporter such as the transporter encoded by NagP otShewanella oneidensis.
  • Examples of combinations of PTS deletions with overexpression of alternative transporters are: 1) the deletion of the glucose PTS system, e.g.
  • ptsG gene combined with the introduction and/or overexpression of a glucose permease (e.g. galP of glcP), 2) the deletion of the fructose PTS system, e.g. one or more of the fruB, fruA, fruK genes, combined with the introduction and/or overexpression of fructose permease, e.g. frul, 3) the deletion of the lactose PTS system, combined with the introduction and/or overexpression of lactose permease, e.g. LacY, and/or 4) the deletion of the sucrose PTS system, combined with the introduction and/or overexpression of a sucrose permease, e.g. cscB.
  • a sucrose permease e.g. cscB.
  • the cell is modified to compensate for the deletion of a PTS system of a carbon source by the introduction of carbohydrate kinases, such as glucokinase (EC 2.7.1.1, EC 2.7.1.2, EC 2.7.1.63), galactokinase (EC 2.7.1.6), and/or fructokinase (EC 2.7.1.3, EC 2.7.1.4).
  • carbohydrate kinases such as glucokinase (EC 2.7.1.1, EC 2.7.1.2, EC 2.7.1.63), galactokinase (EC 2.7.1.6), and/or fructokinase (EC 2.7.1.3, EC 2.7.1.4).
  • the cell is modified by the introduction of or modification in any one or more of the list comprising phosphoenolpyruvate synthase activity (EC: 2.7.9.2 encoded for instance in E. coli by ppsA), phosphoenolpyruvate carboxykinase activity (EC 4.1.1.32 or EC 4.1.1.49 encoded for instance in Corynebacterium glutamicum by PCK or in E. coli by pckA, resp.), phosphoenolpyruvate carboxylase activity (EC 4.1.1.31 encoded for instance in E.
  • phosphoenolpyruvate synthase activity EC: 2.7.9.2 encoded for instance in E. coli by ppsA
  • phosphoenolpyruvate carboxykinase activity EC 4.1.1.32 or EC 4.1.1.49 encoded for instance in Corynebacterium glutamicum by PCK or in E. coli by pckA, resp.
  • coli by ppc oxaloacetate decarboxylase activity
  • EC 4.1.1.112 encoded for instance in E. coli by eda oxaloacetate decarboxylase activity
  • EC 2.7.1.40 encoded for instance in E. coli by pykA and pykF pyruvate carboxylase activity
  • malate dehydrogenase activity EC 1.1.1.38 or EC 1.1.1.40 encoded for instance in E. coli by maeA or maeB, resp.
  • the cell is modified by a reduced activity of phosphoenolpyruvate carboxylase activity, and/or pyruvate kinase activity, preferably a deletion of at least one of the genes encoding for phosphoenolpyruvate carboxylase, the pyruvate carboxylase activity and/or pyruvate kinase.
  • the cell is further modified for reduced degradation of acetyl-CoA and/or its main precursor pyruvate.
  • the cell comprises a modification for reduced production of acetate compared to a non-modified progenitor.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of overexpression of an acetyl-coenzyme A synthetase, a full or partial knock-out or rendered less functional pyruvate dehydrogenase and a full or partial knock-out or rendered less functional lactate dehydrogenase.
  • the cell is modified in the expression or activity of at least one acetyl-coenzyme A synthetase like e.g. acs from E. coli, S. cerevisiae, H. sapiens, M. musculus.
  • at least one acetyl-coenzyme A synthetase like e.g. acs from E. coli, S. cerevisiae, H. sapiens, M. musculus.
  • said acetyl-coenzyme A synthetase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous acetyl-coenzyme A synthetase is overexpressed; alternatively, said acetyl-coenzyme A synthetase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous acetyl-coenzyme A synthetase can have a modified expression in the cell which also expresses a heterologous acetyl-coenzyme A synthetase.
  • the cell is modified in the expression or activity of at least one pyruvate dehydrogenase like e.g. from E. coli, S. cerevisiae, H. sapiens and R. norvegicus.
  • the cell has been modified to have at least one partially or fully knocked out or mutated pyruvate dehydrogenase encoding gene by means generally known by the person skilled in the art resulting in at least one protein with less functional or being disabled for pyruvate dehydrogenase activity.
  • the cell has a full knock-out in the poxB encoding gene resulting in a cell lacking pyruvate dehydrogenase activity.
  • the cell is modified in the expression or activity of at least one lactate dehydrogenase like e.g. from E. coli, S. cerevisiae, H. sapiens and R. norvegicus.
  • the cell has been modified to have at least one partially or fully knocked out or mutated lactate dehydrogenase encoding gene by means generally known by the person skilled in the art resulting in at least one protein with less functional or being disabled for lactate dehydrogenase activity.
  • the cell has a full knock-out in the 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, EI
  • 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 the negatively charged, preferably sialylated, oligosaccharide of present invention.
  • the cell possesses, expresses and/or overexpresses at least one gene selected from the list comprising, consisting of or consisting essentially of genes encoding a disulfide bond isomerase, a thiol oxidase or a chaperone.
  • the cell expresses a membrane transporter protein or a polypeptide having transport activity hereby transporting compounds across the outer membrane of the cell wall.
  • the cell expresses more than one membrane transporter protein or polypeptide having transport activity hereby transporting compounds across the outer membrane of the cell wall.
  • the cell is modified in the expression or activity of said membrane transporter protein or polypeptide having transport activity.
  • Said membrane transporter protein or polypeptide having transport activity is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous membrane transporter protein or polypeptide having transport activity is overexpressed; alternatively said membrane transporter protein or polypeptide having transport activity is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous membrane transporter protein or polypeptide having transport activity can have a modified expression in the cell which also expresses a heterologous membrane transporter protein or polypeptide having transport activity.
  • the membrane transporter protein or polypeptide having transport activity is selected from the list comprising, consisting of or consisting essentially of porters, P-P-bond-hydrolysis-driven transporters, p-barrel porins, auxiliary transport proteins and phosphotransfer-driven group translocators.
  • the porters comprise MFS transporters, sugar efflux transporters (SET) and siderophore exporters.
  • the P-P-bond-hydrolysis-driven transporters comprise ABC transporters and siderophore exporters.
  • the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of said negatively charged, preferably sialylated, oligosaccharide.
  • the membrane transporter protein or polypeptide having transport activity controls the flow over the outer membrane of the cell wall of one or more precursor(s) to be used in said production of said negatively charged, preferably sialylated, oligosaccharide.
  • the membrane transporter protein or polypeptide having transport activity provides improved production of said negatively charged, preferably sialylated, oligosaccharide.
  • the membrane transporter protein or polypeptide having transport activity provides enabled efflux of said negatively charged, preferably sialylated, oligosaccharide. In an alternative and/or additional preferred embodiment of the method and/or cell of the invention, the membrane transporter protein or polypeptide having transport activity provides enhanced efflux of said negatively charged, preferably sialylated, oligosaccharide.
  • the cell is transformed to comprise at least one nucleic acid sequence encoding a protein selected from the group comprising, consisting of or consisting essentially of 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 a negatively charged, preferably sialylated, oligosaccharide 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
  • a glucose transporter e.g. the LacY or Iacl2 permease
  • a glucose transporter e
  • the cell expresses a membrane transporter protein belonging to the family of MFS transporters like e.g., an MdfA polypeptide of the multidrug transporter MdfA family from species comprising E. coli (UniProt ID P0AEY8), Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), Citrobacter youngae (UniProt ID D4BC23) and Yokenella regensburgei (UniProt ID G9Z5F4).
  • MFS transporters e.g., an MdfA polypeptide of the multidrug transporter MdfA family from species comprising E. coli (UniProt ID P0AEY8), Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), Citrobacter youngae (UniProt ID D4BC23) and Yokenella regensburgei (UniProt ID G9Z5F4)
  • the cell expresses a membrane transporter protein belonging to the family of sugar efflux transporters (SET) like e.g., a SetA polypeptide of the SetA family from species comprising E. coli (UniProt ID P31675, sequence version 03 (11 Oct 2004)) and Citrobacter koseri (UniProt ID A0A078LM16).
  • SET sugar efflux transporters
  • the cell expresses a membrane transporter protein belonging to the family of siderophore exporters like e.g., the E. coli entS (UniProt ID P24077, sequence version 02 (01 Nov 1997)), the K.
  • the cell expresses a membrane transporter protein belonging to the family of ABC transporters like e.g., oppF from E. coli (UniProt ID P77737), ImrA from Lactococcus lactissubsp. lactisbv. diacetylactis (UniProt ID A0A1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4).
  • a membrane transporter protein belonging to the family of ABC transporters like e.g., oppF from E. coli (UniProt ID P77737), ImrA from Lactococcus lactissubsp. lactisbv. diacetylactis (UniProt ID A0A1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4).
  • the cell expresses more than one membrane transporter protein selected from the list comprising, consisting of or consisting essentially of a lactose transporter like e.g. the LacY or Iacl2 permease, a fucose transporter, a glucose transporter, a galactose transporter, a transporter for a nucleotide-activated sugar like for example a transporter for UDP-GIcNAc, UDP-Gal and/or GDP-Fuc, the MdfA protein from E.
  • a lactose transporter like e.g. the LacY or Iacl2 permease
  • a fucose transporter e.g. the LacY or Iacl2 permease
  • a fucose transporter e.g. the LacY or Iacl2 permease
  • a fucose transporter e.g. the LacY or Iacl2 permease
  • glucose transporter e.g.
  • the cell is transformed to comprise at least one nucleic acid sequence encoding a membrane transporter protein selected from the list comprising, consisting of or consisting essentially of a siderophore exporter, a major facilitator superfamily (MFS) transporter, an ATP-binding cassette (ABC) transporter or a sugar efflux transporter.
  • a membrane transporter protein selected from the list comprising, consisting of or consisting essentially of a siderophore exporter, a major facilitator superfamily (MFS) transporter, an ATP-binding cassette (ABC) transporter or a sugar efflux transporter.
  • the cell is capable to synthesize N-acetylmannosamine (ManNAc) and/or N-acetylmannosamine-6-phosphate (ManNAc-6-phosphate).
  • ManNAc N-acetylmannosamine
  • ManNAc-6-phosphate N-acetylmannosamine-6-phosphate
  • one or more gene(s) involved in one or more reductive pathway(s) in the cell is/are rendered less functional or is/are knocked-out.
  • a reductive pathway comprise but are not limited to the reductive acetyl-CoA-pathway, the reductive pyrimidine catabolic pathway, the reductive citric acid cycle, the thiol- redox pathway and the reductive glycine pathway.
  • one or more gene(s) involved in one or more reductive pathway(s) is/are rendered less functional by insertion, deletion and/or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) selected from the list comprising, consisting of or consisting essentially of promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said one or more gene(s).
  • said one or more gene(s) may be selected from the list comprising, consisting of or consisting essentially of gene(s) encoding formate dehydrogenase, formate-tetrahydrofolate ligase, methenyltetrahydrofolate cyclohydrolase, glycine dehydrogenase/decarboxylating, glycine cleavage system protein, glutamate dehydrogenase, glycine reductase complex, Co 2 reductase, folate synthetase, folate cyclohydrolase, folate dehydrogenase, folate reductase, methyltransferase, pyruvate synthase, phosphotransacetylase, acetate kinase, ATPase, acetyl-CoA carbonylase/synthase, methylenetetrahydrofolate reductase, methylenetetrahydrolate
  • said one or more genes involved in one or more reductive pathway(s) is/are selected from the list comprising, consisting of or consisting essentially of a glutathione reductase and a thioredoxin reductase.
  • the negatively charged, preferably sialylated, oligosaccharide of present invention is produced by a cell that is cultured in a cell cultivation.
  • the cell cultivation comprises in vitro and/or ex vivo cultivation of cells.
  • the cell cultivation is a fermentation.
  • the cell is cultivated or incubated in a reactor as defined herein.
  • the cell is cultivated or incubated in an incubator as defined herein.
  • the cell is stably cultured in a cultivation or incubation medium.
  • the cell is cultivated in cultivation or incubation medium comprising at least one carbon source selected from the list comprising, consisting of or consisting essentially of a monosaccharide; disaccharide; oligosaccharide; polysaccharide; polyol; glycerol; a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract.
  • said carbon source is selected from the list comprising glucose, N-acetylglucosamine (GIcNAc), 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.
  • GIcNAc N-acetylglucosamine
  • said at least one carbon source selected from the list consisting of glucose, fructose, sucrose, and glycerol.
  • the cultivation or incubation medium is a chemically defined medium.
  • the cultivation or incubation medium is a minimal salt medium comprising sulphate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt, and/or selenium.
  • the cultivation or incubation medium comprises one or more precursor(s) that is/are used for production of said negatively charged, preferably sialylated, oligosaccharide.
  • the cultivation or incubation medium contains at least one compound selected from the list consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid, CMP-sialic acid, CMP- Neu5Ac and CMP-KDO.
  • the method for production of a negatively charged, preferably sialylated, oligosaccharide as described herein 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 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 1
  • the precursor is selected from the list comprising sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose.
  • the acceptor is lactose.
  • the method for production of a negatively charged, preferably sialylated, oligosaccharide as described herein 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 in one pulse or in a discontinuous (pulsed) manner wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter), 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 pulse(s); iii) Adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and/or acceptor feed in one pulse or in a discontinuous (pulsed) manner wherein the total reactor or incubator volume
  • the precursor is selected from the list comprising sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose.
  • the acceptor is lactose.
  • the method for production of a negatively charged, preferably sialylated, oligosaccharide as described herein comprises at least one of the following steps: i) Use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter); ii) Use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter); iii) Adding to the cultivation or incubation medium in a reactor or incubator a precursor feed comprising at least 50, more
  • the precursor is selected from the list comprising sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose.
  • the acceptor is lactose.
  • the method for production of a negatively charged, preferably sialylated, oligosaccharide as described herein comprises at least one of the following steps: i) Use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter); ii) Use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter); iii) Adding to the cultivation or incubation medium in a reactor or incubator a precursor feed comprising at least 50, more
  • the precursor is selected from the list comprising sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose.
  • the acceptor is lactose.
  • the method for the production of a negatively charged, preferably sialylated, oligosaccharide as described herein comprises at least one of the following steps: i) Adding to the cultivation or incubation medium 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 volume wherein the total reactor 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 2-fold of the volume of the cultivation or incubation medium before the addition of said lactose feed; ii) Adding a lactose feed in a continuous manner to the cultivation or incubation medium over the course of 1 day, 2 days, 3 days, 4
  • the lactose feed is accomplished by adding lactose from the beginning of the cultivation or incubation in a concentration of at least ImM, preferably 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 1 mM, preferably 5 mM, 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 or incubation medium for 3 or more days, preferably up to 7 days; and/or provided, in the cultivation or incubation 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 or incubation medium in a continuous manner, so that the final volume of the cultivation or incubation 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 or incubation medium before the culturing.
  • a first phase of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the cultivation or incubation medium before the lactose is added to the cultivation or incubation medium in a second phase.
  • a carbon source preferably glucose or sucrose
  • the lactose is added already in the first phase of exponential growth together with the carbon-based substrate.
  • the cell is selected from the list consisting of prokaryotic cells and eukaryotic cells, preferably from the list consisting of yeast cells, bacterial cells, archaebacterial cells, algae cells, plant cells, fungal cells, animal cells and protozoan cells.
  • the cell is a bacterium, fungus, yeast, a plant cell, an animal cell or a protozoan cell.
  • the latter bacterium preferably belongs to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus or the phylum of Actinobacteria.
  • the latter bacterium belonging to the phylum Proteobacteria belongs preferably to the family Enterobacteriaceae, preferably to the species Escherichia coli.
  • the latter bacterium preferably relates to any strain belonging to the species Escherichia coli such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultivated Escherichia coli strains - designated as E. coli K12 strains - which are well- adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Well-known examples of the E.
  • coli K12 strains are K12 Wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200.
  • the present invention specifically relates to a mutated and/or transformed Escherichia coli cell or strain as indicated above wherein said E. coli strain is a K12 strain. More preferably, the Escherichia coli K12 strain is E. coli MG1655.
  • the latter bacterium belonging to the phylum Firmicutes belongs preferably to the Bacilli, preferably Lactobacilliales, with members such as Lactobacillus lactis, Leuconostoc mesenteroides, or Bacil lales with members such as from the genus Bacillus, such as Bacillus subtilis or, B. amyloliquefaciens.
  • Bacterium belonging to the phylum Actinobacteria preferably belonging to the family of the Corynebacteriaceae, with members Corynebacterium glutamicum or C. afermentans, or belonging to the family of the Streptomycetaceae with members Streptomyces griseus or S. fradiae.
  • the latter bacterium belonging to the phylum Proteobacteria preferably belonging to the family of the Vibrionaceae, with member Vibrio natriegens.
  • the latter yeast preferably belongs to the phylum of the Ascomycota or the phylum of the Basidiomycota or the phylum of the Deuteromycota or the phylum of the Zygomycetes.
  • the latter yeast belongs preferably to the genus Saccharomyces (with members like e.g. Saccharomyces cerevisiae, S. bayanus, S. boulardii), Zygosaccharomyces, Pichia (with members like e.g. Pichia pastoris, P. anomala, P.
  • the latter yeast is preferably selected from Pichia pastoris, Yarrowia lipolytica, 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, rose, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant. More preferably, the latter plant cell is selected from the Rosa family.
  • 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.
  • human and non-human mammalian cells are preferably selected 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
  • COS cell a Chinese hamster ovary (CHO) cell
  • murine myeloma cell like e.g. an 1X120, SP2/0 or YB2/0 cell, an NIH-3T3 cell
  • a non-mammary adult stem cell or derivatives thereof such as described in WO21067641, preferably mesenchymal stem cell or derivates thereof as described in WO21067641
  • a lactocyte derived from mammalian induced pluripotent stem cells preferably human induced pluripotent stem cells
  • a lactocyte as part of mammary-like gland organoids a post-parturition mammary epithelium cell
  • a polarized mammary cell preferably a polarized mammary cell selected from the list comprising live primary mammary epithelial cells, live mammary myoepithelial cells, live mammary progenitor
  • the latter insect cell is preferably derived from Spodoptera frugiperda like e.g., Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae, Trichoplusia ni like e.g., BTI-TN-5B1-4 cells or Drosophila melanogaster like e.g. Drosophila S2 cells.
  • the latter protozoan cell preferably is a Leishmania tarentolae cell.
  • the cell is an E. coll or yeast with a lactose permease positive phenotype, preferably wherein said lactose permease is coded by the gene LacY or LAC12, respectively.
  • the cell is a viable Gram-negative bacterium that comprises a reduced or abolished synthesis of poly-N-acetylglucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), glucosylglycerol, glycan and/or trehalose compared to a non-modified progenitor.
  • PNAG poly-N-acetylglucosamine
  • ECA Enterobacterial Common Antigen
  • OPG Osmoregulated Periplasmic Glucans
  • glucosylglycerol glycan and/or trehalose
  • said reduced or abolished synthesis of poly- N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose is provided by a mutation in any one or more glycosyltransferases involved in the synthesis of any one of said poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans (OPG), Glucosylglycerol, glycan, and/or trehalose, wherein said mutation provides for a deletion or lower expression of any one of said glycosyltransferases.
  • Said glycosyltransferases comprise glycosyltransferase genes encoding poly-N- acetyl-D-glucosamine synthase subunits, UDP-N-acetylglucosamine— undecaprenyl-phosphate N- acetylglucosaminephosphotransferase, Fuc4NAc (4-acetamido-4,6-dideoxy-D-galactose) transferase, UDP-N-acetyl-D-mannosaminuronic acid transferase, the glycosyltransferase genes encoding the cellulose synthase catalytic subunits, the cellulose biosynthesis protein, colanic acid biosynthesis glucuronosyltransferase, colanic acid biosynthesis galactosyltransferase, colanic acid biosynthesis fucosyltransferase, UDP-glucose:undecaprenyl-phosphate glucose-l-phosphat
  • the cell is mutated in any one or more of the glycosyltransferases comprising pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, weal, wcaJ, wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA and yaiP, wherein said mutation provides for a deletion or lower expression of any one of said glycosyltransferases.
  • said reduced or abolished synthesis of poly-N-acetyl-glucosamine is provided by over-expression of a carbon storage regulator encoding gene, deletion of a Na+/H+ antiporter regulator encoding gene and/or deletion of the sensor histidine kinase encoding gene.
  • the cell produces 30 g/L or more of the negatively charged, preferably sialylated, oligosaccharide in the whole broth and/or supernatant and/or wherein said negatively charged, preferably sialylated, oligosaccharide in the whole broth and/or supernatant has a purity of at least 80 % measured on the total amount of negatively charged, preferably sialylated, oligosaccharide and its precursor(s) produced by said cell in the whole broth and/or supernatant, respectively.
  • the cell produces 30 g/L, 31 g/L, 32 g/L, 33 g/L, 34 g/L, 35 g/L, 36 g/L, 37 g/L, 38 g/L, 39 g/L, 40 g/L, 41 g/L, 42 g/L, 43 g/L, 44 g/L, 45 g/L, 46 g/L, 47 g/L, 48 g/L, 49 g/L, 50 g/L, 51 g/L, 52 g/L, 53 g/L, 54 g/L, 55 g/L, 56 g/L, 57 g/L, 58 g/L, 59 g/L, 60 g/L, 61 g/L, 62 g/L, 63 g/L, 64 g/L, 65 g/L, 66 g/L, 67 g/L, 68 g/L, 69
  • the cell produces an oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide as described herein.
  • the cell is capable to produce and/or produces a mixture of two or more oligosaccharides wherein at least one of said two or more oligosaccharides is a negatively charged, preferably sialylated, oligosaccharide as described herein.
  • the cell is capable to produce and/or produces a mixture of one or more disaccharide(s) and one or more oligosaccharide(s) wherein at least one of said one or more oligosaccharide(s) is a negatively charged, preferably sialylated, oligosaccharide as described herein.
  • the cell is capable to produce and/or produces a mixture comprising a negatively charged, preferably sialylated, oligosaccharide and at least one non-charged (neutral) oligosaccharide.
  • the cell is capable to produce and/or produces a mixture comprising 1) a negatively charged, preferably sialylated, oligosaccharide and 2) a disaccharide and/or a non-charged (neutral) oligosaccharide.
  • the negatively charged, preferably sialylated, oligosaccharide produced by a cell of present invention is recovered from said cultivation or incubation medium and/or said cell.
  • said negatively charged, preferably sialylated, oligosaccharide is purified.
  • separating from said cultivation or incubation means harvesting, collecting, or retrieving said negatively charged, preferably sialylated, oligosaccharide from the cell and/or the medium of its growth.
  • the negatively charged, preferably sialylated, oligosaccharide can be separated in a conventional manner from the aqueous culture, cultivation or incubation medium, in which the cell was grown.
  • said negatively charged, preferably sialylated, oligosaccharide can be clarified in a conventional manner.
  • said negatively charged, preferably sialylated, oligosaccharide is clarified by centrifugation, flocculation, decantation and/or filtration.
  • a second step of separating said negatively charged, preferably sialylated, oligosaccharide preferably involves removing substantially all the eventually remaining proteins, peptides, amino acids, RNA, DNA, endotoxins and glycolipids that could interfere with the subsequent separation step, from said negatively charged, preferably sialylated, oligosaccharide, preferably after it has been clarified.
  • remaining proteins and related impurities can be removed from said negatively charged, preferably sialylated, oligosaccharide in a conventional manner.
  • remaining proteins, salts, by-products, colour, endotoxins and other related impurities are removed from said negatively charged, preferably sialylated, oligosaccharide by ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, electrophoresis (e.g. using slab-polyacrylamide or sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands including e.g.
  • the methods as described herein also provide for a further purification of the negatively charged, preferably sialylated, oligosaccharide of present invention.
  • a further purification of said negatively charged, preferably sialylated, oligosaccharide may be accomplished, for example, by use of (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment, 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 negatively charged, preferably 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 negatively charged, preferably sialylated, oligosaccharide.
  • the separation and purification of the negatively charged, preferably sialylated, oligosaccharide is made in a process, comprising the following steps in any order: a) contacting the cultivation 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 negatively charged, preferably sialylated, oligosaccharide and allowing at least a part of the proteins, salts, by-products, colour and other related impurities to pass, b) conducting a diafiltration process on the retentate from step a), using said membrane, with an aqueous solution of an inorganic electrolyte, followed by optional diafiltration with pure water to remove excess of the electrolyte, c) and collecting the retentate enriched in said negatively charged, preferably sialylated, oligosaccharide in the form of a salt from the cation of said electrolyte.
  • MWCO mole
  • the separation and purification of said negatively charged, preferably sialylated, oligosaccharide is made in a process, comprising the following steps in any order: subjecting the cultivation 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 negatively charged, preferably sialylated, oligosaccharide is made in a process, comprising the following steps in any order comprising the step of treating the cultivation or a clarified version thereof with a strong cation exchange resin in H+-form and a weak anion exchange resin in free base form.
  • the separation and purification of said negatively charged, preferably sialylated, oligosaccharide is made in the following way.
  • the cultivation comprising the produced negatively charged, preferably sialylated, oligosaccharide, biomass, medium components and contaminants, and wherein the purity of the produced negatively charged, preferably sialylated, oligosaccharide in the cultivation is ⁇ 80 %, is applied to the following purification steps: i) separation of biomass from the cultivation, 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 negatively charged, preferably sialylated, oligosaccharide at a purity of greater than or equal to 80 % is provided.
  • the purified solution is spray dried.
  • the separation and purification of the negatively charged, preferably sialylated, oligosaccharide is made in a process, comprising the following steps in any order: enzymatic treatment of the cultivation; removal of the biomass from the cultivation; 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 produced negatively charged, preferably sialylated, oligosaccharide which is spray-dried to powder, wherein the spray-dried powder contains ⁇ 15 % -wt. of water, preferably ⁇ 10 % -wt. of water, more preferably ⁇ 7 % -wt. of water, most preferably ⁇ 5 % -wt. of water.
  • the monomeric building blocks e.g. the monosaccharide or glycan unit composition
  • the anomeric configuration of side chains e.g. the anomeric configuration of side chains
  • the presence and location of substituent groups e.g. the degree of polymerization/molecular weight and the linkage pattern
  • 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
  • oligosaccharide methods such as e.g. acid-catalysed hydrolysis, HPLC (high performance liquid chromatography) or GLC (gas-liquid chromatography) (after conversion to alditol acetates) may be used.
  • said negatively charged, preferably sialylated, oligosaccharide 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).
  • GLC/MS gas-liquid chromatography coupled with mass spectrometry
  • said negatively charged, preferably sialylated, oligosaccharide 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 alpha-glucosidase, etc., and NMR may be used to analyse the products.
  • an enzyme that is specific for a particular type of linkage e.g., beta-galactosidase, or alpha-glucosidase, etc.
  • the present invention provides use of a cell as described herein for the production of a negatively charged, preferably sialylated, oligosaccharide as described herein.
  • the present invention provides use of a method as described herein for the production of a negatively charged, preferably sialylated, oligosaccharide as described herein.
  • the present invention provides for a purified negatively charged, preferably sialylated, oligosaccharide, or a purified oligosaccharide mixture comprising a negatively charged, preferably sialylated, oligosaccharide as described herein for use in medicine, preferably for use in prophylaxis or therapy of a gastrointestinal disorder.
  • the present invention provides use of a purified negatively charged, preferably sialylated, oligosaccharide obtained by a method as described herein in a food or feed preparation, in a dietary supplement, in a cosmetic ingredient or in a pharmaceutical ingredient.
  • said negatively charged, preferably sialylated, oligosaccharide is mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine.
  • Said purified negatively charged, preferably sialylated, oligosaccharide 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.
  • the present invention provides use of milk oligosaccharide as described herein as additive in food, preferably as additive in human food and/or pet food, more preferably as additive in human baby food.
  • the food is a human food, preferably infant food, human baby food and/or an infant formula or an infant supplement and the feed is a pet food, animal milk replacer, veterinary product, veterinary feed supplement, nutrition supplement, post weaning feed, or creep feed.
  • 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.
  • 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 said negatively charged, preferably sialylated, oligosaccharide being a prebiotic purified by a method disclosed in this specification, to promote growth of one or more beneficial microorganisms.
  • prebiotic ingredients for dietary supplements include other prebiotic molecules (such as HMOs) and plant polysaccharides (such as inulin, pectin, b-glucan and xylooligosaccharide).
  • a "probiotic" product typically contains live microorganisms that replace or add to gastrointestinal microflora, to the benefit of the recipient. Examples of such microorganisms include Lactobacillus species (for example, L. acidophilus and L. bulgaricus), Bifidobacterium species (for example, B. animalis, B. longum and B. infantis (e.g., Bi-26)), and Saccharomyces boulardii.
  • said negatively charged, preferably sialylated, oligosaccharide produced and/or purified by a method of this specification is orally administered in combination with such microorganism.
  • further ingredients for dietary supplements include oligosaccharides (such as 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose), disaccharides (such as lactose), monosaccharides (such as glucose, galactose, L-fucose, sialic acid, glucosamine and N-acetylglucosamine), thickeners (such as gum arabic), acidity regulators (such as trisodium citrate), water, skimmed milk, and flavourings.
  • oligosaccharides such as 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'
  • said negatively charged, preferably sialylated, oligosaccharide purified by a method as described herein 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 cupfeeding to an infant by mixing with water.
  • the composition of infant formula is typically designed to be roughly mimic human breast milk.
  • said negatively charged, preferably sialylated, oligosaccharide purified by a method as described herein is included in infant formula to provide nutritional benefits similar to those provided by the oligosaccharides in human breast milk.
  • said purified negatively charged, preferably sialylated, oligosaccharide is mixed with one or more ingredients of the infant formula.
  • infant formula ingredients include non-fat milk, carbohydrate sources (e.g., lactose), protein sources (e.g., whey protein concentrate and casein), fat sources (e.g., vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and/or sunflower oil; and fish oils), vitamins (such as vitamins A, 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 (HMOs).
  • carbohydrate sources e.g., lactose
  • protein sources e.g., whey protein concentrate and casein
  • fat sources e.g., vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and
  • the one or more infant formula ingredients comprise non-fat milk, a carbohydrate source, a protein source, a fat source, and/or a vitamin and mineral. In some embodiments, the one or more infant formula ingredients comprise lactose, whey protein concentrate and/or high oleic safflower oil. In some embodiments, the concentration of the negatively charged, preferably sialylated, oligosaccharide in the infant formula is approximately the same concentration as the concentration of the oligosaccharide generally present in human breast milk.
  • a negatively charged, preferably sialylated, oligosaccharide purified by a method as described herein is added to the infant formula with a concentration that is approximately the same concentration as the concentration of the compound generally present in human breast milk.
  • the methods and the cell of the invention preferably provide at least one of the following further surprising advantages when using a hydrolyzing UDP-N-acetyl-D- glucosamine-2-epimerase as described herein:
  • Higher production rate r (g negatively charged, preferably sialylated, oligosaccharide / L/h), Higher cell performance index CPI (g negatively charged, preferably sialylated, oligosaccharide / g X),
  • sucrose Ys g negatively charged, preferably sialylated, oligosaccharide / g sucrose
  • GIcNAc smaller titres of any one or more of GIcNAc, ManNAc, N-acetyllactosamine (LacNAc), lacto-N- biose (LNB), sialylated GIcNAc, sialylated ManNAc, sialylated LacNAc, sialylated LNB (g/L) in a mixture comprising the negatively, preferably sialylated, oligosaccharide,
  • GIcNAc smaller titres of any one or more of GIcNAc, ManNAc, N-acetyllactosamine (LacNAc), lacto-N- biose (LNB), sialylated GIcNAc, sialylated ManNAc, sialylated LacNAc, sialylated LNB (g/L) in a mixture of two or more oligosaccharides comprising the negatively, preferably sialylated, oligosaccharide, and/or
  • GIcNAc smaller titres of any one or more of GIcNAc, ManNAc, N-acetyllactosamine (LacNAc), lacto-N- biose (LNB), sialylated GIcNAc, sialylated ManNAc, sialylated LacNAc, sialylated LNB (g/L) in a mixture of one or more disaccharide(s) and one or more oligosaccharide(s) comprising the negatively, preferably sialylated, oligosaccharide, when compared to a method or a cell using an identical setup or enzymatic or genetic background but lacking the use of a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase as described herein.
  • a cell comprising a pathway for production of a negatively charged, preferably sialylated, oligosaccharide, characterized in that said cell is genetically engineered to possess or express, preferably to over-express, a hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase wherein said hydrolyzing UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising a conserved motif with SEQ ID NO 12 and having hydrolyzing UDP-N-acetyl-D- glucosamine-2-epimerase activity.
  • nucleic acid molecule is operably linked to control sequences recognized by the cell, said nucleic acid molecule further i) being integrated in the genome of said cell and/or ii) presented to said cell on a vector.
  • said pathway for production of said negatively charged, preferably sialylated, oligosaccharide is a sialylation pathway
  • said cell is genetically engineered to comprise said sialylation pathway, more preferably said cell comprises said sialylation pathway wherein said sialylation pathway has been genetically engineered.
  • said cell further comprises a pathway selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway, preferably said cell is genetically engineered to comprise at least one of said pathway(s), more preferably said cell comprises at least one of said pathway(s) wherein at least one of said pathway(s) has/have been genetically engineered.
  • said cell possesses, preferably expresses, more preferably overexpresses, one or more glycosyltransferase(s) selected from the list comprising, consisting of or consisting essentially of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N- acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases,
  • said cell comprises a pathway for the synthesis of a nucleotide-activated sugar selected from the list comprising, consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (U DP-Man NAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP- mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP-glucuronate, UDP-galacturonate, UDP-2- acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acety
  • said cell is capable to produce, preferably produces, N-acetylmannosamine (ManNAc), a sialic acid residue and/or N- acetylglucosamine (GIcNAc), wherein said sialic acid residue is selected from the list comprising 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), preferably said cell is genetically engineered for production of ManNAc, a sialic acid residue and/or GIcNAc.
  • ManNAc N-acetylmannosamine
  • GIcNAc N-acetylglucosamine
  • said negatively charged oligosaccharide is a sialylated oligosaccharide having at least one sialic acid residue selected from the list comprising, consisting of or consisting essentially 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;
  • said negatively charged oligosaccharide is an oligosaccharide selected from the list comprising, consisting of or consisting essentially of a negatively charged, preferably sialylated, milk oligosaccharide, preferably a negatively charged, more preferably sialylated, mammalian milk oligosaccharide (MMO), more preferably a negatively charged, more preferably sialylated, human milk oligosaccharide (HMO); O-antigen; the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an amino-sugar; Lewis-type antigen oligosaccharide; a negatively charged, preferably sialylated, animal oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans;
  • Cell according to any one of previous embodiments, wherein said cell possesses, expresses and/or overexpresses at least one gene selected from the list comprising, consisting of or consisting essentially of genes encoding a disulfide bond isomerase, a thiol oxidase or a chaperone.
  • said cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell
  • said bacterium belongs to a phylum selected from the list comprising, consisting of or consisting essentially of Proteobacteria, Firmicutes, Cyanobacteria, Deinococcus-Thermus and Actinobacteria; more preferably, said bacterium belongs to a family selected from the list comprising, consisting of or consisting essentially of Enterobacteriaceae, Bacillaceae, Lactobacillaceae, Corynebacteriaceae and Vibrionaceae; even more preferably, said bacterium is selected from the list comprising, consisting of or consisting essentially of an Escherichia coli strain, a Bacillus subtilis strain and a Vibrio natriegens strain; even more preferably said Escherichia coli strain is
  • said fungus belongs to a genus selected from the list comprising, consisting of or consisting essentially of Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus, preferably, said yeast belongs to a genus selected from the list comprising, consisting of or consisting essentially of Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces, Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces and Torulaspora; more preferably, said yeast is selected from the list comprising, consisting of or consisting essentially of Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipites, Candida
  • Cell according to any one of previous embodiments, wherein said cell is selected from the list consisting of prokaryotic cells and eukaryotic cells, preferably from the list consisting of yeast cells, bacterial cells, archaebacterial cells, algae cells, and fungal cells.
  • PNAG poly-N-acetylglucosamine
  • ECA Enterobacterial Common Antigen
  • OPG Osmoregulated Periplasmic Glucans
  • glucosylglycerol glycan and/or trehalose compared to a non-modified progenitor.
  • Cell according to any one of previous embodiments wherein said cell produces 30 g/L or more of said negatively charged, preferably sialylated, oligosaccharide in the whole broth and/or supernatant and/or wherein said negatively charged, preferably sialylated, oligosaccharide in the whole broth and/or supernatant has a purity of at least 80% measured on the total amount of said negatively charged, preferably sialylated, oligosaccharide and its precursor(s) produced by said cell in the whole broth and/or supernatant, respectively.
  • Cell according to any one of previous embodiments, wherein the cell is capable to produce and/or produces a mixture of one or more disaccharide(s) and one or more oligosaccharide(s) wherein at least one of said one or more oligosaccharide(s) is said negatively charged, preferably sialylated, oligosaccharide.
  • Cell according to any one of previous embodiments, wherein the cell is capable to produce and/or produces a mixture comprising 1) said negatively charged, preferably sialylated, oligosaccharide and 2) a disaccharide and/or a non-charged (neutral) oligosaccharide.
  • Method for the production of a negatively charged, preferably sialylated, oligosaccharide comprising: i. cultivating and/or incubating a cell of any one of previous embodiments, in cultivation and/or incubation medium under conditions permissive to produce said negatively charged, preferably sialylated, oligosaccharide, ii. preferably, followed by separation and/or purification of said negatively charged, preferably sialylated, oligosaccharide from said cultivation and/or incubation.
  • said negatively charged oligosaccharide is a sialylated oligosaccharide having at least one sialic acid residue selected from the list comprising, consisting of or consisting essentially 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), preferably, said negatively charged oligosaccharide is an oligosaccharide selected from the list comprising, consisting of or consisting essentially of a negatively charged, preferably sialylated, milk oligosaccharide, preferably a negatively charged, more preferably sialylated, mamm
  • cultivation or incubation medium comprises one or more precursor(s) that is/are used for production of said negatively charged, preferably sialylated, oligosaccharide.
  • said cultivation medium or incubation medium contains: at least one carbon source selected from the list comprising, consisting of or consisting essentially of a monosaccharide; disaccharide; oligosaccharide; polysaccharide; polyol; glycerol; a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract; glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose, maltooligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate, and/or at least one compound selected from the list consisting of lac
  • Method according to any one of embodiments 39 to 43 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
  • Method according to any one of embodiments 39 to 44 comprising at least one of the following steps: i) Use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter); ii) Use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of acceptor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter); iii) Adding to the cultivation or incubation medium in a reactor or incubator a precursor feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor
  • Method according to any one of embodiments 39 to 45 wherein said cell produces 30 g/L or more of said negatively charged, preferably sialylated, oligosaccharide in the whole broth and/or supernatant and/or wherein said negatively charged, preferably sialylated, oligosaccharide in the whole broth and/or supernatant has a purity of at least 80 % measured on the total amount of negatively charged, preferably sialylated, oligosaccharide and its precursor(s) produced by said cell in the whole broth and/or supernatant, respectively.
  • Method according to any one of embodiments 39 to 46 wherein the cell produces a mixture of two or more oligosaccharides wherein at least one of said two or more oligosaccharides is said negatively charged, preferably sialylated, oligosaccharide.
  • Method according to any one of embodiments 39 to 47 wherein the cell produces a mixture of one or more disaccharide(s) and one or more oligosaccharide(s) wherein at least one of said one or more oligosaccharide(s) is said negatively charged, preferably sialylated, oligosaccharide.
  • Method according to any one of embodiments 39 to 48 wherein the cell produces a mixture comprising said negatively charged, preferably sialylated, oligosaccharide and at least one noncharged (neutral) oligosaccharide.
  • Method according to any one of embodiments 39 to 49 wherein the cell produces a mixture comprising 1) said negatively charged, preferably sialylated, oligosaccharide and 2) a disaccharide and/or a non-charged (neutral) oligosaccharide.
  • Method according to any one of embodiments 39 to 50 wherein said negatively charged, preferably sialylated, oligosaccharide is recovered from said cultivation or incubation medium and/or said cell, more preferably said negatively charged, preferably sialylated, oligosaccharide is purified.
  • Method according to any one of embodiments 39 to 51 wherein said separation comprises at least one of the following steps: clarification, 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, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography, electrodialysis.
  • Method according to any one of embodiments 39 to 52, wherein said purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange, temperature adjustment, pH adjustment, pH adjustment with an alkaline or acidic solution, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, 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 or vacuum roller drying.
  • 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 cultivation experiments in 96-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.
  • precursor(s) and/or acceptor(s) for saccharide synthesis compounds like e.g., galactose, glucose, fructose, fucose, lactose, LacNAc, LNB, a co-factor and sialic acid could be added to the medium.
  • the minimal medium was set to a pH of 7 with IM KOH.
  • Vitamin solution consisted of 3.6 g/L FeCI 2.4 H 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 H3BO 4 , 0.4 g/L Na 2 EDTA.2H 2 O and 1.01 g/L thiamine. HCI.
  • the molybdate solution contained 0.967 g/L NaMoO 4 .2H 2 O.
  • the selenium solution contained 42 g/L Seo2.
  • 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 pL/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above.
  • 0.30 g/L sialic acid, 0.30 g/L GIcNAc, 20 g/L lactose, 20 g/L LacNAc, 20 g/L LNB, 20 g/L LN3, 20 g/L LNT and/or 20 g/L LNnT 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).
  • 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.
  • 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 H2SO4 and 20% NH 4 OH.
  • 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). The pET28b(+) vector was obtained from Millipore and adapted for Golden Gate cloning. Plasmids were maintained in the host E.
  • coli DH5alpha (F", phi80d/ocZZ!M15, t (lacZYA-argF) U169, deoR, recAl, endAl, hsdR17(rk", mk + ), phoA, supE44, lambda", thi-1, gyrA96, relAl) bought from Invitrogen.
  • Escherichia coli K12 MG1655 [X-, 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). All constitutive promoters, UTRs and terminator sequences originated from the libraries described by Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148), Dunn et al. (Nucleic Acids Res. 1980, 8, 2119- 2132), Edens et al. (Nucleic Acids Res.
  • the mutant strain was derived from E. coli K12 MG1655 comprising a knock-out of the E. coli wcaJ gene and genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E. coli W (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6).
  • a sucrose transporter like e.g. CscB from E. coli W (UniProt ID E0IXR1)
  • a fructose kinase like e.g. Frk originating from Zymomonas mobilis
  • a sucrose phosphorylase like e.g
  • GDP-fucose production can further be optimized in the mutant E. coli strain by genomic knock-outs of any one or more of the E. coli genes comprising glgC, agp, pfkA, pfkB, pgi, arcA, icIR, pgi and Ion as described in WO2016075243 and W02012007481.
  • GDP-fucose production can additionally be optimized comprising genomic knock-ins of constitutive transcriptional units for a mannose-6-phosphate isomerase like e.g. manA from E. coli (UniProt ID P00946), a phosphomannomutase like e.g. manB from E.
  • GDP-fucose production can also be obtained by genomic knock-outs of the E.
  • the mutant GDP-fucose production strain was additionally modified with an expression plasmid comprising a constitutive transcriptional unit for a fucosyltransferase, like e.g. the alpha-1, 2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435) to produce 2'-fucosyllactose (2'FL) or the alpha-1, 3-fucosyltransferase HpFucT from H.
  • a fucosyltransferase like e.g. the alpha-1, 2-fucosyltransferase HpFutC from H.
  • pylori UniProt ID Q9X435
  • the mutant 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.
  • 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 055:1-17.
  • 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 055:1-17.
  • 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. LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from Neisseria meningitidis.
  • LgtB Uniprot ID Q51116, sequence version 02, 01 Dec 2000
  • LN3, LNT and/or LNnT production can further be optimized in the mutant E. coli strains with genomic knock-outs of the E. coli genes comprising any one or more of galT, ushA, IdhA and agp.
  • the mutant 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 E. coli glmS (UniProt ID P17169, sequence version 04, 23 Jan 2007) or a mutant glmS*54 from E. coli with SEQ.
  • 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 mutant E. coli strains can also optionally be adapted with a genomic knock-in of a constitutive transcriptional unit for an UDP-glucose-4-epimerase like e.g. galE from E. coli (UniProt ID P09147), a phosphoglucosamine mutase like e.g. glmM from E.
  • the mutant LN3, LNT and/or LNnT producing E. coli strains can also 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 Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6).
  • the mutant strain was derived from E. coli K12 MG1655 comprising genomic knock-ins of constitutive transcriptional units containing a hydrolyzing UDP-N- acetylglucosamine 2-epimerase like e.g. NeuC from C.
  • sialic acid production can be obtained by 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- acetylglucosamine 2-epimerase like e.g. AGE from Bacteroides ovatus (UniProt ID A7LVG6) and an N- acetylneuraminate synthase like e.g. NeuB from Neisseria meningitidis (UniProt ID E0NCD4).
  • a glucosamine 6-phosphate N- acetyltransferase like e.g. GNA1 from Saccharomyces cerevisiae (UniProt ID P43577)
  • an N- acetylglucosamine 2-epimerase like e.g. AGE from
  • sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03, 23 Jan 2007), an N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase like e.g. glmU from E. coli (UniProt ID P0ACC7), a hydrolyzing UDP-N-acetylglucosamine 2-epimerase like e.g. NeuC from C.
  • a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03, 23 Jan 2007)
  • 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. from Mus musculus (strain C57BL/6J) (UniProt ID Q91WG8), an N- acylneuraminate-9-phosphate synthetase like e.g. from Pseudomonas sp. UW4 (UniProt ID K9NPH9) and an N-acylneuraminate-9-phosphatase like e.g.
  • a bifunctional UDP-GIcNAc 2-epimerase/N- acetylmannosamine kinase like e.g. from Mus musculus (strain C57BL/6J) (UniProt ID Q91WG8)
  • an N- acylneuraminate-9-phosphate synthetase like e.g. from Ps
  • Sialic acid production can further be optimized in the mutant s coli strain with genomic knock-outs of the E. coli genes comprising any one or more of nagA, nagB, nagC, nagD, nagE, nanA, nanE, nanK, manX, manY and manZ as described in WO18122225, and/or genomic knock-outs of the E.
  • coli genes comprising any one or more of nan T, 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. E. coli glmS (UniProt ID P17169, sequence version 04, 23 Jan 2007) or a mutant glmS*54 from E. coli with SEQ ID NO 09 and 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), preferably a phosphatase like any one of e.g. the E.
  • coli 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 YbiU or PsMupP from Pseudomonas putida, ScDOGl from S.
  • sialic acid production strains were further modified to express an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P. multocida (UniProt ID A0A849CI62) and to express a sialyltransferase like e.g. the alpha-2, 3-siayltransferase PmultST3 from P.
  • an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P. multocida (UniProt ID A0A849CI62) and to express a sialyltransferase like e.g. the alpha-2, 3-siayltransferase PmultST3 from P.
  • PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2, 3-sialyltransferase activity or the alpha-2, 6-sialyltransferase PdST6 from P. damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 11) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity.
  • Constitutive transcriptional units of the N-acylneuraminate cytidylyltransferase and the sialyltransferase can be delivered to the mutant strain either via genomic knock-in or via expression plasmids. If the mutant strains producing sialic acid and CMP-sialic acid were intended to make sialylated lactose structures, 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 mutant strains producing sialic acid, CMP-sialic acid and/or sialylated saccharides 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 W (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 W (UniProt ID E0IXR1)
  • a fructose kinase like e.g. Frk originating from Z. mobilis
  • a sucrose phosphorylase like e.g. BaSP from B. adolescent
  • coli strains adapted for LNT production as described herein can also be further modified with a hydrolyzing UDP-N-acetylglucosamine 2-epimerase like e.g. NeuC from C. jejuni (UniProt ID Q93MP8) or any one of SEQ ID NO 01, 02, 03, 04, 05, 06, 07 or 08 and an N-acetylneuraminate synthase like e.g. NeuB from N. meningitidis (UniProt ID E0NCD4) and an expression plasmid comprising containing constitutive expression cassettes for the N-acylneuraminate cytidylyltransferase (NeuA) from P.
  • a hydrolyzing UDP-N-acetylglucosamine 2-epimerase like e.g. NeuC from C. jejuni (UniProt ID Q93MP8) or any one of SEQ ID NO 01, 02, 03, 04, 05, 06, 07 or 08 and
  • multocida (UniProt ID A0A849CI62) and 1) the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide (SEQ ID NO 10) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2, 3-sialyltransferase activity or 2) the alpha-2, 6- sialyltransferase (PdST6) from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 11) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity to produce 1) LSTa (Neu5Ac-a2,3-Gal-pi,3-GlcNA
  • the mutant E. coli strains adapted for LNnT production as described herein can also be further modified with a hydrolyzing UDP-N-acetylglucosamine 2-epimerase like e.g. NeuC from C. jejuni (UniProt ID Q93MP8) or any one of SEQ ID NO 01, 02, 03, 04, 05, 06, 07 or 08 and an N-acetylneuraminate synthase like e.g. NeuB from N. meningitidis (UniProt ID E0NCD4) and an expression plasmid comprising containing constitutive expression cassettes for the N-acylneuraminate cytidylyltransferase (NeuA) from P.
  • a hydrolyzing UDP-N-acetylglucosamine 2-epimerase like e.g. NeuC from C. jejuni (UniProt ID Q93MP8) or any one of SEQ ID NO 01, 02, 03, 04, 05,
  • multocida (UniProt ID A0A849CI62) and 1) the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide (SEQ ID NO 10) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2, 3-sialyltransferase activity or 2) the alpha-2, 6-sialyltransferase (PdST6) from P.
  • P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide (SEQ ID NO 10) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2, 3-sialyltransferase activity or 2) the alpha-2, 6-sialyltransferase (PdST
  • damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 11) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity to produce 1) LSTd (Neu5Ac-a2,3-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc) or 2) LSTc (Neu5Ac-a2,6-Gal-pi,4- GlcNAc-pi,3-Gal-pi,4-Glc), respectively.
  • any one or more of the glycosyltransferases, the proteins involved in nucleotide-activated sugar synthesis and/or the membrane transporter proteins were N- and/or C- terminally fused to a solubility enhancer tag like e.g. a SUMO-tag, an MBP-tag, His, FLAG, Strep-11, Halotag, 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, Halotag, NusA, thioredoxin, GST and/or the F
  • the modified E. coli strains were modified with a genomic knock-in 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).
  • a chaperone protein like e.g., DnaK, DnaJ, GrpE or the GroEL/ES chaperonin system
  • the mutant E. coli strains are modified to create a glycominimized E. coli strain comprising genomic knock-out of any one or more of non-essential glycosyltransferase genes comprising pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, weal, wcaJ, wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA and yaiP.
  • wild-type E. coli K12 MG1655 cells or mutant E. coli K12 MG1655 cells modified for HMO synthesis were modified for protein production by mutation in or genomic knock-out of one or more cytoplasmic reductive pathway genes like e.g., glutathione reductase (gor) and/or thioredoxin reductase (trxB) rendering said genes less functional or eliminated compared to non-modified cells.
  • the mutant cells were further modified with genomic knock-ins of constitutive transcriptional units containing one or more copies of a disulfide bond isomerase, a thiol oxidase and/or a chaperone like e.g., dsbC from E.
  • mutant cells were further modified with genomic knock-outs of one or more gene(s) encoding a protease like e.g. Ion, OmpT and/or a nucleotide-sugar degrading enzyme like e.g. ushA.
  • a protease like e.g. Ion, OmpT
  • a nucleotide-sugar degrading enzyme like e.g. ushA.
  • a co-factor and sialic acid could be added to the medium.
  • 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.
  • a yeast expression plasmid like p2a_2p_Fuc (Chan 2013, Plasmid 70, 2-17) can be used for expression of foreign genes in S. cerevisiae.
  • This plasmid contains an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E. coli and the 2p yeast ori and the Ura3 selection marker for selection and maintenance in yeast.
  • This plasmid is further modified with constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), a GDP-mannose 4,6-dehydratase like e.g. gmd from E.
  • the yeast expression plasmid p2a_2p_Fuc2 can be used as an alternative expression plasmid of the p2a_2p_Fuc plasmid comprising next to the ampicillin resistance gene, the bacterial ori, the 2p yeast ori and the Ura3 selection marker constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), a fucose permease like e.g. fucP from E.
  • a yeast expression plasmid can be derived from the pRS420- plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the HIS3 selection marker and a constitutive transcriptional unit for an UDP-glucose-4-epimerase like e.g. galE from E. coli (UniProt ID P09147).
  • This plasmid can be further modified with constitutive transcriptional units for a lactose permease like e.g. LAC12 from K.
  • lactis (UniProt ID P07921) and a galactoside beta-1, 3-N- acetylglucosaminyltransferase activity like e.g. IgtA from N. meningitidis (UniProt ID Q9JXQ6) to produce LN3.
  • the mutant LN3 producing strains were further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 3- galactosyltransferase like e.g. WbgO (Uniprot ID D3QY14) from E. coli 055:1-17.
  • LN3 derived oligosaccharides like lacto-/V-neotetraose (LNnT, Gal-pi,4-GlcNAc-pi,3-Gal- pi,4-Glc)
  • the mutant LN3 producing strain were further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis.
  • 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. E. coli glmS (UniProt ID P17169 (sequence version 04 (23 Jan 2007)) or a mutant glmS*54 from E. coli with SEQ ID NO 09 and differing from the wild-type E.
  • E. coli glmS UniProt ID P17169 (sequence version 04 (23 Jan 2007)
  • mutant glmS*54 from E. coli with SEQ ID NO 09 and 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), a phosphatase like e.g. SurE from E. coli (UniProt ID P0A840), an N-acylglucosamine 2-epimerase like e.g. AGE from B. ovatus (UniProt ID A7LVG6), an N-acetylneuraminate synthase like e.g. NeuB from N.
  • meningitidis (UniProt ID E0NCD4) and an N-acylneuraminate cytidylyltransferase like e.g. NeuA from P. multocida (UniProt A0A849CI62).
  • N-acylneuraminate cytidylyltransferase like e.g. NeuA from P. multocida
  • a constitutive transcriptional unit for a glucosamine 6- phosphate N-acetyltransferase like e.g. GNA1 from S. cerevisiae (UniProt ID P43577) was added as well.
  • 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. E. coli glmS (UniProt ID P17169 (sequence version 04 (23 Jan 2007)) or a mutant glmS*54 from E. coli with SEQ ID NO 09, a hydrolyzing UDP-N-acetylglucosamine 2-epimerase like e.g. NeuC from C.
  • a constitutive transcriptional unit for a siderophore transporter like e.g. entS from E. coli 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 sialyltransferase like e.g., an alpha-2, 3- sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P.
  • UniProt ID Q9CLP3 or a PmultST3-like polypeptide (SEQ ID NO 10) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g. the alpha-2, 6-sialyltransferase (PdST6) from P.
  • damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 11) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity.
  • 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, Ec
  • Plasmids were maintained in the host E. coli DH5alpha (F", phi80d/ocZdeltaM15, delta(/ocZYA-orgF)U169, deoR, recAl, endAl, hsdR17(rk", mk + ), phoA, supE44, lambda", thi-1, gyrA96, relAl) bought from Invitrogen.
  • Two media are used to cultivate B. subtilis: i.e., a complex medium like 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 (Nl- hSO ⁇ 7.5 g/L KH2PO4, 17.5 g/L K2HPO4, 1.25 g/L Na-citrate, 0.25 g/L MgSO 4 .7H2O, 0.05 g/L tryptophan, from 10 up to 30 g/L glucose (or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose), 10 mL/L trace element mix and 10 mL/L Fe-citrate solution.
  • the medium was set to a pH of 7 with 1 M KOH.
  • precursor(s) and/or acceptor(s) for saccharide synthesis compounds like e.g., galactose, glucose, fructose, fucose, lactose, LacNAc, LNB, a co-factor and sialic acid could be added to the medium.
  • compounds like e.g., galactose, glucose, fructose, fucose, lactose, LacNAc, LNB, a co-factor and sialic acid could be added to the medium.
  • the trace element mix consisted of 0.735 g/L CaCl2.2H2O, 0.1 g/L MnCl2.2H2O, 0.033 g/L CuCl2.2H 2 O, 0.06 g/L COCI2.6H2O, 0.17 g/L ZnCI 2 , 0.0311 g/L H3BO4, 0.4 g/L Na 2 EDTA.2H2O and 0.06 g/L Na2MoO 4 .
  • the Fe-citrate solution contained 0.135 g/L FeCl3.6H2O, 1 g/L Na-citrate (Hoch 1973 PMC1212887).
  • Complex medium e.g., LB
  • LB Complex medium
  • B. subtilis strains were initially grown on LB agar to obtain single colonies. These plates were grown over night at 37°C. Starting from a single colony, a preculture was grown over night in 5 mL at 37°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 37°C with an orbital shaking of 200 rpm for 72h, or shorter of longer.
  • 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.
  • B. 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). Integrative vectors as described by Popp et al. (Sci.
  • 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.
  • Bacillus subtilis mutant strains are created to contain a gene coding for a lactose importer (such as e.g. the E. coli lacY with UniProt ID P02920).
  • a lactose importer such as e.g. the E. coli lacY with UniProt ID P02920.
  • the B. subtilis strain is modified with a genomic knock-in of constitutive transcriptional units comprising a lactose importer (such as e.g. the E. coli lacY with UniProt ID P02920) and a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. LgtA from N. meningitidis (UniProt ID Q9JXQ6).
  • the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g. WbgO from E.
  • the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000).
  • the N-acetylglucosamine beta-1, 3-galactosyltransferase and the N-acetylglucosamine beta-1, 4-galactosyltransferase can be delivered to the strain either via genomic knock-in or from an expression plasmid.
  • the B. subtilis strains are modified with a constitutive transcriptional unit for a fucosyltransferase.
  • a mutant B. subtilis strain is created by overexpressing a fructose- 6-P-aminotransferase like the native fructose-6-P-aminotransferase glmS (UniProt ID P0CI73) to enhance the intracellular glucosamine-6-phosphate pool.
  • a fructose- 6-P-aminotransferase like the native fructose-6-P-aminotransferase glmS (UniProt ID P0CI73) to enhance the intracellular glucosamine-6-phosphate pool.
  • the enzymatic activities of the genes nagA, nagB and gamA are disrupted by genetic knockouts and a glucosamine-6-P-aminotransferase like e.g. GNA1 from S.
  • subtilis strain is created by genetic knockouts of the genes nagA, nagB and gamA and by overexpressing a fructose-6-P-aminotransferase like the native fructose-6-P-aminotransferase glmS (UniProt ID P0CI73), a hydrolyzing UDP-N-acetylglucosamine 2-epimerase like e.g. NeuC from C. jejuni (UniProt ID Q93MP8) or any one of SEQ ID NO 01, 02, 03, 04, 05, 06, 07 or 08, and an N-acetylneuraminate synthase like e.g. NeuB from N.
  • a fructose-6-P-aminotransferase like the native fructose-6-P-aminotransferase glmS (UniProt ID P0CI73)
  • UDP-N-acetylglucosamine 2-epimerase
  • the sialic acid producing strain is further modified with a constitutive transcriptional unit comprising an N- acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P. multocida (UniProt ID A0A849CI62), and a sialyltransferase like e.g., an alpha-2, 3-sialyltransferase like e.g. the alpha-2, 3- sialyltransferase PmultST3 from P.
  • a constitutive transcriptional unit comprising an N- acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P. multocida (UniProt ID A0A849CI62), and a sialyltransferase like e.g., an alpha-2, 3-sialyltransferase like e.g. the alpha-2, 3- sialyl
  • UniProt ID Q9CLP3 or a PmultST3-like polypeptide (SEQ ID NO 10) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha- 2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g. the alpha-2, 6-sialyltransferase (PdST6) from P.
  • damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 11) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity.
  • the mutant strains can additionally be modified with genomic knock-ins of constitutive transcriptional units comprising the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6).
  • a constitutive transcriptional unit for a siderophore transporter like e.g. entS from E. coli (UniProt ID P24077, sequence version 02 (01 Nov 1997)) is added as well.
  • Two different media are used, namely complex medium like e.g., a rich tryptone-yeast extract (TY) medium, and a minimal medium for shake flask (MMsf).
  • the minimal medium uses a lOOOx stock trace element mix. Trace element mix consisted of 10 g/L CaCL, 10 g/L FeSO 4 .7H2O, 10 g/L MnSO 4 .H 2 O, 1 g/L ZnSO 4 .7H2O, 0.2 g/L CuSO 4 , 0.02 g/L NiCh.eHzO, 0.2 g/L biotin (pH 7) and 0.03 g/L protocatechuic acid.
  • the minimal medium for the shake flasks (MMsf) experiments contained 20 g/L (NH 4 )2SO 4 , 5 g/L urea, 1 g/L KH 2 PO 4 , 1 g/L K 2 HPO 4 , 0.25 g/L MgSO 4 .7H 2 O, 42 g/L MOPS, from 10 up to 30 g/L glucose or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose when specified in the examples and 1 ml/L trace element mix.
  • TY medium consisted of 1.6% tryptone (Difco, Erembodegem, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium).
  • TY agar (TYA) plates consisted of the TY media, with 12 g/L agar (Difco, Erembodegem, Belgium) added.
  • Complex medium e.g., TY, 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.
  • a preculture was started from a cryovial or a single colony from a TY plate, in 6 mL TY and was incubated overnight at 37 °C on an orbital shaker at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL MMsf medium. These shake flasks were incubated at 37°C with an orbital shaking of 200 rpm for 72h, or shorter of longer. At the end of the cultivation experiment samples were taken to measure the supernatant concentration (extracellular sugar concentrations, after 5 min.
  • the cell performance index or CPI was determined by dividing the oligosaccharide concentrations, e.g., 3'SL concentrations, measured in the whole broth by the biomass, in relative percentages compared to the reference strain.
  • the biomass is empirically determined to be approximately l/3rd of the optical density measured at 600 nm.
  • Corynebacterium glutamicum was used as available at the American Type Culture Collection (ATCC 13032). Integrative plasmid vectors were made using 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. (Journal of Microbiological Methods 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.
  • C. glutamicum mutant strains are created to contain a gene coding for a lactose importer (such as e.g. the E. coli lacY with UniProt ID P02920).
  • a lactose importer such as e.g. the E. coli lacY with UniProt ID P02920.
  • the C. glutamicum strain is modified with a genomic knock-in of constitutive expression units comprising a lactose importer (such as e.g. the E. coli lacY with UniProt ID P02920) and a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. LgtA from N. meningitidis (UniProt ID Q9JXQ6).
  • the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g. WbgO from E.
  • the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000).
  • the N-acetylglucosamine beta-1, 3-galactosyltransferase and the N-acetylglucosamine beta-1, 4-galactosyltransferase can be delivered to the strain either via genomic knock-in or from an expression plasmid.
  • the mutant C. glutamicum strains are further modified with a constitutive transcriptional unit for a fucosyltransferase.
  • a mutant C. glutamicum strain is created by overexpressing a fructose-6-P-aminotransferase like the native fructose-6-P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 03, 23 Jan 2007) to enhance the intracellular glucosamine-6-phosphate pool.
  • a fructose-6-P-aminotransferase like the native fructose-6-P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 03, 23 Jan 2007) to enhance the intracellular glucosamine-6-phosphate pool.
  • the enzymatic activities of the genes nagA, nagB and gamA are disrupted by genetic knockouts and a glucosamine-6-P-aminotransferase like e.g. GNA1 from S.
  • glutamicum strain is created by genetic knockouts of the genes nagA, nagB and gamA and by overexpressing a fructose-6-P-aminotransferase like the native fructose-6- P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 03, 23 Jan 2007), a hydrolyzing UDP-N- acetylglucosamine 2-epimerase like e.g. NeuC from C. jejuni (UniProt ID Q93MP8) or any one of SEQ ID NO 01, 02, 03, 04, 05, 06, 07 or 08, and an N-acetylneuraminate synthase like e.g. NeuB from N.
  • the sialic acid producing strain is further modified with a constitutive transcriptional unit comprising an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P. multocida (UniProt ID A0A849CI62), and a sialyltransferase like e.g., an alpha-2, 3-sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P.
  • a constitutive transcriptional unit comprising an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P. multocida (UniProt ID A0A849CI62), and a sialyltransferase like e.g., an alpha-2, 3-sialyltransferase like e.g. the alpha-2, 3-sialyl
  • UniProt ID Q9CLP3 or a PmultST3-like polypeptide (SEQ ID NO 10) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g. the alpha-2, 6-sialyltransferase (PdST6) from P.
  • the mutant strains can additionally be modified with genomic knock-ins of constitutive transcriptional units comprising the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6).
  • sucrose transporter CscB
  • E0IXR1 E. coli W
  • Frk fructose kinase
  • BaSP sucrose phosphorylase
  • Chlamydomonas reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7).
  • TAP medium uses a lOOOx stock Hutner's trace element mix.
  • Hutner's trace element mix consisted of 50 g/L Na2EDTA.H2O (Titriplex III), 22 g/L ZnSO4.7H2O, 11.4 g/L H3BO3, 5 g/L MnCI2.4H2O, 5 g/L FeSO4.7H2O, 1.6 g/L CoCI2.6H2O, 1.6 g/L CuSO4.5H2O and 1.1 g/L (NH4)6MoO3.
  • the TAP medium contained 2.42 g/L Tris (tris(hydroxymethyl)aminomethane), 25 mg/L salt stock solution, 0.108 g/L K2HPO4, 0.054 g/L KH2PO4 and 1.0 mL/L glacial acetic acid.
  • the salt stock solution consisted of 15 g/L NH4CL, 4 g/L MgSO4.7H2O and 2 g/L CaCI2.2H2O.
  • precursor(s) and/or acceptor(s) for saccharide synthesis compounds like e.g., galactose, glucose, fructose, fucose, lactose, LacNAc, LNB, a co-factor and sialic acid 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).
  • Cells of C. reinhardtii were cultured in selective TAP-agar plates at 23 +/- 0.5°C under 14/10 h I ight/dark cycles with a light intensity of 8000 Lx. Cells were analysed after 5 to 7 days of cultivation. For high-density cultures, 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 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 the Chlamydomonas Resource Center (https://www.chlamycollection.org) (University of Minnesota, U.S.A) were used.
  • Expression plasmids originated from pSH03, as available from the 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 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) and as described like e.g., in WO22034067 or in WO22034069.
  • the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067.
  • the mutant strain is modified with a transcriptional unit for an additional hydrolyzingUDP-N-acetylglucosamine-2-epimerase like e.g. any one of SEQ ID NO 01, 02, 03, 04, 05, 06, 07 or 08.
  • a transcriptional unit for an additional hydrolyzingUDP-N-acetylglucosamine-2-epimerase like e.g. any one of SEQ ID NO 01, 02, 03, 04, 05, 06, 07 or 08.
  • sialylated oligosaccharides C.
  • reinhardtii cells are modified with a CMP-sialic acid transporter like e.g., CST from Mus musculus (UniProt ID Q61420), and a Golgi-localised sialyltransferase selected from species like e.g., Homo sapiens, Mus musculus, Rattus norvegicus.
  • a CMP-sialic acid transporter like e.g., CST from Mus musculus (UniProt ID Q61420)
  • a Golgi-localised sialyltransferase selected from species like e.g., Homo sapiens, Mus musculus, Rattus norvegicus.
  • the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067.
  • fucosylation C.
  • reinhardtii cells can be modified with an expression plasmid comprising a constitutive transcriptional unit for an alpha-1, 2-fucosyltransferase and/or an alpha-1, 3-fucosyltransferase.
  • the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067.
  • the mutant strain was derived from C.
  • the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g., WbgO (Uniprot ID D3QY14) from E.
  • LgtB Uniprot ID Q51116, sequence version 02, 01 Dec 2000
  • a C. reinhardtii strain is modified for production of GDP-fucose, UDP-galactose, LN3, LNT and/or LNnT as described herein and for expression of one or more compatible fucosyltransferase(s).
  • a C. reinhardtii strain is modified for production of CMP-sialic acid, UDP-galactose, LN3 and LNT as described herein and for expression of one or more compatible sialyltransferase(s) like e.g., an alpha-2, 3-sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P.
  • UniProt ID Q9CLP3 or a PmultST3-like polypeptide (SEQ ID NO 10) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g. the alpha-2, 6-sialyltransferase (PdST6) from P.
  • a C. reinhardtii strain is modified for production of CMP-sialic acid, UDP-galactose, LN3 and LNnT as described herein and for expression of one or more compatible sialyltransferase(s).
  • 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.
  • the culture medium is subsequently replaced with 10% FBS (foetal bovine serum)-supplemented media after the first passage.
  • FBS foetal bovine serum
  • 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 WO21067641, which is incorporated herein by reference in its entirety for all purposes.
  • remodelling systems such as CRISPR/Cas9
  • select genes of interest such as casein, a- lactalbumin to be constitutively on, to allow for the expression of their respective proteins
  • down-regulate and/or knock-out select endogenous genes as described e.g., in WO21067641, which is incorporated herein by reference in its entirety for all purposes.
  • isolated mesenchymal cells re-programmed into mammary-like cells are modified via CRISPR-CAS as described e.g., in WO22034067, W0220
  • 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 DM EM/ Fl 2, 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.
  • 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.
  • the maximal growth rate (pMax) was calculated based on the observed optical densities at 600 nm using the R package grofit.
  • Standards such as but not limited to sucrose, lactose, 3'SL, 6'SL, lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neo-tetraose (LNnT), LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, 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 ELSD detector had a drift tube temperature of 50 °C and the N2 gas pressure was 50 psi, the gain
  • Sialylated oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Refractive Index (Rl) detection.
  • Rl Refractive Index
  • 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.
  • eluent A was ultrapure water with 0.1 % formic acid and wherein eluent B was acetonitrile with 0.1 % formic acid.
  • the oligosaccharides were separated in 55 min using the following gradient: an initial increase from 2 to 12 % of eluent B over 21 min, a second increase from 12 to 40 % of eluent B over 11 min and a third increase from 40 to 100 % of eluent B over 5 min.
  • As a washing step 100 % of eluent B was used for 5 min.
  • the initial condition of 2 % of eluent B was restored in 1 min and maintained for 12 min.
  • 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
  • Lactobionic acid was analysed on a Dionex HPAEC system with pulsed amperometric detection (PAD).
  • a volume of 5 pL of sample was injected on a Dionex CarboPac PA01 column 2 x 250 mm with a Dionex CarboPac PA01 guard column 4 x 50 mm.
  • the column temperature was set to 30 °C.
  • a gradient was used wherein eluent A was ultrapure water, eluent B was 200 mM Sodium hydroxide and eluent C was 500 mM Sodium acetate. Total gradient time was 41 min and started with 50% B and 5% C in the first 7 minutes.
  • An E. coli K-12 MG1655 strain was modified for production of sialic acid comprising genomic knock-ins of constitutive transcriptional units containing nucleotide sequences encoding the mutant glmS*54 from E. coli (SEQ ID NO 09) (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.
  • the strains were further modified with genomic knock-ins of two constitutive transcriptional units containing nucleotide sequences encoding the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and two constitutive transcriptional units containing nucleotide sequences encoding the PdST6-like polypeptide (SEQ ID NO 11) consisting of amino acid residues 108 to 497 of UniProt ID 066375 and having beta-galactoside alpha-2, 6-sialyltransferase activity.
  • SEQ ID NO 11 nucleotide sequences encoding the PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 and having beta-galactoside alpha-2, 6-sialyltransferase activity.
  • the novel strains were evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 1 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. For each strain with a particular hydrolyzing UDP-N-acetylglucosamine-2-epimerase tested, the measured sialic acid and 6'SL concentrations were averaged over all biological replicates and then normalized to the averaged sialic acid and 6'SL concentration measured of the strain expressing the reference NeuC polypeptide from C.
  • Example 3 Evaluation of production of sialic acid and 6'SL with modified E. coli hosts when evaluated in a fed-batch fermentation process with sucrose and lactose
  • Example 2 The mutant E. coli strains as described in Example 2 were selected for further evaluation in fed-batch fermentation processes.
  • Fed-batch fermentations at bioreactor scale were performed as described in Example 1.
  • 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 the production of sialic acid and 6'SL was measured using UPLC as described in Example 1.
  • the mutant E. coli strains as described in Example 2 are further modified with a genomic knock-in of a constitutive transcriptional unit containing nucleotide sequences encoding the galactoside beta-1, 3-N- acetylglucosaminyltransferase LgtA from N. meningitidis (UniProt ID Q9JXQ6) and the N- acetylglucosamine beta-1, 4-galactosyltransferase LgtB from N.
  • meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000) to allow production of lacto-N-neotetraose (LNnT, Gal-pi,4-GlcNAc- pi,3-Gal-pi,4-Glc).
  • LSTc lacto-N-neotetraose
  • the novel strains are evaluated in a growth experiment for production of sialic acid and LSTc (Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc) according to the culture conditions provided in Example 1 in which the strains are cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. The strains are grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested, and production of sialic acid and LSTc is analysed on UPLC.
  • Example 5 Evaluation of production of sialic acid and 6'SL with a modified S. cerevisiae host
  • a S. cerevisiae strain is modified for production of CMP-sialic acid and for expression of a sialyltransferase with a yeast expression plasmid containing the TRP1 selection marker and constitutive transcriptional units containing nucleotide sequences encoding the L-glutamine— D-fructose-6-phosphate aminotransferase glmS from E.
  • coli (UniProt ID P17169, sequence version 04, 23 Jan 2007), a hydrolyzing UDP-N-acetylglucosamine 2-epimerase selected from the list comprising SEQ ID NO 01, 02, 03, 04, 05, 06, 07 and 08, the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4), the N- acylneuraminate cytidylyltransferase NeuA from P. multocida (UniProt A0A849CI62) and the alpha-2, 6- sialyltransferase PdST6 from P. damselae (UniProt ID 066375).
  • UDP-N-acetylglucosamine 2-epimerase selected from the list comprising SEQ ID NO 01, 02, 03, 04, 05, 06, 07 and 08
  • the novel strains are evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 1, in which the appropriate selective medium comprises glucose as carbon source and lactose as precursors.
  • the strains are grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested, and production of sialic acid and 6'SL is analysed on UPLC.
  • a B. subtilis strain is modified with genetic knockouts of the genes nagA, nagB and gamA and with genetic knock-ins of constitutive transcriptional units containing nucleotide sequences encoding the native fructose-6-P-aminotransferase glmS (UniProt ID P0CI73), a hydrolyzing UDP-N- acetylglucosamine 2-epimerase selected from the list comprising SEQ ID NO 01, 02, 03, 04, 05, 06, 07 and 08, and the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4).
  • the strains are transformed with an expression plasmid containing a constitutive transcriptional unit containing nucleotide sequences encoding the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 3-sialyltransferasePmultST3 from Pasteurella multocida (UniProt ID Q9CLP3).
  • the novel strains are evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 1, in which the appropriate selective medium comprises glucose as carbon source and lactose as precursors.
  • the strain is grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested, and production of sialic acid and 3'SL is analysed on UPLC.
  • Example 7 Evaluation of production of sialic acid and 3'SL with a modified C. glutamicum host
  • a mutant C. glutamicum strain is created by genetic knockouts of the genes nagA, nagB and gamA and by overexpressing the native fructose-6-P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 03, 23 Jan 2007), a hydrolyzing UDP-N-acetylglucosamine 2-epimerase selected from the list comprising SEQ ID NO 01, 02, 03, 04, 05, 06, 07 and 08, and the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4).
  • the strains are transformed with an expression plasmid containing a constitutive transcriptional unit containing nucleotide sequences encoding the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3).
  • the novel strains are evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 1, using MMsf medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of sialic acid and 3'SL.
  • Example 8 Evaluation of production of 6'SL and LSTc with a modified E. coli host
  • An E. coli K-12 MG1655 strain was modified for production of LNT and LNnT comprising genomic knock- ins of constitutive transcriptional units containing nucleotide sequences encoding the galactoside beta- 1,3-N-acetylglucosaminyltransferase IgtA from Neisseria meningitidis (UniProt ID Q9JXQ6), the beta-1, 3- galactosyltransferase furA from Pseudogulbenkiania ferrooxidans (UniProt ID B9YZ84), the beta-1, 3- galactosyltransferase wbdO from Salmonella enterica (UniProt ID Q5UHA8) and the beta-1, 4- galactosyltransferase galT from Helicobacter pylori (UniProt ID Q9RHG8).
  • the strain was further modified with genomic knock-ins of constitutive transcriptional units containing nucleotide sequences encoding a hydrolyzing UDP-N-acetylglucosamine-2-epimerase selected from the list comprising SEQ ID NO 01, 02, 03, 04, 06, 08 and the reference polypeptide NeuC from Campylobacter jejuni (UniProt ID Q93MP8), and the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4).
  • a hydrolyzing UDP-N-acetylglucosamine-2-epimerase selected from the list comprising SEQ ID NO 01, 02, 03, 04, 06, 08 and the reference polypeptide NeuC from Campylobacter jejuni (UniProt ID Q93MP8), and the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4).
  • the strains were further modified with a plasmid containing a constitutive transcriptional unit containing nucleotide sequences encoding the N-acylneuraminate cytidylyltransferase neuA from C. jejuni (UniProt ID Q93MP7) and a constitutive transcriptional unit containing nucleotide sequences encoding the PdST6-like polypeptide (SEQ ID NO 11) consisting of amino acid residues 108 to 497 of UniProt ID 066375 and having beta-galactoside alpha-2, 6- sialyltransferase activity.
  • SEQ ID NO 11 PdST6-like polypeptide
  • the novel strains were evaluated in a growth experiment for production of 6'SL, LSTc, 6S(2)- LNnT (Neu5Ac-a2,6-(Gal-bl,4-GlcNAc-bl,3)-Gal-bl,4-Glc), 6S(2)-LNT (Neu5Ac-a2,6-(Gal-bl,3-GlcNAc- bl,3)-Gal-bl,4-Glc) and/or 6S(4)-LNT (Neu5Ac-a2,6-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc) according to the culture conditions provided in Example 1 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. For each strain with a particular hydrolyzing UDP-N-acetylglucosamine-2-epimerase tested, the measured 6'SL and LSTc concentrations were averaged over all biological replicates and then normalized to the averaged 6'SL and LSTc concentration measured of the strain expressing the reference NeuC polypeptide from C. jejuni (UniProt ID Q93MP8).
  • the data further showed small production of 6S(2)-LNT and 6S(4)-LNT in all samples, further comprising zero to small production of 6S(2)-LNnT (Results not shown).
  • the data also showed that said production of 6S(2)-LNnT, 6S(2)-LNT and/or 6S(4)-LNT could be tuned based on the hydrolyzing UDP-N-acetylglucosamine-2- epimerase selected resulting in fine-tuning of the purity of 6'SL in the mixture comprising 6'SL and LSTc.
  • Example 9 Evaluation of production of 3'SL, LSTa and LSTd with a modified E. coli host
  • An E. coli K-12 MG1655 strain was modified for production of LNT and LNnT comprising genomic knock- ins of constitutive transcriptional units containing nucleotide sequences encoding the galactoside beta- 1,3-N-acetylglucosaminyltransferase IgtA from Neisseria meningitidis (UniProt ID Q9JXQ6), the beta-1, 3- galactosyltransferase furA from Pseudogulbenkiania ferrooxidans (UniProt ID B9YZ84), the beta-1, 3- galactosyltransferase wbdO from Salmonella enterica (UniProt ID Q5UHA8) and the beta-1, 4- galactosyltransferase galT from Helicobacter pylori (UniProt ID Q9RHG8).
  • the strain was further modified with genomic knock-ins of constitutive transcriptional units containing nucleotide sequences encoding a hydrolyzing UDP-N-acetylglucosamine-2-epimerase selected from the list comprising SEQ ID NO 01, 02, 03, 04, 06, 08 and the reference polypeptide NeuC from Campylobacter jejuni (UniProt ID Q93MP8), and the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4).
  • a hydrolyzing UDP-N-acetylglucosamine-2-epimerase selected from the list comprising SEQ ID NO 01, 02, 03, 04, 06, 08 and the reference polypeptide NeuC from Campylobacter jejuni (UniProt ID Q93MP8), and the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4).
  • the strains were further modified with a plasmid containing a constitutive transcriptional unit containing nucleotide sequences encoding neuA from C. jejuni (UniProt ID Q93MP7) and a constitutive transcriptional unit containing nucleotide sequences encoding the alpha-2, 3-sialyltransferase PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3).
  • the novel strains were modified for growth on sucrose as in Example 1.
  • the novel strains were evaluated in a growth experiment for production of 3'SL, LSTa and LSTd according to the culture conditions provided in Example 1 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.
  • Example 10 Evaluation of production of a mixture of sialylated and non-charged (non-sialylated) oligosaccharides with a modified E. coli host
  • An E. coli K-12 MG1655 strain was modified for production of LNT and LNnT comprising genomic knock- ins of constitutive transcriptional units containing nucleotide sequences encoding the galactoside beta- 1,3-N-acetylglucosaminyltransferase IgtA from Neisseria meningitidis (UniProt ID Q9JXQ6), the beta-1, 3- galactosyltransferase furA from Pseudogulbenkiania ferrooxidans (UniProt ID B9YZ84), the beta-1, 3- galactosyltransferase wbdO from Salmonella enterica (UniProt ID Q5UHA8) and the beta-1, 4- galactosyltransferase galT from Helicobacter pylori (UniProt ID Q9RHG8).
  • the strain was further modified with genomic knock-ins of constitutive transcriptional units containing nucleotide sequences encoding a hydrolyzing UDP-N-acetylglucosamine-2-epimerase selected from the list comprising SEQ. ID NO 04 and 06 and the reference polypeptide NeuC from Campylobacter jejuni (UniProt ID Q93MP8), and the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4).
  • the strains were further modified with a genomic knock-in containing a constitutive transcriptional unit containing nucleotide sequences encoding neuA from P. multocida (UniProt ID A0A849CI62) and a constitutive transcriptional unit containing nucleotide sequences encoding the alpha-2, 3-sialyltransferase PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3).
  • the strains were modified with a genomic knock-in of constitutive transcriptional units for the phosphomannomutase manB from E.
  • MIT 01-6242 (UniProt ID A0A1B1U4V1) and encoding an alpha-1, 3-fucosyltransferase (3FT) from Porphyromonas catoniae (UniProt ID Z4WWI2).
  • the novel strains were modified for growth on sucrose as in Example 1.
  • the novel strains were evaluated in a growth experiment for production of 2'FL, 3-FL, DiFL, 3'SL, LNT, LNnT, LSTa, LSTd, fucosylated LNT and/or fucosylated LNnT according to the culture conditions provided in Example 1 in which the strains were cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose.
  • each strain was grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed on UPLC. The experiment demonstrated that each strain with a particular transcriptional unit of a hydrolyzing UDP-N-acetylglucosamine-2-epimerase tested produced an oligosaccharide mixture comprising of 5 to 8 % sialylated oligosaccharides and 95 to 92 % non-charged (non-sialylated) oligosaccharides.
  • the fraction of sialylated oligosaccharides comprised 3'SL, LSTa (Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc) and LSTd (Neu5Ac-a2,3-Gal- pi,4-GlcNAc-pi,3-Gal-pi,4-Glc).
  • the fraction of non-charged (non-sialylated) oligosaccharide comprised LNFP-I (Fuc-al,2-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc), LNnFP-l (Fuc-al,2-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4- Glc), LNDFH-I (Fuc-al,4-(Fuc-al,2-Gal-bl,3)-GlcNAc-bl,3-Gal-bl,4-Glc), Fuc-al,2-Gal-bl,4-(Fuc-al,3)- GlcNAc-bl,3-Gal-bl,4-Glc, Fuc-al,2-Gal-bl,4-(Fuc-al,3)- GlcNAc-bl,3-Gal-b
  • coli strains M and N expressing an alpha-1, 2- fucosyltransferase, an alpha-1, 3-fucosyltransferase, an alpha-2, 3-sialyltransferase and a hydrolyzing UDP-N-acetylglucosamine-2-epimerase and producing LNT, LNnT, sialic acid, and GDP-fucose when evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the cultivation medium contained 30 g/L sucrose and 20 g/L lactose, and compared to a reference strain REF2 with the same genetic background but expressing NeuC from C. jejuni (UniProt ID Q93MP8).

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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. L'invention propose une cellule pour la production d'un oligosaccharide chargé négativement, de préférence sialylaté, ladite cellule étant génétiquement modifiée pour posséder ou exprimer, de préférence pour surexprimer une UDP-N-acétyl-D-glucosamine-2-épimérase d'hydrolyse. L'invention propose en outre l'utilisation de ladite cellule dans une culture ou une incubation. L'invention décrit également des procédés de production d'un oligosaccharide chargé négativement, de préférence sialylaté, à l'aide de ladite cellule ainsi que la purification dudit oligosaccharide chargé négativement, de préférence sialylaté.
PCT/EP2024/061529 2023-04-26 2024-04-26 Production d'un oligosaccharide chargé négativement par une cellule Pending WO2024223815A1 (fr)

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CN202480027924.2A CN121079401A (zh) 2023-04-26 2024-04-26 通过细胞生产带负电荷的寡糖
PCT/EP2025/061445 WO2025224348A1 (fr) 2024-04-26 2025-04-25 Production d'un mélange d'oligosaccharides de lait

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