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WO2025088103A1 - Production d'un composé sialylé - Google Patents

Production d'un composé sialylé Download PDF

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Publication number
WO2025088103A1
WO2025088103A1 PCT/EP2024/080201 EP2024080201W WO2025088103A1 WO 2025088103 A1 WO2025088103 A1 WO 2025088103A1 EP 2024080201 W EP2024080201 W EP 2024080201W WO 2025088103 A1 WO2025088103 A1 WO 2025088103A1
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Prior art keywords
gal
neu5ac
hexnac
galnac
cell
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Joeri Beauprez
Romane BREYSSE
Annelies VERCAUTEREN
Tom Verhaeghe
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Inbiose NV
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Inbiose NV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/99—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • C12Y204/99001—Beta-galactoside alpha-2,6-sialyltransferase (2.4.99.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae

Definitions

  • the present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation.
  • the present invention relates to methods for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as well as the purification of said sialylated compound.
  • the present invention also provides a cell for production of said sialylated compound and the use of said cell in a cultivation or incubation.
  • the present invention also relates to methods and a cell for the production of sialyltransferases having alpha-2, 6-sialyltransferase activity on the N-acetylhexosamine (HexNAc) residue of Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R, wherein said sialyltransferases are involved in the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • HexNAc N-acetylhexosamine
  • Saccharides like e.g., disaccharides and 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. Saccharides 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
  • Saccharides occur in free form or in a bound form, like e.g., as part of glycoproteins, glycolipids or glycosphingolipids. Economical production of these different forms of saccharides is of utmost importance to fully benefit of their biological advantages.
  • An important group of free saccharides comprises mammalian milk oligosaccharides (MMOs) and human milk oligosaccharides (HMOs) found in mammalian and human milk, respectively. More than 150 structurally distinct human milk oligosaccharides (HMOs) have been identified to date. Although HMOs represent only a minor amount of total human milk nutrients, their beneficial effects on the development of breast-fed infants became evident over the past decades.
  • sialylated HMOs were observed to support several beneficial effects as described in the art.
  • sialylated oligosaccharides in human milk 3'sialyllactose, 6'sialyllactose, sialyllacto-N- tetraose a (LSTa), sialyl lacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc) and disialyllacto-N- tetraose (DSLNT) are the most prevalent members.
  • said sialylated compounds are found to be a complex structure and their syntheses have been proven challenging: there are extensive difficulties, e.g. control of stereochemistry, formation of specific linkages, availability of feedstocks, etc.
  • sialylated compounds comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, wherein said R is absent or a monosaccharide, a disaccharide, an oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or a glycolipid, is the transfer of the sialic group(s) on the growing saccharide chain within the saccharide compound, said transfer being catalyzed by sialyltransferases.
  • this and other objects are achieved by providing methods and a cell for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein.
  • the present invention also provides methods for the purification of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the present invention provides a cell which is genetically engineered as described herein. This invention also provides a purified sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R by the above-referenced process.
  • the present invention provides methods and a cell for production of a sialyltransferase as described herein wherein said sialyltransferase has alpha-2, 6-sialyltransferase activity on the HexNAc residue of Gal-pi,3-HexNAc- R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R. 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 two", more preferably by “at least three", even more preferably by “at least four", even more preferably by “at least five", even more preferably by “at least six", most preferably by "at least two”.
  • the word “about” or “approximately” when used in association with a numerical value (e.g., “about 10") or with a range (e.g., “about x to approximately y”) preferably means that the value or range is interpreted as being as accurate as the method used to measure it.
  • polynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotide(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triplestranded regions, or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • the term "polynucleotide(s)” also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" according to the present invention.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases are to be understood to be covered by the term “polynucleotides”.
  • polynucleotides DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases.
  • polynucleotides are to be understood to be covered by the term “polynucleotides”.
  • polynucleotide(s) as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells.
  • polynucleotide(s) also embraces short polynucleotides often referred to as oligonucleotide(s).
  • Polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
  • Polypeptide(s) refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids.
  • Polypeptide(s) include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to the skilled person.
  • modification may be present in the same or varying degree at several sites in a given polypeptide.
  • a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid sidechains, and the amino or carboxyl termini.
  • Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulphide bond formation, demethylation, formation of covalent cross-links, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation, selenoylation, transfer-RNA mediated addition
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention.
  • the term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions that also may contain coding and/or non-coding sequences.
  • isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • a “synthetic" sequence as the term is used herein, means any sequence that has been generated synthetically and not directly isolated from a natural source.
  • Synthesized as the term is used herein, means any synthetically generated sequence and not directly isolated from a natural source.
  • Recombinant means genetically engineered DNA prepared by transplanting or splicing genes from one species into the cells of a host organism of a different species. Such DNA becomes part of the host's genetic makeup and is replicated.
  • recombinant or “transgenic” or “metabolically engineered” or “genetically engineered” as used herein with reference to a cell or host cell are used interchangeably and indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence "foreign to said cell” or a sequence "foreign to said location or environment in said cell”).
  • Such cells are described to be transformed with at least one heterologous or exogenous gene or are described to be transformed by the introduction of at least one heterologous or exogenous gene.
  • 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 microorganism cell, wherein techniques may be applied which will depend on the cell and the sequence that is to be introduced.
  • techniques are known to a person skilled in the art and are, e.g., disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
  • the term "mutant" or "engineered” cell or microorganism as used within the context of the present invention refers to a cell or microorganism which is genetically engineered.
  • exogenous within the context of the present disclosure refers to any polynucleotide, polypeptide or protein sequence that is a natural part of a cell and is occurring at its natural location in the cell chromosome and of which the control of expression has not been altered compared to the natural control mechanism acting on its expression.
  • exogenous refers to any polynucleotide, polypeptide or protein sequence which originates from outside the cell under study and not a natural part of the cell or which is not occurring at its natural location in the cell chromosome or plasmid.
  • heterologous when used in reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme refers to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is from a source or derived from a source other than the host organism species.
  • a “homologous" polynucleotide, gene, nucleic acid, polypeptide, or enzyme is used herein to denote a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is derived from the host organism species.
  • heterologous means that the regulatory sequence or auxiliary sequence is not naturally associated with the gene with which the regulatory or auxiliary nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome.
  • a promoter operably linked to a gene to which it is not operably linked to in its natural state i.e.
  • heterologous promoter in the genome of a non- genetically engineered organism 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein.
  • Said modified expression is either a lower or higher expression compared to the wild-type, wherein the term “higher expression” is also defined as "overexpression" of said gene in the case of an endogenous gene or “expression” in the case of a heterologous gene that is not present in the wild-type strain.
  • Lower expression is obtained by means of common well-known technologies for a skilled person (such as the usage of siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon mutagenesis, etc.) which are used to change the genes in such a way that they are "less-able” (i.e., statistically significantly 'less-able' compared to a functional wild-type gene) or completely unable (such as knocked-out genes) to produce functional final products.
  • a skilled person such as the usage of siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon mutagenesis, etc.
  • riboswitch as used herein is defined to be part of the messenger RNA that folds into intricate structures that block expression by interfering with translation. Binding of an effector molecule induces conformational change(s) permitting regulated expression post-transcriptionally.
  • lower expression can also be obtained by changing the transcription unit, the promoter, an untranslated region, the ribosome binding site, the Shine Dalgarno sequence or the transcription terminator.
  • Lower expression or reduced expression can for instance be obtained by mutating one or more base pairs in the promoter sequence or changing the promoter sequence fully to a constitutive promoter with a lower expression strength compared to the wild-type or an inducible promoter which result in regulated expression or a repressible promoter which results in regulated expression.
  • Overexpression or expression is obtained by means of common well-known technologies for a skilled person (such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids), wherein said gene is part of an "expression cassette" that relates to any sequence in which a promoter sequence, untranslated region sequence (containing either a ribosome binding sequence, Shine Dalgarno or Kozak sequence), a coding sequence (for instance a sialyltransferase gene sequence) and optionally a transcription terminator is present, and leading to the expression of a functional active protein. Said expression is either constitutive or conditional or regulated or tuneable.
  • RNA polymerase e.g., the bacterial sigma factors like 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein.
  • non-native as used herein with reference to the activity of a protein indicates that the protein has been modified to have an abolished, impaired, reduced, delayed, higher, accelerated or improved activity compared to the native activity of said protein.
  • a modified activity of a protein is obtained by modified expression of said protein or is obtained by expression of a modified, i.e., mutant form of the protein.
  • a mutant form of the protein can be obtained by expression of a mutant form of the gene encoding the protein, e.g., comprising a deletion, an insertion and/or a mutation of one or more nucleotides compared to the native gene sequence.
  • a mutant form of a gene can be obtained by techniques well-known to a person skilled in the art, such as but not limited to site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis.
  • non-native indicates that said sialylated compound 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R or to have a higher production of the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • 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.
  • Variant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to the persons skilled in the art.
  • the present invention contemplates making functional variants by modifying the structure of an enzyme, like e.g. a sialyltransferase, as used in the present invention.
  • Variants can be produced by amino acid substitution, deletion, addition, or combinations thereof.
  • a variant can be produced as a fusion protein comprising at least one portion of an enzyme, like e.g. a sialyltransferase, 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 50.0 %, 60.0 %, 70.0 %, 80.0 %, 81.0 %, 82.0 %, 83.0 %, 84.0 %, 85.0 %, 86.0 %, 87.0 %, 88.0 %, 89.0 %, 90.0 %, 91.0 %, 92.0 %, 93.0 %, 94.0 %, 95.0 %, 95.5%, 96.0 %, 96.5 %, 97.0 %, 97.5 %, 98.0 %, 98.5 %, 99.0 %, 99.5 %, 100 %, preferably at least 80.0 %, more preferably at least 85.0 %, even more preferably at least 87.0 %, even even more
  • a fragment of a polynucleotide SEQ ID NO preferably means a nucleotide sequence which comprises or consists of said polynucleotide SEQ ID NO, wherein an amount of consecutive nucleotides is missing and wherein said amount is no more than 50.0 %, 40.0 %, 30.0 % of the full-length of said polynucleotide SEQ ID NO, preferably no more than 20.0 %, 15.0 %, 10.0 %, 9.0 %, 8.0 %, 7.0 %, 6.0 %, 5.0 %, 4.5 %, 4.0 %, 3.5 %, 3.0 %, 2.5 %, 2.0 %, 1.5 %, 1.0 %, 0.5 %, more preferably no more than 15.0 %, even more preferably no more than 10.0 %, even more preferably no more than 5.0 %, most
  • “Fragment”, with respect to a polypeptide refers to a subsequence of the polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide.
  • a “subsequence of the polypeptide” or “a stretch of amino acid residues” as described herein refers to a sequence of contiguous amino acid residues derived from the polypeptide.
  • a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription.
  • Fragments can vary in size from as few as 3 amino acid residues to the full length of the intact polypeptide, for example at least about 10 amino acid residues in length, for example at least about 20 amino acid residues in length, for example at least about 30 amino acid residues in length, for example at least about 100 amino acid residues in length, for example at least about 150 amino acid residues in length, for example at least about 200 amino acid residues in length.
  • a fragment of a polypeptide SEQ ID NO preferably means a polypeptide sequence which comprises or consists of said polypeptide SEQ ID NO (or UniProt ID) wherein no more than about 200, 150, 125, 100, 80, 60, 50, 40, 30, 20 or 15 consecutive amino acid residues are missing, preferably no more than about 100 consecutive amino acid residues are missing, more preferably no more than about 50 consecutive amino acid residues are missing, even more preferably no more than about 40 consecutive amino acid residues are missing, and performs at least one biological function of the intact polypeptide in substantially the same manner, preferably to a similar or greater extent, as does the intact polypeptide which can be routinely assessed by the skilled person.
  • a fragment of a polypeptide SEQ ID NO preferably means a polypeptide sequence which comprises or consists of an amount of consecutive amino acid residues from said polypeptide SEQ ID NO (or UniProt ID) and wherein said amount of consecutive amino acid residues is at least 50.0 %, 60.0 %, 70.0 %, 80.0 %, 81.0 %, 82.0 %, 83.0 %, 84.0 %, 85.0 %, 86.0 %, 87.0 %, 88.0 %, 89.0 %, 90.0 %, 91.0 %, 92.0 %, 93.0 %, 94.0 %, 95.0 %, 95.5%, 96.0 %, 96.5 %, 97.0 %, 97.5 %, 98.0 %, 98.5 %, 99.0 %, 99.5 %, 100 %, preferably at least 80.0 %, more preferably at least 85.0 %, even more preferably at least 87.0%
  • a fragment of a polypeptide SEQ ID NO preferably means a polypeptide sequence which comprises or consists of said polypeptide SEQ ID NO (or UniProt ID), wherein an amount of consecutive amino acid residues is missing and wherein said amount is no more than 50.0 %, 40.0 %, 30.0 % of the full-length of said polypeptide SEQ ID NO (or UniProt ID), preferably no more than 20.0 %, 15.0 %, 10.0 %, 9.0 %, 8.0 %, 7.0 %, 6.0 %, 5.0 %, 4.5 %, 4.0 %, 3.5 %, 3.0 %, 2.5 %, 2.0 %, 1.5 %, 1.0 %, 0.5 %, more preferably no more than 15.0 %, even more preferably no more than 10.0 %, even more preferably no more than 5.0 %, most preferably no more than 2.5 %, of the full-length of said polypeptide SEQ ID NO
  • polypeptide SEQ ID NO SEQ ID NO
  • polypeptide UniProt ID 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.
  • 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.
  • each database is fixed at each release and is not to be changed.
  • this specific database receives a new release version with a new release date. All release versions for each database with their corresponding release dates and specific content as annotated at these specific release dates are available and known to those skilled in the art.
  • nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms or by visual inspection.
  • sequence comparison one sequence acts as a reference sequence, to which test sequences are compared.
  • sequence comparison algorithm test and reference sequences are inputted into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • the sequence comparison algorithm calculates the % sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • the percentage of sequence identity can be, preferably is, determined by alignment of the two sequences and identification of the number of positions with identical residues divided by the number of residues in the shorter of the sequences x 100. Percent identity may be calculated globally over the full-length sequence of a given SEQ. ID NO, i.e. the reference sequence, resulting in a global % identity score. Alternatively, % identity may be calculated over a partial sequence of the reference sequence, resulting in a local percent identity score.
  • a partial sequence preferably means at least about 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 92 % 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 92 %, 92.5 %, 93 %, 93.5 %, 94 %, 94.5 %, 95 %, 95.5 %, 96 %, 96.5 %, 97 %, 97.5 %, 98 %, 98.5 %, 99 %, 99.5 %, 99.6 %, 99.7 %, 99.8 %, 99.9 %, 100 % sequence identity to the full-length of the amino acid sequence of the reference polypeptide sequence.
  • a polypeptide comprising, consisting of or having an amino acid sequence having 92 % 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 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 %, more preferably has at least 95 %, even more preferably has at least 97.5 %, 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.
  • sialylated compound refers to the group of sialylated molecules comprising sialylated oligosaccharide, sialylated peptide, glycoprotein comprising a sialylated oligosaccharide, sialylated lipid, and a glycolipid comprising a sialylated oligosaccharide.
  • sialic acid refers to an acidic sugar comprising but not limited to Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulonic acid (KDO).
  • KDO 2-keto-3-deoxymanno-octulonic acid
  • Neu4Ac is also known as 4-O-acetyl-5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid or 4-O-acetyl neuraminic acid and has C11H19NO9 as molecular formula.
  • Neu5Ac is also known as 5- acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, D-glycero-5-acetamido-3,5- dideoxy-D-galacto-non-2-ulo-pyranosonic acid, 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2- nonulopyranosonic acid, 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid, 5- (acetylamino)-3,5-dideoxy-D-glycero-D-galacto-non-2-nonulosonic acid or 5-(acetylamino)-3,5-dideoxy- D-glycero-D-galacto-non-2-ulopyranosonic acid and has C11H19
  • Neu4,5Ac2 is also known as N-acetyl-4-O-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 4-O-acetyl-N- acetylneuraminate, 4-acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonate, 4-acetate 5- (acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonate, 4-acetate 5-acetamido-3,5-dideoxy-D- glycero-D-galacto-nonulosonic acid or 4-acetate 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2- nonulosonic acid and has C13H21NO10 as molecular formula.
  • Neu5,7Ac2 is also known as 7-O-acetyl-N- acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, 7-O-acetyl-N-acetylneuraminate, 7-acetate 5- acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonate, 7-acetate 5-(acetylamino)-3,5-dideoxy-D- glycero-D-galacto-2-nonulosonate, 7-acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonic acid or 7-acetate 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid and has C13H21NO10 as molecular formula.
  • Neu5,8Ac2 is also known as 5-n-acetyl-8-o-acetyl neuraminic acid and has C13H21NO10 as molecular formula.
  • Neu5,9Ac2 is also known as N-acetyl-9-O-acetylneuraminic acid, 9-anana, 9-O-acetylsialic acid, 9-O-acetyl-N-acetylneuraminic acid, 5-n-acetyl-9-O-acetyl neuraminic acid, N,9-O-diacetylneuraminate or N,9-O-diacetylneuraminate and has C13H21NO10 as molecular formula.
  • Neu4,5,9Ac3 is also known as 5-N-acetyl-4,9-di-O-acetylneuraminic acid.
  • Neu5,7,9Ac3 is also known as 5- N-acetyl-7,9-di-O-acetylneuraminic acid.
  • Neu5,8,9Ac3 is also known as 5-N-acetyl-8,9-di-O- acetylneuraminic acid.
  • Neu4,5,7,9Ac4 is also known as 5-N-acetyl-4,7,9-tri-O-acetylneuraminic acid.
  • Neu5,7,8,9Ac4 is also known as 5-N-acetyl-7,8,9-tri-O-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
  • Sialyltransferases are glycosyltransferases that transfer a sialic acid (like Neu5Ac) from a donor (like CMP- Neu5Ac) onto an acceptor.
  • Sialyltransferases comprise alpha-2, 3-sialyltransferases, alpha-2, 6- sialyltransferases and alpha-2, 8-sialyltransferases that catalyse the transfer of a sialic acid onto an acceptor via alpha-glycosidic bonds.
  • Sialyltransferases can be found but are not limited to the GT29, GT42, GT52, GT80, GT97 and GT100 CAZy families.
  • monosaccharide refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed either an aldose or ketose, and contains one or more hydroxyl groups per molecule. Monosaccharides are saccharides containing only one simple sugar.
  • phosphorylated monosaccharide refers to a monosaccharide that is phosphorylated.
  • 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-Neu4,5Ac?, CMP-Neu5,7Ac?, 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 (Glc
  • 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, neutral (non-charged) oligosaccharides, negatively charged oligosaccharides, fucosylated oligosaccharides, sialylated oligosaccharides, N-acetylglucosamine containing oligosaccharides, N- acetyllactosamine containing oligosaccharides, lacto-N-biose containing oligosaccharides, lactose containing oligosaccharides, non-f
  • a 'sialylated oligosaccharide' is to be understood as a negatively charged sialic acid containing oligosaccharide, i.e., an oligosaccharide having a sialic acid residue. It has an acidic nature.
  • sialylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of said monosaccharide subunit is a sialic acid residue.
  • a sialylated oligosaccharide can contain more than one sialic acid residue, e.g., two, three or more.
  • Said more than one sialic acid residue can be two, three or more identical sialic acid residues. Said more than one sialic acid residue can also be two, three or more different sialic acid residues.
  • a sialylated oligosaccharide can contain one or more Neu5Ac residues and one or more KDO residues.
  • 3-SL 3-SL (3'-sialyllactose or 3'SL or Neu5Ac-a2,3-Gal-bl,4-Glc), 3'-sialyllactosamine, 6-SL (6'sialyllactose, 6'-sialyllactose or 6'SL or Neu5Ac-a2,6-Gal-bl,4-Glc), 3,6-disialyllactose (Neu5Ac-a2,3- (Neu5Ac-a2,6)-Gal-bl,4-Glc), 6,6'-disialyllactose (Neu5Ac-a2,6-Gal-bl,4-(Neu5Ac-a2,6)-Glc), 8,3- disialyllactose (Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-bl,4-Glc), 6'-sialyllactosamine,
  • a 'neutral oligosaccharide' or a 'non-charged oligosaccharide' as used herein and as generally understood in the state of the art is an oligosaccharide that has no negative charge originating from a carboxylic acid group.
  • Examples of such neutral oligosaccharide are 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 2', 3- difucosyllactose (diFL), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto- N-fucopentaose I (LNFP I), lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose II (LNFP II), lacto- N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-fucopentaose VI, lacto-N- neofucopentaose V (LNnFP V), lacto-N-
  • Mammalian milk oligosaccharides or MMOs comprise oligosaccharides present in milk found in any phase during lactation including colostrum milk from humans (i.e. human milk oligosaccharides or HMOs) and mammals including but not limited to cows (Bos Taurus), sheep (Ovis aries), goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus), horses (Eguusferus caballus), pigs (Sus scropha), dogs (Canis lupus familiaris), ezo brown bears (Ursus arctos yesoensis), polar bear (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephant (Lo
  • mammalian milk oligosaccharide 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-fucopentaose II
  • mammalian milk oligosaccharide 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-fucopentaose II, lac
  • 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.
  • 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
  • Gaipi-4GlcNAcpi-3Gaipi-4Glc as used in the present invention, are used interchangeably.
  • LSTa LS-Tetrasaccharide a
  • Sialyl-lacto-N-tetraose a sialyllacto-N-tetraose a
  • Neu5Ac-a2,3-Gal- i,3-GlcNAc- i,3-Gal- i,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- i,3-(Neu5Ac-a2,6)-GlcNAc- i,3-Gal- i,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 "sialyllacto-N-tetraose d”
  • sialyllacto-N-neotetraose d or "Neu5Ac-a2,3-Gal-pi,4- GlcNAc-pi,3-Gal-pi,4-Glc" as used in the present invention, are used interchangeably.
  • 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.
  • glycopeptide refers to a peptide that contains one or more saccharide groups, being mono-, di-, oligo-, polysaccharides and/or glycans, that is/are covalently attached to the side chains of the amino acid residues of the peptide.
  • Glycopeptides comprise natural glycopeptide antibiotics such as e.g.
  • glycosylated non-ribosomal peptides produced by a diverse group of soil actinomycetes that target Gram-positive bacteria by binding to the acyl-D-alanyl-D-alanine (D-Ala-D-Ala) terminus of the growing peptidoglycan on the outer surface of the cytoplasmatic membrane, and synthetic glycopeptide antibiotics.
  • the common core of natural glycopeptides is made of a cyclic peptide consisting in 7 amino acids, to which are bound 2 sugars.
  • Examples of glycopeptides comprise vancomycin, teicoplanin, oritavancin, chloroeremomycin, telavancin and dalbavancin.
  • glycoprotein and "glycopolypeptide” are used interchangeably and refer to a polypeptide that contains one or more saccharide groups, being mono-, di-, oligo-, polysaccharides and/or glycans, that is/are covalently attached to the side chains of the amino acid residues of the polypeptide.
  • glycolipid refers to any of the glycolipids which are generally known in the art. Glycolipids (GLs) can be subclassified into Simple (SGLs) and Complex (CGLs) glycolipids. Simple GLs, sometimes called saccharolipids, are two-component (glycosyl and lipid moieties) GLs in which the glycosyl and lipid moieties are directly linked to each other. Examples of SGLs include glycosylated fatty acids, fatty alcohols, carotenoids, hopanoids, sterols or paraconic acids.
  • Bacterially produced SGLs can be classified into rhamnolipids, glucolipids, trehalolipids, other glycosylated (non-trehalose containing) mycolates, trehalose-containing oligosaccharide lipids, glycosylated fatty alcohols, glycosylated macrolactones and macro-lactams, glycomacrodiolides (glycosylated macrocyclic dilactones), glyco-carotenoids and glyco-terpenoids, and glycosylated hopanoids/sterols.
  • CGLs Complex glycolipids
  • CGLs Complex glycolipids
  • glycerol glycoglycerolipids
  • peptide glycopeptidolipids
  • acylated-sphingosine glycosphingolipids
  • lipopolysaccharides phenolic glycolipids, nucleoside lipids
  • 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, p- 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).
  • pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc- R is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as defined herein.
  • Said pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]- HexNAc-R can comprise but is not limited to pathways involved in the synthesis of a nucleotide-activated sugar and the transfer of said nucleotide-activated sugar to an acceptor to create a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R of the present invention.
  • An example of such pathway is a sialylation pathway.
  • pathway comprises but are not limited to a fucosylation, galactosylation, N-acetylglucosaminylation, N-acetylgalactosaminylation, mannosylation, N- acetylmannosaminylation pathway.
  • a 'sialylation pathway' is a biochemical pathway consisting of at least one of the enzymes and their respective genes selected from the list comprising, consisting of or consisting essentially of an L- glutamine— D-fructose-6-phosphate aminotransferase, a phosphoglucosamine mutase, an N- acetylglucosamine-6-P deacetylase, an N-acylglucosamine 2-epimerase, a 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-phosphat
  • 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). Reducing environments may affect the oxidation state of a molecule, thereby altering its solubility.
  • glycosylcholine reductase glutathione reductase (NADPH)
  • glutathione S-reductase glutathione S-reductase
  • GSH reductase glutathione S-reductase
  • GSG reductase oxidized-glutathione oxidoreductase
  • NADPH-glutathione reductase glutathione reductase
  • GR GRase
  • 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.
  • 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R that is produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell.
  • culture medium and “cultivation medium” as used herein are used interchangeably and refer to the medium wherein the cell is cultivated.
  • incubation refers to a mixture wherein a sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R 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 sialylated compound comprising Gal-pi,3- [Neu5Ac-a2,6]-HexNAc-R, 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R 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.
  • cell productivity index refers to the mass of the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R produced by the cells divided by the mass of the cells produced in the culture.
  • precursor refers to substances which are taken up or synthetized by the cell for the specific production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R according to the present invention.
  • a precursor can be an acceptor as defined herein, but can also be another substance, metabolite, which is first modified within the cell as part of the biochemical synthesis route of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • precursor as used herein is also to be understood as a chemical compound that participates in a chemical or enzymatic reaction to produce another compound like e.g. an intermediate or an acceptor as defined herein, as part in the metabolic pathway of a sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R.
  • precursor as used herein is also to be understood as a donor that is used by a glycosyltransferase to modify an acceptor as defined herein with a sugar moiety in a glycosidic bond, as part in the metabolic pathway of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • Such precursors comprise the acceptors as defined herein, and/or dihydroxyacetone, glucosamine, N-acetylglucosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyllactose, phosphorylated sugars or sugar phosphates like e.g.
  • glucose-1- phosphate galactose-l-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6- bisphosphate, mannose-6-phosphate, mannose-l-phosphate, glycerol-3-phosphate, glyceraldehyde-3- phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N-acetylglucosamine-l-phosphate, N-acetylneuraminic acid-9- phosphate and nucleotide-activated sugars like nucleotide diphospho-sugars and nucleotide monophospho-sugars as defined herein like e.g.
  • UDP-glucose UDP-galactose, UDP-N-acetylglucosamine, CMP-sialic acid, GDP-mannose, GDP-4-dehydro-6-deoxy-a-D-mannose, GDP-fucose.
  • the cell is transformed to comprise and to express at least one nucleic acid sequence encoding a protein selected from the 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-
  • acceptor refers to a mono-, di- or oligosaccharide, which can be modified by a glycosyltransferase.
  • acceptors comprise glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, lacto-N-triose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-pentaose (LNP), lacto-N-neopentaose, para lacto-N-pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), para lacto-N-n
  • the present invention provides a method for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, the method comprising contacting a sialyltransferase with a mixture comprising a) a donor comprising a sialic acid residue and b) Gal-pi,3-HexNAc-R and/or Neu5Ac- a2,3-Gal-pi,3-HexNAc-R under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from said donor to the N-acetylhexosamine (HexNAc) residue of said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R in an alpha-2, 6-glycosidic linkage, thereby producing said sialylated compound comprising Gal-pi,3
  • the sialyltransferase is an alpha-2, 6-sialyltransferase having alpha-2, 6-sialyltransferase activity on the HexNAc residue of said Gal- pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R and comprises a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[GS]X[HKR][ST]X[FILMV][HKR]XXX[HFY] with SEQ. ID NO 01 wherein X can be any amino acid residue and wherein n is 10 or 11.
  • the present invention provides a method for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, the method comprising contacting a cell extract comprising a sialyltransferase as described herein with a mixture comprising a) a donor comprising a sialic acid residue, and b) Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from said donor to the N- acetylhexosamine (HexNAc) residue of said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R in an alpha-2, 6-glycosidic linkage, thereby
  • the cell extract is obtained from a cell that possesses said alpha-2, 6- sialyltransferase.
  • said cell expresses said alpha-2, 6-sialyltransferase.
  • said cell overexpresses said alpha-2, 6-sialyltransferase.
  • the sialyltransferase is any one of the sialyltransferase as described herein.
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is produced in a cell-free system.
  • the present invention provides a method for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, wherein said method comprises the steps of: i) providing a cell, preferably a single cell, expressing, preferably heterologously expressing, more preferably overexpressing, even more preferably heterologously overexpressing, a sialyltransferase wherein said sialyltransferase is an alpha-2, 6-sialyltransferase having alpha-2, 6- sialyltransferase activity on the HexNAc residue of Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal- pi,3-HexNAc-R and comprises a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[
  • said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is separated from said cultivation or incubation.
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is produced by a cell, preferably a single cell, wherein said cell expresses, preferably over-expresses, a sialyltransferase as described herein.
  • the cell used can be a metabolically engineered cell as described herein, preferably wherein said cell is metabolically engineered for the production of said sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R.
  • the term "cell for the production of a sialylated oligosaccharide comprising Gal-pi,3- [Neu5Ac-a2,6]-HexNAc-R” is to be understood herein as that the cell itself produces a sialylated oligosaccharide comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R. Said production can be extracellularly and/or intracellularly.
  • the cell has been metabolically engineered to possess, express, heterologously express, overexpress and/or heterologously overexpress a sialyltransferase as described herein that is usable and/or used in the cellular production of said sialylated oligosaccharide comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the sialyltransferase as described herein is a polypeptide comprising an amino acid sequence that is at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, at least 99 % identical to any one of the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • the sialyltransferase as described herein comprises a polypeptide as represented by any one of SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • the sialic acid residue is at least one selected from the list consisting of Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulonic acid (KDO).
  • the sialic acid residue is Neu5Ac.
  • the donor comprising a sialic acid residue is CMP-sialic acid.
  • the donor comprising a sialic acid residue is selected from the list consisting of CMP-Neu5Ac, CMP-Neu4Ac, CMP- Neu5Ac9N 3 , CMP-Neu4,5Ac 2 , CMP-Neu5,7Ac 2 , CMP-Neu5,9Ac 2 , CMP-Neu5,7(8,9)Ac 2 , CMP-N- glycolylneuraminic acid (CMP-Neu5Gc) and CMP-KDO.
  • the donor comprising a sialic acid residue is CMP-Neu5Ac.
  • GIcNAc N-acetylglucosamine
  • GaalNAc N- acetylgalactosamine
  • said sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R is any one of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-R or sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-GalNAc-R; said Gal-pi,3-HexNAc-R is any one of Gal-pi,3-GlcNAc-R or Gal-pi,3-GalNAc-R and/or said Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is any one of Neu5Ac-a2,3-Gal-pi,3-GlcNAc-R or Neu5Ac-a2,3-Gal-pi,3-GalNAc-R.
  • the R group within said i) sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, ii) Gal-pi,3- HexNAc-R and/or iii) Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is absent, a monosaccharide, a disaccharide, an oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or a glycolipid as described herein.
  • the Gal-pi,3-HexNAc-R is a disaccharide, an oligosaccharide, a glycoprotein, a glycopeptide or a glycolipid.
  • Said Gal-pi,3-HexNAc-R can be a charged compound, like e.g., a negatively, preferably sialylated, compound.
  • said Gal-pi,3-HexNAc-R can be a non-charged, i.e., neutral compound.
  • said Gal-pi,3-HexNAc-R is selected from the list comprising, consisting of or consisting essentially of Gal-pi,3-GlcNAc (LNB, lacto-N-biose); Gal-pi,3-GlcNAc-R; Gal-pi,3-GlcNAc-pi,3-R; Gal- pi,3-GlcNAc-pi,3-Gal; Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-R; Gal-pi,3-GlcNAc- pi,3-Gal-pi,4-Glc (LNT, lacto-N-tetraose); Gal-pi,3-GlcNAc-pi,4-R; Gal-pi,3-GalNAc; Gal-pi,3-GalNAc; Gal-pi,3-GalNAc; Gal-pi,3-GalNAc-R; Gal
  • said Gal-pi,3-HexNAc-R is an oligosaccharide, a glycoprotein, a glycopeptide or a glycolipid wherein said Gal residue in said Gal-pi,3-HexNAc-R is glycosidically linked to a monosaccharide like e.g. Neu5Ac, a disaccharide or an oligosaccharide.
  • said Gal-pi,3-HexNAc-R may be Neu5Ac-a2,3-Gal-pi,3-HexNAc-R.
  • the Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is an oligosaccharide, a glycoprotein, a glycopeptide or a glycolipid.
  • said oligosaccharide, glycoprotein, glycopeptide or glycolipid is sialylated.
  • Said Neu5Ac-a2,3- Gal-pi,3-HexNAc-R can have one sialic acid group, being Neu5Ac.
  • said Neu5Ac-a2,3-Gal- pi,3-HexNAc-R can have more than one sialic acid groups, as described herein.
  • said Neu5Ac- a2,3-Gal-pi,3-HexNAc-R is selected from the list comprising, consisting of or consisting essentially of Neu5Ac-a2,3-Gal-pi,3-GlcNAc (3'SLNB, 3' -sialylated LNB); Neu5Ac-a2,3-Gal-pi,3-GlcNAc-R; Neu5Ac- a2,3-Gal-pi,3-GlcNAc-pi,3-R; Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-R; Neu5Ac-a2,3-Gal-pi,3-GlcNAc- pi,3-Gal-pi,4-R; Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-R; Neu5Ac-a2,3
  • said Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is an oligosaccharide, a glycoprotein, a glycopeptide or a glycolipid wherein said Gal residue in said Neu5Ac-a2,3-Gal-pi,3- HexNAc-R is not only glycosidically linked to said Neu5Ac and said HexNAc, but also to a monosaccharide, a disaccharide and/or an oligosaccharide.
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein is a sialylated oligosaccharide, glycoprotein, glycopeptide or glycolipid.
  • Said Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R can have one sialic acid group, being Neu5Ac.
  • said Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R can have more than one sialic acid groups, as described herein.
  • said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is selected from the list comprising Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc; Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-R; Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-pi,3-R; Gal-pi,3-[Neu5Ac-a2,6]- GlcNAc-pi,4-R; Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-pi,3-Gal; Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-pi,3-Gal-R; Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-pi,3-Gal-R; Gal-
  • said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is an oligosaccharide, a glycoprotein, a glycopeptide or a glycolipid wherein said Gal residue in said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]- HexNAc-R is not only glycosidica lly linked to said HexNAc, but also to a monosaccharide, a disaccharide and/or an oligosaccharide.
  • the Gal-pi,3-HexNAc-R is Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc (LNT, lacto-N-tetraose) and the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is Gal-pi,3- [Neu5Ac-a2,6]-GlcNAc-pi,3-Gal-pi,4-Glc (LSTb, sialyllacto-N-tetraose b).
  • the Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is Neu5Ac-a2,3-Gal-pi,3- GlcNAc-pi,3-Gal-pi,4-Glc (LSTa, sialyllacto-N-tetraose a) and the sialylated compound comprising Gal- pi,3-[Neu5Ac-a2,6]-HexNAc-R is Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,3)-GlcNAc-pi,3-Gal-pi,4-Glc (DSLNT, disialyllacto-N-tetraose).
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]- HexNAc-R is an oligosaccharide.
  • said sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R is a sialylated oligosaccharide selected from the list comprising Gal-pi,3-[Neu5Ac-a2,6]- GIcNAc, Gal-pi,3-[Neu5Ac-a2,6]-GalNAc, Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-R and Gal-pi,3-[Neu5Ac- a2,6]-GalNAc-R with R being a disaccharide or an oligosaccharide as described herein.
  • Said sialylated oligosaccharide can have one sialic acid residue, being Neu5Ac.
  • said sialylated oligosaccharide can have more than one sialic acid residues, as described herein.
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is a milk oligosaccharide, a mammalian milk oligosaccharide (MMO) or a human milk oligosaccharide (HMO).
  • the MMO is an oligosaccharide selected from the list comprising Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc- R and Gal-pi,3-[Neu5Ac-a2,6]-GalNAc-R with R being absent, a monosaccharide, a disaccharide or an oligosaccharide as described herein.
  • the HMO is an oligosaccharide selected from the list comprising Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-R and Gal-pi,3-[Neu5Ac-a2,6]-GalNAc-R with R being absent, a monosaccharide, a disaccharide or an oligosaccharide as described herein.
  • permissive conditions are understood to be conditions relating to physical or chemical parameters including but not limited to temperature, pH, pressure, osmotic pressure and product/donor/precursor/acceptor concentration.
  • the permissive conditions may include a temperature-range of about 30 +/- 20 degrees centigrade, a pH-range of 2.0 - 10.0, preferably a pH range of 3.0 - 7.0.
  • the conditions permissive to produce sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein comprise the use of a cultivation or incubation medium comprising at least one precursor and/or acceptor for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein.
  • the cultivation or incubation medium contains at least one precursor and/or acceptor, wherein said precursor is selected from the list comprising, consisting of or consisting essentially of a monosaccharide like e.g.
  • galactose glucose, fucose, sialic acid, GIcNAc, GalNAc
  • a nucleotide-activated sugar like e.g. CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, UDP-Gal, UDP-GIcNAc, GDP-fucose
  • a disaccharide like e.g. lactose
  • an oligosaccharide like e.g.
  • lacto-N-triose LN3
  • lacto-N-tetraose LNT
  • lacto-N-neotetraose LNnT
  • acceptor is selected from the list comprising, consisting of or consisting essentially of a disaccharide like e.g. lactose, an oligosaccharide like e.g. LN3, LNT, LNnT, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid and a glycolipid.
  • said precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, said donor comprising a sialic acid residue, CMP-sialic acid, CMP- Neu5Ac, glucose, galactose, GIcNAc, GalNAc, UDP-GIcNAc, UDP-GalNAc.
  • said acceptor is selected from the list comprising, consisting of or consisting essentially of lactose, GlcNAc-pi,3-Gal-pi,4-Glc (LN3, lacto-N-triose), Gal-pi,3-HexNAc-R, Neu5Ac-a2,3-Gal-pi,3-HexNAc-R, LNT and LSTa, with R as described herein.
  • the conditions permissive to produce said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein comprise the use of a cultivation or incubation medium and adding to said cultivation or incubation medium at least one precursor and/or acceptor feed for the production of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the conditions permissive to produce said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein comprise the use of a cultivation or incubation medium wherein said cultivation or incubation medium lacks any precursor and/or acceptor for the production of said sialylated compound comprising Gal-pi,3- [Neu5Ac-a2,6]-HexNAc-R, and is combined with a further addition to said cultivation or incubation medium of at least one precursor and/or acceptor feed for the production of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the cultivation or incubation is contained in a reactor or incubator, as defined herein.
  • the volume of said reactor or incubator ranges from microlitre (pL) scale to 10.000 m3 (cubic meter). In a preferred embodiment, the volume of said reactor or incubator ranges from 250 mL (millilitre) to 10.000 m3 (cubic meter).
  • the method for the production of a sialylated compound comprising Gal-pi,3- [Neu5Ac-a2,6]-HexNAc-R 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
  • said precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose.
  • said acceptor is lactose.
  • the method for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R 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 in one pulse or in a discontinuous (pulsed) manner wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m 3 (cubic meter), preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than twofold, more preferably less than two-fold of the volume of the cultivation or incubation medium before the addition of said precursor and/or acceptor feed pulse(s); iii) Adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and/
  • said precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose.
  • said acceptor is lactose.
  • the method for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R 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
  • said precursor is selected from the list comprising, consisting of or consisting essentially of sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose.
  • said acceptor is lactose.
  • the method for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R comprises at least one of the following steps: i) Use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter); ii) Adding to the cultivation or incubation medium in a reactor or incubator a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per litre of initial reactor or incubator volume wherein the total reactor or incubator volume ranges from 250 mL (millilitre) to 10.000 m 3 (cubic meter), preferably in
  • the lactose feed is accomplished by adding lactose from the beginning of the cultivation or incubation in a concentration of at least 5 mM, preferably in a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably in a concentration > 300 mM.
  • the lactose feed is accomplished by adding lactose to the cultivation or incubation medium in a concentration, such that throughout the production phase of the cultivation or incubation a lactose concentration of at least 5 mM, preferably 10 mM or 30 mM is obtained.
  • the cells are cultivated or incubated for at least about 60, 80, 100, or about 120 hours or in a continuous manner.
  • a carbon source is provided, preferably sucrose, in the cultivation medium for 3 or more days, preferably up to 7 days; and/or provided, in the cultivation medium, at least 100, advantageously at least 105, more advantageously at least 110, even more advantageously at least 120 grams of sucrose per litre of initial cultivation volume in a continuous manner, so that the final volume of the cultivation medium is not more than three-fold, advantageously not more than two-fold, more advantageously less than two-fold of the volume of the cultivation medium before the cultivation.
  • a first phase of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the cultivation medium before the lactose is added to the cultivation medium in a second phase.
  • a carbon source preferably glucose or sucrose
  • the lactose is added already in the first phase of exponential growth together with the carbon-based substrate.
  • the present invention provides a cell metabolically engineered for expression, preferably overexpression, of a biologically active sialyltransferase, wherein said sialyltransferase is an alpha-2, 6-sialyltransferase having alpha-2, 6-sialyltransferase activity on the HexNAc residue of Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R and comprising a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[GS]X[HKR][ST]X[FILMV][HKR]XXX[HFY] with SEQ ID NO 01 wherein X can be any amino acid residue and wherein n is 10 or 11.
  • said HexNAc and R groups are as described herein.
  • the present invention provides a metabolically engineered cell for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, wherein said cell has been metabolically engineered to possess, preferably to express, more preferably to heterologously express, even more preferably to overexpress, most preferably to heterologously overexpress, a sialyltransferase which has alpha-2, 6-sialyltransferase activity on the HexNAc residue of Gal-pi,3-HexNAc- R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R and comprising a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no
  • said HexNAc and R groups are as described herein.
  • said sialyltransferase used in the cell is a sialyltransferase as described herein.
  • the sialyltransferase is a polypeptide comprising an amino acid sequence that is at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, at least 99 % identical to any one of the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 1.
  • the sialyltransferase comprises a polypeptide as represented by any one of SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • the cell comprises a polynucleotide sequence that encodes a sialyltransferase as described herein, and operably linked to control sequences recognized by the cell, wherein said sequence is foreign to the cell, said sequence further i) being integrated in the genome of said cell and/or ii) presented to said cell on a vector.
  • a cell comprising a pathway for production of said sialylated compound comprising Gal-pi,3- [Neu5Ac-a2,6]-HexNAc-R.
  • the cell is genetically engineered to comprise a pathway for production of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R. More preferably, the cell comprises a pathway for production of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R wherein said pathway has been genetically engineered.
  • Examples of such pathways comprise but are not limited to pathways involved in the synthesis of monosaccharide, phosphorylated monosaccharide, nucleotide- activated sugar, and/or glycosylation pathways like e.g., a fucosylation, sialylation, galactosylation, N- acetylglucosaminylation, N-acetylgalactosaminylation, mannosylation and/or N- acetylmannosaminylation pathway.
  • Said pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R preferably comprises at least one sialyltransferase as described herein.
  • Said cell may further comprise and express at least one further glycosyltransferase that is involved in the production of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the cell comprises one or more pathway(s) for monosaccharide synthesis.
  • the cell is genetically engineered to comprise one or more pathway(s) for monosaccharide synthesis. Said pathways for monosaccharide synthesis comprise enzymes like e.g.
  • carboxylases decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases, dehydrogenases, enzymes involved in the synthesis of one or more nucleoside triphosphate(s) like UTP, GTP, ATP and CTP, enzymes involved in the synthesis of any one or more nucleoside mono- or diphosphates like e.g. UMP and UDP, respectively, and enzymes involved in the synthesis of phosphoenolpyruvate (PEP).
  • 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 phospho
  • the cell comprises one or more pathway(s) for phosphorylated monosaccharide synthesis.
  • the cell is genetically engineered to comprise one or more pathway(s) for phosphorylated monosaccharide synthesis.
  • Said pathways for phosphorylated monosaccharide synthesis comprise enzymes involved in the synthesis of one or more monosaccharide(s), one or more nucleoside mono-, di- and/or triphosphate(s) and enzymes involved in the synthesis of phosphoenolpyruvate (PEP) like e.g., but not limited to PEP synthase, carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases and dehydrogenases.
  • PEP phosphoenolpyruvate
  • the cell comprises one or more pathways for the synthesis of one or more nucleotide-activated sugars.
  • the cell is genetically engineered to comprise 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-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, L-fucokinase/GDP-fucose pyrophosphorylase, L- glutamine— D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2- epimerase, UDP-N
  • the cell is metabolically engineered to comprise a pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R as defined herein.
  • the cell is metabolically engineered to comprise a pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as defined herein, and to have modified expression or activity of a sialyltransferase of present invention.
  • the cell comprises a recombinant sialyltransferase capable of modifying Gal-pi,3-HexNAc-R, Neu5Ac-a2,3-Gal-pi,3-HexNAc- R, and/or another acceptor as defined herein with one or more sialic acid molecules that is/are synthesized by any one or more sialic acid synthases like e.g. Neu5Ac synthases, or by e.g.
  • d-arabinose 5- phosphate isomerase KDO-8P synthase and KDO 8-phosphate phosphatase expressed in the cell, into a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the metabolically engineered cell is modified with one or more expression modules.
  • Said expression modules are also known as transcriptional units and comprise polynucleotides for expression of recombinant genes including coding gene sequences and appropriate transcriptional and/or translational control signals that are operably linked to the coding genes.
  • Said control signals comprise promoter sequences, untranslated regions, ribosome binding sites, terminator sequences.
  • Said expression modules can contain elements for expression of one single recombinant gene but can also contain elements for expression of more recombinant genes or can be organized in an operon structure for integrated expression of two or more recombinant genes.
  • Said polynucleotides may be produced by recombinant DNA technology using techniques well-known in the art.
  • each of said expression modules can be constitutive or is created by a natural or chemical inducer.
  • constitutive expression should be understood as expression of a gene that is transcribed continuously in an organism.
  • Expression that is created by a natural inducer should be understood as a facultative or regulatory expression of a gene that is only expressed upon a certain natural condition of the host (e.g. organism being in labour, or during lactation), as a response to an environmental change (e.g. including but not limited to hormone, heat, cold, pH shifts, light, oxidative or osmotic stress / signalling), or dependent on the position of the developmental stage or the cell cycle of said host cell including but not limited to apoptosis and autophagy.
  • a certain natural condition of the host e.g. organism being in labour, or during lactation
  • an environmental change e.g. including but not limited to hormone, heat, cold, pH shifts, light, oxidative or osmotic stress / signalling
  • Expression that is created by a chemical inducer should be understood as a facultative or regulatory expression of a gene that is only expressed upon sensing of external chemicals (e.g. IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose) via an inducible promoter or via a genetic circuit that either induces or represses the transcription or translation of said polynucleotide to a polypeptide.
  • external chemicals e.g. IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose
  • the expression modules can be integrated in the genome of said cell or can be presented to said cell on a vector.
  • Said vector can be present in the form of a plasmid, cosmid, phage, liposome, or virus, which is to be stably transformed/transfected into said metabolically engineered cell.
  • Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • These vectors may contain selection markers such as but not limited to antibiotic markers, auxotrophic markers, toxinantitoxin markers, RNA sense/antisense markers.
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard.
  • the appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., see above.
  • cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
  • Introduction of a polynucleotide into the cell can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986), and Sambrook et al., 1989, supra.
  • an expression module comprises polynucleotides for expression of at least one recombinant gene.
  • Said recombinant gene is involved in the expression of a polypeptide acting in the synthesis of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R; or said recombinant gene is linked to other pathways in said cell that are not involved in the synthesis of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • Said recombinant genes encode endogenous proteins with a modified expression or activity, preferably said endogenous proteins are overexpressed; or said recombinant genes encode heterologous proteins that are heterogeneously introduced and expressed in said modified cell, preferably overexpressed.
  • the endogenous proteins can have a modified expression in the cell which also expresses a heterologous protein.
  • each of said expression modules present in said metabolically engineered cell is constitutive or tuneable as described herein.
  • the cell is modified in the expression or activity of at least one of said sialyltransferases.
  • said sialyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous sialyltransferase is overexpressed; alternatively said sialyltransferase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous sialyltransferase can have a modified expression in the cell which also expresses a heterologous sialyltransferase.
  • the cell comprises a pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R comprising at least one sialyltransferase according to present invention.
  • said pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R further comprises at least one enzyme selected from the list comprising, consisting of or consisting essentially of 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 phosphatas
  • said pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R further comprises at least one enzyme selected from the list comprising, consisting of or consisting essentially of a d-arabinose 5-phosphate isomerase, a KDO-8P synthase, a KDO 8-phosphate phosphatase and a CMP-KDO synthetase.
  • the cell comprises a pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, wherein said cell expresses at least one enzyme selected from the list comprising, consisting of or consisting essentially of an N-acylglucosamine 2-epimerase like is known e.g. from several species including Bacteroides ovatus, E. coli, Homo sapiens, Rattus norvegicus, a Neu5Ac synthase, a CMP sialic acid synthase like is known e.g.
  • GIcNAc N- acetylglucosamine
  • Such cell producing GIcNAc can express a phosphatase converting GlcNAc-6-phosphate into GIcNAc, like any one or more of e.g. the E.
  • coli HAD-like phosphatase genes comprising, consisting of or consisting essentially of aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU, PsMupP from Pseudomonas putida, ScDOGl from S.
  • the cell is modified to produce GIcNAc. More preferably, the cell is modified for enhanced GIcNAc production. Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knockout of a glucosamine-6-phosphate deaminase, an N-acetylglucosamine-6-phosphate deacetylase and/or an N-acetyl-D-glucosamine kinase and over-expression of an L-glutamine— D-fructose-6-phosphate aminotransferase and/or a glucosamine 6-phosphate N-acetyltransferase.
  • the cell comprises a pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, wherein said cell expresses at least one enzyme selected from the list comprising, consisting of or consisting essentially of an UDP-N- acetylglucosamine 2-epimerase like is known e.g. from several species including Campylobacter jejuni, E.
  • UDP-N-acetylglucosamine can be added to the cell and/or can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing an UDP-GIcNAc can express enzymes converting, e.g.
  • GIcNAc which is to be added to the cell, to UDP-GIcNAc.
  • These enzymes may be any one or more enzymes selected from the list comprising, consisting of or consisting essentially of an N-acetyl-D-glucosamine kinase, an N- acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, and an N- acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli.
  • the cell is modified to produce UDP- GIcNAc. More preferably, the cell is modified for enhanced UDP-GIcNAc production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knock-out of an N-acetylglucosamine-6-phosphate deacetylase, over-expression of an L-glutamine— D-fructose-6- phosphate aminotransferase, over-expression of a phosphoglucosamine mutase, and over-expression of an N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase.
  • the cell comprises a pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R wherein said cell expresses at least one enzyme selected from the list comprising, consisting of or consisting essentially of an N- acetylmannosamine-6-phosphate 2-epimerase like is known e.g. from several species including E. coli, Haemophilus influenzae, Enterobacter sp., Streptomyces sp., an N-acylneuraminate-9-phosphate synthetase, an N-acylneuraminate-9-phosphatase like is known e.g.
  • N-acetyl-D-glucosamine 6-phosphate can be added to the cell and/or can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing GlcNAc-6P can express an enzyme converting, e.g., GlcN6P, which is to be added to the cell, to GIcNAc- 6P.
  • This enzyme may be a glucosamine 6-phosphate N-acetyltransferase from several species including Saccharomyces cerevisiae, Kluyveromyces lactis, Homo sapiens.
  • the cell is modified to produce GlcNAc-6P. More preferably, the cell is modified for enhanced GlcNAc-6P production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knockout of a glucosamine-6-phosphate deaminase, an N-acetylglucosamine-6-phosphate deacetylase and over-expression of an L-glutamine— D-fructose-6-phosphate aminotransferase and/or a glucosamine 6-phosphate N-acetyltransferase.
  • the cell comprises a pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R wherein said cell expresses at least one enzyme selected from the list comprising, consisting of or consisting essentially of a bifunctional UDP-GIcNAc 2-epimerase/kinase like is known e.g. from several species including Homo sapiens, Rattus norvegicus and Mus musculus, an N-acylneuraminate-9-phosphate synthetase, an N-acylneuraminate-9- phosphatase like is known e.g. from Candidatus Magnetomorum sp.
  • HK-1 or Bacteroides thetaiotaomicron, a Neu5Ac synthase, a CMP sialic acid synthase like is known e.g. from Neisseria meningitidis, and a sialyltransferase according to present invention, wherein the enzymes are as defined herein.
  • UDP-N-acetylglucosamine can be added to the cell and/or can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing an UDP-GIcNAc can express enzymes converting, e.g. GIcNAc, which is to be added to the cell, to UDP-GIcNAc.
  • These enzymes may be an N-acetyl-D-glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, and an N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli.
  • the cell is modified to produce UDP-GIcNAc. More preferably, the cell is modified for enhanced UDP-GIcNAc production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knock-out of an N-acetylglucosamine-6- phosphate deacetylase, over-expression of an L-glutamine— D-fructose-6-phosphate aminotransferase, over-expression of a phosphoglucosamine mutase, and over-expression of an N-acetylglucosamine-1- phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase.
  • the cell comprises a pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R wherein said cell expresses at least one enzyme selected from the list comprising, consisting of or consisting essentially of a d-arabinose 5-phosphate isomerase, a KDO-8P synthase, a KDO 8-phosphate phosphatase, a CMP-KDO synthetase from different species like e.g. Escherichia coli, Pseudomonas aeruginosa, Agrobacterium sp.
  • the cell is capable to make CMP-KDO. More preferably, the cell is modified to produce CMP-KDO. More preferably, the cell is modified for enhanced CMP-KDO production. Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of over-expression of a d- arabinose 5-phosphate isomerase, a KDO-8P synthase, a KDO 8-phosphate phosphatase and/or a CMP- KDO synthetase encoding gene.
  • the cell used herein is optionally genetically engineered to import a precursor and/or an acceptor in the cell, by the introduction and/or overexpression of a transporter able to import the respective precursor and/or acceptor in the cell.
  • a transporter is for example a membrane protein belonging to the major facilitator superfamily (MFS), the ATP-binding cassette (ABC) transporter family or the PTS system involved in the uptake of e.g., mono-, di- and/or oligosaccharides.
  • the cell used herein is optionally genetically engineered to produce polyisoprenoid alcohols like e.g., phosphorylated dolichol that can act as lipid carrier.
  • polyisoprenoid alcohols like e.g., phosphorylated dolichol that can act as lipid carrier.
  • the cell used herein is an E. coli or yeast with a lactose permease positive phenotype.
  • said lactose permease is coded by the gene LacY or LAC12, respectively.
  • the cell used herein is optionally genetically engineered to import lactose in the cell, by the introduction and/or overexpression of a lactose permease, like e.g., encoded by the LacY gene or the LAC12 gene.
  • the cell expresses a membrane protein that is a transporter protein involved in transport of compounds, like e.g., a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc- R as defined in present invention out of the cell.
  • a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is preferably produced intracellularly.
  • 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 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the membrane transporter protein or polypeptide having transport activity provides improved production of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the membrane transporter protein or polypeptide having transport activity provides enabled efflux of said sialylated compound comprising Gal- pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the membrane transporter protein or polypeptide having transport activity provides enhanced efflux of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the cell is transformed to comprise at least one nucleic acid sequence encoding a protein selected from the list comprising, consisting of or consisting essentially of a lactose transporter like e.g. the LacY or 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R 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 Iac
  • 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, consisting of or consisting essentially of E. coli (UniProt ID P0AEY8), Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), Citrobacter youngae (UniProt ID D4BC23) and Yokenella regensburgei (UniProt ID G9Z5F4).
  • a membrane transporter protein belonging to the family of MFS transporters like e.g., an MdfA polypeptide of the multidrug transporter MdfA family from species comprising, consisting of or consisting essentially of E. coli (UniProt ID P0AEY8), Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), Citrobacter younga
  • the cell expresses a membrane transporter protein belonging to the family of sugar efflux transporters like e.g., a SetA polypeptide of the SetA family from species comprising E. coli (UniProt ID P31675, sequence version 03 (11 Oct 2004)) and Citrobacter koseri (UniProt ID A0A078LM16).
  • 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 lactis subsp. lactis bv. diacetylactis (UniProt ID A0A1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4).
  • a membrane transporter protein belonging to the family of ABC transporters like e.g., oppF from E. coli (UniProt ID P77737), ImrA from Lactococcus lactis subsp. lactis bv. diacetylactis (UniProt ID A0A1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID B7GPD4).
  • the cell expresses more than one membrane transporter protein selected from the list comprising, consisting of or consisting essentially of a lactose transporter like e.g. the LacY or 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.
  • MFS major facilitator superfamily
  • ABS ATP-binding cassette
  • the cell is capable to synthesize N-acetylmannosamine (ManNAc) and/or N-acetylmannosarnine-6-phosphate (ManNAc-6-phosphate).
  • the cell comprises a pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R comprising a pathway for production of ManNAc.
  • ManNAc can be provided by an enzyme expressed in the cell or by the mechanism of the cell.
  • Such cell producing ManNAc can express an N-acylglucosamine 2-epimerase like is known e.g. from several species including Bacteroides ovatus, E. coli, Homo sapiens, Rattus norvegicus that converts GIcNAc into ManNAc.
  • the cell producing ManNAc can express an UDP-N-acetylglucosamine 2-epimerase like is known e.g. from several species including Campylobacter jejuni, E. coli, Neisseria meningitidis, Bacillus subtilis, Citrobacter rodentium that converts UDP-GIcNAc into ManNAc.
  • GIcNAc and/or UDP-GIcNAc can be added to the cell and/or provided by an enzyme expressed in the cell or by the mechanism of the cell as described herein.
  • the cell is modified for enhanced ManNAc production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knockout of N-acetylmannosamine kinase, over-expression of N-acetylneuraminate lyase.
  • the cell comprises a pathway for production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R comprising a pathway for production of ManNAc-6- phosphate.
  • ManNAc-6-phosphate can be provided by an enzyme expressed in the cell or by the mechanism of the cell.
  • Such cell producing ManNAc-6-phosphate can express a bifunctional UDP-GIcNAc 2-epimerase/kinase like is known e.g. from several species including Homo sapiens, Rattus norvegicus and Mus musculus that converts UDP-GIcNAc into ManNAc-6-phosphate.
  • the cell producing ManNAc-6-phosphate can express an N-acetylmannosamine-6-phosphate 2-epimerase that converts GlcNAc-6-phosphate into ManNAc-6-phosphate.
  • UDP-GIcNAc and/or GlcNAc-6-phosphate can be added to the cell and/or provided by an enzyme expressed in the cell or by the mechanism of the cell as described herein.
  • the cell is modified for enhanced ManNAc-6- phosphate production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of over-expression of N-acetylglucosamine-6-phosphate deacetylase, over-expression of N-acetyl-D-glucosamine kinase, over-expression of phosphoglucosamine mutase, over-expression of N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-1- phosphate acetyltransferase.
  • the cell is capable to synthesize any one or more nucleotide-activated sugars.
  • the cell comprises a pathway for the synthesis of one or more nucleotide-activated sugars 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), UDP- glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy--L-arabino-4-hexulose, UDP-2- aceta
  • the cell is capable to synthesize at least the nucleotide- activated sugar CMP-Neu5Ac.
  • the cell is capable to synthesize at least the nucleotide-activated sugar CMP-KDO.
  • the cell uses at least one of the synthesized nucleotide-activated sugars in the production of a sialylated compound comprising Gal-pi,3- [Neu5Ac-a2,6]-HexNAc-R.
  • the cell is genetically engineered for production of one or more of said nucleotide-activated sugar(s) selected from the list comprising, consisting of or consisting essentially of UDP-GIcNAc, UDP-GalNAc, UDP-ManNAc, UDP-GIc, UDP-Gal, 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-L-RhaNAc, dTDP-N-acetylfucosamine, UDP-L-FucNAc, UDP-L-PneNAC, UDP-N- acetylmuramic acid, UDP-L-QuiNAc, CMP-sialic acid (e.g.
  • 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, and an N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli.
  • the cell is modified to produce UDP-GIcNAc. More preferably, the cell is modified for enhanced UDP-GIcNAc production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knock-out of an N-acetylglucosamine-6- phosphate deacetylase, over-expression of an L-glutamine— D-fructose-6-phosphate aminotransferase, over-expression of a phosphoglucosamine mutase, and over-expression of an N-acetylglucosamine-1- phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase.
  • the cell used herein is optionally genetically engineered to express the de novo synthesis of CMP-Neu5Ac.
  • CMP-Neu5Ac can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing CMP-Neu5Ac can express an enzyme converting, e.g., sialic acid to CMP-Neu5Ac.
  • This enzyme may be a CMP-sialic acid synthetase, like the N-acylneuraminate cytidylyltransferase from several species including Homo sapiens, Neisseria meningitidis, and Pasteurella multocida.
  • the cell is modified to produce CMP-Neu5Ac. More preferably, the cell is modified for enhanced CMP-Neu5Ac production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knock-out of an N-acetylglucosamine-6-phosphate deacetylase, knock-out of a glucosamine-6-phosphate deaminase, over-expression of a CMP-sialic acid synthetase, and over-expression of an N-acetyl-D-glucosamine-2-epimerase encoding gene.
  • CMP-KDO can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing CMP-KDO can express an enzyme converting, e.g., KDO to CMP-KDO.
  • This enzyme may be a CMP-KDO synthetase, like the 3-deoxy-manno-octulosonate cytidylyltransferase kdsB from several species including Escherichia coli, Arabidopsis thaliana, Pseudomonas aeruginosa, Xanthomonas campestris.
  • the cell is modified to produce CMP-KDO. More preferably, the cell is modified for enhanced CMP-KDO production. Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of over-expression of a d-arabinose 5-phosphate isomerase, a KDO-8P synthase, a KDO 8-phosphate phosphatase and/or a CMP-KDO synthetase encoding gene.
  • the cell used herein is optionally genetically engineered to express the de novo synthesis of GDP-fucose.
  • GDP-fucose can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing GDP-fucose can express an enzyme converting, e.g., fucose, which is to be added to the cell, to GDP-fucose.
  • This enzyme may be, e.g., a bifunctional fucose kinase/fucose-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 engineered to express the de novo synthesis of UDP-Gal.
  • UDP-Gal can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing UDP-Gal can express an enzyme converting, e.g. UDP-glucose, to UDP-Gal.
  • This enzyme may be, e.g., the UDP-glucose-4-epimerase GalE like as known from several species including Homo sapiens, Escherichia coli, and Rattus norvegicus.
  • the cell is modified to produce UDP-Gal. More preferably, the cell is modified for enhanced UDP-Gal production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knock-out of a bifunctional 5'-nucleotidase/UDP-sugar hydrolase encoding gene, knock-out of a galactose-l-phosphate uridylyltransferase encoding gene and over-expression of a UDP-glucose-4- epimerase encoding gene.
  • the cell used herein is optionally genetically engineered to express the de novo synthesis of UDP-GalNAc.
  • UDP-GalNAc can be synthesized from UDP-GIcNAc by the action of a single-step reaction using a UDP-N-acetylglucosamine 4-epimerase like e.g. wbgU from Plesiomonas shigelloides, gne from Yersinia enterocolitica or wbpP from Pseudomonas aeruginosa serotype 06.
  • the cell is modified to produce UDP-GalNAc. More preferably, the cell is modified for enhanced UDP-GalNAc production.
  • the cell used herein is optionally genetically engineered to express the de novo synthesis of UDP-ManNAc.
  • UDP-ManNAc can be synthesized directly from UDP-GIcNAc via an epimerization reaction performed by a UDP-GIcNAc 2-epimerase (like e.g. cap5P from Staphylococcus aureus, RffE from E. coli, Cpsl9fK from S. pneumoniae, and RfbC from S. enterica).
  • a UDP-GIcNAc 2-epimerase like e.g. cap5P from Staphylococcus aureus, RffE from E. coli, Cpsl9fK from S. pneumoniae, and RfbC from S. enterica.
  • the cell is modified to produce UDP-ManNAc. More preferably, the cell is modified for enhanced UDP-ManNAc production.
  • the cell 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-1- phosphate guanylyltransferase, L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase
  • 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, 3/4-fucosyltransferase, alpha-1, 4-fucosyltransferase and alpha-1, 6- fucosyltransferase.
  • the sialyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-2, 3- sialyltransferase, alpha-2, 6-sialyltransferase, and alpha-2, 8-sialyltransferase.
  • the galactosyltransferase is selected from the list comprising, consisting of or consisting essentially of beta- 1,3-galactosyltransferase, N-acetylglucosamine beta-1, 3-galactosyltransferase, beta-1, 4- galactosyltransferase, N-acetylglucosamine beta-1, 4-galactosyltransferase, alpha-1,3- galactosyltransferase and alpha-1, 4-galactosyltransferase.
  • the glucosyltransferase is selected from the list comprising, consisting of or consisting essentially of alphaglucosyltransferase, 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-l,3-N-acetylgalactosaminyltransferase.
  • the cell is modified in the expression or activity of at least one of said glycosyltransferases.
  • said glycosyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous glycosyltransferase is overexpressed; alternatively said glycosyltransferase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous glycosyltransferase can have a modified expression in the cell which also expresses a heterologous glycosyltransferase.
  • the cell comprises a fucosylation pathway comprising at least one enzyme 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, fucosyltransferase.
  • a fucosylation pathway comprising at least one enzyme selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase,
  • the cell comprises a galactosylation pathway comprising at least one enzyme selected from the list comprising, consisting of or consisting essentially of galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase, galactosyltransferase.
  • a galactosylation pathway comprising at least one enzyme selected from the list comprising, consisting of or consisting essentially of galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phospho
  • the cell comprises an N-acetylglucosaminylation pathway comprising at least one enzyme selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N- acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase, N- acetylglucosaminyltransferase.
  • the cell is capable to produce phosphoenolpyruvate (PEP).
  • PEP phosphoenolpyruvate
  • the cell comprises a pathway for production of PEP.
  • the cell is modified for enhanced synthesis and/or supply of PEP compared to a non-modified progenitor.
  • one or more PEP-dependent, sugar-transporting phosphotransferase system(s) is/are disrupted such as but not limited to: 1) the N-acetyl-D-glucosamine Npi-phosphotransferase (EC 2.7.1.193), which is for instance encoded by the nagE gene (or the cluster nagABCD) in E.
  • ManXYZ which encodes the Enzyme 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 of Shewanella 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, consisting of or consisting essentially of phosphoenolpyruvate synthase activity (EC: 2.7.9.2 encoded for instance in E. coli by ppsA), phosphoenolpyruvate carboxykinase activity (EC 4.1.1.32 or EC 4.1.1.49 encoded for instance in Corynebacterium glutamicum by PCK or in E. coli by pckA, resp.), phosphoenolpyruvate carboxylase activity (EC 4.1.1.31 encoded for instance in E.
  • coli by ppc oxaloacetate decarboxylase activity
  • EC 4.1.1.112 encoded for instance in E. coli by eda oxaloacetate decarboxylase activity
  • EC 2.7.1.40 encoded for instance in E. coli by pykA and pykF pyruvate carboxylase activity
  • malate dehydrogenase activity EC 1.1.1.38 or EC 1.1.1.40 encoded for instance in E. coli by maeA or maeB, resp.
  • the cell is modified by a reduced activity of phosphoenolpyruvate carboxylase activity, and/or pyruvate kinase activity, preferably a deletion of the genes encoding for phosphoenolpyruvate carboxylase, the pyruvate carboxylase activity and/or pyruvate kinase.
  • the cell 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, consisting of or consisting essentially of beta-galactosidase, galactoside O- acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP- glucose:undecaprenyl-phosphate glucose-l-phosphate transferase, L-fuculokinase, L-fucose isomerase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2- epimerase, E
  • one or more gene(s) involved in one or more reductive pathway(s) in the cell is/are rendered less functional compared to a non-modified progenitor or is/are knocked-out.
  • a reductive pathway comprise but are not limited to the reductive acetyl-CoA-pathway, the reductive pyrimidine catabolic pathway, the reductive citric acid cycle, the thiol-redox pathway and the reductive glycine pathway.
  • one or more gene(s) involved in one or more reductive pathway(s) is/are rendered less functional by insertion, deletion and/or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) selected from the list comprising, consisting of or consisting essentially of promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said one or more gene(s).
  • said one or more gene(s) may be selected from the list comprising, consisting of or consisting essentially of gene(s) encoding formate dehydrogenase, formate-tetrahydrofolate ligase, methenyltetrahydrofolate cyclohydrolase, glycine dehydrogenase/decarboxylating, glycine cleavage system protein, glutamate dehydrogenase, glycine reductase complex, 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 cell expresses, preferably overexpresses, at least one gene selected from the list comprising, consisting of or consisting essentially of genes encoding a disulfide bond isomerase, a thiol oxidase and a chaperone.
  • the cell is capable to produce, preferably produces, said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc- R from one or more precursor(s).
  • said precursor is lactose.
  • the precursor is fed to the cell from the cultivation or incubation medium.
  • the cell is capable to produce, preferably produces, one or more precursor(s) for the synthesis of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the cell is capable to produce, preferably produces, all of said one or more precursor(s) for the synthesis of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the cell is genetically engineered for the production of at least one of said one or more precursor(s) for the synthesis of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R. More preferably, the cell is genetically engineered for the production of all of said one or more precursor(s) for the synthesis of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • At least one of said one or more precursor(s) is internalized in said cell via one or more membrane protein(s).
  • the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the synthesis of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein.
  • the method results in the production of 0.1 g/L or more, preferably 0.5 g/L or more, more preferably 1 g/L or more, more preferably 5 g/L or more, even more preferably 10 g/L or more, even more preferably 20 g/L or more, even more preferably 30 g/L or more, even preferably 40 g/L or more, most preferably 50 g/L or more of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein.
  • the cell produces 25 g/L or more of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein, in the whole broth and/or supernatant.
  • a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R produced in the whole broth and/or supernatant has a purity of at least 80 % measured on the total amount of the sialylated compound comprising Gal-pi,3- [Neu5Ac-a2,6]-HexNAc-R and its precursor produced by the cell in the whole broth and/or supernatant, respectively.
  • the method results in the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R with a purity equal to or greater than 80 % measured on the total amount of the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R and its precursor.
  • the method results in the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R with a purity equal to or greater than 85 % measured on the total amount of the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R and its precursor.
  • the method results in the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc- R with a purity equal to or greater than 91 %, equal to or greater than 92 %, equal to or greater than 93 %, equal to or greater than 94 %, equal to or greater than 95 %, equal to or greater than 96 %, equal to or greater than 97 %, equal to or greater than 98 %, equal to or greater than 99 % measured on the total amount of the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R and its precursor.
  • the method results in the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R with a purity equal to or greater than 90 % measured on the total amount of the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc- R and its precursor.
  • the method results in the production of a mixture comprising a sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R and sialic acid.
  • the method results in the production of a mixture comprising a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, lactose and sialic acid.
  • the method results in the production of a mixture comprising a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, LSTa and/or sialic acid.
  • the method results in the production of a mixture comprising a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, LNT, lactose and/or sialic acid.
  • said mixture is a saccharide mixture consisting essentially of saccharides like e.g., a monosaccharide, a disaccharide and/or an oligosaccharide.
  • the method results in the production of a mixture comprising sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R, together with lactose and sialic acid, wherein said sialylated compound comprising Gal- pi,3-[Neu5Ac-a2,6]-HexNAc-R has a purity equal to or greater than 80 % measured on the total amount of the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, lactose and sialic acid in said mixture and wherein said mixture comprises less than 10 % lactose and/or less than 5 % sialic acid.
  • said mixture comprises less than 9 % lactose. In an even more preferred embodiment, said mixture comprises less than 8 % lactose. In another even more preferred embodiment, said mixture comprises less than 7 %, less than 6 %, less than 5 %, less than 4 %, less than 3 %, less than 2 %, less than 1 % lactose. In an additional and/or alternative more preferred embodiment, said mixture comprises less than 5 % sialic acid. In an even more preferred additional and/or alternative embodiment, said mixture comprises less than 4 %, less than 3 %, less than 2 %, less than 1 %, less than 0.5 %, less than 0.1 % sialic acid. Preferably, said mixture is a saccharide mixture consisting essentially of saccharides like e.g., a monosaccharide, a disaccharide and/or an oligosaccharide.
  • the method results in the production of a mixture comprising sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R and sialic acid, wherein said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]- HexNAc-R has a purity equal to or greater than 80 % measured on the total amount of the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, and sialic acid in said mixture and wherein said mixture comprises less than 5 % sialic acid.
  • said mixture comprises less than 4 %, less than 3 %, less than 2 %, less than 1 %, less than 0.5 %, less than 0.1 % sialic acid.
  • said mixture is a saccharide mixture consisting essentially of saccharides like e.g., a monosaccharide, a disaccharide and/or an oligosaccharide.
  • the cell is capable to synthesize a mixture of oligosaccharides.
  • the cell is capable to synthesize a mixture of di- and oligosaccharides, alternatively, the cell is capable to synthesize a mixture of sialic acid, di- and/or oligosaccharides.
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is produced in and/or by a cell which is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell.
  • the latter bacterium preferably belongs to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus or the phylum of Actinobacteria.
  • the latter bacterium belonging to the phylum Proteobacteria belongs preferably to the family Enterobacteriaceae, preferably to the species Escherichia coli.
  • the latter bacterium preferably relates to any strain belonging to the species Escherichia coli such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultivated Escherichia coli strains - designated as E. coli K12 strains - which are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine.
  • E. coli K12 strains are K12 Wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200.
  • the present invention specifically relates to a mutated and/or transformed Escherichia coli cell or strain as indicated above wherein said E. coli strain is a K12 strain. More preferably, the Escherichia coli K12 strain is E. coli MG1655.
  • the latter bacterium belonging to the phylum Firmicutes belongs preferably to the Bacilli, preferably Lactobacilliales, with members such as Lactobacillus lactis, Leuconostoc mesenteroides, or 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 lipolitica, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha, Kluyveromyces marxianus, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Zygosaccharomyces rouxii, and Zygosaccharomyces bailii.
  • the latter fungus belongs preferably to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus.
  • Plant cells include cells of flowering and non-flowering plants, as well as algal cells, for example Chlamydomonas, Chlorella, etc.
  • said plant is a tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant.
  • the latter animal cell is preferably derived from non-human mammals (e.g.
  • primate e.g., chimpanzee, orangutan, gorilla, monkey (e.g., Old World, New World), lemur)
  • dog cat, rabbit, horse, cow, goat, ox, deer, musk deer, bovid, whale, dolphin, hippopotamus, elephant, rhinoceros, giraffe, zebra, lion, cheetah, tiger, panda, red panda, otter
  • birds e.g. chicken, duck, ostrich, turkey, pheasant
  • fish e.g. swordfish, salmon, tuna, sea bass, trout, catfish
  • invertebrates e.g.
  • Both human and non-human mammalian cells are preferably selected from the list comprising, consisting of or consisting essentially of an epithelial cell like e.g., a mammary epithelial cell, an embryonic kidney cell (e.g., HEK293 or HEK 293T cell), a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell like e.g.
  • an epithelial cell like e.g., a mammary epithelial cell, an embryonic kidney cell (e.g., HEK293 or HEK 293T cell), a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell like e.g.
  • the latter insect cell is preferably derived from Spodoptera frugiperda like e.g., Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae, Trichoplusia ni like e.g., BTI-TN-5B1-4 cells or Drosophila melanogaster like e.g., Drosophila S2 cells.
  • the latter protozoan cell preferably is a Leishmania tarentolae cell.
  • 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, fungal cells, plant cells, animal cells, insect cells and protozoan cells as described herein.
  • sialyltransferase has alpha-2, 6-sialyltransferase activity on the HexNAc residue of Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R and comprises a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[GS]X[HKR][ST]X[FILMV][HKR]XXX[HFY] with SEQ ID NO 01 wherein X can be any amino acid residue and wherein n is 10 or 11.
  • the HexNAc residue in said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3- Gal-pi,3-HexNAc-R is GIcNAc or GalNAc
  • the R group in said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3- Gal-pi,3-HexNAc-R is absent, a monosaccharide, a disaccharide, an oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or a glycolipid, as described herein.
  • the sialyltransferase encoded by said isolated nucleic acid molecule is a polypeptide comprising an amino acid sequence that is at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, at least 99 % identical to any one of the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • the sialyltransferase encoded by said isolated nucleic acid molecule comprises a polypeptide as represented by any one of SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • Another aspect of the present invention provides for a vector comprising an isolated nucleic acid molecule encoding a sialyltransferase as described herein.
  • a further aspect of the present invention provides a method for the production of a biologically active sialyltransferase by a cell, wherein said sialyltransferase is an alpha-2, 6-sialyltransferase having alpha-2, 6- sialyltransferase activity on the HexNAc residue of Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3- HexNAc-R and comprises a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[GS]X[HKR][ST]X[FILMV][HKR]XXX[HFY] with SEQ ID NO 01 wherein X can be any amino acid residue and wherein n is 10 or 11.
  • the HexNAc residue in said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is GIcNAc or GalNAc
  • the R group in said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is absent, a monosaccharide, a disaccharide, an oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or a glycolipid, as described herein.
  • the method comprises cultivation of a cell of present invention under conditions permissive to express said sialyltransferase, and protein extraction from the cell cultivation.
  • the biologically active sialyltransferase produced by said method is a polypeptide comprising an amino acid sequence that is at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, at least 99 % identical to any one of the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • the biologically active sialyltransferase produced by said method comprises a polypeptide as represented by any one of SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • a cell to be stably cultured in a cultivation or incubation medium
  • said cultivation or incubation medium can be any type of growth medium comprising, consisting of or consisting essentially of minimal medium, complex medium or growth medium enriched in certain compounds like, for example, but not limited to, vitamins, trace elements, amino acids.
  • the cell as used herein is capable to grow on a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium or a mixture thereof as the main carbon source.
  • main is meant the most important carbon source for the cell for the production of biologically active sialyltransferase as described herein and/or the sialylated compound comprising Gal-pi,3- [Neu5Ac-a2,6]-HexNAc-R of interest, biomass formation, carbon dioxide and/or by-products formation (such as acids and/or alcohols, such as acetate, lactate, and/or ethanol), i.e.
  • said carbon source is the sole carbon source for said organism, i.e. 100 % of all the required carbon is derived from the above-indicated carbon source.
  • Common main carbon sources comprise but are not limited to glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate.
  • a precursor as defined herein cannot be used as a carbon source for the production of the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R of present invention.
  • the cultivation or incubation medium contains at least one compound selected from the list consisting of lactose, galactose, glucose, sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, UDP-GIcNAc and UDP-GalNAc.
  • the methods as described herein preferably comprises a step of separating the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R of present invention from said cultivation or incubation, otherwise said recovering the sialylated compound comprising Gal- pi,3-[Neu5Ac-a2,6]-HexNAc-R from the cultivation or incubation medium and/or the cell.
  • separating from said cultivation or incubation means harvesting, collecting, or retrieving said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R from the cell and/or the medium of its cultivation or incubation.
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R can be separated in a conventional manner from the aqueous culture medium, in which the cell was cultivated or incubated.
  • said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is still present in the cells producing the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R
  • conventional manners to free or to extract said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R out of the cells can be used, such as cell destruction using high pH, heat shock, sonication, French press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergent, hydrolysis, etc.
  • the cultivation or incubation medium and/or cell extract together and separately can then be further
  • said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R can be clarified in a conventional manner.
  • said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is clarified by centrifugation, flocculation, decantation and/or filtration.
  • Another step of separating said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R preferably involves removing substantially all the eventually remaining proteins, peptides, amino acids, RNA and DNA, and any endotoxins and glycolipids that could interfere with the subsequent separation step, from said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, preferably after it has been clarified.
  • remaining proteins and related impurities can be removed from said sialylated compound comprising Gal- pi,3-[Neu5Ac-a2,6]-HexNAc-R in a conventional manner.
  • sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R of present invention.
  • a further purification of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R may be accomplished, for example, by use of (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange, temperature adjustment, pH adjustment or pH adjustment with an alkaline or acidic solution to remove any remaining DNA, protein, LPS, endotoxins, or other impurity.
  • Alcohols such as ethanol, and aqueous alcohol mixtures can also be used.
  • Another purification step is accomplished by crystallization, evaporation or precipitation of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • the separation and purification of the sialylated compound comprising Gal- pi,3-[Neu5Ac-a2,6]-HexNAc-R is made in a process, comprising the following steps in any order: a) contacting the cultivation or incubation or a clarified version thereof with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da ensuring the retention of the produced sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R 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 sialylated compound comprising
  • MWCO
  • the separation and purification of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is made in a process, comprising the following steps in any order: subjecting the cultivation or incubation or a clarified version thereof to two membrane filtration steps using different membranes, wherein: one membrane has a molecular weight cut-off of between about 300 to about 500 Dalton, and the other membrane as a molecular weight cut-off of between about 600 to about 800 Dalton.
  • the separation and purification of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is made in a process, comprising treating the cultivation or incubation or a clarified version thereof with a strong cation exchange resin in H+-form in a step and with a weak anion exchange resin in free base form in another step, wherein said steps can be performed in any order.
  • the separation and purification of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is made in the following way.
  • the cultivation or incubation comprising the produced sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, biomass, medium components and contaminants is applied to the following purification steps: i) separation of biomass from the cultivation or incubation, ii) cationic ion exchanger treatment for the removal of positively charged material, iii) anionic ion exchanger treatment for the removal of negatively charged material, iv) nanofiltration step and/or electrodialysis step, wherein a purified solution comprising the produced sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R at a purity of greater than or equal to 80 % is provided.
  • the purified solution is dried by any one or more drying steps selected from the list comprising, consisting of or consisting essentially of spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying and vacuum roller drying.
  • the separation and purification of the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is made in a process, comprising the following steps in any order: enzymatic treatment of the cultivation or incubation; removal of the biomass from the cultivation or incubation; ultrafiltration; nanofiltration; and a column chromatography step.
  • enzymatic treatment of the cultivation or incubation 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 sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R which is dried to powder by any one or more drying steps selected from the list comprising, consisting of or consisting essentially of spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying and vacuum roller drying, wherein the dried powder contains ⁇ 15 % -wt. of water, preferably ⁇ 10 % -wt. of water, more preferably ⁇ 7 % -wt. of water, most preferably
  • Another aspect of the present invention provides the use of a sialyltransferase that has alpha-2, 6- sialyltransferase activity on the HexNAc residue of Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3- HexNAc-R and comprises a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[GS]X[HKR][ST]X[FILMV][HKR]XXX[HFY] with SEQ ID NO 01 wherein X can be any amino acid residue and wherein n is 10 or 11.
  • the HexNAc residue in said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is GIcNAc or GalNAc
  • the R group in said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is absent, a monosaccharide, a disaccharide, an oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or a glycolipid, as described herein.
  • the sialyltransferase is a polypeptide comprising an amino acid sequence that is at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, at least 99 % identical to any one of the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • the sialyltransferase comprises a polypeptide as represented by any one of SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • the sialyltransferase is used for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, as described herein, wherein said HexNAc residue in said sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R is GIcNAc or GalNAc and wherein said R in said sialylated compound comprising Gal-pi,3- [Neu5Ac-a2,6]-HexNAc-R is absent, a monosaccharide, a disaccharide, an oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or a glycolipid.
  • Another aspect of the present invention provides the use of an isolated nucleic acid molecule encoding a sialyltransferase of present invention for the production of said sialyltransferase in a biologically active form.
  • a further aspect of the present invention provides the use of a vector comprising an isolated nucleic acid molecule encoding a sialyltransferase of present invention for the production of said sialyltransferase in a biologically active form.
  • Another aspect provides the use of a cell of the invention for the production of a sialyltransferase of present invention. Preferably, the cell produces said sialyltransferase in a biologically active form.
  • Another aspect of the present invention provides the use of a method as described herein for the production of a sialyltransferase of present invention in a biologically active form.
  • Another aspect of the present invention provides the use of an isolated nucleic acid molecule encoding a sialyltransferase of present invention for the production of a sialylated compound comprising Gal-pi,3- [Neu5Ac-a2,6]-HexNAc-R as described herein.
  • a further aspect of the present invention provides the use of a vector comprising an isolated nucleic acid molecule encoding a sialyltransferase of present invention for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein.
  • Another aspect of the present invention provides the use of a cell of the invention for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein.
  • Another aspect of the present invention provides the use of a method of the invention for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R as described herein.
  • the invention also relates to the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]- HexNAc-R obtained by the methods according to the invention.
  • Said sialylated compound comprising Gal- pi,3-[Neu5Ac-a2,6]-HexNAc-R may be used for the manufacture of a preparation, as food additive, prebiotic, symbiotic, for the supplementation of baby food, adult food, infant animal feed, adult animal feed, or as either therapeutically or pharmaceutically active compound or in cosmetic applications.
  • said preparation comprises at least one sialylated compound comprising Gal- pi,3-[Neu5Ac-a2,6]-HexNAc-R that is obtainable, preferably obtained, by the methods as described herein.
  • a preparation is provided that further comprises at least one probiotic microorganism.
  • said preparation is a nutritional composition.
  • said preparation is a medicinal formulation, a dietary supplement, a dairy drink or an infant formula.
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R can easily and effectively be provided, without the need for complicated, time and cost consuming synthetic processes.
  • the monosaccharide or the monomeric building blocks e.g. the monosaccharide or glycan unit composition
  • the anomeric configuration of side chains e.g. the monosaccharide or glycan unit composition
  • the presence and location of substituent groups, degree of polymerization/molecular weight and the linkage pattern can be identified by standard methods known in the art, such as, e.g.
  • methylation analysis methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas chromatography-mass spectrometry), MALDI-MS (Matrix-assisted laser desorption/ionization-mass spectrometry), ESI-MS (Electrospray ionization-mass spectrometry), HPLC (High-Performance Liquid chromatography with ultraviolet or refractive index detection), HPAEC-PAD (High-Performance Anion-Exchange chromatography with Pulsed Amperometric Detection), CE (capillary electrophoresis), IR (infrared)/Raman spectroscopy, and NMR (Nuclear magnetic resonance) spectroscopy techniques.
  • GC-MS gas chromatography-mass spectrometry
  • MALDI-MS Microx-assisted laser desorption/ionization-mass spectrometry
  • ESI-MS Electropray ionization-mass spectrometry
  • 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
  • HPLC high performance liquid chromatography
  • GLC gas-liquid chromatography
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R 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 (gasliquid chromatography coupled with mass spectrometry).
  • a partial depolymerization is carried out using an acid or enzymes to determine the structures.
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R 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.
  • the separated and preferably also purified sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]- HexNAc-R as described herein is incorporated into a food (e.g., human food or feed), dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine.
  • a food e.g., human food or feed
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine.
  • the dietary supplement comprises at least one prebiotic ingredient and/or at least one probiotic ingredient.
  • a "prebiotic” is a substance that promotes growth of microorganisms beneficial to the host, particularly microorganisms in the gastrointestinal tract.
  • a dietary supplement provides multiple prebiotics, including the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R being a prebiotic produced and/or purified by a process disclosed in this specification, to promote growth of one or more beneficial microorganisms.
  • prebiotic ingredients for dietary supplements include other prebiotic molecules (such as HMOs) and plant polysaccharides (such as inulin, pectin, b- glucan and xylooligosaccharide).
  • a "probiotic" product typically contains live microorganisms that replace or add to gastrointestinal microflora, to the benefit of the recipient.
  • microorganisms include Lactobacillus species (for example, L. acidophilus and L. bulgaricus), Bifidobacterium species (for example, B. animalis, B. longum and B. infantis (e.g., Bi-26)), and Saccharomyces boulardii.
  • a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R produced and/or purified by a process of this specification is orally administered in combination with such microorganism.
  • oligosaccharides such as 2'- fucosyllactose, 3-fucosyllactose, 6'-sialyllactose
  • disaccharides such as lactose
  • monosaccharides such as glucose, galactose, L-fucose, sialic acid, glucosamine and N-acetylglucosamine
  • thickeners such as gum arabic
  • acidity regulators such as trisodium citrate
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is incorporated into a human baby food (e.g., infant formula).
  • Infant formula is generally a manufactured food for feeding to infants as a complete or partial substitute for human breast milk.
  • infant formula is sold as a powder and prepared for bottle- or cup-feeding to an infant by mixing with water. The composition of infant formula is typically designed to be roughly mimic human breast milk.
  • a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc- R produced and/or purified by a process in this specification is included in infant formula to provide nutritional benefits similar to those provided by the oligosaccharides in human breast milk.
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R 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, B3, B6, B12, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate) and possibly human milk oligosaccharides (HMOs).
  • carbohydrate sources e.g., lactose
  • protein sources e.g., whey protein concentrate and casein
  • fat sources e.g., vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and/or sunflower oil; and fish oils
  • vitamins such as vitamins A, Bb, Bi2, B3, B6, B12,
  • Such HMOs may include, for example, DiFL, lacto-N-triose II, LNT, LNnT, lacto-N-fucopentaose I, lacto-N-neofucopentaose, lacto-N-fucopentaose II, lacto-N- fucopentaose III, lacto-N-fucopentaose V, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, 6' -galactosyllactose, 3' -galactosyllactose, lacto-N-hexaose and lacto- N-neohexaose.
  • DiFL lacto-N-triose II, LNT, LNnT
  • lacto-N-fucopentaose I lacto
  • the one or more infant formula ingredients comprise non-fat milk, a carbohydrate source, a protein source, a fat source, and/or a vitamin and mineral.
  • the one or more infant formula ingredients comprise lactose, whey protein concentrate and/or high oleic safflower oil.
  • the concentration of the sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R in the infant formula is approximately the same concentration as the concentration of the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R generally present in human breast milk.
  • the sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is incorporated into a feed preparation, wherein said feed is selected from the list comprising, consisting of or consisting essentially of pet food, animal milk replacer, veterinary product, veterinary feed supplement, nutrition supplement, post weaning feed, or creep feed.
  • the methods and the cell of the invention preferably provide at least one of the following further surprising advantages when using a sialyltransferase as described herein:
  • sucrose Ys g sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc- R / g sucrose
  • sialylated compound comprising Gal- pi,3-[Neu5Ac-a2,6]-HexNAc-R, and/or
  • a method for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc- R comprising contacting a sialyltransferase with a mixture comprising: a) a donor comprising a sialic acid residue, and b) Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from said donor to the N-acetylhexosamine (HexNAc) residue of said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3- Gal-pi,3-HexNAc-R in an alpha-2, 6-glycosidic linkage resulting in the production of said sialylated compound comprising Gal-pi,3-[
  • Method according to embodiment 1, wherein said method comprises: i) contacting a cell extract comprising said sialyltransferase wherein said cell extract is obtained from a cell wherein said cell possesses, preferably expresses, more preferably, overexpresses said alpha-2, 6-sialyltransferase, with ii) a mixture comprising a) a donor comprising a sialic acid residue, and b) Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R, under conditions wherein said sialyltransferase catalyses the transfer of a sialic acid residue from said donor to the HexNAc residue of said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R in an alpha-2, 6-glycosidic linkage resulting in the production of said si
  • a method for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc- R comprising the steps of: i) providing a cell, preferably a single cell, expressing, preferably heterologously expressing, more preferably overexpressing, even more preferably heterologously overexpressing, a sialyltransferase wherein said sialyltransferase is an alpha-2, 6-sialyltransferase having alpha-2, 6- sialyltransferase activity on the HexNAc residue of Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal- pi,3-HexNAc-R and comprises a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[GS]X[HKR][ST
  • sialyltransferase is a polypeptide comprising an amino acid sequence that is at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, at least 99 % identical to any one of the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 , or comprises a polypeptide as represented by any one of SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • said sialic acid residue is at least one selected from the list consisting of Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulonic acid (KDO), preferably, said sialic acid residue is Neu5Ac, and/or said donor comprising a sialic acid residue is CMP-sialic acid, preferably said donor comprising a sialic acid residue selected from the list consisting of CMP-Neu5Ac, CMP-Neu4Ac, CMP- Neu5Ac9N 3 , CMP-Neu4,5Ac 2 , CMP-Neu5,7Ac 2 ,
  • Gal-pi,3-HexNAc-R is selected from the list comprising Gal-pi,3-GlcNAc (LNB, lacto-N-biose); Gal- pi,3-GlcNAc-R; Gal-pi,3-GlcNAc-pi,3-R; Gal-pi,3-GlcNAc-pi,3-Gal; Gal-pi,3-GlcNAc-pi,3-Gal-R; Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-R; Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc (LNT, lacto-N-tetraose); Gal-pi,3-GlcNAc-pi,4-R; Gal-pi,3-GalNAc; Gal-pi,3-GalNAc; Gal-pi,3-GalNAc-R;
  • Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is selected from the list comprising Neu5Ac-a2,3-Gal-pi,3- GIcNAc (3'SLNB, 3'-sialylated LNB); Neu5Ac-a2,3-Gal-pi,3-GlcNAc-R; Neu5Ac-a2,3-Gal-pi,3- GlcNAc-pi,3-R; Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-R; Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3- Gal-pi,4-R; Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc (LSTa, sialyllacto-N-tetraose
  • Gal-pi,3-HexNAc-R is Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc (LNT, lacto-N-tetraose) and said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is Gal-pi,3-[Neu5Ac-a2,6]- GlcNAc-pi,3-Gal-pi,4-Glc (LSTb, sialyllacto-N-tetraose b), or
  • Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc (LSTa, sialyllacto-N-tetraose a) and said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]- HexNAc-R is Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,3)-GlcNAc-pi,3-Gal-pi,4-Glc (DSLNT, disialyllacto-N-tetraose).
  • sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is an oligosaccharide, a milk oligosaccharide, a mammalian milk oligosaccharide (MMO) or a human milk oligosaccharide (HMO).
  • the method comprising use of a cultivation or an incubation medium, wherein: i) said cultivation or incubation medium comprises at least one precursor and/or acceptor feed for the production of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, and/or ii) at least one precursor and/or acceptor feed for the production of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is added to said cultivation or incubation medium, preferably, said precursor is selected from the list comprising sialic acid, said donor comprising a sialic acid residue, CMP-sialic acid, CMP-Neu5Ac, glucose, galactose, GIcNAc, GalNAc, UDP-GIcNAc, UDP- GalNAc, preferably, said acceptor is selected from the list comprising lactose, GlcNA
  • said cultivation or incubation medium contains at least one compound selected from the list consisting of lactose, galactose, glucose, sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, UDP-GIcNAc and UDP-GalNAc.
  • said cultivation or incubation medium contains at least one carbon source selected from the list comprising a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract; preferably, wherein said carbon source is selected from the list comprising 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.
  • carbon source is selected from the list comprising glucose, glycerol, fructose, sucrose,
  • Method according to any one of embodiments 12 to 14, the method comprising at least one of the following steps: i) Use of a cultivation or incubation medium comprising at least one precursor and/or acceptor; ii) Adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and/or acceptor feed wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation or incubation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the cultivation or incubation medium before the addition of said precursor and/or acceptor feed; iii) Adding to the cultivation or incubation medium in a reactor or incubator at least one precursor and/or acceptor feed wherein the total reactor or incubator volume ranges from 250 mL to 10.000 m 3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the cultivation
  • Method according to any one of embodiments 12 to 14, the method comprising at least one of the following steps: i) Use of a cultivation or incubation medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of precursor per litre of initial reactor or incubator volume wherein the reactor or incubator volume ranges from 250 mL to 10.000 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 previous embodiments wherein said method results in the production of 0.5 g/L or more, preferably 1 g/L or more, more preferably 5 g/L or more, even more preferably 10 g/L or more, even more preferably 20 g/L or more, even more preferably 30 g/L or more, even preferably 40 g/L or more, most preferably 50 g/L or more of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R.
  • separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated charcoal or carbon treatment, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.
  • said purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration, ion exchange, use of alcohols, use of aqueous alcohol mixtures, crystallization, evaporation, precipitation, drying, spray drying or lyophilization.
  • sialyltransferase is an alpha-2, 6-sialyltransferase having alpha-2, 6-sialyltransferase activity on the HexNAc residue of Gal-pi,3-HexNAc-R and/or Neu5Ac- a2,3-Gal-pi,3-HexNAc-R and comprising a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[GS]X[HKR][ST]X[FILMV][HKR]XXX[HFY] with SEQ ID NO 01 wherein X can be any amino acid residue and wherein n is 10 or 11.
  • a metabolically engineered cell for the production of a sialylated compound comprising Gal-pi,3- [Neu5Ac-a2,6]-HexNAc-R wherein said cell has been metabolically engineered to possess, preferably to express, a sialyltransferase, characterized in that said sialyltransferase is an alpha-2, 6- sialyltransferase having alpha-2, 6-sialyltransferase activity on the HexNAc residue of Gal-pi,3- HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R and comprises a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[GS]X[HKR][ST]X[FILMV][HKR]XXX[HFY] with SEQ ID NO 01 where
  • sialyltransferase is a polypeptide comprising an amino acid sequence that is at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, at least 99 % identical to any one of the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 , or comprises a polypeptide as represented by any one of SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • Cell according to any one of embodiments 20 to 22, 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, fungal cells, plant cells, animal cells, insect cells, protozoan cells.
  • said cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell
  • said bacterium belongs to a phylum selected from the list comprising Proteobacteria, Firmicutes, Cyanobacteria, Deinococcus-Thermus and Actinobacteria; more preferably, said bacterium belongs to a family selected from the list comprising Enterobacteriaceae, Bacillaceae, Lactobacillaceae, Corynebacteriaceae and Vibrionaceae; even more preferably, said bacterium is selected from the list comprising an Escherichia coli strain, a Bacillus subtilis strain, a Vibrio natriegens strain; even more preferably said Escherichia coli strain is a K-12 strain, most preferably said Escherichia coli K-12 strain is E.
  • said fungus belongs to a genus selected from the list comprising Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus, preferably, said yeast belongs to a genus selected from the list comprising Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces, Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces or Torulaspora; more preferably, said yeast is selected from the list consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Tor
  • said cell comprises a pathway for production of said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, preferably said cell is genetically engineered to comprise said pathway, more preferably said cell comprises said pathway wherein said pathway has been genetically engineered.
  • said pathway is selected from the list comprising sialylation pathway, fucosylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N- acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway.
  • Cell according to embodiment 33 wherein said cell is capable to produce, preferably produces, at least one of said one or more precursor(s), preferably said cell is capable to produce, preferably produces, all of said one or more precursor(s).
  • Cell according to any one of embodiment 33 or 34 wherein said cell is genetically engineered for the production of at least one of said one or more precursor(s), preferably said cell is genetically engineered for the production of all of said one or more precursor(s).
  • 36 Cell according to any one of embodiments 33 to 35, wherein at least one of said one or more precursor(s) is internalized in said cell via one or more membrane protein(s).
  • Cell according to any one of embodiments 20 to 40 wherein said cell expresses, preferably overexpresses, at least one gene selected from the list comprising genes encoding a disulfide bond isomerase, a thiol oxidase, a chaperone.
  • Gal-pi,3-HexNAc-R is selected from the list comprising Gal-pi,3-GlcNAc (LNB, lacto-N-biose); Gal- pi,3-GlcNAc-R; Gal-pi,3-GlcNAc-pi,3-R; Gal-pi,3-GlcNAc-pi,3-Gal; Gal-pi,3-GlcNAc-pi,3-Gal-R; Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-R; Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc (LNT, lacto-N-tetraose); Gal-pi,3-GlcNAc-pi,4-R; Gal-pi,3-GalNAc; Gal-pi,3-GalNAc; Gal-pi,3-GalNAc-R;
  • Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is selected from the list comprising Neu5Ac-a2,3-Gal-pi,3- GIcNAc (3'SLNB, 3' -sialylated LNB); Neu5Ac-a2,3-Gal-pi,3-GlcNAc-R; Neu5Ac-a2,3-Gal-pi,3- GlcNAc-pi,3-R; Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-R; Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3- Gal-pi,4-R; Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc (LSTa, sialyllacto-N-tetraose
  • sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is an oligosaccharide, a milk oligosaccharide, a mammalian milk oligosaccharide (MMO), a human milk oligosaccharide (HMO) or is selected from the list comprising Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc; Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-R; Gal-pi,3-[Neu5Ac-a2,6]- GlcNAc-pi,3-R; Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-pi,4-R; Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-pi,3-Gal;
  • sialyltransferase having alpha-2, 6-sialyltransferase activity on the HexNAc residue of Gal-pi,3-HexNAc-R and/or Neu5Ac- a2,3-Gal-pi,3-HexNAc-R and comprises a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[GS]X[HKR][ST]X[FILMV][HKR]XXX[HFY] with SEQ ID NO 01 wherein X can be any amino acid residue and wherein n is 10 or 11, wherein said HexNAc in said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is
  • sialyltransferase is a polypeptide comprising an amino acid sequence that is at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, at least 99 % identical to any one of the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 , or comprises a polypeptide as represented by any one of SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • Method for the production of a biologically active sialyltransferase by a cell comprising: i) cultivation of a cell according to any one of embodiments 20 to 45 under conditions permissive to express said sialyltransferase, and ii) protein extraction from said cultivation, wherein said sialyltransferase is an alpha-2, 6-sialyltransferase having alpha-2, 6-sialyltransferase activity on the HexNAc residue of Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R and comprises a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[GS]X[HKR][ST]X[FILMV][HKR]XXX[HFY] with SEQ ID NO
  • sialyltransferase is a polypeptide comprising an amino acid sequence that is at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, at least 99 % identical to any one of the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 , or comprises a polypeptide as represented by any one of SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • Method for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R wherein said HexNAc in said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is GIcNAc or GalNAc and wherein said R in said sialylated compound comprising Gal-pi,3-[Neu5Ac- a2,6]-HexNAc-R is absent, a monosaccharide, a disaccharide, an oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or a glycolipid, the method comprising: i) cultivating and/or incubating a cell of any one of embodiments 21 to 45, in cultivation and/or incubation medium under conditions permissive to produce said sialylated compound comprising Gal-pi,3-[Neu5
  • sialyltransferase that has alpha-2, 6-sialyltransferase activity on the HexNAc residue of Gal- pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R and comprises a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[GS]X[HKR][ST]X[FILMV][HKR]XXX[HFY] with SEQ ID NO 01 wherein X can be any amino acid residue and wherein n is 10 or 11, wherein said HexNAc residue in said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is GIcNAc or GalNAc, and wherein said R in said Gal-pi,
  • sialyltransferase is a polypeptide comprising an amino acid sequence that is at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 98.5 %, at least 99 % identical to any one of the full-length amino acid sequences as represented by SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 , or comprises a polypeptide as represented by any one of SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.
  • any one of embodiment 52 or 53 for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, wherein said HexNAc residue in said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is GIcNAc or GalNAc and wherein said R in said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is absent, a monosaccharide, a disaccharide, an oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or a glycolipid.
  • an isolated nucleic acid molecule for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, wherein said HexNAc residue in said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is GIcNAc or GalNAc and wherein said R in said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]- HexNAc-R is absent, a monosaccharide, a disaccharide, an oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or a glycolipid.
  • a vector according to embodiment 48 for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R, wherein said HexNAc residue in said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is GIcNAc or GalNAc and wherein said R in said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is absent, a monosaccharide, a disaccharide, an oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or a glycolipid.
  • a cell according to any one of embodiments 20 to 45 for the production of a sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R wherein said HexNAc residue in said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is GIcNAc or GalNAc and wherein said R in said sialylated compound comprising Gal-pi,3-[Neu5Ac-a2,6]-HexNAc-R is absent, a monosaccharide, a disaccharide, an oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or a glycolipid.
  • X can be any amino acid residue and wherein n is 10 or 11, wherein said HexNAc residue in said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R is GIcNAc or N-GalNAc, and wherein said R in said Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3- HexNAc-R is absent, a monosaccharide, a disaccharide, an oligosaccharide, a protein, a glycoprotein, a peptide, a glycopeptide, a lipid or a glycolipid.
  • GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. (1970) 48: 443-453) to find the global (i.e., spanning the full-length sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps.
  • the BLAST algorithm (Altschul et al., J. Mol. Biol. (1990) 215: 403-10) calculates the global percentage sequence identity (i.e., over the full-length sequence) and performs a statistical analysis of the similarity between the two sequences.
  • the software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologs may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity (i.e., spanning the full-length sequences) may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics (2003) 4:29). Minor manual editing may be performed to optimize alignment between conserved motifs, as would be apparent to a person skilled in the art.
  • the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1); 195-7).
  • Genes related to proteins or protein sequences with alpha-2, 6-sialyltransferase activity on the HexNAc residue of Gal-pi,3-HexNAc-R and/or Neu5Ac-a2,3-Gal-pi,3-HexNAc-R can be obtained from sequence databases like Uniprot (https://www.uniprot.org/), NCBI nr or nt databases (https://www.ncbi.nlm.nih.gov/) and others.
  • GT29 glycosyltransferases comprising a polypeptide sequence comprising a conserved domain GX(no S)X[ILV][DEQ]XXXCXXRM[NS]X(no Q)(Xn)[GS]X[HKR][ST]X[FILMV][HKR]XXX[HFY] with SEQ ID NO 01 wherein X can be any amino acid residue and wherein n is 10 or 11.
  • GT29 glycosyltransferases are represented by PFAM domain PF00777 Glyco_transf_29. Members of this family were extracted using the Uniprot sequence database. In total 25885 protein sequences were found (30/06/2023).
  • a regex search with the domain 'G[ A S].[ILV][EDQ]...C..RM[NS][ A Q][A-Z] ⁇ 10,ll ⁇ [GS].[HKR][ST].[FILMV] [HKR]...[HFY]' was performed with a custom script in python (https://www.python.org/; https://docs.python.Org/3/library/re.html) resulting in 2653 identifiers. Amino acid sequences were clustered using CD-HIT (http://weizhongli-lab.org/cd-hit/) with a sequence identity threshold of 80% and further filtered on completeness and source.
  • the representative sequences are the next 196 identifiers: G3PE22, A0A851X5G9, A0A6J0TKG4, L5K3H9, A0A3Q1GR44, A0A3Q1GWI1, A0A4W3I0G7, A0A4W3HU88, A0A5N4E9R9, A0A5N4DDH8, A0A3Q3A9Y9, A0A3Q2V979, A0A8C0ZUJ5, A0A8C2W9I3, A0A6Q2YGB5, A0A3P8YHV0, A0A3P8Z6L8, A0A8C7CU02, A0A8C7HUA8, A0A2I0MED9, A0A851SKS3, Q5NDG1, B0S5A8, A0A8M6YV93, A0A811Z310, A0A096M399, A0A4W5LP
  • the Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium).
  • the minimal medium used in the cultivation experiments in 96-well plates or in shake flasks contained 2.00 g/L NH 4 CI, 5.00 g/L (NH 4 ) 2 SO 4 , 2.993 g/L KH 2 PO 4 , 7.315 g/L K 2 HPO 4 , 8.372 g/L MOPS, 0.5 g/L NaCI, 0.5 g/L MgSO 4 .7H 2 O, 30 g/L sucrose or 30 g/L glycerol, 1 ml/L vitamin solution, 100 pl/L molybdate solution, and 1 mL/L selenium solution.
  • Vitamin solution consisted of 3.6 g/L FeCI 2 .4H 2 O, 5.0 g/L CaCI 2 .2H 2 O, 1.3 g/L MnCI 2 .2H 2 O, 0.38 g/L CuCI 2 .2H 2 O, 0.5 g/L CoCI 2 .6H 2 O, 0.94 g/L ZnCI 2 , 0.0311 g/L H3BO 4 , 0.4 g/L Na 2 EDTA.2H 2 O and 1.01 g/L thiamine.
  • sialic acid and/or 20 g/L lactose were additionally added to the medium.
  • Complex medium was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 pm Sartorius).
  • the medium was made selective by adding an antibiotic: e.g., chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L).
  • an inducer for inducible gene expression like e.g., IPTG or arabinose was added.
  • a preculture for the bioreactor was started from an entire 1 mL cryovial of a certain strain, inoculated in 250 mL or 500 mL minimal medium in a 1 L or 2.5 L shake flask and incubated for 24 h at 37°C on an orbital shaker at 200 rpm.
  • a 5 L bioreactor was then inoculated (250 mL inoculum in 2 L batch medium); the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsoder, Germany). Culturing condition were set to 37 °C, and maximal stirring; pressure gas flow rates were dependent on the strain and bioreactor.
  • the pH was controlled at 6.8 using 0.5 M H2S04 and 20% NH4OH.
  • the exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.
  • a preculture of 96-well microtiter plate experiments was started from a cryovial, in 175 pL LB and was incubated overnight at 37°C on an orbital shaker at 800 rpm. This culture was used as inoculum for a 24-well deep well microtiter plate with 4 mL LB medium by diluting 50x. These final 24-well culture plates were incubated at 16°C or 37°C on an orbital shaker at 200 rpm for 5 to 24 h, or shorter or longer. If necessary, an inducer like e.g., IPTG was added to induce gene expression.
  • an inducer like e.g., IPTG was added to induce gene expression.
  • the 24-well culture plates were centrifuged for 30 min at 4200 rpm and 4°C, the supernatant was discarded, and the pellets were frozen at -80°C for at least one hour.
  • Pellets were subsequently resuspended in 100 mM MES buffer pH 6.5 containing 20 mM MgCL, 1 mg/mL of lysozyme, 10 U/mL DNase and 1 mM protease inhibitor and incubated for 30 min at 37°C.
  • pellets were subsequently resuspended and lysed by sonication.
  • Cell lysates were optionally cleared by centrifuging the plates (30 min at 4200 rpm and 4°C) and transferring the supernatant to new plates.
  • a preculture of a shake flask experiment was started from a cryovial in 5 mL LB medium and was incubated overnight at 37°C on an orbital shaker at 200 rpm. This culture was used as inoculum for a shake flask Erlenmeyer filled with 50 to 250 mL LB medium by diluting 50x. These final cultures were then incubated at 16°C or 37°C on an orbital shaker at 200 rpm for 5 to 24 h, or longer or shorter. Afterwards, cells were harvested by centrifuging the cultures for 30 min at 4200 rpm and 4°C. The supernatant was discarded, and the pellets were frozen at -20°C for at least 2 hours.
  • pellets were resuspended and lysed by sonication.
  • Cell lysates were optionally cleared by centrifuging (30 min at 4200 rpm and 4°C) and the supernatant was transferred to a new recipient.
  • a preculture for a bioreactor was started from an entire 1 mL cryovial of a certain strain, inoculated in 250 mL or 500 mL LB 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 25-37°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 H2S0 4 and 20% NH 4 OH.
  • the exhaust gas was cooled.
  • 10% solution of silicone antifoaming agent was added when foaming raised during the fermentation.
  • a soft release of the product was established by physically disrupting cells by means of sonication.
  • Other commonly used methods known in the art are methods such as, freeze thawing and/or shear stress through mixing, a homogenizer and/or French press.
  • 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.
  • E. coli NiCo21 (DE3) was obtained from New England Biolabs, in November 2017.
  • E. coli SHuffle T7 Express was obtained from New England Biolabs, in December 2021.
  • E. coli Origami 2 (DE3) was obtained from Merck Life Science (Sigma), in December 2021.
  • Gene disruptions, gene introductions and gene replacements were performed using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645).
  • the mutant strain was derived from E. coli K12 MG1655 comprising genomic knock-ins of constitutive transcriptional units containing one or more copies of a glucosamine 6-phosphate N-acetyltransferase like e.g. GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), an N-acylglucosamine 2-epimerase like e.g. AGE from Bacteroides ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase like e.g.
  • GNA1 from Saccharomyces cerevisiae
  • N-acylglucosamine 2-epimerase like e.g. AGE from Bacteroides ovatus (UniProt ID A7LVG6)
  • N-acetylneuraminate synthase like e.g.
  • sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing an UDP-N- acetylglucosamine 2-epimerase like e.g., NeuC from C. jejuni (UniProt ID Q93MP8) and an N- acetylneuraminate synthase like e.g., NeuB from N. meningitidis (UniProt ID E0NCD4) or NeuB from C. jejuni (UniProt ID Q93MP9).
  • an UDP-N- acetylglucosamine 2-epimerase like e.g., NeuC from C. jejuni (UniProt ID Q93MP8) and an N- acetylneuraminate synthase like e.g., NeuB from N. meningitidis (UniProt ID E0NCD4) or NeuB from C. jejuni (UniProt ID Q93MP9).
  • sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, Sequence version 03 (23 Jan 2007)), an N-acetylglucosamine-1- phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase like e.g. glmU from E. coli (UniProt ID P0ACC7), an 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.
  • GNE from Mus musculus (strain C57BL/6J) (UniProt ID Q91WG8), an N-acylneuraminate-9-phosphate synthetase like e.g. NANS from Pseudomonas sp. UW4 (UniProt ID K9NPH9) and an N-acylneuraminate-9-phosphatase like e.g. NANP from Candidatus Magnetomorum sp. HK-1 (UniProt ID KPA15328.1) or NANP from Bacteroides thetaiotaomicron (UniProt ID Q8A712).
  • NANS from Pseudomonas sp. UW4
  • N-acylneuraminate-9-phosphatase like e.g. NANP from Candidatus Magnetomorum sp. HK-1 (UniProt ID KPA15328.1) or NANP from Bacteroides thetaiotaomicron (UniProt ID
  • sialic acid production can be obtained by genomic knock- ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03 (23 Jan 2007)), an N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase like e.g. glmU from E. coli (UniProt ID P0ACC7), a bifunctional UDP-GIcNAc 2-epimerase/N-acetylmannosamine kinase like e.g.
  • a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03 (23 Jan 2007)
  • GNE from M. musculus (strain C57BL/6J) (UniProt ID Q91WG8), an N-acylneuraminate-9-phosphate synthetase like e.g. NANS from Pseudomonas sp. UW4 (UniProt ID K9NPH9) and an N-acylneuraminate-9-phosphatase like e.g. NANP from C. Magnetomorum sp. HK-1 (UniProt ID KPA15328.1) or NANP from B. thetaiotaomicron (UniProt ID Q8A712).
  • NANS from Pseudomonas sp. UW4
  • N-acylneuraminate-9-phosphatase like e.g. NANP from C. Magnetomorum sp. HK-1 (UniProt ID KPA15328.1) or NANP from B. thetaiotaomicron (UniProt ID Q8A712).
  • Sialic acid production can further be optimized in the modified E. coli strain with genomic knock-outs of the E. coli genes comprising any one or more of nagA, nagB, nagC, nagD, nagE, nanA, nanE, nanK, manX, manY and manZ as described in WO18122225, and/or genomic knock-outs of the E.
  • coli genes comprising any one or more of 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. glmS from E. coli (UniProt ID P17169, Sequence version 04 (23 Jan 2007)) and an acetyl-CoA synthetase like e.g. acs from E. coli (UniProt ID P27550, Sequence version 02 (01 Oct 1993)).
  • sialic acid production strains were further modified to express an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from Pasteurella multocida (UniProt ID A0A849CI62) and to express an alpha-2, 3-sialyltransferase like e.g. PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3), an alpha-2, 6-sialyltransferase like e.g.
  • an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from Pasteurella multocida (UniProt ID A0A849CI62) and to express an alpha-2, 3-sialyltransferase like e.g. PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3), an alpha-2, 6-sialy
  • PdbST6 from Photobacterium damselae (UniProt ID 066375) and/or a sialyltransferase selected from the list comprising SEQ ID NOs 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 or a variant thereof comprising a fusion to a His6-tag and an MBP-tag like e.g., selected from the list comprising SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.
  • Constitutive transcriptional units of the N-acylneuraminate cytidylyltransferase and the sialyltransferase(s) can be delivered to the modified strain either via genomic knock-in or via expression plasmids.
  • the strains were additionally modified with genomic knock-outs of the E. coli LacZ, LacY and LacA genes and with a genomic knock-in of a constitutive transcriptional unit for a lactose permease like e.g. E. coli LacY (UniProt ID P02920).
  • All modified strains producing sialic acid, CMP-sialic acid and/or sialylated oligosaccharides could optionally be adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E. coli 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. a
  • sialic acid and/or sialylated oligosaccharide production can further be optimized in the mutant E. coli strains with genomic knock-ins of constitutive transcriptional units comprising a membrane transporter protein like e.g. a sialic acid transporter like e.g. nanT from E. coli K- 12 MG1655 (UniProt ID P41036, sequence version 02 (01 Nov 1995)), nanT from E. coli 06:1-11 (UniProt ID Q8FD59), nanT from E. albertii (UniProt ID B1EFH1) or a porter like e.g. EntS from E.
  • a membrane transporter protein like e.g. a sialic acid transporter like e.g. nanT from E. coli K- 12 MG1655 (UniProt ID P41036, sequence version 02 (01 Nov 1995)
  • nanT from E. coli 06:1-11
  • the modified strain was derived from E. coli K12 MG1655 and modified with a knock-out of the E. coli lacZ, lacY, lacA and nagB genes and with genomic knock-ins of constitutive transcriptional units for a lactose permease like e.g. the E. coli LacY (UniProt ID P02920) and a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. IgtA (UniProt ID Q9JXQ6) from N. meningitidis.
  • 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 modified LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB (Uniprot ID Q51116, Sequence version 02 (01 Dec 2000)) from N. meningitidis.
  • LgtB Uniprot ID Q51116, Sequence version 02 (01 Dec 2000)
  • LN3 derived oligosaccharides like lacto-/V-tetraose (LNT, Gal-pi,3-GlcNAc-pi,3-Gal-pi,4- Glc)
  • the modified LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N- acetylglucosamine beta-1, 3-galactosyltransferase like e.g. WbgO (Uniprot ID D3QY14) from E. coli 055:1-17.
  • LN3 and/or LNnT production can further be optimized in the modified E.
  • the modified LN3 and/or LNnT producing strains can also be optionally modified for enhanced UDP-GIcNAc production with a genomic knock-in of a constitutive transcriptional unit for an L-glutamine— D-fructose-6-phosphate aminotransferase like e.g., glmS from E. coli (UniProt ID P17169, Sequence version 04, 23 Jan 2007).
  • coli strains can also optionally be adapted with a genomic knock-in of a constitutive transcriptional unit for a UDP-glucose-4-epimerase like e.g., galE from E. coli (UniProt ID P09147), a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, Sequence version 03, 23 Jan 2007) and an N-acetylglucosamine-l-phosphate uridylyltransferase / glucosamine-l-phosphate acetyltransferase like e.g. glmU from E. coli (UniProt ID P0ACC7).
  • a genomic knock-in of a constitutive transcriptional unit for a UDP-glucose-4-epimerase like e.g., galE from E. coli (UniProt ID P09147)
  • 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.
  • 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
  • the modified E. coli strains were modified with a genomic knock-ins of a constitutive transcriptional unit encoding a chaperone protein like e.g., DnaK, DnaJ, GrpE or the GroEL/ES chaperonin system (Baneyx F., Palumbo J.L. (2003) Improving Heterologous Protein Folding via Molecular Chaperone and Foldase Co-Expression. In: Vaillancourt P.E. (eds) E. coli Gene Expression Protocols. Methods in Molecular BiologyTM, vol 205. Humana Press).
  • 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.
  • non-essential glycosyltransferase genes compris
  • 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.
  • yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were grown for 2-3 days at 30 °C. Starting from a single colony, a preculture was grown over night in 5 mL at 30 °C, shaking at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL media. These shake flasks were incubated at 30 °C with an orbital shaking of 200 rpm.
  • a yeast expression plasmid was derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the TRP1 selection marker and constitutive transcriptional units for an L-glutamine— D-fructose-6-phosphate aminotransferase like e.g. glmS from E. coli (UniProt ID P17169, sequence version 04 (23 Jan 2007)), a phosphatase like e.g. SurE from E. coli (UniProt ID P0A840), an N-acylglucosamine 2-epimerase like e.g. AGE from B.
  • L-glutamine— D-fructose-6-phosphate aminotransferase like e.g. glmS from E. coli (UniProt ID P17169, sequence version 04 (23 Jan 2007)
  • a phosphatase like e.g. SurE from E. coli (Uni
  • 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 enzyme from P. multocida (UniProt ID A0A849CI62).
  • 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.
  • the plasmid further comprised constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), and a sialyltransferase with SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 1 or a variant thereof comprising a fusion to a His6-tag and a MBP-tag like e.g., selected from the list comprising SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.
  • a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921)
  • a sialyltransferase with SEQ ID NO 02, 03, 04,
  • any one or more of the glycosyltransferases and/or the proteins involved in nucleotide-activated sugar synthesis were N- and/or C -terminally fused to a solubility tag like e.g., a SUMOstar tag (e.g. obtained from pYSUMOstar, Life Sensors, Malvern, PA) or an MBP-tag to enhance their solubility.
  • a solubility tag like e.g., a SUMOstar tag (e.g. obtained from pYSUMOstar, Life Sensors, Malvern, PA) or an MBP-tag to enhance their solubility.
  • the mutant yeast strains were modified with a genomic knock-in of a constitutive transcriptional unit encoding a chaperone protein like e.g.
  • coli DH5alpha (F", phi80d/ocZdeltaM15, delta(/ocZYA-orgF)U169, deoR, recAl, endAl, hsdR17(rk", mk + ), phoA, supE44, lambda", thi-1, gyrA96, relAl) bought from Invitrogen.
  • the Yeast Peptone Dextrose (YPD) medium consisted of 20 g/L tryptone peptone (Difco, Erembodegem, Belgium), 10 g/L yeast extract (Difco), 20 g/L yeast extract (Difco) and 20 g/L glucose. For plates, 20 g/L agarose was also added. The YPD medium was set to a pH of 7.5 with KOH grains.
  • the BMY medium used in cultivation experiments in 96-well plates or in shake flasks contained 10 g/L yeast extract (Difco), 20 g/L peptone (Difco), 20 g/L glucose or 10 mL/L glycerol or 10 mL/L methanol, O.lxPhosphate-buffer (KH2PO4/K2HPO4) pH 6, 13.4 g/L Yeast Nitrogen Base (YNB) and 0.4 g/L biotin.
  • the lOx Phosphate-buffer, the YNB solution and the biotin solution were filter-sterilized (0.22 pm Sartorius) prior to use.
  • Nitrogen and carbohydrate sources were sterilized separately by autoclaving (121°C, 21 min). Final media were prepared by combining sterile nitrogen source, carbohydrate source and all other sterile components listed here above. When necessary, the medium was made selective by adding an antibiotic like e.g., nourseothricine (50 mg/L). When necessary, inducer (e.g., methanol) was added as carbohydrate source in the media or spiked to a final concentration of 10 mL/L.
  • an antibiotic e.g., nourseothricine (50 mg/L).
  • inducer e.g., methanol
  • a strain was streaked on an YPD agrose plate and incubated at 28°C for 2 days to obtain single colonies. Single colonies were used as inoculum for a 24-well deep well microtiter plate, filled with 3 mL BMY medium comprising glycerol. These final 24-well culture plates were then incubated at 28°C on an orbital shaker at 225 rpm for 48 hours. After 48 hours, expression was induced by centrifuging the plates for 10 min at 4200 rpm and replacing media with BMY medium comprising methanol. Alternatively, methanol was directly spiked into the culture. Cultures were further grown at 28°C for 24 or 48 hours.
  • methanol was used to induce expression again after 24 hours.
  • the deep well plates were centrifuged for 30 min at 4200 rpm and 4°C, the supernatant was transferred to 96-well plates, stored at -20°C, and the pellets were frozen at -20°C for at least one hour.
  • pellets were subsequently resuspended in 250 pLY-PERTM Yeast Protein Extraction buffer (ThermoFisher Scientifc) containing lx Complete Protease Inhibitor (Roche), then transferred into 1.5 mL Eppendorf tubes and incubated for 20 min at room temperature.
  • Cell lysates were cleared by centrifuging the cell lysates (30 min at 4200 rpm and 4°C) and transferring the supernatant to new tubes.
  • a single colony was used as inoculum for a 500 mL baffled shake flask filled with 100 mL BMY medium comprising glycerol. These final cultures were then incubated at 28°C on an orbital shaker at 225 rpm. After 48 hours, expression was induced by transferring the culture to sterile 50 mL falcon tubes, centrifuging for 10 min at 4200 rpm and then replacing media with BMY medium comprising methanol. Cultures were further grown at 28°C for 24 or 48 hours.
  • the yeast Komagataella phaffii CBS2612 was obtained from Westerdijk Institute (Netherlands). Culturing for propagation was done at 28°C on YPD or YPD agar (1% Yeast Extract (BD Biosciences), 2% Peptone (BD Biosciences), 2% D-glucose (Merck) and optionally 1.5 % Agar (BD Biosciences)).
  • Plasmids to be used in the K. phaffii strains as described herein comprised the methanol-inducible AOX1 promoter, a cleavable AMF secretion signal in frame with the coding sequences encoding an alpha- sialyltransferase selected from the list comprising SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 or a variant thereof comprising a fusion to a His6-tag and a MBP-tag like e.g., selected from the list comprising SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53, and antibiotic resistance cassettes for subcloning.
  • Plasmids were assembled using MOCLO assembly (as described by Lee et al, 2015, DOI: 10.1021/sb500366v) and propagated in Escherichia coli DH5a which was cultured at 37°C on LB or LB agar (1% Yeast Extract (BD Biosciences), 1% Tryptone (BD Biosciences) and optionally 1.5 % Agar (BD Biosciences)). Antibiotics were used in the following concentrations for selection in E.
  • coli Zeocin (Life Technologies) 50 pg/mL, Nourseothricin (WERNER Bioagents) 50 pg/mL, Hygromycin (J&K scientific) 50 pg/mL, Genetecin G418 (InvivoGen) 200 pg/mL, Kanamycin (Duchefa Biochemie) 50 pg/mL, Chloramphenicol (Duchefa Biochemie) 50 pg/mL and Carbenicillin (Duchefa Biochemie) 50 pg/mL. Antibiotics were used in the following concentrations for selection in K.
  • phaffii Zeocin 100 pg/mL, Nourseothricin 100 pg/mL, Hygromycin 100 pg/mL, Kanamycin 100 pg/mL, Blasticidin 100 pg/mL. Plasmids were linearized using Pmel digestion and DNA was introduced into the AOX1 loci of K. phaffii by homologous recombination. All strains were stored in cryovials at -80°C (overnight YPD culture mixed in a 1:1 ratio with 70% glycerol).
  • B. subtilis i.e. a rich Luria Broth (LB) and a minimal medium for shake flask cultures.
  • the LB medium consisted of 1% tryptone peptone (Difco), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR).
  • Luria Broth agar (LBA) plates consisted of the LB media, with 12 g/L agar (Difco) added.
  • the minimal medium contained 2.00 g/L (NF 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 7.0 with 1 M KOH. Depending on the experiment lactose is added as a precursor.
  • the trace element mix consisted of 0.735 g/L CaCl2.2H 2 O, 0.1 g/L MnCl2.2H 2 O, 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 Na2EDTA.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, 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. zeocin (20mg/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.
  • Plasmids for gene deletion via Cre/lox are constructed as described by Yan et al. (Appl & Environm microbial, Sept 2008, p5556-5562). Gene disruption is done via homologous recombination with linear DNA and transformation via the electroporation as described by Xue et al. (J. microb. Meth. 34 (1999) 183-191). The method of gene knockouts is described by Liu et al. (Metab. Engine. 24 (2014) 61-69). This method uses 1000 bp homologies up- and downstream of the target gene.
  • Integrative vectors as described by Popp et al. are used as expression vector and could be further used for genomic integrations if necessary.
  • a suitable promoter for expression can be derived from the part repository (iGem): sequence id: BBa_K143012, BBa_K823000, BBa_K823002 or BBa_K823003. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.
  • the engineered strain was derived from B. subtilis comprising knockouts of the B. subtilis nagA, nagB and gamA genes and genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03 (23 Jan 2007)), an N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase like e.g. glmU from E.
  • a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03 (23 Jan 2007)
  • an N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransfera
  • Sialic acid production can also be obtained in modified B. subtilis comprising knockouts of the B. subtilis nagA, nagB and gamA genes and genomic knock-ins of constitutive transcriptional units containing an N- acylglucosamine 2-epimerase like e.g., AGE from B.
  • the modified strain can further be modified with a genomic knock-in of one or more constitutive transcriptional units containing a glutamine-fructose-6-P- aminotransferase like e.g., the native glutamine-fructose-6-P-aminotransferase glmS (UniProt ID P0CI73).
  • the strains were also modified for expression of a phosphatase like e.g., SurE from E. coli (UniProt ID P0A840).
  • a phosphatase like e.g., SurE from E. coli (UniProt ID P0A840).
  • the sialic acid production strains further need to express an N-acylneuraminate cytidylyltransferase like e.g. NeuA enzyme from P.
  • multocida (UniProt ID A0A849CI62), and a sialyltransferase with SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 or a variant thereof comprising a fusion to a His6-tag and a MBP-tag like e.g., selected from the list comprising SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.
  • Constitutive transcriptional units of the N-acylneuraminate cytidylyltransferase and the sialyltransferases can be delivered to the engineered strain either via genomic knock-in or via expression plasmids. If the engineered strains producing sialic acid and CMP-sialic acid were intended to make sialylated lactose structures, the strains were additionally modified with a genomic knock-in of a constitutive transcriptional unit for a lactose permease like e.g. the E. coli LacY (UniProt ID P02920).
  • TY tryptone-yeast extract
  • VWR 0.5% sodium chloride
  • TY agar (TYA) plates consisted of the TY media, with 12 g/L agar (Difco) added.
  • the minimal medium for the shake flask experiments contained 20 g/L (Nl- hSO ⁇ 5 g/L urea, 1 g/L KH2PO4, l g/L K2HPO4, 0.25 g/L MgSO4.7H2O, 42 g/L MOPS, from 10 up to 30 g/L glucose (or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose) and 1 mL/L trace element mix. Depending on the experiment lactose is added as a precursor.
  • the trace element mix consisted of 10 g/L CaCI 2 , 10 g/L FeSO 4 .7H 2 O, 10 g/L MnSO 4 .H 2 O, 1 g/L ZnSO 4 .7H 2 O, 0.2 g/L CuSO 4 , 0.02 g/L NiCI 2 .6H 2 O, 0.2 g/L biotin (pH 7.0) and 0.03 g/L protocatechuic acid.
  • Complex medium e.g., TY
  • TY e.g., TY
  • minimal medium e.g., 1%
  • an antibiotic e.g., kanamycin, ampicillin
  • a preculture of 96-well microtiter plate experiments was started from a cryovial or a single colony from a TY plate, in 150 pL TY and was incubated overnight at 37 °C on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, with 400 pL minimal medium by diluting 400x. Each strain was grown in multiple wells of the 96-well plate as biological replicates.
  • 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 ATCC 13032 was used as available at the American Type Culture Collection. Integrative plasmid vectors based on the Cre/loxP technique as described by Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 Apr, 67(2):225-33) and temperature-sensitive shuttle vectors as described by Okibe et al. (J. Microbiol. Meth. 85, 2011, 155-163) are constructed for gene deletions, mutations and insertions. Suitable promoters for (heterologous) gene expression can be derived from Yim et al. (Biotechnol. Bioeng., 2013 Nov, 110(ll):2959-69). Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.
  • the engineered strain was derived from C. glutamicum comprising knockouts of the C. glutamicum Idh, cgl2645 and nagB genes and genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03 (23 Jan 2007)), an N-acetylglucosamine-l-phosphate uridylyltransferase/glucosamine-l-phosphate acetyltransferase like e.g. glmU from E.
  • C. glutamicum comprising knockouts of the C. glutamicum Idh, cgl2645 and nagB genes and genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03 (
  • the modified strain can further be modified with a genomic knock-in of one or more constitutive transcriptional units containing a glutamine-fructose-6-P- aminotransferase like e.g., the native glutamine-fructose-6-P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 02 (23 Jan 2007)).
  • a glutamine-fructose-6-P- aminotransferase like e.g., the native glutamine-fructose-6-P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 02 (23 Jan 2007)).
  • the sialic acid production strains further need to express an N-acylneuraminate cytidylyltransferase like e.g. NeuA enzyme from P.
  • Constitutive transcriptional units of the N-acylneuraminate cytidylyltransferase and the sialyltransferases can be delivered to the engineered strain either via genomic knock-in or via expression plasmids. If the engineered strains producing sialic acid and CMP-sialic acid were intended to make sialylated lactose structures, the strains were additionally modified with a genomic knock-in of a constitutive transcriptional unit for a lactose permease like e.g., the E. coli LacY (UniProt ID P02920).
  • TAP Tris-acetate-phosphate
  • the TAP medium uses a lOOOx stock Hutner's trace element mix.
  • Hutner's trace element mix consisted of 50 g/L Na 2 EDTA.H 2 O (Titriplex III), 22 g/L ZnSO 4 .7H 2 O, 11.4 g/L H3BO3, 5 g/L MnCI 2 .4H 2 O, 5 g/L FeSO 4 .7H 2 O, 1.6 g/L CoCI 2 .6H 2 O, 1.6 g/L CUSO 4 .5H 2 O and 1.1 g/L (NH 4 )gMoO3.
  • the TAP medium contained 2.42 g/L Tris (tris(hydroxymethyl)aminomethane), 25 mg/L salt stock solution, 0.108 g/L K 2 HPO 4 , 0.054 g/L KH 2 PO 4 and 1.0 mL/L glacial acetic acid.
  • the salt stock solution consisted of 15 g/L NH 4 CI, 4 g/L MgSO 4 .7H 2 O and 2 g/L CaCI 2 .2H 2 O.
  • precursors like e.g., galactose, glucose, fructose, fucose, GIcNAc could be added.
  • Medium was sterilized by autoclaving (121°C, 21 min).
  • TAP medium 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.
  • 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).
  • C. reinhardtii wild-type strains 21gr (CC-1690, wild-type, mt+), 6145C (CC-1691, wild-type, mt-), CC-125 (137c, wild-type, mt+), CC-124 (137c, wild-type, mt-) as available from Chlamydomonas Resource Center (https://www.chlamycollection.org), University of Minnesota, U.S.A.
  • Expression plasmids originated from pSH03, as available from Chlamydomonas Resource Center. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. Suitable promoters for (heterologous) gene expression can be derived from e.g., Scranton et al. (Algal Res. 2016, 15: 135-142). Targeted gene modification (like gene knock-out or gene replacement) can be carried using the Crispr-Cas technology as described e.g., by Jiang et al. (Eukaryotic Cell 2014, 13(11): 1465-1469). Transformation via electroporation was performed as described by Wang et al. (Biosci. Rep.
  • C. reinhardtii cells were modified with transcriptional units comprising the gene encoding the galactokinase from Arabidopsis thaliana (KIN, UniProt ID Q9SEE5) and the gene encoding the UDP-sugar pyrophosphorylase (USP) from A. thaliana (UniProt ID Q9C5I1).
  • C. reinhardtii cells were modified with constitutive transcriptional units for a UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase like e.g. GNE from Homo sapiens (UniProt ID Q9Y223) or a mutant form of the human GNE polypeptide comprising the R263L mutation, an N-acylneuraminate-9-phosphate synthetase like e.g.
  • C. reinhardtii cells are modified with a CMP-sialic acid transporter like e.g.
  • CST from Mus musculus (UniProt ID Q61420), and a sialyltransferase with SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 or a variant thereof comprising a fusion to a His6-tag and a MBP-tag like e.g. a sialyltransferase selected from the list comprising SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.
  • Fresh adipose tissue is obtained from slaughterhouses (e.g. cattle, pigs, sheep, chicken, ducks, catfish, snake, frogs) or liposuction (e.g., in case of humans, after informed consent) and kept in phosphate buffer saline supplemented with antibiotics. Enzymatic digestion of the adipose tissue is performed followed by centrifugation to isolate mesenchymal stem cells. The isolated mesenchymal stem cells are transferred to cell culture flasks and grown under standard growth conditions, e.g., 37°C, 5% CO2.
  • the initial culture medium includes DMEM-F12, RPMI, and Alpha-MEM medium (supplemented with 15% foetal bovine serum), and 1% antibiotics.
  • FBS farnesoid bovine serum
  • Ahmad and Shakoori 2013, Stem Cell Regen. Med. 9(2): 29-36, which is incorporated herein by reference in its entirety for all purposes, describes certain variation(s) of the method(s) described herein in this example.
  • This example illustrates isolation of mesenchymal stem cells from milk collected under aseptic conditions from human or any other mammal(s) such as described herein.
  • An equal volume of phosphate buffer saline is added to diluted milk, followed by centrifugation for 20 min.
  • the cell pellet is washed thrice with phosphate buffer saline and cells are seeded in cell culture flasks in DMEM-F12, RPMI, and Alpha-MEM medium supplemented with 10% foetal bovine serum and 1% antibiotics under standard culture conditions.
  • Hassiotou et al. 2012, Stem Cells. 30(10): 2164-2174
  • the mesenchymal cells isolated from adipose tissue of different animals or from milk as described above can be differentiated into mammary-like epithelial and luminal cells in 2D and 3D culture systems. See, for example, Huynh et al. 1991. Exp Cell Res. 197(2): 191 -199; Gibson et al. 1991, In Vitro Cell Dev Biol Anim. 27(7): 585-594; Blatchford et al. 1999; Animal Cell Technology': Basic & Applied Aspects, Springer, Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res 11(3): 26-43; and Arevalo et al. 2015, Am J Physiol Cell Physiol. 310(5): C348 - C356; each of which is incorporated herein by reference in their entireties for all purposes.
  • the isolated cells were initially seeded in culture plates in growth media supplemented with 10 ng/mL epithelial growth factor and 5 pg/mL insulin.
  • growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/mL penicillin, 100 ug/mL streptomycin), and 5 pg/mL insulin for 48h.
  • penicillin-streptomycin 100 U/mL penicillin, 100 ug/mL streptomycin
  • 5 pg/mL insulin for 48h.
  • the cells were fed with complete growth medium containing 5 pg/mL insulin, 1 pg/mL hydrocortisone, 0.65 ng/mL triiodothyronine, 100 nM dexamethasone, and 1 pg/mL prolactin.
  • serum is removed from the complete induction medium.
  • the isolated cells were trypsinized and cultured in Matrigel, hyaluronic acid, or ultra- low attachment surface culture plates for six days and induced to differentiate and lactate by adding growth media supplemented with 10 ng/mL epithelial growth factor and 5 pg/mL insulin.
  • growth media supplemented with 10 ng/mL epithelial growth factor and 5 pg/mL insulin.
  • cells were fed with growth medium supplemented with 2% foetal bovine serum, 1% penicillin-streptomycin (100 U/mL penicillin, 100 ug/mL streptomycin), and 5 pg/mL insulin for 48h.
  • the cells were fed with complete growth medium containing 5 pg/mL insulin, 1 pg/mL hydrocortisone, 0.65 ng/mL triiodothyronine, 100 nM dexamethasone, and 1 pg/mL prolactin. After 24h, serum is removed from the complete induction medium.
  • the cells are brought to induced pluripotency by reprogramming with viral vectors encoding for Oct4, Sox2, Klf4, and c-Myc.
  • the resultant reprogrammed cells are then cultured in Mammocult media (available from Stem Cell Technologies), or mammary cell enrichment media (DMEM, 3% FBS, estrogen, progesterone, heparin, hydrocortisone, insulin, EGF) to make them mammary-like, from which expression of select milk components can be induced.
  • Mammocult media available from Stem Cell Technologies
  • DMEM mammary cell enrichment media
  • epigenetic remodelling is performed using remodelling systems such as CRISPR/Cas9, to activate select genes of interest, such as casein, a- lactalbumin to be constitutively on, to allow for the expression of their respective proteins, and/or to down-regulate and/or knock-out select endogenous genes as described e.g., in WO21067641, which is incorporated herein by reference in its entirety for all purposes.
  • remodelling systems such as CRISPR/Cas9
  • Completed growth media includes high glucose DMEM/F12, 10% FBS, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/mL EGF, and 5 pg/mL hydrocortisone.
  • Completed lactation media includes high glucose DMEM/F12, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/mL EGF, 5 pg/mL hydrocortisone, and 1 pg/mL prolactin (5ug/mL in Hyunh 1991).
  • Cells are seeded at a density of 20,000 cells/cm2 onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media.
  • the cells Upon exposure to the lactation media, the cells start to differentiate and stop growing.
  • 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.
  • Genes that needed to be expressed were it from a plasmid or from the genome were synthetically synthetized with one of the following companies: IDT, Twist Bioscience, DNA2.0 or Gen9. Proteins described in present disclosure are summarized in Table 1. Unless stated otherwise, the UniProt IDs of the proteins described correspond to their sequence version 01 as present in the UniProt Database version release 2021_03 of 09 June 2021. Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Genes were optimized using the tools of the supplier.
  • the column temperature was 50 °C.
  • the mobile phase consisted of a % water and % acetonitrile solution to which 0.2 % triethylamine was added.
  • the method was isocratic with a flow of 0.130 mL/min.
  • the ELS detector had a drift tube temperature of 50 °C and the N2 gas pressure was 50 psi, the gain 200 and the data rate 10 pps.
  • the temperature of the Rl detector was set at 35 °C.
  • 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.
  • samples were diluted two times in acetonitrile and subsequently mixed with 2,5-dihydroxybenzoic acid (DHB, Merck Life Science B.V.) matrix and Girard's Reagent T (GT, Merck Life Science B.V.).
  • Samples were analysed using a 4800 Plus MALDI TOF/TOF Analyser (Applied Biosystems, Germany) with an Nd:YAG laser (200 Hz, 355 nm) controlled by the 4000 Series Explorer software version 3.5.3 (Applied Biosystems, Germany).
  • the instrument was operated in positive ion mode with delayed extraction and an acceleration voltage of 20 kV with a grid of 15.6 kV.
  • eluent A was deionized water
  • eluent B was 200 mM Sodium hydroxide
  • eluent C was 500 mM Sodium acetate.
  • the oligosaccharides were separated in 60 min while maintaining a constant ratio of 25 % of eluent B using the following gradient: an initial isocratic step maintained for 10 min of 75 % of eluent A, an initial increase from 0 to 4 % of eluent C over 8 min, a second isocratic step maintained for 6 min of 71 % of eluent A and
  • a method is used that is compatible with reducing agents, such as reducing sugars or oligosaccharides with a reducing end.
  • reducing agents such as reducing sugars or oligosaccharides with a reducing end.
  • a Bradford assay (Thermo Scientific, Pierce) was used with a linear range between 1 and 1500 pg/mL. The assay was calibrated with a standard curve of BSA.
  • the protein content of dried oligosaccharide products was quantified by dissolving a pre-weighed quantify in 18.2 MO-cm (Millipore, Bedford, MA, USA) de-ionized water (DIW) up to a quantity of 50% (m/v). The amount of protein is measured at 595 nm and converted to concentration with the calibration curve based on BSA.
  • Total DNA is measured by means of a Threshold assay (Molecular Devices), based on an immunoassay allowing to measure as low as 2 pg of DNA in a sample in solution. Double stranded DNA is measured by means of SpectraMax® QuantTM AccuBlueTM Pico dsDNA Assay Kit (Molecular Devices) having a linear range between 5 pg and 3 ng of dsDNA.
  • Threshold assay based on an immunoassay allowing to measure as low as 2 pg of DNA in a sample in solution.
  • Double stranded DNA is measured by means of SpectraMax® QuantTM AccuBlueTM Pico dsDNA Assay Kit (Molecular Devices) having a linear range between 5 pg and 3 ng of dsDNA.
  • Example 4 Expression of biologically active sialyltransferases in E. coli and production of a sialylated oligosaccharide
  • E. coli Origami 2 (DE3) cells or E. coli SHuffle T7 Express cells as described in Example 3 are transformed with an expression plasmid comprising an inducible transcriptional unit encoding a sialyltransferase with SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 or a variant thereof comprising a fusion to a His6-tag and a MBP-tag selected from the list comprising SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.
  • a protein production experiment is performed in a 24-well deep well plate as described in Example 3. Afterwards, cells are harvested, lysed and cleared cell lysates are analysed via Western blot analysis as described in Example 3 for expression of the target sialyltransferase. Next, an activity assay is set up by incubating 1 mM CTP, 1 mM Neu5Ac, 40 ng/pL of purified NeuA (from N.
  • meningitidis with UniProt ID P0A0Z7) 5 U/mL inorganic pyrophosphatase from Baker's yeast (Sigma), 50% (v/v) sialyltransferase cell extract (soluble fraction) and 1 mM acceptor substrate like e.g., Neu5Ac-a2,3-Gal-pi,3-GalNAc-pi,4-Gal- Pl,4-Glc; Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc or Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc in 100 mM MES buffer comprising 20 mM MgCp (pH 6.5) for 20 h at 37°C.
  • a sialylated oligosaccharide like e.g., Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,3)-GalNAc-pi,4-Gal-pi,4-Glc; Neu5Ac-a2,6- (Neu5Ac-a2,3-Gal-pi,3)-GlcNAc-pi,3-Gal-pi,4-Glc or Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc-pi,3-Gal-pi,4-Glc, respectively, is analysed via UPLC as described in Example 3.
  • Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,3)-GalNAc-pi,4-Gal-pi,4-Glc e.g., Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-p
  • E. coli Origami 2 (DE3) cells or E. coli SHuffle T7 Express cells as described in Example 3 are transformed with an expression plasmid comprising an inducible transcriptional unit encoding a sialyltransferase comprising a fusion to a His6-tag and a MBP-tag selected from the list comprising SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.
  • a protein production experiment is performed in a shake flask as described in Example 3.
  • Example 3 cells are harvested, lysed and cleared cell lysates are analysed via Western blot analysis as described in Example 3 for expression of the target sialyltransferase.
  • the sialyltransferases expressing the best based on the Western Blot results are purified as described in example 3.
  • an activity assay is set up by incubating 1 mM CTP, 1 mM Neu5Ac, 40 ng/pL purified NeuA (from N.
  • meningitidis with UniProt ID P0A0Z7 meningitidis with UniProt ID P0A0Z7), 5 U/mL inorganic pyrophosphatase from Baker's yeast (Sigma), 10 to 50% (v/v) purified sialyltransferase (stored at a concentration of 0.5 to 2 mg/mL), and 1 mM acceptor substrate like e.g., Neu5Ac-a2,3-Gal-pi,3-GalNAc- pi,4-Gal-pi,4-Glc; Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gaipi,4-Glc or Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc in 100 mM MES buffer comprising 20 mM MgCp (pH 6.5) for 20 h at 37°C.
  • a sialylated oligosaccharide like e.g., Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,3)-GalNAc-pi,4-Gal-pi,4-Glc; Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,3)-GlcNAc-pi,3-Gal-pi,4-Glc or Gal-pi,3-[Neu5Ac-a2,6]-GlcNAc- pi,3-Gal-pi,4-Glc, respectively, is analysed via UPLC as described in Example 3.
  • Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,3)-GalNAc-pi,4-Gal-pi,4-Glc e.g., Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi
  • An E. coli K-12 MG1655 strain modified for uptake of sucrose and production of sialic acid as described in Example 3 is further modified by knocking out the E. coli genes trxB and gor and by knocking in a constitutive transcriptional unit for the beta-1, 3-N-acetylglucosaminyltransferase IgtA from N. meningitidis (UniProt ID Q9JXQ6) and the beta-1, 3-galactosyltransferase (PfFurA) from Pseudogulbenkiania ferrooxidans with SEQ ID NO 54.
  • the mutant strain is transformed with an expression plasmid comprising constitutive transcriptional units for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 3-sialyltransferase from Bibersteinia trehalosi with SEQ ID NO 55.
  • the mutant strain is transformed with an expression plasmid comprising a constitutive transcriptional unit for an alpha-2, 6-sialyltransferase with SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 or a variant thereof comprising a fusion to a His6-tag and a MBP-tag selected from the list comprising SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.
  • the novel strains are evaluated in a 96-well plate according to the culture conditions provided in Example 3 in which the strains are cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. After 72h of incubation, the culture broth is harvested and analysed for production of DSLNT (Neu5Ac-a2,6-(Neu5Ac- a2,3-Gal-pi,3)-GlcNAc-pi,3-Gal-pi,4-Glc) as described in Example 3.
  • Example 7 Expression of biologically active sialyltransferases in K. phaffii and production of a sialylated oligosaccharide
  • K. phaffii CBS2612 cells as described in Example 3 are transformed with an expression plasmid comprising a methanol inducible transcriptional unit under control of the AOX1 promoter and encoding a fusion protein of a sialyltransferase with SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 or a variant thereof comprising a fusion to a His6-tag and a MBP- tag selected from the list comprising SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 that is additionally fused to the AMF secretion signal, wherein the plasmid is linearized upon genomic integration of said transcriptional unit in the genome of the strain.
  • a protein production experiment is performed for 72 h in a 24-well deep well plate as described in Example 3. Afterwards, cells are harvested, lysed and cleared cell lysates are analysed via Western blot analysis as described in Example 3 for expression of the target sialyltransferase. Next, an activity assay is set up by incubating 1 mM CTP, 1 mM Neu5Ac, 40 ng/pL of purified NeuA (from N.
  • meningitidis with UniProt ID P0A0Z7 5 U/mL inorganic pyrophosphatase from Baker's yeast (Sigma), 50% (v/v) sialyltransferase cell extract (soluble fraction) and 1 mM acceptor substrate like e.g., Neu5Ac-a2,3-Gal- pi,3-GalNAc-pi,4-Gal-pi,4-Glc; Neu5Aca-2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc or Gal-pi,3-GlcNAc- pi,3-Gal-pi,4-Glc in 100 mM MES buffer comprising 20 mM MgCL (pH 6.5) for 20 h at 37°C.
  • MES buffer comprising 20 mM MgCL (pH 6.5) for 20 h at 37°C.
  • Example 8 Production of DSLNT with modified E. coli hosts when evaluated in a fed-batch fermentation process with sucrose and lactose
  • the modified E. coli strains expressing an alpha-2, 6-sialyltransferase with SEQ ID NO 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 and able to produce DSLNT as described in Example 5 are selected for further evaluation in a fed-batch fermentation process.
  • Fed- batch fermentations at bioreactor scale are performed as described in Example 3.
  • Sucrose is used as a carbon source and lactose is added in the batch medium.
  • sucrose is added via an additional feed.
  • Example 9 Production of DSLNT with a modified S. cerevisiae host
  • a S. cerevisiae strain is modified for production of CMP-sialic acid and LNT and for expression of an alpha- 2,3-sialyltransferase and an alpha-2, 6-sialyltransferase as described in Example 3 with a first yeast expression plasmid comprising constitutive transcriptional units for LAC12 from K. lactis (UniProt ID P07921), glmS from E. coli (UniProt ID P17169), the phosphatase SurE from E. coli (UniProt ID P0A840), AGE from B. ovatus (UniProt ID A7LVG6), NeuB from N.
  • meningitidis (UniProt ID E0NCD4), NeuA from P. multocida (UniProt ID A0A849CI62), the alpha-2, 3-sialyltransferase PmultST3 (UniProt ID Q9CLP3) from P. multocida and an alpha-2, 6-sialyltransferase with SEQ ID NO 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 and a second yeast expression plasmid comprising constitutive transcriptional units for galE from E. coli (UniProt ID P09147), LgtA from N.
  • meningitidis (UniProt ID Q9JXQ6) and WbgO from E. coli O55:H7 (UniProt ID D3QY14).
  • the novel strains are evaluated for production of DSLNT when evaluated in a 3-days growth experiment according to the culture conditions provided in Example 3 using appropriate selective medium comprising lactose.
  • a wild-type B. subtilis strain is first modified for production of LNB (lacto-N-biose, Gal-pi,3-GlcNAc) with genomic knockouts of the B. subtilis genes nagB and gamA together with genomic knock-ins of constitutive transcriptional units for glmS from E. coli (UniProt ID P17169), the glucosamine 6-phosphate N-acetyltransferase GNA1 from S. cerevisiae (UniProt ID P43577), the phosphatase AraL from B. subtilis (UniProt ID P94526) and WbgO from E. coli 055:1-17 (UniProt ID D3QY14).
  • LNB lacto-N-biose, Gal-pi,3-GlcNAc
  • the modified strain is transformed with an expression plasmid comprising a constitutive transcriptional unit for an alpha-2, 6-sialyltransferase with SEQ ID NO 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.
  • the novel strains are evaluated for production of sialylated LNB (Gal- pi,3-[Neu5Ac-a2,6]-GlcNAc) when evaluated in a 3-days growth experiment according to the culture conditions provided in Example 3 using appropriate selective medium.
  • a wild-type C. glutamicum strain is first modified for production of LN3 with genomic knockouts of the Idh, cgl2645 and nagB genes together with genomic knock-ins of constitutive transcriptional units for the lactose permease LacYfrom E. coli (UniProt ID P02920) and IgtA from N. meningitidis (UniProt ID Q9JXQ6).
  • the modified C. glutamicum strain is modified for production of LNT with a genomic knock- in of a constitutive transcriptional unit for WbgO from E. coli 055:1-17 (UniProt ID D3QY14).
  • the modified strain is transformed with an expression plasmid comprising constitutive transcriptional units for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 3-sialyltransferase from Bibersteinia trehalosi with SEQ ID NO 55 and an alpha-2, 6-sialyltransferase with SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 1 or a variant thereof comprising a fusion to a His6-tag and a MBP-tag selected from the list comprising SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.
  • the novel strain is evaluated for production of
  • Example 12 Production of DSLNT with a modified C. reinhardtii strain
  • C. reinhardtii cells modified for production of UDP-galactose as described e.g., in WQ22034067 are further modified for CMP-sialic acid synthesis with genomic knock-ins of constitutive transcriptional units comprising the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase GNE from Homo sapiens (UniProt ID Q9Y223), the N-acylneuraminate-9-phosphate synthetase NANS from H. sapiens (UniProt ID Q9NR45, Sequence version 03 (13 Oct 2009)), the N-acylneuraminate cytidylyltransferase CMAS from H.
  • genomic knock-ins of constitutive transcriptional units comprising the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase GNE from Homo sapiens (UniProt
  • the cells are modified with an expression plasmid comprising constitutive transcriptional units comprising the alpha-2, 3- sialyltransferase from Bibersteinia trehalosi with SEQ ID NO 55 and an alpha-2, 6-sialyltransferase with SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 or a variant thereof comprising a fusion to a His6-tag and a MBP-tag selected from the list comprising SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53, the gal
  • Isolated mesenchymal cells and re-programmed into mammary-like cells as described in Example 3 are modified via CRISPR-CAS to express the GlcN6P synthase GFPT1 from Homo sapiens (UniProt ID Q06210), the glucosamine 6-phosphate N-acetyltransferase GNA1 from Homo sapiens (UniProt ID Q96EK6), the phosphoacetylglucosamine mutase PGM3 from Homo sapiens (UniProt ID 095394), the UDP-N- acetylhexosamine pyrophosphorylase UAP1 from Homo sapiens (UniProt ID Q16222, Sequence version 03 (02 Jun 2021)), the galactoside beta-1, 3-N-acetylglucosaminyltransferase LgtA from N.
  • meningitidis (UniProt ID Q9JXQ6), the N-acetylglucosamine beta-1, 3-galactosyltransferase WbgO from E. coli 055:1-17 (UniProt ID D3QY14), the N-acylneuraminate cytidylyltransferases neuA from Mus musculus (UniProt ID Q99KK2), the alpha-2, 3-sialyltransferase from Bibersteinia trehalosi with SEQ ID NO 55 and an alpha-2, 6- sialyltransferase with SEQ ID NO 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 or a variant thereof comprising a fusion to a His6-tag and a MBP-tag selected from the list comprising SEQ ID NOs 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
  • Cells are seeded at a density of 20,000 cells/cm2 onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media for about 7 days. After cultivation as described in Example 3, cells are subjected to UPLC to analyse for production of DSLNT.
  • Example 14 Expression of biologically active sialyltransferases in E. coli and production of Neu5Ac-a2,6- (Neu5Ac-a2,3-Gal-pi,3)-GlcNAc-pi,3-Gal-pi,4-Glc (DSLNT)
  • E. coli Origami 2 (DE3) cells as described in Example 3 were transformed with an expression plasmid comprising an inducible transcriptional unit encoding a fusion protein as represented with SEQ ID NO 05, 06, 09, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25 or 26, each fusion protein comprising an alpha-2, 6- sialyltransferase linked with one or more tags like a His tag, an MBP tag, a NusA tag and/or a TrxA tag.
  • a protein production experiment was performed in a 24-well deep well plate as described in Example 2.
  • meningitidis with UniProt ID P0A0Z7 meningitidis with UniProt ID P0A0Z7), 5 U/mL inorganic pyrophosphatase from Baker's yeast (Sigma), 50% (v/v) sialyltransferase cell extract (soluble fraction) and 2 mM Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc (LSTa) in 100 mM MES buffer comprising 20 mM MgCL (pH 6.5) for 20 h at 37°C.
  • Example 15 Expression of biologically active sialyltransferases in E. coli and production of Gal-pi,3- [Neu5Ac-a2,6]-GlcNAc- i,3-Gal- i,4-Glc (LSTb)
  • E. coli Origami 2 (DE3) cells as described in Example 3 were transformed with an expression plasmid comprising an inducible transcriptional unit encoding a fusion protein as represented with SEQ ID NO 06, 15, 17 and 26, the fusion protein comprising an alpha-2, 6-sialyltransferase linked with a His tag and a MBP tag.
  • a protein production experiment was performed in a 24-well deep well plate as described in Example 2. Afterwards, cells were harvested, lysed and cleared cell lysates were analysed via Western blot analysis as described in Example 2 for expression of the target alpha-2, 6-sialyltransferase.
  • an activity assay was set up by incubating 1 mM CTP, 1 mM Neu5Ac, 40 ng/pL of purified NeuA (from N. meningitidis with UniProt ID P0A0Z7), 5 U/mL inorganic pyrophosphatase from Baker's yeast (Sigma), 50% (v/v) sialyltransferase cell extract (soluble fraction) and 1 mM Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc (LNT) in 100 mM MES buffer comprising 20 mM MgCL (pH 6.5) for 20 h at 37°C.
  • Example 16 Expression of biologically active sialyltransferases in E. coli for increased production of Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-pi,3)-GlcNAc-pi,3-Gal-pi,4-Glc (DSLNT)
  • E. coli Origami 2 (DE3) cells as described in Example 3 were transformed with an expression plasmid comprising an inducible transcriptional unit encoding a fusion protein as represented with SEQ ID NO 17 or 26, each fusion protein comprising an alpha-2, 6-sialyltransferase linked with one or more tags like a His tag, an MBP tag, a NusA tag and/or a TrxA tag.
  • a protein production experiment was performed in a 24-well deep well plate as described in Example 2. Afterwards, cells were harvested, lysed and cleared cell lysates were analysed via Western blot analysis as described in Example 3 for expression of the target alpha-2, 6-sialyltransferases.
  • an activity assay was set up by incubating CTP (5, 10 or 20 mM), Neu5Ac (5, 10 or 20 mM), 40 ng/pL of purified NeuA (from N. meningitidis with UniProt ID P0A0Z7), 5 U/mL inorganic pyrophosphatase from Baker's yeast (Sigma), 50% (v/v) sialyltransferase cell extract (soluble fraction) and Neu5Ac-a2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc (LSTa) (5, 10 or 20 mM) in 100 mM MES buffer comprising 20 mM MgCI2 (pH 6.5) for 48 h at 37°C.

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Abstract

La présente invention relève du domaine technique de la biologie synthétique, de l'ingénierie métabolique et de la culture cellulaire. La présente invention concerne des procédés de production d'un composé sialylé comprenant le Gal-β1,3-[Neu5Ac-α2,6]-HexNAc-R ainsi que la purification dudit composé sialylé. La présente invention concerne également une cellule pour la production dudit composé sialylé et l'utilisation de ladite cellule dans une culture ou une incubation. La présente invention concerne également des procédés et une cellule pour la production de sialyltransférases ayant une activité alpha-2,6-sialyltransférase sur le résidu N-acétylhexosamine (HexNAc) de Gal-β1,3-HexNAc-R et/ou Neu5Ac-α2,3-Gal-β1,3-HexNAc-R, lesdites sialyltransférases étant impliquées dans la production d'un composé sialylé comprenant le Gal-β1,3-[Neu5Ac-α2,6]-HexNAc-R.
PCT/EP2024/080201 2023-10-26 2024-10-25 Production d'un composé sialylé Pending WO2025088103A1 (fr)

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