WO2024165611A1 - Production of a disaccharide and/or milk oligosaccharide by a cell with reduced synthesis of udp-glcnac - Google Patents

Production of a disaccharide and/or milk oligosaccharide by a cell with reduced synthesis of udp-glcnac Download PDF

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WO2024165611A1
WO2024165611A1 PCT/EP2024/053033 EP2024053033W WO2024165611A1 WO 2024165611 A1 WO2024165611 A1 WO 2024165611A1 EP 2024053033 W EP2024053033 W EP 2024053033W WO 2024165611 A1 WO2024165611 A1 WO 2024165611A1
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lacto
milk oligosaccharide
udp
cell
oligosaccharide
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PCT/EP2024/053033
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French (fr)
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Joeri Beauprez
Thomas DECOENE
Annelies VERCAUTEREN
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Inbiose N.V.
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Publication of WO2024165611A1 publication Critical patent/WO2024165611A1/en

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/52—Genes encoding for enzymes or proenzymes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
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    • C12N9/90—Isomerases (5.)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01157—Glucosamine-1-phosphate N-acetyltransferase (2.3.1.157)
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    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • C12Y206/01016—Glutamine-fructose-6-phosphate transaminase (isomerizing) (2.6.1.16), i.e. glucosamine-6-phosphate-synthase
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07023—UDP-N-acetylglucosamine diphosphorylase (2.7.7.23)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/02—Phosphotransferases (phosphomutases) (5.4.2)
    • C12Y504/02003—Phosphoacetylglucosamine mutase (5.4.2.3)

Definitions

  • the present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation.
  • the invention provides a cell for production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide wherein synthesis of UDP-N-acetylglucosamine in said cell is rendered less functional.
  • the invention further provides use of said cell in a cultivation or incubation.
  • the invention also describes methods for the production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide using said cell as well as the purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide.
  • 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.
  • Disaccharides and oligosaccharides are widely distributed in all living organisms and play important roles in a variety of physiological and pathological processes, such as cell metastasis, signal transduction, intercellular adhesion, inflammation, and immune response.
  • oligosaccharides comprises milk oligosaccharides like mammalian milk oligosaccharides (MMOs) and human milk oligosaccharides (HMOs) found in mammalian and human milk, respectively.
  • MMOs mammalian milk oligosaccharides
  • HMOs human milk oligosaccharides
  • a wide variety of synthesis methods have been developed already, ranging from extraction over chemical synthesis to enzymatic synthesis. These methods are currently least applied, whereas biotechnological fermentative production is nowadays pursued and commercialized.
  • Methods for the production of disaccharides and/or oligosaccharides are well known for a person skilled in the art (like e.g. described in Faijes et al (2019), US2010120096A, JP2013201913, W02022/034067).
  • GIcNAc N-acetylglucosamine
  • this and other objects are achieved by providing methods and a cell for the production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide.
  • the present invention also provides methods for the purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide.
  • the present invention provides a cell which is genetically engineered as described herein and wherein synthesis of UDP-GIcNAc is rendered less functional.
  • This invention also provides a purified disaccharide and/or oligosaccharide like a milk oligosaccharide by the above-referenced process. Further benefits of the teachings of this invention will be apparent to one skilled in the art from reading this invention.
  • the features “synthesize”, “synthesized” and “synthesis” are interchangeably used with the features “produce”, “produced” and “production”, respectively.
  • the expressions “capable of... ⁇ verb>” and “capable to... ⁇ verb>” are preferably replaced with the active voice of said verb and vice versa.
  • the expression “capable of expressing” is preferably replaced with “expresses” and vice versa, i.e., “expresses” is preferably replaced with "capable of expressing”.
  • the verb "to comprise”, “to have” and “to contain” and their conjugations are used in their nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • the verb "to comprise” may be replaced by “to consist” or “to consist essentially of” and vice versa.
  • the verb “to consist” may be replaced by "to consist essentially of” meaning that a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • the articles “a” and “an” are preferably replaced by "at least one", more preferably “at least two”, even more preferably by “at least three", even more preferably by “at least four", even more preferably by "at least five", even more preferably by “at least six", most preferably by "at least two”.
  • the word “about” or “approximately” when used in association with a numerical value (e.g., “about 10") or with a range (e.g., "about x to approximately y”) preferably means that the value or range is interpreted as being as accurate as the method used to measure it.
  • polynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, are to be understood to be covered by the term “polynucleotides”. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • polynucleotide(s) as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells.
  • polynucleotide(s) also embraces short polynucleotides often referred to as oligonucleotide(s).
  • Polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
  • Polypeptide(s) refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids.
  • Polypeptide(s) include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques as well known to the skilled person. The same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Furthermore, a given polypeptide may contain many types of modifications.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid sidechains, and the amino or carboxyl termini.
  • Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention.
  • the term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions that also may contain coding and/or non-coding sequences.
  • isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • a “synthetic" sequence as the term is used herein, means any sequence that has been generated synthetically and not directly isolated from a natural source.
  • Synthesized as the term is used herein, means any synthetically generated sequence and not directly isolated from a natural source.
  • Recombinant means genetically engineered DNA prepared by transplanting or splicing genes from one species into the cells of a host organism of a different species. Such DNA becomes part of the host's genetic makeup and is replicated.
  • recombinant or “transgenic” or “metabolically engineered” or “genetically engineered” as used herein with reference to a cell or host cell are used interchangeably and indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence "foreign to said cell” or a sequence "foreign to said location or environment in said cell”).
  • Such cells are described to be transformed with at least one heterologous or exogenous gene or are described to be transformed by the introduction of at least one heterologous or exogenous gene.
  • Recombinant or metabolically engineered or genetically engineered or transgenic cells can contain genes that are not found within the native (non-recombinant) form of the cell.
  • Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means.
  • the terms also encompass cells that contain a nucleic acid endogenous to the cell that has been modified or its expression or activity has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, replacement of a promoter; site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis and related techniques as known to a person skilled in the art.
  • a "recombinant polypeptide” is one which has been produced by a recombinant cell.
  • the terms also encompass cells that have been modified by removing a nucleic acid endogenous to the cell by means of common well-known technologies for a skilled person (like e.g. knocking-out genes).
  • heterologous sequence or a “heterologous nucleic acid”, as used herein, is one that originates from a source foreign to the particular cell (e.g., from a different species), or, if from the same source, is modified from its original form or place in the genome.
  • a heterologous nucleic acid operably linked to a promoter is from a source different from that from which the promoter was derived, or, if from the same source, is modified from its original form or place in the genome.
  • the heterologous sequence may be stably introduced, e.g., by transfection, transformation, conjugation or transduction, into the genome of the host cell, wherein techniques may be applied which will depend on the cell and the sequence that is to be introduced.
  • mutant or "engineered” cell as used within the context of the present invention refers to a cell which is genetically engineered.
  • exogenous within the context of the present disclosure refers to any polynucleotide, polypeptide or protein sequence that is a natural part of a cell and is occurring at its natural location in the cell chromosome and of which the control of expression has not been altered compared to the natural control mechanism acting on its expression.
  • exogenous refers to any polynucleotide, polypeptide or protein sequence which originates from outside the cell under study and not a natural part of the cell or which is not occurring at its natural location in the cell chromosome or plasmid.
  • heterologous when used in reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme refers to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is from a source or derived from a source other than the host organism.
  • a "homologous" polynucleotide, gene, nucleic acid, polypeptide, or enzyme is used herein to denote a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is derived from the host organism.
  • heterologous means that the regulatory sequence or auxiliary sequence is not naturally associated with the gene with which the regulatory or auxiliary nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome.
  • a promoter operably linked to a gene to which it is not operably linked to in its natural state i.e.
  • heterologous promoter in the genome of a non-genetically engineered organism is referred to herein as a "heterologous promoter," even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked.
  • modified expression of a gene relates to a change in expression compared to the wild-type expression of said gene in any phase of the production process of the desired disaccharide and/or oligosaccharide, like a desired milk oligosaccharide.
  • Said modified expression is either a lower or higher expression compared to the wild-type, wherein the term “higher expression” is also defined as “overexpression” of said gene in the case of an endogenous gene or “expression” in the case of a heterologous gene that is not present in the wild-type strain.
  • Lower expression is obtained by means of common well-known technologies for a skilled person (such as the usage of siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon mutagenesis, etc.) which are used to change the genes in such a way that they are "less-able” (i.e., statistically significantly ess-able' compared to a functional wild-type gene) or completely unable (such as knocked-out genes) to produce functional final products.
  • a skilled person such as the usage of siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon mutagenesis, etc.
  • riboswitch as used herein is defined to be part of the messenger RNA that folds into intricate structures that block expression by interfering with translation. Binding of an effector molecule induces conformational change(s) permitting regulated expression post-transcriptionally.
  • lower expression can also be obtained by changing the transcription unit, the promoter, an untranslated region, the ribosome binding site, the Shine Dalgarno sequence or the transcription terminator.
  • Lower expression or reduced expression can for instance be obtained by mutating one or more base pairs in the promoter sequence or changing the promoter sequence fully to a constitutive promoter with a lower expression strength compared to the wild-type or an inducible promoter which result in regulated expression or a repressible promoter which results in regulated expression.
  • Overexpression or expression is obtained by means of common well-known technologies for a skilled person (such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids), wherein said gene is part of an "expression cassette" that relates to any sequence in which a promoter sequence, untranslated region sequence (containing either a ribosome binding sequence, Shine Dalgarno or Kozak sequence), a coding sequence and optionally a transcription terminator is present, and leading to the expression of a functional active protein. Said expression is either constitutive or conditional or regulated or tuneable.
  • RNA polymerase e.g., the bacterial sigma factors like s 70 , s 54 , or related s- factors and the yeast mitochondrial RNA polymerase specificity factor MTF1 that co-associate with the RNA polymerase core enzyme
  • transcription factors are CRP, Lacl, ArcA, Cra, IcIR in E. coli, or, Aft2p, Crzlp, Skn7 in Saccharomyces cerevisiae, or, DeoR, GntR, Fur in B. subtilis.
  • RNA polymerase is the catalytic machinery for the synthesis of RNA from a DNA template. RNA polymerase binds a specific DNA sequence to initiate transcription, for instance via a sigma factor in prokaryotic hosts or via MTFl in yeasts. Constitutive expression offers a constant level of expression with no need for induction or repression.
  • regulated expression is defined as 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 disaccharide and/or oligosaccharide, like a desired milk oligosaccharide.
  • non-native as used herein with reference to the activity of a protein indicates that the protein has been modified to have an abolished, impaired, reduced, delayed, higher, accelerated or improved activity compared to the native activity of said protein.
  • a modified activity of a protein is obtained by modified expression of said protein or is obtained by expression of a modified, i.e., mutant form of the protein.
  • a mutant form of the protein can be obtained by expression of a mutant form of the gene encoding the protein, e.g., comprising a deletion, an insertion and/or a mutation of one or more nucleotides compared to the native gene sequence.
  • a mutant form of a gene can be obtained by techniques well-known to a person skilled in the art, such as but not limited to site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis.
  • non-native indicates that the disaccharide 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 disaccharide or to have a higher production of the disaccharide.
  • non-native indicates that the oligosaccharide is i) not naturally produced or ii) when naturally produced not in the same amounts by the cell; and that the cell has been genetically engineered to be able to produce said oligosaccharide, more particularly said milk oligosaccharide, or to have a higher production of the oligosaccharide, more particularly the milk oligosaccharide.
  • non-native indicates that the disaccharide and the milk oligosaccharide are 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 disaccharide and milk oligosaccharide or to have a higher production of the disaccharide and the milk oligosaccharide.
  • Variant(s) as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to the persons skilled in the art.
  • the present invention contemplates making variants by modifying the structure of an enzyme as used in the present invention.
  • Variants can be produced by amino acid substitution, deletion, addition, or combinations thereof.
  • “Fragment” with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule, particularly a part of a polynucleotide.
  • sequence of a polynucleotide can be represented by a SEQ ID NO or alternatively by a GenelD (Maglott et al (2011) Nucl. Acids Res. 39, Issue suppl_l, D52-D57) or GenBank NO (https://www.ncbi.nlm.nih.gov/genbank/). Therefore, the terms "polynucleotide SEQ ID NO", “polynucleotide GenelD” and “polynucleotide GenBank NO.” can be interchangeably used, unless explicitly stated otherwise.
  • polypeptide SEQ ID NO SEQ ID NO
  • polypeptide UniProt ID polypeptide UniProt ID
  • a domain can be characterized, for example, by a Pfam (El-Gebali et al., Nucleic Acids Res. 47 (2019) D427- D432), an IPR (InterPro domain) (http://ebi.ac.uk/interpro) (Mitchell et aL, Nucleic Acids Res. 47 (2019) D351-D360), a conserveed Domain Database (CDD) designation (https://www.ncbi.nlm.nih.gov/cdd) (Lu et aL, Nucleic Acids Res.
  • Protein or polypeptide sequence information and functional information can be provided by a comprehensive resource for protein sequence and annotation data like e.g., the Universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49(D1), D480-D489).
  • UniProt comprises the expertly and richly curated protein database called the UniProt Knowledgebase (UniProtKB), together with the UniProt Reference Clusters (UniRef) and the UniProt Archive (UniParc).
  • the UniProt identifiers (UniProt ID) are unique for each protein present in the database.
  • sequence of a polypeptide is represented by a SEQ. ID NO or an UniProt ID.
  • the UniProt IDs of the proteins described correspond to their sequence version 01 as present in the UniProt Database (www.uniprot.org) version release 2021_03 and consulted on 09 June 2021.
  • InterPro provides functional analysis of proteins by classifying them into families and predicting domains and important sites. To classify proteins in this way, InterPro uses predictive models, known as signatures, provided by several different databases (referred to as member databases) that make up the InterPro consortium. Protein signatures from these member databases are combined into a single searchable resource, capitalizing on their individual strengths to produce a powerful integrated database and diagnostic tool.
  • member databases predictive models, known as signatures, provided by several different databases (referred to as member databases) that make up the InterPro consortium. Protein signatures from these member databases are combined into a single searchable resource, capitalizing on their individual strengths to produce a powerful integrated database and diagnostic tool.
  • monosaccharide refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed as an aldose, a ketose, a deoxysugar, a deoxy-aminosugar, a uronic acid, an aldonic acid, a ketoaldonic acid, an aldaric acid or a sugar alcohol, and contains one or more hydroxyl groups per molecule.
  • Monosaccharides are saccharides containing only one simple sugar.
  • phosphorylated monosaccharide refers to a monosaccharide which is phosphorylated.
  • phosphorylated monosaccharides include but are not limited to glucose-1- phosphate, glucose-6-phosphate, glucose-l,6-bisphosphate, galactose-l-phosphate, fructose-6- phosphate, fructose-l,6-bisphosphate, fructose-l-phosphate, glucosamine-l-phosphate, glucosamine-6- phosphate, N-acetylglucosamine-l-phosphate, mannose-l-phosphate, mannose-6-phosphate or fucose- 1-phosphate.
  • 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
  • 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, consisting of or consisting essentially of: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N- acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N- glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino- 4,6-dideoxy-N-acetyl-beta-L-altrosamine transa
  • 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 (LNB, Gal- P1,3-GICNAC), N-acetyllactosamine (LacNAc, Gal-pi,4-GlcNAc), LacDiNAc (GalNAc-pi,4-GlcNAc), N- acetylgalactosaminylglucose (GalNAc-pi,4-Glc), Neu5Ac-a2,3-Gal, Neu5Ac-a2,6-Gal and fucopyranosyl- (l-4)-N-glycolylneuraminic acid (Fuc-(l-4)-Neu5Gc), sucrose (Glc-ocl,2-Fru), malto
  • Disaccharides comprise charged disaccharides carrying a negative charge like e.g. sialylated disaccharides like e.g. Neu5Ac-a2,3-Gal, Neu5Ac-a2,6-Gal and Fuc-(l-4)-Neu5Gc, and noncharged, i.e. neutral, disaccharides like e.g. lactose, LNB, LacNAc, sucrose, maltose and melibiose.
  • a negative charge like e.g. sialylated disaccharides like e.g. Neu5Ac-a2,3-Gal, Neu5Ac-a2,6-Gal and Fuc-(l-4)-Neu5Gc
  • noncharged i.e. neutral, disaccharides like e.g. lactose, LNB, LacNAc, sucrose, maltose and melibiose.
  • 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, milk oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigen, enterobacterial common antigen (ECA), the glycan chain present in lipopolysaccharides (LPS), the oligosaccharide repeats present in capsular polysaccharides, peptidoglycan (PG), amino-sugars, antigens of the human ABO blood group system, an animal oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans, a plant oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans, sialylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, fucosylated oligosaccharide,
  • oligosaccharide or “acidic oligosaccharide” are used interchangeably and refer to an oligosaccharide with a negative charge.
  • the negatively charged oligosaccharide is a sialylated oligosaccharide.
  • a 'sialylated oligosaccharide' is to be understood as a negatively charged sialic acid containing oligosaccharide, i.e., an oligosaccharide having one or more sialic acid residue(s). It has an acidic nature.
  • Some examples are 3'SL (3'-sialyllactose, Neu5Ac-a2,3-Gal-pi,4-Glc), 3'-sialyllactosamine, 6'SL (6'sialyllactose, Neu5Ac-a2,6-Gal-pi,4-Glc), 8'SL (8'sialyllactose, Neu5Ac-ot2,8-Gal-pi,4-Glc), 3,6-disialyllactose (Neu5Ac-a2,3-(Neu5Ac-a2,6)-Gal-pi,4- Glc), 6,6'-disialyllactose (Neu5Ac-oc2,6-Gal-pi,4-(Neu5Ac-a2,6)-Glc), 8,3-disialyllactose (Neu5Ac-oc2,8- Neu5Ac-a2,3-Gal-
  • Charged oligosaccharides are oligosaccharide structures that contain one or more negatively charged monosaccharide subunits including N-acetylneuraminic acid (Neu5Ac), commonly known as sialic acid, N- glycolylneuraminic acid (Neu5Gc), glucuronate, galacturonate and 2-keto-3-deoxymanno-octulonic acid (KDO).
  • Neu5Ac N-acetylneuraminic acid
  • Ne5Gc N- glycolylneuraminic acid
  • KDO 2-keto-3-deoxymanno-octulonic acid
  • Charged oligosaccharides are also referred to as acidic oligosaccharides.
  • neutral (noncharged) oligosaccharides are non-sialylated oligosaccharides, and thus do not contain an acidic monosaccharide subunit.
  • Neutral oligosaccharides comprise non-charged fucosylated oligosaccharides that contain one or more fucose subunits in their glycan structure as well as non-charged non-fucosylated oligosaccharides that lack any fucose subunit.
  • Other examples of charged oligosaccharides are sulphated chitosans and deacetylated chitosans.
  • 'neutral oligosaccharide' and 'non-charged' oligosaccharide as used herein are used interchangeably and refer, as generally understood in the state of the art, to an oligosaccharide that has no negative charge originating from a carboxylic acid group.
  • Examples of such neutral oligosaccharide are 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyl lactose (6FL), 2', 3- difucosyllactose (diFL), lacto-N-triose II (LN3, GlcNAc i-3Gaipi-4Glc), lacto-N-tetraose (LNT, Gaipi- 3GlcNAc i-3Gaipi-4Glc), lacto-N-neotetraose (LNnT, Gaipi-4GlcNAcpi-3Gaipi-4Glc), lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose
  • a 'fucosylated oligosaccharide' as used herein and as generally understood in the state of the art is an oligosaccharide that is carrying a fucose-residue.
  • Such fucosylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of said monosaccharide subunit is a fucose.
  • a fucosylated oligosaccharide can contain more than one fucose residue, e.g., two, three or more.
  • a fucosylated oligosaccharide can be a neutral oligosaccharide or a charged oligosaccharide e.g., also comprising sialic acid structures. Fucose can be linked to other monosaccharide subunits comprising glucose, galactose, GIcNAc via alpha-glycosidic bonds comprising alpha-1,2 alpha-1,3, alpha-1,4, alpha-1,6 linkages.
  • Examples comprise 2'-fucosyl lactose (2' FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL), Lacto-N- fucopentaose I (LNFP I), Gal-al,3-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc (Gal-LNFP I), GalNAc-al,3- (Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc (GalNAc-LNFP I), Lacto-N-fucopentaose II (LNFP II), Lacto- N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (
  • Mammalian milk oligosaccharides comprise oligosaccharides present in milk found in any phase during lactation including colostrum milk from humans and mammals including but not limited to cows (Bos Taurus), sheep (Ovis aries), goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus), horses (Equus ferus caballus), pigs (Sus scropha), dogs (Canis lupus familiaris), ezo brown bears (Ursus arctos yesoensis), polar bear (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephant (Loxodonta africana), giant anteater (Myrmecophaga tri
  • 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, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose II,
  • human milk oligosaccharide or "HMO” refer to oligosaccharides found in human breast milk, including preterm human milk, colostrum and term human milk. HMOs comprise fucosylated oligosaccharides, non-fucosylated neutral oligosaccharides and sialylated oligosaccharides.
  • HMOs comprise 3-fucosyllactose, 2'-fucosyllactose, 2',3-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, LN3, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N- fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N- tetraose b, sialyllacto-N-tetraose a, difucosyllacto-N-tetraose, lacto-N-hexao
  • N-acetylglucosamine 1-phosphate uridylyltransferase "N-acetylglucosamine-l-phosphate uridyltransferase”
  • UDP-N-acetylglucosamine diphosphorylase "UDP-N-acetylglucosamine pyrophosphorylase”
  • uridine diphosphoacetylglucosamine pyrophosphorylase "UTP:2-acetamido-2- deoxy-alpha-D-glucose-l-phosphate uridylyltransferase”
  • UDP-GIcNAc pyrophosphorylase "GlmU uridylyltransferase”
  • Acetylglucosamine 1-phosphate uridylyltransferase "UDP-acetylglucosamine pyrophosphorylase”
  • uridine diphosphate-N-acetylglucosamine pyrophosphorylase "
  • glucosamine-l-phosphate acetyltransferase refers to an enzyme that catalyses the transfer of the acetyl group from acetyl coenzyme A to glucosamine-l-phosphate (GlcN-1-P) to produce N- acetylglucosamine-l-phosphate (GlcNAc-1-P).
  • glycosmU refers to a bifunctional enzyme that has both N-acetylglucosamine-l-phosphate uridyltransferase and glucosamine-l-phosphate acetyltransferase activity and that catalyses two sequential reactions in the de novo biosynthetic pathway for UDP-GIcNAc.
  • the C-terminal domain catalyses the transfer of acetyl group from acetyl coenzyme A to GlcN-1-P to produce GlcNAc-1-P, which is converted into UDP-GIcNAc by the transfer of uridine 5-monophosphate, a reaction catalysed by the N- terminal domain.
  • bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase refers to a polypeptide comprising both N-acetylglucosamine-1- phosphate uridyltransferase activity as well as glucosamine-l-phosphate acetyltransferase activity.
  • L-glutamine— D-fructose-6-phosphate aminotransferase D-fructose-6-phosphate aminotransferase
  • glutamine — fructose-6-phosphate transaminase (isomerizing) hexosephosphate aminotransferase
  • glucosamine-6-phosphate isomerase glutamine-forming
  • glutamine-fructose-6-phosphate transaminase (isomerizing) "D- fructose-6-phosphate amidotransferase
  • fructose-5-phosphate aminotransferase "glucosaminephosphate isomerase
  • glucosamine 6-phosphate synthase synthase
  • GlcN6P synthase GFA
  • glms glmS
  • glmS*54 are used interchangeably and refer to an enzyme that catalyses the conversion of D-fructose-6-phosphate into
  • pathway for production of a disaccharide is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of a disaccharide as defined herein.
  • Said pathway for production of a disaccharide 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 disaccharide of the present invention.
  • pathway for production of an oligosaccharide is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of an oligosaccharide as defined herein, like e.g., a milk oligosaccharide.
  • Said pathway for production of an oligosaccharide like e.g., a milk oligosaccharide can comprise but is not limited to pathways involved in the synthesis of a nucleotide-activated sugar and the transfer of said nucleotide-activated sugar to an acceptor to create an oligosaccharide of the present invention, like e.g., a milk oligosaccharide.
  • pathway for production of a disaccharide and a milk oligosaccharide is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of a disaccharide and a milk oligosaccharide as defined herein.
  • Said pathway for production of a disaccharide and a milk oligosaccharide can comprise but is not limited to pathways involved in the synthesis of a nucleotide-activated sugar and the transfer of said nucleotide-activated sugar to an acceptor to create a disaccharide and a milk oligosaccharide of the present invention.
  • Examples of such pathways comprise but are not limited to a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway and an N-acetylmannosaminylation pathway.
  • Said pathway for production of a disaccharide may comprise a pathway for synthesis and/or import of a co-factor used in said pathway for production of said disaccharide.
  • Said pathway for production of an oligosaccharide like a milk oligosaccharide may comprise a pathway for synthesis and/or import of a co-factor used in said pathway for production of said oligosaccharide like said milk oligosaccharide.
  • Said pathway for production of a disaccharide and a milk oligosaccharide may comprise a pathway for synthesis and/or import of a co-factor used in said pathway for production of said disaccharide and a milk oligosaccharide.
  • 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, 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 disaccharide and/or oligosaccharide, like e.g., a milk oligosaccharide.
  • 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 disaccharide and/or oligosaccharide like a milk oligosaccharide or an oligosaccharide mixture like a milk oligosaccharide mixture 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 disaccharide and/or an oligosaccharide like a milk oligosaccharide or an oligosaccharide mixture like a milk oligosaccharide mixture 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 i) a disaccharide and/or an oligosaccharide like a milk oligosaccharide or ii) an oligosaccharide mixture like a milk oligosaccharide mixture, 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 disaccharide and/or an oligosaccharide, like a milk oligosaccharide, or an oligosaccharide mixture like a milk oligosaccharide mixture 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 disaccharide produced by the cells divided by the mass of the cells produced in the culture.
  • cell productivity index (CPI) also refers to the mass of the oligosaccharide like the milk oligosaccharide produced by the cells divided by the mass of the cells produced in the culture.
  • CPI cell productivity index
  • the term “CPI” as used herein is also to be understood as mass of the mixture of a disaccharide and a milk oligosaccharide produced by the cells divided by the mass of the cells produced in the culture.
  • CPI as used herein is also to be understood as mass of the oligosaccharide mixture like a milk oligosaccharide mixture produced by the cells divided by the mass of the cells produced in the culture.
  • 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).
  • mammalian mammary-like cell(s) may be engineered to remove at least one undesired genetic component and/or to include at least one predetermined genetic construct that is typical of a mammalian mammary cell.
  • mammalian mammary-like cell (s) may include mammalian mammary epithelial-like cell(s), mammalian mammary epithelial luminal-like cell(s), mammalian non-mammary cell(s) that exhibits one or more characteristics of a cell of a mammalian mammary cell lineage, or any combination thereof.
  • mammalian mammary-like cell(s) may include mammalian cell(s) having a phenotype similar (or substantially similar) to natural mammalian mammary cell (s), or more particularly a phenotype similar (or substantially similar) to natural mammalian mammary epithelial cell(s).
  • a mammalian cell with a phenotype or that exhibits at least one characteristic similar to (or substantially similar to) a natural mammalian mammary cell or a mammalian mammary epithelial cell may comprise a mammalian cell (e.g., derived from a mammary cell lineage or a non-mammary cell lineage) that exhibits either naturally, or has been engineered to, be capable of expressing at least one milk component.
  • the term "non- mammary cell(s)" may generally include any mammalian cell of non-mammary lineage.
  • a non-mammary cell can be any mammalian cell capable of being engineered to express at least one milk component.
  • non-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.
  • precursor refers to substances which are taken up or synthetized by the cell for the specific production of a disaccharide and/or an oligosaccharide, like a milk oligosaccharide, or an oligosaccharide mixture like a milk oligosaccharide mixture according to the present invention.
  • a precursor can be an acceptor as defined herein, but can also be another substance, metabolite, co-factor, which is first modified within the cell as part of the biochemical synthesis route of a disaccharide and/or oligosaccharide, like a milk oligosaccharide or an oligosaccharide mixture like a milk oligosaccharide mixture.
  • precursor as used herein is also to be understood as a chemical compound that participates in an incubation or an enzymatic reaction to produce another compound like e.g., an intermediate or an acceptor as defined herein, as part in the metabolic pathway of a disaccharide and/or oligosaccharide like a milk oligosaccharide or an oligosaccharide mixture like a milk oligosaccharide mixture.
  • 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 disaccharide and/or oligosaccharide like a milk oligosaccharide or an oligosaccharide mixture like a milk oligosaccharide mixture.
  • Such precursors comprise the acceptors as defined herein, and/or dihydroxyacetone, glucosamine, N-acetylglucosamine, N- acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyllactose, phosphorylated sugars or sugar phosphates like e.g.
  • glucose-l-phosphate galactose-l-phosphate, glucose-6- phosphate, fructose-6-phosphate, fructose-l,6-bisphosphate, mannose-6-phosphate, mannose-1- phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N- acetylglucosamine-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.
  • the cell used to produce the saccharide, like a disaccharide and/or a milk oligosaccharide is transformed to comprise and to express at least one nucleic acid sequence encoding a protein selected from the group consisting of lactose transporter, N-acetylneuraminic acid transporter, fucose transporter, glucose transporter, galactose transporter, transporter for a nucleotide-activated sugar wherein said transporter internalizes a to the medium added precursor for the synthesis of the saccharide like the disaccharide and/or milk oligosaccharide of present invention.
  • a protein selected from the group consisting of lactose transporter, N-acetylneuraminic acid transporter, fucose transporter, glucose transporter, galactose transporter, transporter for a nucleotide-activated sugar wherein said transporter internalizes a to the medium added precursor for the synthesis of the saccharide like the disaccharide and
  • 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, lactulose, lactobionic acid (LBA), lacto-N-triose, lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-pentaose (LN P), lacto-N-neopentaose, para lacto-N- pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N-hexaose (LNH), lacto-N- neohex
  • the present invention provides a cell capable of synthesizing and/or synthesizing UDP-N-acetylglucosamine (UDP-GIcNAc) and genetically engineered for the production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide, wherein the cell comprises a pathway for production of said disaccharide and/or oligosaccharide like a milk oligosaccharide, characterized in that UDP-GIcNAc synthesis in said cell is rendered less functional.
  • UDP-GIcNAc UDP-N-acetylglucosamine
  • the present invention provides a method for the production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide, wherein the method comprises cultivating and/or incubating a cell as described herein, in cultivation and/or incubation medium under conditions permissive to produce a disaccharide and/or oligosaccharide like a milk oligosaccharide.
  • permissive conditions are understood to be conditions relating to physical or chemical parameters including but not limited to temperature, pH, pressure, osmotic pressure and product/precursor/acceptor concentration.
  • the permissive conditions may include a temperature-range of 30 +/- 20 degrees centigrade, a pH-range of 7 +/- 3.
  • the disaccharide and/or oligosaccharide like a milk oligosaccharide is/are separated from said cultivation and/or incubation.
  • the disaccharide and/or oligosaccharide like a milk oligosaccharide is/are purified.
  • the cell is capable of synthesizing and/or synthesizing UDP-GIcNAc.
  • UDP-GIcNAc can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • UDP-GIcNAc is formed from fructose-6-phophate (Fru-6-P) in a four-step version of the Leloir pathway.
  • the four successive reactions comprise: (a) conversion of fructose-6-P (Fru-6-P) into glucosamine-6-phosphate (GlcN-6-P); (b) acetylation of GlcN-6-P to GlcNAc-6- P; (c) isomerization of GlcNAc-6-P to GlcNAc-1-P; and (d) uridylation of GlcNAc-1-P to give UDPGIcNAc
  • the first and the last step are essentially the same as in eukaryotes, but GlcN-6-P is first isomerized to give GlcN-1-P, which is subsequently N-acetylated.
  • Each step of the eukaryotic pathway is catalysed by a separate enzyme, while in bacteria there are only three enzymatic proteins involved in UDP-GIcNAc biosynthesis.
  • These enzymes may be any one or more of the list comprising an N-acetyl-D- glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, and an N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase.
  • the cell synthesizes UDP-GIcNAc.
  • the cell comprises a pathway for production of UDP-GIcNAc. Said pathway for production of UDP-GIcNAc consists of the enzymes and their respective genes involved in the synthesis of UDP-GIcNAc.
  • Enzymes involved in the synthesis of UDP-GIcNAc comprise but are not limited to bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, an N-acetylglucosamine-l-phosphate uridyltransferase and a glucosamine-1- phosphate acetyltransferase.
  • Synthesis of UDP-GIcNAc may make use of one or more co-factor(s). Examples of co-factors comprise but are not limited to Mg 2+ , Co 2+ , Mn 2+ , Ca 2+ , Zn 2+ , Ni 2+ and FAD.
  • Said pathway for production of UDP-GIcNAc may comprise a pathway for synthesis and/or for import of a cofactor used in a pathway for production of UDP-GIcNAc.
  • the cell of present invention may comprise a pathway for the production and/or import of any one or more Mg 2+ , Co 2+ , Mn 2+ , Ca 2+ , Zn 2+ , Ni 2+ and FAD.
  • the cell of present invention does not synthesize a co-factor that is necessary in the synthesis of UDP-GIcNAc but has all other enzymes necessary for UDP-GIcNAc synthesis.
  • Said cell may synthesize UDP-GIcNAc upon supplementation with one or more co-factor(s).
  • UDP-GIcNAc synthesis in the cell is rendered less functional.
  • a cell wherein UDP-GIcNAc synthesis is rendered less functional is to be understood as that said cell has lower production of UDP-GIcNAc compared to a cell wherein UDP-GIcNAc synthesis is not rendered less functional.
  • synthesis of UDP-GIcNAc is rendered less functional in a cell by making one or more genes involved in the pathway for production of UDP-GIcNAc less functional.
  • synthesis of UDP-GIcNAc is rendered less functional in a cell by making one or more genes involved in the pathway for production and/or the import of one or more co-factor(s) that are used in the synthesis of UDP-GIcNAc less functional.
  • synthesis of UDP-GIcNAc is rendered less functional in a cell by making one or more genes involved in the pathway for production and/or the import of one or more co-factor(s) selected from the list comprising Mg 2+ , Co 2+ , Mn 2+ , Ca 2+ , Zn 2+ , Ni 2+ and FAD less functional.
  • Rendering a gene less functional is to be understood as rendering a gene 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 a functional final product.
  • a gene can be made less functional by means of common well-known technologies for a skilled person, by e.g., any one or more of insertion, deletion and/or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) selected from the list comprising promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said gene so that said gene is made less-able to produce a functional final product.
  • UDP-GIcNAc synthesis possesses at least one gene selected from the list comprising genes encoding bifunctional N- acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, N- acetylglucosamine-l-phosphate uridyltransferase and glucosamine-l-phosphate acetyltransferase, and wherein said at least one gene is rendered less functional as described herein.
  • UDP-GIcNAc synthesis is obtained by expression of at least one gene selected from the list comprising genes encoding bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, N-acetylglucosamine-l-phosphate uridyltransferase and glucosamine-l-phosphate acetyltransferase, and wherein said at least one gene is rendered less functional as described herein.
  • At least one gene involved in the synthesis and/or import of a co-factor that is involved in UDP-GIcNAc synthesis is rendered less functional or knocked out, preferably said co-factor is selected from the list comprising Mg 2+ , Co 2+ , Mn 2+ , Ca 2+ , Zn 2+ , Ni 2+ and FAD.
  • At least one of said genes involved in UDP- GIcNAc synthesis and/or involved in the synthesis and/or import of a co-factor that is involved in UDP- GIcNAc synthesis is rendered less functional by insertion, deletion and/or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) selected from the list comprising promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said at least one gene. It is to be understood herein that by rendering at least one of said genes less functional in the cell renders the cell with a less functional synthesis of UDP-GIcNAc.
  • the cell produces less UDP-GIcNAc compared to a cell wherein no one of said genes is rendered less functional.
  • the term "less UDP-GIcNAc" is, however, not to be understood to comprise 0 g/L UDP-GIcNAc since then the cell is no longer viable.
  • UDP-GIcNAc synthesis is obtained by expression of at least two genes selected from the list comprising genes encoding bifunctional N- acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, N- acetylglucosamine-l-phosphate uridyltransferase and glucosamine-l-phosphate acetyltransferase, and wherein i) at least one of said at least two genes is rendered less functional, ii) at least two of said at least two genes is rendered less functional or iii) all of said at least two genes are rendered less functional, as described herein.
  • the at least one gene encodes an enzyme wherein the enzyme is selected from an enzyme class selected from the list comprising EC:2.7.7.23, EC:2.3.1.157 and EC:5.4.2.3.
  • the at least one gene encodes an enzyme wherein the enzyme comprises a polypeptide sequence comprising an IPR domain selected from the list comprising IPR001451, IPR002618, IPR005175, IPR005835, IPR005843, IPR005844, IPR005882, IPR011004, IPR016055, IPR016066, IPR016657, IPR018357, IPR023915, IPR025877, IPR029044, IPR036900 and IPR038009 as defined by InterPro 90.0 as released on 4 th August 2022.
  • the at least one gene encodes an enzyme wherein the enzyme comprises a polypeptide sequence comprising a PFAM domain selected from the list comprising PF00132, PF00408, PF00465, PF00483, PF01070, PF01565, PF01704, PF02878, PF02879, PF02880, PF03479, PF04030, PF05199, PF12146, PF12804, PF13562 and PF14602 as defined by InterPro 90.0 as released on 4 th August 2022.
  • the enzyme comprises a polypeptide sequence comprising a PFAM domain selected from the list comprising PF00132, PF00408, PF00465, PF00483, PF01070, PF01565, PF01704, PF02878, PF02879, PF02880, PF03479, PF04030, PF05199, PF12146, PF12804, PF13562 and
  • the at least one gene encodes an enzyme wherein the enzyme comprises a polypeptide sequence comprising the conserved protein domain selected from the list comprising cd03086 and cd03353 as defined by InterPro 90.0 as released on 4th August 2022.
  • the at least one gene encodes an enzyme wherein the enzyme is part of a NOG family selected from the list comprising COG1109 and COG4284 as defined by eggNOG5.0 as released in 2019.
  • the at least one gene encodes an enzyme wherein the enzyme uses a cofactor selected from the list comprising Mg 2+ , Co 2+ , Mn 2+ , Ca 2+ , Zn 2+ , Ni 2+ and FAD.
  • the at least one gene encodes an enzyme wherein the enzyme is selected from the enzyme class EC:2.3.1.157, comprises a polypeptide sequence comprising the IPR domains IPR001451, IPR005175, IPR005882, IPR011004, IPR018357, IPR025877, IPR029044 and IPR038009 as defined by InterPro 90.0 as released on 4 th August 2022, comprises a polypeptide sequence comprising the PFAM domains PF00132, PF00465, PF00483, PF01070, PF01565, PF03479, PF04030, PF05199, PF12804, PF13562 and PF14602 as defined by InterPro 90.0 as released on 4 th August 2022, comprises a polypeptide sequence comprising the conserved protein domain cd03353 as defined by InterPro 90.0 as released on 4th August 2022, and is part of the NOG family COG4284 as defined by eggNOG
  • the at least one gene encodes an enzyme wherein the enzyme is selected from the enzyme class EC:5.4.2.3, comprises a polypeptide sequence comprising the IPR domains IPR005843, IPR005844, IPR016055, IPR016066, IPR016657 and IPR036900 as defined by InterPro 90.0 as released on 4 th August 2022, comprises a polypeptide sequence comprising the PFAM domains PF00408, PF02878, PF02879 and PF02880 as defined by InterPro 90.0 as released on 4 th August 2022, comprises a polypeptide sequence comprising the conserved protein domain cd03086 as defined by InterPro 90.0 as released on 4th August 2022, and is part of the NOG family COG1109 as defined by eggNOG5.0 as released in 2019 and uses a cofactor selected from the list comprising Mg 2+ , Ni 2+ and FAD.
  • the at least one gene encodes an enzyme wherein the enzyme is selected from the enzyme class EC:2.7.7.23, comprises a polypeptide sequence comprising the IPR domains IPR001451, IPR002618, IPR005175, IPR005835, IPR005882, IPR011004, IPR018357, IPR023915, IPR025877, IPR029044, IPR038009 as defined by InterPro 90.0 as released on 4 th August 2022, comprises a polypeptide sequence comprising the PFAM domains PF00132, PF00483, PF01704, PF03479, PF12146, PF12804, PF13562 and PF14602 as defined by InterPro 90.0 as released on 4 th August 2022, comprises a polypeptide sequence comprising the conserved protein domain cd03353 as defined by InterPro 90.0 as released on 4th August 2022, and is part of the NOG family COG4284 as defined by eggNOG5.0
  • the cell is genetically engineered for production of a disaccharide, wherein the cell comprises a pathway for production of said disaccharide.
  • the cell is genetically engineered for production of two or more disaccharides.
  • the cell is genetically engineered for an enhanced production of a disaccharide, an enhanced uptake of one or more precursor(s) and/or acceptor(s) that is/are used in the synthesis of a disaccharide, a better efflux of the disaccharide, a decreased production of by-products like e.g. acids, an increased availability of co-factors like e.g.
  • ATP ATP, NADP, NADPH, and/or better metabolic flux through any one of the sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N- acetylgalactosaminylation, mannosylation, and/or N-acetylmannosaminylation pathway present in the cell.
  • the cell is genetically engineered for production of an oligosaccharide like a milk oligosaccharide, wherein the cell comprises a pathway for production of said oligosaccharide.
  • the cell is genetically engineered for production of two or more oligosaccharides like e.g. two or more milk oligosaccharides.
  • the cell is genetically engineered for an enhanced production of an oligosaccharide like a milk oligosaccharide, an enhanced uptake of one or more precursor(s) and/or acceptor(s) that is/are used in the synthesis of an oligosaccharide like a milk oligosaccharide, a better efflux of the oligosaccharide like a milk oligosaccharide, a decreased production of by-products like e.g. acids, an increased availability of co-factors like e.g.
  • ATP ATP, NADP, NADPH, and/or better metabolic flux through any one of the sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N- acetylgalactosaminylation, mannosylation, and/or N-acetylmannosaminylation pathway present in the cell.
  • the cell is genetically engineered for production of a disaccharide and a milk oligosaccharide, wherein the cell comprises a pathway for production of said disaccharide and milk oligosaccharide.
  • the cell is genetically engineered for production of one disaccharide and one milk oligosaccharide.
  • the cell is genetically engineered for production of a) two or more disaccharides and b) one milk oligosaccharide.
  • the cell is genetically engineered for production of a) one disaccharide and b) two or more milk oligosaccharides.
  • the cell is genetically engineered for production of a) two or more disaccharides and b) two or more milk oligosaccharides.
  • the cell is genetically engineered for an enhanced production of a disaccharide and a milk oligosaccharide, an enhanced uptake of one or more precursor(s) and/or acceptor(s) that is/are used in the synthesis of a disaccharide and a milk oligosaccharide, a better efflux of the disaccharide and the milk oligosaccharide, a decreased production of by-products like e.g. acids, an increased availability of co-factors like e.g.
  • ATP ATP, NADP, NADPH, and/or better metabolic flux through any one of the sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N- acetylgalactosaminylation, mannosylation, and/or N-acetylmannosaminylation pathway present in the cell.
  • the genetically engineered cell is modified with gene expression modules wherein the expression from any one of said expression modules is constitutive or is tuneable.
  • Said expression modules are also known as transcriptional units and comprise polynucleotides for expression of recombinant genes including coding gene sequences and appropriate transcriptional and/or translational control signals that are operably linked to the coding genes.
  • Said control signals comprise promoter sequences, untranslated regions, ribosome binding sites, terminator sequences.
  • Said expression modules can contain elements for expression of one single recombinant gene but can also contain elements for expression of more recombinant genes or can be organized in an operon structure for integrated expression of two or more recombinant genes.
  • Said polynucleotides may be produced by recombinant DNA technology using techniques well-known in the art.
  • the cell is modified with one or more expression modules.
  • the expression modules can be integrated in the genome of said cell or can be presented to said cell on a vector.
  • Said vector can be present in the form of a plasmid, cosmid, phage, liposome, or virus, which is to be stably transformed/transfected into said metabolically engineered cell.
  • Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • These vectors may contain selection markers such as but not limited to antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA sense/antisense markers.
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard.
  • the appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., see above.
  • cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
  • Introduction of a polynucleotide into the cell can be effected by methods described in many standard laboratory manuals, such as e.g. Sambrook et aL, 1989, supra.
  • an expression module comprises polynucleotides for expression of at least one recombinant gene.
  • Said recombinant gene is involved in the pathway for production of a disaccharide and/or oligosaccharide like a milk oligosaccharide; or said recombinant gene is linked to other pathways in said cell that are not involved in the synthesis of a disaccharide and/or oligosaccharide like a milk oligosaccharide.
  • Said recombinant genes encode endogenous proteins with a modified expression or activity, preferably said endogenous proteins are overexpressed; or said recombinant genes encode heterologous proteins that are heterogeneously introduced and expressed in said modified cell, preferably overexpressed.
  • the endogenous proteins can have a modified expression in the cell which also expresses a heterologous protein.
  • each of said expression modules is constitutive or tuneable as defined herein.
  • the pathway for production of said disaccharide and/or oligosaccharide like a milk oligosaccharide is selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway.
  • the cell is genetically engineered to comprise at least one of said pathway(s).
  • the cell comprises at least one of said pathway(s) wherein at least one of said pathway(s) has/have been genetically engineered.
  • the cell comprises a fucosylation pathway.
  • the cell is metabolically engineered to comprise a fucosylation pathway.
  • the cell has been metabolically engineered to comprise a fucosylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-1- phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase and fucosyltransferase has/have a modified and/or enhanced expression.
  • the cell comprises a sialyation pathway.
  • the cell is metabolically engineered to comprise a sialylation pathway.
  • the cell has been metabolically engineered to comprise a sialylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6- phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N- acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6- phosphate 2-epimerase, UDP-GIcNAc 2-epimerase/kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglu
  • the cell comprises a galactosylation pathway.
  • the cell is metabolically engineered to comprise a galactosylation pathway.
  • the cell has been metabolically engineered to comprise a galactosylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of galactose-l-epimerase, galactokinase, glucokinase, galactose-1- phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase and galactosyltransferase has/have a modified and/or enhanced expression.
  • the cell comprises an 'N- acetylglucosaminylation' pathway.
  • the cell is metabolically engineered to comprise an N-acetylglucosaminylation pathway.
  • the cell has been metabolically engineered to comprise an N-acetylglucosaminylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N- acetylglucosamine-l-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, and a glycosyltransferase transferring GIcNAc has/have a modified and/or enhanced expression.
  • the cell comprises an 'N- acetylgalactosaminylation' pathway.
  • the cell is metabolically engineered to comprise an N-acetylgalactosaminylation pathway.
  • the cell has been metabolically engineered to comprise an N-acetylgalactosaminylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, UDP-N-acetylglucosamine 4-epimerase, UDP-glucose 4-epimerase, N-acetylgalactosamine kinase and/or UDP-N-acetylgalact
  • the cell comprises a 'mannosylation' pathway.
  • the cell is metabolically engineered to comprise a mannosylation pathway.
  • the cell has been metabolically engineered to comprise a mannosylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase and/or mannose-l-phosphate guanylyltransferase and mannosyltransferase has/have a modified and/or enhanced expression.
  • the cell comprises an 'N- acetylmannosaminylation' pathway.
  • the cell is metabolically engineered to comprise an N-acetylmannosaminylation pathway.
  • the cell has been metabolically engineered to comprise an N-acetylmannosaminylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N- acetylglucosamine-5-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase, N- acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine
  • the cell comprises one or more pathway(s) for monosaccharide synthesis.
  • Said pathways for monosaccharide synthesis comprise, consist of or consist essentially of 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
  • PEP phosphoenolpyruvate
  • the cell comprises one or more pathway(s) for phosphorylated monosaccharide synthesis.
  • Said pathways for phosphorylated monosaccharide synthesis comprise, consist of or consist essentially of enzymes involved in the synthesis of one or more monosaccharide(s), one or more nucleoside mono-, di- and/or triphosphate(s) and enzymes involved in the synthesis of phosphoenolpyruvate (PEP) like e.g.
  • the cell comprises one or more pathways for the synthesis of one or more nucleotide-activated sugars.
  • Said pathways for nucleotide-activated sugar synthesis comprise, consist of or consist essentially of 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-acetylglucosamine epimerase, UDP-
  • the cell possesses, preferably expresses, more preferably overexpresses, one or more glycosyltransferase(s) selected from the list comprising, consisting of or consisting essentially of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N- acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N- glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases
  • the fucosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1, -fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-1, 4-fucosyltransferase and alpha-1, 6-fucosyltransferase.
  • the sialyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-2, 3- sialyltransferase, alpha-2, 6-sialyltransferase, and alpha-2, 8-sialyltransferase.
  • the galactosyltransferase is selected from the list comprising, consisting of or consisting essentially of beta- 1,3-galactosyltransferase, N-acetylglucosamine beta-1, 3-galactosyltransferase, beta-1, 4- galactosyltransferase, N-acetylglucosamine beta-1, 4-galactosyltransferase, alpha-1, 3- galactosyltransferase and alpha-1, 4-galactosyltransferase.
  • the glucosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-glucosyltransferase, beta-1, 2- glucosyltransferase, beta-1, 3-glucosyltransferase and beta-1, 4-glucosyltransferase.
  • the mannosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha- 1,2-mannosyltransferase, alpha-1, 3-mannosyltransferase and alpha-1, 6-mannosyltransferase.
  • the N-acetylglucosaminyltransferase is selected from the list comprising, consisting of or consisting essentially of galactoside beta-1, 3-N-acetylglucosaminyltransferase and beta-1, 6-N-acetylglucosaminyltransferase.
  • the N-acetylgalactosaminyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1, 3-N-acetylgalactosaminyltransferase.
  • the cell is modified in the expression or activity of at least one of said glycosyltransferases.
  • said glycosyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous glycosyltransferase is overexpressed; alternatively said glycosyltransferase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed.
  • Said endogenous glycosyltransferase can have a modified expression in the cell which also expresses a heterologous glycosyltransferase.
  • the cell is capable to produce, preferably produces, one or more nucleotide-activated sugars, preferably said cell is genetically engineered for production of one or more of said nucleotide-activated sugar(s).
  • said one or more nucleotide-activated sugar(s) is/are selected from the list comprising, consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP- N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP-glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6- dideoxy— L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy— L-lyxo-4-hexulose, UDP-N-acetyl-L- rhamnosamine (UDP-L
  • the cell comprises a pathway for the synthesis of a nucleotide-activated sugar selected from the list comprising, consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP- GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP-glucuronate, UDP-galacturonate, UDP-2- acetamido-2,6-dideoxy— L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy— L-lyxo-4-hexulose, UDP- N-ace
  • UDP-GIcNAc can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing an UDP-GIcNAc can express enzymes converting, e.g. GIcNAc, which is to be added to the cell, to UDP-GIcNAc.
  • These enzymes may be any one or more of the list comprising, consisting of or consisting essentially of an N-acetyl-D-glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, N-acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, and a bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli.
  • the cell is modified to produce UDP-GIcNAc.
  • the cell used herein is optionally genetically modified to express the de novo synthesis of CMP-Neu5Ac.
  • CMP-Neu5Ac can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing CMP-Neu5Ac can express an enzyme converting, e.g., sialic acid to CMP-Neu5Ac.
  • This enzyme may be a CMP-sialic acid synthetase, like the N-acylneuraminate cytidylyltransferase from several species including Homo sapiens, Neisseria meningitidis, and Pasteurella multocida.
  • the cell is modified to produce CMP-Neu5Ac.
  • the cell is modified for enhanced CMP-Neu5Ac production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knock-out of an N-acetylglucosamine-6-phosphate deacetylase, knock-out of a glucosamine-6-phosphate deaminase, over-expression of a CMP-sialic acid synthetase, and over-expression of an N-acetyl-D-glucosamine-2-epimerase encoding gene.
  • the cell used herein is optionally genetically modified to express the de novo synthesis of GDP-fucose.
  • GDP-fucose can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing GDP-fucose can express an enzyme converting, e.g., fucose, which is to be added to the cell, to GDP-fucose.
  • This enzyme may be, e.g., a bifunctional fucose kinase/fucose-l-phosphate guanylyltransferase, like Fkp from Bacteroidesfragilis, or the combination of one separate fucose kinase together with one separate fucose-l-phosphate guanylyltransferase like they are known from several species including Homo sapiens, Sus scrofa and Rattus norvegicus.
  • the cell is modified to produce GDP-fucose. More preferably, the cell is modified for enhanced GDP-fucose production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knock-out of an UDP-glucose:undecaprenyl-phosphate glucose-l-phosphate transferase encoding gene, over-expression of a GDP-L-fucose synthase encoding gene, over-expression of a GDP-mannose 4,6-dehydratase encoding gene, over-expression of a mannose-l-phosphate guanylyltransferase encoding gene, over-expression of a phosphomannomutase encoding gene and overexpression of a mannose-6-phosphate isomerase encoding gene.
  • the cell used herein is optionally genetically modified to express the de novo synthesis of UDP-Gal.
  • UDP-Gal can be provided by an enzyme expressed in the cell or by the metabolism of the cell.
  • Such cell producing UDP-Gal can express an enzyme converting, e.g. UDP-glucose, to UDP-Gal.
  • This enzyme may be, e.g., the UDP-glucose-4-epimerase GalE like as known from several species including Homo sapiens, Escherichia coli, and Rattus norvegicus.
  • the cell is modified to produce UDP- Gal. More preferably, the cell is modified for enhanced UDP-Gal production.
  • Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knock-out of a bifunctional 5'-nucleotidase/UDP-sugar hydrolase encoding gene, knock-out of a galactose-l-phosphate uridylyltransferase encoding gene and over-expression of an UDP-glucose-4-epimerase encoding gene.
  • the cell used herein is optionally genetically modified to express the de novo synthesis of UDP-GalNAc.
  • UDP-GalNAc can be synthesized from UDP-GIcNAc by the action of a single-step reaction using an UDP-N-acetylglucosamine 4-epimerase like e.g. wbgU from Plesiomonas shigelloides, gne from Yersinia enterocolitica or wbpP from Pseudomonas aeruginosa serotype 06.
  • the cell is modified to produce UDP-GalNAc. More preferably, the cell is modified for enhanced UDP-GalNAc production.
  • the cell used herein is optionally genetically modified to express the de novo synthesis of UDP-ManNAc.
  • UDP-ManNAc can be synthesized directly from UDP-GIcNAc via an epimerization reaction performed by an UDP-GIcNAc 2-epimerase (like e.g. cap5P from Staphylococcus aureus, RffE from E. coli, Cpsl9fK from S. pneumoniae, and RfbC from S. enterica).
  • an UDP-GIcNAc 2-epimerase like e.g. cap5P from Staphylococcus aureus, RffE from E. coli, Cpsl9fK from S. pneumoniae, and RfbC from S. enterica.
  • the cell is modified to produce UDP-ManNAc. More preferably, the cell is modified for enhanced UDP-ManNAc production.
  • the cell possesses, preferably expresses, one or more genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N- acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6- phosphate 2-ep
  • the cell overexpresses one or more genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L- glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N- acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2- epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GIcNAc 2-epimerase/
  • the cell is genetically engineered for production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide wherein said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N- acetyllactosamine (LacNAc); lacto-N-biose (LNB); mammalian milk oligosaccharide; human milk oligosaccharide; neutral (non-charged) milk oligosaccharide; a negatively charged, preferably sia lylated, milk oligosaccharide ; fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mamm
  • the oligosaccharide is a mammalian milk oligosaccharide (MMO) as described herein. In another more preferred embodiment, the oligosaccharide is a human milk oligosaccharide (HMO) as described herein.
  • MMO mammalian milk oligosaccharide
  • HMO human milk oligosaccharide
  • the fucosylated milk oligosaccharide is selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4- fucosyllactose (4FL), 6-fucosyllactose (5FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N- neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N- fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaos
  • the sialylated milk oligosaccharide is selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N- hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexao
  • the N- acetylglucosamine containing neutral (non-charged) milk oligosaccharide is selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'- galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose.
  • LN3 lacto-N-triose II
  • LNT lacto-N-tetraose
  • LNnT lacto-N-neotetraose
  • 6'- galactosyllactose 3'-galactosyllactose
  • the oligosaccharide like the milk oligosaccharide is selected from the list comprising, consisting of or consisting essentially of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N- neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N- fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II,
  • the oligosaccharide in the context of the present invention is preferably in free form, i.e., the oligosaccharide does not contain any protective group.
  • the cell is capable to produce, preferably produces, said disaccharide and/or oligosaccharide like a milk oligosaccharide from one or more precursor(s) as defined herein.
  • the precursor is lactose.
  • said one or more precursor(s) is/are fed to the cell from the culture or cultivation medium or the incubation.
  • the cell is capable to produce, preferably produces, at least one of said one or more precursor(s).
  • the cell is capable to produce, preferably produces, all of said one or more precursor(s).
  • the cell is genetically engineered for the production of at least one of said one or more precursor(s). In an even more preferred embodiment, the cell is genetically engineered for the production of all of said one or more precursor(s). In another more preferred embodiment, at least one of said one or more precursor(s) is internalized in said cell via one or more membrane protein(s). In another preferred embodiment, the precursor(s) that is/are used by the cell for the production of said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are completely converted into said disaccharide and/or oligosaccharide like a milk oligosaccharide, respectively.
  • the cell is further genetically engineered to possess, preferably to express, a glutamine— fructose-6- phosphate aminotransferase. In a more preferred embodiment, the cell is further genetically engineered to overexpress a glutamine— fructose-6-phosphate aminotransferase.
  • the glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and is selected from the enzyme class EC:2.6.1.16.
  • the glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and comprises a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of IPR001347, IPR005855, IPR017932, IPR029055, IPR035466, IPR035490, IPR036291, IPR046348 and IPR047084 as defined by InterPro 90.0 as released on 4 th August 2022.
  • the glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and comprises a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of PF00310, PF01380, PF01408, PF13230, PF13537 and PF13580 as defined by InterPro 90.0 as released on 4 th August 2022.
  • the glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and comprises a polypeptide sequence comprising a conserved protein domain selected from the list consisting of or consisting essentially of cd00714, cd05007, cd05008, cd05009, cd05013 and cd05710 as defined by InterPro 90.0 as released on 4 th August 2022.
  • glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and is part of the NOG family COG0449 as defined by eggNOG5.0 as released in 2019.
  • the cell contains a nucleic acid molecule which comprises a polynucleotide sequence which encodes a glutamine— fructose-6-phosphate aminotransferase as described herein.
  • the nucleic acid molecule is operably linked to control sequences recognized by the cell, said nucleic acid molecule further i) being integrated in the genome of said cell and/or ii) presented to said cell on a vector.
  • the nucleic acid molecule is foreign to the cell.
  • the cell is modified for enhanced synthesis and/or supply of phosphoenolpyruvate (PEP).
  • PEP phosphoenolpyruvate
  • the cell is further modified for reduced degradation of acetyl-CoA and/or its main precursor pyruvate.
  • the oligosaccharide of present invention is produced by a cell that is cultured in a cell cultivation.
  • the cell cultivation comprises in vitro and/or ex vivo cultivation of cells.
  • the cell cultivation is a fermentation.
  • the cell is cultivated or incubated in a reactor as defined herein.
  • the cell is cultivated or incubated in an incubator as defined herein.
  • the cell is cultivated in culture or cultivation medium comprising a carbon source comprising, consisting of or consisting essentially of a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract.
  • a carbon source comprising, consisting of or consisting essentially of a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract.
  • said carbon source is selected from the list comprising, consisting of or consisting essentially of glucose, N-acetylglucosamine (GIcNAc), glycerol, fructose, sucrose, maltose, lactose, arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate.
  • GIcNAc N-acetylglucosamine
  • the cultivation medium contains at least one carbon source selected from the list consisting of glucose, fructose, sucrose and glycerol.
  • the cultivation or incubation medium contains at least one compound selected from the list consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid and CMP-sialic acid.
  • the culture or cultivation medium is a chemically defined medium.
  • the culture or cultivation medium is a minimal salt medium comprising sulphate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt, and/or selenium.
  • the cultivation or incubation medium comprises one or more precursor(s) that is/are used for production of said disaccharide and/or oligosaccharide like a milk oligosaccharide.
  • the cultivation or incubation medium comprises one or more co-factor(s) selected from the list comprising, consisting of or consisting essentially of Mg 2+ , Co 2+ , Mn 2+ , Ca 2+ , Zn 2+ , Ni 2+ and FAD.
  • the method for production of a disaccharide as described herein comprises at least one of the following steps: i) Adding to the culture or cultivation medium in a reactor at least one precursor and/or acceptor feed wherein the total reactor volume ranges from 250 ml (millilitre) to 10.000 m 3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture or cultivation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture or cultivation medium before the addition of said precursor and/or acceptor feed; ii) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; iii) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days
  • the method for production of a disaccharide as described herein comprises at least one of the following steps: i) Adding to the culture or cultivation medium at least one precursor and/or acceptor in one pulse or in a discontinuous (pulsed) manner wherein the total reactor volume ranges from 250 ml_ (millilitre) to 10.000 m 3 (cubic meter), preferably so that the final volume of the culture or cultivation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture or cultivation medium before the addition of said precursor and/or acceptor feed pulse(s); ii) Adding at least one precursor and/or acceptor feed in a discontinuous (pulsed) manner to the culture or cultivation medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; iii) Adding
  • the method for the production of a disaccharide as described herein comprises at least one of the following steps: i) Adding to the culture or cultivation medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per litre of initial reactor volume wherein the total reactor volume ranges from 250 mL (millilitre) to 10.000 m 3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture or cultivation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture or cultivation medium before the addition of said lactose feed; ii) Adding a lactose feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; iii) Adding a lactose feed in a continuous
  • the method for production of an oligosaccharide like a milk oligosaccharide as described herein comprises at least one of the following steps: i) Adding to the culture or cultivation medium in a reactor at least one precursor and/or acceptor feed wherein the total reactor volume ranges from 250 m L (millilitre) to 10.000 m 3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture or cultivation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture or cultivation medium before the addition of said precursor and/or acceptor feed; ii) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; iii) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture or cultivation
  • the method for production of an oligosaccharide like a milk oligosaccharide as described herein comprises at least one of the following steps: i) Adding to the culture or cultivation medium at least one precursor and/or acceptor in one pulse or in a discontinuous (pulsed) manner wherein the total reactor volume ranges from 250 mL (millilitre) to 10.000 m 3 (cubic meter), preferably so that the final volume of the culture or cultivation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture or cultivation medium before the addition of said precursor and/or acceptor feed pulse(s); ii) Adding at least one precursor and/or acceptor feed in a discontinuous (pulsed) manner to the culture or cultivation medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of
  • the method for the production of an oligosaccharide like a milk oligosaccharide as described herein comprises at least one of the following steps: i) Adding to the culture or cultivation medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per litre of initial reactor volume wherein the total reactor volume ranges from 250 mL (millilitre) to 10.000 m 3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture or cultivation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture or cultivation medium before the addition of said lactose feed; ii) Adding a lactose feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a
  • the lactose feed is accomplished by adding lactose from the beginning of the cultivating in a concentration of at least ImM, preferably at least 5 mM, preferably in a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably in a concentration > 300 mM.
  • the lactose feed is accomplished by adding lactose to the cultivation medium in a concentration, such that throughout the production phase of the cultivation a lactose concentration of at least 1 mM, preferably 5 mM, 10 mM or 30 mM is obtained.
  • the cells are cultivated 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 culture or cultivation medium for 3 or more days, preferably up to 7 days; and/or provided, in the culture or 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 culture volume in a continuous manner, so that the final volume of the culture or 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 culturing or cultivation medium before the culturing.
  • a first phase of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the culture or cultivation medium before the lactose is added to the culture or 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 cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the synthesis of said disaccharide and/or oligosaccharide like a milk oligosaccharide.
  • the cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably from the group consisting of yeast cells, bacterial cells, archaebacterial cells, algae cells, plant cells, fungal cells, animal cells and protozoan cells.
  • the cell is a bacterium, fungus, yeast, a plant cell, an animal cell or a protozoan cell.
  • the latter bacterium preferably belongs to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus or the phylum of Actinobacteria.
  • the latter bacterium belonging to the phylum Proteobacteria belongs preferably to the family Enterobacteriaceae, preferably to the species Escherichia coli.
  • the latter bacterium preferably relates to any strain belonging to the species Escherichia coli such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultivated Escherichia coli strains - designated as E. coli K12 strains - which are well- adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Well-known examples of the E.
  • coli K12 strains are K12 Wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200.
  • the present invention specifically relates to a mutated and/or transformed Escherichia coli cell or strain as indicated above wherein said E. coli strain is a K12 strain. More preferably, the Escherichia coli K12 strain is E. coli MG1655.
  • the latter bacterium belonging to the phylum Firmicutes belongs preferably to the Bacilli, preferably Lactobacilliales, with members such as Lactobacillus lactis, Leuconostoc mesenteroides, or Bacil lales with members such as from the genus Bacillus, such as Bacillus subtilis or, B. amyloliquefaciens.
  • Bacterium belonging to the phylum Actinobacteria preferably belonging to the family of the Corynebacteriaceae, with members Corynebacterium glutamicum or C. afermentans, or belonging to the family of the Streptomycetaceae with members Streptomyces griseus or S. fradiae.
  • the latter bacterium belonging to the phylum Proteobacteria preferably belonging to the family of the Vibrionaceae, with member Vibrio natriegens.
  • the latter yeast preferably belongs to the phylum of the Ascomycota or the phylum of the Basidiomycota or the phylum of the Deuteromycota or the phylum of the Zygomycetes.
  • the latter yeast belongs preferably to the genus Saccharomyces (with members like e.g. Saccharomyces cerevisiae, S. bayanus, S. boulardii), Zygosaccharomyces, Pichia (with members like e.g. Pichia pastoris, P. anomala, P.
  • the latter yeast is preferably selected from Pichia pastoris, Yarrowia lipolytica, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha, Kluyveromyces marxianus, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Zygosaccharomyces rouxii, and Zygosaccharomyces bailii.
  • the latter fungus belongs preferably to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus.
  • Plant cells include cells of flowering and non-flowering plants, as well as algal cells, for example Chlamydomonas, Chlorella, etc.
  • said plant is a tobacco, rose, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant. More preferably, the latter plant cell is selected from the Rosa family.
  • the latter animal cell is preferably derived from non-human mammals (e.g.
  • primate e.g., chimpanzee, orangutan, gorilla, monkey (e.g., Old World, New World), lemur)
  • dog cat, rabbit, horse, cow, goat, ox, deer, musk deer, bovid, whale, dolphin, hippopotamus, elephant, rhinoceros, giraffe, zebra, lion, cheetah, tiger, panda, red panda, otter
  • birds e.g. chicken, duck, ostrich, turkey, pheasant
  • fish e.g. swordfish, salmon, tuna, sea bass, trout, catfish
  • invertebrates e.g.
  • human and non-human mammalian cells are preferably selected from the list comprising an epithelial cell like e.g. a mammary epithelial cell, an embryonic kidney cell (e.g.
  • HEK293 or HEK 293T cell a fibroblast cell
  • COS cell a Chinese hamster ovary (CHO) cell
  • murine myeloma cell like e.g. an N20, SP2/O or YB2/0 cell, an NIH-3T3 cell
  • a non-mammary adult stem cell or derivatives thereof such as described in WO21067641, preferably mesenchymal stem cell or derivates thereof as described in WO21067641, a lactocyte derived from mammalian induced pluripotent stem cells, preferably human induced pluripotent stem cells, a lactocyte as part of mammary-like gland organoids, a post-parturition mammary epithelium cell, a polarized mammary cell, preferably a polarized mammary cell selected from the group comprising live primary mammary epithelial cells, live mammary myoepithelial cells, live mammary progenitor cells
  • 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 82 cells.
  • the latter protozoan cell preferably is a Leishmania tarentolae cell.
  • the cell is an E. coli or yeast with a lactose permease positive phenotype, preferably wherein said lactose permease is coded by the gene LacY or LAC12, respectively.
  • the cell produces 30 g/L or more of said disaccharide in the whole broth and/or supernatant and/or wherein said disaccharide in the whole broth and/or supernatant has a purity of at least 80 % measured on the total amount of disaccharide and its precursor(s) produced by said cell in the whole broth and/or supernatant, respectively.
  • the cell produces 30 g/L, 31 g/L, 32 g/L, 33 g/L, 34 g/L, 35 g/L, 36 g/L, 37 g/L, 38 g/L, 39 g/L, 40 g/L, 41 g/L, 42 g/L, 43 g/L, 44 g/L, 45 g/L, 46 g/L, 47 g/L, 48 g/L, 49 g/L, 50 g/L, 51 g/L, 52 g/L, 53 g/L, 54 g/L, 55 g/L, 56 g/L, 57 g/L, 58 g/L, 59 g/L, 60 g/L, 61 g/L, 62 g/L, 63 g/L, 64 g/L, 65 g/L, 66 g/L, 67 g/L, 68 g/L, 69
  • the cell produces 30 g/L or more of said oligosaccharide like a milk oligosaccharide in the whole broth and/or supernatant and/or wherein said oligosaccharide like a milk oligosaccharide in the whole broth and/or supernatant has a purity of at least 80 % measured on the total amount of oligosaccharide like a milk oligosaccharide and its precursor(s) produced by said cell in the whole broth and/or supernatant, respectively.
  • the cell produces 30 g/L, 31 g/L, 32 g/L, 33 g/L, 34 g/L, 35 g/L, 36 g/L, 37 g/L, 38 g/L, 39 g/L, 40 g/L, 41 g/L, 42 g/L, 43 g/L, 44 g/L, 45 g/L, 46 g/L, 47 g/L, 48 g/L, 49 g/L, 50 g/L, 51 g/L, 52 g/L, 53 g/L, 54 g/L, 55 g/L, 56 g/L, 57 g/L, 58 g/L, 59 g/L, 60 g/L, 61 g/L, 62 g/L, 63 g/L, 64 g/L, 65 g/L, 66 g/L, 67 g/L, 68 g/L, 69
  • said less functional synthesis of UDP-GIcNAc confers unaffected and/or enhanced i) disaccharide and/or oligosaccharide like a milk oligosaccharide formation, ii) productivity, iii) biomass production, iv) cell growth and/or v) yield of the produced disaccharide and/or oligosaccharide like a milk oligosaccharide, relative to a corresponding non-modified or non-engineered cell.
  • the cell produces a disaccharide and/or oligosaccharide like a milk oligosaccharide as described herein.
  • the cell produces an oligosaccharide mixture like a milk oligosaccharide mixture.
  • the disaccharide and/or oligosaccharide like a milk oligosaccharide produced by a cell of present invention is/are recovered from said cultivation or incubation medium and/or said cell.
  • said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are purified.
  • separating from said cultivation or incubation means harvesting, collecting, or retrieving said disaccharide and/or oligosaccharide like a milk oligosaccharide from the cell and/or the medium of its growth.
  • the disaccharide and the oligosaccharide like a milk oligosaccharide can be separated in a conventional manner from the aqueous culture or cultivation medium, in which the cell was grown.
  • disaccharide and/or oligosaccharide like a milk oligosaccharide is/are still present in the cells producing the disaccharide and/or oligosaccharide like a milk oligosaccharide
  • conventional manners to free or to extract said disaccharide and/or oligosaccharide like a milk oligosaccharide out of the cells can be used, such as cell destruction using high pH, heat shock, sonication, French press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergent, hydrolysis,...
  • the culture or cultivation medium and/or cell extract together and separately can then be further used for separating said disaccharide and/or oligosaccharide like a milk oligosaccharide.
  • This preferably involves clarifying said disaccharide and/or oligosaccharide like a milk oligosaccharide to remove suspended particulates and contaminants, particularly cells, cell components, insoluble metabolites and debris produced by culturing the genetically engineered cell.
  • said disaccharide and/or oligosaccharide like a milk oligosaccharide can be clarified in a conventional manner.
  • said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are clarified by centrifugation, flocculation, decantation and/or filtration.
  • a second step of separating said disaccharide and/or oligosaccharide like a milk oligosaccharide preferably involves removing substantially all the eventually remaining proteins, peptides, amino acids, RNA, DNA, endotoxins and glycolipids that could interfere with the subsequent separation step, from said disaccharide and/or oligosaccharide like a milk oligosaccharide, preferably after it/they has/have been clarified.
  • remaining proteins and related impurities can be removed from said disaccharide and/or oligosaccharide like a milk oligosaccharide in a conventional manner.
  • remaining proteins, salts, by-products, colour, endotoxins and other related impurities are removed from said disaccharide and/or oligosaccharide like a milk oligosaccharide by ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, electrophoresis (e.g.
  • affinity chromatography using affinity ligands including e.g. DEAE-sepharose, poly-L-lysine and polymyxin-B, endotoxin-selective adsorber matrices), ion exchange chromatography (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange, inside-out ligand attachment), hydrophobic interaction chromatography and/or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography. With the exception of size exclusion chromatography, remaining proteins and related impurities are retained by a chromatography medium or a selected membrane.
  • affinity chromatography using affinity chromatography (using affinity ligands including e.g. DEAE-sepharose, poly-L-lysine and polymyxin-B, endotoxin-selective adsorber matrices), i
  • the methods as described herein also provide for a further purification of the disaccharide and/or oligosaccharide like a milk oligosaccharide of present invention.
  • a further purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide may be accomplished, for example, by use of (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange 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 disaccharide and/or oligosaccharide like a milk oligosaccharide.
  • Another purification step is to dry, e.g. spray dry or lyophilize the produced disaccharide and/or oligosaccharide like a milk oligosaccharide.
  • the separation and purification of the disaccharide and/or oligosaccharide like a milk oligosaccharide is made in a process, comprising the following steps in any order: a) contacting the cultivation or a clarified version thereof with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da ensuring the retention of the produced disaccharide and/or oligosaccharide like a milk oligosaccharide and allowing at least a part of the proteins, salts, by-products, colour and other related impurities to pass, b) conducting a diafiltration process on the retentate from step a), using said membrane, with an aqueous solution of an inorganic electrolyte, followed by optional diafiltration with pure water to remove excess of the electrolyte, c) and collecting the retentate enriched in said disaccharide and/or oligosaccharide like a milk oligosaccharide
  • the separation and purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide is made in a process, comprising the following steps in any order: subjecting the cultivation or a clarified version thereof to two membrane filtration steps using different membranes, wherein one membrane has a molecular weight cut-off of between about 300 to about 500 Dalton, and the other membrane as a molecular weight cut-off of between about 600 to about 800 Dalton.
  • the separation and purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide is made in a process, comprising the following steps in any order comprising the step of treating the cultivation or a clarified version thereof with a strong cation exchange resin in H+-form and a weak anion exchange resin in free base form.
  • the separation and purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide is made in the following way.
  • the cultivation comprising i) the produced disaccharide and/or oligosaccharide like a milk oligosaccharide, 2) biomass, 3) medium components and 4) contaminants, and wherein the purity of the produced disaccharide and/or oligosaccharide like a milk oligosaccharide in the cultivation is ⁇ 80 %, is applied to the following purification steps: i) separation of biomass from the cultivation, ii) cationic ion exchanger treatment for the removal of positively charged material, iii) anionic ion exchanger treatment for the removal of negatively charged material, iv) nanofiltration step and/or electrodialysis step, wherein a purified solution comprising the produced disaccharide and/or oligosaccharide like a milk oligosaccharide at a purity of
  • the separation and purification of the disaccharide and/or oligosaccharide like a milk oligosaccharide is made in a process, comprising the following steps in any order: enzymatic treatment of the cultivation; removal of the biomass from the cultivation; ultrafiltration; nanofiltration; and a column chromatography step.
  • a column chromatography step is a single column or a multiple column.
  • the column chromatography step is simulated moving bed chromatography.
  • Such simulated moving bed chromatography preferably comprises i) at least 4 columns, wherein at least one column comprises a weak or strong cation exchange resin; and/or ii) four zones I, II, III and IV with different flow rates; and/or iii) an eluent comprising water; and/or iv) an operating temperature of 15 degrees to 60 degrees centigrade.
  • the present invention provides the produced disaccharide and/or oligosaccharide like a milk oligosaccharide which is/are spray-dried to powder, wherein the spray-dried powder contains ⁇ 15 % -wt. of water, preferably ⁇ 10 % -wt. of water, more preferably ⁇ 7 % -wt. of water, most preferably ⁇ 5 % -wt. of water.
  • the monomeric building blocks e.g. the monosaccharide or glycan unit composition
  • the anomeric configuration of side chains e.g. the anomeric configuration of side chains
  • the presence and location of substituent groups e.g. the degree of polymerization/molecular weight and the linkage pattern
  • degree of polymerization/molecular weight and the linkage pattern can be identified by standard methods known in the art, such as, e.g.
  • the crystal structure can be solved using, e.g., solid-state NMR, FT-IR (Fourier transform infrared spectroscopy), and WAXS (wide-angle X-ray scattering).
  • the degree of polymerization (DP), the DP distribution, and polydispersity can be determined by, e.g., viscosimetry and SEC (SEC-HPLC, high performance size-exclusion chromatography).
  • SEC-HPLC high performance size-exclusion chromatography
  • HPLC high performance liquid chromatography
  • GLC gas-liquid chromatography
  • a partial depolymerization is carried out using an acid or enzymes to determine the structures.
  • said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are subjected to enzymatic analysis, e.g., it is contacted with an enzyme that is specific for a particular type of linkage, e.g., beta-galactosidase, or alphaglucosidase, etc., and NMR may be used to analyse the products.
  • the present invention provides use of a cell as described herein for the production of a disaccharide and/or oligosaccharide like a milk oligosaccharide wherein said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N- acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; a negatively charged, preferably sialylated, milk oligosaccharide; mammalian milk oligosaccharide (MMO); a human milk oligosaccharide (HMO); neutral (non-charged) milk oligosaccharide; fucosylated milk oligosaccharide; non-fucosylated neutral (non
  • the present invention provides use of a method as described herein for the production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide, wherein said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N- acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; a negatively charged, preferably sialylated, milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); neutral (non-charged) milk oligosaccharide; fucosylated milk oligosaccharide; non-fucosylated neutral (non
  • the present invention provides for a purified disaccharide and/or a purified oligosaccharide like a milk oligosaccharide, or a purified oligosaccharide mixture like a purified milk oligosaccharide mixture, a purified mixture of disaccharides, or a purified mixture of one or more disaccharide(s) and one or more milk oligosaccharide(s) as described herein for use in medicine, preferably for use in prophylaxis or therapy of a gastrointestinal disorder.
  • the present invention provides use of a purified disaccharide and/or oligosaccharide like a milk oligosaccharide obtained by a method as described herein in a food or feed preparation, in a dietary supplement, in a cosmetic ingredient or in a pharmaceutical ingredient.
  • said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine.
  • Said purified disaccharide and/or oligosaccharide like a milk oligosaccharide may be used for the manufacture of a preparation, as food additive, prebiotic, symbiotic, for the supplementation of baby food, adult food, infant animal feed, adult animal feed, or as either therapeutically or pharmaceutically active compound or in cosmetic applications.
  • the present invention provides use of a disaccharide and/or a milk oligosaccharide as described herein as additive in food, preferably as additive in human food and/or pet food, more preferably as additive in human baby food.
  • the food is a human food, preferably infant food, human baby food and/or an infant formula or an infant supplement and the feed is a pet food, animal milk replacer, veterinary product, veterinary feed supplement, nutrition supplement, post weaning feed, or creep feed.
  • a preparation is provided that further comprises at least one probiotic microorganism.
  • said preparation is a nutritional composition.
  • said preparation is a medicinal formulation, a dietary supplement, a dairy drink or an infant formula.
  • a "prebiotic” is a substance that promotes growth of microorganisms beneficial to the host, particularly microorganisms in the gastrointestinal tract.
  • a dietary supplement provides multiple prebiotics, including said disaccharide and/or oligosaccharide like a milk oligosaccharide being a prebiotic purified by a method disclosed in this specification, to promote growth of one or more beneficial microorganisms.
  • prebiotic ingredients for dietary supplements include other prebiotic molecules (such as HMDs) and plant polysaccharides (such as inulin, pectin, b-glucan and xylooligosaccharide).
  • a "probiotic" product typically contains live microorganisms that replace or add to gastrointestinal microflora, to the benefit of the recipient. Examples of such microorganisms include Lactobacillus species (for example, L. acidophilus and L. bulgaricus), Bifidobacterium species (for example, B. animalis, B. longum and B. infantis (e.g., Bi-26)), and Saccharomyces boulardii.
  • said disaccharide and/or oligosaccharide like a milk oligosaccharide produced and/or purified by a method of this specification is/are orally administered in combination with such microorganism.
  • further ingredients for dietary supplements include oligosaccharides (such as 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose), disaccharides (such as lactose), monosaccharides (such as glucose, galactose, L-fucose, sialic acid, glucosamine and N-acetylglucosamine), thickeners (such as gum arabic), acidity regulators (such as trisodium citrate), water, skimmed milk, and flavourings.
  • oligosaccharides such as 2'-fucosyllactose, 3-fucosyllactose, 3'-sia
  • said disaccharide and/or oligosaccharide like a milk oligosaccharide purified by a method as described herein is/are 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.
  • said disaccharide and/or oligosaccharide like a milk oligosaccharide purified by a method as described herein is/are included in infant formula to provide nutritional benefits similar to those provided by the disaccharides and/or oligosaccharides in human breast milk.
  • said purified disaccharide and/or oligosaccharide like a milk oligosaccharide is/are mixed with one or more ingredients of the infant formula.
  • infant formula ingredients include non-fat milk, carbohydrate sources (e.g., lactose), protein sources (e.g., whey protein concentrate and casein), fat sources (e.g., vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and/or sunflower oil; and fish oils), vitamins (such as vitamins A, Bb, Bi2, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate) and possibly human milk oligosaccharides (HMDs).
  • carbohydrate sources e.g., lactose
  • protein sources e.g., whey protein concentrate and casein
  • fat sources e.g., vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and/or sunflower oil; and fish oils
  • vitamins such as vitamins A, Bb, Bi2, C and D
  • minerals such as potassium citrate, calcium cit
  • the one or more infant formula ingredients comprise non-fat milk, a carbohydrate source, a protein source, a fat source, and/or a vitamin and mineral. In some embodiments, the one or more infant formula ingredients comprise lactose, whey protein concentrate and/or high oleic safflower oil. In some embodiments, the concentration of the disaccharide and/or oligosaccharide like a milk oligosaccharide in the infant formula is/are approximately the same concentration as the concentration of the disaccharide and/or oligosaccharide generally present in human breast milk.
  • a disaccharide and/or an oligosaccharide like a milk oligosaccharide purified by a method as described herein is/are added to the infant formula with a concentration that is approximately the same concentration as the concentration of the compound generally present in human breast milk.
  • UDP-GIcNAc UDP-N-acetylglucosamine
  • said pathway for production of said oligosaccharide is chosen from the list comprising fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway, preferably said cell is genetically engineered to comprise at least one of said pathway(s), more preferably said cell comprises at least one of said pathway(s) wherein at least one of said pathway(s) has/have been genetically engineered.
  • nucleotide-activated sugar chosen from the list comprising UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP- ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP-glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy--L-arabino-4- hexulose, UDP-2-acetamido-2,6-dideoxy--L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine
  • UDP-GIcNAc synthesis possesses at least one gene chosen from the list comprising genes encoding bifunctional N-acetylglucosamine-1- phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, an N-acetylglucosamine-1- phosphate uridyltransferase and a glucosamine-l-phosphate acetyltransferase, and wherein said at least one gene is rendered less functional.
  • At least one gene involved in the synthesis and/or import of a co-factor that is involved in UDP-GIcNAc synthesis is rendered less functional, preferably said co-factor is chosen from the list comprising Mg 2+ , Co 2+ , Mn 2+ , Ca 2+ , Zn 2+ , Ni 2+ and FAD.
  • said at least one gene is rendered less functional by insertion, deletion and/or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) chosen from the list comprising promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said at least one gene.
  • oligosaccharide is chosen from the list comprising neutral (non-charged) oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide, milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMD); sialylated milk oligosaccharide, neutral (noncharged) milk oligosaccharide, fucosylated milk oligosaccharide, non-fucosylated neutral (non- charged) milk oligosaccharide, sialylated mammalian milk oligosaccharide, neutral (non-charged) mammalian milk oligosaccharide, fucosylated mammalian milk oligosaccharide, non-fucosylated neutral (non-charged) mammalian milk oligosaccharide, non-fucosyl
  • nucleic acid molecule is operably linked to control sequences recognized by the cell, said nucleic acid molecule further i) being integrated in the genome of said cell and/or ii) presented to said cell on a vector.
  • said cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell
  • said bacterium belongs to a phylum chosen from the group comprising Proteobacteria, Firmicutes, Cyanobacteria, Deinococcus-Thermus and Actinobacteria; more preferably, said bacterium belongs to a family chosen from the group comprising Enterobacteriaceae, Bacillaceae, Lactobacillaceae, Corynebacteriaceae and Vibrionaceae; even more preferably, said bacterium is chosen 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 chosen from the group comprising Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus, preferably, said yeast belongs to a genus chosen from the group comprising Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces, Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces or Torulaspora; more preferably, said yeast is selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Tor
  • Method for the production of an oligosaccharide comprising: i. cultivating and/or incubating a cell of any one of previous embodiments, in cultivation and/or incubation medium under conditions permissive to produce said oligosaccharide, ii. preferably, separating said oligosaccharide from said cultivation and/or incubation.
  • cultivation medium contains at least one carbon source selected from the group consisting of glucose, fructose, sucrose, and glycerol.
  • oligosaccharide is chosen from the list comprising neutral (non-charged) oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide, milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); sialylated milk oligosaccharide, neutral (non-charged) milk oligosaccharide, fucosylated milk oligosaccharide, non-fucosylated neutral (non-charged) milk oligosaccharide, sialylated mammalian milk oligosaccharide, neutral (non-charged) mammalian milk oligosaccharide, fucosylated mammalian milk oligosaccharide, fucosylated mammalian milk oligosaccharide, fucosylated mammalian milk oligosacchari
  • oligosaccharide is chosen from the list comprising neutral (noncharged) oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide, milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); sialylated milk oligosaccharide, neutral (non-charged) milk oligosaccharide, fucosylated milk oligosaccharide, non-fucosylated neutral (non-charged) milk oligosaccharide, sialylated mammalian milk oligosaccharide, neutral (non-charged) mammalian milk oligosaccharide, fucosylated mammalian milk oligosaccharide, non-f
  • the present invention relates to the following preferred specific embodiments:
  • UDP-GIcNAc UDP-N-acetylglucosamine
  • said pathway for production of said disaccharide and/or milk oligosaccharide is selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway
  • said pathway for production of said disaccharide and/or milk oligosaccharide is selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway
  • said cell is genetically engineered to comprise at least one of said pathway(s) and/or said cell comprises at least one of said pathway
  • glycosyltransferase(s) selected from the list consisting of or consisting essentially of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N- acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N- acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amin
  • UDP-GIcNAc synthesis possesses at least one gene selected from the list consisting of or consisting essentially of genes encoding bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-1- phosphate acetyltransferase, an N-acetylglucosamine-l-phosphate uridyltransferase and a glucosamine-l-phosphate acetyltransferase, and wherein said at least one gene is rendered less functional.
  • disaccharide and/or milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; a negatively charged milk oligosaccharide; sialylated milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide;
  • Cell according to any one of previous preferred embodiments wherein said cell: is capable to produce and/or produces said disaccharide and/or milk oligosaccharide from one or more precursor(s), is capable to produce and/or produces said disaccharide and/or milk oligosaccharide from lactose, is capable to produce and/or produces at least one precursor that is used to produce said disaccharide and/or milk oligosaccharide, is capable to produce and/or produces all precursors that are used to produce said disaccharide and/or milk oligosaccharide, is genetically engineered for the production of at least one precursor that is used to produce said disaccharide and/or milk oligosaccharide, and/or is genetically engineered for the production of all precursors that are used to produce said disaccharide and/or milk oligosaccharide.
  • Cell according to preferred embodiment 11, wherein said glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and: is selected from the enzyme class EC:2.6.1.16, comprises a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of IPR001347, IPR005855, IPR017932, IPR029055, IPR035466, IPR035490, IPR036291, IPR046348 and IPR047084 as defined by InterPro 90.0 as released on 4 th August 2022, comprises a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of PF00310, PF01380, PF01408, PF13230, PF13537 and PF13580 as defined by InterPro 90.0 as released on 4 th August 2022, comprises a polypeptide sequence comprising a conserved protein domain selected from the list consisting of
  • PEP phosphoenolpyruvate
  • cell according to any one of previous preferred embodiments, wherein said cell is: a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell, an E. coli or yeast with a lactose permease positive phenotype, and/or an E. coli or yeast with a lactose permease positive phenotype wherein said lactose permease is coded by the gene LacY or LAC12, respectively.
  • Method for the production of a disaccharide and/or milk oligosaccharide comprising: i. cultivating and/or incubating a cell of any one of previous preferred embodiments, in cultivation and/or incubation medium under conditions permissive to produce said disaccharide and/or milk oligosaccharide, and/or ii. separating said disaccharide and/or milk oligosaccharide from said cultivation and/or incubation.
  • cultivation or incubation medium comprises one or more precursor(s) that is/are used for production of said disaccharide and/or milk oligosaccharide
  • cultivation medium contains at least one carbon source selected from the list consisting of glucose, fructose, sucrose, and glycerol
  • cultivation or incubation medium contains at least one compound selected from the list consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid and CMP-sialic acid.
  • disaccharide and/or milk oligosaccharide are/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; negatively charged milk oligosaccharide; sialylated milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non
  • disaccharide and/or milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; negatively charged milk oligosaccharide; sialylated milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charge
  • the Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium).
  • the minimal medium used in cultivation experiments in 96-well plates or in shake flasks contained 2.00 g/L NH 4 CI, 5.00 g/L (NH 4 )2SO 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 pil/L molybdate solution, and 1 mL/L selenium solution.
  • precursor(s) and/or acceptor(s) for saccharide like disaccharide and/or milk oligosaccharide
  • synthesis compounds like e.g., galactose, glucose, fructose, fucose, lactose, a co-factor could be added to the medium.
  • the minimal medium was set to a pH of 7 with IM KOH.
  • Vitamin solution consisted of 3.6 g/L FeCI 2.4 H 2 O, 5.0 g/LCaCI 2 .2H 2 0, 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 H 3 BO 4 , 0.4 g/L Na 2 EDTA.2H 2 O and 1.01 g/L thiamine.HCL
  • the molybdate solution contained 0.967 g/L NaMoO 4 .2H 2 O.
  • the selenium solution contained 42 g/L Seo2.
  • the minimal medium for fermentations contained 6.75 g/L NH 4 CI, 1.25 g/L (NH 4 ) 2 SO 4 , 2.93 g/L KH 2 PO 4 and 7.31 g/L KH 2 PO 4 , 0.5 g/L NaCI, 0.5 g/L M SO 4 .7H2O, 30 g/L sucrose or 30 g/L lycerol, 1 mL/L vitamin solution, 100 pL/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above. As specified in the respective examples, 20 g/L lactose and/or 20 g/L glucose were additionally added to the medium.
  • Complex medium was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 pm Sartorius). When necessary, the medium was made selective by adding an antibiotic: e.g., chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L).
  • an antibiotic e.g., chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L).
  • a preculture of 96-well microtiter plate experiments was started from a cryovial, in 150 pL LB and was incubated overnight at 37 °C on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96well square microtiter plate, with 400 pL minimal medium by diluting 400x.
  • 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, Melsungen, 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 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.
  • Escherichia coli K12 MG1655 [A-, F-, rph-1] was obtained from the Coli Genetic Stock Center (US), CGSC Strain#: 7740, in March 2007.
  • Gene disruptions, gene introductions and gene replacements were performed using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). All constitutive promoters, UTRs and terminator sequences originated from the libraries described by Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148), Dunn et al. (Nucleic Acids Res. 1980, 8, 2119- 2132), Edens et al. (Nucleic Acids Res.
  • the mutant strain was derived from E. coli K12 MG1655 comprising a knock-out of the E. coli wcaJgene and genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E. coli ⁇ N (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6).
  • a sucrose transporter like e.g. CscB from E. coli ⁇ N (UniProt ID E0IXR1)
  • a fructose kinase like e.g. Frk originating from Zymomonas mobilis
  • a sucrose phosphorylase
  • GDP-fucose production can further be optimized in the mutant E. coli strain by genomic knock-outs of any one or more of the E. coli genes comprising glgC, agp, pfkA, pfkB, pgi, arcA, icIR, pgi and Ion as described in WO2016075243 and W02012007481.
  • GDP-fucose production can additionally be optimized comprising genomic knock-ins of constitutive transcriptional units for a mannose-6-phosphate isomerase like e.g. manA from E. coli (UniProt ID P00946), a phosphomannomutase like e.g. manB from E.
  • GDP-fucose production can also be obtained by genomic knock-outs of the E.
  • the mutant GDP-fucose production strain was additionally modified with an expression plasmid comprising a constitutive transcriptional unit for a fucosyltransferase, like e.g. the alpha-1, 2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435) to produce 2'-fucosyllactose (2' FL) or the alpha-1, 3-fucosyltransferase HpFucT from H.
  • a fucosyltransferase like e.g. the alpha-1, 2-fucosyltransferase HpFutC from H.
  • pylori UniProt ID Q9X435
  • the mutant strain was derived from E. coli K12 MG1655 and modified with a knock-out of the E. coli lacZ, lacY, lacA and nagB genes and with genomic knock-ins of constitutive transcriptional units for a lactose permease like e.g. the E. coli LacY (UniProt ID P02920) and a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. IgtA (UniProt ID Q9JXQ6) from N. meningitidis.
  • a lactose permease like e.g. the E. coli LacY (UniProt ID P02920) and a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. IgtA (UniProt ID Q9JXQ6) from N. meningitidis.
  • the mutant LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g. wbgO (Uniprot ID D3QY14) from E. coli 055:1-17.
  • a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g. wbgO (Uniprot ID D3QY14) from E. coli 055:1-17.
  • the mutant LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from Neisseria meningitidis.
  • LgtB Uniprot ID Q51116, sequence version 02, 01 Dec 2000
  • LN3, LNT and/or LNnT production can further be optimized in the mutant E. coli strains with genomic knock-outs of the E. coli genes comprising any one or more of galT, ushA, IdhA and agp.
  • the mutant LN3, LNT and/or LNnT producing strains can also be optionally modified for enhanced UDP-GIcNAc production with a genomic knock-in of a constitutive transcriptional unit for an L-glutamine— D-fructose-6-phosphate aminotransferase like e.g. the E. coli glmS (UniProt ID P17169, sequence version 04, 23 Jan 2007) or a mutant glmS*54 from E. coli with SEQ.
  • E. coli glmS having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006).
  • the mutant E. coli strains can also optionally be adapted with a genomic knock-in of a constitutive transcriptional unit for an UDP-glucose-4-epimerase like e.g. galE from E. coli (UniProt ID P09147), a phosphoglucosamine mutase like e.g. glmM from E.
  • the mutant LN3, LNT and/or LNnT producing E. coli strains can also optionally be adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E.
  • 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-acetylglucosamine 2-epimerase like e.g. AGE from Bacteroides ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase like e.g. NeuB from Neisseria meningitidis (UniProt ID E0NCD4).
  • 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).
  • 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).
  • sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03, 23 Jan 2007), an N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase like e.g. glmU from E. coli (UniProt ID P0ACC7), an UDP-N-acetylglucosamine 2-epimerase like e.g. NeuCfrom C. jejuni (UniProt ID Q93MP8) and an N-acetylneuraminate synthase like e.g. NeuB from N. meningitidis (UniProt ID E0NCD4).
  • a phosphoglucosamine mutase like e.g. glm
  • sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a bifunctional UDP-GIcNAc 2-epimerase/N- acetylmannosamine kinase like e.g. from Mus musculus (strain C57BL/6J) (UniProt ID Q91WG8), an N- acylneuraminate-9-phosphate synthetase like e.g. from Pseudomonas sp. UW4 (UniProt ID K9NPH9) and an N-acylneuraminate-9-phosphatase like e.g. from Bacteroides thetaiotaomicron (UniProt ID Q8A712).
  • a bifunctional UDP-GIcNAc 2-epimerase/N- acetylmannosamine kinase like e.g. from Mus musculus (strain C57BL/6J) (UniProt ID Q91WG8)
  • Sialic acid production can further be optimized in the mutant E. co// strain with genomic knock-outs of the E. coli genes comprising any one or more of nagA, nagB, nagC, nagD, nagE, nanA, nanE, nanK, manX, manY and manZ as described in WO18122225, and/or genomic knock-outs of the E.
  • coli genes comprising any one or more of nanT, poxB, IdhA, adbE, aldB, pflA, pfIC, ybiY, ackA and/or pta and with genomic knock- ins of constitutive transcriptional units comprising one or more copies of an L-glutamine— D-fructose-6- phosphate aminotransferase like e.g. E. coli glmS (UniProt ID P17169, sequence version 04, 23 Jan 2007) or a mutant glmS*54 from E. coli with SEQ ID NO 01 and differing from the wild-type E.
  • coli glmS having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006), preferably a phosphatase like any one of e.g. the E.
  • coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida, ScDOGl from S.
  • sialic acid production strains were further modified to express an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P. multocida (UniProt ID A0A849CI62) and to express a sialyltransferase like e.g. the alpha-2, 3-siayltransferase PmultST3 from P.
  • an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P. multocida (UniProt ID A0A849CI62) and to express a sialyltransferase like e.g. the alpha-2, 3-siayltransferase PmultST3 from P.
  • PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2, 3-sialyltransferase activity or the alpha-2, 6-sialyltransferase PdST6 from P. damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity.
  • Constitutive transcriptional units of the N-acylneuraminate cytidylyltransferase and the sialyltransferase can be delivered to the mutant strain either via genomic knock-in or via expression plasmids. If the mutant strains producing sialic acid and CMP-sialic acid were intended to make sialylated lactose structures, the strains were additionally modified with genomic knock-outs of the E. coli LacZ, LacY and LacA genes and with a genomic knock-in of a constitutive transcriptional unit for a lactose permease like e.g. E. coli LacY (UniProt ID P02920).
  • All mutant strains producing sialic acid, CMP-sialic acid and/or sialylated saccharides could optionally be adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E. coli W (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Z. mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP from 8. 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 8. adolescentis (
  • coli strains adapted for LNT production as described herein can also be further modified with one or more copies of a glucosamine 6-phosphate N-acetyltransferase like e.g. GNA1 from S. cerevisiae (UniProt ID P43577), an N-acetylglucosamine 2-epimerase like e.g. AGE from B. ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase like e.g. NeuB from N.
  • a glucosamine 6-phosphate N-acetyltransferase like e.g. GNA1 from S. cerevisiae (UniProt ID P43577)
  • an N-acetylglucosamine 2-epimerase like e.g. AGE from B. ovatus
  • N-acetylneuraminate synthase like e.g. NeuB from N.
  • meningitidis (UniProt ID E0NCD4) and an expression plasmid comprising containing constitutive expression cassettes for the N-acylneuraminate cytidylyltransferase (NeuA) from P. multocida (UniProt ID A0A849CI62) and 1) the alpha-2, 3-sialyltransferase PmultST3 from P.
  • NeA N-acylneuraminate cytidylyltransferase
  • P. multocida (UniProt ID A0A849CI62) and 1) the alpha-2, 3-sialyltransferase PmultST3 from P.
  • PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having betagalactoside alpha-2, 3-sialyltransferase activity or 2) the alpha-2, 6-sialyltransferase (PdST6) from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity to produce 1) LSTa (Neu5Ac-oc2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc) or 2) LSTb (Gal-pi,3- (Neu5Ac-oc2,6)-
  • mutant E. coli strains adapted for LNnT production as described herein can also be further modified with one or more copies of a glucosamine 6-phosphate N-acetyltransferase like e.g. GNA1 from S. cerevisiae (UniProt ID P43577), an N-acetylglucosamine 2-epimerase like e.g. AGE from B. ovatus (UniProt ID A7LVG6) and an N- acetylneuraminate synthase like e.g. NeuB from N.
  • a glucosamine 6-phosphate N-acetyltransferase like e.g. GNA1 from S. cerevisiae (UniProt ID P43577)
  • an N-acetylglucosamine 2-epimerase like e.g. AGE from B. ovatus
  • N- acetylneuraminate synthase like e.g. Neu
  • meningitidis (UniProt ID E0NCD4) and an expression plasmid comprising containing constitutive expression cassettes for the N-acylneuraminate cytidylyltransferase (NeuA) from P. multocida (UniProt ID A0A849CI62) and 1) the alpha-2, 3- sialyltransferase PmultST3 from P.
  • NeA N-acylneuraminate cytidylyltransferase
  • P. multocida (UniProt ID A0A849CI62) and 1) the alpha-2, 3- sialyltransferase PmultST3 from P.
  • UniProt ID Q9CLP3 or a PmultST3-like polypeptide (SEQ ID NO 02) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha- 2,3-sialyltransferase activity or 2) the alpha-2, 6-sialyltransferase (PdST6) from P.
  • damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity to produce 1) LSTd (Neu5Ac-a2,3-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc) or 2) LSTc (Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal- pi,4-Glc), respectively.
  • saccharide like disaccharide and/or milk oligosaccharide
  • synthesis compounds like e.g., galactose, glucose, fructose, fucose, lactose, a co-factor could be added to the medium.
  • yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were grown for 2-3 days at 30°C. Starting from a single colony, a preculture was grown over night in 5 mL at 30°C, shaking at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL media. These shake flasks were incubated at 30°C with an orbital shaking of 200 rpm.
  • a yeast expression plasmid like p2a_2p_Fuc (Chan 2013, Plasmid 70, 2-17) can be used for expression of foreign genes in S. cerevisiae.
  • This plasmid contains an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E. coli and the 2p yeast ori and the Ura3 selection marker for selection and maintenance in yeast.
  • This plasmid is further modified with constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), a GDP-mannose 4,6-dehydratase like e.g. gmd from E.
  • the yeast expression plasmid p2a_2p_Fuc2 can be used as an alternative expression plasmid of the p2a_2p_Fuc plasmid comprising next to the ampicillin resistance gene, the bacterial ori, the 2p yeast ori and the Ura3 selection marker constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), a fucose permease like e.g. fucP from E.
  • a yeast expression plasmid can be derived from the pRS420- plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the HIS3 selection marker and a constitutive transcriptional unit for an UDP-glucose-4-epimerase like e.g. galE from E. coli (UniProt ID P09147).
  • This plasmid can be further modified with constitutive transcriptional units for a lactose permease like e.g. LAC12 from K.
  • lactis (UniProt ID P07921) and a galactoside beta-1, 3-N- acetylglucosaminyltransferase activity like e.g. IgtA from N. meningitidis (UniProt ID Q9JXQ6) to produce LN3.
  • the mutant LN3 producing strains were further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 3- galactosyltransferase like e.g. WbgO (Uniprot ID D3QY14) from E. coli O55:H7.
  • LN3 derived oligosaccharides like lacto-A/-neotetraose (LNnT, Gal-pi,4-GlcNAc-pi,3-Gal- pi,4-Glc)
  • the mutant LN3 producing strain were further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis.
  • a yeast expression plasmid was derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the TRP1 selection marker and constitutive transcriptional units for an L-glutamine— D-fructose-6-phosphate aminotransferase like e.g. E. coli lmS (UniProt ID P17169 (sequence version 04 (23 Jan 2007)) or a mutant glmS*54 from E. coli with SEQ. ID NO 01 and differing from the wild-type E.
  • E. coli lmS UniProt ID P17169 (sequence version 04 (23 Jan 2007)
  • mutant glmS*54 from E. coli with SEQ. ID NO 01 and differing from the wild-type E.
  • coli glmS having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006), a phosphatase like e.g. SurE from E. coli (UniProt ID P0A840), an N-acylglucosamine 2-epimerase like e.g. AGE from B. ovatus (UniProt ID A7LVG6), an N-acetylneuraminate synthase like e.g. NeuB from N.
  • meningitidis (UniProt ID E0NCD4) and an N-acylneuraminate cytidylyltransferase like e.g. NeuA from P. multocida (UniProt A0A849CI62).
  • a constitutive transcriptional unit for a glucosamine 6- phosphate N-acetyltransferase like e.g. GNA1 from S. cerevisiae (UniProt ID P43577) was added as well.
  • a constitutive transcriptional unit for a siderophore transporter like e.g. entS from E. coli (UniProt ID P24077, sequence version 02 (01 Nov 1997)) was added as well.
  • the plasmid further comprised constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), and a sialyltransferase like e.g., an alpha-2, 3- sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P.
  • a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921)
  • a sialyltransferase like e.g., an alpha-2, 3- sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P.
  • UniProt ID Q9CLP3 or a PmultST3-like polypeptide (SEQ ID NO 02) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g. the alpha-2, 6-sialyltransferase (PdST6) from P.
  • damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity.
  • any one or more of the glycosyltransferases and/or the proteins involved in nucleotide-activated sugar synthesis were N- and/or C-terminally fused to a SUMOstar tag (e.g. obtained from pYSU MOstar, Life Sensors, Malvern, PA) to enhance their solubility.
  • a SUMOstar tag e.g. obtained from pYSU MOstar, Life Sensors, Malvern, PA
  • mutant yeast strains were modified with a genomic knock-in of a constitutive transcriptional unit encoding a chaperone protein like e.g. Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssbl, Ssel, Sse2, Ssal, Ssa2, Ssa3, Ssa4, Ssb2, EcmlO, Sscl, Ssql, Sszl, Lhsl, Hsp82, Hsc82, Hsp78, Hspl04, Tcpl, Cct4, Cct8, Cct2, Cct3, Cct5, Cct6 or Cct7. Plasmids were maintained in the host E.
  • a genomic knock-in of a constitutive transcriptional unit encoding a chaperone protein like e.g. Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssbl, Ssel, Sse2, Ssal, Ssa2, S
  • coli DH5alpha (F _ , phi80d/acZdeltaM15, delta(/acZYA-argF)U169, deoR, recAl, endAl, hsdR17(rk', mk + ), phoA, supE44, lambda', thi-1, gyrA96, re I Al) bought from Invitrogen.
  • Two media are used to cultivate B. subtilis: i.e., a complex medium like a rich Luria Broth (LB) and a minimal medium for shake flask cultures.
  • the LB medium consisted of 1% tryptone peptone (Difco), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR).
  • Luria Broth agar (LBA) plates consisted of the LB media, with 12 g/Lagar (Difco) added.
  • the minimal medium contained 2.00 g/L (NEUhSO ⁇ 7.5 g/L KH2PO4, 17.5 g/L K2HPO4, 1.25 g/L Na-citrate, 0.25 g/L MgSO 4 .7H2O, 0.05 g/L tryptophan, from 10 up to 30 g/L glucose (or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose), 10 mL/L trace element mix and 10 mL/L Fe-citrate solution.
  • the medium was set to a pH of 7 with 1 M KOH.
  • precursor(s) and/or acceptor(s) for saccharide like disaccharide and/or milk oligosaccharide
  • synthesis compounds like e.g., galactose, glucose, fructose, fucose, lactose, a co-factor could be added to the medium.
  • the trace element mix consisted of 0.735 g/L CaCl2.2H 2 O, 0.1 g/L MnCI 2 .2H 2 O, 0.033 g/L CuCI 2 .2H 2 O, 0.06 g/L CoCI 2 .6H 2 O, 0.17 g/L ZnCI 2 , 0.0311 g/L H3BO4, 0.4 g/L Na 2 EDTA.2H2O and 0.06 g/L Na 2 MoO 4 .
  • the Fe-citrate solution contained 0.135 g/L FeCl3.6H 2 O, 1 g/L Na- citrate (Hoch 1973 PMC1212887).
  • Complex medium e.g., LB
  • LB Complex medium
  • B. subtilis strains were initially grown on LB agar to obtain single colonies. These plates were grown over night at 37°C. Starting from a single colony, a preculture was grown over night in 5 m L at 37°C, shaking at 200 rpm. Subsequent 125 m L shake flask experiments were inoculated with 2% of this preculture, in 25 m L media. These shake flasks were incubated at 37°C with an orbital shaking of 200 rpm for 72h, or shorter of longer.
  • B. subtilis 168 is used as available at the Bacillus Genetic Stock Center (Ohio, USA). Plasmids for gene deletion via Cre/lox are constructed as described by Yan et al. (Appl & Environm microbial, Sept 2008, p5556-5562). Gene disruption is done via homologous recombination with linear DNA and transformation via the electroporation as described by Xue et al. (J. microb. Meth. 34 (1999) 183-191). The method of gene knockouts is described by Liu et al. (Metab. Engine. 24 (2014) 61-69). Integrative vectors as described by Popp et al. (Sci.
  • a suitable promoter for expression can be derived from the part repository (iGem): sequence id: BBa_K143012, BBa_K823000, BBa_K823002 or BBa_K823003. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.
  • Bacillus subtilis mutant strains are created to contain a gene coding for a lactose importer (e.g. the E. coli lacY with UniProt ID P02920).
  • a lactose importer e.g. the E. coli lacY with UniProt ID P02920
  • the B. subtilis strain is modified with a genomic knock-in of constitutive transcriptional units comprising a lactose importer (such as e.g. the E. coli lacY with UniProt ID P02920) and a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. LgtA from N.
  • the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g. WbgO from E. coli O55:H7 (UniProt ID D3QY14).
  • the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000).
  • the N-acetylglucosamine beta-1, 3-galactosyltransferase and the N-acetylglucosamine beta-1, 4-galactosyltransferase can be delivered to the strain either via genomic knock-in or from an expression plasmid.
  • the B. subtilis strains are modified with a constitutive transcriptional unit for a fucosyltransferase.
  • sialic acid production a mutant B.
  • subtilis strain is created by overexpressing a fructose-6-P-aminotransferase like the native fructose-6-P-aminotransferase glmS (UniProt ID P0CI73) to enhance the intracellular glucosamine-6-phosphate pool.
  • a fructose-6-P-aminotransferase like the native fructose-6-P-aminotransferase glmS (UniProt ID P0CI73) to enhance the intracellular glucosamine-6-phosphate pool.
  • the enzymatic activities of the genes nagA, nagB and gamA are disrupted by genetic knockouts and a glucosamine-6-P- aminotransferase like e.g. GNA1 from S. cerevisiae (UniProt ID P43577), an N-acetylglucosamine-2- epimerase like e.g. from B.
  • the sialic acid producing strain is further modified with a constitutive transcriptional unit comprising an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P. multocida (UniProt ID A0A849CI62), and a sialyltransferase like e.g., an alpha-2, 3-sialyltransferase like e.g.
  • P. multocida UniProt ID Q9CLP3
  • SEQ ID NO 02 PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having betagalactoside alpha-2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g. the alpha-2, 6- si
  • the mutant strains can additionally be modified with genomic knock-ins of constitutive transcriptional units comprising the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from 7. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B.
  • sucrose transporter CscB
  • E. coli W UniProt ID E0IXR1
  • Frk fructose kinase
  • BaSP sucrose phosphorylase
  • adolescentis (UniProt ID A0ZZH6).
  • a constitutive transcriptional unit for a siderophore transporter like e.g. entS from E. coli (UniProt ID P24077, sequence version 02 (01 Nov 1997)) is added as well.
  • MMsf shake flask
  • complex medium like e.g., a rich tryptone-yeast extract (TY) medium
  • MMsf minimal medium for shake flask
  • the minimal medium uses a lOOOx stock trace element mix.
  • Trace element mix consisted of 10 g/L CaCL, 10 g/L FeSO ⁇ THjO, 10 g/L MnSO ⁇ HjO, 1 g/L ZnSOi.THjO, 0.2 g/L CuSO 4 , 0.02 g/L NiCh.6H2O, 0.2 g/L biotin (pH 7) and 0.03 g/L protocatechuic acid.
  • the minimal medium for the shake flasks (MMsf) experiments contained 20 g/L (NH ⁇ SO ⁇ , 5 g/L urea, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.25 g/L MgSO 4 .7H 2 O, 42 g/L MOPS, from 10 up to 30 g/L glucose or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose when specified in the examples and 1 ml/L trace element mix.
  • TY medium consisted of 1.6% tryptone (Difco, Erembodegem, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium).
  • TY agar (TYA) plates consisted of the TY media, with 12 g/L agar (Difco, Erembodegem, Belgium) added.
  • Complex medium e.g., TY
  • TY Complex medium
  • autoclaving 121°C, 21 min
  • minimal medium by filtration 0.22 pm Sartorius
  • the medium was made selective by adding an antibiotic.
  • a preculture was started from a cryovial or a single colony from a TY plate, in 6 mL TY and was incubated overnight at 37 °C on an orbital shaker at 200 rpm.
  • Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL MMsf medium. These shake flasks were incubated at 37°C with an orbital shaking of 200 rpm for 72h, or shorter of longer.
  • Corynebacterium glutamicum was used as available at the American Type Culture Collection (ATCC 13032). Integrative plasmid vectors were made using the Cre/loxP technique as described by Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 Apr, 67(2):225-33) and temperature-sensitive shuttle vectors as described by Okibe et al. (Journal of Microbiological Methods 85, 2011, 155-163) are constructed for gene deletions, mutations and insertions. Suitable promoters for (heterologous) gene expression can be derived from Yim et al. (Biotechnol. Bioeng., 2013 Nov, 110(ll):2959-69).
  • Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.
  • C. glutamicum mutant strains are created to contain a gene coding for a lactose importer (such as e.g. the E. coli lacY with UniProt ID P02920).
  • the C. glutamicum strain is modified with a genomic knock-in of constitutive expression units comprising a lactose importer (such as e.g. the E.
  • the LN3 producing strain is further modified with a constitutive transcriptional unit for an N- acetylglucosamine beta-1, 3-galactosyltransferase like e.g. WbgO from E. coli O55:H7 (UniProt ID D3QY14).
  • the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000).
  • the N-acetylglucosamine beta-1, 3-galactosyltransferase and the N-acetylglucosamine beta-1, 4-galactosyltransferase can be delivered to the strain either via genomic knock-in or from an expression plasmid.
  • the mutant C To further produce a fucosylated saccharide, the mutant C.
  • glutamicum strains are further modified with a constitutive transcriptional unit for a fucosyltransferase.
  • a mutant C. glutamicum strain is created by overexpressing a fructose-6-P-aminotransferase like the native fructose-6-P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 03, 23 Jan 2007) to enhance the intracellular glucosamine-6-phosphate pool.
  • the enzymatic activities of the genes nagA, nagB and gamA are disrupted by genetic knockouts and a glucosamine-6-P-aminotransferase like e.g.
  • GNA1 from S. cerevisiae (UniProt ID P43577), an N- acetylglucosamine-2-epimerase like e.g. from B. ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase like e.g. from N. meningitidis (UniProt ID E0NCD4) are overexpressed on the genome.
  • the sialic acid producing strain is further modified with a constitutive transcriptional unit comprising an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P.
  • multocida (UniProt ID A0A849CI62), and a sialyltransferase like e.g., an alpha-2, 3-sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide (SEQ ID NO 02) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having betagalactoside alpha-2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g.
  • a sialyltransferase like e.g., an alpha-2, 3-sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P. multocida
  • the mutant strains can additionally be modified with genomic knock-ins of constitutive transcriptional units comprising the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6).
  • CscB sucrose transporter
  • E0IXR1 E. coli W
  • Frk fructose kinase
  • Z. mobilis UniProt ID Q03417
  • BaSP sucrose phosphorylase
  • Chlamydomonas reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7).
  • TAP medium uses a lOOOx stock Hutner's trace element mix.
  • Hutner's trace element mix consisted of 50 g/L Na2EDTA.H2O (Titriplex III), 22 g/L ZnSO4.7H2O, 11.4 g/L H3BO3, 5 g/L MnCI2.4H2O, 5 g/L FeSO4.7H2O, 1.6 g/L CoCI2.6H2O, 1.6 g/L CuSO4.5H2O and 1.1 g/L (NH4)6MoO3.
  • the TAP medium contained 2.42 g/L Tris (tris(hydroxymethyl)aminomethane), 25 mg/L salt stock solution, 0.108 g/L K2HPO4, 0.054 g/L KH2PO4 and 1.0 mL/L glacial acetic acid.
  • the salt stock solution consisted of 15 g/L NH4CL, 4 g/L MgSO4.7H2O and 2 g/L CaCI2.2H2O.
  • precursor(s) and/or acceptor(s) for saccharide like disaccharide and/or milk oligosaccharide, synthesis, compounds like e.g., galactose, glucose, fructose, fucose, lactose, a co-factor could be added.
  • Medium was sterilized by autoclaving (121°C, 21 min).
  • TAP medium was used containing 1% agar (of purified high strength, 1000 g/cm2).
  • Cells of C. reinhardtii were cultured in selective TAP-agar plates at 23 +/- 0.5°C under 14/10 h I ight/dark cycles with a light intensity of 8000 Lx. Cells were analysed after 5 to 7 days of cultivation. For high-density cultures, cells could be cultivated in closed systems like e.g., vertical or horizontal tube photobioreactors, stirred tank photobioreactors or flat panel photobioreactors as described by Chen et aL (Bioresour. TechnoL 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827).
  • closed systems like e.g., vertical or horizontal tube photobioreactors, stirred tank photobioreactors or flat panel photobioreactors as described by Chen et aL (Bioresour. TechnoL 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827).
  • C. reinhardtii wild-type strains 21gr (CC-1690, wild-type, mt+), 6145C (CC-1691, wild-type, mt-), CC-125 (137c, wild-type, mt+), CC-124 (137c, wild-type, mt-) as available from the Chlamydomonas Resource Center (https://www.chlamycollection.org) (University of Minnesota, U.S.A) were used.
  • Expression plasmids originated from pSH03, as available from the Chlamydomonas Resource Center. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.
  • Suitable promoters for (heterologous) gene expression can be derived from e.g., Scranton et al. (Algal Res. 2016, 15: 135-142). Targeted gene modification (like gene knock-out or gene replacement) can be carried using the Crispr-Cas technology as described e.g., by Jiang et al. (Eukaryotic Cell 2014, 13(11): 1465-1469). Transformation via electroporation was performed as described by Wang et al. (Biosci. Rep. 2019, 39:
  • BSR2018210 and as described like e.g., in WO22034067 or in WO22034069.
  • the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067.
  • C. reinhardtii cells are modified with a CMP-sialic acid transporter like e.g., CST from Mus musculus (UniProt ID Q61420), and a Golgi-localised sialyltransferase selected from species like e.g., Homo sapiens, Mus musculus, Rattus norvegicus.
  • the mutant strain was derived from C.
  • C. reinhardtii cells can be modified with an expression plasmid comprising a constitutive transcriptional unit for an alpha-1, 2-fucosyltransferase and/or an alpha-1, 3-fucosyltransferase.
  • the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067.
  • the mutant strain was derived from C.
  • the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g., WbgO (Uniprot ID D3QY14) from E.
  • LgtB Uniprot ID Q51116, sequence version 02, 01 Dec 2000
  • a C. reinhardtii strain is modified for production of GDP-fucose, UDP-galactose, LN3, LNT and/or LNnT as described herein and for expression of one or more compatible fucosyltransferase(s).
  • a C. reinhardtii strain is modified for production of CMP-sialic acid, UDP-galactose, LN3 and LNT as described herein and for expression of one or more compatible sialyltransferase(s) like e.g., an alpha-2, 3-sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P.
  • UniProt ID Q9CLP3 or a PmultST3-like polypeptide (SEQ ID NO 02) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having betagalactoside alpha-2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g. the alpha-2, 6- sialyltransferase (PdST6) from P.
  • a C. reinhardtii strain is modified for production of CMP-sialic acid, UDP-galactose, LN3 and LNnT as described herein and for expression of one or more compatible sialyltransferase(s).
  • Fresh adipose tissue is obtained from slaughterhouses (e.g., cattle, pigs, sheep, chicken, ducks, catfish, snake, frogs) or liposuction (e.g., in case of humans, after informed consent) and kept in phosphate buffer saline supplemented with antibiotics. Enzymatic digestion of the adipose tissue is performed followed by centrifugation to isolate mesenchymal stem cells. The isolated mesenchymal stem cells are transferred to cell culture flasks and grown under standard growth conditions, e.g., 37°C, 5% CO2.
  • the initial culture medium includes DMEM-F12, RPMI, and Alpha-MEM medium (supplemented with 15% foetal bovine serum), and 1% antibiotics.
  • the culture medium is subsequently replaced with 10% FBS (foetal bovine serum)-supplemented media after the first passage.
  • FBS foetal bovine serum
  • This example illustrates isolation of mesenchymal stem cells from milk collected under aseptic conditions from human or any other mammal(s) such as described herein.
  • An equal volume of phosphate buffer saline is added to diluted milk, followed by centrifugation for 20 min.
  • the cell pellet is washed thrice with phosphate buffer saline and cells are seeded in cell culture flasks in DMEM-F12, RPMI, and Alpha-MEM medium supplemented with 10% foetal bovine serum and 1% antibiotics under standard culture conditions.
  • Hassiotou et al. 2012, Stem Cells. 30(10): 2164-2174
  • the mesenchymal cells isolated from adipose tissue of different animals or from milk as described above can be differentiated into mammary-like epithelial and luminal cells in 2D and 3D culture systems. See, for example, Huynh et al. 1991. Exp Cell Res. 197(2): 191 -199; Gibson et al. 1991, In Vitro Cell Dev Biol Anim. 27(7): 585-594; Blatchford et al. 1999; Animal Cell Technology': Basic & Applied Aspects, Springer, Dordrecht. 141-145; Williams et al. 2009, Breast Cancer Res 11(3): 26-43; and Arevalo et al. 2015, Am J Physiol Cell Physiol. 310(5): C348 - C356; each of which is incorporated herein by reference in their entireties for all purposes.
  • the isolated cells were initially seeded in culture plates in growth media supplemented with 10 ng/mL epithelial growth factor and 5 pg/mL insulin.
  • growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100 U/mL penicillin, 100 ug/mL streptomycin), and 5 pg/mL insulin for 48h.
  • penicillin-streptomycin 100 U/mL penicillin, 100 ug/mL streptomycin
  • 5 pg/mL insulin for 48h.
  • the cells were fed with complete growth medium containing 5 pg/mL insulin, 1 pg/mL hydrocortisone, 0.65 ng/mL triiodothyronine, 100 nM dexamethasone, and 1 pg/mL prolactin.
  • serum is removed from the complete induction medium.
  • the isolated cells were trypsinized and cultured in Matrigel, hyaluronic acid, or ultra- low attachment surface culture plates for six days and induced to differentiate and lactate by adding growth media supplemented with 10 ng/mL epithelial growth factor and 5 pg/mL insulin.
  • growth media supplemented with 10 ng/mL epithelial growth factor and 5 pg/mL insulin.
  • cells were fed with growth medium supplemented with 2% foetal bovine serum, 1% penicillin-streptomycin (100 U/mL penicillin, 100 ug/mL streptomycin), and 5 pg/mL insulin for 48h.
  • the cells were fed with complete growth medium containing 5 pg/mL insulin, 1 pg/mL hydrocortisone, 0.65 ng/mL triiodothyronine, 100 nM dexamethasone, and 1 pg/mL prolactin. After 24h, serum is removed from the complete induction medium.
  • the cells are brought to induced pluripotency by reprogramming with viral vectors encoding for Oct4, Sox2, Klf4, and c-Myc.
  • the resultant reprogrammed cells are then cultured in Mammocult media (available from Stem Cell Technologies), or mammary cell enrichment media (DMEM, 3% FBS, estrogen, progesterone, heparin, hydrocortisone, insulin, EGF) to make them mammary-like, from which expression of select milk components can be induced.
  • Mammocult media available from Stem Cell Technologies
  • DMEM mammary cell enrichment media
  • epigenetic remodelling is performed using remodelling systems such as CRISPR/Cas9, to activate select genes of interest, such as casein, a- lactalbumin to be constitutively on, to allow for the expression of their respective proteins, and/or to down-regulate and/or knock-out select endogenous genes as described e.g., in WO21067641, which is incorporated herein by reference in its entirety for all purposes.
  • remodelling systems such as CRISPR/Cas9
  • select genes of interest such as casein, a- lactalbumin to be constitutively on, to allow for the expression of their respective proteins
  • down-regulate and/or knock-out select endogenous genes as described e.g., in WO21067641, which is incorporated herein by reference in its entirety for all purposes.
  • isolated mesenchymal cells re-programmed into mammary-like cells are modified via CRISPR-CAS as described e.g., in WO22034067, W0220
  • Completed growth media includes high glucose DMEM/F12, 10% FBS, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/mL EGF, and 5 pg/mL hydrocortisone.
  • Completed lactation media includes high glucose 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.
  • the maximal growth rate (pMax) was calculated based on the observed optical densities at 600 nm using the R package grofit.
  • Heterologous and homologous expression Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: IDT or Twist Bioscience. Proteins described in present disclosure are summarized in 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.
  • Standards such as but not limited to sucrose, lactose, 3'SL, 6'SL, lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neo-tetraose (LNnT), LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LSTa, LSTc and LSTd were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with in-house made standards.
  • Neutral oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Evaporative Light Scattering Detector (ELSD) or a Refractive Index (Rl) detection.
  • ELSD Evaporative Light Scattering Detector
  • Rl Refractive Index
  • a volume of 0.7 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 A;1.7 pm) column with an Acquity UPLC BEH Amide VanGuard column, 130 A, 2. lx 5 mm.
  • the column temperature was 50 °C.
  • the mobile phase consisted of a % water and % acetonitrile solution to which 0.2 % triethylamine was added.
  • the method was isocratic with a flow of 0.130 mL/min.
  • the ELSD detector had a drift tube temperature of 50 °C and the N2 gas pressure was 50 psi, the gain
  • Sialylated oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Refractive Index (Rl) detection.
  • a volume of 0. 5 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 A;1.7 pm). The column temperature was 50 °C.
  • the mobile phase consisted of a mixture of 70 % acetonitrile, 26 % ammonium acetate buffer (150 mM) and 4 % methanol to which 0.05 % pyrrolidine was added.
  • the method was isocratic with a flow of 0.150 mL/min.
  • the temperature of the Rl detector was set at 35 °C.
  • a Waters Xevo TQ-MS with Electron Spray Ionisation (ESI) was used with a desolvation temperature of 450 °C, a nitrogen desolvation gas flow of 650 L/h and a cone voltage of 20 V.
  • the MS was operated in selected ion monitoring (SIM) in negative mode for all oligosaccharides. Separation was performed on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1 x 100 mm; 3 pm) on 35 °C.
  • eluent A was ultrapure water with 0.1 % formic acid and wherein eluent B was acetonitrile with 0.1 % formic acid.
  • the oligosaccharides were separated in 55 min using the following gradient: an initial increase from 2 to 12 % of eluent B over 21 min, a second increase from 12 to 40 % of eluent B over 11 min and a third increase from 40 to 100 % of eluent B over 5 min.
  • As a washing step 100 % of eluent B was used for 5 min.
  • the initial condition of 2 % of eluent B was restored in 1 min and maintained for 12 min.
  • Both neutral and sialylated sugars at low concentrations were analyzed on a Dionex HPAEC system with pulsed amperometric detection (PAD).
  • a volume of 5 pL of sample was injected on a Dionex CarboPac PA200 column 4 x 250 mm with a Dionex CarboPac PA200 guard column 4 x 50 mm.
  • the column temperature was set to 30 °C.
  • a gradient was used wherein eluent A was deionized water, wherein eluent B was 200 mM Sodium hydroxide and wherein eluent C was 500 mM Sodium acetate.
  • the oligosaccharides were separated in 60 min while maintaining a constant ratio of 25 % of eluent B using the following gradient: an initial isocratic step maintained for 10 min of 75 % of eluent A, an initial increase from 0 to 4 % of eluent C over 8 min, a second isocratic step maintained for 6 min of 71 % of eluent A and
  • Lactobionic acid was analysed on a Dionex HPAEC system with pulsed amperometric detection (PAD).
  • a volume of 5 pL of sample was injected on a Dionex CarboPac PA01 column 2 x 250 mm with a Dionex CarboPac PA01 guard column 4 x 50 mm.
  • the column temperature was set to 30 °C.
  • a gradient was used wherein eluent A was ultrapure water, eluent B was 200 mM Sodium hydroxide and eluent C was 500 mM Sodium acetate. Total gradient time was 41 min and started with 50% B and 5% C in the first 7 minutes.
  • Example 2 Evaluation of production of sialic acid and 6'SL with a modified E. coli host having the glmU gene rendered less functional
  • E. coli K-12 MG1655 strain was modified for production of sialic acid comprising genomic knock-ins of constitutive transcriptional units containing the mutant glmS*54 from E. coli (SEQ ID NO 01) (differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006)), the glucosamine 6-phosphate N- acetyltransferase GNA1 from S. cerevisiae (UniProt ID P43577), the N-acylglucosamine 2-epimerase AGE from B.
  • SEQ ID NO 01 mutant glmS*54 from E. coli
  • the strain was further modified with constitutive transcriptional units for the N-acylneuraminate cytidylyltransferases NeuA from Campylobacter jejuni (UniProt ID Q93MP7) and NeuA from Haemophilus influenzae (SEQ ID NO 04), and with two constitutive transcriptional units for expression of the PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 and having beta-galactoside alpha-2, 6-sialyltransferase activity.
  • SEQ ID NO 03 constitutive transcriptional units for expression of the PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 and having beta-galactoside alpha-2, 6-sialyltransferase activity.
  • the strain was modified with a genomic knock-in for overexpression of the acetyl-CoA synthetase acs from E. coli (UniProt ID P27550) and with a genomic knock-out of the O-antigen gene cluster deleting all the genes between wbbK and wcaN including wbbK and wcaN.
  • the strain was modified for growth on sucrose as described in Example 1.
  • the mutant strain A thus obtained was then further engineered to create five new strains (B, C, D, E and F) having a glmU gene (UniProt ID P0ACC7) that was less functional compared to the native glmU gene present in strain A.
  • Each strain was created by genomic knock-in of a different constitutive transcriptional unit comprising the native glmU gene from E. coli (UniProt ID P0ACC7) that was introduced with different promoter (P), untranslated (UTR) and terminator (T) sequences (Table 3) at a foreign location in the E. coli cell's genome, i.e. at the insN locus, and by subsequent genomic knock-out of the native glmU gene from E. coli (locus 3,913,830 ⁇ - 3,915,200).
  • P promoter
  • UTR untranslated
  • T terminator sequences
  • the novel strains were evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 1 in which the strains were cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. The strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed on UPLC.
  • the measured sialic acid concentration, sum of sialylated molecules (sialic acid and 6'SL) and biomass concentration was averaged over all biological replicates and then normalized to the averaged sialic acid concentration, sum of sialylated molecules and biomass concentration of the reference strain A having the same genetic make-up but lacking any modification to the native glmU gene.
  • the experiment showed that each knockdown of glmU in the five strains resulted in higher production of sialic acid and sialylated molecules (sialic acid and 6'SL) compared to the reference strain A (Table 2).
  • Promoter, untranslated region (UTR) and terminator sequences used to express the glmU enzyme from E. coli (Uniprot ID POACC7) from a knockdown transcriptional unit integrated in the mutant E. coli strains B, C, D, E, F, H, I and J as given in Table 2 and Table 4.
  • Example 3 Evaluation of production of sialic acid and 6'SL with a modified E. coli host having the glmU gene rendered less functional and overexpressing E. coli glmS (UniProt ID P17169)
  • the mutant E. coli strain F was further modified to overexpress its native E. coli glmS gene (UniProt ID P17169) by integrating a strong constitutive promoter sequence on the native glmU locus (locus 3,913,830 ⁇ - 3,915,200) 5' of the E. coli glmS gene.
  • a strong constitutive promoter sequence on the native glmU locus (locus 3,913,830 ⁇ - 3,915,200) 5' of the E. coli glmS gene.
  • three new strains were created, each expressing the E. coli glmS from a different strong constitutive promoter sequence (Table 4).
  • the new strains were further transformed with an expression plasmid comprising a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P.
  • strains were evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 1, in which the strains were cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. The strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed on UPLC. For each strain with a particular constitutive promoter sequence controlling over-expression of the E.
  • Example 4 Evaluation of production of sialic acid and 6'SL with modified E. coli hosts when evaluated in a fed-batch fermentation process with sucrose and lactose
  • the mutant E. coli strains G, H and I as described in Example 3 were selected for further evaluation in fed- batch fermentation processes.
  • Fed-batch fermentations at bioreactor scale were performed as described in Example 1.
  • Sucrose was used as a carbon source and lactose was added in the batch medium.
  • sucrose was added via an additional feed.
  • regular broth samples were taken at several time points during the fermentation process and the production of sialic acid and 6'SL was measured using UPLC as described in Example 1.
  • Example 5 Evaluation of production of sialic acid and 6'SL with a modified S. cerevisiae host
  • a 5. cerevisiae strain is modified for production of CMP-sialic acid and for expression of a sialyltransferase as described in Example 1.
  • This strain is further modified via homologous recombination as described in Example 1 to integrate a very weak constitutive yeast promoter 5' of the 5. cerevisiae native PCM1 gene (UniProt ID P38628, sequence version 02 (07 April 2021)) which is located on chromosome V and to integrate a strong constitutive yeast promoter 5' of the S. cerevisiae GFA1 gene (UniProt ID P14742, sequence version 04 (02 June 2021)) which is located on chromosome XI.
  • the strain is transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 6-sialyltransferase PdST6 from P. damselae (UniProt ID 066375).
  • the novel strain is evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 1, in which the appropriate selective medium comprises glucose as carbon source and lactose as precursors.
  • the strain is grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested, and production of sialic acid and 6'SL is analysed on UPLC.
  • Example 6 Evaluation of production of sialic acid and 3'SL with a modified B. subtilis host
  • a B. subtilis strain is modified for production of CMP-sialic acid and for expression of a sialyltransferase as described in Example 1.
  • This strain is further modified as described in Example 1 to integrate a very weak constitutive promoter 5' of the B. subtilis glmU gene (UniProt ID P14192) and to integrate a strong constitutive promoter s' of the B. subtilis glmS gene (UniProt ID P0CI73).
  • native expression of the B. subtilis glmU gene is downregulated and the native glmS gene is overexpressed.
  • the strain is transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3).
  • the novel strain is evaluated in a growth experiment for production of sialic acid and 3'SL according to the culture conditions provided in Example 1, in which the appropriate selective medium comprises glucose as carbon source and lactose as precursors.
  • the strain is grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested, and production of sialic acid and 3'SL is analysed on UPLC.
  • Example 7 Evaluation of production of sialic acid and 3'SL with a modified C. glutamicum host
  • a C. glutamicum strain is modified for production of CMP-sialic acid and for expression of a sialyltransferase as described in Example 1.
  • This strain is further modified as described in Example 1 to integrate a very weak constitutive promoter 5' of the C. glutamicum glmU gene (UniProt ID Q8NRU8) and to integrate a strong constitutive promoter 5' of the C. glutamicum glmS gene (UniProt ID Q8NND3).
  • native expression of the C. glutamicum glmU gene is downregulated and the native glmS gene is overexpressed.
  • the strain is transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3).
  • the novel strain is evaluated in a growth experiment for production of sialic acid and 3'SL according to the culture conditions provided in Example 1, using MMsf medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of sialic acid and 3'SL.
  • the mutant E. coli strains B to F are further modified to overexpress the native E. coli glmS gene (UniProt ID P17169) by integrating a strong constitutive promoter sequence on the native glmU locus (locus 3,913,830 ⁇ - 3,915,200) 5' of the E. coli glmS gene.
  • the new strains are further transformed with an expression plasmid comprising a constitutive transcriptional unit for either the N- acetylglucosamine beta-1, 3-galactosyltransferase wbgO (Uniprot ID D3QY14) from E.
  • LNB N-acetylglucosamine beta-1, 4-galactosyltransferase LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from Neisseria meningitidis. All strains comprising the wbgO gene are evaluated in a growth experiment for production of LNB according to the culture conditions provided in Example 1, in which the strains are cultivated in minimal medium with 30 g/L sucrose, and 20 g/L galactose. The strains are grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broths are harvested and production of LNB is analysed on UPLC.
  • strains comprising the IgtB gene are evaluated in a growth experiment for production of LacNAc according to the culture conditions provided in Example 1, in which the strains are cultivated in minimal medium with 30 g/L sucrose, and 20 g/L galactose. The strains are grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broths are harvested and production of LacNAc is analysed on UPLC.
  • Example 9 Evaluation of production of a mixture comprising LNB and 6'SL with a modified E. coli host having the glmU gene rendered less functional and overexpressing E. coli glmS (UniProt ID P17169)
  • the mutant E. coli strains B to F are further modified to overexpress the native E. coli glmS ene (UniProt ID P17169) by integrating a strong constitutive promoter sequence on the native glmU locus (locus 3,913,830 ⁇ - 3,915,200) 5' of the E. coli glmS gene.
  • the new strains are further transformed with an expression plasmid comprising a constitutive transcriptional unit for the N- acetylglucosamine beta-1, 3-galactosyltransferase wbgO (Uniprot ID D3QY14) from E.
  • Example 10 Evaluation of production of 3*-sialyl-3-fucosyllactose with a modified E. coli host having the glmU gene rendered less functional and overexpressing E. coli glmS (UniProt ID P17169)
  • the mutant E. coli strain F as described in Example 2, is further modified to overexpress the native E. coli glmS gene (UniProt ID P17169) by integrating a strong constitutive promoter sequence on the native glmU locus (locus 3,913,830 ⁇ - 3,915,200) 5' of the E. coli glmS gene.
  • the strain is modified with a knockout of the E. coli wcaJ, fucK and fuel genes and genomic knock-ins of constitutive transcriptional units comprising the mannose-6-phosphate isomerase manA from E. coli (UniProt ID P00946), the phosphomannomutase manB from E.
  • odolescentis (UniProt ID A0ZZH6).
  • the new strain is further transformed with an expression plasmid comprising a constitutive transcriptional unit for the N- acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62), the alpha-2, 3- sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) and the alpha-1, 3-fucosyltransferase HpFucT from H. pylori (UniProt ID 030511).
  • the new strain is evaluated in a growth experiment for production of 3'-sialyl-3-fucosyllactose according to the culture conditions provided in Example 1, in which the strain is cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. The strain is grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested, and production of 3'-sialyl-3-fucosyllactose, 3'FL and 3'SL is analysed on UPLC.
  • Example 11 Evaluation of production of a mixture comprising LacNAc and 3'SL with a modified S. cerevisiae host
  • a S. cerevisiae strain is modified for production of CMP-sialic acid and for expression of a sialyltransferase as described in Example 1.
  • This strain is further modified via homologous recombination as described in Example 1 to integrate a very weak constitutive yeast promoter 5' of the S. cerevisiae native PCM1 gene (UniProt ID P38628, sequence version 02 (07 April 2021)) which is located on chromosome V and to integrate a strong constitutive yeast promoter 5' of the S. cerevisiae GFA1 gene (UniProt ID P14742, sequence version 04 (02 June 2021)) which is located on chromosome XI.
  • native expression of the 5 is a very weak constitutive yeast promoter 5' of the S. cerevisiae native PCM1 gene (UniProt ID P38628, sequence version 02 (07 April 2021)) which is located on chromosome V
  • the strain is transformed with an expression plasmid containing a constitutive transcriptional unit for the phosphatase surE from E. coli (UniProt ID P0A840), the N- acetylglucosamine beta-1, 4-galactosyltransferase LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis, the N-acylneuraminate cytidylyltransferase neuA from P.
  • the novel strain is evaluated in a growth experiment for production of a mixture comprising LacNAc and 3'SL according to the culture conditions provided in Example 1, in which the appropriate selective medium comprises glucose as carbon source and lactose as precursors.
  • the strain is grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested and production of a mixture comprising LacNAc and 3'SL is analysed on UPLC. Also the production of sialylated LacNAc in said mixture is evaluated.

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Abstract

The present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation. The invention provides a cell for production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide wherein synthesis of UDP-N-acetylglucosamine in said cell is rendered less functional. The invention further provides use of said cell in a cultivation or incubation. The invention also describes methods for the production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide using said cell as well as the purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide.

Description

Production of a disaccharide and/or milk oligosaccharide by a cell with reduced synthesis of
UDP-GIcNAc
Field of the invention
The present invention is in the technical field of synthetic biology, metabolic engineering and cell cultivation. The invention provides a cell for production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide wherein synthesis of UDP-N-acetylglucosamine in said cell is rendered less functional. The invention further provides use of said cell in a cultivation or incubation. The invention also describes methods for the production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide using said cell as well as the purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide.
Background
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. Disaccharides and oligosaccharides are widely distributed in all living organisms and play important roles in a variety of physiological and pathological processes, such as cell metastasis, signal transduction, intercellular adhesion, inflammation, and immune response. Economical production of these disaccharides and oligosaccharides is of utmost importance to fully benefit of their biological advantages. An important group of oligosaccharides comprises milk oligosaccharides like mammalian milk oligosaccharides (MMOs) and human milk oligosaccharides (HMOs) found in mammalian and human milk, respectively. A wide variety of synthesis methods have been developed already, ranging from extraction over chemical synthesis to enzymatic synthesis. These methods are currently least applied, whereas biotechnological fermentative production is nowadays pursued and commercialized. Methods for the production of disaccharides and/or oligosaccharides are well known for a person skilled in the art (like e.g. described in Faijes et al (2019), US2010120096A, JP2013201913, W02022/034067).
N-acetylglucosamine (GIcNAc) is an important monosaccharide that is used in its free form as well as in its phosphorylated form, i.e., GlcNAc-6-phosphate and in its activated form UDP-GIcNAc in the synthesis of a disaccharide and/or oligosaccharide. A balanced availability of said different forms of GIcNAc often determines the efficacy of a disaccharide and/or oligosaccharide synthesis reaction in a cell.
Description
Summary of the invention
It is an object of the present invention to provide for tools and methods by means of which a disaccharide and/or an oligosaccharide like a milk oligosaccharide can be produced, preferably in an efficient, time and cost-effective way and which yields high amounts of the desired disaccharide and/or oligosaccharide like a milk oligosaccharide. According to the invention, this and other objects are achieved by providing methods and a cell for the production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide. The present invention also provides methods for the purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide. Furthermore, the present invention provides a cell which is genetically engineered as described herein and wherein synthesis of UDP-GIcNAc is rendered less functional. This invention also provides a purified disaccharide and/or oligosaccharide like a milk oligosaccharide by the above-referenced process. Further benefits of the teachings of this invention will be apparent to one skilled in the art from reading this invention.
Definitions
The words used in this specification to describe the invention and its various embodiments are to be understood not only in the sense of their commonly defined meanings, but to include by special definition in this specification structure, material or acts beyond the scope of the commonly defined meanings. Thus, if an element can be understood in the context of this specification as including more than one meaning, then its use in a claim must be understood as being generic to all possible meanings supported by the specification and by the word itself.
The various aspects and embodiments of the invention disclosed herein are to be understood not only in the order and context specifically described in this specification, but to include any order and any combination thereof. Each embodiment as identified herein may be combined together unless otherwise indicated. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Unless specifically stated otherwise, all words used in the singular number shall be deemed to include the plural and vice versa. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described herein are those well- known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications.
In the specification, there have been disclosed embodiments of the invention, and although specific terms are employed, the terms are used in a descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims. It must be understood that the illustrated embodiments have been set forth only for the purposes of example and that it should not be taken as limiting the invention. It will be apparent to those skilled in the art that alterations, other embodiments, improvements, details and uses can be made consistent with the letter and spirit of the disclosure herein and within the scope of this disclosure, which is limited only by the claims, construed in accordance with the patent law, including the doctrine of equivalents. In the claims that follow, reference characters used to designate claim steps are provided for convenience of description only, and are not intended to imply any particular order for performing the steps, unless specifically stated otherwise.
Throughout the application, unless explicitly stated otherwise, the features "synthesize", "synthesized" and "synthesis" are interchangeably used with the features "produce", "produced" and "production", respectively. Throughout the application, unless explicitly stated otherwise, the expressions "capable of...<verb>" and "capable to...<verb>" are preferably replaced with the active voice of said verb and vice versa. For example, the expression "capable of expressing" is preferably replaced with "expresses" and vice versa, i.e., "expresses" is preferably replaced with "capable of expressing". In this document and in its claims, 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. Throughout the application, the verb "to comprise" may be replaced by "to consist" or "to consist essentially of" and vice versa. In addition, 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. In this document and in its claims, unless specifically stated otherwise, the verbs "to comprise", "to have" and "to contain", and their conjugations, may be replaced by "to consist of" (and its conjugations) or "to consist essentially of" (and its conjugations) and vice versa. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
Throughout the document and in the claims, unless explicitly stated otherwise, the articles "a" and "an" are preferably replaced by "at least one", more preferably "at least two", even more preferably by "at least three", even more preferably by "at least four", even more preferably by "at least five", even more preferably by "at least six", most preferably by "at least two". The word "about" or "approximately" when used in association with a numerical value (e.g., "about 10") or with a range (e.g., "about x to approximately y") preferably means that the value or range is interpreted as being as accurate as the method used to measure it. If no error margins are specified, the expression "about" or "approximately" when used in association with a numerical value is interpreted as having the same round-off as the given value. Throughout this document and its claims, unless otherwise stated, the expression "from x to y", wherein x and y represent numerical values, refers to a range of numerical values wherein x is the lower value of the range and y is the upper value of the range. Herein, x and y are also included in the range.
According to the present invention, the term "polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, are to be understood to be covered by the term "polynucleotides". It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. The term "polynucleotide(s)" also embraces short polynucleotides often referred to as oligonucleotide(s).
"Polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. "Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques as well known to the skilled person. The same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Furthermore, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid sidechains, and the amino or carboxyl termini. Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions that also may contain coding and/or non-coding sequences.
"Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Similarly, a "synthetic" sequence, as the term is used herein, means any sequence that has been generated synthetically and not directly isolated from a natural source. "Synthesized", as the term is used herein, means any synthetically generated sequence and not directly isolated from a natural source.
"Recombinant" means genetically engineered DNA prepared by transplanting or splicing genes from one species into the cells of a host organism of a different species. Such DNA becomes part of the host's genetic makeup and is replicated.
The terms "recombinant" or "transgenic" or "metabolically engineered" or "genetically engineered" as used herein with reference to a cell or host cell are used interchangeably and indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence "foreign to said cell" or a sequence "foreign to said location or environment in said cell"). Such cells are described to be transformed with at least one heterologous or exogenous gene or are described to be transformed by the introduction of at least one heterologous or exogenous gene. Recombinant or metabolically engineered or genetically engineered or transgenic cells can contain genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means. The terms also encompass cells that contain a nucleic acid endogenous to the cell that has been modified or its expression or activity has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, replacement of a promoter; site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis and related techniques as known to a person skilled in the art. Accordingly, a "recombinant polypeptide" is one which has been produced by a recombinant cell. The terms also encompass cells that have been modified by removing a nucleic acid endogenous to the cell by means of common well-known technologies for a skilled person (like e.g. knocking-out genes).
A "heterologous sequence" or a "heterologous nucleic acid", as used herein, is one that originates from a source foreign to the particular cell (e.g., from a different species), or, if from the same source, is modified from its original form or place in the genome. Thus, a heterologous nucleic acid operably linked to a promoter is from a source different from that from which the promoter was derived, or, if from the same source, is modified from its original form or place in the genome. The heterologous sequence may be stably introduced, e.g., by transfection, transformation, conjugation or transduction, into the genome of the host cell, wherein techniques may be applied which will depend on the cell and the sequence that is to be introduced. Various techniques are known to a person skilled in the art and are, e.g., disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The term "mutant" or "engineered" cell as used within the context of the present invention refers to a cell which is genetically engineered.
The term "endogenous" within the context of the present disclosure refers to any polynucleotide, polypeptide or protein sequence that is a natural part of a cell and is occurring at its natural location in the cell chromosome and of which the control of expression has not been altered compared to the natural control mechanism acting on its expression. The term "exogenous" refers to any polynucleotide, polypeptide or protein sequence 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.
The term "heterologous" when used in reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme refers to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is from a source or derived from a source other than the host organism. In contrast a "homologous" polynucleotide, gene, nucleic acid, polypeptide, or enzyme is used herein to denote a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is derived from the host organism. When referring to a gene regulatory sequence or to an auxiliary nucleic acid sequence used for maintaining or manipulating a gene sequence (e.g. a promoter, a 5' untranslated region, 3' untranslated region, poly A addition sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genome homology region, recombination site, etc.), "heterologous" means that the regulatory sequence or auxiliary sequence is not naturally associated with the gene with which the regulatory or auxiliary nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome. Thus, a promoter operably linked to a gene to which it is not operably linked to in its natural state (i.e. in the genome of a non-genetically engineered organism) is referred to herein as a "heterologous promoter," even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked.
The term "modified expression" of a gene relates to a change in expression compared to the wild-type expression of said gene in any phase of the production process of the desired disaccharide and/or oligosaccharide, like a desired milk oligosaccharide. Said modified expression is either a lower or higher expression compared to the wild-type, wherein the term "higher expression" is also defined as "overexpression" of said gene in the case of an endogenous gene or "expression" in the case of a heterologous gene that is not present in the wild-type strain. Lower expression is obtained by means of common well-known technologies for a skilled person (such as the usage of siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon mutagenesis, etc.) which are used to change the genes in such a way that they are "less-able" (i.e., statistically significantly ess-able' compared to a functional wild-type gene) or completely unable (such as knocked-out genes) to produce functional final products. The term "riboswitch" as used herein is defined to be part of the messenger RNA that folds into intricate structures that block expression by interfering with translation. Binding of an effector molecule induces conformational change(s) permitting regulated expression post-transcriptionally. Next to changing the gene of interest in such a way that lower expression is obtained as described above, lower expression can also be obtained by changing the transcription unit, the promoter, an untranslated region, the ribosome binding site, the Shine Dalgarno sequence or the transcription terminator. Lower expression or reduced expression can for instance be obtained by mutating one or more base pairs in the promoter sequence or changing the promoter sequence fully to a constitutive promoter with a lower expression strength compared to the wild-type or an inducible promoter which result in regulated expression or a repressible promoter which results in regulated expression. Overexpression or expression is obtained by means of common well-known technologies for a skilled person (such as the usage of artificial transcription factors, de novo design of a promoter sequence, ribosome engineering, introduction or re-introduction of an expression module at euchromatin, usage of high-copy-number plasmids), wherein said gene is part of an "expression cassette" that relates to any sequence in which a promoter sequence, untranslated region sequence (containing either a ribosome binding sequence, Shine Dalgarno or Kozak sequence), a coding sequence and optionally a transcription terminator is present, and leading to the expression of a functional active protein. Said expression is either constitutive or conditional or regulated or tuneable.
The term "constitutive expression" is defined as expression that is not regulated by transcription factors other than the subunits of RNA polymerase (e.g., the bacterial sigma factors like s70, s54, or related s- factors and the yeast mitochondrial RNA polymerase specificity factor MTF1 that co-associate with the RNA polymerase core enzyme) under certain growth conditions. Non-limiting examples of such transcription factors are CRP, Lacl, ArcA, Cra, IcIR in E. coli, or, Aft2p, Crzlp, Skn7 in Saccharomyces cerevisiae, or, DeoR, GntR, Fur in B. subtilis. These transcription factors bind on a specific sequence and may block or enhance expression in certain growth conditions. The RNA polymerase is the catalytic machinery for the synthesis of RNA from a DNA template. RNA polymerase binds a specific DNA sequence to initiate transcription, for instance via a sigma factor in prokaryotic hosts or via MTFl in yeasts. Constitutive expression offers a constant level of expression with no need for induction or repression.
The term "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.
The term "control sequences" refers to sequences recognized by the cells transcriptional and translational systems, allowing transcription and translation of a polynucleotide sequence to a polypeptide. Such DNA sequences are thus necessary for the expression of an operably linked coding sequence in a particular host cell, cell or organism. Such control sequences can be, but are not limited to, promoter sequences, ribosome binding sequences, Shine Dalgarno sequences, Kozak sequences, transcription terminator sequences. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. DNA for a presequence or secretory leader may be operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Said control sequences can furthermore be controlled with external chemicals, such as, but not limited to, IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose via an inducible promoter or via a genetic circuit that either induces or represses the transcription or translation of said polynucleotide to a polypeptide.
Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. The term "wildtype" refers to the commonly known genetic or phenotypical situation as it occurs in nature.
The term "modified expression of a protein" as used herein refers to i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein, iii) expression and/or overexpression of a variant protein that has a higher activity compared to the wild-type (i.e. native in the expression host) protein, iv) reduced expression of an endogenous protein or v) expression and/or overexpression of a variant protein that has a reduced activity compared to the wild-type (i.e. native in the expression host) protein. Preferably, the term "modified expression of a protein" as used herein refers to i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein or iii) expression and/or overexpression of a variant protein that has a higher activity compared to the wild-type (i.e. native in the expression host) protein.
The term "modified activity" of a protein relates to a non-native activity of the protein in any phase of the production process of the desired disaccharide and/or oligosaccharide, like a desired milk oligosaccharide. The term "non-native", as used herein with reference to the activity of a protein indicates that the protein has been modified to have an abolished, impaired, reduced, delayed, higher, accelerated or improved activity compared to the native activity of said protein. A modified activity of a protein is obtained by modified expression of said protein or is obtained by expression of a modified, i.e., mutant form of the protein. A mutant form of the protein can be obtained by expression of a mutant form of the gene encoding the protein, e.g., comprising a deletion, an insertion and/or a mutation of one or more nucleotides compared to the native gene sequence. A mutant form of a gene can be obtained by techniques well-known to a person skilled in the art, such as but not limited to site-specific mutation; CrispR; riboswitch; recombineering; ssDNA mutagenesis; transposon mutagenesis.
The term "non-native", as used herein with reference to a cell producing a disaccharide indicates that the disaccharide 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 disaccharide or to have a higher production of the disaccharide.
The term "non-native", as used herein with reference to a cell producing an oligosaccharide like a milk oligosaccharide, indicates that the oligosaccharide is i) not naturally produced or ii) when naturally produced not in the same amounts by the cell; and that the cell has been genetically engineered to be able to produce said oligosaccharide, more particularly said milk oligosaccharide, or to have a higher production of the oligosaccharide, more particularly the milk oligosaccharide.
The term "non-native", as used herein with reference to a cell producing a disaccharide and a milk oligosaccharide indicates that the disaccharide and the milk oligosaccharide are 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 disaccharide and milk oligosaccharide or to have a higher production of the disaccharide and the milk oligosaccharide. "Variant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to the persons skilled in the art.
In some embodiments, the present invention contemplates making variants by modifying the structure of an enzyme as used in the present invention. Variants can be produced by amino acid substitution, deletion, addition, or combinations thereof.
"Fragment", with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule, particularly a part of a polynucleotide.
Throughout the application, the sequence of a polynucleotide can be represented by a SEQ ID NO or alternatively by a GenelD (Maglott et al (2011) Nucl. Acids Res. 39, Issue suppl_l, D52-D57) or GenBank NO (https://www.ncbi.nlm.nih.gov/genbank/). Therefore, the terms "polynucleotide SEQ ID NO", "polynucleotide GenelD" and "polynucleotide GenBank NO." can be interchangeably used, unless explicitly stated otherwise.
Throughout the application, the sequence of a polypeptide can be represented by a SEQ ID NO or alternatively by an UniProt ID. Therefore, the terms "polypeptide SEQ ID NO" and "polypeptide UniProt ID" can be interchangeably used, unless explicitly stated otherwise.
A domain can be characterized, for example, by a Pfam (El-Gebali et al., Nucleic Acids Res. 47 (2019) D427- D432), an IPR (InterPro domain) (http://ebi.ac.uk/interpro) (Mitchell et aL, Nucleic Acids Res. 47 (2019) D351-D360), a Conserved Domain Database (CDD) designation (https://www.ncbi.nlm.nih.gov/cdd) (Lu et aL, Nucleic Acids Res. 48 (2020) D265-D268) or a PTHR domain (http://www.pantherdb.org) (Mi et aL, Nucleic Acids. Res. 41 (2013) D377-D386; Thomas et al., Genome Research 13 (2003) 2129-2141). 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. Throughout the application, the sequence of a polypeptide is represented by a SEQ. ID NO or an UniProt ID. Unless stated otherwise, the UniProt IDs of the proteins described correspond to their sequence version 01 as present in the UniProt Database (www.uniprot.org) version release 2021_03 and consulted on 09 June 2021.
InterPro provides functional analysis of proteins by classifying them into families and predicting domains and important sites. To classify proteins in this way, InterPro uses predictive models, known as signatures, provided by several different databases (referred to as member databases) that make up the InterPro consortium. Protein signatures from these member databases are combined into a single searchable resource, capitalizing on their individual strengths to produce a powerful integrated database and diagnostic tool.
It should be understood forthose skilled in the art that for the databases used herein, comprising InterPro 90.0 (released on 4th August 2022) and eggNOG5.0 (released in 2019), the content of each database is fixed at each release and is not to be changed. When the content of a specific database is changed, this specific database receives a new release version with a new release date. All release versions for each database with their corresponding release dates and specific content as annotated at these specific release dates are available and known to those skilled in the art.
The term "monosaccharide" as used herein refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed as an aldose, a ketose, a deoxysugar, a deoxy-aminosugar, a uronic acid, an aldonic acid, a ketoaldonic acid, an aldaric acid or a sugar alcohol, and contains one or more hydroxyl groups per molecule. Monosaccharides are saccharides containing only one simple sugar.
The term "phosphorylated monosaccharide" as used herein refers to a monosaccharide which is phosphorylated. Examples of phosphorylated monosaccharides include but are not limited to glucose-1- phosphate, glucose-6-phosphate, glucose-l,6-bisphosphate, galactose-l-phosphate, fructose-6- phosphate, fructose-l,6-bisphosphate, fructose-l-phosphate, glucosamine-l-phosphate, glucosamine-6- phosphate, N-acetylglucosamine-l-phosphate, mannose-l-phosphate, mannose-6-phosphate or fucose- 1-phosphate.
The terms "activated monosaccharide”, "nucleotide-activated sugar”, "nucleotide-sugar", "activated sugar", "nucleoside" or "nucleotide donor" are used herein interchangeably and refer to activated forms of monosaccharides. Examples of activated monosaccharides include but are not limited to UDP-N- acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), UDP- glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2- acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2- acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2- acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L- QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinovose, CMP-sialic acid (CMP-Neu5Ac or CMP-N-acetylneuraminic acid), GDP-fucose (GDP-Fuc), GDP-rhamnose and UDP-xylose. Nucleotidesugars act as glycosyl donors in glycosylation reactions. Glycosylation reactions are reactions that are catalysed by glycosyltransferases.
The term "glycosyltransferase" as used herein refers to an enzyme capable to catalyse the transfer of a sugar moiety of a donor to a specific acceptor, forming glycosidic bonds. Said donor can be a precursor as defined herein. A classification of glycosyltransferases using nucleotide diphospho-sugar, nucleotide monophospho-sugar and sugar phosphates and related proteins into distinct sequence-based families has been described (Campbell et al., Biochem. J. 326, 929-939 (1997)) and is available on the CAZy (CArbohydrate-Active EnZymes) website (www.cazy.org). As used herein the glycosyltransferase can be selected from the list comprising, consisting of or consisting essentially of: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N- acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N-acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N- glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino- 4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases.
The term "disaccharide" as used herein refers to a saccharide polymer containing two simple sugars, i.e., monosaccharides. Examples of disaccharides comprise lactose (Gal-pi,4-Glc), lacto-N-biose (LNB, Gal- P1,3-GICNAC), N-acetyllactosamine (LacNAc, Gal-pi,4-GlcNAc), LacDiNAc (GalNAc-pi,4-GlcNAc), N- acetylgalactosaminylglucose (GalNAc-pi,4-Glc), Neu5Ac-a2,3-Gal, Neu5Ac-a2,6-Gal and fucopyranosyl- (l-4)-N-glycolylneuraminic acid (Fuc-(l-4)-Neu5Gc), sucrose (Glc-ocl,2-Fru), maltose (Glc-al,4-Glc) and melibiose (Gal-al,6-Glc). Disaccharides comprise charged disaccharides carrying a negative charge like e.g. sialylated disaccharides like e.g. Neu5Ac-a2,3-Gal, Neu5Ac-a2,6-Gal and Fuc-(l-4)-Neu5Gc, and noncharged, i.e. neutral, disaccharides like e.g. lactose, LNB, LacNAc, sucrose, maltose and melibiose. "Oligosaccharide" as the term is used herein and as generally understood in the state of the art, 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.) between two sugar units can be expressed, for example, as 1,4, l->4, or (1-4), used interchangeably herein. For example, the terms "Gal-bl,4-Glc", "Gal-pi,4-Glc", "b-Gal-(l->4)-Glc", "P-Gal- (l->4)-Glc", "Galbetal-4-Glc", "Gal-b(l-4)-Glc" and "Gal-P(l-4)-Glc" have the same meaning, i.e. a beta- glycosidic bond links carbon-1 of galactose (Gal) with the carbon-4 of glucose (Glc). Each monosaccharide can be in the cyclic form (e.g., pyranose or furanose form). Linkages between the individual monosaccharide units may include alpha l->2, alpha l->3, alpha l->4, alpha l->6, alpha 2->l, alpha 2->3, alpha 2->4, alpha 2->6, beta l->2, beta l->3, beta l->4, beta l->6, beta 2->l, beta 2->3, beta 2->4, and beta 2->6. An oligosaccharide can contain both alpha- and beta-glycosidic bonds or can contain only alpha- glycosidic or only beta-glycosidic bonds. The term "polysaccharide" refers to a compound consisting of a large number, typically more than twenty, of monosaccharides linked glycosidically. Examples of oligosaccharides include but are not limited to Lewis-type antigen oligosaccharides, milk oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigen, enterobacterial common antigen (ECA), the glycan chain present in lipopolysaccharides (LPS), the oligosaccharide repeats present in capsular polysaccharides, peptidoglycan (PG), amino-sugars, antigens of the human ABO blood group system, an animal oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans, a plant oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans, sialylated oligosaccharide, neutral (non-charged) oligosaccharide, negatively charged oligosaccharide, fucosylated oligosaccharide, N-acetylglucosamine containing oligosaccharides, lacto-N-biose containing oligosaccharides, N-acetyllactosamine-containing oligosaccharides, N-acetylglucosamine containing sialylated oligosaccharides, N-acetylglucosamine containing neutral (non-charged) oligosaccharides, N- acetylglucosamine containing negatively charged oligosaccharides, N-acetylglucosamine containing fucosylated oligosaccharides, N-acetylglucosamine containing non-fucosylated oligosaccharides, lacto-N- biose containing sialylated oligosaccharides, lacto-N-biose containing neutral (non-charged) oligosaccharides, lacto-N-biose containing negatively charged oligosaccharides, lacto-N-biose containing fucosylated oligosaccharides, lacto-N-biose containing non-fucosylated oligosaccharides, N- acetyllactosamine containing sialylated oligosaccharides, N-acetyllactosamine containing neutral (noncharged) oligosaccharides, N-acetyllactosamine containing negatively charged oligosaccharides, N- acetyllactosamine containing fucosylated oligosaccharides, N-acetyllactosamine containing non- fucosylated oligosaccharides, chitosan, chitosan comprising oligosaccharide, heparosan, chondroitin sulphate, glycosaminoglycan oligosaccharide, heparin, heparan sulphate, dermatan sulphate, hyaluronan, hyaluronic acid, keratan sulphate, erlose (Glc-al,4-Glc-otl,2-Fru), lactul-N-triose II (GlcNAc-pi,3-Gal-pi,4- Fru), lactul-N-tetraose, lactul-N-neotetraose and globotriose.
The terms "negatively charged oligosaccharide" or "acidic oligosaccharide" are used interchangeably and refer to an oligosaccharide with a negative charge. In a preferred embodiment, the negatively charged oligosaccharide is a sialylated oligosaccharide. As used herein, a 'sialylated oligosaccharide' is to be understood as a negatively charged sialic acid containing oligosaccharide, i.e., an oligosaccharide having one or more sialic acid residue(s). It has an acidic nature. Some examples are 3'SL (3'-sialyllactose, Neu5Ac-a2,3-Gal-pi,4-Glc), 3'-sialyllactosamine, 6'SL (6'sialyllactose, Neu5Ac-a2,6-Gal-pi,4-Glc), 8'SL (8'sialyllactose, Neu5Ac-ot2,8-Gal-pi,4-Glc), 3,6-disialyllactose (Neu5Ac-a2,3-(Neu5Ac-a2,6)-Gal-pi,4- Glc), 6,6'-disialyllactose (Neu5Ac-oc2,6-Gal-pi,4-(Neu5Ac-a2,6)-Glc), 8,3-disialyllactose (Neu5Ac-oc2,8- Neu5Ac-a2,3-Gal-pi,4-Glc), 6'-sialyllactosamine, 3'-sialyllactosamine, oligosaccharides comprising 6'sialyllactose, SGG hexasaccharide (Neu5Aca-2,3Gaip-l,3GalNacp-l,3Gala-l,4Gaip-l,4Gal), sialylated tetrasaccharide, sialylated pentasaccharide, sialylated lacto-N-triose, sialylated lacto-N-tetraose, sialyllacto-N-neotetraose, LSTc (Neu5Ac-cx2,6-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), LSTd (Neu5Ac-a2,3- Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a (LSTa, Neu5Ac-oc2,3-Gal-pi,3- GlcNAc-pi,3-Gal-pi,4-Glc), disialyllacto-N-hexaose I, sialyllacto-N-tetraose b (LSTb, Gal- i,3-(Neu5Ac- a2,6)-GlcNAc-pi,3-Gal-pi,4-Glc), 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyl lacto-N- fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose, Neu5Ac-a2,3-Gal- bl,4-GlcNAc-bl,3-Gal, Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'- KDO-6'sialyllactose, 3’KDO-8-sialyllactose, KDO-2,3Gaip-l,3GalNacp-l,3Gala-l,4Gaip-l,4Gal, KDO- 2,3Gaip-l,3GlcNacP-l,3Gaip-l,4Glc, KDO-2,3Gaip-l,4GlcNacP-l,3Gaip-l,4Glc, 3'-KDO-3-fucosyllactose, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal, 3'-Sialyl-2'-fucosyllactose, 6'-Sialyl-2'- fucosyllactose, 6'-Sialyl-3-fucosyllactose, 3'-Sialyl-3-fucosyllactose, Neu5Ac-a2,6-(Neu5Ac-a2,3-)Gal-bl,4- Glc, 3'-Sialyl-3-fucosyllactosamine, Fuc-al,4-(Neu5Ac-a2,3-Gal-bl,3-)GlcNAc, 6'-Sialyllacto-N-biose, 3'- Sialyllacto-N-biose, Neu5Ac-a2,6-(GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,4-(Fuc-al,3-)GlcNAc- bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,3-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac- a2,6-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,4-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,6-Gal-bl,3-GlcNAc-bl,3-Gal- bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac-a2,3-(Fuc-al,2- )Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Fuc-al,2-Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc, Fuc-al,4-(Neu5Ac-a2,3-Gal-bl,3-)GlcNAc- bl,3-Gal-bl,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-(Fuc-al,3-)Glc, Neu5Ac- a2,6-(Neu5Ac-a2,6-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc, Neu5Ac-a2,6-(Gal-bl,3-GlcNAc-bl,3- )Gal-bl,4-Glc, Neu5Ac-a2,6-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc and oligosaccharides bearing one or several sialic acid residue(s), including but not limited to: oligosaccharide moieties of the gangliosides selected from GM3 (3'sialyl lactose, Neu5Aca-2,3Gaip-4Glc) and oligosaccharides comprising the GM3 motif, GD3 Neu5Aca-2,8Neu5Aca-2,3Gaip-l,4Glc GT3 (Neu5Aca-2,8Neu5Aca-2,8Neu5Aca-2,3Gaip- l,4Glc); GM2 GalNAcP-l,4(Neu5Aca-2,3)Gaip-l,4Glc, GM1 Gaip-l,3GalNAcP-l,4(Neu5Aca-2,3)Gaip- l,4Glc, GDla Neu5Aca-2,3Gaip-l,3GalNAcP-l,4(Neu5Aca-2,3)Gaip-l,4Glc, GTla Neu5Aca-2,8Neu5Aca- 2,3Gaip-l,3GalNAcP-l,4(Neu5Aca-2,3)Gaip-l,4Glc, GD2 GalNAcP-l,4(Neu5Aca-2,8Neu5Aca2,3)Gaip- l,4Glc, GT2 GalNAc -l,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GDlb, Gaip-l,3GalNAcp- !,4(Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GTlb Neu5Aca-2,3Gaip-l,3GalNAcP-l,4(Neu5Aca-
2,8Neu5Aca2,3)Gaip-l,4Glc, GQlb Neu5Aca-2,8Neu5Aca-2,3Gaip-l,3GalNAc P -l,4(Neu5Aca-
2,8Neu5Aca2,3)Gaip-l,4Glc, GTlc Gaip-l,3GalNAcP-l,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Gaip- l,4Glc, GQ.1C Neu5Aca-2,3Gaip-l,3GalNAc -l,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Gaip-l,4Glc, GPlc Neu5Aca-2,8Neu5Aca-2,3Gaip-l,3GalNAc -l,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Gaip- l,4Glc, GDla Neu5Aca-2,3Gaip-l,3(Neu5Aca-2,6)GalNAcP -l,4Gaip-l,4Glc, Fucosyl-GMl Fuca-l,2Gaip- l,3GalNAcP -l,4(Neu5Aca-2,3)Gal -l,4Glc; all of which may be extended to the production of the corresponding gangliosides by reacting the above oligosaccharide moieties with ceramide or synthetizing the above oligosaccharides on a ceramide.
"Charged oligosaccharides" are oligosaccharide structures that contain one or more negatively charged monosaccharide subunits including N-acetylneuraminic acid (Neu5Ac), commonly known as sialic acid, N- glycolylneuraminic acid (Neu5Gc), glucuronate, galacturonate and 2-keto-3-deoxymanno-octulonic acid (KDO). Charged oligosaccharides are also referred to as acidic oligosaccharides. In contrast, neutral (noncharged) oligosaccharides are non-sialylated oligosaccharides, and thus do not contain an acidic monosaccharide subunit. Neutral oligosaccharides comprise non-charged fucosylated oligosaccharides that contain one or more fucose subunits in their glycan structure as well as non-charged non-fucosylated oligosaccharides that lack any fucose subunit. Other examples of charged oligosaccharides are sulphated chitosans and deacetylated chitosans.
The terms 'neutral oligosaccharide' and 'non-charged' oligosaccharide as used herein are used interchangeably and refer, as generally understood in the state of the art, to an oligosaccharide that has no negative charge originating from a carboxylic acid group. Examples of such neutral oligosaccharide are 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyl lactose (6FL), 2', 3- difucosyllactose (diFL), lacto-N-triose II (LN3, GlcNAc i-3Gaipi-4Glc), lacto-N-tetraose (LNT, Gaipi- 3GlcNAc i-3Gaipi-4Glc), lacto-N-neotetraose (LNnT, Gaipi-4GlcNAcpi-3Gaipi-4Glc), lacto-N- fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N- difucohexaose II, Fuc-al,2-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,2-Gal-bl,4-GlcNAc-bl,3- Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,2-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-Glc, Gal-bl,4-(Fuc-al,3- )GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,2-Gal-bl,4-(Fuc-al,3-)GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, Fuc-al,4-(Fuc-al,2-Gal-bl,3-)GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, monofucosyllacto-N-hexaose-lll, difucosyllacto-N-hexaose (a), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N- neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, difucosyl-lacto-N-hexaose, difucosyl-lacto- N-neohexaose, trifucosyllacto-N-hexaose, al,3-galactosyl-3-fucosyllactose, Gal-al,3-(Fuc-al,2-)Gal-bl,4- (Fuc-al,3-)Glc, GalNAc-al,3-(Fuc-al,2-)Gal-bl,4-(Fuc-al,3-)Glc, 2-fucosyllactulose, 3-fucosyl-N- acetyllactosamine, 2'-fucosyl-N-acetyllactosamine, difucosyl-N-acetyllactosamine, 4-fucosyllacto-N- biose, 2'-fucosyllacto-N-biose, difucosyllacto-N-biose and GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3-)Glc, GIcNAc- bl,6-(GlcNAc-bl,3-)Gal-bl,4-Glc, lacto-N-pentaose (LN5), lacto-N-neopentaose, para lacto-N-pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N-heptaose (LN7), lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N-neooctaose, iso lacto-N- octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N- neooctaose (pLNnO), iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose (LN9), lacto-N- decaose, iso lacto-N-decaose, novo lacto-N-decaose, lacto-N-neodecaose, para lacto-N-neodecaose (pLNnD), al,3-galactosyllacto-N-neotetraose, GlcNAc-bl,3-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc, GIcNAc- bl,6-(Gal-bl,4-GlcNAc-bl,3-)Gal-bl,4-Glc and GlcNAc-bl,6-(Gal-bl,3-GlcNAc-bl,3-)Gal-bl,4-Glc.
A 'fucosylated oligosaccharide' as used herein and as generally understood in the state of the art is an oligosaccharide that is carrying a fucose-residue. Such fucosylated oligosaccharide is a saccharide structure comprising at least three monosaccharide subunits linked to each other via glycosidic bonds, wherein at least one of said monosaccharide subunit is a fucose. A fucosylated oligosaccharide can contain more than one fucose residue, e.g., two, three or more. A fucosylated oligosaccharide can be a neutral oligosaccharide or a charged oligosaccharide e.g., also comprising sialic acid structures. Fucose can be linked to other monosaccharide subunits comprising glucose, galactose, GIcNAc via alpha-glycosidic bonds comprising alpha-1,2 alpha-1,3, alpha-1,4, alpha-1,6 linkages. Examples comprise 2'-fucosyl lactose (2' FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL), Lacto-N- fucopentaose I (LNFP I), Gal-al,3-(Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc (Gal-LNFP I), GalNAc-al,3- (Fuc-al,2-)Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc (GalNAc-LNFP I), Lacto-N-fucopentaose II (LNFP II), Lacto- N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), lacto-N-fucopentaose VI (LNFP VI), lacto- N-neofucopentaose I, lacto-N-difucohexaose I (LDFH I), lacto-N-difucohexaose II (LDFH II), Monofucosyllacto-N-hexaose III (MFLNH III), difucosyllacto-N-hexaose (a), Difucosyllacto-N-hexaose, difucosyl-lacto-N-neohexaose, trifucosyllacto-N-hexaose, al,3-galactosyl-3-fucosyllactose, Gal-al,3-(Fuc- al,2-)Gal-bl,4-(Fuc-al,3-)Glc, GalNAc-al,3-(Fuc-al,2-)Gal-bl,4-(Fuc-al,3-)Glc, 2-fucosyllactulose, 3- fucosyl-N-acetyllactosamine, 2'-fucosyl-N-acetyllactosamine, difucosyl-N-acetyllactosamine, 4- fucosyllacto-N-biose, 2'-fucosyllacto-N-biose, difucosyllacto-N-biose and GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3- )Glc, 3'-Sialyl-2'-fucosyllactose, 6'-Sialyl-2'-fucosyllactose, 6'-Sialyl-3-fucosyllactose, 3'-sialyl-3- fucosyllactose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II, monofucosyldisialyllacto-N-tetraose.
Mammalian milk oligosaccharides comprise oligosaccharides present in milk found in any phase during lactation including colostrum milk from humans and mammals including but not limited to cows (Bos Taurus), sheep (Ovis aries), goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus), horses (Equus ferus caballus), pigs (Sus scropha), dogs (Canis lupus familiaris), ezo brown bears (Ursus arctos yesoensis), polar bear (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephant (Loxodonta africana), giant anteater (Myrmecophaga tridactyla), common bottlenose dolphins (Tursiops truncates), northern minke whales (Balaenoptera acutorostrata), tammar wallabies (Macropus eugenii), red kangaroos (Macropus rufus), common brushtail possum (Trichosurus Vulpecula), koalas (Phascolarctos cinereus), eastern quolls (Dasyurus viverrinus), platypus (Ornithorhynchus anatinus). As used herein, "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, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto- N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, lacto-N- difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosylmonosialyllacto-N-tetraose c, monofucosyl para-lacto-N-hexaose, monofucosyllacto-N- hexaose III, isomeric fucosylated lacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose I, sialyllacto- N-hexaose, sialyllacto-N-neohexaose II, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, difucosyllacto-N-hexaose a, difucosyllacto-N-hexaose c, galactosylated chitosan, fucosylated oligosaccharides, neutral oligosaccharides and/or sialylated oligosaccharides.
The terms "human milk oligosaccharide" or "HMO" refer to oligosaccharides found in human breast milk, including preterm human milk, colostrum and term human milk. HMOs comprise fucosylated oligosaccharides, non-fucosylated neutral oligosaccharides and sialylated oligosaccharides. Examples of HMOs comprise 3-fucosyllactose, 2'-fucosyllactose, 2',3-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, LN3, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N- fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose c, sialyllacto-N- tetraose b, sialyllacto-N-tetraose a, difucosyllacto-N-tetraose, lacto-N-hexaose, lacto-N-difucohexaose I, lacto-N-difucohexaose II, disialyllacto-N-tetraose, fucosyllacto-N-hexaose, difucosyllacto-N-hexaose, fucodisialyllacto-N-hexaose, disialyllacto-N-hexaose.
The terms "N-acetylglucosamine 1-phosphate uridylyltransferase", "N-acetylglucosamine-l-phosphate uridyltransferase", "UDP-N-acetylglucosamine diphosphorylase", "UDP-N-acetylglucosamine pyrophosphorylase", "uridine diphosphoacetylglucosamine pyrophosphorylase", "UTP:2-acetamido-2- deoxy-alpha-D-glucose-l-phosphate uridylyltransferase", "UDP-GIcNAc pyrophosphorylase”, "GlmU uridylyltransferase", "Acetylglucosamine 1-phosphate uridylyltransferase", "UDP-acetylglucosamine pyrophosphorylase", "uridine diphosphate-N-acetylglucosamine pyrophosphorylase", "uridine diphosphoacetylglucosamine phosphorylase", and "acetylglucosamine 1-phosphate uridylyltransferase" are used interchangeably and refer to an enzyme that catalyses the conversion of N-acetylglucosamine 1- phosphate (GlcNAc-1-P) into UDP-N-acetylglucosamine (UDP-GIcNAc) by the transfer of uridine 5- monophosphate (from uridine 5-triphosphate (UTP)).
The term glucosamine-l-phosphate acetyltransferase refers to an enzyme that catalyses the transfer of the acetyl group from acetyl coenzyme A to glucosamine-l-phosphate (GlcN-1-P) to produce N- acetylglucosamine-l-phosphate (GlcNAc-1-P).
The term "glmU" refers to a bifunctional enzyme that has both N-acetylglucosamine-l-phosphate uridyltransferase and glucosamine-l-phosphate acetyltransferase activity and that catalyses two sequential reactions in the de novo biosynthetic pathway for UDP-GIcNAc. The C-terminal domain catalyses the transfer of acetyl group from acetyl coenzyme A to GlcN-1-P to produce GlcNAc-1-P, which is converted into UDP-GIcNAc by the transfer of uridine 5-monophosphate, a reaction catalysed by the N- terminal domain.
The term "bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase" as used herein refers to a polypeptide comprising both N-acetylglucosamine-1- phosphate uridyltransferase activity as well as glucosamine-l-phosphate acetyltransferase activity.
The terms "L-glutamine— D-fructose-6-phosphate aminotransferase", "glutamine — fructose-6-phosphate transaminase (isomerizing)", "hexosephosphate aminotransferase", "glucosamine-6-phosphate isomerase (glutamine-forming)", "glutamine-fructose-6-phosphate transaminase (isomerizing)", "D- fructose-6-phosphate amidotransferase", "fructose-5-phosphate aminotransferase", "glucosaminephosphate isomerase", "glucosamine 6-phosphate synthase", "GlcN6P synthase", "GFA", "glms", "glmS" and "glmS*54" are used interchangeably and refer to an enzyme that catalyses the conversion of D-fructose-6-phosphate into D-glucosamine-6-phosphate using L-glutamine.
The term "pathway for production of a disaccharide" as used herein is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of a disaccharide as defined herein. Said pathway for production of a disaccharide 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 disaccharide of the present invention. The term "pathway for production of an oligosaccharide" as used herein is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of an oligosaccharide as defined herein, like e.g., a milk oligosaccharide. Said pathway for production of an oligosaccharide like e.g., a milk oligosaccharide can comprise but is not limited to pathways involved in the synthesis of a nucleotide-activated sugar and the transfer of said nucleotide-activated sugar to an acceptor to create an oligosaccharide of the present invention, like e.g., a milk oligosaccharide. The term "pathway for production of a disaccharide and a milk oligosaccharide" as used herein is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of a disaccharide and a milk oligosaccharide as defined herein. Said pathway for production of a disaccharide and a milk oligosaccharide can comprise but is not limited to pathways involved in the synthesis of a nucleotide-activated sugar and the transfer of said nucleotide-activated sugar to an acceptor to create a disaccharide and a milk oligosaccharide of the present invention. Examples of such pathways comprise but are not limited to a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway and an N-acetylmannosaminylation pathway.
Said pathway for production of a disaccharide may comprise a pathway for synthesis and/or import of a co-factor used in said pathway for production of said disaccharide.
Said pathway for production of an oligosaccharide like a milk oligosaccharide may comprise a pathway for synthesis and/or import of a co-factor used in said pathway for production of said oligosaccharide like said milk oligosaccharide.
Said pathway for production of a disaccharide and a milk oligosaccharide may comprise a pathway for synthesis and/or import of a co-factor used in said pathway for production of said disaccharide and a milk oligosaccharide.
The term "purified" refers to material that is substantially or essentially free from components that interfere with the activity of the biological molecule. For cells, saccharides, nucleic acids, and polypeptides, the term "purified" refers to material that is substantially or essentially free from components that normally accompany the material as found in its native state. Typically, purified saccharides, oligosaccharides, proteins or nucleic acids of the invention are at least about 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 % or 85 % pure, usually at least about 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99.0 % pure as measured by band intensity on a silver-stained gel or other method for determining purity. Purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized. For di- and oligosaccharides, purity can be determined using methods such as but not limited to thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis or mass spectroscopy. Further herein, the terms "contaminants" and "impurities" preferably mean particulates, cells, cell components, metabolites, cell debris, proteins, peptides, amino acids, nucleic acids, glycolipids and/or endotoxins which can be present in an aqueous medium like e.g., a cultivation or an incubation.
The term "clarifying" as used herein refers to the act of treating an aqueous medium like e.g., a cultivation, 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 disaccharide and/or oligosaccharide, like e.g., a milk oligosaccharide. Such treatment can be carried out in a conventional manner by centrifugation, flocculation, flocculation with optional ultrasonic treatment, gravity filtration, microfiltration, foam separation or vacuum filtration (e.g., through a ceramic filter which can include a Celite™ filter aid).
The term "cultivation" refers to the culture medium wherein the cell is cultivated, or fermented, the cell itself, and a disaccharide and/or oligosaccharide like a milk oligosaccharide or an oligosaccharide mixture like a milk oligosaccharide mixture that is produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell. The terms "culture medium" and "cultivation medium" as used herein are used interchangeably and refer to the medium wherein the cell is cultivated.
The term "incubation" refers to a mixture wherein a disaccharide and/or an oligosaccharide like a milk oligosaccharide or an oligosaccharide mixture like a milk oligosaccharide mixture 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 i) a disaccharide and/or an oligosaccharide like a milk oligosaccharide or ii) an oligosaccharide mixture like a milk oligosaccharide mixture, 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 disaccharide and/or an oligosaccharide, like a milk oligosaccharide, or an oligosaccharide mixture like a milk oligosaccharide mixture that is produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell. Said incubation can also be the cultivation as defined herein. The terms "reactor" and "incubator" refer to the recipient filled with the cultivation or incubation. Examples of reactors and incubators comprise but are not limited to microfluidic devices, well plates, tubes, shake flasks, fermenters, bioreactors, process vessels, cell culture incubators, CO2 incubators.
As used herein, the term "cell productivity index (CPI)" refers to the mass of the disaccharide produced by the cells divided by the mass of the cells produced in the culture. The term "cell productivity index (CPI)" also refers to the mass of the oligosaccharide like the milk oligosaccharide produced by the cells divided by the mass of the cells produced in the culture. The term "CPI" as used herein is also to be understood as mass of the mixture of a disaccharide and a milk oligosaccharide produced by the cells divided by the mass of the cells produced in the culture. The term "CPI" as used herein is also to be understood as mass of the oligosaccharide mixture like a milk oligosaccharide mixture produced by the cells divided by the mass of the cells produced in the culture.
As used herein, the term "mammary cell(s)" generally refers to mammalian mammary epithelial cell(s), mammalian mammary-epithelial luminal cell(s), or mammalian epithelial alveolar cell(s), or any combination thereof. As used herein, the term "mammary-like cell(s)" generally refers to mammalian cell(s) having a phenotype/genotype similar (or substantially similar) to natural mammalian mammary cell(s) but is/are derived from mammalian non-mammary cell source(s). Such mammalian mammary-like cell(s) may be engineered to remove at least one undesired genetic component and/or to include at least one predetermined genetic construct that is typical of a mammalian mammary cell. Non-limiting examples of mammalian mammary-like cell (s) may include mammalian mammary epithelial-like cell(s), mammalian mammary epithelial luminal-like cell(s), mammalian non-mammary cell(s) that exhibits one or more characteristics of a cell of a mammalian mammary cell lineage, or any combination thereof. Further nonlimiting examples of mammalian mammary-like cell(s) may include mammalian cell(s) having a phenotype similar (or substantially similar) to natural mammalian mammary cell (s), or more particularly a phenotype similar (or substantially similar) to natural mammalian mammary epithelial cell(s). A mammalian cell with a phenotype or that exhibits at least one characteristic similar to (or substantially similar to) a natural mammalian mammary cell or a mammalian mammary epithelial cell may comprise a mammalian cell (e.g., derived from a mammary cell lineage or a non-mammary cell lineage) that exhibits either naturally, or has been engineered to, be capable of expressing at least one milk component. As used herein, the term "non- mammary cell(s)" may generally include any mammalian cell of non-mammary lineage. In the context of the invention, a non-mammary cell can be any mammalian cell capable of being engineered to express at least one milk component. Non-limiting examples of such non-mammary cell(s) include hepatocyte(s), blood cell(s), kidney cell(s), cord blood cell(s), epithelial cell(s), epidermal cell(s), myocyte(s), fibroblast(s), mesenchymal cell(s), or any combination thereof. In some instances, molecular biology and genome editing techniques can be engineered to eliminate, silence, or attenuate myriad genes simultaneously. The term "precursor" as used herein refers to substances which are taken up or synthetized by the cell for the specific production of a disaccharide and/or an oligosaccharide, like a milk oligosaccharide, or an oligosaccharide mixture like a milk oligosaccharide mixture according to the present invention. In this sense a precursor can be an acceptor as defined herein, but can also be another substance, metabolite, co-factor, which is first modified within the cell as part of the biochemical synthesis route of a disaccharide and/or oligosaccharide, like a milk oligosaccharide or an oligosaccharide mixture like a milk oligosaccharide mixture. The term "precursor" as used herein is also to be understood as a chemical compound that participates in an incubation or an enzymatic reaction to produce another compound like e.g., an intermediate or an acceptor as defined herein, as part in the metabolic pathway of a disaccharide and/or oligosaccharide like a milk oligosaccharide or an oligosaccharide mixture like a milk oligosaccharide mixture. The term "precursor" as used herein is also to be understood as a donor that is used by a glycosyltransferase to modify an acceptor as defined herein with a sugar moiety in a glycosidic bond, as part in the metabolic pathway of a disaccharide and/or oligosaccharide like a milk oligosaccharide or an oligosaccharide mixture like a milk oligosaccharide mixture. Examples of such precursors comprise the acceptors as defined herein, and/or dihydroxyacetone, glucosamine, N-acetylglucosamine, N- acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyllactose, phosphorylated sugars or sugar phosphates like e.g. but not limited to glucose-l-phosphate, galactose-l-phosphate, glucose-6- phosphate, fructose-6-phosphate, fructose-l,6-bisphosphate, mannose-6-phosphate, mannose-1- phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N- acetylglucosamine-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.
Optionally, the cell used to produce the saccharide, like a disaccharide and/or a milk oligosaccharide is transformed to comprise and to express at least one nucleic acid sequence encoding a protein selected from the group consisting of lactose transporter, N-acetylneuraminic acid transporter, fucose transporter, glucose transporter, galactose transporter, transporter for a nucleotide-activated sugar wherein said transporter internalizes a to the medium added precursor for the synthesis of the saccharide like the disaccharide and/or milk oligosaccharide of present invention. The term "acceptor" as used herein refers to a mono-, di- or oligosaccharide, which can be modified by a glycosyltransferase. Examples of such acceptors comprise glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, lactulose, lactobionic acid (LBA), lacto-N-triose, lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-pentaose (LN P), lacto-N-neopentaose, para lacto-N- pentaose, para lacto-N-neopentaose, lacto-N-novopentaose I, lacto-N-hexaose (LNH), lacto-N- neohexaose (LNnH), para lacto-N-neohexaose (pLNnH), para lacto-N-hexaose (pLNH), lacto-N-heptaose, lacto-N-neoheptaose, para lacto-N-neoheptaose, para lacto-N-heptaose, lacto-N-octaose (LNO), lacto-N- neooctaose, iso lacto-N-octaose, para lacto-N-octaose, iso lacto-N-neooctaose, novo lacto-N-neooctaose, para lacto-N-neooctaose, iso lacto-N-nonaose, novo lacto-N-nonaose, lacto-N-nonaose, lacto-N-decaose, iso lacto-N-decaose, novo lacto-N-decaose, lacto-N-neodecaose, and oligosaccharide containing 1 or more N-acetyllactosamine units and/or 1 or more lacto-N-biose units or an intermediate into oligosaccharide, fucosylated and sialylated versions thereof, ceramide, N-acylated sphingoid, glucosylceramide, lactosylceramide, sphingosine, phytosphingosine, sphingosine synthons, peptide backbones with beta-GIcNAc-Asn residues, glycoproteins with terminal GIcNAc and Gal residues, immunoglobulins.
Detailed description of the invention
According to a first aspect, the present invention provides a cell capable of synthesizing and/or synthesizing UDP-N-acetylglucosamine (UDP-GIcNAc) and genetically engineered for the production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide, wherein the cell comprises a pathway for production of said disaccharide and/or oligosaccharide like a milk oligosaccharide, characterized in that UDP-GIcNAc synthesis in said cell is rendered less functional.
According to a second aspect, the present invention provides a method for the production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide, wherein the method comprises cultivating and/or incubating a cell as described herein, in cultivation and/or incubation medium under conditions permissive to produce a disaccharide and/or oligosaccharide like a milk oligosaccharide.
In the scope of the present invention, permissive conditions are understood to be conditions relating to physical or chemical parameters including but not limited to temperature, pH, pressure, osmotic pressure and product/precursor/acceptor concentration.
In a particular embodiment, the permissive conditions may include a temperature-range of 30 +/- 20 degrees centigrade, a pH-range of 7 +/- 3.
In a preferred embodiment, the disaccharide and/or oligosaccharide like a milk oligosaccharide is/are separated from said cultivation and/or incubation. In another and/or additional preferred embodiment, the disaccharide and/or oligosaccharide like a milk oligosaccharide is/are purified. In a specific embodiment of the method and/or cell of present invention, the cell is capable of synthesizing and/or synthesizing UDP-GIcNAc. UDP-GIcNAc can be provided by an enzyme expressed in the cell or by the metabolism of the cell. UDP-GIcNAc is formed from fructose-6-phophate (Fru-6-P) in a four-step version of the Leloir pathway. In eukaryotic cells the four successive reactions comprise: (a) conversion of fructose-6-P (Fru-6-P) into glucosamine-6-phosphate (GlcN-6-P); (b) acetylation of GlcN-6-P to GlcNAc-6- P; (c) isomerization of GlcNAc-6-P to GlcNAc-1-P; and (d) uridylation of GlcNAc-1-P to give UDPGIcNAc In prokaryotic cells, the first and the last step are essentially the same as in eukaryotes, but GlcN-6-P is first isomerized to give GlcN-1-P, which is subsequently N-acetylated. Each step of the eukaryotic pathway is catalysed by a separate enzyme, while in bacteria there are only three enzymatic proteins involved in UDP-GIcNAc biosynthesis. These enzymes may be any one or more of the list comprising an N-acetyl-D- glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, and an N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase.
In a preferred embodiment of the method and/or cell, the cell synthesizes UDP-GIcNAc. In a more preferred embodiment, the cell comprises a pathway for production of UDP-GIcNAc. Said pathway for production of UDP-GIcNAc consists of the enzymes and their respective genes involved in the synthesis of UDP-GIcNAc. Enzymes involved in the synthesis of UDP-GIcNAc comprise but are not limited to bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, an N-acetylglucosamine-l-phosphate uridyltransferase and a glucosamine-1- phosphate acetyltransferase. Synthesis of UDP-GIcNAc may make use of one or more co-factor(s). Examples of co-factors comprise but are not limited to Mg2+, Co2+, Mn2+, Ca2+, Zn2+, Ni2+ and FAD. Said pathway for production of UDP-GIcNAc may comprise a pathway for synthesis and/or for import of a cofactor used in a pathway for production of UDP-GIcNAc. In other words, the cell of present invention may comprise a pathway for the production and/or import of any one or more Mg2+, Co2+, Mn2+, Ca2+, Zn2+, Ni2+ and FAD. Alternatively, the cell of present invention does not synthesize a co-factor that is necessary in the synthesis of UDP-GIcNAc but has all other enzymes necessary for UDP-GIcNAc synthesis. Said cell may synthesize UDP-GIcNAc upon supplementation with one or more co-factor(s).
In an additional specific embodiment, UDP-GIcNAc synthesis in the cell is rendered less functional. Herein, a cell wherein UDP-GIcNAc synthesis is rendered less functional is to be understood as that said cell has lower production of UDP-GIcNAc compared to a cell wherein UDP-GIcNAc synthesis is not rendered less functional. According to present invention, synthesis of UDP-GIcNAc is rendered less functional in a cell by making one or more genes involved in the pathway for production of UDP-GIcNAc less functional. Alternatively, and/or additionally, synthesis of UDP-GIcNAc is rendered less functional in a cell by making one or more genes involved in the pathway for production and/or the import of one or more co-factor(s) that are used in the synthesis of UDP-GIcNAc less functional. Preferably, synthesis of UDP-GIcNAc is rendered less functional in a cell by making one or more genes involved in the pathway for production and/or the import of one or more co-factor(s) selected from the list comprising Mg2+, Co2+, Mn2+, Ca2+, Zn2+, Ni2+ and FAD less functional. Rendering a gene less functional is to be understood as rendering a gene 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 a functional final product. A gene can be made less functional by means of common well-known technologies for a skilled person, by e.g., any one or more of insertion, deletion and/or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) selected from the list comprising promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said gene so that said gene is made less-able to produce a functional final product. Methods like e.g. siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutating genes and transposon mutagenesis could be used herein. Alternatively, and/or additionally, synthesis of UDP-GIcNAc is rendered less functional in a cell by replacement of the native pathway for UDP-GIcNAc production that is present in a cell by another pathway for UDP-GIcNAc production that gives lower UDP-GIcNAc production compared to the cell's native UDP-GIcNAc production pathway.
In a preferred embodiment of the method and/or cell of present invention, UDP-GIcNAc synthesis possesses at least one gene selected from the list comprising genes encoding bifunctional N- acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, N- acetylglucosamine-l-phosphate uridyltransferase and glucosamine-l-phosphate acetyltransferase, and wherein said at least one gene is rendered less functional as described herein. In other words, UDP-GIcNAc synthesis is obtained by expression of at least one gene selected from the list comprising genes encoding bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, N-acetylglucosamine-l-phosphate uridyltransferase and glucosamine-l-phosphate acetyltransferase, and wherein said at least one gene is rendered less functional as described herein. In another preferred embodiment of the method and/or cell of present invention, at least one gene involved in the synthesis and/or import of a co-factor that is involved in UDP-GIcNAc synthesis is rendered less functional or knocked out, preferably said co-factor is selected from the list comprising Mg2+, Co2+, Mn2+, Ca2+, Zn2+, Ni2+ and FAD. In a more preferred embodiment, at least one of said genes involved in UDP- GIcNAc synthesis and/or involved in the synthesis and/or import of a co-factor that is involved in UDP- GIcNAc synthesis is rendered less functional by insertion, deletion and/or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) selected from the list comprising promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said at least one gene. It is to be understood herein that by rendering at least one of said genes less functional in the cell renders the cell with a less functional synthesis of UDP-GIcNAc. As a result, the cell produces less UDP-GIcNAc compared to a cell wherein no one of said genes is rendered less functional. The term "less UDP-GIcNAc" is, however, not to be understood to comprise 0 g/L UDP-GIcNAc since then the cell is no longer viable. In a more preferred embodiment, UDP-GIcNAc synthesis is obtained by expression of at least two genes selected from the list comprising genes encoding bifunctional N- acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, N- acetylglucosamine-l-phosphate uridyltransferase and glucosamine-l-phosphate acetyltransferase, and wherein i) at least one of said at least two genes is rendered less functional, ii) at least two of said at least two genes is rendered less functional or iii) all of said at least two genes are rendered less functional, as described herein.
In another and/or additional more preferred embodiment of the method and/or cell of present invention, the at least one gene encodes an enzyme wherein the enzyme is selected from an enzyme class selected from the list comprising EC:2.7.7.23, EC:2.3.1.157 and EC:5.4.2.3.
In another and/or additional more preferred embodiment of the method and/or cell of present invention, the at least one gene encodes an enzyme wherein the enzyme comprises a polypeptide sequence comprising an IPR domain selected from the list comprising IPR001451, IPR002618, IPR005175, IPR005835, IPR005843, IPR005844, IPR005882, IPR011004, IPR016055, IPR016066, IPR016657, IPR018357, IPR023915, IPR025877, IPR029044, IPR036900 and IPR038009 as defined by InterPro 90.0 as released on 4th August 2022.
In another and/or additional more preferred embodiment of the method and/or cell of present invention, the at least one gene encodes an enzyme wherein the enzyme comprises a polypeptide sequence comprising a PFAM domain selected from the list comprising PF00132, PF00408, PF00465, PF00483, PF01070, PF01565, PF01704, PF02878, PF02879, PF02880, PF03479, PF04030, PF05199, PF12146, PF12804, PF13562 and PF14602 as defined by InterPro 90.0 as released on 4th August 2022.
In another and/or additional more preferred embodiment of the method and/or cell of present invention, the at least one gene encodes an enzyme wherein the enzyme comprises a polypeptide sequence comprising the conserved protein domain selected from the list comprising cd03086 and cd03353 as defined by InterPro 90.0 as released on 4th August 2022.
In another and/or additional more preferred embodiment of the method and/or cell of present invention, the at least one gene encodes an enzyme wherein the enzyme is part of a NOG family selected from the list comprising COG1109 and COG4284 as defined by eggNOG5.0 as released in 2019.
In another and/or additional more preferred embodiment of the method and/or cell of present invention, the at least one gene encodes an enzyme wherein the enzyme uses a cofactor selected from the list comprising Mg2+, Co2+, Mn2+, Ca2+, Zn2+, Ni2+ and FAD. In an even more preferred embodiment, the at least one gene encodes an enzyme wherein the enzyme is selected from the enzyme class EC:2.3.1.157, comprises a polypeptide sequence comprising the IPR domains IPR001451, IPR005175, IPR005882, IPR011004, IPR018357, IPR025877, IPR029044 and IPR038009 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising the PFAM domains PF00132, PF00465, PF00483, PF01070, PF01565, PF03479, PF04030, PF05199, PF12804, PF13562 and PF14602 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising the conserved protein domain cd03353 as defined by InterPro 90.0 as released on 4th August 2022, and is part of the NOG family COG4284 as defined by eggNOG5.0 as released in 2019 and uses a cofactor selected from the list comprising Mg2+, Co2+, Mn2+, Ca2+ and Zn2+.
In another even more preferred embodiment, the at least one gene encodes an enzyme wherein the enzyme is selected from the enzyme class EC:5.4.2.3, comprises a polypeptide sequence comprising the IPR domains IPR005843, IPR005844, IPR016055, IPR016066, IPR016657 and IPR036900 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising the PFAM domains PF00408, PF02878, PF02879 and PF02880 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising the conserved protein domain cd03086 as defined by InterPro 90.0 as released on 4th August 2022, and is part of the NOG family COG1109 as defined by eggNOG5.0 as released in 2019 and uses a cofactor selected from the list comprising Mg2+, Ni2+ and FAD.
In another even more preferred embodiment, the at least one gene encodes an enzyme wherein the enzyme is selected from the enzyme class EC:2.7.7.23, comprises a polypeptide sequence comprising the IPR domains IPR001451, IPR002618, IPR005175, IPR005835, IPR005882, IPR011004, IPR018357, IPR023915, IPR025877, IPR029044, IPR038009 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising the PFAM domains PF00132, PF00483, PF01704, PF03479, PF12146, PF12804, PF13562 and PF14602 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising the conserved protein domain cd03353 as defined by InterPro 90.0 as released on 4th August 2022, and is part of the NOG family COG4284 as defined by eggNOG5.0 as released in 2019 and uses a cofactor selected from the list comprising Mg2+, Co2+, Mn2+, Ca2+, Zn2+.
In an additional and/or alternative specific embodiment, the cell is genetically engineered for production of a disaccharide, wherein the cell comprises a pathway for production of said disaccharide. In a preferred embodiment, the cell is genetically engineered for production of two or more disaccharides. In another and/or additional preferred embodiment, the cell is genetically engineered for an enhanced production of a disaccharide, an enhanced uptake of one or more precursor(s) and/or acceptor(s) that is/are used in the synthesis of a disaccharide, a better efflux of the disaccharide, a decreased production of by-products like e.g. acids, an increased availability of co-factors like e.g. ATP, NADP, NADPH, and/or better metabolic flux through any one of the sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N- acetylgalactosaminylation, mannosylation, and/or N-acetylmannosaminylation pathway present in the cell.
In an additional and/or alternative specific embodiment, the cell is genetically engineered for production of an oligosaccharide like a milk oligosaccharide, wherein the cell comprises a pathway for production of said oligosaccharide. In a preferred embodiment, the cell is genetically engineered for production of two or more oligosaccharides like e.g. two or more milk oligosaccharides. In another and/or additional preferred embodiment, the cell is genetically engineered for an enhanced production of an oligosaccharide like a milk oligosaccharide, an enhanced uptake of one or more precursor(s) and/or acceptor(s) that is/are used in the synthesis of an oligosaccharide like a milk oligosaccharide, a better efflux of the oligosaccharide like a milk oligosaccharide, a decreased production of by-products like e.g. acids, an increased availability of co-factors like e.g. ATP, NADP, NADPH, and/or better metabolic flux through any one of the sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N- acetylgalactosaminylation, mannosylation, and/or N-acetylmannosaminylation pathway present in the cell.
In an additional and/or alternative specific embodiment, the cell is genetically engineered for production of a disaccharide and a milk oligosaccharide, wherein the cell comprises a pathway for production of said disaccharide and milk oligosaccharide. In a preferred embodiment, the cell is genetically engineered for production of one disaccharide and one milk oligosaccharide. In another preferred embodiment, the cell is genetically engineered for production of a) two or more disaccharides and b) one milk oligosaccharide. In another preferred embodiment, the cell is genetically engineered for production of a) one disaccharide and b) two or more milk oligosaccharides. In another preferred embodiment, the cell is genetically engineered for production of a) two or more disaccharides and b) two or more milk oligosaccharides.
In another and/or additional preferred embodiment, the cell is genetically engineered for an enhanced production of a disaccharide and a milk oligosaccharide, an enhanced uptake of one or more precursor(s) and/or acceptor(s) that is/are used in the synthesis of a disaccharide and a milk oligosaccharide, a better efflux of the disaccharide and the milk oligosaccharide, a decreased production of by-products like e.g. acids, an increased availability of co-factors like e.g. ATP, NADP, NADPH, and/or better metabolic flux through any one of the sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N- acetylgalactosaminylation, mannosylation, and/or N-acetylmannosaminylation pathway present in the cell.
In a preferred aspect of the method and/or cell of the invention, the genetically engineered cell is modified with gene expression modules wherein the expression from any one of said expression modules is constitutive or is tuneable.
Said expression modules are also known as transcriptional units and comprise polynucleotides for expression of recombinant genes including coding gene sequences and appropriate transcriptional and/or translational control signals that are operably linked to the coding genes. Said control signals comprise promoter sequences, untranslated regions, ribosome binding sites, terminator sequences. Said expression modules can contain elements for expression of one single recombinant gene but can also contain elements for expression of more recombinant genes or can be organized in an operon structure for integrated expression of two or more recombinant genes. Said polynucleotides may be produced by recombinant DNA technology using techniques well-known in the art. Methods which are well known to those skilled in the art to construct expression modules include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989 and yearly updates).
According to a preferred aspect of the present invention, the cell is modified with one or more expression modules. The expression modules can be integrated in the genome of said cell or can be presented to said cell on a vector. Said vector can be present in the form of a plasmid, cosmid, phage, liposome, or virus, which is to be stably transformed/transfected into said metabolically engineered cell. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. These vectors may contain selection markers such as but not limited to antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA sense/antisense markers. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., see above. For recombinant production, cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the cell can be effected by methods described in many standard laboratory manuals, such as e.g. Sambrook et aL, 1989, supra.
As used herein an expression module comprises polynucleotides for expression of at least one recombinant gene. Said recombinant gene is involved in the pathway for production of a disaccharide and/or oligosaccharide like a milk oligosaccharide; or said recombinant gene is linked to other pathways in said cell that are not involved in the synthesis of a disaccharide and/or oligosaccharide like a milk oligosaccharide. Said recombinant genes encode endogenous proteins with a modified expression or activity, preferably said endogenous proteins are overexpressed; or said recombinant genes encode heterologous proteins that are heterogeneously introduced and expressed in said modified cell, preferably overexpressed. The endogenous proteins can have a modified expression in the cell which also expresses a heterologous protein.
According to a preferred aspect of the present invention, the expression of each of said expression modules is constitutive or tuneable as defined herein.
In a preferred embodiment of the method and/or cell of the present invention, the pathway for production of said disaccharide and/or oligosaccharide like a milk oligosaccharide is selected from the list comprising, consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway. In a more preferred embodiment, the cell is genetically engineered to comprise at least one of said pathway(s). In an even more preferred embodiment, the cell comprises at least one of said pathway(s) wherein at least one of said pathway(s) has/have been genetically engineered.
In another and/or additional preferred embodiment of the method and/or cell, the cell comprises a fucosylation pathway. In a more preferred additional and/or alternative embodiment, the cell is metabolically engineered to comprise a fucosylation pathway. In another even more preferred additional and/or alternative embodiment, the cell has been metabolically engineered to comprise a fucosylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-1- phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase and fucosyltransferase has/have a modified and/or enhanced expression.
In another and/or additional preferred embodiment of the method and/or cell, the cell comprises a sialyation pathway. In an even more preferred embodiment, the cell is metabolically engineered to comprise a sialylation pathway. In another even more preferred embodiment, the cell has been metabolically engineered to comprise a sialylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6- phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N- acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6- phosphate 2-epimerase, UDP-GIcNAc 2-epimerase/kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N- acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase and sialyltransferase has/have a modified and/or enhanced expression.
In another and/or additional preferred embodiment of the method and/or cell, the cell comprises a galactosylation pathway. In a more preferred additional and/or alternative embodiment, the cell is metabolically engineered to comprise a galactosylation pathway. In another even more preferred additional and/or alternative embodiment, the cell has been metabolically engineered to comprise a galactosylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of galactose-l-epimerase, galactokinase, glucokinase, galactose-1- phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase and galactosyltransferase has/have a modified and/or enhanced expression.
In another and/or additional preferred embodiment of the method and/or cell, the cell comprises an 'N- acetylglucosaminylation' pathway. In a more preferred additional and/or alternative embodiment, the cell is metabolically engineered to comprise an N-acetylglucosaminylation pathway. In another even more preferred additional and/or alternative embodiment, the cell has been metabolically engineered to comprise an N-acetylglucosaminylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N- acetylglucosamine-l-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, and a glycosyltransferase transferring GIcNAc has/have a modified and/or enhanced expression.
In another and/or additional preferred embodiment of the method and/or cell, the cell comprises an 'N- acetylgalactosaminylation' pathway. In a more preferred additional and/or alternative embodiment, the cell is metabolically engineered to comprise an N-acetylgalactosaminylation pathway. In another even more preferred additional and/or alternative embodiment, the cell has been metabolically engineered to comprise an N-acetylgalactosaminylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, UDP-N-acetylglucosamine 4-epimerase, UDP-glucose 4-epimerase, N-acetylgalactosamine kinase and/or UDP-N-acetylgalactosamine pyrophosphorylase and a glycosyltransferase transferring GalNAc has/have a modified and/or enhanced expression.
In another and/or additional preferred embodiment of the method and/or cell, the cell comprises a 'mannosylation' pathway. In a more preferred additional and/or alternative embodiment, the cell is metabolically engineered to comprise a mannosylation pathway. In another even more preferred additional and/or alternative embodiment, the cell has been metabolically engineered to comprise a mannosylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase and/or mannose-l-phosphate guanylyltransferase and mannosyltransferase has/have a modified and/or enhanced expression.
In another and/or additional preferred embodiment of the method and/or cell, the cell comprises an 'N- acetylmannosaminylation' pathway. In a more preferred additional and/or alternative embodiment, the cell is metabolically engineered to comprise an N-acetylmannosaminylation pathway. In another even more preferred additional and/or alternative embodiment, the cell has been metabolically engineered to comprise an N-acetylmannosaminylation pathway wherein any one or more of the genes selected from the list comprising, consisting of or consisting essentially of L-glutamine— D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N- acetylglucosamine-5-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase, N- acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, UDP-GIcNAc 2-epimerase and/or ManNAc kinase and a glycosyltransferase transferring ManNAc has/have a modified and/or enhanced expression. In another and/or additional preferred embodiment of the method and/or cell of present invention, the cell comprises one or more pathway(s) for monosaccharide synthesis. Said pathways for monosaccharide synthesis comprise, consist of or consist essentially of 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).
In another and/or additional preferred embodiment of the method and/or cell of present invention, the cell comprises one or more pathway(s) for phosphorylated monosaccharide synthesis. Said pathways for phosphorylated monosaccharide synthesis comprise, consist of or consist essentially of enzymes involved in the synthesis of one or more monosaccharide(s), one or more nucleoside mono-, di- and/or triphosphate(s) and enzymes involved in the synthesis of phosphoenolpyruvate (PEP) like e.g. but not limited to PEP synthase, carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases and dehydrogenases. In another and/or additional preferred embodiment of the method and/or cell of present invention, the cell comprises one or more pathways for the synthesis of one or more nucleotide-activated sugars. Said pathways for nucleotide-activated sugar synthesis comprise, consist of or consist essentially of 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-acetylglucosamine epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylglucosamine-6P 2-epimerase, glucosamine 6- phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, N-acetylmannosamine- 5-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N- acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphate phosphatase, CMP-sialic acid synthase, galactose-1- epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4- epimerase, glucose-l-phosphate uridylyltransferase, glucophosphomutase and/or N-acetylglucosamine- 1-phosphate uridylyltransferase.
In another and/or additional preferred embodiment of the method and/or cell, 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, UDP-4-amino- 4,6-dideoxy-N-acetyl-beta-L-altrosamine transaminases, UDP-A/-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases.
In a more preferred embodiment of the method and/or cell of the invention, the fucosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1, -fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-1, 4-fucosyltransferase and alpha-1, 6-fucosyltransferase. In an alternative and/or additional more preferred embodiment of the method and/or cell of the invention, 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. In an alternative and/or additional more preferred embodiment of the method and/or cell of the invention, 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. In an alternative and/or additional more preferred embodiment of the method and/or cell of the invention, the glucosyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-glucosyltransferase, beta-1, 2- glucosyltransferase, beta-1, 3-glucosyltransferase and beta-1, 4-glucosyltransferase. In an alternative and/or additional more preferred embodiment of the method and/or cell of the invention, 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. In an alternative and/or additional more preferred embodiment of the method and/or cell of the invention, 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. In an alternative and/or additional more preferred embodiment of the method and/or cell of the invention, the N-acetylgalactosaminyltransferase is selected from the list comprising, consisting of or consisting essentially of alpha-1, 3-N-acetylgalactosaminyltransferase.
In an alternative and/or additional more preferred embodiment of the method and/or cell of the invention, the cell is modified in the expression or activity of at least one of said glycosyltransferases. In a preferred embodiment, said glycosyltransferase is an endogenous protein of the cell with a modified expression or activity, preferably said endogenous glycosyltransferase is overexpressed; alternatively said glycosyltransferase is a heterologous protein that is heterogeneously introduced and expressed in said cell, preferably overexpressed. Said endogenous glycosyltransferase can have a modified expression in the cell which also expresses a heterologous glycosyltransferase.
In another and/or additional preferred embodiment of the method and/or cell, the cell is capable to produce, preferably produces, one or more nucleotide-activated sugars, preferably said cell is genetically engineered for production of one or more of said nucleotide-activated sugar(s). Herein, said one or more nucleotide-activated sugar(s) is/are selected from the list comprising, consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP- N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP-glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6- dideoxy— L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy— L-lyxo-4-hexulose, UDP-N-acetyl-L- rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L- pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), GDP-rhamnose and UDP-xylose. In another and/or additional preferred embodiment of the method and/or cell, the cell comprises a pathway for the synthesis of a nucleotide-activated sugar selected from the list comprising, consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP- GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP-glucuronate, UDP-galacturonate, UDP-2- acetamido-2,6-dideoxy— L-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy— L-lyxo-4-hexulose, UDP- N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N- acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L- galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6- dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), GDP-rhamnose and UDP-xylose.
The cell used herein is optionally genetically modified to express the de novo synthesis of UDP-GIcNAc. UDP-GIcNAc can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing an UDP-GIcNAc can express enzymes converting, e.g. GIcNAc, which is to be added to the cell, to UDP-GIcNAc. These enzymes may be any one or more of the list comprising, consisting of or consisting essentially of an N-acetyl-D-glucosamine kinase, an N-acetylglucosamine-6-phosphate deacetylase, a phosphoglucosamine mutase, N-acetylglucosamine-l-phosphate uridyltransferase, glucosamine-l-phosphate acetyltransferase, and a bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase from several species including Homo sapiens, Escherichia coli. Preferably, the cell is modified to produce UDP-GIcNAc.
Additionally, or alternatively, the cell used herein is optionally genetically modified to express the de novo synthesis of CMP-Neu5Ac. CMP-Neu5Ac can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing CMP-Neu5Ac can express an enzyme converting, e.g., sialic acid to CMP-Neu5Ac. This enzyme may be a CMP-sialic acid synthetase, like the N-acylneuraminate cytidylyltransferase from several species including Homo sapiens, Neisseria meningitidis, and Pasteurella multocida. Preferably, the cell is modified to produce CMP-Neu5Ac. More preferably, the cell is modified for enhanced CMP-Neu5Ac production. Said modification can be any one or more 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.
Additionally, or alternatively, the cell used herein is optionally genetically modified to express the de novo synthesis of GDP-fucose. GDP-fucose can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing GDP-fucose can express an enzyme converting, e.g., fucose, which is to be added to the cell, to GDP-fucose. This enzyme may be, e.g., a bifunctional fucose kinase/fucose-l-phosphate guanylyltransferase, like Fkp from Bacteroidesfragilis, or the combination of one separate fucose kinase together with one separate fucose-l-phosphate guanylyltransferase like they are known from several species including Homo sapiens, Sus scrofa and Rattus norvegicus. Preferably, the cell is modified to produce GDP-fucose. More preferably, the cell is modified for enhanced GDP-fucose production. Said modification can be any one or more 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.
Additionally, or alternatively, the cell used herein is optionally genetically modified to express the de novo synthesis of UDP-Gal. UDP-Gal can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cell producing UDP-Gal can express an enzyme converting, e.g. UDP-glucose, to UDP-Gal. This enzyme may be, e.g., the UDP-glucose-4-epimerase GalE like as known from several species including Homo sapiens, Escherichia coli, and Rattus norvegicus. Preferably, the cell is modified to produce UDP- Gal. More preferably, the cell is modified for enhanced UDP-Gal production. Said modification can be any one or more selected from the list comprising, consisting of or consisting essentially of knock-out of a bifunctional 5'-nucleotidase/UDP-sugar hydrolase encoding gene, knock-out of a galactose-l-phosphate uridylyltransferase encoding gene and over-expression of an UDP-glucose-4-epimerase encoding gene.
Additionally, or alternatively, the cell used herein is optionally genetically modified to express the de novo synthesis of UDP-GalNAc. UDP-GalNAc can be synthesized from UDP-GIcNAc by the action of a single-step reaction using an UDP-N-acetylglucosamine 4-epimerase like e.g. wbgU from Plesiomonas shigelloides, gne from Yersinia enterocolitica or wbpP from Pseudomonas aeruginosa serotype 06. Preferably, the cell is modified to produce UDP-GalNAc. More preferably, the cell is modified for enhanced UDP-GalNAc production.
Additionally, or alternatively, the cell used herein is optionally genetically modified to express the de novo synthesis of UDP-ManNAc. UDP-ManNAc can be synthesized directly from UDP-GIcNAc via an epimerization reaction performed by an UDP-GIcNAc 2-epimerase (like e.g. cap5P from Staphylococcus aureus, RffE from E. coli, Cpsl9fK from S. pneumoniae, and RfbC from S. enterica). Preferably, the cell is modified to produce UDP-ManNAc. More preferably, the cell is modified for enhanced UDP-ManNAc production.
In another and/or additional preferred embodiment of the method and/or cell, the cell possesses, preferably expresses, one or more genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N- acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6- phosphate 2-epimerase, UDP-GIcNAc 2-epimerase/kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N- acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase, UDP-N- acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase. In a more preferred embodiment, the cell overexpresses one or more genes selected from the list comprising, consisting of or consisting essentially of mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L- glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N- acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2- epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GIcNAc 2-epimerase/kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, Neu5Ac synthase, N-acetylneuraminate lyase, N-acylneuraminate-9- phosphate synthase, N-acylneuraminate-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N- acetylgalactosamine pyrophosphorylase.
In a specific embodiment of the method and/or cell, the cell is genetically engineered for production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide wherein said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N- acetyllactosamine (LacNAc); lacto-N-biose (LNB); mammalian milk oligosaccharide; human milk oligosaccharide; neutral (non-charged) milk oligosaccharide; a negatively charged, preferably sia lylated, milk oligosaccharide ; fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide; non-fucosylated neutral (non-charged) mammalian milk oligosaccharide; sialylated human milk oligosaccharide; neutral (non-charged) human milk oligosaccharide; fucosylated human milk oligosaccharide; non-fucosylated neutral (non-charged) human milk oligosaccharide; N-acetylglucosamine containing neutral (non-charged) milk oligosaccharide; N-acetylglucosamine containing milk oligosaccharide; N-acetyllactosamine containing milk oligosaccharide; fucosylated N-acetyllactosamine containing milk oligosaccharide; sialylated N- acetyllactosamine containing milk oligosaccharide; lacto-N-biose containing milk oligosaccharide; fucosylated lacto-N-biose containing milk oligosaccharide and sialylated lacto-N-biose containing milk oligosaccharide.
In a more preferred embodiment, the oligosaccharide is a mammalian milk oligosaccharide (MMO) as described herein. In another more preferred embodiment, the oligosaccharide is a human milk oligosaccharide (HMO) as described herein. In another more preferred embodiment, the fucosylated milk oligosaccharide is selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4- fucosyllactose (4FL), 6-fucosyllactose (5FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N- neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N- fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N-neohexaose. In another more preferred embodiment, the sialylated milk oligosaccharide is selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N- hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto- N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N- fucopentaose II and monofucosyldisialyllacto-N-tetraose. In another more preferred embodiment, the N- acetylglucosamine containing neutral (non-charged) milk oligosaccharide is selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'- galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose.
In an even more preferred embodiment, the oligosaccharide like the milk oligosaccharide is selected from the list comprising, consisting of or consisting essentially of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N- neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N- fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose, difucosyl-lacto-N-neohexaose, lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N- neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto- N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose ll7 monosialyllacto-N-neohexaose I, monosialyllacto-N- neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose.
The oligosaccharide in the context of the present invention is preferably in free form, i.e., the oligosaccharide does not contain any protective group.
In another and/or additional preferred embodiment of the method and/or cell of present invention, the cell is capable to produce, preferably produces, said disaccharide and/or oligosaccharide like a milk oligosaccharide from one or more precursor(s) as defined herein. In a more preferred embodiment, the precursor is lactose. Preferably, said one or more precursor(s) is/are fed to the cell from the culture or cultivation medium or the incubation. In another more preferred embodiment, the cell is capable to produce, preferably produces, at least one of said one or more precursor(s). In an even more preferred embodiment, the cell is capable to produce, preferably produces, all of said one or more precursor(s). In another more preferred embodiment, the cell is genetically engineered for the production of at least one of said one or more precursor(s). In an even more preferred embodiment, the cell is genetically engineered for the production of all of said one or more precursor(s). In another more preferred embodiment, at least one of said one or more precursor(s) is internalized in said cell via one or more membrane protein(s). In another preferred embodiment, the precursor(s) that is/are used by the cell for the production of said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are completely converted into said disaccharide and/or oligosaccharide like a milk oligosaccharide, respectively.
In another and/or additional preferred embodiment of the method and/or cell of present invention, the cell is further genetically engineered to possess, preferably to express, a glutamine— fructose-6- phosphate aminotransferase. In a more preferred embodiment, the cell is further genetically engineered to overexpress a glutamine— fructose-6-phosphate aminotransferase.
In another more preferred embodiment, the glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and is selected from the enzyme class EC:2.6.1.16.
In another and/or additional more preferred embodiment the glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and comprises a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of IPR001347, IPR005855, IPR017932, IPR029055, IPR035466, IPR035490, IPR036291, IPR046348 and IPR047084 as defined by InterPro 90.0 as released on 4th August 2022. In another and/or additional more preferred embodiment the glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and comprises a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of PF00310, PF01380, PF01408, PF13230, PF13537 and PF13580 as defined by InterPro 90.0 as released on 4th August 2022.
In another and/or additional more preferred embodiment the glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and comprises a polypeptide sequence comprising a conserved protein domain selected from the list consisting of or consisting essentially of cd00714, cd05007, cd05008, cd05009, cd05013 and cd05710 as defined by InterPro 90.0 as released on 4th August 2022.
In another and/or additional more preferred embodiment the glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and is part of the NOG family COG0449 as defined by eggNOG5.0 as released in 2019.
In another and/or additional preferred embodiment of the method and/or cell of present invention, the cell contains a nucleic acid molecule which comprises a polynucleotide sequence which encodes a glutamine— fructose-6-phosphate aminotransferase as described herein. In a more preferred embodiment, the nucleic acid molecule is operably linked to control sequences recognized by the cell, said nucleic acid molecule further i) being integrated in the genome of said cell and/or ii) presented to said cell on a vector. In another and/or additional more preferred embodiment, the nucleic acid molecule is foreign to the cell.
In another and/or additional preferred embodiment of the method and/or cell of present invention, the cell is modified for enhanced synthesis and/or supply of phosphoenolpyruvate (PEP).
In another and/or additional preferred embodiment of the method and/or cell of present invention, the cell is further modified for reduced degradation of acetyl-CoA and/or its main precursor pyruvate.
In a preferred embodiment, the oligosaccharide of present invention is produced by a cell that is cultured in a cell cultivation. Within the context of present invention, the cell cultivation comprises in vitro and/or ex vivo cultivation of cells. In another and/or additional more preferred embodiment, the cell cultivation is a fermentation. In an alternative and/or additional more preferred embodiment, the cell is cultivated or incubated in a reactor as defined herein. In an alternative and/or additional more preferred embodiment, the cell is cultivated or incubated in an incubator as defined herein. In another and/or additional preferred embodiment, the cell is cultivated in culture or cultivation medium comprising a carbon source comprising, consisting of or consisting essentially of a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium including molasses, corn steep liquor, peptone, tryptone or yeast extract. Preferably, said carbon source is selected from the list comprising, consisting of or consisting essentially of glucose, N-acetylglucosamine (GIcNAc), glycerol, fructose, sucrose, maltose, lactose, arabinose, malto-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemi-cellulose, molasses, corn-steep liquor, high-fructose syrup, acetate, citrate, lactate and pyruvate. In a more preferred embodiment, the cultivation medium contains at least one carbon source selected from the list consisting of glucose, fructose, sucrose and glycerol. In another and/or additional preferred embodiment, the cultivation or incubation medium contains at least one compound selected from the list consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid and CMP-sialic acid. In another and/or additional more preferred embodiment, the culture or cultivation medium is a chemically defined medium. In another and/or additional preferred embodiment, the culture or cultivation medium is a minimal salt medium comprising sulphate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt, and/or selenium. In another and/or additional preferred embodiment, the cultivation or incubation medium comprises one or more precursor(s) that is/are used for production of said disaccharide and/or oligosaccharide like a milk oligosaccharide. In a more preferred embodiment, the cultivation or incubation medium comprises one or more co-factor(s) selected from the list comprising, consisting of or consisting essentially of Mg2+, Co2+, Mn2+, Ca2+, Zn2+, Ni2+ and FAD.
In another and/or additional preferred embodiment of the method of present invention, the method for production of a disaccharide as described herein comprises at least one of the following steps: i) Adding to the culture or cultivation medium in a reactor at least one precursor and/or acceptor feed wherein the total reactor volume ranges from 250 ml (millilitre) to 10.000 m3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture or cultivation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture or cultivation medium before the addition of said precursor and/or acceptor feed; ii) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; iii) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein preferably, the pH of said feeding solution is set between 3 and 7 and wherein preferably, the temperature of said feeding solution is kept between 20°C and 80°C; said method resulting in a disaccharide with a concentration of at least 30 g/L in the final volume of said culture or cultivation medium.
In another and/or additional preferred embodiment of the method of present invention, the method for production of a disaccharide as described herein comprises at least one of the following steps: i) Adding to the culture or cultivation medium at least one precursor and/or acceptor in one pulse or in a discontinuous (pulsed) manner wherein the total reactor volume ranges from 250 ml_ (millilitre) to 10.000 m3 (cubic meter), preferably so that the final volume of the culture or cultivation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture or cultivation medium before the addition of said precursor and/or acceptor feed pulse(s); ii) Adding at least one precursor and/or acceptor feed in a discontinuous (pulsed) manner to the culture or cultivation medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; iii) Adding at least one precursor and/or acceptor feed in a discontinuous (pulsed) manner to the culture or cultivation medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein preferably, the pH of said feeding solution is set between 3 and 7 and wherein preferably, the temperature of said feeding solution is kept between 20°C and 80°C; said method resulting in a disaccharide with a concentration of at least 30 g/L in the final volume of said culture or cultivation medium.
In a further, more preferred embodiment, the method for the production of a disaccharide as described herein comprises at least one of the following steps: i) Adding to the culture or cultivation medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per litre of initial reactor volume wherein the total reactor volume ranges from 250 mL (millilitre) to 10.000 m3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture or cultivation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture or cultivation medium before the addition of said lactose feed; ii) Adding a lactose feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; iii) Adding a lactose feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein the concentration of said lactose feeding solution is 25 g/L, preferably 50 g/L, more preferably 75 g/L, more preferably 100 g/L, more preferably 125 g/L, more preferably 150 g/L, more preferably 175 g/L, more preferably 200 g/L, more preferably 225 g/L, more preferably 250 g/L, more preferably 275 g/L, more preferably 300 g/L, more preferably 325 g/L, more preferably 350 g/L, more preferably 375 g/L, more preferably, 400 g/L, more preferably 450 g/L, more preferably 500 g/L, even more preferably, 550 g/L, most preferably 600 g/L; and wherein preferably the pH of said solution is set between 3 and 7 and wherein preferably the temperature of said feed solution is kept between 20°C and 80°C; said method resulting in a disaccharide with a concentration of at least 30 g/L in the final volume of said culture or cultivation medium.
In another and/or additional preferred embodiment of the method of present invention, the method for production of an oligosaccharide like a milk oligosaccharide as described herein comprises at least one of the following steps: i) Adding to the culture or cultivation medium in a reactor at least one precursor and/or acceptor feed wherein the total reactor volume ranges from 250 m L (millilitre) to 10.000 m3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture or cultivation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture or cultivation medium before the addition of said precursor and/or acceptor feed; ii) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; iii) Adding at least one precursor and/or acceptor feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein preferably, the pH of said feeding solution is set between 3 and 7 and wherein preferably, the temperature of said feeding solution is kept between 20°C and 80°C; said method resulting in an oligosaccharide like a milk oligosaccharide with a concentration of at least 30 g/L in the final volume of said culture or cultivation medium.
In another and/or additional preferred embodiment of the method of present invention, the method for production of an oligosaccharide like a milk oligosaccharide as described herein comprises at least one of the following steps: i) Adding to the culture or cultivation medium at least one precursor and/or acceptor in one pulse or in a discontinuous (pulsed) manner wherein the total reactor volume ranges from 250 mL (millilitre) to 10.000 m3 (cubic meter), preferably so that the final volume of the culture or cultivation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture or cultivation medium before the addition of said precursor and/or acceptor feed pulse(s); ii) Adding at least one precursor and/or acceptor feed in a discontinuous (pulsed) manner to the culture or cultivation medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; iii) Adding at least one precursor and/or acceptor feed in a discontinuous (pulsed) manner to the culture or cultivation medium over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein preferably, the pH of said feeding solution is set between 3 and 7 and wherein preferably, the temperature of said feeding solution is kept between 20°C and 80°C; said method resulting in an oligosaccharide like a milk oligosaccharide with a concentration of at least 30 g/L in the final volume of said culture or cultivation medium.
In a further, more preferred embodiment, the method for the production of an oligosaccharide like a milk oligosaccharide as described herein comprises at least one of the following steps: i) Adding to the culture or cultivation medium a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 gram of lactose per litre of initial reactor volume wherein the total reactor volume ranges from 250 mL (millilitre) to 10.000 m3 (cubic meter), preferably in a continuous manner, and preferably so that the final volume of the culture or cultivation medium is not more than three-fold, preferably not more than two-fold, more preferably less than 2-fold of the volume of the culture or cultivation medium before the addition of said lactose feed; ii) Adding a lactose feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution; iii) Adding a lactose feed in a continuous manner to the culture or cultivation medium over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feeding solution and wherein the concentration of said lactose feeding solution is 25 g/L, preferably 50 g/L, more preferably 75 g/L, more preferably 100 g/L, more preferably 125 g/L, more preferably 150 g/L, more preferably 175 g/L, more preferably 200 g/L, more preferably 225 g/L, more preferably 250 g/L, more preferably 275 g/L, more preferably 300 g/L, more preferably 325 g/L, more preferably 350 g/L, more preferably 375 g/L, more preferably, 400 g/L, more preferably 450 g/L, more preferably 500 g/L, even more preferably, 550 g/L, most preferably 600 g/L; and wherein preferably the pH of said solution is set between 3 and 7 and wherein preferably the temperature of said feed solution is kept between 20°C and 80°C; said method resulting in an oligosaccharide like a milk oligosaccharide with a concentration of at least 30 g/L in the final volume of said culture or cultivation medium.
Preferably the lactose feed is accomplished by adding lactose from the beginning of the cultivating in a concentration of at least ImM, preferably at least 5 mM, preferably in a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably in a concentration > 300 mM.
In another aspect the lactose feed is accomplished by adding lactose to the cultivation medium in a concentration, such that throughout the production phase of the cultivation a lactose concentration of at least 1 mM, preferably 5 mM, 10 mM or 30 mM is obtained.
In a further embodiment of the methods described herein the cells are cultivated for at least about 60, 80, 100, or about 120 hours or in a continuous manner.
In a preferred embodiment, a carbon source is provided, preferably sucrose, in the culture or cultivation medium for 3 or more days, preferably up to 7 days; and/or provided, in the culture or 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 culture volume in a continuous manner, so that the final volume of the culture or 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 culturing or cultivation medium before the culturing.
Preferably, when performing the method as described herein, a first phase of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the culture or cultivation medium before the lactose is added to the culture or cultivation medium in a second phase.
In an alternative preferable embodiment, in the method as described herein, the lactose is added already in the first phase of exponential growth together with the carbon-based substrate.
In another and/or additional preferred embodiment of the method and/or cell of present invention, the cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the synthesis of said disaccharide and/or oligosaccharide like a milk oligosaccharide.
In another and/or additional preferred embodiment of the method and/or cell of present invention, the cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably from the group consisting of yeast cells, bacterial cells, archaebacterial cells, algae cells, plant cells, fungal cells, animal cells and protozoan cells. In another preferred embodiment, the cell is a bacterium, fungus, yeast, a plant cell, an animal cell or a protozoan cell.
The latter bacterium preferably belongs to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus or the phylum of Actinobacteria. The latter bacterium belonging to the phylum Proteobacteria belongs preferably to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacterium preferably relates to any strain belonging to the species Escherichia coli such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultivated Escherichia coli strains - designated as E. coli K12 strains - which are well- adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Well-known examples of the E. coli K12 strains are K12 Wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Hence, the present invention specifically relates to a mutated and/or transformed Escherichia coli cell or strain as indicated above wherein said E. coli strain is a K12 strain. More preferably, the Escherichia coli K12 strain is E. coli MG1655. The latter bacterium belonging to the phylum Firmicutes belongs preferably to the Bacilli, preferably Lactobacilliales, with members such as Lactobacillus lactis, Leuconostoc mesenteroides, or Bacil lales with members such as from the genus Bacillus, such as Bacillus subtilis or, B. amyloliquefaciens. The latter Bacterium belonging to the phylum Actinobacteria, preferably belonging to the family of the Corynebacteriaceae, with members Corynebacterium glutamicum or C. afermentans, or belonging to the family of the Streptomycetaceae with members Streptomyces griseus or 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. kluyveri), Komagataella, Hansenula, Kluyveromyces (with members like e.g. Kluyveromyces lactis, K. marxianus, K. thermotolerans), Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces, Torulaspora, Yarrowia (like e.g. Yarrowia lipolytica) or Starmerella (like e.g. Starmerella bombicola). The latter yeast is preferably selected from Pichia pastoris, Yarrowia lipolytica, Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha, Kluyveromyces marxianus, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Zygosaccharomyces rouxii, and Zygosaccharomyces bailii. The latter fungus belongs preferably to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus. Plant cells include cells of flowering and non-flowering plants, as well as algal cells, for example Chlamydomonas, Chlorella, etc. Preferably, said plant is a tobacco, rose, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant. More preferably, the latter plant cell is selected from the Rosa family. The latter animal cell is preferably derived from non-human mammals (e.g. cattle, buffalo, pig, sheep, mouse, rat, primate (e.g., chimpanzee, orangutan, gorilla, monkey (e.g., Old World, New World), lemur), dog, cat, rabbit, horse, cow, goat, ox, deer, musk deer, bovid, whale, dolphin, hippopotamus, elephant, rhinoceros, giraffe, zebra, lion, cheetah, tiger, panda, red panda, otter), birds (e.g. chicken, duck, ostrich, turkey, pheasant), fish (e.g. swordfish, salmon, tuna, sea bass, trout, catfish), invertebrates (e.g. lobster, crab, shrimp, clams, oyster, mussel, sea urchin), reptiles (e.g. snake, alligator, turtle), amphibians (e.g. frogs) or insects (e.g. fly, nematode) or is a genetically modified cell line derived from human cells excluding embryonic stem cells. Both human and non-human mammalian cells are preferably selected from the list comprising an epithelial cell like e.g. a mammary epithelial cell, an embryonic kidney cell (e.g. HEK293 or HEK 293T cell), a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell like e.g. an N20, SP2/O or YB2/0 cell, an NIH-3T3 cell, a non-mammary adult stem cell or derivatives thereof such as described in WO21067641, preferably mesenchymal stem cell or derivates thereof as described in WO21067641, a lactocyte derived from mammalian induced pluripotent stem cells, preferably human induced pluripotent stem cells, a lactocyte as part of mammary-like gland organoids, a post-parturition mammary epithelium cell, a polarized mammary cell, preferably a polarized mammary cell selected from the group comprising live primary mammary epithelial cells, live mammary myoepithelial cells, live mammary progenitor cells, live immortalized mammary epithelial cells, live immortalized mammary myoepithelial cells, live immortalized mammary progenitor cells, a non-mammary adult stem cell or derivatives thereof as well-known to the person skilled in the art from e.g. WO2021/219634, WO 2022/054053, WO 2021/141762, WO 2021/142241, WO 2021/067641 and WO2021/242866. The latter insect cell is preferably derived from Spodoptera frugiperda like e.g., Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae, Trichoplusia ni like e.g., BTI-TN-5B1-4 cells or Drosophila melanogaster like e.g. Drosophila 82 cells. The latter protozoan cell preferably is a Leishmania tarentolae cell.
In another and/or additional preferred embodiment, the cell is an E. coli or yeast with a lactose permease positive phenotype, preferably wherein said lactose permease is coded by the gene LacY or LAC12, respectively.
In another and/or additional preferred embodiment of the method and/or cell of present invention, the cell produces 30 g/L or more of said disaccharide in the whole broth and/or supernatant and/or wherein said disaccharide in the whole broth and/or supernatant has a purity of at least 80 % measured on the total amount of disaccharide and its precursor(s) produced by said cell in the whole broth and/or supernatant, respectively. In a more preferred embodiment, the cell produces 30 g/L, 31 g/L, 32 g/L, 33 g/L, 34 g/L, 35 g/L, 36 g/L, 37 g/L, 38 g/L, 39 g/L, 40 g/L, 41 g/L, 42 g/L, 43 g/L, 44 g/L, 45 g/L, 46 g/L, 47 g/L, 48 g/L, 49 g/L, 50 g/L, 51 g/L, 52 g/L, 53 g/L, 54 g/L, 55 g/L, 56 g/L, 57 g/L, 58 g/L, 59 g/L, 60 g/L, 61 g/L, 62 g/L, 63 g/L, 64 g/L, 65 g/L, 66 g/L, 67 g/L, 68 g/L, 69 g/L, 70 g/L, 71 g/L, 72 g/L, 73 g/L, 74 g/L, 75 g/L, 76 g/L, 77 g/L, 78 g/L, 79 g/L, 80 g/L, 81 g/L, 82 g/L, 83 g/L, 84 g/L, 85 g/L or more than 85 g/L of said disaccharide in the whole broth and/or supernatant. In another and/or additional preferred embodiment of the method and/or cell of present invention, the cell produces 30 g/L or more of said oligosaccharide like a milk oligosaccharide in the whole broth and/or supernatant and/or wherein said oligosaccharide like a milk oligosaccharide in the whole broth and/or supernatant has a purity of at least 80 % measured on the total amount of oligosaccharide like a milk oligosaccharide and its precursor(s) produced by said cell in the whole broth and/or supernatant, respectively. In a more preferred embodiment, the cell produces 30 g/L, 31 g/L, 32 g/L, 33 g/L, 34 g/L, 35 g/L, 36 g/L, 37 g/L, 38 g/L, 39 g/L, 40 g/L, 41 g/L, 42 g/L, 43 g/L, 44 g/L, 45 g/L, 46 g/L, 47 g/L, 48 g/L, 49 g/L, 50 g/L, 51 g/L, 52 g/L, 53 g/L, 54 g/L, 55 g/L, 56 g/L, 57 g/L, 58 g/L, 59 g/L, 60 g/L, 61 g/L, 62 g/L, 63 g/L, 64 g/L, 65 g/L, 66 g/L, 67 g/L, 68 g/L, 69 g/L, 70 g/L, 71 g/L, 72 g/L, 73 g/L, 74 g/L, 75 g/L, 76 g/L, 77 g/L, 78 g/L, 79 g/L, 80 g/L, 81 g/L, 82 g/L, 83 g/L, 84 g/L, 85 g/L or more than 85 g/L of said oligosaccharide like a milk oligosaccharide in the whole broth and/or supernatant.
In another and/or additional preferred embodiment of the method and/or cell of present invention, said less functional synthesis of UDP-GIcNAc confers unaffected and/or enhanced i) disaccharide and/or oligosaccharide like a milk oligosaccharide formation, ii) productivity, iii) biomass production, iv) cell growth and/or v) yield of the produced disaccharide and/or oligosaccharide like a milk oligosaccharide, relative to a corresponding non-modified or non-engineered cell.
In another aspect of present invention, the cell produces a disaccharide and/or oligosaccharide like a milk oligosaccharide as described herein. In a preferred embodiment of the method and/or cell of present invention, the cell produces an oligosaccharide mixture like a milk oligosaccharide mixture.
In another aspect of present invention, the disaccharide and/or oligosaccharide like a milk oligosaccharide produced by a cell of present invention is/are recovered from said cultivation or incubation medium and/or said cell. In a preferred embodiment, said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are purified.
The terms "separating from said cultivation or incubation" means harvesting, collecting, or retrieving said disaccharide and/or oligosaccharide like a milk oligosaccharide from the cell and/or the medium of its growth. The disaccharide and the oligosaccharide like a milk oligosaccharide can be separated in a conventional manner from the aqueous culture or cultivation medium, in which the cell was grown. In case said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are still present in the cells producing the disaccharide and/or oligosaccharide like a milk oligosaccharide, conventional manners to free or to extract said disaccharide and/or oligosaccharide like a milk oligosaccharide out of the cells can be used, such as cell destruction using high pH, heat shock, sonication, French press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergent, hydrolysis,... The culture or cultivation medium and/or cell extract together and separately can then be further used for separating said disaccharide and/or oligosaccharide like a milk oligosaccharide. This preferably involves clarifying said disaccharide and/or oligosaccharide like a milk oligosaccharide to remove suspended particulates and contaminants, particularly cells, cell components, insoluble metabolites and debris produced by culturing the genetically engineered cell. In this step, said disaccharide and/or oligosaccharide like a milk oligosaccharide can be clarified in a conventional manner. Preferably, said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are clarified by centrifugation, flocculation, decantation and/or filtration. A second step of separating said disaccharide and/or oligosaccharide like a milk oligosaccharide preferably involves removing substantially all the eventually remaining proteins, peptides, amino acids, RNA, DNA, endotoxins and glycolipids that could interfere with the subsequent separation step, from said disaccharide and/or oligosaccharide like a milk oligosaccharide, preferably after it/they has/have been clarified. In this step, remaining proteins and related impurities can be removed from said disaccharide and/or oligosaccharide like a milk oligosaccharide in a conventional manner. Preferably, remaining proteins, salts, by-products, colour, endotoxins and other related impurities are removed from said disaccharide and/or oligosaccharide like a milk oligosaccharide by ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high-performance filtration, tangential flow ultrafiltration, electrophoresis (e.g. using slab-polyacrylamide or sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands including e.g. DEAE-sepharose, poly-L-lysine and polymyxin-B, endotoxin-selective adsorber matrices), ion exchange chromatography (such as but not limited to cation exchange, anion exchange, mixed bed ion exchange, inside-out ligand attachment), hydrophobic interaction chromatography and/or gel filtration (i.e., size exclusion chromatography), particularly by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography. With the exception of size exclusion chromatography, remaining proteins and related impurities are retained by a chromatography medium or a selected membrane.
In a further preferred embodiment, the methods as described herein also provide for a further purification of the disaccharide and/or oligosaccharide like a milk oligosaccharide of present invention. A further purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide may be accomplished, for example, by use of (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange 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 disaccharide and/or oligosaccharide like a milk oligosaccharide. Another purification step is to dry, e.g. spray dry or lyophilize the produced disaccharide and/or oligosaccharide like a milk oligosaccharide. In an exemplary embodiment, the separation and purification of the disaccharide and/or oligosaccharide like a milk oligosaccharide is made in a process, comprising the following steps in any order: a) contacting the cultivation or a clarified version thereof with a nanofiltration membrane with a molecular weight cut-off (MWCO) of 600-3500 Da ensuring the retention of the produced disaccharide and/or oligosaccharide like a milk oligosaccharide and allowing at least a part of the proteins, salts, by-products, colour and other related impurities to pass, b) conducting a diafiltration process on the retentate from step a), using said membrane, with an aqueous solution of an inorganic electrolyte, followed by optional diafiltration with pure water to remove excess of the electrolyte, c) and collecting the retentate enriched in said disaccharide and/or oligosaccharide like a milk oligosaccharide in the form of a salt from the cation of said electrolyte.
In an alternative exemplary embodiment, the separation and purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide is made in a process, comprising the following steps in any order: subjecting the cultivation or a clarified version thereof to two membrane filtration steps using different membranes, wherein one membrane has a molecular weight cut-off of between about 300 to about 500 Dalton, and the other membrane as a molecular weight cut-off of between about 600 to about 800 Dalton.
In an alternative exemplary embodiment, the separation and purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide is made in a process, comprising the following steps in any order comprising the step of treating the cultivation or a clarified version thereof with a strong cation exchange resin in H+-form and a weak anion exchange resin in free base form.
In an alternative exemplary embodiment, the separation and purification of said disaccharide and/or oligosaccharide like a milk oligosaccharide is made in the following way. The cultivation comprising i) the produced disaccharide and/or oligosaccharide like a milk oligosaccharide, 2) biomass, 3) medium components and 4) contaminants, and wherein the purity of the produced disaccharide and/or oligosaccharide like a milk oligosaccharide in the cultivation is < 80 %, is applied to the following purification steps: i) separation of biomass from the cultivation, ii) cationic ion exchanger treatment for the removal of positively charged material, iii) anionic ion exchanger treatment for the removal of negatively charged material, iv) nanofiltration step and/or electrodialysis step, wherein a purified solution comprising the produced disaccharide and/or oligosaccharide like a milk oligosaccharide at a purity of greater than or equal to 80 % is provided. Optionally the purified solution is spray dried.
In an alternative exemplary embodiment, the separation and purification of the disaccharide and/or oligosaccharide like a milk oligosaccharide is made in a process, comprising the following steps in any order: enzymatic treatment of the cultivation; removal of the biomass from the cultivation; ultrafiltration; nanofiltration; and a column chromatography step. Preferably such column chromatography is a single column or a multiple column. Further preferably the column chromatography step is simulated moving bed chromatography. Such simulated moving bed chromatography preferably comprises i) at least 4 columns, wherein at least one column comprises a weak or strong cation exchange resin; and/or ii) four zones I, II, III and IV with different flow rates; and/or iii) an eluent comprising water; and/or iv) an operating temperature of 15 degrees to 60 degrees centigrade.
In a specific embodiment, the present invention provides the produced disaccharide and/or oligosaccharide like a milk oligosaccharide which is/are spray-dried to powder, wherein the spray-dried powder contains < 15 % -wt. of water, preferably < 10 % -wt. of water, more preferably < 7 % -wt. of water, most preferably < 5 % -wt. of water.
For identification of said disaccharide and/or oligosaccharide like a milk oligosaccharide as described herein, the monomeric building blocks (e.g. the monosaccharide or glycan unit composition), the anomeric configuration of side chains, the presence and location of substituent groups, degree of polymerization/molecular weight and the linkage pattern can be identified by standard methods known in the art, such as, e.g. methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas chromatographymass spectrometry), MALDI-MS (Matrix-assisted laser desorption/ionization-mass spectrometry), ESI-MS (Electrospray ionization-mass spectrometry), HPLC (High-Performance Liquid chromatography with ultraviolet or refractive index detection), HPAEC-PAD (High-Performance Anion-Exchange chromatography with Pulsed Amperometric Detection), CE (capillary electrophoresis), IR (infrared)/Raman spectroscopy, and NMR (Nuclear magnetic resonance) spectroscopy techniques. The crystal structure can be solved using, e.g., solid-state NMR, FT-IR (Fourier transform infrared spectroscopy), and WAXS (wide-angle X-ray scattering). The degree of polymerization (DP), the DP distribution, and polydispersity can be determined by, e.g., viscosimetry and SEC (SEC-HPLC, high performance size-exclusion chromatography). To identify the monomeric components of the disaccharide and/or oligosaccharide like a milk oligosaccharide, methods such as e.g. acid-catalysed hydrolysis, HPLC (high performance liquid chromatography) or GLC (gas-liquid chromatography) (after conversion to alditol acetates) may be used. To determine the glycosidic linkages, said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are methylated with methyl iodide and strong base in DMSO, hydrolysis is performed, a reduction to partially methylated alditols is achieved, an acetylation to methylated alditol acetates is performed, and the analysis is carried out by GLC/MS (gas-liquid chromatography coupled with mass spectrometry). To determine the glycan sequence, a partial depolymerization is carried out using an acid or enzymes to determine the structures. To identify the anomeric configuration, said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are subjected to enzymatic analysis, e.g., it is contacted with an enzyme that is specific for a particular type of linkage, e.g., beta-galactosidase, or alphaglucosidase, etc., and NMR may be used to analyse the products.
In another aspect, the present invention provides use of a cell as described herein for the production of a disaccharide and/or oligosaccharide like a milk oligosaccharide wherein said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N- acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; a negatively charged, preferably sialylated, milk oligosaccharide; mammalian milk oligosaccharide (MMO); a human milk oligosaccharide (HMO); neutral (non-charged) milk oligosaccharide; fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide; non-fucosylated neutral (non-charged) mammalian milk oligosaccharide; sialylated human milk oligosaccharide; neutral (non-charged) human milk oligosaccharide; fucosylated human milk oligosaccharide; non-fucosylated neutral (non-charged) human milk oligosaccharide; fucosylated milk oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4- fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N- neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N- fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N-neohexaose; sialylated milk oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d ( LSTd), disialyllacto- N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N-acetylglucosamine containing neutral (non-charged) milk oligosaccharide; N-acetylglucosamine containing neutral (non-charged) milk oligosaccharide preferably selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N- hexaose, para-lacto-N-neohexaose; N-acetylglucosamine containing milk oligosaccharide; N- acetyllactosamine containing milk oligosaccharide; fucosylated N-acetyllactosamine containing milk oligosaccharide; sialylated N-acetyllactosamine containing milk oligosaccharide; lacto-N-biose containing milk oligosaccharide; fucosylated lacto-N-biose containing milk oligosaccharide and sialylated lacto-N- biose containing milk oligosaccharide.
In another aspect, the present invention provides use of a method as described herein for the production of a disaccharide and/or an oligosaccharide like a milk oligosaccharide, wherein said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N- acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; a negatively charged, preferably sialylated, milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); neutral (non-charged) milk oligosaccharide; fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide; non-fucosylated neutral (non-charged) mammalian milk oligosaccharide; sialylated human milk oligosaccharide; neutral (non-charged) human milk oligosaccharide; fucosylated human milk oligosaccharide; non-fucosylated neutral (non-charged) human milk oligosaccharide; fucosylated milk oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4- fucosyllactose (4FL), 6-fucosyllactose (5FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N- neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N- fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N-neohexaose; sialylated milk oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto- N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N-acetylglucosamine containing neutral (non-charged) oligosaccharide; N-acetylglucosamine containing neutral (non-charged) milk oligosaccharide selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'- galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose; N-acetylglucosamine containing milk oligosaccharide; N-acetyllactosamine containing milk oligosaccharide; fucosylated N-acetyllactosamine containing milk oligosaccharide; sialylated N-acetyllactosamine containing milk oligosaccharide; lacto-N-biose containing milk oligosaccharide; fucosylated lacto-N-biose containing milk oligosaccharide and sialylated lacto-N-biose containing milk oligosaccharide. In another aspect, the present invention provides for a purified disaccharide and/or a purified oligosaccharide like a milk oligosaccharide, or a purified oligosaccharide mixture like a purified milk oligosaccharide mixture, a purified mixture of disaccharides, or a purified mixture of one or more disaccharide(s) and one or more milk oligosaccharide(s) as described herein for use in medicine, preferably for use in prophylaxis or therapy of a gastrointestinal disorder.
In another aspect, the present invention provides use of a purified disaccharide and/or oligosaccharide like a milk oligosaccharide obtained by a method as described herein in a food or feed preparation, in a dietary supplement, in a cosmetic ingredient or in a pharmaceutical ingredient. In some embodiments, said disaccharide and/or oligosaccharide like a milk oligosaccharide is/are mixed with one or more ingredients suitable for food, feed, dietary supplement, pharmaceutical ingredient, cosmetic ingredient or medicine. Said purified disaccharide and/or oligosaccharide like a milk oligosaccharide may be used for the manufacture of a preparation, as food additive, prebiotic, symbiotic, for the supplementation of baby food, adult food, infant animal feed, adult animal feed, or as either therapeutically or pharmaceutically active compound or in cosmetic applications. In another aspect, the present invention provides use of a disaccharide and/or a milk oligosaccharide as described herein as additive in food, preferably as additive in human food and/or pet food, more preferably as additive in human baby food. In the context of present invention, the food is a human food, preferably infant food, human baby food and/or an infant formula or an infant supplement and the feed is a pet food, animal milk replacer, veterinary product, veterinary feed supplement, nutrition supplement, post weaning feed, or creep feed.
In another preferred embodiment, a preparation is provided that further comprises at least one probiotic microorganism. In another preferred embodiment of present invention, said preparation is a nutritional composition. In a more preferred embodiment, said preparation is a medicinal formulation, a dietary supplement, a dairy drink or an infant formula. A "prebiotic" is a substance that promotes growth of microorganisms beneficial to the host, particularly microorganisms in the gastrointestinal tract. In some embodiments, a dietary supplement provides multiple prebiotics, including said disaccharide and/or oligosaccharide like a milk oligosaccharide being a prebiotic purified by a method disclosed in this specification, to promote growth of one or more beneficial microorganisms. Examples of prebiotic ingredients for dietary supplements include other prebiotic molecules (such as HMDs) and plant polysaccharides (such as inulin, pectin, b-glucan and xylooligosaccharide). A "probiotic" product typically contains live microorganisms that replace or add to gastrointestinal microflora, to the benefit of the recipient. Examples of such microorganisms include Lactobacillus species (for example, L. acidophilus and L. bulgaricus), Bifidobacterium species (for example, B. animalis, B. longum and B. infantis (e.g., Bi-26)), and Saccharomyces boulardii. In some embodiments, said disaccharide and/or oligosaccharide like a milk oligosaccharide produced and/or purified by a method of this specification is/are orally administered in combination with such microorganism. Examples of further ingredients for dietary supplements include oligosaccharides (such as 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose), disaccharides (such as lactose), monosaccharides (such as glucose, galactose, L-fucose, sialic acid, glucosamine and N-acetylglucosamine), thickeners (such as gum arabic), acidity regulators (such as trisodium citrate), water, skimmed milk, and flavourings.
In some embodiments, said disaccharide and/or oligosaccharide like a milk oligosaccharide purified by a method as described herein is/are incorporated into a human baby food (e.g., infant formula). Infant formula is generally a manufactured food for feeding to infants as a complete or partial substitute for human breast milk. In some embodiments, infant formula is sold as a powder and prepared for bottle- or cup-feeding to an infant by mixing with water. The composition of infant formula is typically designed to be roughly mimic human breast milk. In some embodiments, said disaccharide and/or oligosaccharide like a milk oligosaccharide purified by a method as described herein is/are included in infant formula to provide nutritional benefits similar to those provided by the disaccharides and/or oligosaccharides in human breast milk. In some embodiments, said purified disaccharide and/or oligosaccharide like a milk oligosaccharide is/are mixed with one or more ingredients of the infant formula. Examples of infant formula ingredients include non-fat milk, carbohydrate sources (e.g., lactose), protein sources (e.g., whey protein concentrate and casein), fat sources (e.g., vegetable oils - such as palm, high oleic safflower oil, rapeseed, coconut and/or sunflower oil; and fish oils), vitamins (such as vitamins A, Bb, Bi2, C and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate and calcium phosphate) and possibly human milk oligosaccharides (HMDs). In some embodiments, the one or more infant formula ingredients comprise non-fat milk, a carbohydrate source, a protein source, a fat source, and/or a vitamin and mineral. In some embodiments, the one or more infant formula ingredients comprise lactose, whey protein concentrate and/or high oleic safflower oil. In some embodiments, the concentration of the disaccharide and/or oligosaccharide like a milk oligosaccharide in the infant formula is/are approximately the same concentration as the concentration of the disaccharide and/or oligosaccharide generally present in human breast milk. In some embodiments, a disaccharide and/or an oligosaccharide like a milk oligosaccharide purified by a method as described herein is/are added to the infant formula with a concentration that is approximately the same concentration as the concentration of the compound generally present in human breast milk.
Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described above and below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, purification steps are performed according to the manufacturer's specifications.
Further advantages follow from the specific embodiments and the examples. It goes without saying that the abovementioned features and the features which are still to be explained below can be used not only in the respectively specified combinations, but also in other combinations or on their own, without departing from the scope of the present invention.
Moreover, the present invention relates to the following specific embodiments:
1. A cell capable of synthesizing, preferably synthesizing, UDP-N-acetylglucosamine (UDP-GIcNAc), said cell comprising a pathway for production of an oligosaccharide, said cell genetically engineered for the production of said oligosaccharide, characterized in that said UDP-GIcNAc synthesis in said cell is rendered less functional.
2. Cell according to embodiment 1, wherein said pathway for production of said oligosaccharide is chosen from the list comprising fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway, preferably said cell is genetically engineered to comprise at least one of said pathway(s), more preferably said cell comprises at least one of said pathway(s) wherein at least one of said pathway(s) has/have been genetically engineered.
3. Cell according to any one of embodiment 1 or 2, wherein said cell: possesses, preferably expresses, more preferably overexpresses, one or more glycosyltransferase(s) chosen from the list comprising fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N- acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N- acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl- beta-L-altrosamine transaminases, UDP-/V-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases, and/or is capable to produce, preferably produces, one or more nucleotide-activated sugars, preferably said cell is genetically engineered for production of one or more of said nucleotide-activated sugar(s).
4. Cell according to any one of previous embodiments, wherein said cell comprises a pathway for the synthesis of a nucleotide-activated sugar chosen from the list comprising UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP- ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP-glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy--L-arabino-4- hexulose, UDP-2-acetamido-2,6-dideoxy--L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L- RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N- acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L- pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), GDP- rhamnose and UDP-xylose. Cell according to any one of previous embodiments, wherein said cell possesses, preferably expresses, one or more genes chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L- glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N- acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2- epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GIcNAc 2-epimerase/kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, Neu5Ac synthase, N- acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9- phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4- epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, more preferably overexpresses one or more genes chosen from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6- dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N- acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GIcNAc 2- epimerase/kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, Neu5Ac synthase, N-acetylneuraminate lyase, N- acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-l-epimerase, galactokinase, glucokinase, galactose- l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase. Cell according to any one of previous embodiments, wherein said UDP-GIcNAc synthesis possesses at least one gene chosen from the list comprising genes encoding bifunctional N-acetylglucosamine-1- phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, an N-acetylglucosamine-1- phosphate uridyltransferase and a glucosamine-l-phosphate acetyltransferase, and wherein said at least one gene is rendered less functional. Cell according to embodiment 6, wherein said at least one gene encodes an enzyme, wherein said enzyme: is chosen from an enzyme class chosen from the list comprising EC:2.7.7.23, EC:2.3.1.157 and EC:5.4.2.3, comprises a polypeptide sequence comprising an IPR domain chosen from the list comprising IPR001451, IPR002618, IPR005175, IPR005835, IPR005843, IPR005844, IPR005882, IPR011004, IPR016055, IPR016066, IPR016657, IPR018357, IPR023915, IPR025877, IPR029044, IPR036900 and IPR038009 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising a PFAM domain chosen from the list comprising PF00132, PF00408, PF00465, PF00483, PF01070, PF01565, PF01704, PF02878, PF02879, PF02880, PF03479, PF04030, PF05199, PF12146, PF12804, PF13552 and PF14602 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising a conserved protein domain chosen from the list comprising cd03086 and cd03353 as defined by InterPro 90.0 as released on 4th August 2022, is part of a NOG family chosen from the list comprising COG1109, COG4284 as defined by eggNOG5.0 as released in 2019, and/or uses a cofactor chosen from the list comprising Mg2+, Co2+, Mn2+, Ca2+, Zn2+, Ni2+ and FAD. Cell according to any one of previous embodiments, wherein at least one gene involved in the synthesis and/or import of a co-factor that is involved in UDP-GIcNAc synthesis is rendered less functional, preferably said co-factor is chosen from the list comprising Mg2+, Co2+, Mn2+, Ca2+, Zn2+, Ni2+ and FAD. Cell according to any one of embodiments 6 to 8, wherein said at least one gene is rendered less functional by insertion, deletion and/or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) chosen from the list comprising promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said at least one gene. Cell according to any one of previous embodiments, wherein said oligosaccharide is chosen from the list comprising neutral (non-charged) oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide, milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMD); sialylated milk oligosaccharide, neutral (noncharged) milk oligosaccharide, fucosylated milk oligosaccharide, non-fucosylated neutral (non- charged) milk oligosaccharide, sialylated mammalian milk oligosaccharide, neutral (non-charged) mammalian milk oligosaccharide, fucosylated mammalian milk oligosaccharide, non-fucosylated neutral (non-charged) mammalian milk oligosaccharide, sialylated human milk oligosaccharide, neutral (non-charged) human milk oligosaccharide, fucosylated human milk oligosaccharide, non- fucosylated neutral (non-charged) human milk oligosaccharide; O-antigen; enterobacterial common antigen (ECA); the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an amino-sugar; Lewis-type antigen oligosaccharide; an antigen of the human ABO blood group system; an animal oligosaccharide, preferably selected from the group consisting of N-glycans and O-glycans; a plant oligosaccharide, preferably selected from the group consisting of N-glycans and O-glycans; fucosylated oligosaccharide, preferably selected from the group comprising 2'-fucosyllactose (2' FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N- difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N- neohexaose; sialylated oligosaccharide, preferably selected from the group comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto- N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N- neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'- sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N-acetylglucosamine containing neutral (non-charged) oligosaccharide, preferably selected from the group lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N- neohexaose; N-acetylglucosamine containing oligosaccharide; N-acetyllactosamine containing oligosaccharide; lacto-N-biose containing oligosaccharide; non-fucosylated neutral (non-charged) oligosaccharide; chitosan; chitosan comprising oligosaccharide; heparosan; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan sulphate; dermatan sulphate; hyaluronan; hyaluronic acid; and keratan sulphate.
11. Cell according to any one of previous embodiments, wherein said cell is capable to produce, preferably produces, said oligosaccharide from one or more precursor(s), preferably said precursor is lactose.
12. Cell according to embodiment 11, 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).
13. Cell according to any one of embodiment 11 or 12, 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).
14. Cell according to any one of embodiments 11 to 13, wherein at least one of said one or more precursor(s) is internalized in said cell via one or more membrane protein(s).
15. Cell according to any one of previous embodiments, wherein said cell is further genetically engineered to possess, preferably to express, more preferably to over-express, a glutamine— fructose-6-phosphate aminotransferase.
16. Cell according to embodiment 15, wherein said glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and: is chosen from the enzyme class EC:2.6.1.16, comprises a polypeptide sequence comprising an IPR domain chosen from the list comprising IPR001347, IPR005855, IPR017932, IPR029055, IPR035466, IPR035490, IPR036291, IPR046348 and IPR047084 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising a PFAM domain chosen from the list comprising PF00310, PF01380, PF01408, PF13230, PF13537 and PF13580 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising a conserved protein domain chosen from the list comprising cd00714, cd05007, cd05008, cd05009, cd05013 and cd05710 as defined by InterPro 90.0 as released on 4th August 2022, and/or is part of the NOG family COG0449 as defined by eggNOG5.0 as released in 2019.
17. Cell according to any one of embodiment 15 or 16, wherein said cell contains a nucleic acid molecule which comprises a polynucleotide sequence which encodes said glutamine— fructose-6-phosphate aminotransferase.
18. Cell according to embodiment 17, wherein said nucleic acid molecule is operably linked to control sequences recognized by the cell, said nucleic acid molecule further i) being integrated in the genome of said cell and/or ii) presented to said cell on a vector.
19. Cell according to any one of embodiment 17 or 18, wherein said nucleic acid molecule is foreign to said cell.
20. Cell according to any one of previous embodiments, wherein said cell is modified for enhanced synthesis and/or supply of phosphoenolpyruvate (PEP).
21. Cell according to any one of previous embodiments, wherein said cell is further modified for reduced degradation of acetyl-CoA and/or its main precursor pyruvate.
22. Cell according to any one of previous embodiments, wherein said cell comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the synthesis of said oligosaccharide.
23. Cell according to any one of previous embodiments, wherein said cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell, preferably, said bacterium belongs to a phylum chosen from the group comprising Proteobacteria, Firmicutes, Cyanobacteria, Deinococcus-Thermus and Actinobacteria; more preferably, said bacterium belongs to a family chosen from the group comprising Enterobacteriaceae, Bacillaceae, Lactobacillaceae, Corynebacteriaceae and Vibrionaceae; even more preferably, said bacterium is chosen 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. coli MG1655, preferably, said fungus belongs to a genus chosen from the group comprising Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus, preferably, said yeast belongs to a genus chosen from the group comprising Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces, Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces or Torulaspora; more preferably, said yeast is selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipites, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Yarrowia lipolytica, Zygosaccharomyces rouxii, and Zygosaccharomyces bailii, preferably, said plant cell is an algal cell or is derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soy, maize, or corn plant, preferably, said animal cell is derived from insects, amphibians, reptiles, invertebrates, fish, birds or mammalian cells excluding human embryonic stem cells, more preferably said mammalian cell is chosen from the list comprising an epithelial cell, an embryonic kidney cell, a fibroblast cell, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell, an NIH-3T3 cell, a lactocyte derived from mammalian induced pluripotent stem cells, more preferably said mammalian induced pluripotent stem cells are human induced pluripotent stem cells, a post-parturition mammary epithelium cell, a polarized mammary cell, more preferably said polarized mammary cell is selected from the group comprising live primary mammary epithelial cells, live mammary myoepithelial cells, live mammary progenitor cells, live immortalized mammary epithelial cells, live immortalized mammary myoepithelial cells, live immortalized mammary progenitor cells, a non-mammary adult stem cell or derivatives thereof, more preferably said insect cell is derived from Spodoptera frugiperda, Bombyx mori, Mamestra brassicae, Trichoplusia ni or Drosophila melanogaster, preferably, said protozoan cell is a Leishmania tarentolae cell.
24. Cell according to any one of previous embodiments, wherein said cell is a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell. 25. Cell according to any one of previous embodiments, wherein said cell is an E. coli or yeast with a lactose permease positive phenotype, preferably wherein said lactose permease is coded by the gene LacY or LAC12, respectively.
26. Method for the production of an oligosaccharide, the method comprising: i. cultivating and/or incubating a cell of any one of previous embodiments, in cultivation and/or incubation medium under conditions permissive to produce said oligosaccharide, ii. preferably, separating said oligosaccharide from said cultivation and/or incubation.
27. Method according to embodiment 26, wherein said cultivation or incubation medium comprises one or more precursor(s) that is/are used for production of said oligosaccharide.
28. Method according to any one of embodiment 26 or 27, wherein said cultivation medium contains at least one carbon source selected from the group consisting of glucose, fructose, sucrose, and glycerol.
29. Method according to any one of embodiments 26 to 28, wherein said cultivation or incubation medium contains at least one compound selected from the group consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid and CMP-sialic acid.
30. Method according to any one of embodiments 26 to 29, wherein said cell produces 30 g/L or more of said oligosaccharide in the whole broth and/or supernatant and/or wherein said oligosaccharide in the whole broth and/or supernatant has a purity of at least 80 % measured on the total amount of oligosaccharide and its precursor(s) produced by said cell in the whole broth and/or supernatant, respectively.
31. Method according to any one of embodiments 26 to 30, wherein said less functional synthesis of UDP-GIcNAc confers unaffected and/or enhanced i) oligosaccharide formation, ii) productivity, iii) biomass production, iv) cell growth and/or v) yield of the produced oligosaccharide, relative to a corresponding non-engineered cell.
32. Method according to any one of embodiments 26 to 31, wherein said oligosaccharide is recovered from said cultivation or incubation medium and/or said cell, more preferably said oligosaccharide is purified.
33. Use of a cell according to any one of embodiments 1 to 25 for the production of an oligosaccharide, wherein said oligosaccharide is chosen from the list comprising neutral (non-charged) oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide, milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); sialylated milk oligosaccharide, neutral (non-charged) milk oligosaccharide, fucosylated milk oligosaccharide, non-fucosylated neutral (non-charged) milk oligosaccharide, sialylated mammalian milk oligosaccharide, neutral (non-charged) mammalian milk oligosaccharide, fucosylated mammalian milk oligosaccharide, non-fucosylated neutral (non-charged) mammalian milk oligosaccharide, sialylated human milk oligosaccharide, neutral (non-charged) human milk oligosaccharide, fucosylated human milk oligosaccharide, non-fucosylated neutral (non-charged) human milk oligosaccharide; O-antigen; enterobacterial common antigen (ECA); the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an amino-sugar; Lewis-type antigen oligosaccharide; an antigen of the human ABO blood group system; an animal oligosaccharide, preferably selected from the group consisting of N-glycans and O-glycans; a plant oligosaccharide, preferably selected from the group consisting of N-glycans and O-glycans; fucosylated oligosaccharide, preferably selected from the group comprising 2'-fucosyllactose (2'FL), 3- fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto- N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N- difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N- neohexaose; sialylated oligosaccharide, preferably selected from the group comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto- N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N- neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'- sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N-acetylglucosamine containing neutral (non-charged) oligosaccharide, preferably selected from the group lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N- neohexaose; N-acetylglucosamine containing oligosaccharide; N-acetyllactosamine containing oligosaccharide; lacto-N-biose containing oligosaccharide; non-fucosylated neutral (non-charged) oligosaccharide; chitosan; chitosan comprising oligosaccharide; heparosan; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan sulphate; dermatan sulphate; hyaluronan; hyaluronic acid; and keratan sulphate. Use of a method according to any one of embodiments 26 to 32 for the production of an oligosaccharide, wherein said oligosaccharide is chosen from the list comprising neutral (noncharged) oligosaccharide, a negatively charged, preferably sialylated, oligosaccharide, milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); sialylated milk oligosaccharide, neutral (non-charged) milk oligosaccharide, fucosylated milk oligosaccharide, non-fucosylated neutral (non-charged) milk oligosaccharide, sialylated mammalian milk oligosaccharide, neutral (non-charged) mammalian milk oligosaccharide, fucosylated mammalian milk oligosaccharide, non-fucosylated neutral (non-charged) mammalian milk oligosaccharide, sialylated human milk oligosaccharide, neutral (non-charged) human milk oligosaccharide, fucosylated human milk oligosaccharide, non-fucosylated neutral (non-charged) human milk oligosaccharide; O-antigen; enterobacterial common antigen (ECA); the oligosaccharide repeats present in capsular polysaccharides; peptidoglycan; an amino-sugar; Lewis-type antigen oligosaccharide; an antigen of the human ABO blood group system; an animal oligosaccharide, preferably selected from the group consisting of N-glycans and O-glycans; a plant oligosaccharide, preferably selected from the group consisting of N-glycans and O-glycans; fucosylated oligosaccharide, preferably selected from the group comprising 2'-fucosyllactose (2'FL), 3- fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto- N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N- difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N- neohexaose; sialylated oligosaccharide, preferably selected from the group comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto- N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N- neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'- sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N-acetylglucosamine containing neutral (non-charged) oligosaccharide, preferably selected from the group lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N- neohexaose; N-acetylglucosamine containing oligosaccharide; N-acetyllactosamine containing oligosaccharide; lacto-N-biose containing oligosaccharide; non-fucosylated neutral (non-charged) oligosaccharide; chitosan; chitosan comprising oligosaccharide; heparosan; chondroitin sulphate; glycosaminoglycan oligosaccharide; heparin; heparan sulphate; dermatan sulphate; hyaluronan; hyaluronic acid; and keratan sulphate.
More specifically, the present invention relates to the following preferred specific embodiments:
1. A cell capable of synthesizing and/or synthesizing UDP-N-acetylglucosamine (UDP-GIcNAc), said cell comprising a pathway for production of a disaccharide and/or milk oligosaccharide, said cell genetically engineered for the production of said disaccharide and/or milk oligosaccharide, characterized in that said UDP-GIcNAc synthesis in said cell is rendered less functional.
2. Cell according to preferred embodiment 1, wherein: said pathway for production of said disaccharide and/or milk oligosaccharide is selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway, and/or said pathway for production of said disaccharide and/or milk oligosaccharide is selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway and wherein said cell is genetically engineered to comprise at least one of said pathway(s) and/or said cell comprises at least one of said pathway(s) wherein at least one of said pathway(s) has/have been genetically engineered. according to any one of preferred embodiment 1 or 2, wherein said cell: possesses, expresses and/or overexpresses one or more glycosyltransferase(s) selected from the list consisting of or consisting essentially of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N- acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N- acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl- beta-L-altrosamine transaminases, UDP-/V-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases, is capable to produce and/or produces one or more nucleotide-activated sugars, is genetically engineered for production of one or more nucleotide-activated sugar(s), comprises a pathway for the synthesis of a nucleotide-activated sugar selected from the list consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N- acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP- glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2- acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N- acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP- Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), GDP-rhamnose and UDP-xylose, possesses and/or expresses one or more genes selected from the list consisting of or consisting essentially of mannose-5-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,5-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N- acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6- phosphate 2-epimerase, UDP-GIcNAc 2-epimerase/kinase, glucosamine 6-phosphate N- acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine- 1-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminate lyase, N- acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-1- phosphate uridylyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N- acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, more preferably overexpresses one or more genes selected from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2- epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2- epimerase, UDP-GIcNAc 2-epimerase/kinase, glucosamine 6-phosphate N-acetyltransferase, N- acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, Neu5Ac synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N- acylneuraminate-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase, UDP- N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N- acetylgalactosamine pyrophosphorylase, and/or comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the synthesis of said disaccharide and/or milk oligosaccharide.
4. Cell according to any one of previous preferred embodiments, wherein said UDP-GIcNAc synthesis possesses at least one gene selected from the list consisting of or consisting essentially of genes encoding bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-1- phosphate acetyltransferase, an N-acetylglucosamine-l-phosphate uridyltransferase and a glucosamine-l-phosphate acetyltransferase, and wherein said at least one gene is rendered less functional.
5. Cell according to preferred embodiment 4, wherein said at least one gene encodes an enzyme, wherein said enzyme: is selected from an enzyme class selected from the list consisting of or consisting essentially of EC:2.7.7.23, EC:2.3.1.157 and EC:5.4.2.3, comprises a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of IPR001451, IPR002618, IPR005175, IPR005835, IPR005843, IPR005844, IPR005882, IPR011004, IPR016055, IPR016066, IPR016657, IPR018357, IPR023915, IPR025877, IPR029044, IPR036900 and IPR038009 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of PF00132, PF00408, PF00465, PF00483, PF01070, PF01565, PF01704, PF02878, PF02879, PF02880, PF03479, PF04030, PF05199, PF12146, PF12804, PF13562 and PF14602 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising a conserved protein domain selected from the list consisting of or consisting essentially of cd03086 and cd03353 as defined by InterPro 90.0 as released on 4th August 2022, is part of a NOG family selected from the list consisting of or consisting essentially of COG1109 and COG4284 as defined by eggNOG5.0 as released in 2019, and/or uses a cofactor selected from the list consisting of or consisting essentially of Mg2+, Co2+, Mn2+, Ca2+, Zn2+, Ni2+ and FAD.
6. Cell according to any one of previous preferred embodiments, wherein at least one gene involved in the synthesis and/or import of: a co-factor that is involved in UDP-GIcNAc synthesis is rendered less functional, and/or a co-factor that is involved in UDP-GIcNAc synthesis is rendered less functional and that is selected from the list consisting of or consisting essentially of Mg2+, Co2+, Mn2+, Ca2+, Zn2+, Ni2+ and FAD.
7. Cell according to any one of preferred embodiments 4 to 6, wherein said at least one gene is rendered less functional by insertion, deletion and/or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) selected from the list comprising promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said at least one gene.
8. Cell according to any one of previous preferred embodiments, wherein said disaccharide and/or milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; a negatively charged milk oligosaccharide; sialylated milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide; non-fucosylated neutral (noncharged) mammalian milk oligosaccharide; sialylated human milk oligosaccharide; neutral (noncharged) human milk oligosaccharide; fucosylated human milk oligosaccharide; non-fucosylated neutral (non-charged) human milk oligosaccharide; fucosylated milk oligosaccharide selected from the list comprising 2'-fucosyl lactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6- fucosyllactose (6F L), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N- hexaose and difucosyl-lacto-N-neohexaose; sialylated milk oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N- tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N- tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N-acetylglucosamine containing neutral (non-charged) milk oligosaccharide; N-acetylglucosamine containing neutral (non-charged) milk oligosaccharide selected from the list of lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'- galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose and para-lacto-N-neohexaose; N-acetylglucosamine containing milk oligosaccharide; N- acetyllactosamine containing milk oligosaccharide; fucosylated N-acetyllactosamine containing milk oligosaccharide; sialylated N-acetyllactosamine containing milk oligosaccharide; lacto-N-biose containing milk oligosaccharide; fucosylated lacto-N-biose containing milk oligosaccharide and sialylated lacto-N-biose containing milk oligosaccharide.
9. Cell according to any one of previous preferred embodiments, wherein said cell: is capable to produce and/or produces said disaccharide and/or milk oligosaccharide from one or more precursor(s), is capable to produce and/or produces said disaccharide and/or milk oligosaccharide from lactose, is capable to produce and/or produces at least one precursor that is used to produce said disaccharide and/or milk oligosaccharide, is capable to produce and/or produces all precursors that are used to produce said disaccharide and/or milk oligosaccharide, is genetically engineered for the production of at least one precursor that is used to produce said disaccharide and/or milk oligosaccharide, and/or is genetically engineered for the production of all precursors that are used to produce said disaccharide and/or milk oligosaccharide.
10. Cell according to preferred embodiment 9, wherein at least one of said one or more precursor(s) is internalized in said cell via one or more membrane protein(s).
11. Cell according to any one of previous preferred embodiments, wherein said cell is further genetically engineered to possess, to express and/or to over-express a glutamine— fructose-6-phosphate aminotransferase.
12. Cell according to preferred embodiment 11, wherein said glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and: is selected from the enzyme class EC:2.6.1.16, comprises a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of IPR001347, IPR005855, IPR017932, IPR029055, IPR035466, IPR035490, IPR036291, IPR046348 and IPR047084 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of PF00310, PF01380, PF01408, PF13230, PF13537 and PF13580 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising a conserved protein domain selected from the list consisting of or consisting essentially of cd00714, cd05007, cd05008, cd05009, cd05013 and cd05710 as defined by InterPro 90.0 as released on 4th August 2022, and/or is part of the NOG family COG0449 as defined by eggNOG5.0 as released in 2019.
13. Cell according to any one of preferred embodiment 11 or 12, wherein said cell contains a nucleic acid molecule which comprises a polynucleotide sequence which encodes said glutamine— fructose-6- phosphate aminotransferase and wherein said nucleic acid molecule is: operably linked to control sequences recognized by the cell, said nucleic acid molecule further i) being integrated in the genome of said cell and/or ii) presented to said cell on a vector, and/or foreign to said cell.
14. Cell according to any one of previous preferred embodiments, wherein said cell is: modified for enhanced synthesis and/or supply of phosphoenolpyruvate (PEP), and/or further modified for reduced degradation of acetyl-CoA and/or its main precursor pyruvate.
15. Cell according to any one of previous preferred embodiments, wherein said cell is: a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell, an E. coli or yeast with a lactose permease positive phenotype, and/or an E. coli or yeast with a lactose permease positive phenotype wherein said lactose permease is coded by the gene LacY or LAC12, respectively.
16. Method for the production of a disaccharide and/or milk oligosaccharide, the method comprising: i. cultivating and/or incubating a cell of any one of previous preferred embodiments, in cultivation and/or incubation medium under conditions permissive to produce said disaccharide and/or milk oligosaccharide, and/or ii. separating said disaccharide and/or milk oligosaccharide from said cultivation and/or incubation.
17. Method according to preferred embodiment 16, wherein said: cultivation or incubation medium comprises one or more precursor(s) that is/are used for production of said disaccharide and/or milk oligosaccharide, cultivation medium contains at least one carbon source selected from the list consisting of glucose, fructose, sucrose, and glycerol, and/or cultivation or incubation medium contains at least one compound selected from the list consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid and CMP-sialic acid.
18. Method according to any one of preferred embodiment 16 or 17, wherein said cell produces 30 g/L or more of said disaccharide and/or milk oligosaccharide in the whole broth and/or supernatant and/or wherein said disaccharide and/or milk oligosaccharide in the whole broth and/or supernatant has a purity of at least 80 % measured on the total amount of disaccharide and/or milk oligosaccharide and its/their precursor(s) produced by said cell in the whole broth and/or supernatant, respectively.
19. Method according to any one of preferred embodiments 16 to 18, wherein said less functional synthesis of UDP-GIcNAc confers unaffected and/or enhanced i) disaccharide and/or milk oligosaccharide formation, ii) productivity, iii) biomass production, iv) cell growth and/or v) yield of the produced disaccharide and/or milk oligosaccharide, relative to a corresponding non-engineered cell.
20. Method according to any one of preferred embodiments 16 to 19, wherein said: disaccharide and/or milk oligosaccharide is/are recovered from said cultivation or incubation medium and/or said cell, and/or disaccharide and/or milk oligosaccharide is/are purified.
21. Use of a cell according to any one of preferred embodiments 1 to 15 for the production of a disaccharide and/or milk oligosaccharide, wherein said disaccharide and/or milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; negatively charged milk oligosaccharide; sialylated milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide; non-fucosylated neutral (non-charged) mammalian milk oligosaccharide; sialylated human milk oligosaccharide; neutral (non-charged) human milk oligosaccharide; fucosylated human milk oligosaccharide; non-fucosylated neutral (non-charged) human milk oligosaccharide; fucosylated milk oligosaccharide selected from the list comprising 2'- fucosyl lactose (2'FL), 3-fucosyl lactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2', 3- difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto- N-neohexaose; sialylated milk oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyl lacto-N- tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N- neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3- fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N- fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N- acetylglucosamine containing neutral (non-charged) milk oligosaccharide; N-acetylglucosamine containing neutral (non-charged) milk oligosaccharide selected from the list of lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto- N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose and para-lacto-N-neohexaose; N- acetylglucosamine containing milk oligosaccharide; N-acetyllactosamine containing milk oligosaccharide; fucosylated N-acetyllactosamine containing milk oligosaccharide; sialylated N- acetyllactosamine containing milk oligosaccharide; lacto-N-biose containing milk oligosaccharide; fucosylated lacto-N-biose containing milk oligosaccharide and sialylated lacto-N-biose containing milk oligosaccharide. Use of a method according to any one of preferred embodiments 16 to 20 for the production of a disaccharide and/or milk oligosaccharide, wherein said disaccharide and/or milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; negatively charged milk oligosaccharide; sialylated milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide; non-fucosylated neutral (non-charged) mammalian milk oligosaccharide; sialylated human milk oligosaccharide; neutral (non-charged) human milk oligosaccharide; fucosylated human milk oligosaccharide; non-fucosylated neutral (non-charged) human milk oligosaccharide; fucosylated milk oligosaccharide selected from the list comprising 2'- fucosyl lactose (2'FL), 3-fucosyl lactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2', 3- difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto- N-neohexaose; sialylated milk oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyl lacto-N- tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N- neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3- fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N- fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N- acetylglucosamine containing neutral (non-charged) milk oligosaccharide; N-acetylglucosamine containing neutral (non-charged) milk oligosaccharide selected from the list of lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto- N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose and para-lacto-N-neohexaose; N- acetylglucosamine containing milk oligosaccharide; N-acetyllactosamine containing milk oligosaccharide; fucosylated N-acetyllactosamine containing milk oligosaccharide; sialylated N- acetyllactosamine containing milk oligosaccharide; lacto-N-biose containing milk oligosaccharide; fucosylated lacto-N-biose containing milk oligosaccharide and sialylated lacto-N-biose containing milk oligosaccharide.
The invention will be described in more detail in the examples. The following examples will serve as further illustration and clarification of the present invention and are not intended to be limiting.
Examples
Example 1. Materials and Methods
A. Escherichia coli
Media and cultivation
The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). The minimal medium used in cultivation experiments in 96-well plates or in shake flasks contained 2.00 g/L NH4CI, 5.00 g/L (NH4)2SO4, 2.993 g/L KH2PO4, 7.315 g/L K2HPO4, 8.372 g/L MOPS, 0.5 g/L NaCI, 0.5 g/L MgSO4.7H2O, 30 g/L sucrose or 30 g/L glycerol, 1 ml/L vitamin solution, 100 pil/L molybdate solution, and 1 mL/L selenium solution. As precursor(s) and/or acceptor(s) for saccharide, like disaccharide and/or milk oligosaccharide, synthesis, compounds like e.g., galactose, glucose, fructose, fucose, lactose, a co-factor could be added to the medium. The minimal medium was set to a pH of 7 with IM KOH. Vitamin solution consisted of 3.6 g/L FeCI2.4H2O, 5.0 g/LCaCI2.2H20, 1.3 g/L MnCI2.2H2O, 0.38 g/L CuCI2.2H2O, 0.5 g/L CoCI2.6H2O, 0.94 g/L ZnCI2, 0.0311 g/L H3BO4, 0.4 g/L Na2EDTA.2H2O and 1.01 g/L thiamine.HCL The molybdate solution contained 0.967 g/L NaMoO4.2H2O. The selenium solution contained 42 g/L Seo2. The minimal medium for fermentations contained 6.75 g/L NH4CI, 1.25 g/L (NH4)2SO4, 2.93 g/L KH2PO4 and 7.31 g/L KH2PO4, 0.5 g/L NaCI, 0.5 g/L M SO4.7H2O, 30 g/L sucrose or 30 g/L lycerol, 1 mL/L vitamin solution, 100 pL/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above. As specified in the respective examples, 20 g/L lactose and/or 20 g/L glucose were additionally added to the medium. Complex medium was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 pm Sartorius). When necessary, the medium was made selective by adding an antibiotic: e.g., chloramphenicol (20 mg/L), carbenicillin (100 mg/L), spectinomycin (40 mg/L) and/or kanamycin (50 mg/L). A preculture of 96-well microtiter plate experiments was started from a cryovial, in 150 pL LB and was incubated overnight at 37 °C on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96well square microtiter plate, with 400 pL minimal medium by diluting 400x. These final 96-well culture plates were then incubated at 37°C on an orbital shaker at 800 rpm for 72h, or shorter, or longer. To measure sugar concentrations at the end of the cultivation experiment whole broth samples were taken from each well by boiling the culture broth for 15 min at 60°C before spinning down the cells (= average of intra- and extracellular sugar concentrations). 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, Melsungen, 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.
Strains and mutations
Escherichia coli K12 MG1655 [A-, F-, rph-1] was obtained from the Coli Genetic Stock Center (US), CGSC Strain#: 7740, in March 2007. Gene disruptions, gene introductions and gene replacements were performed using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). All constitutive promoters, UTRs and terminator sequences originated from the libraries described by Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148), Dunn et al. (Nucleic Acids Res. 1980, 8, 2119- 2132), Edens et al. (Nucleic Acids Res. 1975, 2, 1811-1820), Kim and Lee (FEBS Letters 1997, 407, 353-356) and Mutalik et al. (Nat. Methods 2013, No. 10, 354-360). Genes were ordered synthetically at Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and the codon usage was adapted using the tools of the supplier. All strains were stored in cryovials at -80°C (overnight LB culture mixed in a 1:1 ratio with 70% glycerol).
In an example for GDP-fucose production, the mutant strain was derived from E. coli K12 MG1655 comprising a knock-out of the E. coli wcaJgene and genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E. coli \N (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6). GDP-fucose production can further be optimized in the mutant E. coli strain by genomic knock-outs of any one or more of the E. coli genes comprising glgC, agp, pfkA, pfkB, pgi, arcA, icIR, pgi and Ion as described in WO2016075243 and W02012007481. GDP-fucose production can additionally be optimized comprising genomic knock-ins of constitutive transcriptional units for a mannose-6-phosphate isomerase like e.g. manA from E. coli (UniProt ID P00946), a phosphomannomutase like e.g. manB from E. coli (UniProt ID P24175), a mannose- 1-phosphate guanylyltransferase like e.g. manC from E. coli (UniProt ID P24174), a GDP-mannose 4,6- dehydratase like e.g. gmd from E. coli (UniProt ID P0AC88) and a GDP-L-fucose synthase like e.g. fcl from E. coli (UniProt ID P32055). GDP-fucose production can also be obtained by genomic knock-outs of the E. coli fucK and fuel genes and genomic knock-ins of constitutive transcriptional units containing a fucose permease like e.g. fucP from E. coli (UniProt ID P11551) and a bifunctional enzyme with fucose kinase/fucose-l-phosphate guanylyltransferase activity like e.g. fkp from Bacteroides fragilis (UniProt ID SUV40286.1). All mutant strains can be 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. the E. coli LacY (UniProt ID P02920). For production of a fucosylated saccharide, the mutant GDP-fucose production strain was additionally modified with an expression plasmid comprising a constitutive transcriptional unit for a fucosyltransferase, like e.g. the alpha-1, 2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435) to produce 2'-fucosyllactose (2' FL) or the alpha-1, 3-fucosyltransferase HpFucT from H. pylori (UniProt ID 030511) to produce 3-fucosyl lactose (3-FL), or both the alpha-1, 2- fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435) and the alpha-1, 3-fucosyltransferase HpFucT from H. pylori (UniProt ID 030511) to produce difucosyllactose (DiFL).
In an example to produce lacto-N-triose (LN3, GlcNAc-pi,3-Gal-pi,4-Glc), the mutant strain was derived from E. coli K12 MG1655 and modified with a knock-out of the E. coli lacZ, lacY, lacA and nagB genes and with genomic knock-ins of constitutive transcriptional units for a lactose permease like e.g. the E. coli LacY (UniProt ID P02920) and a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. IgtA (UniProt ID Q9JXQ6) from N. meningitidis. In an example for production of LN3 derived oligosaccharides like lacto-/V- tetraose (LNT, Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc), the mutant LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g. wbgO (Uniprot ID D3QY14) from E. coli 055:1-17. In an example for production of LN3 derived oligosaccharides like lacto-A/- neotetraose (LNnT, Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc), the mutant LN3 producing strain was further modified with a constitutive transcriptional unit delivered to the strain either via genomic knock-in or from an expression plasmid for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from Neisseria meningitidis. LN3, LNT and/or LNnT production can further be optimized in the mutant E. coli strains with genomic knock-outs of the E. coli genes comprising any one or more of galT, ushA, IdhA and agp. The mutant LN3, LNT and/or LNnT producing strains can also be optionally modified for enhanced UDP-GIcNAc production with a genomic knock-in of a constitutive transcriptional unit for an L-glutamine— D-fructose-6-phosphate aminotransferase like e.g. the E. coli glmS (UniProt ID P17169, sequence version 04, 23 Jan 2007) or a mutant glmS*54 from E. coli with SEQ. ID NO 01 and differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006). The mutant E. coli strains can also optionally be adapted with a genomic knock-in of a constitutive transcriptional unit for an UDP-glucose-4-epimerase like e.g. galE from E. coli (UniProt ID P09147), a phosphoglucosamine mutase like e.g. glmM from E. 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). The mutant LN3, LNT and/or LNnT producing E. coli strains can also optionally be adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E. coli \N (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Zymomonas mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP originating from Bifidobacterium adolescentis (UniProt ID A0ZZH6). In an example for sialic acid production, 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-acetylglucosamine 2-epimerase like e.g. AGE from Bacteroides ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase like e.g. NeuB from Neisseria meningitidis (UniProt ID E0NCD4).
Alternatively, and/or additionally, 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).
Alternatively and/or additionally, sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a phosphoglucosamine mutase like e.g. glmM from E. coli (UniProt ID P31120, sequence version 03, 23 Jan 2007), an N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase like e.g. glmU from E. coli (UniProt ID P0ACC7), an UDP-N-acetylglucosamine 2-epimerase like e.g. NeuCfrom C. jejuni (UniProt ID Q93MP8) and an N-acetylneuraminate synthase like e.g. NeuB from N. meningitidis (UniProt ID E0NCD4).
Alternatively, and/or additionally, sialic acid production can be obtained by genomic knock-ins of constitutive transcriptional units containing a bifunctional UDP-GIcNAc 2-epimerase/N- acetylmannosamine kinase like e.g. from Mus musculus (strain C57BL/6J) (UniProt ID Q91WG8), an N- acylneuraminate-9-phosphate synthetase like e.g. from Pseudomonas sp. UW4 (UniProt ID K9NPH9) and an N-acylneuraminate-9-phosphatase like e.g. from Bacteroides thetaiotaomicron (UniProt ID Q8A712).
Sialic acid production can further be optimized in the mutant E. co// strain with genomic knock-outs of the E. coli genes comprising any one or more of nagA, nagB, nagC, nagD, nagE, nanA, nanE, nanK, manX, manY and manZ as described in WO18122225, and/or genomic knock-outs of the E. coli genes comprising any one or more of nanT, poxB, IdhA, adbE, aldB, pflA, pfIC, ybiY, ackA and/or pta and with genomic knock- ins of constitutive transcriptional units comprising one or more copies of an L-glutamine— D-fructose-6- phosphate aminotransferase like e.g. E. coli glmS (UniProt ID P17169, sequence version 04, 23 Jan 2007) or a mutant glmS*54 from E. coli with SEQ ID NO 01 and differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006), preferably a phosphatase like any one of e.g. the E. coli genes comprising aphA, Cof, HisB, OtsB, SurE, Yaed, YcjU, YedP, YfbT, YidA, YigB, YihX, YniC, YqaB, YrbL, AppA, Gph, SerB, YbhA, YbiV, YbjL, Yfb, YieH, YjgL, YjjG, YrfG and YbiU or PsMupP from Pseudomonas putida, ScDOGl from S. cerevisiae or BsAraL from Bacillus subtilis as described in WO18122225 and an acetyl-CoA synthetase like e.g. acs from E. coli (UniProt ID P27550).
For sialylated oligosaccharide production, said sialic acid production strains were further modified to express an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P. multocida (UniProt ID A0A849CI62) and to express a sialyltransferase like e.g. the alpha-2, 3-siayltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide (SEQ ID NO 02) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2, 3-sialyltransferase activity or the alpha-2, 6-sialyltransferase PdST6 from P. damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity. Constitutive transcriptional units of the N-acylneuraminate cytidylyltransferase and the sialyltransferase can be delivered to the mutant strain either via genomic knock-in or via expression plasmids. If the mutant strains producing sialic acid and CMP-sialic acid were intended to make sialylated lactose structures, the strains were additionally modified with genomic knock-outs of the E. coli LacZ, LacY and LacA genes and with a genomic knock-in of a constitutive transcriptional unit for a lactose permease like e.g. E. coli LacY (UniProt ID P02920). All mutant strains producing sialic acid, CMP-sialic acid and/or sialylated saccharides could optionally be adapted for growth on sucrose via genomic knock-ins of constitutive transcriptional units containing a sucrose transporter like e.g. CscB from E. coli W (UniProt ID E0IXR1), a fructose kinase like e.g. Frk originating from Z. mobilis (UniProt ID Q03417) and a sucrose phosphorylase like e.g. BaSP from 8. adolescentis (UniProt ID A0ZZH6). In another example, the mutant E. coli strains adapted for LNT production as described herein can also be further modified with one or more copies of a glucosamine 6-phosphate N-acetyltransferase like e.g. GNA1 from S. cerevisiae (UniProt ID P43577), an N-acetylglucosamine 2-epimerase like e.g. AGE from B. ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase like e.g. NeuB from N. meningitidis (UniProt ID E0NCD4) and an expression plasmid comprising containing constitutive expression cassettes for the N-acylneuraminate cytidylyltransferase (NeuA) from P. multocida (UniProt ID A0A849CI62) and 1) the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide (SEQ ID NO 02) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having betagalactoside alpha-2, 3-sialyltransferase activity or 2) the alpha-2, 6-sialyltransferase (PdST6) from Photobacterium damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity to produce 1) LSTa (Neu5Ac-oc2,3-Gal-pi,3-GlcNAc-pi,3-Gal-pi,4-Glc) or 2) LSTb (Gal-pi,3- (Neu5Ac-oc2,6)-GlcNAc-pi,3-Gal-pi,4-Glc), respectively. In another example, the mutant E. coli strains adapted for LNnT production as described herein can also be further modified with one or more copies of a glucosamine 6-phosphate N-acetyltransferase like e.g. GNA1 from S. cerevisiae (UniProt ID P43577), an N-acetylglucosamine 2-epimerase like e.g. AGE from B. ovatus (UniProt ID A7LVG6) and an N- acetylneuraminate synthase like e.g. NeuB from N. meningitidis (UniProt ID E0NCD4) and an expression plasmid comprising containing constitutive expression cassettes for the N-acylneuraminate cytidylyltransferase (NeuA) from P. multocida (UniProt ID A0A849CI62) and 1) the alpha-2, 3- sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide (SEQ ID NO 02) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha- 2,3-sialyltransferase activity or 2) the alpha-2, 6-sialyltransferase (PdST6) from P. damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity to produce 1) LSTd (Neu5Ac-a2,3-Gal-pi,4-GlcNAc-pi,3-Gal-pi,4-Glc) or 2) LSTc (Neu5Ac-a2,6-Gal-pi,4-GlcNAc-pi,3-Gal- pi,4-Glc), respectively.
B. Saccharomyces cerevisiae Media and cultivation
Strains were grown on Synthetic Defined yeast medium with Complete Supplement Mixture (SD CSM) or CSM drop-out (SD CSM-Ura, SD CSM-Trp, SD CSM-His) containing 6.7 g/L Yeast Nitrogen Base without amino acids (YNB w/o AA, Difco), 20 g/L agar (Difco) (solid cultures), 22 g/L glucose monohydrate or 20 g/L lactose and 0.79 g/L CSM or 0.77 g/L CSM-Ura, 0.77 g/L CSM-Trp, or 0.77 g/L CSM-His (MP Biomedicals). As precursor(s) and/or acceptor(s) for saccharide, like disaccharide and/or milk oligosaccharide, synthesis, compounds like e.g., galactose, glucose, fructose, fucose, lactose, a co-factor could be added to the medium. In general, yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were grown for 2-3 days at 30°C. Starting from a single colony, a preculture was grown over night in 5 mL at 30°C, shaking at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL media. These shake flasks were incubated at 30°C with an orbital shaking of 200 rpm.
Strains, plasmids and mutations
S. cerevisiae BY4742 created by Brachmann et al. (Yeast (1998) 14:115-32) was used, available in the Euroscarf culture collection. All mutant strains were created by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11:355-360, 1995). Genes were expressed using synthetic constitutive promoters, as described by e.g., Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012), Redden and Alper (Nat. Common. 2015, 6, 7810), Liu et al. (Microb. Cell Fact. 2020, 19, 38), Xu et al. (Microb. Cell Fact.2021, 20, 148) and Lee et al. (ACS Synth. Biol. 2015, 4(9), 975-986).
In an example to produce GDP-fucose, a yeast expression plasmid like p2a_2p_Fuc (Chan 2013, Plasmid 70, 2-17) can be used for expression of foreign genes in S. cerevisiae. This plasmid contains an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E. coli and the 2p yeast ori and the Ura3 selection marker for selection and maintenance in yeast. This plasmid is further modified with constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), a GDP-mannose 4,6-dehydratase like e.g. gmd from E. coli (UniProt ID P0AC88) and a GDP-L-fucose synthase like e.g. fcl from E. coli (UniProt ID P32055). The yeast expression plasmid p2a_2p_Fuc2 can be used as an alternative expression plasmid of the p2a_2p_Fuc plasmid comprising next to the ampicillin resistance gene, the bacterial ori, the 2p yeast ori and the Ura3 selection marker constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), a fucose permease like e.g. fucP from E. coli (UniProt ID P11551) and a bifunctional enzyme with fucose kinase/fucose-l-phosphate guanylyltransferase activity like e.g. fkp from Bacteroides fragilis (UniProt ID SUV40286.1). To further produce a fucosylated saccharide, the p2a_2p_Fuc and its variant the p2a_2p_Fuc2, additionally contained a constitutive transcriptional unit for a fucosyltransferase.
In an example to produce UDP-galactose, a yeast expression plasmid can be derived from the pRS420- plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the HIS3 selection marker and a constitutive transcriptional unit for an UDP-glucose-4-epimerase like e.g. galE from E. coli (UniProt ID P09147). This plasmid can be further modified with constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921) and a galactoside beta-1, 3-N- acetylglucosaminyltransferase activity like e.g. IgtA from N. meningitidis (UniProt ID Q9JXQ6) to produce LN3. In an example to further produce LN3-derived oligosaccharides like LNT, the mutant LN3 producing strains were further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 3- galactosyltransferase like e.g. WbgO (Uniprot ID D3QY14) from E. coli O55:H7. In an example for production of LN3 derived oligosaccharides like lacto-A/-neotetraose (LNnT, Gal-pi,4-GlcNAc-pi,3-Gal- pi,4-Glc), the mutant LN3 producing strain were further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis.
In an example to produce sialic acid and CMP-sialic acid, a yeast expression plasmid was derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122) containing the TRP1 selection marker and constitutive transcriptional units for an L-glutamine— D-fructose-6-phosphate aminotransferase like e.g. E. coli lmS (UniProt ID P17169 (sequence version 04 (23 Jan 2007)) or a mutant glmS*54 from E. coli with SEQ. ID NO 01 and differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006), a phosphatase like e.g. SurE from E. coli (UniProt ID P0A840), an N-acylglucosamine 2-epimerase like e.g. AGE from B. ovatus (UniProt ID A7LVG6), an N-acetylneuraminate synthase like e.g. NeuB from N. meningitidis (UniProt ID E0NCD4) and an N-acylneuraminate cytidylyltransferase like e.g. NeuA from P. multocida (UniProt A0A849CI62). Optionally, 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. Also optionally, a constitutive transcriptional unit for a siderophore transporter like e.g. entS from E. coli (UniProt ID P24077, sequence version 02 (01 Nov 1997)) was added as well. In an example to produce a sialylated saccharide, the plasmid further comprised constitutive transcriptional units for a lactose permease like e.g. LAC12 from K. lactis (UniProt ID P07921), and a sialyltransferase like e.g., an alpha-2, 3- sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide (SEQ ID NO 02) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g. the alpha-2, 6-sialyltransferase (PdST6) from P. damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity.
Preferably but not necessarily, any one or more of the glycosyltransferases and/or the proteins involved in nucleotide-activated sugar synthesis were N- and/or C-terminally fused to a SUMOstar tag (e.g. obtained from pYSU MOstar, Life Sensors, Malvern, PA) to enhance their solubility.
Optionally, the mutant yeast strains were modified with a genomic knock-in of a constitutive transcriptional unit encoding a chaperone protein like e.g. Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssbl, Ssel, Sse2, Ssal, Ssa2, Ssa3, Ssa4, Ssb2, EcmlO, Sscl, Ssql, Sszl, Lhsl, Hsp82, Hsc82, Hsp78, Hspl04, Tcpl, Cct4, Cct8, Cct2, Cct3, Cct5, Cct6 or Cct7. Plasmids were maintained in the host E. coli DH5alpha (F_, phi80d/acZdeltaM15, delta(/acZYA-argF)U169, deoR, recAl, endAl, hsdR17(rk', mk+), phoA, supE44, lambda', thi-1, gyrA96, re I Al) bought from Invitrogen.
C. Bacillus subtilis
Media and cultivation
Two media are used to cultivate B. subtilis: i.e., a complex medium like a rich Luria Broth (LB) and a minimal medium for shake flask cultures. The LB medium consisted of 1% tryptone peptone (Difco), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR). Luria Broth agar (LBA) plates consisted of the LB media, with 12 g/Lagar (Difco) added. The minimal medium contained 2.00 g/L (NEUhSO^ 7.5 g/L KH2PO4, 17.5 g/L K2HPO4, 1.25 g/L Na-citrate, 0.25 g/L MgSO4.7H2O, 0.05 g/L tryptophan, from 10 up to 30 g/L glucose (or another carbon source including but not limited to fructose, maltose, sucrose, glycerol and maltotriose), 10 mL/L trace element mix and 10 mL/L Fe-citrate solution. The medium was set to a pH of 7 with 1 M KOH. As precursor(s) and/or acceptor(s) for saccharide, like disaccharide and/or milk oligosaccharide, synthesis, compounds like e.g., galactose, glucose, fructose, fucose, lactose, a co-factor could be added to the medium. The trace element mix consisted of 0.735 g/L CaCl2.2H2O, 0.1 g/L MnCI2.2H2O, 0.033 g/L CuCI2.2H2O, 0.06 g/L CoCI2.6H2O, 0.17 g/L ZnCI2, 0.0311 g/L H3BO4, 0.4 g/L Na2EDTA.2H2O and 0.06 g/L Na2MoO4. 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. B. subtilis strains were initially grown on LB agar to obtain single colonies. These plates were grown over night at 37°C. Starting from a single colony, a preculture was grown over night in 5 m L at 37°C, shaking at 200 rpm. Subsequent 125 m L shake flask experiments were inoculated with 2% of this preculture, in 25 m L media. These shake flasks were incubated at 37°C with an orbital shaking of 200 rpm for 72h, or shorter of longer. At the end of the cultivation experiment samples were taken to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 15 min at 90°C or for 60 min at 60°C before spinning down the cells (= whole broth concentration, i.e., intra- and extracellular sugar concentrations).
Strains, plasmids and mutations
B. subtilis 168 is used as available at the Bacillus Genetic Stock Center (Ohio, USA). Plasmids for gene deletion via Cre/lox are constructed as described by Yan et al. (Appl & Environm microbial, Sept 2008, p5556-5562). Gene disruption is done via homologous recombination with linear DNA and transformation via the electroporation as described by Xue et al. (J. microb. Meth. 34 (1999) 183-191). The method of gene knockouts is described by Liu et al. (Metab. Engine. 24 (2014) 61-69). Integrative vectors as described by Popp et al. (Sci. Rep., 2017, 7, 15158) are used as expression vector and could be further used for genomic integrations if necessary. A suitable promoter for expression can be derived from the part repository (iGem): sequence id: BBa_K143012, BBa_K823000, BBa_K823002 or BBa_K823003. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.
In an example for the production of lactose-based oligosaccharides, Bacillus subtilis mutant strains are created to contain a gene coding for a lactose importer (e.g. the E. coli lacY with UniProt ID P02920). In an example for the production of LN3, the B. subtilis strain is modified with a genomic knock-in of constitutive transcriptional units comprising a lactose importer (such as e.g. the E. coli lacY with UniProt ID P02920) and a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. LgtA from N. meningitidis (UniProt ID Q9JXQ6). For LNT production, the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g. WbgO from E. coli O55:H7 (UniProt ID D3QY14). For LNnT production, the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000). The N-acetylglucosamine beta-1, 3-galactosyltransferase and the N-acetylglucosamine beta-1, 4-galactosyltransferase can be delivered to the strain either via genomic knock-in or from an expression plasmid. To produce a fucosylated saccharide, the B. subtilis strains are modified with a constitutive transcriptional unit for a fucosyltransferase. In an example for sialic acid production, a mutant B. subtilis strain is created by overexpressing a fructose-6-P-aminotransferase like the native fructose-6-P-aminotransferase glmS (UniProt ID P0CI73) to enhance the intracellular glucosamine-6-phosphate pool. Further on, the enzymatic activities of the genes nagA, nagB and gamA are disrupted by genetic knockouts and a glucosamine-6-P- aminotransferase like e.g. GNA1 from S. cerevisiae (UniProt ID P43577), an N-acetylglucosamine-2- epimerase like e.g. from B. ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase like e.g. from N. meningitidis (UniProt ID E0NCD4) are overexpressed on the genome. To allow sialylated saccharide production, the sialic acid producing strain is further modified with a constitutive transcriptional unit comprising an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P. multocida (UniProt ID A0A849CI62), and a sialyltransferase like e.g., an alpha-2, 3-sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide (SEQ ID NO 02) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having betagalactoside alpha-2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g. the alpha-2, 6- sialyltransferase (PdST6) from P. damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6- sialyltransferase activity. For growth on sucrose, the mutant strains can additionally be modified with genomic knock-ins of constitutive transcriptional units comprising the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from 7. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6). Optionally, a constitutive transcriptional unit for a siderophore transporter like e.g. entS from E. coli (UniProt ID P24077, sequence version 02 (01 Nov 1997)) is added as well.
D. Corynebacterium glutamicum
Media and cultivation
Two different media are used, namely complex medium like e.g., a rich tryptone-yeast extract (TY) medium, and a minimal medium for shake flask (MMsf). The minimal medium uses a lOOOx stock trace element mix. Trace element mix consisted of 10 g/L CaCL, 10 g/L FeSO^THjO, 10 g/L MnSO^HjO, 1 g/L ZnSOi.THjO, 0.2 g/L CuSO4, 0.02 g/L NiCh.6H2O, 0.2 g/L biotin (pH 7) and 0.03 g/L protocatechuic acid. The minimal medium for the shake flasks (MMsf) experiments contained 20 g/L (NH^SO^, 5 g/L urea, 1 g/L KH2PO4, 1 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 when specified in the examples and 1 ml/L trace element mix. As precursor(s) and/or acceptor(s) for saccharide, like disaccharide and/or milk oligosaccharide, synthesis, compounds like e.g., galactose, glucose, fructose, fucose, lactose, a co-factor could be added to the medium. The TY medium consisted of 1.6% tryptone (Difco, Erembodegem, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). TY agar (TYA) plates consisted of the TY media, with 12 g/L agar (Difco, Erembodegem, Belgium) added. Complex medium, e.g., TY, was sterilized by autoclaving (121°C, 21 min) and minimal medium by filtration (0.22 pm Sartorius). When necessary, the medium was made selective by adding an antibiotic. A preculture was started from a cryovial or a single colony from a TY plate, in 6 mL TY and was incubated overnight at 37 °C on an orbital shaker at 200 rpm. Subsequent 125 mL shake flask experiments were inoculated with 2% of this preculture, in 25 mL MMsf medium. These shake flasks were incubated at 37°C with an orbital shaking of 200 rpm for 72h, or shorter of longer. At the end of the cultivation experiment samples were taken to measure the supernatant concentration (extracellular sugar concentrations, after 5 min. spinning down the cells), or by boiling the culture broth for 15 min at 90°C or for 60 min at 60°C before spinning down the cells (= whole broth concentration, i.e., intra- and extracellular sugar concentrations).
Strains and mutations
Corynebacterium glutamicum was used as available at the American Type Culture Collection (ATCC 13032). Integrative plasmid vectors were made using the Cre/loxP technique as described by Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 Apr, 67(2):225-33) and temperature-sensitive shuttle vectors as described by Okibe et al. (Journal of Microbiological Methods 85, 2011, 155-163) are constructed for gene deletions, mutations and insertions. Suitable promoters for (heterologous) gene expression can be derived from Yim et al. (Biotechnol. Bioeng., 2013 Nov, 110(ll):2959-69). Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. In an example for the production of lactose-based oligosaccharides, C. glutamicum mutant strains are created to contain a gene coding for a lactose importer (such as e.g. the E. coli lacY with UniProt ID P02920). In an example for the production of LN3, the C. glutamicum strain is modified with a genomic knock-in of constitutive expression units comprising a lactose importer (such as e.g. the E. coli lacY with UniProt ID P02920) and a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g. LgtAfrom N. meningitidis (UniProt ID Q9JXQ6). For LNT production, the LN3 producing strain is further modified with a constitutive transcriptional unit for an N- acetylglucosamine beta-1, 3-galactosyltransferase like e.g. WbgO from E. coli O55:H7 (UniProt ID D3QY14). For LNnT production, the LN3 producing strain is further modified with a constitutive transcriptional unit for an N-acetylglucosamine beta-1, 4-galactosyltransferase like e.g. LgtB from N. meningitidis (UniProt ID Q51116, sequence version 02, 01 Dec 2000). The N-acetylglucosamine beta-1, 3-galactosyltransferase and the N-acetylglucosamine beta-1, 4-galactosyltransferase can be delivered to the strain either via genomic knock-in or from an expression plasmid. To further produce a fucosylated saccharide, the mutant C. glutamicum strains are further modified with a constitutive transcriptional unit for a fucosyltransferase. In an example for sialic acid production, a mutant C. glutamicum strain is created by overexpressing a fructose-6-P-aminotransferase like the native fructose-6-P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 03, 23 Jan 2007) to enhance the intracellular glucosamine-6-phosphate pool. Further on, the enzymatic activities of the genes nagA, nagB and gamA are disrupted by genetic knockouts and a glucosamine-6-P-aminotransferase like e.g. GNA1 from S. cerevisiae (UniProt ID P43577), an N- acetylglucosamine-2-epimerase like e.g. from B. ovatus (UniProt ID A7LVG6) and an N-acetylneuraminate synthase like e.g. from N. meningitidis (UniProt ID E0NCD4) are overexpressed on the genome. To allow sialylated saccharide production, the sialic acid producing strain is further modified with a constitutive transcriptional unit comprising an N-acylneuraminate cytidylyltransferase like e.g. the NeuA enzyme from P. multocida (UniProt ID A0A849CI62), and a sialyltransferase like e.g., an alpha-2, 3-sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide (SEQ ID NO 02) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having betagalactoside alpha-2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g. the alpha-2, 6- sialyltransferase (PdST6) from P. damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6- sialyltransferase activity. For growth on sucrose, the mutant strains can additionally be modified with genomic knock-ins of constitutive transcriptional units comprising the sucrose transporter (CscB) from E. coli W (UniProt ID E0IXR1), the fructose kinase (Frk) from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase (BaSP) from B. adolescentis (UniProt ID A0ZZH6).
E. Chlamydomonas reinhardtii
Media and cultivation
Chlamydomonas reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7). The TAP medium uses a lOOOx stock Hutner's trace element mix. Hutner's trace element mix consisted of 50 g/L Na2EDTA.H2O (Titriplex III), 22 g/L ZnSO4.7H2O, 11.4 g/L H3BO3, 5 g/L MnCI2.4H2O, 5 g/L FeSO4.7H2O, 1.6 g/L CoCI2.6H2O, 1.6 g/L CuSO4.5H2O and 1.1 g/L (NH4)6MoO3. The TAP medium contained 2.42 g/L Tris (tris(hydroxymethyl)aminomethane), 25 mg/L salt stock solution, 0.108 g/L K2HPO4, 0.054 g/L KH2PO4 and 1.0 mL/L glacial acetic acid. The salt stock solution consisted of 15 g/L NH4CL, 4 g/L MgSO4.7H2O and 2 g/L CaCI2.2H2O. As precursor(s) and/or acceptor(s) for saccharide, like disaccharide and/or milk oligosaccharide, synthesis, compounds like e.g., galactose, glucose, fructose, fucose, lactose, a co-factor could be added. Medium was sterilized by autoclaving (121°C, 21 min). For stock cultures on agar slants TAP medium was used containing 1% agar (of purified high strength, 1000 g/cm2).
Cells of C. reinhardtii were cultured in selective TAP-agar plates at 23 +/- 0.5°C under 14/10 h I ight/dark cycles with a light intensity of 8000 Lx. Cells were analysed after 5 to 7 days of cultivation. For high-density cultures, cells could be cultivated in closed systems like e.g., vertical or horizontal tube photobioreactors, stirred tank photobioreactors or flat panel photobioreactors as described by Chen et aL (Bioresour. TechnoL 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827).
Strains, plasmids and mutations
C. reinhardtii wild-type strains 21gr (CC-1690, wild-type, mt+), 6145C (CC-1691, wild-type, mt-), CC-125 (137c, wild-type, mt+), CC-124 (137c, wild-type, mt-) as available from the Chlamydomonas Resource Center (https://www.chlamycollection.org) (University of Minnesota, U.S.A) were used. Expression plasmids originated from pSH03, as available from the Chlamydomonas Resource Center. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation. Suitable promoters for (heterologous) gene expression can be derived from e.g., Scranton et al. (Algal Res. 2016, 15: 135-142). Targeted gene modification (like gene knock-out or gene replacement) can be carried using the Crispr-Cas technology as described e.g., by Jiang et al. (Eukaryotic Cell 2014, 13(11): 1465-1469). Transformation via electroporation was performed as described by Wang et al. (Biosci. Rep. 2019, 39:
BSR2018210) and as described like e.g., in WO22034067 or in WO22034069.
In an example for sialic acid and CMP-sialic acid production, the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067. In an example for production of sialylated oligosaccharides, C. reinhardtii cells are modified with a CMP-sialic acid transporter like e.g., CST from Mus musculus (UniProt ID Q61420), and a Golgi-localised sialyltransferase selected from species like e.g., Homo sapiens, Mus musculus, Rattus norvegicus. In an example for GDP-fucose synthesis, the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067. In an example for fucosylation, C. reinhardtii cells can be modified with an expression plasmid comprising a constitutive transcriptional unit for an alpha-1, 2-fucosyltransferase and/or an alpha-1, 3-fucosyltransferase. In an example for UDP-galactose synthesis, the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067. In an example for LN3 production, the mutant strain was derived from C. reinhardtii and modified as described e.g., in WO22034067 to comprise a transcriptional unit for a galactoside beta-1, 3-N-acetylglucosaminyltransferase like e.g., LgtA from N. meningitidis (UniProt ID Q9JXQ6). In an example for LNT or LNnT production, the LN3 producing strain is further modified with a constitutive transcriptional unit comprising an N-acetylglucosamine beta-1, 3-galactosyltransferase like e.g., WbgO (Uniprot ID D3QY14) from E. coli 055:1-17 or an N-acetylglucosamine beta-1, 4- galactosyltransferase like e.g., LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis, respectively. In an example to produce one or more fucosylated non-charged oligosaccharide(s), a C. reinhardtii strain is modified for production of GDP-fucose, UDP-galactose, LN3, LNT and/or LNnT as described herein and for expression of one or more compatible fucosyltransferase(s). In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTa and LSTb, a C. reinhardtii strain is modified for production of CMP-sialic acid, UDP-galactose, LN3 and LNT as described herein and for expression of one or more compatible sialyltransferase(s) like e.g., an alpha-2, 3-sialyltransferase like e.g. the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide (SEQ ID NO 02) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having betagalactoside alpha-2, 3-sialyltransferase activity or an alpha-2, 6-sialyltransferase like e.g. the alpha-2, 6- sialyltransferase (PdST6) from P. damselae (UniProt ID 066375) or a PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6- sialyltransferase activity. In an example to produce one or more sialylated oligosaccharide(s) like e.g., LSTc and LSTd, a C. reinhardtii strain is modified for production of CMP-sialic acid, UDP-galactose, LN3 and LNnT as described herein and for expression of one or more compatible sialyltransferase(s).
F. Animal cells
Isolation of mesenchymal stem cells from adipose tissue of different animals
Fresh adipose tissue is obtained from slaughterhouses (e.g., cattle, pigs, sheep, chicken, ducks, catfish, snake, frogs) or liposuction (e.g., in case of humans, after informed consent) and kept in phosphate buffer saline supplemented with antibiotics. Enzymatic digestion of the adipose tissue is performed followed by centrifugation to isolate mesenchymal stem cells. The isolated mesenchymal stem cells are transferred to cell culture flasks and grown under standard growth conditions, e.g., 37°C, 5% CO2. The initial culture medium includes DMEM-F12, RPMI, and Alpha-MEM medium (supplemented with 15% foetal bovine serum), and 1% antibiotics. The culture medium is subsequently replaced with 10% FBS (foetal bovine serum)-supplemented media after the first passage. For example, 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.
Isolation of mesenchymal stem cells from milk
This example illustrates isolation of mesenchymal stem cells from milk collected under aseptic conditions from human or any other mammal(s) such as described herein. An equal volume of phosphate buffer saline is added to diluted milk, followed by centrifugation for 20 min. The cell pellet is washed thrice with phosphate buffer saline and cells are seeded in cell culture flasks in DMEM-F12, RPMI, and Alpha-MEM medium supplemented with 10% foetal bovine serum and 1% antibiotics under standard culture conditions. For example, Hassiotou et al. (2012, Stem Cells. 30(10): 2164-2174), 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.
Differentiation of stem cells using 2D and 3D culture systems
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.
For 2D culture, 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. At confluence, cells were fed with 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. To induce differentiation, 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.
For 3D culture, 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. At confluence, 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. To induce differentiation, the cells were fed with complete growth medium containing 5 pg/mL insulin, 1 pg/mL hydrocortisone, 0.65 ng/mL triiodothyronine, 100 nM dexamethasone, and 1 pg/mL prolactin. After 24h, serum is removed from the complete induction medium.
Method of making mammary-like cells
In a next step, the cells are brought to induced pluripotency by reprogramming with viral vectors encoding for Oct4, Sox2, Klf4, and c-Myc. The resultant reprogrammed cells are then cultured in Mammocult media (available from Stem Cell Technologies), or mammary cell enrichment media (DMEM, 3% FBS, estrogen, progesterone, heparin, hydrocortisone, insulin, EGF) to make them mammary-like, from which expression of select milk components can be induced. Alternatively, 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. In an example for production of one or more oligosaccharide(s), isolated mesenchymal cells re-programmed into mammary-like cells are modified via CRISPR-CAS as described e.g., in WO22034067, W022034070 and WO22034075.
Cultivation
Completed growth media includes high glucose DMEM/F12, 10% FBS, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/mL EGF, and 5 pg/mL hydrocortisone. Completed lactation media includes high glucose DMEM/F12, 1% NEAA, 1% pen/strep, 1% ITS-X, 1% F-Glu, 10 ng/mL EGF, 5 pg/mL hydrocortisone, and 1 pg/mL prolactin (5ug/mL in Hyunh 1991). Cells are seeded at a density of 20,000 cells/cm2 onto collagen coated flasks in completed growth media and left to adhere and expand for 48 hours in completed growth media, after which the media is switched out for completed lactation media. Upon exposure to the lactation media, the cells start to differentiate and stop growing. Within about a week, the cells start secreting lactation product(s) such as milk lipids, lactose, casein and whey into the media. A desired concentration of the lactation media can be achieved by concentration or dilution by ultrafiltration. A desired salt balance of the lactation media can be achieved by dialysis, for example, to remove unwanted metabolic products from the media. Hormones and other growth factors used can be selectively extracted by resin purification, for example the use of nickel resins to remove His-tagged growth factors, to further reduce the levels of contaminants in the lactated product.
G. Optical density
Cell density of the cultures was frequently monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium or with a Spark 10M microplate reader, Tecan, Switzerland). The maximum growth speed (mumax) was calculated based on the observed optical densities at 600nm using the R package grofit. H. Growth rate/speed measurement
The maximal growth rate (pMax) was calculated based on the observed optical densities at 600 nm using the R package grofit.
/. Heterologous and homologous expression Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: IDT or Twist Bioscience. Proteins described in present disclosure are summarized in 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.
Table 1. Overview of proteins with corresponding SEQ ID NOs or UniProt IDs (sequence version 01,
UniProt Database 2021_03 of 09 June 2021) as described in present disclosure
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
■ Sequence version 02 (01 Feb 2005) as present in the UniProt Database 2021_03 of 09 June 2021; ■■ Sequence version 03 (10 Feb 2009) as present in the UniProt Database 2021_03 of 09 June 2021; ■■■ Sequence version 03 (05 Jul 2005) as present in the UniProt Database 2021JD3 of 09 June 2021; * Sequence version 02 (01 Nov 1997) as present in the UniProt Database 2021_03 of 09 June 2021; ** Sequence version 03 (23 Jan 2007) as present in the UniProt Database 2021J33 of 09 June 2021; *** Sequence version 03 (02 Dec 2020) as present in the UniProt Database 2021J33 of 09 June 2021; **** Sequence version 03 (23 Jan 2007) as present in the UniProt Database 2021_03 of 09 June 2021; ***** Sequence version 02 (23 Jan 2007) as present in the UniProt Database 2021_03 of 09 June 2021; ° Sequence version 04 (23 Jan 2007) as present in the UniProt Database 2021_03 of 09 June 2021; " Sequence version 02 (01 Dec 2000) as present in the UniProt Database 2021_03 of 09 June 2021; °°° Sequence version 03 (19 July 2003) as present in the UniProt Database 2021_03 of 09 June 2021; °°°° Sequence version 03 (13 Oct 2009) as present in the UniProt Database 2021_03 of 09 June 2021; Sequence version 02 (01 Feb 2005) as present in the UniProt Database 2021_03 of 09 June 2021; “ Sequence version 02 (07 April 2021) as present in the UniProt Database 2021_03 of 09 June 2021; 0000 Sequence version 04 (02 June 2021) as present in the UniProt Database 2021_03 of 09 June 2021
J. Analytical analysis
Standards such as but not limited to sucrose, lactose, 3'SL, 6'SL, lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neo-tetraose (LNnT), LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LSTa, LSTc and LSTd were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with in-house made standards.
Neutral oligosaccharides were analyzed on a Waters Acquity H-class UPLC with Evaporative Light Scattering Detector (ELSD) or a Refractive Index (Rl) detection. A volume of 0.7 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 A;1.7 pm) column with an Acquity UPLC BEH Amide VanGuard column, 130 A, 2. lx 5 mm. The column temperature was 50 °C. The mobile phase consisted of a % water and % acetonitrile solution to which 0.2 % triethylamine was added. The method was isocratic with a flow of 0.130 mL/min. The ELSD detector had a drift tube temperature of 50 °C and the N2 gas pressure was 50 psi, the gain 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. 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.
Both neutral and sialylated sugars were analyzed on a Waters Acquity H-class UPLC with Refractive Index (Rl) detection. A volume of 0.5 pL sample was injected on a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm;130 A;1.7 pm). The column temperature was 50°C. The mobile phase consisted of a mixture of 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which 0.1% triethylamine was added. The method was isocratic with a flow of 0.260 mL/min. The temperature of the Rl detector was set at 35°C.
For analysis on a mass spectrometer, a Waters Xevo TQ-MS with Electron Spray Ionisation (ESI) was used with a desolvation temperature of 450 °C, a nitrogen desolvation gas flow of 650 L/h and a cone voltage of 20 V. The MS was operated in selected ion monitoring (SIM) in negative mode for all oligosaccharides. Separation was performed on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1 x 100 mm; 3 pm) on 35 °C. A gradient was used wherein eluent A was ultrapure water with 0.1 % formic acid and wherein eluent B was acetonitrile with 0.1 % formic acid. The oligosaccharides were separated in 55 min using the following gradient: an initial increase from 2 to 12 % of eluent B over 21 min, a second increase from 12 to 40 % of eluent B over 11 min and a third increase from 40 to 100 % of eluent B over 5 min. As a washing step 100 % of eluent B was used for 5 min. For column equilibration, the initial condition of 2 % of eluent B was restored in 1 min and maintained for 12 min.
Both neutral and sialylated sugars at low concentrations (below 50 mg/L) were analyzed on a Dionex HPAEC system with pulsed amperometric detection (PAD). A volume of 5 pL of sample was injected on a Dionex CarboPac PA200 column 4 x 250 mm with a Dionex CarboPac PA200 guard column 4 x 50 mm. The column temperature was set to 30 °C. A gradient was used wherein eluent A was deionized water, wherein eluent B was 200 mM Sodium hydroxide and wherein eluent C was 500 mM Sodium acetate. The oligosaccharides were separated in 60 min while maintaining a constant ratio of 25 % of eluent B using the following gradient: an initial isocratic step maintained for 10 min of 75 % of eluent A, an initial increase from 0 to 4 % of eluent C over 8 min, a second isocratic step maintained for 6 min of 71 % of eluent A and
4 % of eluent C, a second increase from 4 to 12 % of eluent C over 2.6 min, a third isocratic step maintained for 3.4 min of 63 % of eluent A and 12 % of eluent C and a third increase from 12 to 48 % of eluent C over
5 min. As a washing step 48 % of eluent C was used for 3 min. For column equilibration, the initial condition of 75 % of eluent A and 0 % of eluent C was restored in 1 min and maintained for 11 min. The applied flow was 0.5 mL/min.
Lactobionic acid was analysed on a Dionex HPAEC system with pulsed amperometric detection (PAD). A volume of 5 pL of sample was injected on a Dionex CarboPac PA01 column 2 x 250 mm with a Dionex CarboPac PA01 guard column 4 x 50 mm. The column temperature was set to 30 °C. A gradient was used wherein eluent A was ultrapure water, eluent B was 200 mM Sodium hydroxide and eluent C was 500 mM Sodium acetate. Total gradient time was 41 min and started with 50% B and 5% C in the first 7 minutes. During the next 18 minutes, concentration slowly changed to 50% B and 40% C at 25 minutes, followed by a rinse step with 100% C for 7 minutes. For column equilibration, the initial condition with 50% B and 5% C was restored in 9 minutes. The applied flow was 0.25 mL/min.
Example 2. Evaluation of production of sialic acid and 6'SL with a modified E. coli host having the glmU gene rendered less functional
An E. coli K-12 MG1655 strain was modified for production of sialic acid comprising genomic knock-ins of constitutive transcriptional units containing the mutant glmS*54 from E. coli (SEQ ID NO 01) (differing from the wild-type E. coli glmS, having UniProt ID P17169, by an A39T, an R250C and an G472S mutation as described by Deng et al. (Biochimie 88, 419-29 (2006)), the glucosamine 6-phosphate N- acetyltransferase GNA1 from S. cerevisiae (UniProt ID P43577), the N-acylglucosamine 2-epimerase AGE from B. ovatus (UniProt ID A7LVG6) and the N-acetylneuraminate synthase NeuB from N. meningitidis (UniProt ID E0NCD4). To allow production of 6'SL, the strain was further modified with constitutive transcriptional units for the N-acylneuraminate cytidylyltransferases NeuA from Campylobacter jejuni (UniProt ID Q93MP7) and NeuA from Haemophilus influenzae (SEQ ID NO 04), and with two constitutive transcriptional units for expression of the PdST6-like polypeptide (SEQ ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 and having beta-galactoside alpha-2, 6-sialyltransferase activity. Furthermore, the strain was modified with a genomic knock-in for overexpression of the acetyl-CoA synthetase acs from E. coli (UniProt ID P27550) and with a genomic knock-out of the O-antigen gene cluster deleting all the genes between wbbK and wcaN including wbbK and wcaN. Next, the strain was modified for growth on sucrose as described in Example 1. The mutant strain A thus obtained was then further engineered to create five new strains (B, C, D, E and F) having a glmU gene (UniProt ID P0ACC7) that was less functional compared to the native glmU gene present in strain A. Each strain was created by genomic knock-in of a different constitutive transcriptional unit comprising the native glmU gene from E. coli (UniProt ID P0ACC7) that was introduced with different promoter (P), untranslated (UTR) and terminator (T) sequences (Table 3) at a foreign location in the E. coli cell's genome, i.e. at the insN locus, and by subsequent genomic knock-out of the native glmU gene from E. coli (locus 3,913,830 <- 3,915,200). The novel strains were evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 1 in which the strains were cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. The strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed on UPLC. For each strain with a particular transcriptional unit of glmU, the measured sialic acid concentration, sum of sialylated molecules (sialic acid and 6'SL) and biomass concentration was averaged over all biological replicates and then normalized to the averaged sialic acid concentration, sum of sialylated molecules and biomass concentration of the reference strain A having the same genetic make-up but lacking any modification to the native glmU gene. The experiment showed that each knockdown of glmU in the five strains resulted in higher production of sialic acid and sialylated molecules (sialic acid and 6'SL) compared to the reference strain A (Table 2). Surprisingly, the experiment also showed that each knockdown of glmU in the five strains resulted in lower biomass production of all five strains B, C, D, E and F compared to the reference strain A (Table 2) resulting to higher CPI for sialic acid and for sialylated molecules.
Table 2. Relative production of sialic acid (SA) (%), sum of sialylated molecules (SA + 6'SL, %), biomass (%), CPI of SA {%) and CPI of sum sialylated molecules (SA + 6'SL, %) with modified E. coli strains B, C, D, E and F expressing a knockdown transcriptional unit of the E. coli glmU integrated at the insN locus and having a genomic knock-out of the native glmU gene, compared to a reference strain A lacking genetic engineering of its native glmU gene. Strains were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contained 30 g/L sucrose and 20 g/L lactose.
Figure imgf000091_0001
*See Table 3 for details regarding the promoter (P), untranslated region (UTR) and terminator (T sequences used to express the E. coli glmU gene (UniProt ID P0ACC7)
Table 3. Promoter, untranslated region (UTR) and terminator sequences used to express the glmU enzyme from E. coli (Uniprot ID POACC7) from a knockdown transcriptional unit integrated in the mutant E. coli strains B, C, D, E, F, H, I and J as given in Table 2 and Table 4.
Figure imgf000091_0002
Figure imgf000092_0001
Example 3. Evaluation of production of sialic acid and 6'SL with a modified E. coli host having the glmU gene rendered less functional and overexpressing E. coli glmS (UniProt ID P17169)
The mutant E. coli strain F, as described in Example 2, was further modified to overexpress its native E. coli glmS gene (UniProt ID P17169) by integrating a strong constitutive promoter sequence on the native glmU locus (locus 3,913,830 <- 3,915,200) 5' of the E. coli glmS gene. As such, three new strains were created, each expressing the E. coli glmS from a different strong constitutive promoter sequence (Table 4). The new strains were further transformed with an expression plasmid comprising a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62), a PdST6-like polypeptide (SEQ. ID NO 03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 having beta-galactoside alpha-2, 6-sialyltransferase activity and the siderophore transporter entS from E. coli (UniProt ID P24077) resulting in strains H, I and J (Table 4). Also strain F was transformed with said expression plasmid, resulting in the reference strain G (Table 4). All strains were evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 1, in which the strains were cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. The strains were grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth was harvested, and the sugars were analysed on UPLC. For each strain with a particular constitutive promoter sequence controlling over-expression of the E. coli glmS, the measured sialic acid concentration, sum of sialylated molecules (sialic acid and 6'SL) and biomass concentration was averaged over all biological replicates and then normalized to the averaged sialic acid concentration, sum of sialylated molecules and biomass concentration of a reference strain lacking a constitutive promoter in front of glmS. The experiment showed that expression of the E. coli glmS from a different strong constitutive promoter sequence resulted in higher production of sialic acid and sialylated molecules (sialic acid and 6'SL) compared to the reference strain G (Table 4). No effect was seen on the biomass production.
Table 4. Relative production of sialic acid (%), sum of sialylated molecules (sialic acid + 6'SL, %) and biomass (%) with a modified E. coli strain H, I and J expressing a knockdown transcriptional unit of the E. coli glmU (i.e., Plll-UTR64-glmU-T10, Table 2) and overexpressing the native glmS gene (UniProt ID P17169) from a strong constitutive promoter sequence compared to a reference strain G lacking genetic engineering of its native glmS gene. Strains were evaluated in a growth experiment according to the culture conditions provided in Example 1, in which the culture medium contained 30 g/L sucrose and 20 g/L lactose.
Figure imgf000093_0001
*See Table 3 for details regarding the promoter (P), untranslated region (UTR) and terminator (T) sequences used to express the E. coli glmU gene (UniProt ID P0ACC7)
Example 4. Evaluation of production of sialic acid and 6'SL with modified E. coli hosts when evaluated in a fed-batch fermentation process with sucrose and lactose
The mutant E. coli strains G, H and I as described in Example 3 were selected for further evaluation in fed- batch fermentation processes. Fed-batch fermentations at bioreactor scale were performed as described in Example 1. Sucrose was used as a carbon source and lactose was added in the batch medium. During fed-batch, sucrose was added via an additional feed. In contrast to the cultivation experiments that are described herein and wherein only end samples were taken at the end of cultivation (i.e., 72 hours as described herein), regular broth samples were taken at several time points during the fermentation process and the production of sialic acid and 6'SL was measured using UPLC as described in Example 1. As shown in Table 5, besides the use of a less functional glmU gene in all strains G, H and I, the use of a strong constitutive promoter to over-express the native E. coli glmS gene in strains H and I resulted in higher production of sialic acid as well as of total sialylated molecules (sialic acid and 6'SL).
Table 5. Relative production of sialic acid (%), sum of sialylated molecules (sialic acid + 6'SL, with a modified E. coli strain H and I expressing a knockdown transcriptional unit of the E. coli glmU (i.e., Plll- UTR64-glmU-T10, Table 2) and overexpressing the native glmS gene (UniProt ID P17169) from a strong constitutive promoter sequence compared to a reference strain G lacking genetic engineering of its native glmS gene. Strains were evaluated in a fed-batch fermentation process according to the culture conditions provided in Example 1, in which the culture medium contained sucrose and lactose.
Figure imgf000093_0002
Figure imgf000094_0001
Example 5. Evaluation of production of sialic acid and 6'SL with a modified S. cerevisiae host
In a first step, a 5. cerevisiae strain is modified for production of CMP-sialic acid and for expression of a sialyltransferase as described in Example 1. This strain is further modified via homologous recombination as described in Example 1 to integrate a very weak constitutive yeast promoter 5' of the 5. cerevisiae native PCM1 gene (UniProt ID P38628, sequence version 02 (07 April 2021)) which is located on chromosome V and to integrate a strong constitutive yeast promoter 5' of the S. cerevisiae GFA1 gene (UniProt ID P14742, sequence version 04 (02 June 2021)) which is located on chromosome XI. As such, native expression of the S. cerevisiae PCM1 gene is downregulated and the native GFA1 gene is overexpressed. In a next step, the strain is transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 6-sialyltransferase PdST6 from P. damselae (UniProt ID 066375). The novel strain is evaluated in a growth experiment for production of sialic acid and 6'SL according to the culture conditions provided in Example 1, in which the appropriate selective medium comprises glucose as carbon source and lactose as precursors. The strain is grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested, and production of sialic acid and 6'SL is analysed on UPLC.
Example 6. Evaluation of production of sialic acid and 3'SL with a modified B. subtilis host
In a first step, a B. subtilis strain is modified for production of CMP-sialic acid and for expression of a sialyltransferase as described in Example 1. This strain is further modified as described in Example 1 to integrate a very weak constitutive promoter 5' of the B. subtilis glmU gene (UniProt ID P14192) and to integrate a strong constitutive promoter s' of the B. subtilis glmS gene (UniProt ID P0CI73). As such, native expression of the B. subtilis glmU gene is downregulated and the native glmS gene is overexpressed. In a next step, the strain is transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3). The novel strain is evaluated in a growth experiment for production of sialic acid and 3'SL according to the culture conditions provided in Example 1, in which the appropriate selective medium comprises glucose as carbon source and lactose as precursors. The strain is grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested, and production of sialic acid and 3'SL is analysed on UPLC.
Example 7. Evaluation of production of sialic acid and 3'SL with a modified C. glutamicum host
In a first step, a C. glutamicum strain is modified for production of CMP-sialic acid and for expression of a sialyltransferase as described in Example 1. This strain is further modified as described in Example 1 to integrate a very weak constitutive promoter 5' of the C. glutamicum glmU gene (UniProt ID Q8NRU8) and to integrate a strong constitutive promoter 5' of the C. glutamicum glmS gene (UniProt ID Q8NND3). As such, native expression of the C. glutamicum glmU gene is downregulated and the native glmS gene is overexpressed. In a next step, the strain is transformed with an expression plasmid containing a constitutive transcriptional unit for the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3). The novel strain is evaluated in a growth experiment for production of sialic acid and 3'SL according to the culture conditions provided in Example 1, using MMsf medium comprising lactose. Regular samples are taken and evaluated via UPLC for production of sialic acid and 3'SL.
Example s. Evaluation of production of LNB or LacNAc with a modified E. coll host having the glmU gene rendered less functional and overexpressing E. coll glmS (UniProt ID P17169)
The mutant E. coli strains B to F, as described in Example 2, are further modified to overexpress the native E. coli glmS gene (UniProt ID P17169) by integrating a strong constitutive promoter sequence on the native glmU locus (locus 3,913,830 <- 3,915,200) 5' of the E. coli glmS gene. The new strains are further transformed with an expression plasmid comprising a constitutive transcriptional unit for either the N- acetylglucosamine beta-1, 3-galactosyltransferase wbgO (Uniprot ID D3QY14) from E. coli 055:1-17 or the N-acetylglucosamine beta-1, 4-galactosyltransferase LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from Neisseria meningitidis. All strains comprising the wbgO gene are evaluated in a growth experiment for production of LNB according to the culture conditions provided in Example 1, in which the strains are cultivated in minimal medium with 30 g/L sucrose, and 20 g/L galactose. The strains are grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broths are harvested and production of LNB is analysed on UPLC. All strains comprising the IgtB gene are evaluated in a growth experiment for production of LacNAc according to the culture conditions provided in Example 1, in which the strains are cultivated in minimal medium with 30 g/L sucrose, and 20 g/L galactose. The strains are grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broths are harvested and production of LacNAc is analysed on UPLC. Example 9. Evaluation of production of a mixture comprising LNB and 6'SL with a modified E. coli host having the glmU gene rendered less functional and overexpressing E. coli glmS (UniProt ID P17169)
The mutant E. coli strains B to F, as described in Example 2, are further modified to overexpress the native E. coli glmS ene (UniProt ID P17169) by integrating a strong constitutive promoter sequence on the native glmU locus (locus 3,913,830 <- 3,915,200) 5' of the E. coli glmS gene. The new strains are further transformed with an expression plasmid comprising a constitutive transcriptional unit for the N- acetylglucosamine beta-1, 3-galactosyltransferase wbgO (Uniprot ID D3QY14) from E. coli O55:H7, the N- acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI52) and the alpha-2, 6- sialyltransferase PdST6 from P. damselae (UniProt ID 066375). All strains are evaluated in a growth experiment for production of a mixture comprising LNB and 6'SL according to the culture conditions provided in Example 1, in which the strains are cultivated in minimal medium with 30 g/L sucrose, 20 g/L galactose and 20 g/L lactose. The strains are grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broths are harvested, and production of a mixture comprising LNB and 6'SL in each sample is analysed on UPLC. Also, the production of sialylated LNB in said mixture is evaluated.
Example 10. Evaluation of production of 3*-sialyl-3-fucosyllactose with a modified E. coli host having the glmU gene rendered less functional and overexpressing E. coli glmS (UniProt ID P17169)
The mutant E. coli strain F, as described in Example 2, is further modified to overexpress the native E. coli glmS gene (UniProt ID P17169) by integrating a strong constitutive promoter sequence on the native glmU locus (locus 3,913,830 <- 3,915,200) 5' of the E. coli glmS gene. Next, the strain is modified with a knockout of the E. coli wcaJ, fucK and fuel genes and genomic knock-ins of constitutive transcriptional units comprising the mannose-6-phosphate isomerase manA from E. coli (UniProt ID P00946), the phosphomannomutase manB from E. coli (UniProt ID P24175), the mannose-l-phosphate guanylyltransferase manC from E. coli (UniProt ID P24174), the GDP-mannose 4,6-dehydratase gmd from E. coli (UniProt ID P0AC88) and the GDP-L-fucose synthase fcl from E. coli (UniProt ID P32055), the sucrose transporter like from E. coli\N (UniProt ID E0IXR1), the fructose kinase from Z. mobilis (UniProt ID Q03417) and the sucrose phosphorylase from B. odolescentis (UniProt ID A0ZZH6). The new strain is further transformed with an expression plasmid comprising a constitutive transcriptional unit for the N- acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62), the alpha-2, 3- sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3) and the alpha-1, 3-fucosyltransferase HpFucT from H. pylori (UniProt ID 030511). The new strain is evaluated in a growth experiment for production of 3'-sialyl-3-fucosyllactose according to the culture conditions provided in Example 1, in which the strain is cultivated in minimal medium with 30 g/L sucrose and 20 g/L lactose. The strain is grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested, and production of 3'-sialyl-3-fucosyllactose, 3'FL and 3'SL is analysed on UPLC. Example 11. Evaluation of production of a mixture comprising LacNAc and 3'SL with a modified S. cerevisiae host
In a first step, a S. cerevisiae strain is modified for production of CMP-sialic acid and for expression of a sialyltransferase as described in Example 1. This strain is further modified via homologous recombination as described in Example 1 to integrate a very weak constitutive yeast promoter 5' of the S. cerevisiae native PCM1 gene (UniProt ID P38628, sequence version 02 (07 April 2021)) which is located on chromosome V and to integrate a strong constitutive yeast promoter 5' of the S. cerevisiae GFA1 gene (UniProt ID P14742, sequence version 04 (02 June 2021)) which is located on chromosome XI. As such, native expression of the 5. cerevisiae PCMl gene is downregulated and the native GFA1 gene is overexpressed. In a next step, the strain is transformed with an expression plasmid containing a constitutive transcriptional unit for the phosphatase surE from E. coli (UniProt ID P0A840), the N- acetylglucosamine beta-1, 4-galactosyltransferase LgtB (Uniprot ID Q51116, sequence version 02, 01 Dec 2000) from N. meningitidis, the N-acylneuraminate cytidylyltransferase neuA from P. multocida (UniProt ID A0A849CI62) and the alpha-2, 3-sialyltransferase PmultST3 from P. multocida (UniProt ID Q9CLP3). The novel strain is evaluated in a growth experiment for production of a mixture comprising LacNAc and 3'SL according to the culture conditions provided in Example 1, in which the appropriate selective medium comprises glucose as carbon source and lactose as precursors. The strain is grown in four biological replicates in a 96-well plate. After 72h of incubation, the culture broth is harvested and production of a mixture comprising LacNAc and 3'SL is analysed on UPLC. Also the production of sialylated LacNAc in said mixture is evaluated.

Claims

Claims
1. A cell capable of synthesizing and/or synthesizing UDP-N-acetylglucosamine (UDP-GIcNAc), said cell comprising a pathway for production of a disaccharide and/or milk oligosaccharide, said cell genetically engineered for the production of said disaccharide and/or milk oligosaccharide, characterized in that said UDP-GIcNAc synthesis in said cell is rendered less functional.
2. Cell according to claim 1, wherein: said pathway for production of said disaccharide and/or milk oligosaccharide is selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway, and/or said pathway for production of said disaccharide and/or milk oligosaccharide is selected from the list consisting of or consisting essentially of fucosylation pathway, sialylation pathway, galactosylation pathway, N-acetylglucosaminylation pathway, N-acetylgalactosaminylation pathway, mannosylation pathway and N-acetylmannosaminylation pathway and wherein said cell is genetically engineered to comprise at least one of said pathway(s) and/or said cell comprises at least one of said pathway(s) wherein at least one of said pathway(s) has/have been genetically engineered.
3. Cell according to any one of claim 1 or 2, wherein said cell: possesses, expresses and/or overexpresses one or more glycosyltransferase(s) selected from the list consisting of or consisting essentially of fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N- acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, N- acetylmannosaminyltransferases, xylosyltransferases, glucuronyltransferases, galacturonyltransferases, glucosaminyltransferases, N-glycolylneuraminyltransferases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4,6-dideoxy-N-acetyl- beta-L-altrosamine transaminases, UDP-/V-acetylglucosamine enolpyruvyl transferases and fucosaminyltransferases, is capable to produce and/or produces one or more nucleotide-activated sugars, is genetically engineered for production of one or more nucleotide-activated sugar(s), comprises a pathway for the synthesis of a nucleotide-activated sugar selected from the list consisting of or consisting essentially of UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N- acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-GIc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose, (GDP-Fuc), UDP- glucuronate, UDP-galacturonate, UDP-2-acetamido-2,6-dideoxy-L-arabino-4-hexulose, UDP-2- acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-pneumosamine (UDP-L-PneNAC or UDP-2-acetamido-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N- acetyl-L-quinovosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), CMP-sialic acid (CMP-Neu5Ac), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP- Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), GDP-rhamnose and UDP-xylose, possesses and/or expresses one or more genes selected from the list consisting of or consisting essentially of mannose-5-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N- acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6- phosphate 2-epimerase, UDP-GIcNAc 2-epimerase/kinase, glucosamine 6-phosphate N- acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridylyltransferase, glucosamine-l-phosphate acetyltransferase, bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine- 1-phosphate acetyltransferase, Neu5Ac synthase, N-acetylneuraminate lyase, N- acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-1- phosphate uridylyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N- acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, more preferably overexpresses one or more genes selected from the list comprising mannose-6-phosphate isomerase, phosphomannomutase, mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-l-phosphate guanylyltransferase, L-glutamine— D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2- epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2- epimerase, UDP-GIcNAc 2-epimerase/kinase, glucosamine 6-phosphate N-acetyltransferase, N- acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, Neu5Ac synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N- acylneuraminate-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-l-epimerase, galactokinase, glucokinase, galactose-l-phosphate uridylyltransferase, UDP-glucose 4-epimerase, glucose-l-phosphate uridylyltransferase, phosphoglucomutase, UDP- N-acetylglucosamine 4-epimerase, N-acetylgalactosamine kinase and UDP-N- acetylgalactosamine pyrophosphorylase, and/or comprises a catabolic pathway for selected mono-, di- or oligosaccharides which is at least partially inactivated, the mono-, di-, or oligosaccharides being involved in and/or required for the synthesis of said disaccharide and/or milk oligosaccharide.
4. Cell according to any one of previous claims, wherein said UDP-GIcNAc synthesis possesses at least one gene selected from the list consisting of or consisting essentially of genes encoding bifunctional N-acetylglucosamine-l-phosphate uridyltransferase/glucosamine-l-phosphate acetyltransferase, an N-acetylglucosamine-l-phosphate uridyltransferase and a glucosamine-l-phosphate acetyltransferase, and wherein said at least one gene is rendered less functional.
5. Cell according to claim 4, wherein said at least one gene encodes an enzyme, wherein said enzyme: is selected from an enzyme class selected from the list consisting of or consisting essentially of EC:2.7.7.23, EC:2.3.1.157 and EC:5.4.2.3, comprises a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of IPR001451, IPR002618, IPR005175, IPR005835, IPR005843, IPR005844, IPR005882, IPR011004, IPR016055, IPR016066, IPR016657, IPR018357, IPR023915, IPR025877, IPR029044, IPR036900 and IPR038009 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of PF00132, PF00408, PF00465, PF00483, PF01070, PF01565, PF01704, PF02878, PF02879, PF02880, PF03479, PF04030, PF05199, PF12146, PF12804, PF13562 and PF14602 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising a conserved protein domain selected from the list consisting of or consisting essentially of cd03086 and cd03353 as defined by InterPro 90.0 as released on 4th August 2022, is part of a NOG family selected from the list consisting of or consisting essentially of COG1109 and COG4284 as defined by eggNOG5.0 as released in 2019, and/or uses a cofactor selected from the list consisting of or consisting essentially of Mg2+, Co2+, Mn2+, Ca2+, Zn2+, Ni2+ and FAD.
5. Cell according to any one of previous claims, wherein at least one gene involved in the synthesis and/or import of: a co-factor that is involved in UDP-GIcNAc synthesis is rendered less functional, and/or a co-factor that is involved in UDP-GIcNAc synthesis is rendered less functional and that is selected from the list consisting of or consisting essentially of Mg2+, Co2+, Mn2+, Ca2+, Zn2+, Ni2+ and FAD.
7. Cell according to any one of claims 4 to 6, wherein said at least one gene is rendered less functional by insertion, deletion and/or modification of one or more nucleotide(s) in one or more polynucleotide sequence(s) selected from the list comprising promoter sequence, ribosome binding site, untranslated region, coding sequence and transcription terminator sequence of said at least one gene.
8. Cell according to any one of previous claims, wherein said disaccharide and/or milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; a negatively charged milk oligosaccharide; sialylated milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide; non-fucosylated neutral (non-charged) mammalian milk oligosaccharide; sialylated human milk oligosaccharide; neutral (non-charged) human milk oligosaccharide; fucosylated human milk oligosaccharide; non-fucosylated neutral (non-charged) human milk oligosaccharide; fucosylated milk oligosaccharide selected from the list comprising 2'- fucosyl lactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2', 3- difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto- N-neohexaose; sialylated milk oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyl lacto-N- tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N- neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3’-sialyl-3- fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N- fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N- acetylglucosamine containing neutral (non-charged) milk oligosaccharide; N-acetylglucosamine containing neutral (non-charged) milk oligosaccharide selected from the list of lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto- N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose and para-lacto-N-neohexaose; N- acetylglucosamine containing milk oligosaccharide; N-acetyllactosamine containing milk oligosaccharide; fucosylated N-acetyllactosamine containing milk oligosaccharide; sialylated N- acetyllactosamine containing milk oligosaccharide; lacto-N-biose containing milk oligosaccharide; fucosylated lacto-N-biose containing milk oligosaccharide and sialylated lacto-N-biose containing milk oligosaccharide.
9. Cell according to any one of previous claims, wherein said cell: is capable to produce and/or produces said disaccharide and/or milk oligosaccharide from one or more precursor(s), is capable to produce and/or produces said disaccharide and/or milk oligosaccharide from lactose, is capable to produce and/or produces at least one precursor that is used to produce said disaccharide and/or milk oligosaccharide, is capable to produce and/or produces all precursors that are used to produce said disaccharide and/or milk oligosaccharide, is genetically engineered for the production of at least one precursor that is used to produce said disaccharide and/or milk oligosaccharide, and/or is genetically engineered for the production of all precursors that are used to produce said disaccharide and/or milk oligosaccharide.
10. Cell according to claim 9, wherein at least one of said one or more precursor(s) is internalized in said cell via one or more membrane protein(s).
11. Cell according to any one of previous claims, wherein said cell is further genetically engineered to possess, to express and/or to over-express a glutamine— fructose-6-phosphate aminotransferase.
12. Cell according to claim 11, wherein said glutamine— fructose-6-phosphate aminotransferase has glutamine— fructose-6-phosphate aminotransferase activity and: is selected from the enzyme class EC:2.6.1.16, comprises a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of IPR001347, IPR005855, IPR017932, IPR029055, IPR035466, IPR035490, IPR036291, IPR046348 and IPR047084 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of PF00310, PF01380, PF01408, PF13230, PF13537 and PF13580 as defined by InterPro 90.0 as released on 4th August 2022, comprises a polypeptide sequence comprising a conserved protein domain selected from the list consisting of or consisting essentially of cd00714, cd05007, cd05008, cd05009, cd05013 and cd05710 as defined by InterPro 90.0 as released on 4th August 2022, and/or is part of the NOG family COG0449 as defined by eggNOG5.0 as released in 2019.
13. Cell according to any one of claim 11 or 12, wherein said cell contains a nucleic acid molecule which comprises a polynucleotide sequence which encodes said glutamine— fructose-6-phosphate aminotransferase and wherein said nucleic acid molecule is: operably linked to control sequences recognized by the cell, said nucleic acid molecule further i) being integrated in the genome of said cell and/or ii) presented to said cell on a vector, and/or foreign to said cell.
14. Cell according to any one of previous claims, wherein said cell is: modified for enhanced synthesis and/or supply of phosphoenolpyruvate (PEP), and/or further modified for reduced degradation of acetyl-CoA and/or its main precursor pyruvate.
15. Cell according to any one of previous claims, wherein said cell is: a bacterium, fungus, yeast, a plant cell, an animal cell, or a protozoan cell, an E. coli or yeast with a lactose permease positive phenotype, and/or an E. coli or yeast with a lactose permease positive phenotype wherein said lactose permease is coded by the gene LacY or LAC12, respectively.
16. Method for the production of a disaccharide and/or milk oligosaccharide, the method comprising: iii. cultivating and/or incubating a cell of any one of previous claims, in cultivation and/or incubation medium under conditions permissive to produce said disaccharide and/or milk oligosaccharide, and/or iv. separating said disaccharide and/or milk oligosaccharide from said cultivation and/or incubation.
17. Method according to claim 16, wherein said: cultivation or incubation medium comprises one or more precursor(s) that is/are used for production of said disaccharide and/or milk oligosaccharide, cultivation medium contains at least one carbon source selected from the list consisting of glucose, fructose, sucrose, and glycerol, and/or cultivation or incubation medium contains at least one compound selected from the list consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid and CMP-sialic acid.
18. Method according to any one of claim 16 or 17, wherein said cell produces 30 g/L or more of said disaccharide and/or milk oligosaccharide in the whole broth and/or supernatant and/or wherein said disaccharide and/or milk oligosaccharide in the whole broth and/or supernatant has a purity of at least 80 % measured on the total amount of disaccharide and/or milk oligosaccharide and its/their precursor(s) produced by said cell in the whole broth and/or supernatant, respectively.
19. Method according to any one of claims 16 to 18, wherein said less functional synthesis of UDP-GIcNAc confers unaffected and/or enhanced i) disaccharide and/or milk oligosaccharide formation, ii) productivity, iii) biomass production, iv) cell growth and/or v) yield of the produced disaccharide and/or milk oligosaccharide, relative to a corresponding non-engineered cell.
20. Method according to any one of claims 16 to 19, wherein said: disaccharide and/or milk oligosaccharide is/are recovered from said cultivation or incubation medium and/or said cell, and/or disaccharide and/or milk oligosaccharide is/are purified.
21. Use of a cell according to any one of claims 1 to 15 for the production of a disaccharide and/or milk oligosaccharide, wherein said disaccharide and/or milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; negatively charged milk oligosaccharide; sialylated milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide; non-fucosylated neutral (non-charged) mammalian milk oligosaccharide; sialylated human milk oligosaccharide; neutral (non-charged) human milk oligosaccharide; fucosylated human milk oligosaccharide; non-fucosylated neutral (non-charged) human milk oligosaccharide; fucosylated milk oligosaccharide selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 5-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose II, lacto-N- fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N- neohexaose; sialylated milk oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyl lacto-N- tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N- neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3- fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N- fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N- acetylglucosamine containing neutral (non-charged) milk oligosaccharide; N-acetylglucosamine containing neutral (non-charged) milk oligosaccharide selected from the list of lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto- N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose and para-lacto-N-neohexaose; N- acetylglucosamine containing milk oligosaccharide; N-acetyllactosamine containing milk oligosaccharide; fucosylated N-acetyllactosamine containing milk oligosaccharide; sialylated N- acetyllactosamine containing milk oligosaccharide; lacto-N-biose containing milk oligosaccharide; fucosylated lacto-N-biose containing milk oligosaccharide and sialylated lacto-N-biose containing milk oligosaccharide.
22. Use of a method according to any one of claims 16 to 20 for the production of a disaccharide and/or milk oligosaccharide, wherein said disaccharide and/or milk oligosaccharide is/are selected from the list consisting of or consisting essentially of milk disaccharide; mammalian milk disaccharide; human milk disaccharide; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (non-charged) milk oligosaccharide; negatively charged milk oligosaccharide; sialylated milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); fucosylated milk oligosaccharide; non-fucosylated neutral (non-charged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (non-charged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide; non-fucosylated neutral (non-charged) mammalian milk oligosaccharide; sialylated human milk oligosaccharide; neutral (non-charged) human milk oligosaccharide; fucosylated human milk oligosaccharide; non-fucosylated neutral (non-charged) human milk oligosaccharide; fucosylated milk oligosaccharide selected from the list comprising 2’-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyl lactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentaose I, lacto-N-neofucopentaose I, lacto-N-fucopentaose ll7 lacto-N- fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-neofucopentaose V, lacto-N-difucohexaose I, lacto-N-difucohexaose II, difucosyl-lacto-N-hexaose and difucosyl-lacto-N- neohexaose; sialylated milk oligosaccharide selected from the list comprising 3'sialyllactose (3'SL), 6'sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyl lacto-N- tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, disialyllacto-N-hexaose II, monosialyllacto-N- neohexaose I, monosialyllacto-N-neohexaose II, disialyllacto-N-neohexaose, 3'-sialyl-3- fucosyllactose, fucodisialyllacto-N-hexaose, disialomonofucosyllacto-N-neohexaose, sialyllacto-N- fucohexaose II, disialyllacto-N-fucopentaose II and monofucosyldisialyllacto-N-tetraose; N- acetylglucosamine containing neutral (non-charged) milk oligosaccharide; N-acetylglucosamine containing neutral (non-charged) milk oligosaccharide selected from the list of lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyllactose, 3'-galactosyllactose, lacto- N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose and para-lacto-N-neohexaose; N- acetylglucosamine containing milk oligosaccharide; N-acetyllactosamine containing milk oligosaccharide; fucosylated N-acetyllactosamine containing milk oligosaccharide; sialylated N- acetyllactosamine containing milk oligosaccharide; lacto-N-biose containing milk oligosaccharide; fucosylated lacto-N-biose containing milk oligosaccharide and sialylated lacto-N-biose containing milk oligosaccharide.
PCT/EP2024/053033 2023-02-07 2024-02-07 Production of a disaccharide and/or milk oligosaccharide by a cell with reduced synthesis of udp-glcnac WO2024165611A1 (en)

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