Production of negatively charged oligosaccharides by cells

Abstract

The invention relates to the technical field of synthetic biology, metabolic engineering and cell culture. The present invention provides a cell for the production of a negatively charged, preferably sialylated, oligosaccharide wherein the cell is genetically engineered to have or express, preferably overexpress, a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase. The invention further provides the use of said cells in culture or incubation. Also described are methods of producing negatively charged, preferably sialylated oligosaccharides using the cells, and purification of the negatively charged, preferably sialylated oligosaccharides.

Description

TECHNICAL FIELD

[0001] The present invention is in the field of synthetic biology, metabolic engineering and cell cultivation. The present invention provides a cell for producing a negatively charged, preferably sialylated oligosaccharide, wherein the cell is genetically engineered to have or express, preferably overexpress, a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase. The present invention further provides the use of the cell in cultivation or incubation. The present invention also describes a method for producing a negatively charged, preferably sialylated oligosaccharide using the cell, as well as the purification of the negatively charged, preferably sialylated oligosaccharide. BACKGROUND

[0002] Oligosaccharides are highly diverse in chemical structure, composed of a variety of monosaccharides such as, for example, glucose, galactose, N-acetylglucosamine, xylose, rhamnose, fucose, mannose, N-acetylneuraminic acid, N-acetylgalactosamine, galactosamine, glucosamine, glucuronic acid, galacturonic acid, etc. Oligosaccharides are widely distributed in all living organisms and play important roles in a variety of physiological and pathological processes such as cell migration, signal transduction, cell-cell adhesion, inflammation and immune response. In order to fully exploit the biological advantages of oligosaccharides, it is of great importance to achieve the economical production of these oligosaccharides. An important class of oligosaccharides includes mammalian milk oligosaccharides (MMOs) and human milk oligosaccharides (HMOs), which are present in mammalian milk and human milk, respectively. The importance of milk oligosaccharides for the nutrition of animal and human infants is directly related to their biological activities, including protection of newborns from pathogenic agents, support of the development of the immune system and cognitive abilities of infants. In addition, milk oligosaccharides can serve as substrates for beneficial bacteria such as bifidobacteria or lactobacilli. Milk oligosaccharides are also thought to act as “decoys” by binding to human cell surface glycoproteins, reducing the risk of bacterial and viral pathogens adhering to human cells leading to infection. In addition, various milk oligosaccharides have anti-inflammatory effects and act as immunomodulators (e.g. reducing the risk of developing food allergies). Many of these milk oligosaccharides are negatively charged oligosaccharides. Among the negatively charged HMOs, sialylated HMOs (SHMOs) have been observed to have a variety of beneficial effects as described in the prior art. Among the sialylated oligosaccharides in human milk, 3’-sialyllactose, 6’-sialyllactose, sialyllactose-N-tetraose a, sialyllactose-N-tetraose b, sialyllactose-N-tetraose c and disialyllactose-N-tetraose are the most common members.

[0003] Negatively charged oligosaccharides were found to be a complex structure, the chemical or (chemo-)enzymatic synthesis of which has proven to be challenging: there are a wide range of difficulties, such as stereochemical control, formation of specific bonds, availability of starting materials, etc. Therefore, alternative production methods have been developed, in which attempts have been made to engineer microorganisms by metabolic engineering to produce negatively charged oligosaccharides. SUMMARY

[0004] It is an object of the present invention to provide tools and methods by which negatively charged, preferably sialylated oligosaccharides can be produced, preferably in an efficient, time- and cost-saving manner and in a way that allows for obtaining large amounts of the desired oligosaccharides.

[0005] According to the present invention, the above and other objects are achieved by providing methods and cells for producing negatively charged, preferably sialylated oligosaccharides. The present invention also provides methods for purifying said negatively charged, preferably sialylated oligosaccharides. Furthermore, the present invention provides a genetically engineered cell as described herein, which has or expresses, preferably overexpresses, a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase, wherein said hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising a conserved motif as set forth in SEQ ID NO 12 and has the activity of a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase. The present invention also provides purified negatively charged, preferably sialylated oligosaccharides by the above process. Further benefits resulting from the teachings of the present invention will be apparent to the skilled person from the present invention.

[0006] Definitions

[0007] The words used in this specification to describe the invention, its various embodiments and the operations thereof are to be understood not only in the sense of their commonly defined meanings, but to include by special definition in this regard set forth in this specification. Thus if an element can be understood in the context of this specification as including more than one meaning, then its use herein shall not be confined to the meaning for which it is defined in this context.

[0008] The various aspects and embodiments of the invention disclosed herein should be construed in connection with the order and context in which they are specifically described in this specification, but should also be construed to include any order and any combination thereof. Unless otherwise indicated, the various embodiments identified herein can be combined. 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. Where context requires, all words used herein in the singular tense shall be treated as though also used in the plural, and vice versa, unless otherwise specifically indicated. Unless otherwise defined, all technical and scientific terms used herein 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 experimental procedures described herein in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well-known and commonly used 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. The specific terminology of this document has been chosen for reasons of readability and inclusion only, and is not intended to limit the scope of the application. It will be apparent to those skilled in the art that various modifications, other embodiments, improvements, details and uses can be made in the spirit of the disclosure, and these are intended to be within the scope of the disclosure, which is limited only by the claims, interpreted in accordance with the patent statutes, including the doctrine of equivalents. In the appended claims, the reference characters used to indicate steps are provided for ease of understanding only, and are not intended to imply any particular order of execution of the steps, unless otherwise specifically indicated.

[0009] In this specification, although specific terms have been used to describe the embodiments of the invention, these terms have been used in a descriptive sense only and should not be construed to limit the scope of the invention, which is defined by the appended claims. It should be understood that the illustrated embodiments are given only for illustrative purposes and should not be considered as limiting the application. It will be apparent to those skilled in the art that modifications, other embodiments, improvements, details and uses can be made in the spirit of the disclosure, and these are intended to be within the scope of the disclosure, which is limited only by the claims, interpreted in accordance with the patent statutes, including the doctrine of equivalents. In the appended claims, the reference characters used to indicate steps are provided for ease of understanding only, and are not intended to imply any particular order of execution of the steps, unless otherwise specifically indicated.

[0010] Throughout the application, the features "synthesizing", "synthesized" and "synthesis" are used interchangeably with the features "producing", "produced" and "production", respectively, unless explicitly stated otherwise. Throughout the application, the expressions "capable of <verb>" and "capable of performing <verb>" are preferably replaced by the active voice of the verb in question, and vice versa. For example, the expression "capable of expressing" is preferably replaced by "expressing", and vice versa, i.e. "expressing" is preferably replaced by "capable of expressing". In this document and its claims, the verbs "comprise", "have" and "contain" and their conjugates, are used in their non-limiting sense to mean that the elements following the verb are included, but not exclusively. Throughout the application, the verb "comprise" can be replaced by "consist of" or "consist essentially of", and vice versa. Furthermore, the verb "consist of" can be replaced by "consist essentially of", which means that the composition defined herein can include additional components other than the specifically identified components, which do not alter the unique characteristics of the invention. In this document and its claims, the verbs "comprise", "have" and "contain" and their conjugates, unless explicitly stated otherwise, can be replaced by "consist of" (and conjugates thereof) or "consist essentially of" (and conjugates thereof), and vice versa. Furthermore, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of this element is present, unless the context clearly requires only one of the elements. Thus, the indefinite article "a" or "an" generally means "at least one".

[0011] Throughout the document and claims, the indefinite articles "a" and "an" are preferably replaced by "at least one", more preferably by "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 meaning of the word "about" or "approximately" is preferably that the numerical value or range is interpreted as the same as the accuracy of the method used to measure it, when used in connection with a numerical value (e.g. "about 10") or a range (e.g. "about x to about y"). If no error range is specified, the expression "about" or "approximately" used in connection with a numerical value is interpreted as having the same rounding as the given value. Throughout this document and its claims, unless otherwise stated, the expression "from x to y", where x and y represent numerical values, means a range of numerical values, where x is the lower limit of the range and y is the upper limit of the range. Here, x and y are also included in the range.

[0012] According to the present application, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. DNA or RNA containing unusual bases such as inosine, or modified bases such as tritylated bases also is encompassed by the term "polynucleotide." It is understood that DNA and RNA undergo extensive modification by a variety of enzymes in living organisms, and that such chemically, enzymatically, or metabolically modified forms of polynucleotides are included in the term "polynucleotide" as used herein. The term "polynucleotide" as used herein includes such chemically, enzymatically, or metabolically modified forms of polynucleotides as well as the chemical forms of DNA and RNA found in nature, including, for example, simple cells and complex cells. The term "polynucleotide" also includes short nucleic acid sequences generally referred to as oligonucleotide.

[0013] "Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to one another by peptide or modified peptide bonds. "Polypeptide" refers to short chains as well as longer chains of amino acids, and is inclusive of what is commonly referred to as peptides, oligopeptides and proteins. Polypeptides can comprise amino acids other than the 20 gene-encoded amino acids. "Polypeptide" includes those that have been modified by natural processes, such as post- translational and other modifications, or by chemical modification techniques well known in the art. The same type of modification can occur at several different sites on a given polypeptide, and a given polypeptide can contain a variety of types of modifications. Modifications can occur anywhere on the polypeptide, including the peptide backbone, the amino acid side chains, and the amine or carboxyl termini. Polypeptides can be branched or cyclic, and can have modifications that are not naturally occurring. Cyclic, branched, and branched cyclic polypeptides can be produced by synthetic methods or by post- translational natural processes.

[0014] The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that comprise a sequence, which directly or indirectly encodes a polypeptide of the application. The term also encompasses polynucleotides that comprise a single continuous region or discontinuous regions encoding the polypeptide (e.g., by way of an integrative phage or an intervening sequence or editing interruption) and can further comprise additional regions encoding and / or non-coding sequences.

[0015] "Isolated" means "artificially" altered from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. When the term is used herein, for example, a polynucleotide or polypeptide that naturally occurs within a living organism is not "isolated," but the same polynucleotide or polypeptide that is separated from coexisting materials of its natural state is "isolated." Similarly, a "synthetic" sequence, when the term is used herein, means any sequence that is synthetically produced rather than directly isolated from a natural source. "Synthetic," when the term is used herein, means a sequence that is synthetically produced rather than directly isolated from a natural source.

[0016] "Recombinant" means DNA that has been engineered by grafting or splicing a gene from one species into the cell of a host organism of another species. Such DNA becomes part of the host's genetic makeup and is replicated.

[0017] The terms "recombinant" or "transgenic" or "metabolically engineered" or "genetically engineered" as used herein are used interchangeably when referring to a cell or host cell and mean 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 the cell" or a "sequence foreign to the location or environment within the cell"). Such a cell is described as transformed with at least one heterologous or foreign gene or as transformed by the introduction of at least one heterologous or foreign gene. A recombinant or metabolically engineered or genetically engineered or transgenic cell can contain a gene that is not present in the native (non-recombinant) form of that cell. A recombinant cell can also contain a gene that is present in the native form of that cell, wherein the gene is modified and re-introduced into the cell by artificial means. The term also encompasses a cell that contains a nucleic acid endogenous to the cell that has been modified, or whose expression or activity has been modified, without removing the nucleic acid from the cell; such modifications include modifications obtained by gene replacement, promoter replacement, site-directed mutagenesis, CrispR, riboswitches, recombineering, ssDNA mutagenesis, transposon mutagenesis, and related techniques known to those skilled in the art. Thus, a "recombinant polypeptide" is a polypeptide produced by a recombinant cell. The term also encompasses a cell that has been modified by removal of an endogenous nucleic acid of the cell by means of techniques generally well known to the skilled artisan (e.g., gene knockout).

[0018] As used herein, "heterologous sequence" or "heterologous nucleic acid" refers to a sequence or nucleic acid derived from a source other than the particular cell (e.g., from a different species), or if from the same source, is modified from its original form or changed in its location in the genome. Thus, a heterologous nucleic acid operably linked to a promoter is from a different source than the source of the promoter, or, if from the same source, is modified from its original form or changed in its location in the genome. Heterologous sequences can be stably introduced into the genome of a host cell by transfection, transformation, conjugation, or transduction, among other means, where the techniques that can be employed will depend on the cell and sequence to be introduced. Various techniques are well known to those skilled in the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Within the context of the present application, the term "mutated" or "engineered" cell refers to a cell that has been genetically engineered.

[0019] The term "endogenous" within the context of the present disclosure refers to any polynucleotide, polypeptide, or protein sequence that is a natural part of the cell and occurs at its natural location in the cell's chromosome, with the control mechanisms for its expression unchanged as compared to the natural control mechanisms acting on its expression. The term "exogenous" refers, in contrast, to any polynucleotide, polypeptide, or protein sequence that is derived from a source other than the cell under study and is not a natural part of the cell, or does not occur at its natural location in the cell's chromosome or plasmid.

[0020] The term "heterologous," when used in reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme, indicates a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that originates from a source other than the host organism, or, if the host organism, is a eukaryote, source other than the cell's own genome. In contrast, "homologous" polynucleotides, genes, nucleic acids, polypeptides, or enzymes are used herein to indicate polynucleotides, genes, nucleic acids, polypeptides, or enzymes that originate from the host organism. When referring to a gene regulatory sequence or ancillary nucleic acid sequence used to maintain or manipulate a gene sequence (e.g., a promoter, 5' untranslated region, 3' untranslated region, polyadenylation sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genomic homology region, recombination site, etc.), "heterologous" indicates that the regulatory or ancillary sequence is not naturally associated with the gene with which it is present in the construct, genome, chromosome, or episome. Thus, a promoter that is not operably linked with a gene in the natural state of the promoter (i.e., in the genome of a non-genetically engineered organism) is referred to herein as a "heterologous promoter" when operably linked with the gene, even though the promoter can originate from the same species (or in some cases the same organism) as the gene with which it is linked.

[0021] The term "altered expression" of a gene relates to a change in expression relative to the wild type expression of said gene at any stage of the production process of the desired negatively charged, preferably sialylated oligosaccharides. The altered expression is a lower expression or a higher expression relative to the wild type, wherein the term "higher expression" is defined as "overexpression" of the gene in case of an endogenous gene, and as "expression" in case of a heterologous gene which is not present in the wild type strain. Lower expression is obtained by techniques generally known to the skilled person (such as using siRNA, CrispR, CrispRi, riboswitches, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutated genes, gene knock-out, transposon mutagenesis, etc.) which are used to alter the gene in such a way that it is "less capable" (i.e. statistically significantly "less capable" relative to a functional wild type gene) or completely loses its capability (such as a gene knock-out) to produce the functional end product. The term "riboswitch", as used herein, is defined as a part of a messenger RNA which folds into a complex structure that blocks expression by interfering with translation. Binding of an effector molecule induces a conformational change, allowing regulated expression post-transcriptionally. Lower expression can also be obtained by altering the transcriptional unit, the promoter, the untranslated regions, the ribosome binding site, the Shine Dalgarno sequence or the transcriptional terminator, in addition to altering the gene of interest in a way as described above. Lower expression or reduced expression can for example be obtained by mutating one or more base pairs in the promoter sequence, or by completely replacing the promoter sequence by a constitutive promoter with a lower expression strength compared to the wild type, or by replacing it by an inducible promoter which leads to regulated expression, or by replacing it by a repressible promoter which leads to regulated expression. Overexpression or expression is obtained by techniques generally known to the skilled person (such as using artificial transcription factors, de novo design of promoter sequences, ribosome engineering, introduction or re-introduction of expression modules at euchromatin, using high copy number plasmids), wherein the gene is part of an "expression cassette" which relates to any sequence wherein a promoter sequence, an untranslated region sequence (comprising a ribosome binding sequence, Shine Dalgarno or Kozak sequence), a coding sequence, and optionally a transcriptional terminator are present, and which leads to expression of a functionally active protein. The expression is constitutive, or conditional, or regulated or regulatable.

[0022] The term "constitutive expression" is defined as expression which is not regulated by transcriptional factors other than the RNA polymerase subunits (e.g. bacterial sigma factors like s 70 , s 54or related s factors, and the yeast mitochondrial RNA polymerase specific factor MTF1). Non-limiting examples of such transcription factors are CRP, LacI, ArcA, Cra, IclR in E. coli, or Aft2p, Crzlp, Skn7 in S. cerevisiae, or DeoR, GntR, Fur in B. subtilis. These transcription factors bind to specific sequences and can block or enhance expression under certain growth conditions. RNA polymerase is the catalytic machine that synthesizes RNA from a DNA template. RNA polymerase binds to specific DNA sequences to initiate transcription, for example through a sigma factor in prokaryotic hosts, or through MTF1 in yeast. Constitutive expression provides a constant level of expression without the need for induction or repression.

[0023] The term "regulated expression" is defined as expression that is regulated by a transcription factor other than a RNA polymerase subunit, for example a bacterial sigma factor, under certain growth conditions. Examples of such transcription factors are described above. Typically, expression regulation is obtained by an inducer, such as but not limited to IPTG, arabinose, rhamnose, fucose, allo-lactose, or a change in pH, or a change in temperature, or carbon source depletion, or substrate or produced product.

[0024] The term "control sequence" means a sequence of nucleotides that is recognized by the transcription and translation machinery of the cell for the purpose of transcribing and translating a polynucleotide sequence into a polypeptide. Thus, the control sequence is necessary for the expression of an operably linked coding sequence in a particular host cell, cell or organism. Such control sequences can include, but are not limited to, promoter sequences, ribosome binding sequences, Shine Dalgarno sequences, Kozak sequences, transcription terminator sequences. Control sequences appropriate for prokaryotic hosts include, for example, promoters, optional operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. If DNA encoding a pre- or secretion signal sequence is expressed as a preprotein involved in the secretion of the polypeptide, it can be operably linked to the DNA encoding the polypeptide; if a promoter or enhancer affects the transcription of the sequence, it is operably linked to the coding sequence; or if a ribosome binding site is positioned to facilitate translation, it is operably linked to the coding sequence. The control sequences can also be controlled by exogenous chemicals, such as but not limited to IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose, inducing or repressing the transcription or translation of the polynucleotide into a polypeptide by an inducible promoter or by a genetic circuit.

[0025] Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory signal sequence, are in reading frame. However, enhancers need not be contiguous.

[0026] The term "wild type" refers to the genetic or phenotypic state as it normally occurs in nature.

[0027] The term "altered expression of a protein", as used herein, means: i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein, iii) expression and / or overexpression of a variant protein having a higher activity relative to the wild type (i.e. naturally occurring in the expression host) protein, iv) reduced expression of an endogenous protein, or v) expression and / or overexpression of a variant protein having a reduced activity relative to the wild type (i.e. naturally occurring in the expression host) protein. Preferably, the term "altered expression of a protein", as used herein, means: 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 having a higher activity relative to the wild type (i.e. naturally occurring in the expression host) protein.

[0028] The term "altered activity" of a protein means a non-natural activity of the protein in any of the stages of the production process of the negatively charged, preferably sialylated, oligosaccharide of interest. The term "non-natural", as used herein when referring to the activity of a protein, means that the protein has been modified to have an abrogated, attenuated, reduced, delayed, higher, accelerated or improved activity relative to the natural activity of the protein. The altered activity of a protein is obtained by altered expression of the protein, or by expression of a modified form (i.e. a mutant form) of the protein. The mutant form of the protein can be obtained by expression of a mutant form of the gene encoding the protein, e.g. comprising one or more deletions, insertions and / or mutations of nucleotides relative to the natural gene sequence. The mutant form of the gene can be obtained by techniques well known to the person skilled in the art, such as but not limited to site-specific mutagenesis; CrispR; riboswitches; recombineering; ssDNA mutagenesis; transposon mutagenesis.

[0029] The term "non-natural", as used herein when referring to a cell producing a negatively charged, preferably sialylated, oligosaccharide, means that the negatively charged, preferably sialylated, oligosaccharide: i) is not naturally produced, or ii) when naturally produced, is not produced in the same amount by the cell; and the cell has been genetically engineered to be able to produce the negatively charged, preferably sialylated, oligosaccharide, or has a higher production of the negatively charged, preferably sialylated, oligosaccharide.

[0030] The term "variant," as used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, by an alteration of one or more nucleotides or amino acids. Typical polynucleotide variants differ by one or more nucleotide substitutions, additions or deletions from another reference polynucleotide. The nucleotide changes in the variant can or can not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in substitutions, additions, deletions, fusions and truncations of the polypeptide encoded by the reference sequence, as described below. Typical polypeptide variants differ by one or more substitutions, additions, deletions, or any combination thereof in the amino acid sequence as compared to another reference polypeptide. Usually, these differences are limited to substitutions, additions or deletions of only a few amino acids. The reference polypeptide and the variant are typically highly similar in sequence overall and identical in many regions. Variants can differ from the reference polypeptide in amino acid sequence by as few as one amino acid difference. Variants of a polynucleotide or polypeptide can be naturally occurring, such as allelic variants, or they can be non-naturally occurring variants. Non-naturally occurring variants of polynucleotides and polypeptides can be produced by mutagenesis techniques, direct synthesis, and other recombinant methods known to one of skill in the art.

[0031] In some embodiments, the present application contemplates the production of functional variants by altering the structure of the enzymes used in the present application. The variants can be produced by substitution, deletion, addition, or a combination thereof of amino acids. For example, the variants can be produced as fusion proteins comprising at least a portion of an enzyme of the present application fused to at least a portion of a peptide tag. The peptide tag can be used to aid in protein folding of the enzyme, to aid in purification after expression, to protect the enzyme from degradation enzymes, and / or to aid in the passage of the enzyme through a cell membrane. Examples of the peptide tag include, for example, a SUMO tag, an MBP tag, a His tag, a FLAG tag, a Strep-II tag, a Halo tag, a NusA tag, a thioredoxin, a GST, and / or a Fh8 tag. The fusion protein can be designed to comprise at least one cleavable peptide linker so that the enzyme of interest can be subsequently recovered from the fusion protein. The fusion protein can be designed to comprise multiple inclusion body tags, a cleavable peptide linker, and a region encoding the enzyme of interest. In the case of amino acid substitutions, it is reasonably contemplated that the substitution of a leucine for an isoleucine or valine, an aspartic acid for a glutamic acid, a threonine for a serine, or similarly, one amino acid for a structurally related amino acid (e.g., conservative mutations) will not have a major impact on the biological activity of the resulting molecule. A conservative substitution is one that occurs within a family of amino acids with related side chains. Whether a change in the amino acid sequence of a polypeptide of the present application results in a functional homolog can be readily determined by assessing the ability of the variant polypeptide to produce a response in a cell in a manner similar to the wild-type polypeptide.

[0032] The term "fragment", with respect to a polynucleotide, refers to any portion or part of a cloned or polynucleotide molecule, and in particular to a portion of a polynucleotide that retains useful, functional characteristics of the full-length polynucleotide molecule. Useful fragments include oligonucleotides and polynucleotides useful for hybridization or amplification techniques, or for regulating replication, transcription, or translation. A "polynucleotide fragment" refers to any subsequence of a polynucleotide SEQ ID NO, typically comprising or consisting of at least about 9, 10, 11, 12 contiguous nucleotides from the polynucleotide SEQ ID NO, for example at least about 30 nucleotides or at least about 50 nucleotides of any of the polynucleotide sequences provided herein. Exemplary fragments can additionally or alternatively include fragments comprising, consisting essentially of, or consisting of a region encoding a conserved family domain of a polypeptide. Exemplary fragments can additionally or alternatively include fragments comprising a conserved domain of a polypeptide. Thus, a fragment of a polynucleotide SEQ ID NO preferably refers to a nucleotide sequence comprising or consisting of the polynucleotide SEQ ID NO, wherein no more than about 200, 150, 100, 50, or 25 contiguous nucleotides are deleted, preferably no more than about 50 contiguous nucleotides are deleted, and functional characteristics (e.g., activities) useful of the full-length polynucleotide molecule are retained, which can be assessed by the skilled person by routine experimentation. Alternatively, a fragment of a polynucleotide SEQ ID NO preferably refers to a nucleotide sequence comprising or consisting of an amount of contiguous nucleotides of the polynucleotide SEQ ID NO, wherein the amount of contiguous nucleotides is at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100%, preferably at least 80%, more preferably at least 85%, even more preferably at least 87%, even more preferably at least 90%, even more preferably at least 95%, most preferably at least 97%, of the full length of the polynucleotide SEQ ID NO, and functional characteristics (e.g., activities) useful of the full-length polynucleotide molecule are retained, which can be assessed by the skilled person by routine experimentation.Thus, a fragment of the polynucleotide SEQ ID NO preferably means a nucleotide sequence comprising or consisting of said polynucleotide SEQ ID NO, wherein an amount of consecutive nucleotides is deleted, and said amount is not more than 50%, 40%, 30%, preferably not more than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, more preferably not more than 15%, even more preferably not more than 10%, even more preferably not more than 5%, most preferably not more than 2.5% of the full length of said polynucleotide SEQ ID NO, and said fragment retains a functional feature (e.g. activity) available to the full length polynucleotide molecule, which feature can be assessed by the skilled person by routine experimentation.

[0033] The term "fragment", with respect to a polypeptide, refers to a subsequence of the polypeptide which performs at least one biological function of the full polypeptide in essentially the same manner or to a similar extent. As described herein, a "subsequence of a polypeptide" or "segment of amino acid residues" refers to a sequence of contiguous amino acid residues derived from the polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain, such as a DNA binding site or domain that binds to a promoter region of DNA, an activation domain, or a domain for protein-protein interaction, and can initiate transcription. Fragments can vary in size from as few as 3 amino acid residues to the full length of the complete polypeptide, e.g., at least about 10 amino acid residues in length, e.g., at least about 20 amino acid residues in length, e.g., at least about 30 amino acid residues in length, e.g., at least about 100 amino acid residues in length, e.g., at least about 150 amino acid residues in length, e.g., at least about 200 amino acid residues in length. Thus, a fragment of a polypeptide SEQ ID NO (or UniProt ID) preferably refers to a polypeptide sequence comprising or consisting of the polypeptide SEQ ID NO (or UniProt ID) with a deletion of no more than about 200, 150, 125, 100, 80, 60, 50, 40, 30, 20, or 15 contiguous amino acid residues, preferably no more than about 100 contiguous amino acid residues, more preferably no more than about 50 contiguous amino acid residues, even more preferably no more than about 40 contiguous amino acid residues, and performs at least one biological function of the full polypeptide in essentially the same manner, preferably to a similar or higher extent, which can be assessed by the skilled person by routine experimentation. Alternatively, a fragment of a polypeptide SEQ ID NO (or UniProt ID) preferably refers to a polypeptide sequence comprising or consisting of an amount of contiguous amino acid residues from the polypeptide SEQ ID NO (or UniProt ID) which is at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100%, preferably at least 80%, more preferably at least 85%, even more preferably at least 87%, even more preferably at least 90%, even more preferably at least 95%, most preferably at least 97%, of the full length of the polypeptide SEQ ID NO (or UniProt ID), and performs at least one biological function of the full polypeptide in essentially the same manner, preferably to a similar or higher extent, which can be assessed by the skilled person by routine experimentation.A fragment of a polypeptide SEQ ID NO (or UniProt ID) thus preferably refers to a polypeptide sequence which comprises or consists of said polypeptide SEQ ID NO (or UniProt ID), wherein an amount of consecutive amino acid residues is deleted, and wherein said amount is not more than 50%, 40%, 30% of the full length of said polypeptide SEQ ID NO (or UniProt ID), preferably not more than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, more preferably not more than 15%, even more preferably not more than 10%, even more preferably not more than 5%, most preferably not more than 2.5% of the full length of said polypeptide SEQ ID NO (or UniProt ID), and which fragment performs at least one biological function of the full polypeptide in essentially the same manner, preferably to a similar or higher extent, as can be assessed by the skilled person by routine experimentation.

[0034] Throughout the application, sequences of polypeptides can be indicated by SEQ ID NOs, or alternatively by UniProt IDs. Thus, unless explicitly stated otherwise, the terms "polypeptide SEQ ID NO" and "polypeptide UniProt ID" can be used interchangeably.

[0035] A "functional fragment" of a polypeptide has at least one property or activity of the polypeptide from which it is derived, preferably to a similar or higher extent. A functional fragment may, for example, include a functional or conserved domain of the polypeptide. It is understood that a polypeptide or fragment thereof can have conservative amino acid substitutions which have essentially no effect on the activity of the polypeptide. A conservative substitution refers to the replacement of one hydrophobic amino acid by another, or one polar amino acid by another, or one acidic amino acid by another, or one basic amino acid by another, etc. Preferably, a conservative substitution refers to the following combinations: such as glycine replaced by alanine, and vice versa; valine, isoleucine and leucine replaced by methionine, and vice versa; aspartic acid replaced by glutamic acid, and vice versa; asparagine replaced by glutamine, and vice versa; serine replaced by threonine, and vice versa; lysine replaced by arginine, and vice versa; cysteine replaced by methionine, and vice versa; and phenylalanine and tyrosine replaced by tryptophan, and vice versa.

[0036] As used herein, homologous sequences refer to nucleotide sequences that have sequence similarity and encode polypeptides that share at least one functional characteristic, such as a biochemical activity. More specifically, as used herein, the term "functional homolog" refers to polypeptides that have both sequence similarity, in other words, homology, and at the same time at least one functional similarity, such as a biochemical activity (Altenhoff et al., PLoS Comput. Biol. 8 (2012) e1002514).

[0037] Homologs can be identified by nucleotide and polypeptide sequence alignment analysis. For example, a query in a nucleotide or polypeptide sequence database can identify homologs of a nucleotide or polypeptide of interest. Sequence analysis can include BLAST, Reciprocal BLAST, or PSI-BLAST analysis of a non-redundant database using the amino acid sequence of a reference polypeptide sequence. In some cases, the amino acid sequence is deduced from a nucleotide sequence. Typically, polypeptides in the database that have greater than 40% sequence identity to the polypeptide of interest are further evaluated as candidates for being homologous polypeptides. Amino acid sequence similarity allows for conservative amino acid substitutions, such as one hydrophobic residue for another, or one polar residue for another, or one acidic amino acid for another, or one basic amino acid for another, and the like. Preferably, conservative substitutions refer to the following combinations: such as glycine for alanine, and vice versa; valine, isoleucine, and leucine for methionine, and vice versa; aspartic acid for glutamic acid, and vice versa; asparagine for glutamine, and vice versa; serine for threonine, and vice versa; lysine for arginine, and vice versa; cysteine for methionine, and vice versa; phenylalanine and tyrosine for tryptophan, and vice versa. If desired, these candidates can be subjected to manual inspection to narrow the number of candidates for further evaluation.

[0038] Domains can be characterized by, for example, Pfam (El-Gebali et al., Nucleic Acids Res. 47 (2019) D427-D432), IPR (InterPro domains) (http: / / ebi.ac.uk / interpro) (Mitchell et al., Nucleic Acids Res. 47 (2019) D351-D360), PRINTS domains (Attwood et al., Nucleic Acids Res. 31 (2003) 400-402), SUBFAM domains (Gough et al., J. Mol. Biol. 313 (2001) 903-919), TIGRFAM domains (Selengut et al., Nucleic Acids Res. 35 (2007) D260-D264), Conserved Domain Database (CDD) annotations (https: / / www.ncbi.nlm.nih.gov / cdd) (Lu et al., Nucleic Acids Res. 48 (2020) D265-D268), PTHR domains (http: / / www.pantherdb.org) (Mi et al., Nucleic Acids. Res. 41 (2013) D377-D386; Thomas et al., Genome Research 13 (2003) 2129-2141), or PATRIC identifiers or PATRIC DB global family domains (https: / / www.patricbrc.org / ) (Davis et al., Nucleic Acids Res. 48(D1) (2020) D606-D612). Protein or polypeptide sequence information and functional information can be provided through comprehensive protein sequence and annotation data resources, such as, for example, the Universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49(D1), D480-D489). UniProt includes a database of expertly curated proteins, called the UniProt Knowledgebase (UniProtKB), as well as UniProt Reference Clusters (UniRef) and UniProt Archive (UniParc). The UniProt identifier (UniProtID) is unique to each protein present in the database.Throughout the application, the sequences of polypeptides are denoted by SEQ ID NO or UniProt ID. Unless otherwise stated, the UniProt ID of the protein corresponds to the sequence version 01 of its presence in the UniProt database (www.uniprot.org) in the 2021_03 release version and consulted on June 9, 2021.

[0039] InterPro provides functional analysis of proteins by classifying proteins into families and predicting domains and important sites. To classify proteins in this way, InterPro uses prediction models (called signatures) that are provided by several different databases (called member databases) that make up the InterPro consortium. Protein signatures from these member databases are integrated into a single searchable resource, taking advantage of each other’s strengths, forming a powerful integrated database and diagnostic tool.

[0040] It is understood that, for the person skilled in the art, the databases used herein include Pfam 32.0 (released in September 2018), CDD v3.17 (released on April 3, 2019), eggnogdb 4.5.1 (released in September 2016), InterPro 90.0 (released on August 4, 2022), PANTHER 17.0 (released on February 23, 2022) and PATRIC 3.6.9 (released in March 2020), the content of each database being fixed in the respective release version and not modifiable. When the content of a certain database changes, this certain database receives a new release version and a new release date. All release versions of each database and their corresponding release dates and the specific content annotated at these specific release dates are available and known to the person skilled in the art.

[0041] The terms "identical" or "percent identity," or "% identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For sequence comparison, one sequence is used as a reference sequence, to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the % sequence identity for the test sequence against the reference sequence, based on the designated program parameters. The percentage of sequence identity can be determined by comparing two sequences over a window of comparison of at least about 20 consecutive residues, which 20 residue comparison window can operate over the aligned sequences, wherein the portion of the sequences in the comparison window can comprise additions or deletions of one or more residues, relative to the reference sequence for fast and optimal alignment of the comparison window. The percentage of sequence identity is calculated as the number of positions in the comparison window that match divided by the number of positions in the comparison window times 100. The percentage of sequence identity can be calculated globally over the entire length of a given SEQ ID NO (i.e., the reference sequence), resulting in a global % identity score. Alternatively, the % identity can be calculated over a portion of the reference sequence, resulting in a local percentage identity score. The portion of the reference sequence preferably refers to at least about 50%, 60%, 70%, 80%, 90%, or 95% of the full length of the reference sequence. In another preferred embodiment, the portion of the reference polypeptide sequence refers to a segment of at least 150 amino acid residues up to the total number of amino acid residues of the reference polypeptide sequence. In another more preferred embodiment, the portion of the reference polypeptide sequence refers to a segment of at least 200 amino acid residues up to the total number of amino acid residues of the reference polypeptide sequence. The use of the full length of the reference sequence in a local sequence alignment results in a global percentage identity score between the test sequence and the reference sequence.

[0042] The percentage identity can be determined using different algorithms, such as, for example, BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25: 17, 3389-402), the Clustal Omega method (Sievers et al., 2011, Mol. Syst. Biol. 7:539), the MatGAT method (Campanella et al., 2003, BMC Bioinformatics, 4:29) or the EMBOSS Needle.

[0043] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 80% or more sequence identity over a segment of at least 150 amino acid residues of a reference polypeptide sequence shall be understood as said amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity over a segment of at least 150 amino acid residues of the reference polypeptide sequence.

[0044] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 80% or more sequence identity over a segment of at least 200 amino acid residues of a reference polypeptide sequence shall be understood as said amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity over a segment of at least 200 amino acid residues of the reference polypeptide sequence.

[0045] As used herein, a polypeptide comprising or consisting of an amino acid sequence having 80% or more sequence identity over a segment of at least 200 amino acid residues of a reference polypeptide sequence shall be understood as said amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity over a segment of at least 200 amino acid residues of the reference polypeptide sequence.

[0046] Throughout the application, unless explicitly stated otherwise, a polypeptide comprising, consisting of, or having an amino acid sequence having 80% or more sequence identity to the full-length amino acid sequence of a reference polypeptide, typically denoted by a SEQ ID NO or UniProt ID, preferably having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the full-length reference sequence, more preferably having at least 85% sequence identity, even more preferably having at least 90% sequence identity, is intended. In addition, unless explicitly stated otherwise, a polynucleotide sequence comprises, consists of, or has a nucleotide sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the full-length reference sequence, more preferably having at least 85% sequence identity, even more preferably having at least 90% sequence identity.

[0047] For the purposes of the present application, sequence identity is determined using MatGAT 2.01 (Campanella et al., 2003, BMC Bioinformatics 4:29). For proteins, the following default parameters are employed: (1) Gap opening cost: 12, extension: 2; (2) The matrix used is BLOSUM50. In preferred embodiments, sequence identity is calculated based on the full-length sequence of a given SEQ ID NO (i.e. the reference sequence) or a portion thereof. The portion preferably refers to at least 50%, 60%, 70%, 80%, 90%, or 95% of the complete reference sequence.

[0048] The terms "sialic acid", "N-acetylneuraminic acid salt", "N-acylneuraminic acid salt", "N-acetylneuraminic acid" are used interchangeably and refer to an acidic sugar including, but not limited to, Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulosonic acid (KDO).

[0049] Neu4Ac is also known as 4-O-acetyl-5-amino-3,5-dideoxy-D-glycero-D-galacto- nonulosonate or 4-O-acetylneuraminic acid and has the molecular formula C11H19NO9. Neu5Ac is also known as 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonic acid, D-glycero-5-acetamido-3,5-dideoxy-D-galacto-nonulosonic acid, 5-(acetamido)-3,5- dideoxy-D-glycero-D-galacto-2-ketopyranononic acid, 5-(acetamido)-3,5-dideoxy-D- glycero-D-galacto-2-ketopyranononic acid, 5-(acetamido)-3,5-dideoxy-D-glycero-D- galacto-2-ketopyranononic acid, or 5-(acetamido)-3,5-dideoxy-D-glycero-D-galacto-2- pyranononic acid and has the molecular formula C11H19NO9. Neu4,5Ac2 is also known as N-acetyl-4-O-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid salt, 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonic acid 4-acetate, 5-(acetamido)-3,5-dideoxy-D-glycero-D-galacto-2-ketopyranononic acid 4-acetate, 4-acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulosonic acid, or 4-acetate 5-(acetamido)-3,5-dideoxy-D-glycero-D-galacto-2-ketopyranononic acid and has the molecular formula C13H21NO10. Neu5,7Ac2 is also known as 7-O-acetyl-N-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, 7-O-acetyl-N-acetylneuraminic acid salt, 5-acetamido-3,5-dideoxy-D-glycero-D- galacto-nonulosonic acid 7-acetate, 5-(acetamido)-3,5-dideoxy-D-glycero-D-galacto-2- ketopyranononic acid 7-acetate, 7-acetate 5-acetamido-3,5-dideoxy-D-glycero-D-galacto- nonulosonic acid, or 7-acetate 5-(acetamido)-3,5-dideoxy-D-glycero-D-galacto-2- ketopyranononic acid and has the molecular formula C13H21NO10. Neu5,8Ac2 is also known as 5-n-acetyl-8-o-acetylneuraminic acid and has the molecular formula C13H21NO10. Neu5,9Ac2 is also known as N-acetyl-9-O-acetylneuraminic acid, 9-anana, 9-O-acetyl sialic acid, 9-O-acetyl-N-acetylneuraminic acid, 5-n-acetyl-9-O-acetylneuraminic acid, N,9-O- diacetylneuraminic acid, or N,9-O-diacetylneuraminic acid salt and has the molecular formula C13H21NO10. Neu4,5,9Ac3 is also known as 5-N-acetyl-4,9-di-O-acetylneuraminic acid.Neu5,7,9Ac3 is also known as 5-N-acetyl-7,9-di-O-acetylneuraminic acid. Neu5,8,9Ac3 is also known as 5-N-acetyl-8,9-di-O-acetylneuraminic acid. Neu4,5,7,9Ac4 is also known as 5-N-acetyl-4,7,9-tri-O-acetylneuraminic acid. Neu5,7,8,9Ac4 is also known as 5-N-acetyl-7,8,9-tri-O-acetylneuraminic acid. Neu4,5,7,8,9Ac5 is also known as 5-N-acetyl-4,7,8,9-tetra-O-acetylneuraminic acid. Neu5Gc is also known as N-glycolyl-neuraminic acid, N-glycolylneuraminic acid, N-glycolylneuraminic acid salt, N-glycolyl- neuraminic acid salt, N-glycolyl-neuraminic acid salt, N-glycolylneuraminic acid, N-glycolylneuraminic acid, 3,5-dideoxy-5-((hydroxyacyl)amino)-D-glycero-D-galacto-2- ketononulosonic acid, 3,5-dideoxy-5-(glycolylamino)-D-glycero-D-galacto-2- ketopyranonulosonic acid, 3,5-dideoxy-5-(glycolylamino)-D-glycero-D-galacto-non-2- pyranonulosonic acid, 3,5-dideoxy-5-[(hydroxyacetyl)amino]-D-glycero-D-galacto-non-2- pyranonulosonic acid, D-glycero-5-glycolylamino-3,5-dideoxy-D-galacto-non-2-pyranonulosonic acid, and has the molecular formula C11H19NO10. 2-keto-3-deoxymannose-octulosonic acid is also known as KDO, Kdo, kdo, 2-keto-3-deoxy-D-manno-octulosonic acid, 2-oxo-3-deoxy-D-mannooctulosonic acid, 3-deoxy-D-manno-2-octulosonic acid, 3-deoxy-D-manno-oct-2-enonic acid, 3-deoxy-D-manno-oct-2-enonic acid, 3-deoxy-D-manno-octulosonic acid, 3-deoxy-D-manno-oct-2-enonic acid, 3-deoxy-D-manno-octulosonic acid, keto-deoxyoctulosonic acid, keto-deoxyoctulosonic acid, (6R)-6-(hydroxymethyl)-1-carboxy-2-deoxy-D-lyxo-pyranohexose, keto-deoxy-octulosonic acid, and has the molecular formula C8H14O8.

[0050] The term "glycosyltransferase", as used herein, refers to an enzyme capable of catalyzing the transfer of a sugar moiety of a donor to a specific acceptor and forming a glycosidic bond. The donor can be a precursor as defined herein. Glycosyltransferases are classified using nucleotide sugar diphosphates, nucleotide sugar monophosphates and sugar phosphates and related proteins, and have been divided into different sequence-based families that have been described (Campbell et al., Biochem. J. 326, 929-939 (1997)) and are available on the CAZy (CArbohydrate-Active EnZymes) website (www.cazy.org). As used herein, the glycosyltransferase can be selected from the list comprising, but not limited to, the following: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, N-acetylmannosaminyltransferase, xylosyltransferase, glucuronosyltransferase, galacturonosyltransferase, glucosaminyltransferase, N- acetylneuraminic acid transferase, rhamnosyltransferase, N-acetyl rhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-athobiose aminotransferase, UDP-N-acetylglucosamine enolpyruvyltransferase, and fucosaminyltransferase.

[0051] A sialyltransferase is a glycosyltransferase that transfers sialic acid (e.g., Neu5Ac) from a donor (e.g., CMP-Neu5Ac) to an acceptor. Sialyltransferases include alpha-2,3-sialyltransferases, alpha-2,6-sialyltransferases, and alpha-2,8-sialyltransferases, which catalyze the transfer of sialic acid to an acceptor through an alpha-glycosidic linkage. Sialyltransferases can be found in, but are not limited to, the GT29, GT42, GT52, GT80, GT97, and GT100 CAZy families.

[0052] The terms "alpha-2,3-sialyltransferase", "alpha 2,3 sialyltransferase", "3-sialyltransferase", "a-2,3-sialyltransferase", "a 2,3 sialyltransferase", "3 sialyltransferase", "3-ST", "3ST", or "a23-ST", as used herein, can be used interchangeably and refer to a glycosyltransferase that catalyzes the transfer of sialic acid from a donor CMP-sialic acid to an acceptor molecule in an alpha-2,3-linkage.

[0053] The terms "alpha-2,6-sialyltransferase," "alpha 2,6 sialyltransferase," "6-sialyltransferase," "a-2,6-sialyltransferase," "a 2,6 sialyltransferase," "6 sialyltransferase," "6-ST," "6ST," or "a26-ST," as used herein, can be used interchangeably and refer to a glycosyltransferase that catalyzes the transfer of sialic acid from the donor CMP-sialic acid to an acceptor molecule in an alpha-2,6-linkage.

[0054] The terms "alpha-2,8-sialyltransferase," "alpha 2,8 sialyltransferase," "8-sialyltransferase," "a-2,8-sialyltransferase," "a 2,8 sialyltransferase," "8 sialyltransferase," "8-ST," "8ST," or "a28-ST," as used herein, can be used interchangeably and refer to a glycosyltransferase that catalyzes the transfer of sialic acid from the donor CMP-sialic acid to an acceptor molecule in an alpha-2,8-linkage.

[0055] The term "monosaccharide," as used herein, refers to a sugar that cannot be broken down into simpler sugars by hydrolysis, which is classified as an aldose, a ketose, a deoxy sugar, a deoxynucleoside, a uronic acid, an aldonic acid, a ketonic aldonic acid, an aldaric acid, or a sugar alcohol, and contains one or more hydroxyl groups per molecule. A monosaccharide is a saccharide that contains only one simple sugar.

[0056] The term "phosphorylated monosaccharide," as used herein, refers to a monosaccharide that is phosphorylated. Examples of phosphorylated monosaccharides include, but are not limited to: glucose-1-phosphate, glucose-6-phosphate, glucose-1,6-diphosphate, galactose-1-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, fructose-1-phosphate, glucosamine-1-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-1-phosphate, mannose-1-phosphate, mannose-6-phosphate, or fucose-1-phosphate. Some, but not all, of these phosphorylated monosaccharides are precursors or intermediates in the production of activated monosaccharides.

[0057] The terms "activated monosaccharide," "nucleotide-activated sugar," "nucleotide-sugar," "activated sugar," "nucleoside," or "nucleotide donor," as used herein, can be used interchangeably and refer to an activated form of a monosaccharide. Examples of activated monosaccharides include, but are not limited to: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2,6-dideoxy-L-arabinose-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-xylose-4-hexulose, UDP-N-acetyl-L-rhamnoseamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acofucosamine, UDP-N-acofucosamine (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-quinosamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), GDP-L-quinosamine, CMP-sialic acid, GDP-fucose (GDP-Fuc), GDP-rhamnose, and UDP-xylose. Nucleotide-sugars function as glycosyl donors in glycosylation reactions. Glycosylation reactions are catalyzed by glycosyltransferases.

[0058] The term "CMP-sialic acid," as used herein, refers to a nucleotide-activated form of sialic acid, including, but not limited to: CMP-Neu5Ac, CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), and CMP-KDO.

[0059] The term "disaccharide," as used herein, refers to a sugar polymer containing two simple sugars (i.e., monosaccharides). Examples of disaccharides include, but are not limited to: lactose (Gal-β1,4-Glc), lacto-N-biose (Gal-β1,3-GlcNAc), N-acetyllactosamine (Gal-β1,4-GlcNAc), LacDiNAc (GalNAc-β1,4-GlcNAc), N-acetylgalactosaminylglucose (GalNAc-β1,4-Glc), Neu5Ac-α2,3-Gal, Neu5Ac-α2,6-Gal, fucopyranosyl-(1-4)-N- glycolylneuraminic acid (Fuc-(1-4)-Neu5Gc), sucrose (Glc-α1,2-Fru), maltose (Glc-α1,4-Glc), and melibiose (Gal-α1,6-Glc).

[0060] The term "oligosaccharide," as used herein, and as generally understood in the art, refers to a sugar polymer containing a small number (typically 3 to 20, preferably 3 to 10) of simple sugars (i.e., monosaccharides). Oligosaccharides as used in the present application can be linear structures, or can comprise branches. The bond (e.g., glycosidic bond, galactosidic bond, glucosidic bond, etc.) between two sugar units can be represented as 1,4, 1→4, or (1-4), which can be used interchangeably herein. For example, the terms "Gal-b1,4-Glc," "Gal-β1,4-Glc," "b-Gal-(1→4)-Glc," "β-Gal-(1→4)-Glc," "Galbeta1-4-Glc," "Gal-b(1-4)-Glc," and "Gal-β(1-4)-Glc" have the same meaning, i.e., a β-glycosidic bond links the 1-position carbon of galactose (Gal) to the 4-position carbon of glucose (Glc). Each monosaccharide can be in a cyclic form (e.g., pyranose form or furanose form). The bond between individual monosaccharide units can include a 1→2, a 1→3, a 1→4, a 1→6, a 2→1, a 2→3, a 2→4, a 2→6, a 1→2, a 1→3, a 1→4, a 1→6, a 2→1, a 2→3, a 2→4, and a 2→6. Oligosaccharides can contain both a- and β-glycosidic bonds, or can contain only a-glycosidic bonds, or only β-glycosidic bonds. The term "polysaccharide" refers to a compound made up of a large number of monosaccharides (typically more than 20) linked by glycosidic bonds.

[0061] Examples of oligosaccharides include, but are not limited to, Lewis-type antigen oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigens, enterobacterial common antigen (ECA), sugar chains present in lipopolysaccharides (LPS), oligosaccharide repeat units present in capsular polysaccharides, peptidoglycan (PG), amino sugars, human ABO blood group system antigens, animal oligosaccharides (preferably selected from the list consisting of N-glycans and O-glycans), plant oligosaccharides (preferably selected from the list consisting of N-glycans and O-glycans), sialylated oligosaccharides, neutral (uncharged) oligosaccharides, negatively charged oligosaccharides, fucosylated oligosaccharides, N-acetylglucosamine-containing oligosaccharides, lactosamine-containing oligosaccharides, N-acetyllactosamine-containing oligosaccharides, N-acetylglucosamine-containing sialylated oligosaccharides, N-acetylglucosamine-containing neutral (uncharged) oligosaccharides, N-acetylglucosamine-containing negatively charged oligosaccharides, N-acetylglucosamine-containing fucosylated oligosaccharides, N-acetylglucosamine-containing non-fucosylated oligosaccharides, lactosamine-containing sialylated oligosaccharides, lactosamine-containing neutral (uncharged) oligosaccharides, lactosamine-containing negatively charged oligosaccharides, lactosamine-containing fucosylated oligosaccharides, lactosamine-containing non-fucosylated oligosaccharides, N-acetyllactosamine-containing sialylated oligosaccharides, N-acetyllactosamine-containing neutral (uncharged) oligosaccharides, N-acetyllactosamine-containing negatively charged oligosaccharides, N-acetyllactosamine-containing fucosylated oligosaccharides, N-acetyllactosamine-containing non-fucosylated oligosaccharides, chitosan, chitosan containing oligosaccharides, proheparin, chondroitin sulfate, glycosaminoglycan oligosaccharides, heparin, heparan sulfate, dermatan sulfate, hyaluronan (hyaluronic acid), and keratan sulfate.

[0062] The terms "negatively charged oligosaccharide" or "acidic oligosaccharide", as used herein, can be used interchangeably and refer to a negatively charged oligosaccharide. In preferred embodiments, the negatively charged oligosaccharide is a sialylated oligosaccharide. As used herein, "sialylated oligosaccharide" is understood to mean an oligosaccharide containing a negatively charged sialic acid, i.e. an oligosaccharide containing one or more sialic acid residues. It has an acidic character. Such sialylated oligosaccharides are sugar structures comprising at least three monosaccharide subunits which are interconnected by glycosidic bonds, wherein at least one of the monosaccharide subunits is a sialic acid residue as described herein. A sialylated oligosaccharide can contain more than one sialic acid residue, e.g. two, three or more. The more than one sialic acid residue can be two, three or more identical sialic acid residues. The more than one sialic acid residue can also be two, three or more different sialic acid residues. For example, a sialylated oligosaccharide can contain one or more Neu5Ac residues and one or more KDO residues. Some examples are 3-SL (3'-sialyllactose or 3'SL or Neu5Ac-a2,3-Gal- b1,4-Glc), 3'-sialyllactosamine, 6-SL (6'sialyllactose, 6'-sialyllactose or 6'SL or Neu5Ac-a2,6-Gal- b1,4-Glc), 3,6-disialyllactose (Neu5Ac-a2,3-(Neu5Ac-a2,6)-Gal- b1,4-Glc), 6,6'-disialyllactose (Neu5Ac-a2,6-Gal- b1,4-(Neu5Ac-a2,6)-Glc), 8,3-disialyllactose (Neu5Ac-a2,8-Neu5Ac-a2,3-Gal- b1,4-Glc), 6'-sialyllactosamine, an oligosaccharide comprising 6'sialyllactose (also known as 6'-sialyllactose, 6'SL and 6'-SL), SGG hexasaccharide (Neu5Ac a-2,3 Gal b-1,3 GalNac b-1,3 Gal a-1,4 Gal b-1,4 Gal), sialylated tetrasaccharide, sialylated pentasaccharide, sialylated lacto-N-triose, sialylated lacto-N-tetraose, sialylated lacto-N-neotetraose, LSTc (Neu5Ac-a2,6-Gal- b1,4-GlcNAc- b1,3-Gal- b1,4-Glc), LSTd (Neu5Ac-a2,3-Gal- b1,4-GlcNAc- b1,3-Gal- b1,4-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-a2,3-Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc), disialyllacto-N-hexaose I, sialyllacto-N-tetraose b (LSTb, Gal-β1,3-(Neu5Ac-a2,6)-GlcNAc-β1,3-Gal-β1,4-Glc), 3’-sialyl-3-fucosyllactose, fucosidiasialyllacto-N-hexaose, disialylmonofucosyllacto-N-neohexaose, monofucosidemonosialyllacto-N-octaose (sialyl Lea), sialyllacto-N-fucopentaose II, disialyllacto-N-fucopentaose II, monofucosiddisialyllacto-N-tetraose, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal, Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal, 3’-KDO-lactose, 3’-KDO-lactosamine, 3’-KDO-lacto-N-biose, 3’-KDO-6’ sialyllactose, 3’ KDO-8-sialyllactose, KDO-2,3Galβ-1,3GalNacβ-1,3Galα-1,4Galβ-1,4Gal, KDO-2,3Galβ-1,3GlcNacβ-1,3Galβ-1,4Glc, KDO-2,3Galβ-1,4GlcNacβ-1,3Galβ-1,4Glc, 3’-KDO-3-fucosyllactose, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal, 3’-sialyl-2’-fucosyllactose, 6’-sialyl-2’-fucosyllactose, 6’-sialyl-3-fucosyllactose, Neu5Ac-a2,6-(Neu5Ac-a2,3-)Gal-b1,4-Glc, 3’-sialyl-3-fucosyllactosamine, Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc, 6’-sialyllacto-N-biose, 3’-sialyllacto-N-biose, Neu5Ac-a2,6-(GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc,Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,3-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3) -Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, 6’-KDO-lactose, 6’-KDO-lactosamine, 6’-KDO-lactose-N-disaccharide, KDO-lactose-N-trisaccharide, KDO-lactose-N-tetrasaccharide, and oligosaccharides carrying one or several sialic acid residues, including but not limited to: the oligosaccharide portion of the gangliosides selected from the group consisting of GM3 (3’ sialyllactose, Neu5Ac a-2,3 Gal b-4 Glc) and oligosaccharides comprising the GM3 motif, GD3 Neu5Ac a-2,8 Neu5Ac a-2,3 Gal b-1,4 Glc GT3 (Neu5Ac a-2,8 Neu5Ac a-2,8 Neu5Ac a-2,3 Gal b-1,4 Glc); GM2 GalNAc b-1,4 (Neu5Ac a-2,3) Gal b-1,4 Glc, GM1 Gal b-1,3 GalNAc b-1,4 (Neu5Ac a-2,3) Gal b-1,4 Glc, GD1a Neu5Ac a-2,3 Gal b-1,3 GalNAc b-1,4 (Neu5Ac a-2,3) Gal b-1,4 Glc, GT1a Neu5Ac a-2,8 Neu5Ac a-2,3 Gal b-1,3 GalNAc b-1,4 (Neu5Ac a-2,3) Gal b-1,4Glc, GD2 GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc, GT2 GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc, GD1b, Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc, GT1b Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc, GQ1b Neu5Acα-2,8Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc, GT1c Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc, GQ1c Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc, GP1c Neu5Acα-2,8Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc, GD1a Neu5Acα-2,3Galβ-1,3(Neu5Acα-2,6)GalNAcβ-1,4Galβ-1,4Glc, Fucosyl-GM1 Fucα-1,2Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc; All of these oligosaccharides can be extended for the preparation of the corresponding gangliosides by reacting the above oligosaccharide moieties with ceramide, or synthesizing the oligosaccharides on ceramide.

[0063] The term "charged oligosaccharide" is an oligosaccharide structure containing one or more negatively charged monosaccharide subunits, including sialic acid, glucuronic acid, and galacturonic acid. Charged oligosaccharides are also referred to as acidic oligosaccharides. In contrast, neutral (uncharged) oligosaccharides are non-sialylated oligosaccharides, and thus do not contain acidic monosaccharide subunits. Neutral oligosaccharides include uncharged fucosylated oligosaccharides, which contain one or more fucose subunits in their sugar chain structure, and uncharged non-fucosylated oligosaccharides, which do not contain any fucose subunits in their sugar chain structure. Other examples of charged oligosaccharides include sulfated chitosan and deacetylated chitosan.

[0064] The terms“3’ sialyllactose,”“3’-sialyllactose,”“alpha-2,3-sialyllactose,”“alpha 2,3 sialyllactose,”“a-2,3-sialyllactose,”“a 2,3 sialyllactose,”“3SL,” or“3’SL,” as used herein, can be used interchangeably and refer to the product obtained by the transfer of the sialic acid group in CMP-Neu5Ac to lactose in an a-2,3-linkage catalyzed by an alpha-2,3-fucosyltransferase. The terms“6’ sialyllactose,”“6’-sialyllactose,”“alpha-2,6-sialyllactose,”“alpha 2,6 sialyllactose,”“a-2,6-sialyllactose,”“a 2,6 sialyllactose,”“6SL,” or“6’SL,” as used herein, can be used interchangeably and refer to the product obtained by the transfer of the sialic acid group in CMP-Neu5Ac to lactose in an a-2,6-linkage catalyzed by an alpha-2,6-fucosyltransferase. The terms“LSTa,”“LS-tetrasaccharide a,”“sialyl-lactose-N-tetrasaccharide a,”“sialyllactose-N-tetrasaccharide a,” or“Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc,” as used herein, can be used interchangeably. The terms“LSTb,”“LS-tetrasaccharide b,”“sialyl-lactose-N-tetrasaccharide b,”“sialyllactose-N-tetrasaccharide b,” or“Gal-b1,3-(Neu5Ac-a2,6)-GlcNAc-b1,3-Gal-b1,4-Glc,” as used herein, can be used interchangeably. The terms“LSTc,”“LS-tetrasaccharide c,”“sialyl-lactose-N-tetrasaccharide c,”“sialyllactose-N-tetrasaccharide c,”“sialyllactose-N-neotetrasaccharide c,” or“Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc,” as used herein, can be used interchangeably. The terms“LSTd,”“LS-tetrasaccharide d,”“sialyl-lactose-N-tetrasaccharide d,”“sialyllactose-N-tetrasaccharide d,”“sialyllactose-N-neotetrasaccharide d,” or“Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc,” as used herein, can be used interchangeably. The terms“DSLNnT” and“disialyllactose-N-neotetrasaccharide,” as used herein, can be used interchangeably and refer to Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-[Neu5Ac-a2,6]-Gal-b1,4-Glc.The terms "DSLNT," "DS-LNT," and "disialyllacto-N-tetraose," as used herein, can be used interchangeably and refer to Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-βl,3)-GlcNAc-βl,3-Gal-βl,4-Glc.

[0065] The terms "neutral oligosaccharide" and "uncharged" oligosaccharide as used herein can be used interchangeably and refer to oligosaccharides that do not contain a negative charge derived from a carboxylic acid group, as is generally understood in the art. Examples of such neutral oligosaccharides are: 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2', 3-difucosyllactose (diFL), lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP I), lacto-N-neofucopentaose I (LNnFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFPV), lacto-N-fucopentaose VI, lacto-N-neofucopentaose V (LNnFP V), lacto-N-difucohexaose I (LNDFHI), lacto-N-difucohexaose II (LNDFH II), Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Fuc-a1,2-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Fuc-a1,2-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Fuc-a1,2-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Fuc-a1,4-(Fuc-a1,2-Gal-b1,3-)GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, mono-fucosyllacto-N-hexaose-III, di-fucosyllacto-N-hexaose (a), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, fucosyl-lacto-N-hexaose, di-fucosyl-lacto-N-hexaose, di-fucosyl-lacto-N-neohexaose (LNnDFH II), di-fucosyl-para-lacto-N-neohexaose, tri-fucosyllacto-N-hexaose, para-lacto-N-fucosylheptaose, a1,3-galactosyl-3-fucosyllactose, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc,3-) Glc, GalNAc-a1,3-(Fuc-a1,2-) Gal-b1,4-(Fuc-a1,3-) Glc, 2-fucosyllactose, 3-fucosyl-N-acetyllactosamine, 2'-fucosyl-N-acetyllactosamine, difucosyl-N-acetyllactosamine, 4-fucosyllactose-N-biose, 2'-fucosyllactose-N-biose, difucosyllactose-N-biose, GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-) Glc, GlcNAc-b1,6-(GlcNAc-b1,3-) Gal-b1,4-Glc, lacto-N-pentaose (LN5), lacto-N-neopentaose, para-lacto-N-pentaose, para-lacto-N-neopentaose, lacto-N-neopentaose 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, neolacto-N-neooctaose, para-lacto-N-neooctaose (pLNnO), iso-lacto-N-nonaose, neolacto-N-nonaose, lacto-N-nonaose (LN9), lacto-N-decaose, iso-lacto-N-decaose, neolacto-N-decaose, lacto-N-neodecaose, para-lacto-N-neodecaose (pLNnD), a1,3-galactosyllacto-N-neotetraose, GlcNAc-b1,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, GlcNAc-b1,6-(Gal-b1,4-GlcNAc-b1,3-) Gal-b1,4-Glc, and GlcNAc-b1,6-(Gal-b1,3-GlcNAc-b1,3-) Gal-b1,4-Glc.

[0066] The terms "LNT II", "LNT-II", "LN3", "lacto-N-triose II", "lacto-N-triose II", "lacto-N-triose", "lacto-N-triose", or "GlcNAcβ1-3Galβ1-4Glc" are used interchangeably in the present invention. The terms "LNT", "lacto-N-tetraose", "lacto-N-tetraose", or "Galβ1-3GlcNAcβ1-3Galβ1-4Glc" are used interchangeably in the present invention. The terms "LNnT", "lacto-N-neotetraose", "lacto-N-neotetraose", "neo-LNT", or "Galβ1-4GlcNAcβ1-3Galβ1-4Glc" are used interchangeably in the present invention. The terms "LNB" and "lacto-N-biose" are used interchangeably and both refer to the disaccharide Gal-b1,3-GlcNAc. The terms "LacNAc" and "N-acetyl lactosamine" are used interchangeably and both refer to the disaccharide Gal-b1,4-GlcNAc.

[0067] As used herein and as generally understood in the art, "fucosylated oligosaccharide" refers to an oligosaccharide bearing a fucose residue. Such fucosylated oligosaccharides are sugar structures comprising at least three monosaccharide subunits connected to each other by glycosidic bonds, wherein at least one of the monosaccharide subunits is fucose. A fucosylated oligosaccharide can comprise 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 connected to other monosaccharide subunits (including glucose, galactose, GlcNAc) by alpha-glycosidic bonds comprising alpha-1,2, alpha-1,3, alpha-1,4, alpha-1,6 bonds.

[0068] As used herein, the term "O antigen" refers to the repeating glycan component of the surface lipopolysaccharide (LPS) of Gram-negative bacteria. The term "lipopolysaccharide" or "LPS" refers to a glycolipid found in the outer membrane of Gram-negative bacteria, consisting of lipid A, a core oligosaccharide, and an O antigen. The term "capsular polysaccharide" refers to a long chain polysaccharide with an oligosaccharide repeating structure that is present in the bacterial capsule, the latter being a polysaccharide layer located outside the cell envelope. The term "peptidoglycan" or "murein" refers to an essential structural element in the cell wall of most bacteria, consisting of sugars and amino acids, with the sugar component consisting of alternating residues of β-1,4 linked GlcNAc and N-acetylmuramic acid. As used herein, the term "amino sugar" refers to a sugar molecule in which a hydroxyl group is replaced by an amine group. As used herein, human ABO blood group system antigens are oligosaccharides. Such human ABO blood group system antigens are not limited to human structures. The structures include the A determinant GalNAc-α1,3(Fuc-α1,2)-Gal-, the B determinant Gal-α1,3(Fuc-α1,2)-Gal-, and the H determinant Fuc-α1,2-Gal-, which are present on a disaccharide core structure comprising Gal-β1,3-GlcNAc, Gal-β1,4-GlcNAc, Gal-β1,3-GalNAc, and Gal-β1,4-Glc.

[0069] Mammalian milk oligosaccharides include oligosaccharides present in milk at any stage of lactation, including colostrum from humans (i.e., human milk oligosaccharides or HMOs) and mammals including, but not limited to, Bos Taurus, Ovisaries, Capra aegagrus hircus, Camelus bactrianus, Equus feruscaballus, Sus scropha, Canis lupus amilis, Ursus arctos yesoensis, Ursus maritimus, Ursus thibetanus japonicus, Mephitis mephitis, Cystophora cristata, Elephas maximus, Loxodonta africana, Myrmecophaga tridactyla, Tursiops truncates, Balaenoptera acutorostrata, Macropus eugenii, Macropus rufus, Trichosurus Vulpecula, Phascolarctos cinereus, Dasyurus viverrinus, Ornithorhynchus anatinus.As used herein, “mammalian milk oligosaccharide” or “MMO” refers to an oligosaccharide 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-disialyllactose- N-tetraose, lactodifucotetraose, 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, monofucosylpara-lacto-N- hexaose, monofucosyllacto-N-hexaose III, isomeric fucosyllacto-N-hexaose III, isomeric fucosyllacto-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 chitopentaose, fucosylated oligosaccharide, neutral oligosaccharide, and / or sialylated oligosaccharide.

[0070] “Human milk oligosaccharide” or “HMO” refers to an oligosaccharide found in human breast milk, including preterm infant milk, colostrum, and term milk. HMOs include fucosylated oligosaccharides, non-fucosylated neutral oligosaccharides, and sialylated oligosaccharides (see, e.g., Chen X., Chapter Four: Human Milk Oligosaccharides (HMOS): Structure, Function, and Enzyme-Catalyzed Synthesis in Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). Examples of HMOs include: 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, sialyl-lacto-N-tetraose c, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose a, difucosyllacto-N-tetraose, lacto-N-hexaose, lacto-N-difucohexaose I, lacto-N-difucohexaose II, disialyl-lacto-N-tetraose, fucosyl-lacto-N-hexaose, difucosyl-lacto-N-hexaose, fucosyl-disialyl-lacto-N-hexaose, disialyl-lacto-N-hexaose.

[0071] As used herein, the term "mammary cell" generally refers to a mammalian mammary epithelial cell, a mammalian mammary epithelial luminal cell, or a mammalian epithelial alveolar cell, or any combination thereof. As used herein, the term "mammary-like cell" generally refers to a mammalian cell that has a similar (or substantially similar) phenotype / genotype to a native mammalian mammary cell, but is derived from a mammalian non-mammary cell source. Such a mammalian mammary-like cell can be engineered to remove at least one unwanted genetic element and / or to comprise at least one predetermined genetic construct specific to a mammalian mammary cell. Non-limiting examples of a mammalian mammary-like cell can include a mammalian mammary epithelial-like cell, a mammalian mammary epithelial luminal-like cell, a mammalian non-mammary cell that exhibits one or more characteristics of a cell of a mammalian mammary cell lineage, or any combination thereof. Further non-limiting examples of a mammalian mammary-like cell can include a mammalian cell that has a similar (or substantially similar) phenotype to a native mammalian mammary cell, or more specifically, a mammalian cell that has a similar (or substantially similar) phenotype to a native mammalian mammary epithelial cell. A mammalian cell that has a similar (or substantially similar) phenotype to a native mammalian mammary cell or a mammalian mammary epithelial cell, or that exhibits at least one characteristic similar (or substantially similar) to a native mammalian mammary cell or a mammalian mammary epithelial cell, can include a native or engineered mammalian cell capable of expressing at least one milk component (e.g., derived from a mammary cell lineage or a non-mammary cell lineage).

[0072] As used herein, the term "non-mammary cell" generally can include any mammalian cell of a non-mammary lineage. In the context of the present application, a non-mammary cell can be any mammalian cell that can be engineered to express at least one milk component. Non-limiting examples of such a non-mammary cell include a hepatocyte, a blood cell, a kidney cell, a cord blood cell, an epithelial cell, an epidermal cell, a muscle cell, a fibroblast, a mesenchymal cell, or any combination thereof. In certain instances, molecular biology and genome editing techniques can be engineered to simultaneously eliminate, silence, or attenuate multiple genes.

[0073] The terms "hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase", "hydrolytic UDP-N-acetylglucosamine 2-epimerase", "hydrolytic UDP-GlcNAc-2-epimerase", "hydrolytic UDP-N-acetyl-D-glucosamine 2-epimerase", "hydrolytic UDP-N-acetylglucosamine-2-epimerase", "hydrolytic UDP-N-acetylglucosamine 2-epimerase", "hydrolytic uridine diphospho-N-acetylglucosamine-2'-epimerase", and "hydrolytic uridine diphosphoacetylglucosamine 2'-epimerase" are used interchangeably and refer to an enzyme catalyzing the reaction UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) = N-acetylmannosamine (ManNAc). The nnaA polypeptide is an example of a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase.

[0074] The term "non-hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase" refers to an enzyme catalyzing the reaction UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) = UDP-N-acetylmannosamine (UDP-ManNAc).

[0075] The "sialylation pathway" is a biochemical pathway consisting of at least one enzyme selected from the list comprising L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GlcNAc 2-epimerase / kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1 -phosphate urtidyltransferase, glucosamine-1 -phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphatase, D-arabinose 5-phosphate isomerase, KDO-8P synthase, KDO 8-phosphatase, CMP-KDO synthetase, sialate transport protein, and CMP-sialate synthase, in combination with a sialyltransferase, producing a2,3; a2,6 and / or a2,8 sialylated oligosaccharides.

[0076] The pathway for producing a negatively charged, preferably sialylated, oligosaccharide can comprise a pathway for the synthesis and / or import of co-factors and / or precursors used in the pathway for producing the negatively charged, preferably sialylated, oligosaccharide.

[0077] As used herein, the term“reductive pathway” refers to a biochemical pathway that results in a reduction of the cytosolic environment of a cell, where the reduced environment negatively impacts the proper folding of proteins and hinders disulfide bond formation. Examples of reductive pathways include, but are not limited to, the reductive acetyl-CoA pathway, the reductive pyrimidine catabolism pathway, the reductive citrate cycle, the thiol-redox pathway, and the reductive glycine pathway. A reductive cytosol refers to, for example, a low NADP+:NADPH ratio and / or a high level of glutathione (GSH) (e.g., 10 mM) in the cytosolic environment. The reduced environment can affect the oxidation state of molecules, thereby altering their solubility.

[0078] The terms“glutathione reductase,”“glutathione reductase (NADPH),”“glutathione S-reductase,”“GSH reductase,”“GSSG reductase,”“NADPH: oxidized glutathione oxidoreductase,”“NADPH-glutathione reductase,”“NADPH-GSSG reductase,”“gor,”“GR,” and“GRase” are used interchangeably to refer to an enzyme that catalyzes the reaction 2 glutathione + NADP+ = glutathione disulfide + H+ + NADPH.

[0079] The terms“thioredoxin reductase,”“thioredoxin disulfide reductase,”“NADPH: oxidized thioredoxin oxidoreductase,”“NADPH-thioredoxin reductase,”“NADP-thioredoxin reductase,”“thioredoxin reductase (NADPH),”“trxB,” and“TRXR” are used interchangeably to refer to an enzyme that catalyzes the reaction [thioredoxin]-dithiol + NADP+ = [thioredoxin]-disulfide + H+ + NADPH.

[0080] The terms“disulfide bond isomerase,”“protein disulfide bond isomerase,”“S-S rearranging enzyme,” and“PDI” are used interchangeably to refer to an enzyme that catalyzes the rearrangement of disulfide bonds in proteins.

[0081] The terms“disulfide bond oxidoreductase,”“disulfide bond oxidoreductase 2,”“thiol: disulfide bond oxidoreductase,”“dsbC,” and“xprA” are used interchangeably to refer to an enzyme that is reported to be involved in the rearrangement and / or formation of protein disulfide bonds.

[0082] The terms“thiol oxidase” and“mercaptan oxidase” are used interchangeably to refer to an enzyme that catalyzes the reaction O2+ 2R’C(R)SH = H2O2 + R’C(R)S-S(R)CR’.

[0083] The term "chaperone" refers to enzymes that assist in protein folding. Examples are PDI, SecB, ERp57, heat shock proteins or Hsps, such as Hsp 10, Hsp 60, Hsp 70, Hsp 90. The terms "pyruvate dehydrogenase", "pyruvate oxidase", "POX", "poxB", and "pyruvate:ubiquinone-8 oxidoreductase" are used interchangeably to refer to an enzyme that catalyzes the oxidative decarboxylation of pyruvate to acetic acid and carbon dioxide.

[0084] The terms "lactate dehydrogenase", "D-lactate dehydrogenase", "ldhA", "hslI", "htpH", "D-LDH", "fermentative lactate dehydrogenase", and "D-specific 2-hydroxy acid dehydrogenase" are used interchangeably to refer to an enzyme that catalyzes the conversion of lactate to pyruvate with the generation of NADH.

[0085] The term "membrane transport protein" as used herein refers to a protein that constitutes part of or interacts with the cell membrane and controls the flow of molecules and information across the cell. Thus, membrane proteins are involved in transport, whether import into the cell or export out of the cell. Such membrane transport proteins can be, but are not limited to, transporters, P-P-bond-hydrolysis-driven transporters, beta-barrel porins, accessory transporters, and phosphotransfer-driven group transporters (Forrest et al., Biochim. Biophys. Acta 1807 (2011) 167-188; Lengeler, J. Mol. Microbiol. Biotechnol. 25 (2015) 79-93; Moraes and Reithmeier, Biochim. Biophys. Acta 1818 (2012), 2687-2706; Saier et al., Nucleic Acids Res. 44 (2016) D372-D379).

[0086] The term "siderophore" as used herein refers to secondary metabolites of various microorganisms that are primarily iron ion-specific chelators. These molecules are classified as catecholate, hydroxamate, carboxylate, and mixed types. Siderophores are generally synthesized by either a non-ribosomal peptide synthetase (NRPS)-dependent pathway or a NRPS-independent pathway (NIS). The most important precursor in the NRPS-dependent siderophore biosynthetic pathway is chorismate. 2, 3-DHBA can be formed from chorismate via a three-step reaction catalyzed by isochorismate synthase, isochorismate lyase, and 2,3-dihydroxybenzoate-2,3-dehydrogenase. Siderophores can also be formed from salicylate, which in turn is formed from isochorismate by isochorismate pyruvate lyase catalysis. When ornithine is used as a precursor for siderophores, its biosynthesis depends on the hydroxylation of ornithine catalyzed by L-ornithine N5-monooxygenase. In the NIS pathway, an important step in siderophore biosynthesis is N(6)-hydroxylysine synthase. Transporters are required to export siderophores to the extracellular space. To date, four membrane protein superfamilies have been identified in this process: the Major Facilitator Superfamily (MFS); the Multidrug / Oligosaccharide-lipid / Poly-saccharide Flippase Superfamily (MOP); the Resistance, Nodulation, and Cell Division Superfamily (RND); and the ABC Superfamily. Typically, genes involved in siderophore export are clustered with genes for siderophore biosynthesis. The term "siderophore export protein" as used herein refers to such transporters required to export siderophores to the extracellular space.

[0087] The ATP-binding cassette (ABC) superfamily includes uptake and efflux transport systems, members of these two groups of transporters are generally loosely clustered together. ATP hydrolysis provides the energy for transport without protein phosphorylation. The ABC superfamily contains dozens of families, families are generally associated with substrate specificity. Members are classified according to Class 3.A.1 as defined by the Transporter Classification Database (accessible at www.tcdb.org) operated by the Saier Lab Bioinformatics Group, which provides a functional and phylogenetic classification of membrane transporters.

[0088] The Major Facilitator Superfamily (MFS) is a class of membrane transport protein superfamily that catalyze uniport, symport of solutes: cations (H+, but rarely Na+), and / or antiport of solutes: H+ or solutes: solutes. Most MFS proteins are 400-600 aminoacyl residues in length and have 12, 14, or occasionally 24 transmembrane alpha helix spanners (TMS) as defined by the Transporter Classification Database (www.tcdb.org) operated by the Saier Lab Bioinformatics Group.

[0089] “SET” or “sugar efflux transporter” as used herein refers to a membrane protein of the SET family, which are proteins having the InterPro domain IPR004750, and / or proteins belonging to the eggNOGv4.5 family ENOG410XTE9. The InterPro domain can be identified using the online tool at https: / / www.ebi.ac.uk / interpro / or the standalone version of InterProScan (https: / / www.ebi.ac.uk / interpro / download.html) using the default values. The identification of orthologous families in eggNOGv4.5 can be done using the online version or the standalone version of eggNOG-mapper v1 (http: / / eggnogdb.embl.de / # / app / home).

[0090] The term “activated efflux” refers to the transport activity of introducing solutes across the cytoplasmic membrane and / or cell wall. The transport can be achieved by introducing and / or increasing the expression of a membrane transport protein as described in the present application. The term “enhanced efflux” refers to the improved transport activity of solutes across the cytoplasmic membrane and / or cell wall. The transport of solutes across the cytoplasmic membrane and / or cell wall can be enhanced by introducing and / or increasing the expression of a membrane transport protein as described in the present application. “Expression” of a membrane transport protein is defined as “overexpression” of a gene encoding said membrane transport protein if the gene is an endogenous gene, and as “expression” of a gene encoding said membrane transport protein if the gene is a heterologous gene not present in the wild type strain or cell.

[0091] The term "purified" refers to a material that is substantially or essentially free from components that interfere with the activity of the biological molecule. For cells, carbohydrates, nucleic acids, and polypeptides, the term "purified" refers to material that is substantially or essentially free from components that normally accompany the material in its native state. Typically, a purified carbohydrate, oligosaccharide, protein, or nucleic acid of the application is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% pure, and often 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 purity determination method. Purity or homogeneity can be indicated by a number of methods known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample, followed by staining and visualization after dye binding. For some purposes, high resolution is required, and thus HPLC or similar purification methods are required. For disaccharides 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 spectrometry. In addition, the terms "contaminant" and "impurity" herein preferably refer to particles, cells, cell components, metabolites, cell debris, proteins, peptides, amino acids, nucleic acids, glycolipids, and / or endotoxins that can be present in an aqueous medium (e.g., a culture or incubation).

[0092] The term "clarification" as used herein refers to the operation of treating an aqueous medium (e.g., a culture or incubation) to remove suspended particles and contaminants (e.g., cells, cell components, insoluble metabolites, and debris) produced during production that can interfere with the final purification of the negatively charged oligosaccharide, preferably sialylated oligosaccharide. Such treatment can be performed in a conventional manner by centrifugation, flocculation, flocculation (optionally with sonication), gravity filtration, microfiltration, foam separation, or vacuum filtration (e.g., through a ceramic filter that can include a Celite™ filter aid).

[0093] The term "culture" refers to the medium in which cells are cultured or fermented, the cells themselves, and the negatively charged oligosaccharide, preferably sialylated oligosaccharide, produced by the cells in whole broth, i.e., both inside (intracellular) and outside (extracellular) the cells. The terms "medium" and "culture medium" are used interchangeably herein and both refer to the medium in which cells are cultured.

[0094] The term "incubation" refers to a mixture in which negatively charged oligosaccharides, preferably sialylated oligosaccharides, are produced. The mixture can comprise one or more enzymes, one or more precursors and one or more acceptors as defined herein, which are present in a buffered solution and are incubated at a temperature for a time such that the one or more enzymes are able to catalyze the production of negatively charged oligosaccharides, preferably sialylated oligosaccharides, using the one or more precursors and the one or more acceptors in the mixture. The mixture can also comprise i) cells obtained after incubation or incubation, optionally the cells are lysed; ii) a buffered solution or a culture medium or an incubation medium in which the cells are incubated or fermented; and iii) negatively charged oligosaccharides, preferably sialylated oligosaccharides, produced by the cells in whole broth, i.e. inside the cells (intracellularly) and outside the cells (extracellularly). The incubation can also be an incubation as defined herein.

[0095] The terms "reactor" and "incubator" refer to a vessel in which an incubation or an incubation is contained. Examples of reactors and incubators include, but are not limited to, microfluidic devices, well plates, test tubes, shake flasks, fermenters, bioreactors, process vessels, cell culture incubators, carbon dioxide incubators.

[0096] The term "cell productivity index (CPI)" as used herein refers to the mass of negatively charged, preferably sialylated, oligosaccharides produced by the cells divided by the mass of cells produced in the culture.

[0097] The term "precursor" as used herein refers to a substance which is taken up or synthesized by a cell for the specific production of a negatively charged, preferably sialylated oligosaccharide according to the present application. In this sense, a precursor can be an acceptor as defined herein, but also another substance, metabolite, co-factor, which is first modified within the cell as part of the biochemical synthesis pathway of a negatively charged, preferably sialylated oligosaccharide. The term "precursor" as used herein is also to be understood as a chemical compound which participates in an incubation, chemical or enzymatic reaction to produce another compound, such as, for example, an intermediate or an acceptor as defined herein, as part of the metabolic pathway of a negatively charged, preferably sialylated oligosaccharide. The term "precursor" as used herein is also to be understood as a donor which is used by a glycosyltransferase to modify an acceptor as defined herein with a sugar moiety in a glycosidic bond, as part of the metabolic pathway of a negatively charged, preferably sialylated oligosaccharide. Examples of such precursors include: an acceptor as defined herein, and / or dihydroxyacetone, glucosamine, N-acetylglucosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyl lactose, phosphorylated sugars or sugar phosphates, such as, but not limited to, glucose-1 -phosphate, galactose-1 -phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, mannose-6-phosphate, mannose-1 -phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N-acetylglucosamine-1 -phosphate, N-acetylneuraminic acid-9-phosphate and nucleotide-activated sugars as defined herein, such as, for example, UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, CMP-sialic acid, GDP-mannose, GDP-4-dehydro-6-deoxy-alpha-D-mannose, GDP-fucose.

[0098] Optionally, the cell used for the production of a negatively charged, preferably sialylated oligosaccharide is transformed to comprise and express at least one nucleotide sequence encoding a protein selected from the list consisting of: a lactose transporter protein, a N-acetylneuraminic acid transporter protein, a fucose transporter protein, a glucose transporter protein, a galactose transporter protein, a transporter protein for nucleotide-activated sugars, wherein the transporter protein internalizes a precursor added to the culture medium for the synthesis of a negatively charged, preferably sialylated oligosaccharide according to the present application.

[0099] The term "acceptor" as used herein refers to a monosaccharide, disaccharide or oligosaccharide that can be modified by a glycosyltransferase. Examples of such acceptors include glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, N-acetylglucosamine, maltose, sucrose, lactose, lactulose, lactobionic acid (LBA), N-acetyllactosamine, lactose-N-disaccharide, lactose-N-trisaccharide, lactose-N-tetrasaccharide (LNT), lactose-N-neotetraose (LNnT), lactose-N-pentasaccharide (LNP), lactose-N-neopentasaccharide, para-lactose-N-pentasaccharide, para-lactose-N-neopentasaccharide, lactose-N-neotype pentasaccharide I, lactose-N-hexasaccharide (LNH), lactose-N-neohexasaccharide (LNnH), para-lactose-N-neohexasaccharide (pLNnH), para-lactose-N-hexasaccharide (pLNH), lactose-N-heptasaccharide, lactose-N-neoheptasaccharide, para-lactose-N-neoheptasaccharide, para-lactose-N-heptasaccharide, lactose-N-octasaccharide (LNO), lactose-N-neooctasaccharide, iso-lactose-N-octasaccharide, para-lactose-N-octasaccharide, iso-lactose-N-neooctasaccharide, neotype lactose-N-neooctasaccharide, para-lactose-N-neooctasaccharide, iso-lactose-N-nonosaccharide, neotype lactose-N-nonosaccharide, lactose-N-nonosaccharide, lactose-N-decasaccharide, iso-lactose-N-decasaccharide, neotype lactose-N-decasaccharide, lactose-N-neodecasaccharide, LSTa, LSTb, LSTc, and oligosaccharides containing one or more N-acetyllactosamine units and / or one or more lactose-N-disaccharide units or intermediates that are converted to oligosaccharides, their fucosylated and sialylated forms, ceramides, N-acylsphingosines, glucosylceramides, lactosylceramides, sphingosines, phytosphingosines, sphingosine synthases, peptide backbones with a beta-GlcNAc-Asn residue, glycoproteins with terminal GlcNAc and Gal residues, immunoglobulins. DETAILED DESCRIPTION

[0100] According to a first aspect, the present application provides a cell comprising a pathway for the production of a negatively charged, preferably sialylated oligosaccharide, which cell is genetically engineered to have or express, preferably overexpress, a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase, wherein the hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising a conserved motif having SEQ ID NO: 12 and has hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase activity.

[0101] According to a second aspect, the present application provides a method for producing a negatively charged, preferably sialylated oligosaccharide, wherein the method comprises culturing and / or incubating a cell as described herein in a culture medium and / or an incubation medium under conditions allowing the production of a negatively charged, preferably sialylated oligosaccharide.

[0102] Within the scope of the present application, permissive conditions are understood as conditions related to physical or chemical parameters including, but not limited to, temperature, pH, pressure, osmotic pressure and product / precursor / acceptor concentration.

[0103] In a particular embodiment, the permissive conditions can include a temperature range of 30 + / - 20 degrees Celsius, a pH range of 7 + / - 3.

[0104] In a preferred embodiment, the negatively charged oligosaccharides, preferably sialylated oligosaccharides, are isolated from said culture and / or incubation. In another and / or additional preferred embodiment, the oligosaccharides are purified from said culture and / or incubation.

[0105] In a particular embodiment, the cell comprises a pathway for the production of negatively charged, preferably sialylated, oligosaccharides. The term "pathway for the production of negatively charged, preferably sialylated, oligosaccharides" as used herein refers to a biochemical pathway consisting of enzymes involved in the synthesis of negatively charged, preferably sialylated, oligosaccharides as defined herein and their respective genes. Said pathway for the production of negatively charged, preferably sialylated, oligosaccharides can include, but is not limited to, pathways involved in the synthesis of nucleotide-activated sugars and the transfer of said nucleotide-activated sugars to acceptors to produce the negatively charged, preferably sialylated, oligosaccharides of the present application. Examples of such pathways include, but are not limited to, the fucosylation pathway, the sialylation pathway, the galactosylation pathway, the N-acetylglucosaminylation pathway, the N-acetylgalactosaminylation pathway, the mannosylation pathway and the N-acetylmannosaminylation pathway.

[0106] In another particular embodiment, the cell is genetically engineered to have or express a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase as described herein. In a preferred embodiment, the cell is genetically engineered to overexpress a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase as described herein.

[0107] In a preferred aspect of the methods and / or cells of the present application, the genetically engineered cell is modified with gene expression modules, wherein the expression from any of said expression modules is constitutive or regulatable.

[0108] The expression module, also referred to as a transcription unit, comprises polynucleotides for expressing a recombinant gene, including the coding gene sequence and appropriate transcriptional and / or translational control signals operably linked to the coding gene. The control signals include promoter sequences, untranslated regions, ribosome binding sites, and terminator sequences. The expression module can comprise elements for expressing a single recombinant gene, can comprise elements for expressing multiple recombinant genes, or can be organized into operon structures to integrate expression of two or more recombinant genes. The polynucleotides can be prepared by recombinant DNA technology using techniques well known in the art. Methods of constructing expression modules well known to those skilled in the art include, for example, in vitro recombinant DNA technology, synthetic techniques, and in vivo gene recombination. See, for example, Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rdedition, 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).

[0109] According to a preferred aspect of the application, the cell is modified with one or more expression modules. The expression modules can be integrated into the genome of the cell, or presented to the cell by a vector. The vector can be in the form of a plasmid, cosmid, phage, liposome or virus, which will be stably transformed / transfected into the metabolically engineered cell. Such vectors include inter alia chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, and vectors derived from combinations thereof, such as plasmid and bacteriophage genetic ele¬ments, such as cosmids and phagemids. These vectors can contain a selection marker, such as but not limited to an antibiotic marker, an auxotrophic marker, a toxin-antitoxin marker, an RNA sense / antisense marker. The expression system construct can contain control regions that regulate and induce expression. In general, any system or vector that is suitable for maintaining, propagating, or expressing polynucleotides and / or expressing polypeptides in a host can be used for such expression. Suitable DNA sequences can be inserted by any of a variety of well-known and routine techniques, such as, for example, those described by Sambrook et al., supra. For recombinant production, the cells can be genetically engineered by the incorporation of an expression system or portion thereof or polynucleotide of the application. The polynucleotides can be introduced into the cells by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, (1986), and Sambrook et al., 1989, supra.

[0110] As used herein, an expression module comprises a polynucleotide for expressing at least one recombinant gene. The recombinant gene is involved in a pathway for producing a negatively charged oligosaccharide, preferably a sialylated oligosaccharide; or the recombinant gene is linked to other pathways in the cell that are not involved in the synthesis of a negatively charged oligosaccharide, preferably a sialylated oligosaccharide. The recombinant gene encodes an endogenous protein with altered expression or activity, preferably the endogenous protein is overexpressed; or the recombinant gene encodes a heterologous protein that is heterologously introduced and expressed, preferably overexpressed, in the modified cell. The endogenous protein can have altered expression in the cell that also expresses a heterologous protein.

[0111] According to a preferred aspect of the application, the expression of each of the expression modules is constitutive or regulatable, as defined herein.

[0112] In a preferred embodiment of the method and / or cell of the application, the cell is modified in the expression or activity of a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase as described herein. Preferably, the hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase is an endogenous protein of the cell whose expression or activity is modified, preferably the endogenous hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase is overexpressed; alternatively, the hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase is a heterologous protein which is heterologously introduced and expressed in the cell, preferably overexpressed. In cells which also express a heterologous hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase, the endogenous hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase can have altered expression.

[0113] Within the scope of the present application, a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising the conserved motif [ACILM][GST]N[ST][ST]XXXX[DE][ACGILMST][ACDEGIMPQSTV] having SEQ ID NO 12, wherein X can be any amino acid residue, and has the activity of a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase catalyzing the reaction UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) = N-acetylmannosamine (ManNAc). X present in the conserved motif means that any single amino acid is possible. Herein, the amino acid can be one of the 20 common amino acids encoded in the genetic code of life (i.e. A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V), or can be a modified amino acid (e.g. due to decomposition of a common amino acid such as, for example, L-ornithine, or by modification such as, for example, hydroxylation, carboxylation, phosphorylation, methylation, acetylation, glycosylation, ADP-ribosylation or ubiquitination of a common amino acid). If there are multiple X's, as in SEQ ID NO 12, each X is an amino acid resulting from a new, independent selection from the list of possible amino acid residues. In this way, each X present in the conserved motif having SEQ ID NO 12 refers to the same amino acid or to a different amino acid.

[0114] In a preferred embodiment of the method and / or cell of the application, the hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase comprises, consists of or consists essentially of an amino acid sequence which is at least 80% identical to any one of the amino acid sequences shown in SEQ ID NO 01, 02, 03, 04, 05, 06, 07 or 08 over a segment of at least 150 amino acid residues. In a more preferred embodiment, the hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase comprises, consists of or consists essentially of an amino acid sequence which is at least 80% identical to any one of the amino acid sequences shown in SEQ ID NO 01, 02, 03, 04, 05, 06, 07 or 08 over a segment of at least 200 amino acid residues.

[0115] In another and / or further preferred embodiment of the method and / or cell of the application, the hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising an IPR domain selected from the list comprising IPR003331, IPR020004 and IPR029767 as defined in InterPro 90.0 released on August 4, 2022.

[0116] In another and / or further preferred embodiment of the method and / or cell of the application, the hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising a PF02350 motif as defined in PFAM 32.0 released on September 2018.

[0117] In another and / or further preferred embodiment of the method and / or cell of the application, the hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising a cd03786 motif as defined in CDD v3.17 released on September 2016.

[0118] In another and / or further preferred embodiment of the method and / or cell of the application, the hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising a Panther domain selected from the list comprising PTHR43174 and PTHR43174:SF3 as defined in PANTHER 17.0 released on February 23, 2022.

[0119] In another and / or additional preferred embodiment of the method and / or cell of the application, the hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, or at least 99% identical to any one of the full-length amino acid sequences set forth in SEQ ID NO 01, 02, 03, 04, 05, 06, 07, or 08.

[0120] In another and / or additional preferred embodiment of the method and / or cell of the application, the hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, or at least 99% identical to any one of the full-length amino acid sequences set forth in SEQ ID NO 01, 02, 03, 04, 05, 06, 07, or 08.

[0121] In another and / or additional preferred embodiment of the method and / or cell of the application, the hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, or at least 99% identical to any one of the full-length amino acid sequences set forth in SEQ ID NO 01, 02, 03, 04, 05, 06, 07, or 08.

[0122] In another and / or additional preferred embodiment of the method and / or cell of the application, the hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase is a nnaA polypeptide.

[0123] In a preferred embodiment of the method and / or cell of the application, the cell comprises a nucleic acid molecule comprising a polynucleotide sequence encoding a hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase as described herein. In a more preferred embodiment, the nucleic acid molecule is operably linked to a control sequence recognized by the cell, the nucleic acid molecule is further i) integrated into the genome of the cell and / or ii) presented to the cell by a vector. In a further and / or alternative more preferred embodiment, the nucleic acid molecule is exogenous to the cell.

[0124] In preferred embodiments of the methods and / or cells of the application, the cell is genetically engineered for the production of a negatively charged oligosaccharide, preferably a sialylated oligosaccharide, wherein the cell comprises a pathway for the production of the oligosaccharide. In more preferred embodiments, the cell is genetically engineered for the production of two or more negatively charged oligosaccharides, preferably sialylated oligosaccharides. In another and / or additional preferred embodiments, the cell is genetically engineered to enhance the production of negatively charged, preferably sialylated, oligosaccharides, to enhance the uptake of one or more precursors and / or acceptors for the synthesis of negatively charged, preferably sialylated, oligosaccharides, to better and / or enhance the efflux of negatively charged, preferably sialylated, oligosaccharides, to reduce the production of byproducts such as acids, to increase the availability of co-factors such as ATP, NADP, NADPH, and / or to better and / or enhance the metabolic flux through any one of the sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N-acetylgalactosaminylation, mannosylation, and / or N-acetylmannosaminylation pathways present in the cell.

[0125] In preferred embodiments of the methods and / or cells of the application, the pathway for the production of the negatively charged, preferably sialylated, oligosaccharide is a sialylation pathway. In more preferred embodiments, the cell is genetically engineered to comprise the sialylation pathway. In even more preferred embodiments, the cell comprises the sialylation pathway, wherein the sialylation pathway has been genetically engineered. In another even more preferred embodiment, the cell has been metabolically engineered to comprise a sialylation pathway, wherein one or more 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-GlcNAc 2-epimerase / kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine 1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, bifunctional N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphatase, D-arabinose-5-phosphate isomerase, KDO-8P synthase, KDO 8-phosphatase, CMP-KDO synthetase, sialate transport protein, CMP kinase, CMP-sialate synthase, and sialate transferase have modified and / or enhanced expression.

[0126] In another and / or additional preferred embodiments of the method and / or cell, the cell further comprises a pathway selected from the list comprising, consisting of, or consisting essentially of: a fucosylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N- acetylgalactosaminylation pathway, a mannosylation pathway, and an N-acetylmannosaminylation pathway. In more preferred embodiments, the cell is genetically engineered to comprise at least one pathway selected from the list comprising, consisting of, or consisting essentially of: the fucosylation pathway, the galactosylation pathway, the N-acetylglucosaminylation pathway, the N-acetylgalactosaminylation pathway, the mannosylation pathway, and the N-acetylmannosaminylation pathway. In even more preferred embodiments, the cell comprises at least one pathway selected from the list comprising, consisting of, or consisting essentially of: the fucosylation pathway, the galactosylation pathway, the N-acetylglucosaminylation pathway, the N-acetylgalactosaminylation pathway, the mannosylation pathway, and the N-acetylmannosaminylation pathway, wherein at least one of the fucosylation pathway, the galactosylation pathway, the N-acetylglucosaminylation pathway, the N-acetylgalactosaminylation pathway, the mannosylation pathway, and the N-acetylmannosaminylation pathway has been genetically engineered.

[0127] In another and / or additional preferred embodiments of the method and / or cell, the cell comprises a fucosylation pathway. In more preferred additional and / or alternative embodiments, the cell is metabolically engineered to comprise a fucosylation pathway. In another even more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise a fucosylation pathway, wherein any one or more genes selected from the list comprising, consisting of, or consisting essentially of: mannose-6-phosphate isomerase, phosphomannomutase, mannose-1 -phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-1 -phosphate guanylyltransferase, and fucosyltransferase have modified and / or enhanced expression.

[0128] 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 is metabolically engineered to comprise a galactosylation pathway, wherein any one or more genes selected from the list consisting of, consisting essentially of, or consisting of: galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, and galactosyltransferase have modified and / or enhanced expression.

[0129] 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 genes selected from the list consisting of, consisting essentially of, or consisting of: L-glutamine-D-fructose-6-phosphate amidotransferase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-1 -phosphate uridyltransferase, glucosamine-1 -phosphate acetyltransferase, bifunctional N-acetylglucosamine-1 -phosphate uridyltransferase / glucosamine-1 -phosphate acetyltransferase, and GlcNAc-transferring glycosyltransferase have modified and / or enhanced expression.

[0130] 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 is metabolically engineered to comprise an N- acetylgalactosaminylation pathway, wherein any one or more genes selected from the list consisting of, consisting essentially of, or consisting of: L-glutamine-D-fructose-6-phosphate amidotransferase, phosphoglucosamine mutase, N-acetylglucosamine 1 -phosphate uridyltransferase, glucosamine-1 -phosphate acetyltransferase, bifunctional N-acetylglucosamine-1 -phosphate uridyltransferase / glucosamine-1 -phosphate acetyltransferase, UDP-N-acetylglucosamine 4-epimerase, UDP-glucose 4-epimerase, N-acetylgalactosamine kinase, and / or UDP-N-acetylgalactosamine pyrophosphorylase, and GalNAc-transferring glycosyltransferase have modified and / or enhanced expression.

[0131] 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 is metabolically engineered to comprise a mannosylation pathway, wherein any one or more genes selected from the list comprising, consisting of or consisting essentially of: mannose-6-phosphate isomerase, phosphomannomutase, and / or mannosyl-l-phosphate guanylyltransferase and mannosyltransferases have modified and / or enhanced expression.

[0132] 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 more preferred additional and / or alternative embodiment, the cell has been metabolically engineered to comprise an N- acetylmannosaminylation pathway, wherein any one or more genes selected from the list comprising, consisting of or consisting essentially of: L-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate N-acetyltransferase, N-acetylglucosamine- 1 -phosphate uridyltransferase, glucosamine- 1 -phosphate acetyltransferase, glucosamine- 1 -phosphate acetyltransferase, bifunctional N-acetylglucosamine- 1 -phosphate uridyltransferase / glucosamine- 1 -phosphate acetyltransferase, UDP-GlcNAc 2-epimerase, and / or ManNAc kinase, and glycosyltransferases that transfer ManNAc have modified and / or enhanced expression.

[0133] In another and / or additional preferred embodiment of the method and / or cell of the present application, the cell comprises one or more pathways for monosaccharide synthesis. The pathways for monosaccharide synthesis include enzymes such as carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, kinases, phosphatases, aldolases, hydrolases, dehydrogenases; enzymes involved in the synthesis of one or more nucleoside triphosphates (e.g., UTP, GTP, ATP, and CTP); enzymes involved in the synthesis of one or more nucleoside monophosphates or diphosphates (e.g., UMP and UDP), respectively; and enzymes involved in the synthesis of phosphoenolpyruvate (PEP).

[0134] In another and / or additional preferred embodiments of the methods and / or cells of the application, the cells comprise one or more pathways for the synthesis of phosphorylated monosaccharides. The pathways for the synthesis of phosphorylated monosaccharides include enzymes involved in the synthesis of one or more monosaccharides, one or more monophosphate, diphosphate, and / or triphosphate nucleosides, and enzymes involved in the synthesis of phosphoenolpyruvate (PEP), such as, but not limited to, PEP synthase, carboxylase, decarboxylase, isomerase, epimerase, reductase, enolase, phosphorylase, carboxykinase, kinase, phosphatase, aldolase, hydrolase, and dehydrogenase. In another and / or additional preferred embodiments of the methods and / or cells of the application, the cells comprise one or more pathways for the synthesis of one or more nucleotide-activated sugars. The pathways for the synthesis of nucleotide-activated sugars include enzymes, such as PEP synthase, carboxylase, decarboxylase, isomerase, epimerase, reductase, enolase, phosphorylase, carboxykinase, kinase, phosphatase, aldolase, hydrolase, dehydrogenase, mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6- dehydratase, GDP-L-fucose synthase, L-fucokinase / GDP-fucose pyrophosphorylase, L-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acetylglucosamine epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylglucosamine-6P 2-epimerase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, N-acetylmannosamine-6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, bifunctional N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphate phosphatase, D-arabinose-5-phosphate isomerase, KDO-8P synthase, KDO 8-phosphate phosphatase, CMP-KDO synthase, CMP-sialic acid synthase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, glucose phosphate mutase, and / or N-acetylglucosamine-1-phosphate uridyltransferase.

[0135] In another and / or additional preferred embodiment of the method and / or cell, the cell has, preferably expresses, more preferably overexpresses one or more glycosyltransferases selected from the list comprising, consisting of or consisting essentially of a fucosyltransferase, a sialyltransferase, a galactosyltransferase, a glucosyltransferase, a mannosyltransferase, an N-acetylglucosaminyltransferase, an N-acetylgalactosaminyltransferase, an N-acetylmannosaminyltransferase, a xylosyltransferase, a glucuronosyltransferase, a galacturonosyltransferase, a glucosaminyltransferase, a N- acetylneuraminyltransferase, a rhamnosyltransferase, an N-acetyl rhamnosyltransferase, a UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L-athosamine transaminase, a UDP-N-acetylglucosamine enolpyruvate transferase and a fucosaminyltransferase.

[0136] In a more preferred embodiment of the method and / or cell of the application, the fucosyltransferase is selected from the list comprising, consisting of or consisting essentially of an alpha-1,2-fucosyltransferase, an alpha-1,3-fucosyltransferase, an alpha-1,4-fucosyltransferase and an alpha-1,6-fucosyltransferase.

[0137] In an alternative and / or additional more preferred embodiment of the method and / or cell of the application, the further sialyltransferase is selected from the list comprising, consisting of or consisting essentially of an alpha-2,3-sialyltransferase, an alpha-2,6-sialyltransferase and an alpha-2,8-sialyltransferase.

[0138] In an alternative and / or additional more preferred embodiment of the method and / or cell of the application, the galactosyltransferase is selected from the list comprising, consisting of or consisting essentially of a beta-1,3-galactosyltransferase, an N-acetylglucosamine beta-1,3-galactosyltransferase, a beta-1,4-galactosyltransferase, an N-acetylglucosamine beta-1,4-galactosyltransferase, an alpha-1,3-galactosyltransferase and an alpha-1,4-galactosyltransferase.

[0139] In an alternative and / or additional more preferred embodiment of the method and / or cell of the application, the glucosyltransferase is selected from the list comprising, consisting of or consisting essentially of an alpha-glucosyltransferase, a beta-1,2-glucosyltransferase, a beta-1,3-glucosyltransferase and a beta-1,4-glucosyltransferase.

[0140] In an alternative and / or additional more preferred embodiment of the method and / or cell of the application, the mannosyltransferase is selected from the list comprising, consisting of or consisting essentially of an alpha-1,2-mannosyltransferase, an alpha-1,3-mannosyltransferase and an alpha-1,6-mannosyltransferase.

[0141] In alternative and / or additional more preferred embodiments of the method and / or cell of the application, the N-acetylglucosaminyltransferase is selected from the list comprising, consisting of or consisting essentially of a galactoside beta-1,3-N-acetylglucosaminyltransferase and a beta-1,6-N-acetylglucosaminyltransferase.

[0142] In alternative and / or additional more preferred embodiments of the method and / or cell of the application, the N-acetylgalactosaminyltransferase is selected from the list comprising, consisting of or consisting essentially of an alpha-1,3-N-acetylgalactosaminyltransferase.

[0143] In alternative and / or additional more preferred embodiments of the method and / or cell of the application, the cell is modified in the expression or activity of at least one of said glycosyltransferases. In preferred embodiments, the glycosyltransferase is an endogenous protein of the cell whose expression or activity is modified, preferably the endogenous glycosyltransferase is overexpressed; alternatively, the glycosyltransferase is a heterologous protein that is heterologously introduced and expressed in the cell, preferably overexpressed. The endogenous glycosyltransferase can have an altered expression in cells that also express a heterologous glycosyltransferase.

[0144] In another and / or additional preferred embodiment of the method and / or cell, the cell is capable of producing, preferably producing, one or more nucleotide activated sugars, preferably the cell is genetically engineered to produce one or more of said nucleotide activated sugars. Herein, the one or more nucleotide activated sugars is selected from the list comprising, consisting of or consisting essentially of: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2,6-dideoxy-L-arabinose-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-xylose-4-hexulose, UDP-N-acetyl-L-rhamnoseamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetyl-fucosamine, UDP-N-acetyl-fucosamine (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), CMP-2-keto-3-deoxymanno-octulosonic acid (CMP-KDO), GDP-rhamnose, and UDP-xylose.

[0145] In another and / or an 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-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2,6-dideoxy-L-arabinose-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-xylose-4-hexulose, UDP-N-acetyl-L-rhamnoseamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetyl-fucosamine, UDP-N-acetyl-fucosamine (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), CMP-2-keto-3-deoxymanno-octulosonic acid (CMP-KDO), GDP-rhamnose, and UDP-xylose.

[0146] The cells used herein are optionally genetically modified to express de novo synthesis of UDP-GlcNAc. UDP-GlcNAc can be provided by enzymes expressed within the cell or cell metabolism. Cells that produce UDP-GlcNAc can express enzymes that convert, for example, GlcNAc to be added to the cell into UDP-GlcNAc. These enzymes can be any one or more of the list comprising, consisting of, or consisting essentially of: N-acetyl-D-glucosamine kinase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-1 -phosphate uridyltransferase, glucosamine-1 -phosphate acetyltransferase, and bifunctional N-acetylglucosamine-1 -phosphate uridyltransferase / glucosamine-1 -phosphate acetyltransferase from a plurality of species including Homo sapiens, Escherichia coli. Preferably, the cells are modified to produce UDP-GlcNAc.

[0147] Additionally, or alternatively, the cells used herein are optionally genetically modified to express de novo synthesis of CMP-Neu5Ac. CMP-Neu5Ac can be provided by enzymes expressed within the cell or cell metabolism. Such cells that produce CMP-Neu5Ac can express, for example, enzymes that convert sialic acid into CMP-Neu5Ac. This enzyme can be a CMP-sialic acid synthetase, for example, N-acylneuraminate cytidylyltransferase from a plurality of species including Homo sapiens, Neisseria meningitidis, and Pasteurella multocida. Preferably, the cells are modified to produce CMP-Neu5Ac. More preferably, the cells are modified to increase production of CMP-Neu5Ac. The modification can be any one or more selected from the list comprising, consisting of, or consisting essentially of: knocking out N-acetylglucosamine-6-phosphate deacetylase, knocking out glucosamine-6-phosphate deaminase, overexpressing CMP-sialic acid synthetase, and overexpressing N-acetyl-D-glucosamine-2-epimerase encoding genes.

[0148] Additionally, or alternatively, the cells used herein are optionally genetically modified to express de novo synthesis of GDP-fucose. GDP-fucose can be provided by enzymes expressed within the cell or cell metabolism. Such cells producing GDP-fucose can express enzymes that convert, for example, fucose to be added to the cell into GDP-fucose. The enzyme can be, for example, a bifunctional fucose kinase / fucose-1 -phosphate guanylyltransferase, such as Fkp from Bacteroides fragilis, or a combination of a separate fucose kinase and a separate fucose-1 -phosphate guanylyltransferase, as they are known in multiple species including Homo sapiens, Sus scrofa, and Rattus norvegicus. Preferably, the cells are modified to produce GDP-fucose. More preferably, the cells are modified to enhance production of GDP-fucose. The modification can be any one or more selected from the list comprising, consisting of, or consisting essentially of, a knockout of a UDP-glucose: undecaprenyl-phosphate glucose-1 -phosphate transferase encoding gene, overexpression of a GDP-L-fucose synthase encoding gene, overexpression of a GDP-mannose 4,6- dehydratase encoding gene, overexpression of a mannose-1 -phosphate guanylyltransferase encoding gene, overexpression of a phosphomannomutase encoding gene, and overexpression of a mannose-6-phosphate isomerase encoding gene.

[0149] Additionally, or alternatively, the cells used herein are optionally genetically modified to express de novo synthesis of UDP-Gal. UDP-Gal can be provided by enzymes expressed within the cell or cell metabolism. Such cells producing UDP-Gal can express enzymes that convert, for example, UDP-glucose into UDP-Gal. The enzyme can be, for example, a UDP-glucose-4-epimerase GalE, as it is known in multiple species including Homo sapiens, Escherichia coli, and Rattus norvegicus. Preferably, the cells are modified to produce UDP-Gal. More preferably, the cells are modified to enhance production of UDP-Gal. The modification can be any one or more selected from the list comprising, consisting of, or consisting essentially of, a knockout of a bifunctional 5’-nucleotidase / UDP-sugar hydrolase encoding gene, a knockout of a galactose-1 -phosphate uridyltransferase encoding gene, and overexpression of a UDP-glucose-4-epimerase encoding gene.

[0150] Alternatively, or in addition, the cells used herein are optionally genetically modified to express de novo synthesis of UDP-GalNAc. UDP-GalNAc can be synthesized from UDP-GlcNAc in a one-step reaction using a UDP-N-acetylglucosamine 4-epimerase, such as wbgU from Plesiomonas shigelloides, gne from Yersinia enterocolitica, or wbpP from Pseudomonas aeruginosa serotype O6. Preferably, the cells are modified to produce UDP-GalNAc. More preferably, the cells are modified to enhance production of UDP-GalNAc.

[0151] Alternatively, or in addition, the cells used herein are optionally genetically modified to express de novo synthesis of UDP-ManNAc. UDP-ManNAc can be synthesized directly from UDP-GlcNAc by a epimerization reaction with a UDP-GlcNAc 2-epimerase (such as cap5P from Staphylococcus aureus, RffE from Escherichia coli, Cps19fK from Streptococcus pneumoniae, and RfbC from Salmonella enterica). Preferably, the cells are modified to produce UDP-ManNAc. More preferably, the cells are modified to enhance production of UDP-ManNAc.

[0152] In another and / or additional preferred embodiment of the method and / or cell, the cell has, preferably expresses, one or more genes selected from the list comprising, consisting of or consisting essentially of: mannose-6-phosphate isomerase, phosphomannomutase, mannose-1 -phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-1 -phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine-2-epimerase, UDP-N-acetylglucosamine-2-epimerase, N-acetylmannosamine-6-phosphate-2-epimerase, UDP-GlcNAc 2-epimerase / kinase, glucosamine-6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1 -phosphate uridyltransferase, glucosamine-1 -phosphate acetyltransferase, bifunctional N-acetylglucosamine-1 -phosphate uridyltransferase / glucosamine-1 -phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphatase, D-arabinose-5-phosphate isomerase, KDO-8P synthase, KDO-8-phosphate phosphatase, CMP-KDO synthetase, sialate transport protein, CMP kinase, CMP-sialate synthase, galactose-1 -epimerase, galactokinase, glucokinase, galactose-1 -phosphate uridyltransferase, UDP-glucose-4-epimerase, glucose-1 -phosphate uridyltransferase, 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-1 -phosphate guanylyltransferase, GDP-mannose 4,6- dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-1 - phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N- acetylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N- acetylmannosamine-6-phosphate 2-epimerase, UDP-GlcNAc 2-epimerase / kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, sialic acid synthase, N- acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate- 9-phosphatase, D-arabinose-5-phosphate isomerase, KDO-8P synthase, KDO 8- phosphate phosphatase, CMP-KDO synthetase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, galactose-1 -epimerase, galactokinase, glucokinase, galactose-1 - phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1 -phosphate uridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epimerase, N- acetylgalactosamine kinase, and UDP-N-acetylgalactosamine pyrophosphorylase.

[0153] In another and / or further preferred embodiment of the method and / or cell, the cell is capable of producing, preferably producing, N-acetylmannosamine (ManNAc), a sialic acid residue and / or N-acetylglucosamine (GlcNAc), wherein the sialic acid residue is selected from the list comprising Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulosonic acid (KDO). In a more preferred embodiment, the cell comprises a pathway for producing ManNAc, a sialic acid residue and / or GlcNAc. In another and / or further more preferred embodiment, the cell is genetically engineered for producing ManNAc, a sialic acid residue and / or GlcNAc.

[0154] In another and / or additional preferred embodiment of the method and / or cell, the negatively charged oligosaccharide produced by the cell of the application is a sialylated oligosaccharide having at least one sialic acid residue selected from the list comprising, consisting of or essentially consisting of: Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulosonic acid (KDO).

[0155] In a more preferred embodiment, the negatively charged oligosaccharide is selected from the list comprising, consisting of or essentially consisting of: a negatively charged, preferably sialylated milk oligosaccharide, preferably a negatively charged, more preferably sialylated mammalian milk oligosaccharide (MMO), more preferably a negatively charged, more preferably sialylated human milk oligosaccharide (HMO); an O-antigen; an oligosaccharide repeat unit found in a capsular polysaccharide; a peptidoglycan; an amino sugar; a Lewis-type antigen oligosaccharide; a negatively charged, preferably sialylated animal oligosaccharide; a negatively charged, preferably sialylated plant oligosaccharide; a negatively charged, preferably sialylated oligosaccharide containing N-acetyl lactosamine and a negatively charged, preferably sialylated oligosaccharide containing lacto-N-biose. In an even more preferred embodiment, the negatively charged oligosaccharide is a mammalian milk oligosaccharide (MMO) as described herein. In another more preferred embodiment, the negatively charged oligosaccharide is a human milk oligosaccharide (HMO) as described herein. In another more preferred embodiment, the negatively charged oligosaccharide is an animal oligosaccharide selected from the list consisting of N-glycans and O-glycans. In another more preferred embodiment, the negatively charged oligosaccharide is a plant oligosaccharide selected from the list consisting of N-glycans and O-glycans. In the context of the present application, N-glycans and O-glycans refer to oligosaccharide structures known to the person skilled in the art, wherein the structures are not linked to a protein or a peptide.

[0156] In even more preferred embodiments, the negatively charged oligosaccharide is selected from the list comprising: 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), 3'-sialyllactose amine, 3,6-disialyllactose, 8,3-disialyllactose, sialylated lacto-N-triose, sialyllacto-N-tetraose a (LSTa), KDOa-2,3Galb-1,3GlcNAcb-1,3Galb-1,4Glc, 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, fucodiisialyllacto-N-hexaose, disialylmonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyllacto-N-fuclohexaose II, disialyllacto-N-fuclopentaose II, monofucosyl disialyllacto-N-tetraose, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal, Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'-KDO-lacto-N-disaccharide, 3'-KDO-6' sialyllactose, 3'KDO-8-sialyllactose, KDO-2,3Galb-1,3GalNacb-1,3Galb-1,4Gal, KDOa-2,3Galb-1,4GlcNacb-1,3Galb-1,4Glc, 3'-KDO-3-fucosyllactose, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal, 3'-sialyl-2'-fucosyllactose, 6'-sialyl-2'-fucosyllactose, 6'-sialyl-3-fucosyllactose, Neu5Ac-a2,6-(Neu5Ac-a2,3-)Gal-b1,4-Glc, 3'-sialyl-3-fucosyllactosamine, Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc, 6'-sialyllacto-N-disaccharide, 3'-sialyllacto-N-disaccharide, Neu5Ac-a2,6-(GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,3-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, 6'-KDO-lactose, 6'-KDO-lactosamine, 6'-KDO-lactose-N-disaccharide, KDO-lactose-N-trisaccharide, KDO-lactose-N-tetrasaccharide, KDO-lactose-N-tetrasaccharide, and combinations thereof.

[0157] In the context of the present application, the oligosaccharide is preferably in free form, i.e. the oligosaccharide does not contain any protecting groups.

[0158] In another and / or additional preferred embodiment of the method and / or cell of the application, the cell is capable of producing, preferably producing, one or more precursors from which the negatively charged, preferably sialylated, oligosaccharide is produced, preferably produced, as defined herein. In a more preferred embodiment, the precursor is lactose. Preferably, the one or more precursors are fed to the cell from the culture medium or incubation. In another more preferred embodiment, the cell is capable of producing, preferably producing, at least one of the one or more precursors. In an even more preferred embodiment, the cell is capable of producing, preferably producing, all of the one or more precursors. In another more preferred embodiment, the cell is capable of producing, preferably producing, lactose. In another more preferred embodiment, the cell is genetically engineered for producing at least one of the one or more precursors. In an even more preferred embodiment, the cell is genetically engineered for producing all of the one or more precursors. In another more preferred embodiment, the cell is genetically engineered for producing lactose. In another more preferred embodiment, at least one of the one or more precursors is internalized within the cell by one or more membrane proteins, preferably the membrane transporters described herein. In another preferred embodiment, the cell is used for producing precursors of the negatively charged, preferably sialylated, oligosaccharide that are completely converted to the negatively charged, preferably sialylated, oligosaccharide.

[0159] In another and / or additional preferred embodiment of the method and / or cell of the application, the cell is capable of producing, preferably producing, phosphoenolpyruvate (PEP). Preferably, the cell comprises a pathway for producing PEP.

[0160] In another and / or additional preferred embodiment of the method and / or cell of the application, the cell is modified to enhance the synthesis and / or supply of phosphoenolpyruvate (PEP) as compared to an unmodified progenitor cell.

[0161] In preferred embodiments, and as a means to enhance PEP production and / or supply, one or more PEP-dependent sugar transport phosphotransferase systems are disrupted, such as but not limited to: 1) N-acetyl-D-glucosamine Npi-phosphotransferase (EC 2.7.1.193), e.g. encoded by the nagE gene (or cluster nagABCD) in E. coli or Bacillus spp., 2) ManXYZ, which encodes the enzyme II Man complex (mannose PTS permease, protein-Npi-phosphohistidine-D-mannose phosphotransferase), which imports exogenous hexoses (mannose, glucose, glucosamine, fructose, 2-deoxyglucose, mannosamine, N-acetylglucosamine, etc.) and releases the phosphate into the cytoplasm, 3) glucose-specific PTS transporter proteins (e.g. encoded by PtsG / Crr), which take up glucose and form glucose-6-phosphate in the cytoplasm, 4) sucrose-specific PTS transporter proteins, which take up sucrose and form sucrose-6-phosphate in the cytoplasm, 5) fructose-specific PTS transporter proteins (e.g. encoded by genes fruA and fruB and kinase fruK), which take up fructose and form fructose-1-phosphate in a first step and fructose-1,6-bisphosphate in a second step, 6) lactose PTS transporter proteins (e.g. encoded by lacE in Lactococcus lactis), which take up lactose and form lactose-6-phosphate, 7) galactitol-specific PTS enzymes, which take up galactitol and / or sorbitol and form galactitol-1-phosphate or sorbitol-6-phosphate, respectively, 8) mannitol-specific PTS enzymes, which take up mannitol and / or sorbitol and form mannitol-1-phosphate or sorbitol-6-phosphate, respectively, and 9) trehalose-specific PTS enzymes, which take up trehalose and form trehalose-6-phosphate.

[0162] In another and / or additional preferred embodiments, and as a means to enhance PEP production and / or supply, the entire PTS system is disrupted by disrupting the PtsIH / Crr gene cluster.

[0163] In another and / or additional preferred embodiments, the cells are further modified to compensate for the deletion of the carbon source PTS system by introducing and / or overexpressing a corresponding permease. These are, for example, permeases or ABC transporters including, but not limited to, transporters specific for the import of lactose, such as the transporter encoded by the LacY gene of E. coli; sucrose transporters, such as the transporter encoded by the cscB gene of E. coli; glucose transporters, such as the transporter encoded by the galP gene of E. coli; fructose transporters, such as the transporter encoded by the frul gene of S. mutans; or sorbitol / mannitol ABC transporters, such as the transporters encoded by the SmoEFGK cluster of Rhodobacter sphaeroides, trehalose / sucrose / maltose transporters, such as the transporters encoded by the gene cluster ThuEFGK of Sinorhizobium meliloti and N-acetylglucosamine / galactose / glucose transporters, such as the transporter encoded by NagP of Shewanella oneidensis. Examples for the combination of a PTS deletion with the overexpression of an alternative transporter are: 1) deletion of the glucose PTS system, e.g. the ptsG gene, in combination with the introduction and / or overexpression of a glucose permease (e.g. galP or glcP), 2) deletion of the fructose PTS system, e.g. one or more of the fruB, fruA, fruK genes, in combination with the introduction and / or overexpression of a fructose permease, e.g. frul, 3) deletion of the lactose PTS system in combination with the introduction and / or overexpression of a lactose permease, e.g. LacY, and / or 4) deletion of the sucrose PTS system in combination with the introduction and / or overexpression of a sucrose permease, e.g. cscB.

[0164] In further preferred embodiments, the cells are modified to compensate for the deletion of the carbon source PTS system by introducing a carbohydrate kinase, such as a glucose kinase (EC 2.7.1.1, EC 2.7.1.2, EC 2.7.1.63), a galactose kinase (EC 2.7.1.6) and / or a fructose kinase (EC 2.7.1.3, EC 2.7.1.4).

[0165] In another and / or additional preferred embodiment, and as a means to enhance the production and / or supply of PEP, the cell is modified by introducing or modifying any one or more of the list comprising: phosphoenolpyruvate synthase activity (EC: 2.7.9.2, e.g. encoded by ppsA in E. coli), phosphoenolpyruvate carboxykinase activity (EC 4.1.1.32 or EC 4.1.1.49, e.g. encoded by PCK in C. glutamicum or pckA in E. coli, respectively), phosphoenolpyruvate carboxylase activity (EC 4.1.1.31, e.g. encoded by ppc in E. coli), oxaloacetate decarboxylase activity (EC 4.1.1.112, e.g. encoded by eda in E. coli), pyruvate kinase activity (EC 2.7.1.40, e.g. encoded by pykA and pykF in E. coli), pyruvate carboxylase activity (EC 6.4.1.1, e.g. encoded by pyc in B. subtilis), and malate dehydrogenase activity (EC 1.1.1.38 or EC 1.1.1.40, e.g. encoded by maeA or maeB in E. coli, respectively).

[0166] In another and / or additional preferred embodiment, and as a means to enhance the production and / or supply of PEP, the cell is modified by reducing phosphoenolpyruvate carboxylase activity and / or pyruvate kinase activity, preferably by deleting at least one of the genes encoding phosphoenolpyruvate carboxylase, pyruvate carboxylase activity and / or pyruvate kinase.

[0167] In another and / or additional preferred embodiment of the method and / or cell of the application, the cell is further modified to reduce the degradation of acetyl-CoA and / or its main precursor pyruvate.

[0168] In another and / or additional preferred embodiment of the method and / or cell of the application, the cell comprises a modification that reduces the production of acetate compared to the unmodified progenitor cell. The modification can be one or more modifications selected from the list comprising, consisting of or consisting essentially of: overexpression of acetyl-CoA synthase, complete or partial knock-out or functional reduction of pyruvate dehydrogenase, and complete or partial knock-out or functional reduction of lactate dehydrogenase.

[0169] In another and / or additional preferred embodiment of the method and / or cell of the application, the cell is modified in the expression or activity of at least one acetyl-CoA synthase, such as acs from E. coli, S. cerevisiae, Homo sapiens, Mus musculus. In a preferred embodiment, the acetyl-CoA synthase is an endogenous protein of the cell whose expression or activity is modified, preferably the endogenous acetyl-CoA synthase is overexpressed; alternatively, the acetyl-CoA synthase is a heterologous protein that is introduced and expressed heterologously in the cell, preferably overexpressed. The endogenous acetyl-CoA synthase can have altered expression in cells that also express a heterologous acetyl-CoA synthase.

[0170] In an alternative and / or additional preferred embodiment of the method and / or cell of the application, the cell is modified in the expression or activity of at least one pyruvate dehydrogenase, such as pyruvate dehydrogenase from E. coli, S. cerevisiae, Homo sapiens, and Rattus norvegicus. In a preferred embodiment, the cell has been modified by methods generally known to those skilled in the art to have at least one partially or completely knocked-out or mutated pyruvate dehydrogenase-encoding gene, resulting in a reduction of function of at least one protein or loss of pyruvate dehydrogenase activity. In a more preferred embodiment, the poxB-encoding gene of the cell is completely knocked out, resulting in a cell that lacks pyruvate dehydrogenase activity.

[0171] In an alternative and / or additional preferred embodiment of the method and / or cell of the application, the cell is modified in the expression or activity of at least one lactate dehydrogenase, such as lactate dehydrogenase from E. coli, S. cerevisiae, Homo sapiens, and Rattus norvegicus. In a preferred embodiment, the cell has been modified by methods generally known to those skilled in the art to have at least one partially or completely knocked-out or mutated lactate dehydrogenase-encoding gene, resulting in a reduction of function of at least one protein or loss of lactate dehydrogenase activity. In a more preferred embodiment, the ldhA-encoding gene of the cell is completely knocked out, resulting in a cell that lacks lactate dehydrogenase activity.

[0172] According to another and / or additional preferred embodiment of the method and / or cell of the application, the cell comprises reduced or decreased expression and / or impaired, decreased or delayed activity of any one or more of the following proteins: beta-galactosidease, galactoside O-acetyltransferase, N-acetylglucosamine-6-phosphatdeacetylase, glucosamine-6-phosphatdeaminase, N-acetylglucosamine repressor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase, L-fucokinase, L-fuculose isomerase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epimerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridyltransferase, glucose-1-phosphate adenylyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcriptional repressor IclR, ion protease, glucose-specific translocating phosphotransferase IIBC component ptsG, glucose-specific translocating phosphotransferase (PTS) enzyme IIBC component malX, enzyme IIA Glc , beta-glucoside-specific PTS enzyme II, fructose-specific PTS polyphosphate transfer protein FruA and FruB, alcohol dehydrogenase aldehyde dehydrogenase, pyruvate-formate lyase, acetate kinase, phosphoacyltransferase, phosphoacetyltransferase, pyruvate decarboxylase.

[0173] In another and / or additional preferred embodiment of the method and / or cell of the application, the cell comprises at least partially inactivated catabolic pathways of selected monosaccharides, disaccharides or oligosaccharides involved in and / or required for the synthesis of the negatively charged, preferably sialylated oligosaccharides of the application.

[0174] In another and / or additional preferred embodiment of the method and / or cell of the application, the cell has, expresses and / or overexpresses at least one gene selected from the list consisting of, or consisting essentially of, genes encoding disulfide bond isomerases, thiol oxidases or chaperones.

[0175] In another and / or additional preferred embodiment of the method and / or cell of the application, the cell expresses a membrane transport protein or polypeptide having transport activity to transport the compound across the outer cell wall membrane. In another and / or additional preferred embodiment of the method and / or cell of the application, the cell expresses more than one membrane transport protein or polypeptide having transport activity to transport the compound across the outer cell wall membrane. In a more preferred embodiment, the cell is modified in the expression or activity of the membrane transport protein or polypeptide having transport activity. The membrane transport protein or polypeptide having transport activity is an endogenous protein of the cell whose expression or activity is modified, preferably the endogenous membrane transport protein or polypeptide having transport activity is overexpressed; alternatively, the membrane transport protein or polypeptide having transport activity is a heterologous protein introduced and expressed in the cell, preferably overexpressed. In a cell that also expresses a heterologous membrane transport protein or polypeptide having transport activity, the endogenous membrane transport protein or polypeptide having transport activity can have altered expression.

[0176] In another and / or additional more preferred embodiment, the membrane transport protein or polypeptide having transport activity is selected from the list comprising, consisting of or consisting essentially of: transporters, P-P bond hydrolysis driven transporters, beta-barrel porins, accessory transporters, and phosphotransfer driven group transporters. In an even more preferred embodiment, transporters include MFS transporters, sugar efflux transporters (SETs), and siderophore efflux proteins. In another more preferred embodiment, P-P bond hydrolysis driven transporters include ABC transporters and siderophore efflux proteins.

[0177] In another and / or additional preferred embodiment of the method and / or cell of the application, the membrane transport protein or polypeptide having transport activity controls the flow of the negatively charged, preferably sialylated, oligosaccharide across the outer cell wall membrane. In another and / or additional preferred embodiment of the method and / or cell of the application, the membrane transport protein or polypeptide having transport activity controls the flow of one or more precursors for the production of the negatively charged, preferably sialylated, oligosaccharide across the outer cell wall membrane.

[0178] In another preferred embodiment of the method and / or cell of the application, the membrane transport protein or polypeptide with transport activity increases the production of the negatively charged, preferably sialylated oligosaccharide. In an alternative and / or additional preferred embodiment of the method and / or cell of the application, the membrane transport protein or polypeptide with transport activity provides efflux of the negatively charged, preferably sialylated oligosaccharide. In an alternative and / or additional preferred embodiment of the method and / or cell of the application, the membrane transport protein or polypeptide with transport activity increases the efflux of the negatively charged, preferably sialylated oligosaccharide.

[0179] Preferably, the cell is transformed to comprise at least one nucleic acid sequence encoding a protein selected from the group comprising, consisting of or consisting essentially of a lactose transporter (e.g. LacY or lac12 permease), a glucose transporter, a galactose transporter, a transporter of nucleotide-activated sugars (e.g. a transporter of UDP-GlcNAc), a transporter involved in the transport of the negatively charged, preferably sialylated oligosaccharide out of the cell.

[0180] In another and / or additional preferred embodiment of the method and / or cell of the application, the cell expresses a membrane transporter belonging to the MFS transporter family, such as a MdfA polypeptide from the MdfA family of multidrug transporters MdfA from the species including Escherichia coli (UniProt ID P0AEY8), Cronobacter muytjensii (UniProt ID A0A2T7ANQ9), Citrobacter youngae (UniProt ID D4BC23), and Yokenella regensburgei (UniProt ID G9Z5F4). In another preferred embodiment of the method and / or cell of the application, the cell expresses a membrane transporter belonging to the Sugar efflux transporter family (SET), such as a SetA polypeptide from the SetA family of the species including Escherichia coli (UniProt ID P31675, sequence version 03 (2004-10-11)) and Citrobacter freundii (UniProt ID A0A078LM16). In another preferred embodiment of the method and / or cell of the application, the cell expresses a membrane transporter belonging to the Siderophore efflux protein family, such as from Escherichia coli entS (UniProt ID P24077, sequence version 02 (1997-11-01)), K. ascorbata entS (UniProt ID A0A378GQ13), and Escherichia coli iceT (UniProt ID A0A024L207). In another preferred embodiment of the method and / or cell of the application, the cell expresses a membrane transporter belonging to the ABC transporter family, such as oppF from Escherichia coli (UniProt ID P77737), lmrA from Lactococcus lactis subsp. lactis bv. diacetylactis (UniProt ID A0A1V0NEL4), and Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID A0A1V0NEL4).In a more preferred embodiment of the method and / or cell of the application, the cell expresses one or more membrane transporters selected from the list comprising, consisting or consisting essentially of: a lactose transporter, such as LacY or lac12 permease; a fucose transporter; a glucose transporter; a galactose transporter; a nucleotide-activated sugar transporter, such as a transporter for UDP-GlcNAc, UDP-Gal and / or GDP-Fuc; MdfA protein from Escherichia coli (UniProt ID P0AEY8); MdfA protein from Morganella morganii (UniProt ID A0A2T7 ANQ9), MdfA protein from Citrobacter youngae (UniProt ID D4BC23), MdfA protein from Yokenia regensburgei (UniProt ID G9Z5F4), SetA protein from Escherichia coli (UniProt ID P31675, sequence version 03 (October 11, 2004)), SetA protein from Citrobacter koseri (UniProt ID A0A078 LM16), entS protein from Escherichia coli (UniProt ID P24077, sequence version 02 (November 1, 1997)), entS protein from Kluyvera ascorbata (UniProt ID A0A378GQ13), iceT protein from Escherichia coli (UniProt ID A0A024L207), oppF protein from Escherichia coli (UniProt ID P77737), lmrA protein from Lactococcus lactis subsp. lactis biovar. diacetylactis (UniProt ID A0A1V0NEL4) and Blon_2475 from Bifidobacterium longum subsp. infantis (UniProt ID A0A1V0NEL4). Preferably, the cell is transformed to comprise at least one nucleic acid sequence encoding a membrane transporter selected from the list consisting of, consisting essentially of or consisting essentially of a siderophore efflux protein, a Major Facilitator Superfamily (MFS) transporter protein, an ATP-binding cassette (ABC) transporter protein or a sugar efflux transporter.

[0181] According to another preferred aspect of the method and / or cell of the application, the cell is capable of synthesizing N-acetylmannosamine (ManNAc) and / or N-acetylmannosamine-6-phosphate (ManNAc-6-phosphate).

[0182] In another and / or an additional preferred embodiment of the methods and / or cells of the application, the function of one or more genes involved in one or more reductive pathways in the cell is reduced or knocked out. Examples of reductive pathways include, but are not limited to, the reductive acetyl-CoA pathway, the reductive pyrimidine catabolism pathway, the reductive citric acid cycle, the thiol redox pathway, and the reductive glycine pathway. In another and / or an additional preferred embodiment of the methods and / or cells of the application, the function of one or more genes involved in one or more reductive pathways is reduced by inserting, deleting, and / or modifying one or more nucleotides in one or more polynucleotide sequences selected from the list consisting of, consisting essentially of, or consisting of promoter sequences, ribosome binding sites, untranslated regions, coding sequences, and transcription terminator sequences of the one or more genes. In this context, the one or more genes can be selected from the list consisting of, consisting essentially of, or consisting of genes encoding formate dehydrogenase, formate-tetrahydrofolate ligase, methenyltetrahydrofolate cyclization-hydrolysing enzyme, glycine dehydrogenase / decarboxylase, glycine cleavage system protein, glutamate dehydrogenase, glycine reductase complex, Co 2 formate dehydrogenase, formate-tetrahydrofolate ligase, methenyltetrahydrofolate cyclization-hydrolysing enzyme, methenyltetrahydrofolate dehydrogenase, methyltetrahydrofolate:corrinoid / iron-sulfur protein methyltransferase, methylene tetrahydrofolate reductase, methylene tetrahydrofolate dehydrogenase, methyltetrahydrofolate:corrinoid / iron-sulfur protein methyltransferase, methenyltetrahydrofolate cyclization-hydrolysing enzyme, pyruvate:ferredoxin oxidoreductase, serine hydroxymethyltransferase, dihydropyrimidine dehydrogenase, dihydropyrimidinase, beta-ureidopropionase, ribulose-1,5-bisphosphate carboxylase, ATP citrate lyase, citrate synthase, aconitase, isocitrate dehydrogenase, 2-ketoglutarate dehydrogenase, 2-ketoglutarate ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA-acetoacetate-CoA transferase, fumarate reductase, succinate dehydrogenase, fumarase, malate dehydrogenase, isocitrate lyase, malate synthase, phosphoenolpyruvate carboxykinase, phosphoenolpyruvate carboxylase, malic enzyme, pyruvate carboxylase / oxaloacetate decarboxylase, pyruvate dehydrogenase, pyruvate ferredoxin oxidoreductase, KatG catalase, alkyl hydroperoxide reductase, DNA-binding ferritin-like protein, glutaredoxin, iron homeostasis regulator, manganese transport protein, OxyS, OxyR, glutathione reductase, thioredoxin reductase, thioredoxin disulfide reductase, and glutamine synthetase. Preferably, the one or more genes involved in one or more reductive pathways are selected from the list consisting of, consisting essentially of, or consisting of glutathione reductase and thioredoxin reductase.

[0183] In a preferred embodiment, the negatively charged, preferably sialylated oligosaccharides of the present application are produced by cells cultivated in cell culture. In the context of the present application, cell culture includes in vitro and / or ex vivo cell culture. In another and / or further preferred embodiment, the cell culture is fermentation. In an alternative and / or further preferred embodiment, the cells are cultivated or incubated in a reactor as defined herein. In an alternative and / or further preferred embodiment, the cells are cultivated or incubated in an incubator as defined herein.

[0184] In another and / or further preferred embodiment, the cells are stably cultivated in a cultivation or incubation medium.

[0185] In another and / or further preferred embodiment, the cells are cultivated in a cultivation or incubation medium comprising at least one carbon source selected from the list comprising, consisting of or consisting essentially of monosaccharides; disaccharides; oligosaccharides; polysaccharides; polyols; glycerol; complex media comprising molasses, corn steep liquor, peptone, tryptone or yeast extract. Preferably, the carbon source is selected from the list comprising glucose, N-acetylglucosamine (GlcNAc), glycerol, fructose, sucrose, maltose, lactose, arabinose, malt- oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn steep liquor, high fructose syrup, acetate, citrate, lactate and pyruvate. More preferably, the at least one carbon source is selected from the list consisting of glucose, fructose, sucrose and glycerol. In a more preferred embodiment, the cultivation or incubation medium is a chemically defined medium. In another preferred embodiment, the cultivation or incubation medium is a basal salt medium comprising sulfate, phosphate, chloride, ammonium salt, calcium salt, magnesium salt, sodium salt, potassium salt, iron salt, copper salt, zinc salt, manganese salt, cobalt salt and / or selenium salt. In another and / or further preferred embodiment, the cultivation or incubation medium comprises one or more precursors for the production of the negatively charged, preferably sialylated oligosaccharides. In another and / or further preferred embodiment, the cultivation or incubation medium comprises at least one compound selected from the list consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid, CMP-sialic acid, CMP-Neu5Ac and CMP-KDO.

[0186] In another and / or further preferred embodiment of the method of the present application, the method for producing the negatively charged, preferably sialylated oligosaccharides as described herein comprises at least one of the following steps:

[0187] i) using a cultivation or incubation medium comprising at least one precursor and / or acceptor;

[0188] ii) adding at least one precursor and / or acceptor feed to the cultivation or incubation medium in a reactor or incubator, wherein the total volume of the reactor or incubator ranges from 250 mL to 10,000 m3(cubic meters), preferably in a continuous manner, and preferably such that the final volume of the cultivation or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the cultivation or incubation medium before the addition of the precursor and / or acceptor feed;

[0189] iii) adding at least one precursor and / or acceptor feed to the cultivation or incubation medium in a reactor or incubator, wherein the total volume of the reactor or incubator ranges from 250 mL to 10,000 m3(cubic meters), preferably in a continuous manner, and preferably such that the final volume of the cultivation or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the cultivation or incubation medium before the addition of the precursor and / or acceptor feed, and wherein preferably the pH of the precursor and / or acceptor feed is set between 2.0 and 10.0, and wherein preferably the temperature of the precursor and / or acceptor feed is maintained between 20 °C and 80 °C.

[0190] iv) adding at least one precursor and / or acceptor feed to the cultivation or incubation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days, by a precursor and / or acceptor feed solution;

[0191] v) adding at least one precursor and / or acceptor feed to the cultivation or incubation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days, by a precursor and / or acceptor feed solution, and wherein the concentration of the precursor and / or acceptor feed solution is 50 g / L, 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 the precursor and / or acceptor feed solution is set between 2.0 and 10.0, and wherein preferably the temperature of the precursor and / or acceptor feed solution is maintained between 20 °C and 80 °C;

[0192] The method is such that the concentration of the negatively charged, preferably sialylated oligosaccharide in the final volume of the culture or incubation is at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L. Preferably, the precursor is selected from the list comprising sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose. Preferably, the acceptor is lactose.

[0193] In another and / or additional preferred embodiment of the method of the present application, the method for producing a negatively charged, preferably sialylated oligosaccharide as described herein comprises at least one of the following steps:

[0194] i) using a culture or incubation medium comprising at least one precursor and / or acceptor;

[0195] ii) feeding at least one precursor and / or acceptor to the culture or incubation medium in the reactor or incubator in a single pulse or discontinuous (pulsed) manner, wherein the total volume of the reactor or incubator ranges from 250 mL to 10,000 m3 (cubic meters), and preferably such that the final volume of the culture or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture or incubation medium prior to the pulse of the precursor and / or acceptor feed;

[0196] iii) feeding at least one precursor and / or acceptor to the culture or incubation medium in the reactor or incubator in a single pulse or discontinuous (pulsed) manner, wherein the total volume of the reactor or incubator ranges from 250 mL to 10,000 m3 (cubic meters), and preferably such that the final volume of the culture or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture or incubation medium prior to the pulse of the precursor and / or acceptor feed, and wherein preferably the pH of the precursor and / or acceptor feed is set between 2.0 and 10.0, and wherein preferably the temperature of the precursor and / or acceptor feed is maintained between 20 °C and 80 °C.

[0197] iv) feeding at least one precursor and / or acceptor to the culture or incubation medium by a precursor and / or acceptor feed solution in a discontinuous (pulsed) manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days;

[0198] v) adding at least one precursor and / or acceptor feed to said cultivation or incubation medium in a discontinuous (pulsed) manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a precursor and / or acceptor feed solution, and wherein the concentration of said precursor and / or acceptor feed solution is 50 g / L, 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 precursor and / or acceptor feed solution is set between 2.0 and 10.0, and wherein preferably the temperature of said precursor and / or acceptor feed solution is maintained between 20 °C and 80 °C;

[0199] The method is such that the concentration of said negatively charged, preferably sialylated oligosaccharides in the final volume of said cultivation or incubation is at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L. Preferably, said precursor is selected from the list comprising sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose. Preferably, said acceptor is lactose.

[0200] In another and / or additional preferred embodiment of the method of the present application, the method for producing negatively charged, preferably sialylated oligosaccharides as described herein comprises at least one of the following steps:

[0201] i) using a cultivation or incubation medium comprising at least 50 grams, more preferably at least 75 grams, more preferably at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of precursor per liter of initial reactor or incubator volume, wherein said reactor or incubator volume ranges from 250 mL to 10,000 m3 (cubic meters);

[0202] ii) using a cultivation or incubation medium comprising at least 50 grams, more preferably at least 75 grams, more preferably at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of acceptor per liter of initial reactor or incubator volume, wherein said reactor or incubator volume ranges from 250 mL to 10,000 m3 (cubic meters);

[0203] iii) adding to the cultivation or incubation broth in the reactor or incubator a precursor feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of precursor per liter of initial reactor or incubator volume, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m3(cubic meters), preferably in a continuous manner, and preferably such that the final volume of the cultivation or incubation broth is not more than three times, preferably not more than two times, more preferably less than two times the volume of the cultivation or incubation broth prior to the addition of the precursor feed;

[0204] iv) adding to the cultivation or incubation broth in the reactor or incubator a recipient feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of recipient per liter of initial reactor or incubator volume, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m3(cubic meters), preferably in a continuous manner, and preferably such that the final volume of the cultivation or incubation broth is not more than three times, preferably not more than two times, more preferably less than two times the volume of the cultivation or incubation broth prior to the addition of the recipient feed;

[0205] v) adding to the cultivation or incubation broth a precursor feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of precursor per liter of initial reactor or incubator volume, wherein the reactor or incubator total volume ranges from 250 mL to 10,000 m3(cubic meters), preferably in a continuous manner, and preferably such that the final volume of the cultivation or incubation broth is not more than three times, preferably not more than two times, more preferably less than two times the volume of the cultivation or incubation broth prior to the addition of the precursor feed, and wherein preferably the pH of the precursor feed is set between 2.0 and 10.0, and wherein preferably the temperature of the precursor feed is maintained between 20 °C and 80 °C;

[0206] vi) adding to the culture or incubation medium a recipient feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of recipient per liter of initial reactor or incubator volume, wherein the reactor or incubator total volume ranges from 250 mL to 10,000 m3(cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture or incubation medium prior to the addition of the recipient feed, and wherein preferably the pH of the recipient feed is set between 2.0 and 10.0, and wherein preferably the temperature of the recipient feed is maintained between 20 °C and 80 °C;

[0207] vii) adding to the culture or incubation medium a precursor and / or recipient feed solution in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days;

[0208] viii) adding to the culture or incubation medium a precursor feed solution in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days, and wherein the concentration of the precursor feed solution is 50 g / L, 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 the precursor feed solution is set between 2.0 and 10.0, and wherein preferably the temperature of the precursor feed solution is maintained between 20 °C and 80 °C;

[0209] ix) adding a recipient feed to the cultivation or incubation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days by a recipient feed solution, and wherein the concentration of the recipient feed solution is 50 g / L, 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 the recipient feed solution is set between 2.0 and 10.0, and preferably the temperature of the recipient feed solution is kept between 20 °C and 80 °C;

[0210] The method is such that the concentration of the negatively charged, preferably sialylated oligosaccharides in the final volume of the cultivation or incubation is at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L. Preferably, the precursor is selected from the list comprising sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose. Preferably, the recipient is lactose.

[0211] In another and / or additional preferred embodiment of the method of the present application, the method for producing negatively charged, preferably sialylated oligosaccharides as described herein comprises at least one of the following steps:

[0212] i) using a cultivation or incubation medium comprising at least 50 grams, more preferably at least 75 grams, more preferably at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of precursor per liter of initial reactor or incubator volume, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m3(cubic meters);

[0213] ii) using a cultivation or incubation medium comprising at least 50 grams, more preferably at least 75 grams, more preferably at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of recipient per liter of initial reactor or incubator volume, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m3(cubic meters);

[0214] iii) adding a precursor feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of precursors per liter of initial reactor or incubator volume in a single pulse or discontinuous (pulsed) manner to the culture or incubation medium in the reactor or incubator, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m3(cubic meters), and preferably such that the final volume of the culture or incubation medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture or incubation medium prior to the addition of the precursor feed;

[0215] iv) adding a recipient feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of recipients per liter of initial reactor or incubator volume in a single pulse or discontinuous (pulsed) manner to the culture or incubation medium in the reactor or incubator, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m3(cubic meters), and preferably such that the final volume of the culture or incubation medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture or incubation medium prior to the addition of the recipient feed;

[0216] v) adding a precursor feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of precursors per liter of initial reactor or incubator volume in a single pulse or discontinuous (pulsed) manner to the culture or incubation medium, wherein the reactor or incubator total volume ranges from 250 mL to 10,000 m3(cubic meters), and preferably such that the final volume of the culture or incubation medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture or incubation medium prior to the addition of the precursor feed, and wherein preferably the pH of the precursor feed is set between 2.0 and 10.0, and wherein preferably the temperature of the precursor feed is maintained between 20 °C and 80 °C;

[0217] vi) adding to the culture or incubation medium a recipient feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of recipient per liter of initial reactor or incubator volume in a single pulse or discontinuous (pulsed) manner, wherein the reactor or incubator total volume ranges from 250 mL to 10,000 m3(cubic meters), and preferably such that the final volume of the culture or incubation medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture or incubation medium prior to the addition of the recipient feed, and wherein preferably the pH of the recipient feed is set between 2.0 and 10.0, and wherein preferably the temperature of the recipient feed is maintained between 20 °C and 80 °C;

[0218] vii) adding to the culture or incubation medium a precursor and / or recipient feed solution in a discontinuous (pulsed) manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days;

[0219] viii) adding to the culture or incubation medium a precursor feed solution in a discontinuous (pulsed) manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days, and wherein the concentration of the precursor feed solution is 50 g / L, 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 the precursor feed solution is set between 2.0 and 10.0, and wherein preferably the temperature of the precursor feed solution is maintained between 20 °C and 80 °C;

[0220] ix) adding a recipient feed to the cultivation or incubation medium in a discontinuous (pulsed) manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days by a recipient feed solution, and wherein the concentration of the recipient feed solution is 50 g / L, 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 the recipient feed solution is set between 2.0 and 10.0, and preferably the temperature of the recipient feed solution is kept between 20 °C and 80 °C;

[0221] The method is such that the concentration of the negatively charged, preferably sialylated oligosaccharides in the final volume of the cultivation or incubation is at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L. Preferably, the precursor is selected from the list comprising sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose. Preferably, the recipient is lactose.

[0222] In a further more preferred embodiment, the method for producing negatively charged, preferably sialylated oligosaccharides as described herein comprises at least one of the following steps:

[0223] i) adding a lactose feed to the cultivation or incubation medium comprising at least 50 grams, more preferably at least 75 grams, more preferably at least 100 grams, more preferably at least 120 grams, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor volume ranges from 250 mL (milliliter) to 10,000 m³ (cubic meter), preferably in a continuous manner, and preferably such that the final volume of the cultivation or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the cultivation or incubation medium before the lactose feed was added;

[0224] ii) adding a lactose feed to the cultivation or incubation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days by a feed solution;

[0225] iii) feeding of lactose to the culture or incubation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days by means of a feed solution, and wherein the concentration of the lactose feed 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 the solution is set between 3 and 7; and

[0226] wherein preferably the temperature of the feed solution is kept between 20 °C and 80 °C;

[0227] The method is such that the concentration of the negatively charged, preferably sialylated oligosaccharides in the final volume of the culture or incubation is at least 30 g / L, at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L.

[0228] Preferably, the lactose feed is accomplished by adding lactose at a concentration of at least 1 mM, preferably at least 5 mM, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150 mM, more preferably at a concentration > 300 mM from the beginning of the culture or incubation.

[0229] In another aspect, the lactose feed is achieved by adding lactose to the culture or incubation medium at a concentration such that a lactose concentration of at least 1 mM, preferably 5 mM, 10 mM or 30 mM is obtained throughout the production phase of the culture or incubation.

[0230] In a further embodiment of the method described herein, the cells are cultured or incubated for at least about 60, 80, 100 or about 120 hours or in a continuous manner.

[0231] In a preferred embodiment, the carbon source, preferably sucrose, is provided in the culture or incubation medium for 3 days or more, preferably for up to 7 days; and / or at least 100 g, advantageously at least 105 g, more advantageously at least 110 g, even more advantageously at least 120 g of sucrose per liter of initial culture or incubation medium is provided in a continuous manner to the culture or incubation medium, such that the final volume of the culture or incubation medium is not more than three times, advantageously not more than two times, more advantageously less than two times the volume of the culture or incubation medium before the cultivation.

[0232] Preferably, when performing the methods described herein, the first phase of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the culture or incubation medium, and then in a second phase lactose is added to the culture or incubation medium.

[0233] In an alternative preferred embodiment, in the methods described herein, lactose is already added together with the carbon substrate in the first phase of exponential growth.

[0234] In another and / or further preferred embodiment of the methods and / or cells of the application, the cell is selected from the list consisting of prokaryotic cells and eukaryotic cells, preferably from the list consisting of yeast cells, bacterial cells, archaeal cells, algal cells, plant cells, fungal cells, animal cells and protozoan cells. In another preferred embodiment, the cell is a bacterial, fungal, yeast, plant cell, animal cell or protozoan cell.

[0235] The latter bacteria are preferably of the phylum Proteobacteria, of the phylum Firmicutes, of the phylum Cyanobacteria, of the phylum Deinococcus-Thermus or of the phylum Actinobacteria. The latter bacteria of the phylum Proteobacteria are preferably of the family Enterobacteriaceae, preferably of the species Escherichia coli. The latter bacteria preferably relate to any strain of 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 cultured Escherichia coli strains - designated as Escherichia coli K12 strains - that are well adapted to the laboratory environment and that, unlike wild-type strains, have lost the ability to grow in the gut. Common examples of Escherichia coli K12 strains are K12 wild-type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Thus, the present application specifically relates to a mutated and / or transformed Escherichia coli cell or strain as described above, wherein the Escherichia coli strain is a K12 strain. More preferably, the Escherichia coli K12 strain is Escherichia coli MG1655. The latter bacteria are of the phylum Firmicutes, preferably of the class Bacilli, preferably of the order Lactobacillales, members of which are such as Lactobacillus lactis, Leuconostoc mesenteroides, or of the order Bacillales, members of which are such as Bacillus, such as Bacillus subtilis or B. amyloliquefaciens. The latter bacteria are of the phylum Actinobacteria, preferably of the family Corynebacteriaceae, members of which are such as Corynebacterium glutamicum or C. afermentans, or of the family Streptomycetaceae, members of which are such as Streptomyces griseus or S. fradiae. The latter bacteria are of the phylum Proteobacteria, preferably of the family Vibrionaceae, members of which are such as Vibrio natriegens. The latter yeast is preferably of the phylum Ascomycota or of the phylum Basidiomycota or of the phylum Deuteromycota or of the phylum Zygomycota.The latter yeast is preferably selected from the genus Saccharomyces (members of which are, for example, Saccharomyces cerevisiae, S. bayanus, S. boulardii), Zygosaccharomyces, Pichia (members of which are, for example, Pichia pastoris, P. anomala, P. kluyveri), Komagataella, Hansenula, Kluyveromyces (members of which are, for example, Kluyveromyces lactis, K. marxianus, K. thermotolerans), Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces, Torulaspora, Yarrowia (for example Yarrowia lipolytica) or Starmerella (for example 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 is preferably from the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus. The plant cell comprises cells of flowering and non-flowering plants, and algal cells, such as Chlamydomonas, Chlorella, etc. Preferably, the plant is tobacco, rose, alfalfa, rice, tomato, cotton, rapeseed, soybean, maize or corn plant. More preferably, the latter plant cell is selected from the family Rosaceae. The latter animal cell is preferably derived from a non-human mammal (e.g. a bovine, a buffalo, a pig, a sheep, a mouse, a rat, a primate (e.g. chimpanzee, orangutan, gorilla, monkey (e.g. Old World, New World), lemur), a dog, a cat, a rabbit, a horse, a cow, a goat, a cow, a deer, a musk deer, a bovine, a whale, a dolphin, a hippopotamus, an elephant, a rhinoceros, a giraffe, a zebra, a lion, a cheetah, a tiger, a panda, a red panda, a otter), a bird (e.g. chicken, duck, ostrich, turkey, pheasant), a fish (e.g. barracuda, salmon, tuna, sea bass, trout, catfish), an invertebrate (e.g. lobster, crab, shrimp, clam, oyster, mussel, sea urchin), a reptile (e.g. snake, alligator, turtle), an amphibian (e.g. frog) or an insect (e.g. fly, nematode), or is a transgenic cell line from a human cell (excluding embryonic stem cells). Both human and non-human mammalian cells are preferably selected from the list comprising: an epithelial cell, 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, e.g. N20, SP2 / 0 or YB2 / 0 cell, a NIH-3T3 cell, a non-mammary adult stem cell or derivative thereof (such as described in WO21067641), preferably a mesenchymal stem cell or derivative thereof (as described in WO21067641), a mammary cell derived from a mammalian induced pluripotent stem cell (preferably a human induced pluripotent stem cell), a mammary cell that is part of a mammary-like gland organoid, a post-partum mammary epithelial cell, a polarized mammary cell, preferably a polarized mammary cell selected from the list comprising: a live primary mammary epithelial cell, a live mammary myoepithelial cell, a live mammary progenitor cell, a live immortalized mammary epithelial cell, a live immortalized mammary myoepithelial cell, a live immortalized mammary progenitor cell, a list of non-mammary adult stem cells or derivatives thereof, as well known to the person skilled in the art, e.g. WO2021 / 219634, WO 2022 / 054053, WO 2021 / 141762, WO 2021 / 142241, WO 2021 / 067641 and WO2021 / 242866.The latter insect cells are preferably derived from Spodoptera frugiperda, e.g. Sf9 or Sf21 cells, Bombyx mori, Mamestra brassicae, Trichoplusia ni, e.g. BTI-TN-5B1-4 cells, or Drosophila melanogaster, e.g. Drosophila S2 cells. The latter protozoan cells are preferably Leishmania tarentolae cells.

[0236] In another and / or additional preferred embodiment, the cell is E. coli or yeast having a lactose permease positive phenotype, preferably wherein the lactose permease is encoded by the genes LacY or LAC12, respectively.

[0237] In another and / or additional preferred embodiment, the cell is a live Gram-negative bacterium comprising reduced or eliminated synthesis of poly-N-acetylglucosamine (PNAG), enterobacterial common antigen (ECA), cellulose, colanic acid, core oligosaccharide, osmoregulated periplasmic glucan (OPG), glycerol glucoside, glycocalyx, and / or trehalose, as compared to the unmodified progenitor cell.

[0238] In more preferred embodiments of the method and / or cell, the reduction or elimination of synthesis of poly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen (ECA), cellulose, colanic acid, core oligosaccharide, osmoregulated periplasmic glucan (OPG), glycerol glucoside, glycans, and / or trehalose is provided by a mutation in any one or more glycosyltransferases involved in the synthesis of any one of poly-N-acetyl-glucosamine (PNAG), enterobacterial common antigen (ECA), cellulose, colanic acid, core oligosaccharide, osmoregulated periplasmic glucan (OPG), glycerol glucoside, glycans, and / or trehalose, wherein the mutation provides for a deletion or lower expression of any one of the glycosyltransferases. The glycosyltransferases include glycosyltransferase genes encoding poly-N-acetyl-D-glucosamine synthase subunits, UDP-N-acetylglucosamine- undecaprenyl phosphate N-acetylglucosamine phosphotransferase, Fuc4NAc (4-acetamido-4,6-dideoxy-D-galactose) transferase, UDP-N-acetyl-D-mannosamine uronate transferase, glycosyltransferase genes encoding cellulose synthase catalytic subunits, cellulose biosynthesis proteins, colanic acid biosynthesis glucuronosyltransferases, colanic acid biosynthesis galactosyltransferases, colanic acid biosynthesis fucosyltransferases, UDP-glucose:undecaprenyl phosphate glucose-1-phosphate transferase, putative colanic acid biosynthesis glycosyltransferases, UDP-glucuronate:LPS (Hep III) glycosyltransferase, ADP-heptose-LPS heptosyltransferase 2, ADP-heptose:LPS heptosyltransferase 1, putative ADP-heptose:LPS heptosyltransferase 4, lipopolysaccharide core biosynthesis proteins, UDP-glucose:(glucosyl)LPS alpha-1,2-glucosyltransferase, UDP-D-glucose:(glucosyl)LPS alpha-1,3-glucosyltransferase, UDP-D-galactose:(glucosyl)lipopolysaccharide-1,6-D-galactosyltransferase, lipopolysaccharide glucosyltransferase I, lipopolysaccharide core heptosyltransferase 3, beta-1,6-galactofuranosyltransferase, undecaprenyl phosphate 4-deoxy-4-formamido-L-arabinose transferase, lipid IVA 4-amino-4-deoxy-L-arabinosyltransferase, bacterial terpene alcohol glucosyltransferase, putative glycosyltransferase family 2, osmoregulated periplasmic glucan (OPG) biosynthesis protein G, OPG biosynthesis protein H, glucosylglycerate phosphorphydrase, glycogen synthase, 1,4-alpha-glucan branching enzyme, 4-alpha-glucan transferase, and trehalose-6-phosphate synthase.In exemplary embodiments, the cell is mutated in any one or more of the glycosyltransferases comprising pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcaI, wcaJ, wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA, and yaiP, wherein the mutation provides for a loss or reduction in expression of any of the glycosyltransferases.

[0239] In alternative and / or additional preferred embodiments of the method and / or cell, the reduction or elimination of poly-N-acetylglucosamine (PNAG) synthesis is achieved by overexpression of a carbon storage regulator-encoding gene, deletion of a Na+ / H+ antiporter regulator-encoding gene, and / or deletion of a sensor histidine kinase-encoding gene.

[0240] In another and / or additional preferred embodiment of the method and / or cell of the present application, the cell produces 30 g / L or more of the negatively charged, preferably sialylated oligosaccharide in whole broth and / or supernatant, and / or wherein the negatively charged, preferably sialylated oligosaccharide in the whole broth and / or supernatant has a purity of at least 80%, measured against the total amount of negatively charged, preferably sialylated oligosaccharide and its precursors produced by the cell in whole broth and / or supernatant, respectively. In more preferred embodiments, 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 the negatively charged, preferably sialylated oligosaccharide in whole broth and / or supernatant.

[0241] In another aspect of the application, the cell produces a negatively charged, preferably sialylated oligosaccharide as described herein.

[0242] In a preferred embodiment of the method and / or cell of the application, the cell produces a mixture of oligosaccharides comprising a negatively charged, preferably sialylated oligosaccharide as described herein. In another and / or additional preferred embodiment of the method and / or cell of the application, the cell is capable of producing and / or produces a mixture of two or more oligosaccharides, wherein at least one of the two or more oligosaccharides is a negatively charged, preferably sialylated oligosaccharide as described herein. In another and / or additional preferred embodiment of the method and / or cell of the application, the cell is capable of producing and / or produces a mixture of one or more disaccharides and one or more oligosaccharides, wherein at least one of the one or more oligosaccharides is a negatively charged, preferably sialylated oligosaccharide as described herein. In another and / or additional preferred embodiment of the method and / or cell of the application, the cell is capable of producing and / or produces a mixture comprising a negatively charged, preferably sialylated oligosaccharide and at least one uncharged (neutral) oligosaccharide. In another and / or additional preferred embodiment of the method and / or cell of the application, the cell is capable of producing and / or produces a mixture comprising 1) a negatively charged, preferably sialylated oligosaccharide and 2) a disaccharide and / or an uncharged (neutral) oligosaccharide.

[0243] In another aspect of the application, a negatively charged, preferably sialylated oligosaccharide produced by the cell of the application is recovered from the culture medium or incubation medium and / or the cell. In preferred embodiments, the negatively charged, preferably sialylated oligosaccharide is purified.

[0244] The term "isolating from the culture or incubation" means harvesting, collecting or recovering the negatively charged, preferably sialylated oligosaccharide from the cells and / or their growth medium. The negatively charged, preferably sialylated oligosaccharide can be isolated from the aqueous culture, incubation or incubation medium in which the cells were grown using conventional methods. If the negatively charged, preferably sialylated oligosaccharide is still present in the cells that produced the negatively charged, preferably sialylated oligosaccharide, the oligosaccharide can be released or extracted from the cells using conventional methods for cell disruption, such as using high pH, heat shock, sonication, French press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergent hydrolysis, etc. The culture, incubation or incubation medium and / or cell extract can then be further used together and separately to isolate the negatively charged, preferably sialylated oligosaccharide. This preferably includes clarifying the negatively charged, preferably sialylated oligosaccharide to remove suspended particles and contaminants, in particular cells, cellular components, insoluble metabolites and debris produced by the genetically engineered cells of the culture. In this step, the negatively charged, preferably sialylated oligosaccharide can be clarified in a conventional manner. Preferably, the negatively charged, preferably sialylated oligosaccharide is clarified by centrifugation, flocculation, decantation and / or filtration. A second step of isolating the negatively charged, preferably sialylated oligosaccharide preferably includes removing substantially all of the final remaining proteins, peptides, amino acids, RNA, DNA, endotoxins and glycolipids from the negatively charged, preferably sialylated oligosaccharide, preferably after its clarification. In this step, the remaining proteins and associated impurities can be removed from the negatively charged, preferably sialylated oligosaccharide in a conventional manner. Preferably, the remaining proteins, salts, by-products, color, endotoxins and other associated impurities are removed from the negatively charged, preferably sialylated oligosaccharide by ion exchange chromatography, or hydrophobic interaction chromatography or ligand exchange chromatography, in particular chromatography, more specifically by ion exchange chromatography, or hydrophobic interaction chromatography. In addition to size exclusion chromatography, the remaining proteins and associated impurities are retained by the chromatography media or selected membranes.

[0245] In a further preferred embodiment, the process described herein also provides for a further purification of the negatively charged, preferably sialylated oligosaccharides of the application. Said further purification of the negatively charged, preferably sialylated oligosaccharides can be achieved by, for example, using (active) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment, ion exchange, temperature adjustment, pH adjustment or adjustment of the pH with basic or acidic solutions to remove any residual DNA, proteins, LPS, endotoxins or other impurities. Alcohols such as ethanol and aqueous alcohol mixtures can also be used. Another purification step is accomplished by crystallization, evaporation or precipitation of the negatively charged, preferably sialylated oligosaccharides. Another purification step is drying, for example spray drying, freeze drying, spray freeze drying, freeze spray drying, belt drying, band drying, vacuum belt drying, vacuum band drying, drum drying, roller drying, vacuum drum drying or vacuum roller drying of the negatively charged, preferably sialylated oligosaccharides.

[0246] In an exemplary embodiment, the negatively charged, preferably sialylated oligosaccharides are isolated and purified in a process comprising the following steps in any order:

[0247] a) contacting the culture or its clarification with a nanofiltration membrane having a molecular weight cut-off (MWCO) of 600-3500 Daltons to ensure that the negatively charged, preferably sialylated oligosaccharides produced are retained and at least part of the proteins, salts, by-products, color and other associated impurities are allowed to pass through;

[0248] b) using said membrane, subjecting the retentate from step a) to a diafiltration process with an aqueous inorganic electrolyte solution, followed by optionally diafiltration with pure water to remove excess electrolyte;

[0249] c) collecting the retentate enriched in said negatively charged, preferably sialylated oligosaccharides in the form of a salt from said electrolyte cation.

[0250] In an alternative exemplary embodiment, the negatively charged, preferably sialylated oligosaccharides are isolated and purified in a process comprising the following steps in any order: subjecting the culture or its clarification to two membrane filtration steps using different membranes, wherein

[0251] - one membrane has a molecular weight cut-off of about 300 to about 500 Daltons, and

[0252] - the other membrane has a molecular weight cut-off of about 600 to about 800 Daltons.

[0253] In an alternative exemplary embodiment, the negatively charged, preferably sialylated oligosaccharides are isolated and purified in a method comprising the following steps in any order: a step comprising treating the culture or a clarified thereof with a strong cation exchange resin in the H+ form and a weak anion exchange resin in the free base form.

[0254] In an alternative exemplary embodiment, the isolation and purification of the negatively charged, preferably sialylated oligosaccharides is performed by the following. The culture comprises the produced negatively charged, preferably sialylated oligosaccharides, biomass, medium components and contaminants, and wherein the purity of the produced negatively charged, preferably sialylated oligosaccharides in the culture is < 80%, which is used in the following purification steps:

[0255] i) isolating the biomass from the culture,

[0256] ii) a cation exchanger treatment for removing positively charged species,

[0257] iii) an anion exchanger treatment for removing negatively charged species,

[0258] iv) a nanofiltration step and / or an electrodialysis step,

[0259] wherein a purified solution is provided, which comprises the produced negatively charged, preferably sialylated oligosaccharides, with a purity of more than or equal to 80%. Optionally, the purified solution is spray dried.

[0260] In an alternative exemplary embodiment, the negatively charged, preferably sialylated oligosaccharides are isolated and purified in a method comprising the following steps in any order: an enzymatic treatment of the culture; removal of biomass from the culture; ultrafiltration; nanofiltration; and a column chromatography step. Preferably, such column chromatography is single column or multi-column. More preferably, the column chromatography step is a simulated moving bed chromatography. Such simulated moving bed chromatography preferably comprises: i) at least 4 columns, wherein at least one column comprises a weak cation exchange resin or a 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 to 60 degrees Celsius.

[0261] In one particular embodiment, the present application provides the produced negatively charged, preferably sialylated oligosaccharides, which are spray dried into a powder, wherein the spray dried powder contains < 15% by weight of water, preferably < 10% by weight of water, more preferably < 7% by weight of water, most preferably < 5% by weight of water.

[0262] For the identification of the herein described negatively charged, preferably sialylated oligosaccharides, the monomeric building blocks (e.g. monosaccharide or glycan unit composition), the anomeric configuration of side chains, the presence and position of substituents, the degree of polymerization / molecular weight and the linkage pattern can be identified by standard methods known in the art, e.g. methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas chromatography-mass spectrometry), MALDI-MS (matrix-assisted laser desorption / ionization-mass spectrometry), ESI-MS (electrospray ionization-mass spectrometry), HPLC (high performance liquid chromatography with UV 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. Crystal structures 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), DP distribution and polydispersity can be determined by, e.g., viscosimetry and SEC (SEC-HPLC, high performance size exclusion chromatography). For the identification of the monomeric constituents of the negatively charged, preferably sialylated oligosaccharides, methods such as acid catalyzed hydrolysis, HPLC (high performance liquid chromatography) or GLC (gas liquid chromatography) after conversion to glycal acetates can be used. For the determination of the glycosidic linkages, the negatively charged, preferably sialylated oligosaccharides are methylated with iodomethane and strong base in DMSO, hydrolyzed, reduced to partially methylated glycal, acetylated to methylated glycal acetate and analyzed by GLC / MS (gas chromatography-mass spectrometry). For the determination of the glycan sequence, partial depolymerization using acids or enzymes is used to determine the structure. For the identification of the anomeric configuration, the negatively charged, preferably sialylated oligosaccharides are subjected to enzymatic analysis, e.g. by contacting them with enzymes specific for a particular type of linkage (e.g. beta-galactosidase or alpha-glucosidase etc.) and the products can then be analyzed using NMR.

[0263] In another aspect, the present application provides the use of the cells described herein for the production of the negatively charged, preferably sialylated oligosaccharides described herein.

[0264] In another aspect, the present application provides the use of the methods described herein for the production of the negatively charged, preferably sialylated oligosaccharides described herein.

[0265] In another aspect, the present application provides the use of the purified negatively charged, preferably sialylated oligosaccharides, or of the purified oligosaccharide mixture comprising the negatively charged, preferably sialylated oligosaccharides described herein, for use in medicine, preferably for the prevention or treatment of gastrointestinal diseases.

[0266] In another aspect, the present application provides the use of the purified negatively charged, preferably sialylated oligosaccharides obtained by the methods described herein in a food or feed preparation, a dietary supplement, a cosmetic ingredient or a pharmaceutical ingredient. In some embodiments, the negatively charged, preferably sialylated oligosaccharides are mixed with one or more ingredients suitable for use in food, feed, dietary supplements, pharmaceutical ingredients, cosmetic ingredients or medicaments. The purified negatively charged, preferably sialylated oligosaccharides can be used in the manufacture of a preparation, as a food additive, a prebiotic, a synbiotic, for supplementing infant food, adult food, young animal feed, adult animal feed, or as a therapeutically or pharmaceutically active compound or for cosmetic applications. In another aspect, the present application provides the use of the milk oligosaccharides described herein as a food additive, preferably as an additive for human food and / or pet food, more preferably as an additive for human infant food. In the context of the present application, food is human food, preferably infant food, human infant food and / or infant formula or infant supplement, feed is pet food, animal milk replacer, veterinary product, veterinary feed supplement, nutritional supplement, post-weaning feed or young stock feed.

[0267] In another preferred embodiment, a formulation is provided that further comprises at least one probiotic microorganism. In another preferred embodiment of the present application, the formulation is a nutritional composition. In a more preferred embodiment, the formulation is a pharmaceutical formulation, a dietary supplement, a milk beverage, or an infant formula. "Prebiotic" refers to a substance that promotes the growth of microorganisms (particularly gastrointestinal microorganisms) that are beneficial to the host. In some embodiments, a dietary supplement provides a plurality of prebiotics, including the negatively charged, preferably sialylated oligosaccharides, which are prebiotics purified by the methods disclosed in the present specification, to promote the growth of one or more beneficial microorganisms. Examples of prebiotic ingredients in dietary supplements include other prebiotic molecules (such as HMOs) and plant polysaccharides (such as inulin, pectin, beta-glucan, and xylo-oligosaccharides). "Probiotic" products typically contain live microorganisms that replace or supplement the gut flora, benefiting the recipient. Examples of such microorganisms include Lactobacillus species (e.g., Lactobacillus acidophilus and Lactobacillus bulgaricus), Bifidobacterium species (e.g., Bifidobacterium animalis, Bifidobacterium longum, and Bifidobacterium infantis (e.g., Bi-26)), and Saccharomyces boulardii. In some embodiments, the negatively charged, preferably sialylated oligosaccharides produced and / or purified by the methods of the present specification are administered orally in combination with such microorganisms. 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), thickening agents (such as acacia gum), acidity regulators (such as trisodium citrate), water, skim milk, and flavorings.

[0268] In some embodiments, the negatively charged, preferably sialylated oligosaccharides purified by the methods described herein are incorporated into a human infant food product, such as an infant formula. Infant formula is generally a processed food for feeding infants, either wholly or partially as a replacement for breast milk. In some embodiments, infant formula is sold in powder form and prepared for bottle or cup feeding by mixing with water. The ingredients of infant formula are generally designed to roughly mimic human milk. In some embodiments, the negatively charged, preferably sialylated oligosaccharides purified by the methods described herein are included in infant formula to provide similar nutritional benefits as the oligosaccharides provided in human breast milk. In some embodiments, the purified negatively charged, preferably sialylated oligosaccharides are mixed with one or more ingredients of infant formula. Examples of infant formula ingredients include skimmed milk, a carbohydrate source (e.g., lactose), a protein source (e.g., whey protein concentrate and casein), a fat source (e.g., vegetable oils - such as palm oil, high oleic safflower oil, rapeseed oil, coconut oil, and / or sunflower oil; and fish oil), vitamins (such as vitamins A, Bb, Bi2, C, and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate, and calcium phosphate), and possibly human milk oligosaccharides (HMOs). In some embodiments, one or more infant formula ingredients include skimmed milk, a carbohydrate source, a protein source, a fat source, and / or vitamins and minerals. In some embodiments, one or more infant formula ingredients include lactose, whey protein concentrate, and / or high oleic safflower oil. In some embodiments, the concentration of negatively charged, preferably sialylated oligosaccharides in infant formula is roughly the same as the concentration of oligosaccharides normally found in human breast milk. In some embodiments, the negatively charged, preferably sialylated oligosaccharides purified by the methods described herein are added to infant formula at a concentration that is roughly the same as the concentration of the compound normally found in human breast milk.

[0269] As shown in the examples herein, the methods and cells of the application preferably provide at least one of the following more surprising advantages when using the hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerases described herein compared to methods or cells using the same setup or enzymes or genetic background but not using the hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerases described herein:

[0270] - higher titer of negatively charged, preferably sialylated oligosaccharides as described herein (g / L),

[0271] - higher purity of negatively charged, preferably sialylated oligosaccharides (g / L),

[0272] - the purity (g / L) of the negatively charged, preferably sialylated, oligosaccharide is equal to or greater than 80%, preferably equal to or greater than 85%, more preferably equal to or greater than 90%, even more preferably equal to or greater than 91%, even more preferably equal to or greater than 92%, even more preferably equal to or greater than 93%, even more preferably equal to or greater than 94%, even more preferably equal to or greater than 95%, even more preferably equal to or greater than 96%, even more preferably equal to or greater than 97%, even more preferably equal to or greater than 98%, even more preferably equal to or greater than 99%,

[0273] - the lactose conversion is higher, resulting in a lower lactose concentration at the end of the fermentation (g / L lactose),

[0274] - the sialic acid formation is lower (g / L),

[0275] - the production rate r (g negatively charged, preferably sialylated, oligosaccharide / L / h) is higher,

[0276] - the cellular performance index CPI (g negatively charged, preferably sialylated, oligosaccharide / g X) is higher,

[0277] - the specific production rate Qp (g negatively charged, preferably sialylated, oligosaccharide / g X / h) is higher,

[0278] - the yield of used carbon source Y (g negatively charged, preferably sialylated, oligosaccharide / g used carbon source) is higher,

[0279] - the sucrose yield Ys (g negatively charged, preferably sialylated, oligosaccharide / g sucrose) is higher,

[0280] - the uptake / conversion rate of used carbon source Q (g carbon source / g X / h) is higher,

[0281] - the sucrose uptake / conversion rate Qs (g sucrose / g X / h) is higher,

[0282] - the lactose conversion / consumption rate rs (g lactose / h) is higher,

[0283] - the secretion, excretion or extracellular transport of the negatively charged, preferably sialylated, oligosaccharide is higher,

[0284] - the production host grows faster,

[0285] - the titer (g / L) of the negatively charged, preferably sialylated, oligosaccharide is better fine-tuned in a mixture of two or more oligosaccharides,

[0286] - in a mixture of one or more disaccharides and one or more oligosaccharides, the titre (g / L) of negatively charged, preferably sialylated, oligosaccharides is better fine-tuned,

[0287] - in a mixture of two or more oligosaccharides, the purity (g / L) of negatively charged, preferably sialylated, oligosaccharides is higher,

[0288] - in a mixture of one or more disaccharides and one or more oligosaccharides, the purity (g / L) of negatively charged, preferably sialylated, oligosaccharides is higher,

[0289] - in a mixture comprising two or more oligosaccharides, the titre (g / L) of any one or more of GlcNAc, ManNAc, N-acetyl lactosamine (LacNAc), lacto-N-biose (LNB), sialylated GlcNAc, sialylated ManNAc, sialylated LacNAc, sialylated LNB is lower, and / or

[0290] - in a mixture comprising two or more oligosaccharides, the purity (g / L) of any one or more of GlcNAc, ManNAc, N-acetyl lactosamine (LacNAc), lacto-N-biose (LNB), sialylated GlcNAc, sialylated ManNAc, sialylated LacNAc, sialylated LNB is higher, and / or

[0291] - in a mixture of one or more disaccharides and one or more oligosaccharides comprising negatively charged, preferably sialylated, oligosaccharides, the titre (g / L) of any one or more of GlcNAc, ManNAc, N-acetyl lactosamine (LacNAc), lacto-N-biose (LNB), sialylated GlcNAc, sialylated ManNAc, sialylated LacNAc, sialylated LNB is lower.

[0292] Unless otherwise defined, 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 application belongs. Generally, the nomenclature used herein, and the laboratory procedures in cell culture, molecular genetics, organic chemistry, nucleic acid chemistry, and hybridization described below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Purification steps are generally performed according to the manufacturer's instructions.

[0293] Further advantages are embodied in the specific embodiments and examples. It is self-evident that the above-mentioned features, as well as the features to be explained below, can be used not only in the combinations specified, but also in other combinations or alone, without leaving the scope of the application.

[0294] Furthermore, the present application relates to the following specific embodiments:

[0295] 1. A cell comprising a pathway for the production of a negatively charged, preferably sialylated oligosaccharide, characterized in that the cell is genetically engineered to have or express, preferably overexpress, a hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase, wherein the hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence comprising a conserved motif having SEQ ID NO: 12 and has hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase activity.

[0296] 2. The cell according to embodiment 1, wherein the hydrolytic UDP-N-acetyl-D- glucosamine-2-epimerase comprises, consists of or consists essentially of an amino acid sequence which is at least 80% identical to any one of the amino acid sequences set forth in SEQ ID NO: 01, 02, 03, 04, 05, 06, 07 or 08 over a stretch of at least 150 amino acid residues, preferably at least 200 amino acid residues.

[0297] 3. The cell according to any one of embodiments 1 or 2, wherein the hydrolytic UDP-N-acetyl-D-glucosamine-2-epimerase comprises an amino acid sequence which:

[0298] - comprises an IPR domain selected from the list comprising IPR003331, IPR020004 and IPR029767 as defined by InterPro 90.0 released on August 4, 2022,

[0299] - comprises a PF02350 motif as defined by PFAM 32.0 released on September 2018,

[0300] - comprises a cd03786 motif as defined by CDD v3.17 released on September 2016,

[0301] - comprises a panther domain selected from the list comprising PTHR43174 and PTHR43174:SF3 as defined by PANTHER 17.0 released on February 23, 2022,

[0302] - is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, or at least 99% identical to a segment of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 amino acid residues of any one of the amino acid sequences set forth in SEQ ID NO: 01, 02, 03, 04, 05, 06, 07, or 08,

[0303] - is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, or at least 99% identical to any one of the full-length amino acid sequences set forth in SEQ ID NO: 01, 02, 03, 04, 05, 06, 07, or 08, and / or

[0304] - is as set forth in any one of SEQ ID NO: 01, 02, 03, 04, 05, 06, 07, or 08.

[0305] 4. The cell according to any one of the preceding embodiments, wherein the hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase is a nnaA polypeptide.

[0306] 5. The cell according to any one of the preceding embodiments, wherein the cell comprises a nucleic acid molecule comprising a polynucleotide sequence encoding the hydrolytic type UDP-N-acetyl-D-glucosamine-2-epimerase.

[0307] 6. The cell according to embodiment 5, wherein the nucleic acid molecule is operably linked to a control sequence recognized by the cell, the nucleic acid molecule being further i) integrated in the genome of the cell and / or ii) presented to the cell on a vector.

[0308] 7. The cell according to any one of embodiments 5 or 6, wherein the nucleic acid molecule is foreign to the cell.

[0309] 8. The cell according to any one of the preceding embodiments, wherein the pathway for the production of the negatively charged, preferably sialylated oligosaccharide is a sialylation pathway, preferably the cell is genetically engineered to comprise the sialylation pathway, more preferably the cell comprises the sialylation pathway, wherein the sialylation pathway has been genetically engineered.

[0310] 9. The cell according to any one of the preceding embodiments, wherein the cell further comprises a pathway selected from the list comprising, consisting of or consisting essentially of: a fucosylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N- acetylgalactosaminylation pathway, a mannosylation pathway and an N-acetylmannosaminylation pathway, preferably the cell is genetically engineered to comprise at least one of said pathways, more preferably the cell comprises at least one of said pathways, wherein at least one of said pathways has been genetically engineered.

[0311] 10. The cell according to any one of the preceding embodiments, wherein the cell:

[0312] - has, preferably expresses, more preferably overexpresses one or more glycosyltransferases selected from the list comprising, consisting of or consisting essentially of: a fucosyltransferase, a sialyltransferase, a galactosyltransferase, a glucosyltransferase, a mannosyltransferase, an N-acetylglucosaminyltransferase, an N-acetylgalactosaminyltransferase, an N-acetylmannosaminyltransferase, a xylosyltransferase, a glucuronosyltransferase, a galacturonosyltransferase, a glucosaminyltransferase, a N-glycolylneuraminyltransferase, a rhamnosyltransferase, an N-acetyl rhamnosyltransferase, a UDP-4-amino-4,6-dideoxy-N-acetyl-beta-L- altrosamine aminotransferase, a UDP-N-acetylglucosamine enolpyruvate transferase and a fucosaminyltransferase, and / or

[0313] - is capable of producing, preferably produces, one or more nucleotide-activated sugars, preferably the cell is genetically engineered for the production of one or more of said nucleotide-activated sugars.

[0314] 11. The cell of any of the preceding embodiments, wherein the cell comprises a pathway for synthesizing a nucleotide-activated sugar selected from the list consisting of, consisting of, or consisting essentially of: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2,6-dideoxy-L-arabinose-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-xylose-4-hexulose, UDP-N-acetyl-L-rhamnoseamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetyl-fucosamine, UDP-N-acetyl-fucosamine (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), CMP-2-keto-3-deoxymanno-octulosonic acid (CMP-KDO), GDP-rhamnose, and UDP-xylose.

[0315] 12. The cell according to any of the preceding embodiments, wherein the cell has, preferably expresses, one or more genes selected from the list comprising, consisting of or consisting essentially of: mannose-6-phosphate isomerase, phosphomannomutase, mannose-1 -phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-1 -phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine-2-epimerase, UDP-N-acetylglucosamine-2-epimerase, N-acetylmannosamine-6-phosphate-2-epimerase, UDP-GlcNAc 2-epimerase / kinase, glucosamine-6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1 -phosphate uridyltransferase, glucosamine-1 -phosphate acetyltransferase, bifunctional N-acetylglucosamine-1 -phosphate uridyltransferase / glucosamine-1 -phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphatase, sialate transport protein, CMP kinase, CMP-sialate synthase, D-arabinose-5-phosphate isomerase, KDO-8P synthase, KDO-8-phosphate phosphatase, CMP-KDO synthetase, galactose-1 -epimerase, galactokinase, glucokinase, galactose-1 -phosphate uridyltransferase, UDP-glucose-4-epimerase, glucose-1 -phosphate uridyltransferase, 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-1 -phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-1 -phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine-2-epimerase, UDP-N-acetylglucosamine-2-epimerase, N-acetylmannosamine-6-phosphate-2-epimerase, UDP-GlcNAc 2-epimerase / kinase, glucosamine-6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1 -phosphate uridyltransferase, glucosamine-1 -phosphate acetyltransferase, bifunctional N-acetylglucosamine-1 -phosphate uridyltransferase / glucosamine-1 -phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N-acylneuraminate-9-phosphatase, sialate transport protein, CMP kinase, CMP-sialate synthase, D-arabinose-5-phosphate isomerase, KDO-8P synthase, KDO-8-phosphate phosphatase, CMP-KDO synthetase, galactose-1 -epimerase, galactokinase, glucokinase, galactose-1 -phosphate uridyltransferase, UDP-glucose-4-epimerase, glucose-1 -phosphate uridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine-4-epimerase, N-acetylgalactosamine kinase, and UDP-N-acetylgalactosamine pyrophosphorylase.6-dehydratase, GDP-L-fucose synthase, fucose permease, fucose kinase, fucose-1- phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate amidotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N- acetylglucosamine-2-epimerase, UDP-N-acetylglucosamine-2-epimerase, N- acetylmannosamine-6-phosphate-2-epimerase, UDP-GlcNAc 2-epimerase / kinase, glucosamine-6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, sialic acid synthase, N- acetylneuraminate lyase, N-acylneuraminate-9-phosphate synthase, N- acylneuraminate-9-phosphatase, sialate transport protein, CMP kinase, CMP- sialate synthase, D-arabinose-5-phosphate isomerase, KDO-8P synthase, KDO-8- phosphate phosphatase, CMP-KDO synthetase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose-4- epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, UDP-N- acetylglucosamine-4-epimerase, N-acetylgalactosamine kinase, and UDP-N- acetylgalactosamine pyrophosphorylase.

[0316] 13. The cell according to any one of the preceding embodiments, wherein the cell is capable of producing, preferably producing, N-acetylmannosamine (ManNAc), a sialic acid residue and / or N-acetylglucosamine (GlcNAc), wherein the sialic acid residue is selected from the list comprising Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymannooctulosonic acid (KDO), preferably the cell is genetically engineered for producing ManNAc, a sialic acid residue and / or GlcNAc.

[0317] 14. The cell according to any one of the preceding embodiments, wherein the negatively charged oligosaccharide is a sialylated oligosaccharide having at least one sialic acid residue selected from the list consisting of, consisting of or consisting essentially of: Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulosonic acid (KDO),

[0318] Preferably, the negatively charged oligosaccharide is an oligosaccharide selected from the list consisting of, consisting of or consisting essentially of: a negatively charged, preferably sialylated, milk oligosaccharide, preferably a negatively charged, more preferably sialylated, mammalian milk oligosaccharide (MMO), more preferably a negatively charged, more preferably sialylated, human milk oligosaccharide (HMO); an O-antigen; an oligosaccharide repeat unit present in a capsular polysaccharide; a peptidoglycan; an amino sugar; a Lewis-type antigen oligosaccharide; a negatively charged, preferably sialylated, animal oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans; a negatively charged, preferably sialylated, plant oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans; a sialylated oligosaccharide; a negatively charged, preferably sialylated, N-acetyl lactosamine containing oligosaccharide and a negatively charged, preferably sialylated, lacto-N-biose containing oligosaccharide;

[0319] More preferably, the negatively charged oligosaccharide is selected from the list consisting of, consisting of, or consisting essentially of: 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), 3'-sialyllactose amine, 3,6-disialyllactose, 8,3-disialyllactose, sialylated lacto-N-triose, sialyllacto-N-tetraose a (LSTa), KDOa-2,3Galb-1,3GlcNAcb-1,3Galb-1,4Glc, 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, fucodiisialyllacto-N-hexaose, disialylmonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyllacto-N-fuclohexaose II, disialyllacto-N-fuclopentaose II, monofucosyl disialyllacto-N-tetraose, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal, Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'-KDO-lacto-N-disaccharide, 3'-KDO-6' sialyllactose, 3'KDO-8-sialyllactose, KDO-2,3Galb-1,3GalNacb-1,3Galb-1,4Gal, KDOa-2,3Galb-1,4GlcNacb-1,3Galb-1,4Glc, 3'-KDO-3-fucosyllactose, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal, 3'-sialyl-2'-fucosyllactose, 6'-sialyl-2'-fucosyllactose, 6'-sialyl-3-fucosyllactose, Neu5Ac-a2,6-(Neu5Ac-a2,3-)Gal-b1,4-Glc, 3'-sialyl-3-fucosyllactosamine, Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc, 6'-sialyllacto-N-disaccharide, 3'-sialyllacto-N-disaccharide, Neu5Ac-a2,6-(GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,3-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, 6'-KDO-lactose, 6'-KDO-lactosamine, 6'-KDO-lactose-N-disaccharide, KDO-lactose-N-trisaccharide, KDO-lactose-N-tetrasaccharide, KDO-lactose-N-tetrasaccharide, and combinations thereof.

[0320] 15. The cell according to any one of the preceding embodiments, wherein the cell is capable of producing, preferably producing, the one or more precursors, preferably the precursors are lactose.

[0321] 16. The cell according to embodiment 15, wherein the cell is capable of producing, preferably producing, at least one of the one or more precursors, preferably the cell is capable of producing, preferably producing, all of the one or more precursors.

[0322] 17. The cell according to any one of embodiments 15 or 16, wherein the cell is genetically engineered to produce at least one of the one or more precursors, preferably the cell is genetically engineered to produce all of the one or more precursors.

[0323] 18. The cell according to any one of embodiments 15 to 17, wherein the cell is capable of producing, preferably producing, lactose.

[0324] 19. The cell according to any one of embodiments 15 to 18, wherein at least one of the one or more precursors is internalized into the cell by one or more membrane transporters.

[0325] 20. The cell according to any one of the preceding embodiments, wherein the cell is capable of producing and / or produces phosphoenolpyruvate (PEP).

[0326] 21. The cell according to any one of the preceding embodiments, wherein the cell is modified to enhance the synthesis and / or supply of phosphoenolpyruvate (PEP) compared to an unmodified progenitor cell.

[0327] 22. The cell according to any one of the preceding embodiments, wherein the cell is further modified to reduce the degradation of acetyl-CoA and / or its main precursor, pyruvate.

[0328] 23. The cell according to any one of the preceding embodiments, wherein the cell comprises a modification for reducing acetate production compared to an unmodified progenitor cell.

[0329] 24. The cell according to any one of the preceding embodiments, wherein the cell has, expresses and / or overexpresses at least one gene selected from the list comprising, consisting or consisting essentially of genes encoding a disulfide isomerase, a thiol oxidase or a chaperone.

[0330] 25. The cell according to any one of the preceding embodiments, wherein the cell comprises an at least partially inactivated catabolic pathway of a selected monosaccharide, disaccharide or oligosaccharide involved in and / or required for the synthesis of the negatively charged, preferably sialylated oligosaccharide.

[0331] 26. The cell according to any one of the preceding embodiments, wherein the cell has, expresses and / or overexpresses a membrane transporter.

[0332] 27. The cell according to any one of the preceding embodiments, wherein one or more genes involved in one or more reductive pathways in the cell are attenuated or knocked out.

[0333] 28. The cell according to embodiment 27, wherein the one or more genes involved in one or more reduction pathways are attenuated in function by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list comprising, consisting or consisting essentially of a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcriptional terminator sequence.

[0334] 29. The cell according to any one of the preceding embodiments, wherein the cell is a bacterial, fungal, yeast, plant cell, animal cell or protozoan cell,

[0335] - preferably, the bacteria belong to a phylum selected from the list comprising, consisting or consisting essentially of the following: Proteobacteria, Firmicutes, Cyanobacteria, Deinococcus- Thermus and Actinobacteria; more preferably, the bacteria belong to a family selected from the list comprising, consisting or consisting essentially of the following: Enterobacteriaceae, Bacillaceae, Lactobacillaceae, Corynebacteriaceae and Vibrionaceae; even more preferably, the bacteria are selected from the list comprising, consisting or consisting essentially of the following: an Escherichia coli strain, a Bacillus subtilis strain and a Vibrio strain; even more preferably, the Escherichia coli strain is a K-12 strain, most preferably, the Escherichia coli K-12 strain is Escherichia coli MG1655,

[0336] - preferably, the fungi belong to a genus selected from the list comprising, consisting or consisting essentially of the following: Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus,

[0337] - preferably, the yeast belongs to a genus selected from the list comprising, consisting or consisting essentially of the following: Saccharomyces, Zygosaccharomyces, Pichia, Komagataella, Hansenula, Yarrowia, Starmerella, Kluyveromyces, Debaryomyces, Candida, Schizosaccharomyces, Schwanniomyces and Torulopsis; more preferably, the yeast is selected from the list comprising, consisting or consisting essentially of the following: Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipitis, Candida boidinii, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Torulopsis debaryana, Yarrowia lipolytica, Zygosaccharomyces rouxii and Zygosaccharomyces bailii,

[0338] - preferably, the plant cell is an algal cell or is derived from a tobacco, alfalfa, rice, tomato, cotton, rapeseed, soybean, maize or corn plant,

[0339] - Preferably, said animal cell is derived from an insect, an amphibian, a reptile, an invertebrate, a fish, a bird or a mammal cell other than a human embryonic stem cell, more preferably said mammal cell is selected from the list comprising, consisting or consisting essentially of: an epithelial cell, an embryonic kidney cell, a fibroblast, a COS cell, a Chinese hamster ovary (CHO) cell, a murine myeloma cell, a NIH-3T3 cell, a mammary cell derived from a mammal induced pluripotent stem cell, more preferably said mammal induced pluripotent stem cell is a human induced pluripotent stem cell, a postpartum mammary epithelial cell, a polarized mammary cell, more preferably said polarized mammary cell is selected from the list comprising, consisting or consisting essentially of: a live primary mammary epithelial cell, a live mammary myoepithelial cell, a live mammary progenitor cell, a live immortalized mammary epithelial cell, a live immortalized mammary myoepithelial cell, a live immortalized mammary progenitor cell, a non-mammary adult stem cell or a derivative thereof, more preferably said insect cell is derived from Spodoptera frugiperda, Bombyx mori, Mamestra brassicae, Trichoplusia ni or Drosophila melanogaster,

[0340] - Preferably, said protozoan cell is a Leishmania tarentolae cell.

[0341] 30. The cell according to any one of the preceding embodiments, wherein said cell is selected from the list consisting of a prokaryotic cell and a eukaryotic cell, preferably selected from the list consisting of a yeast cell, a bacterial cell, an archaeal cell, an algal cell and a fungal cell.

[0342] 31. The cell according to any one of the preceding embodiments, wherein said cell is an E. coli or a yeast with a lactose permease positive phenotype, preferably wherein said lactose permease is encoded by the gene LacY or LAC12, respectively.

[0343] 32. The cell according to any one of the preceding embodiments, wherein said cell is a live Gram-negative bacterium comprising reduced or eliminated synthesis of poly-N-acetylglucosamine (PNAG), enterobacterial common antigen (ECA), cellulose, colanic acid, core oligosaccharide, osmoregulated periplasmic glucan (OPG), glycerol glucoside, glycans and / or trehalose compared to an unmodified progenitor cell.

[0344] 33. The cell according to any one of the preceding embodiments, wherein said cell produces 30 g / L or more of said negatively charged, preferably sialylated oligosaccharide in whole broth and / or supernatant, and / or wherein the purity of said negatively charged, preferably sialylated oligosaccharide in whole broth and / or supernatant is at least 80%, said purity being measured based on the total amount of said negatively charged, preferably sialylated oligosaccharide and its precursors produced by said cell in whole broth and / or supernatant, respectively.

[0345] 34. The cell according to any one of the preceding embodiments, wherein the cell is stably cultured in a culture or incubation medium.

[0346] 35. The cell according to any one of the preceding embodiments, wherein the cell is capable of producing and / or produces a mixture of two or more oligosaccharides, wherein at least one of the two or more oligosaccharides is the negatively charged, preferably sialylated, oligosaccharide.

[0347] 36. The cell according to any one of the preceding embodiments, wherein the cell is capable of producing and / or produces a mixture of one or more disaccharides and one or more oligosaccharides, wherein at least one of the one or more oligosaccharides is the negatively charged, preferably sialylated, oligosaccharide.

[0348] 37. The cell according to any one of the preceding embodiments, wherein the cell is capable of producing and / or produces a mixture comprising the negatively charged, preferably sialylated, oligosaccharide and at least one uncharged (neutral) oligosaccharide.

[0349] 38. The cell according to any one of the preceding embodiments, wherein the cell is capable of producing and / or produces a mixture comprising 1) the negatively charged, preferably sialylated, oligosaccharide, and 2) a disaccharide and / or an uncharged (neutral) oligosaccharide.

[0350] 39. A method of producing a negatively charged, preferably sialylated, oligosaccharide, the method comprising:

[0351] i. culturing and / or incubating a cell according to any one of the preceding embodiments in a culture and / or incubation medium under conditions that allow for the production of the negatively charged, preferably sialylated, oligosaccharide,

[0352] ii. optionally, subsequently isolating and / or purifying the negatively charged, preferably sialylated, oligosaccharide from the culture and / or incubation.

[0353] 40. The method of embodiment 39, wherein the negatively charged oligosaccharide is a sialylated oligosaccharide having at least one sialic acid residue selected from the list consisting of, consisting of or consisting essentially of: Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymanno-octulosonic acid (KDO),

[0354] Preferably, the negatively charged oligosaccharide is an oligosaccharide selected from the list consisting of, consisting of or consisting essentially of: a negatively charged, preferably sialylated, milk oligosaccharide, preferably a negatively charged, more preferably sialylated, mammalian milk oligosaccharide (MMO), more preferably a negatively charged, more preferably sialylated, human milk oligosaccharide (HMO); an O-antigen; an oligosaccharide repeat unit present in a capsular polysaccharide; a peptidoglycan; an amino sugar; a Lewis-type antigen oligosaccharide; a negatively charged, preferably sialylated, animal oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans; a negatively charged, preferably sialylated, plant oligosaccharide, preferably selected from the list consisting of N-glycans and O-glycans; a sialylated oligosaccharide; a negatively charged, preferably sialylated, N-acetyl lactosamine containing oligosaccharide and a negatively charged, preferably sialylated, lactose-N-bioside containing oligosaccharide;

[0355] More preferably, the negatively charged oligosaccharide is selected from the list consisting of, consisting of, or consisting essentially of: 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), 3'-sialyllactose amine, 3,6-disialyllactose, 8,3-disialyllactose, sialylated lacto-N-triose, sialyllacto-N-tetraose a (LSTa), KDOa-2,3Galb-1,3GlcNAcb-1,3Galb-1,4Glc, 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, fucodiisialyllacto-N-hexaose, disialylmonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N-octaose (sialyl Lea), sialyllacto-N-fuclohexaose II, disialyllacto-N-fuclopentaose II, monofucosyl disialyllacto-N-tetraose, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal, Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'-KDO-lacto-N-disaccharide, 3'-KDO-6' sialyllactose, 3'KDO-8-sialyllactose, KDO-2,3Galb-1,3GalNacb-1,3Galb-1,4Gal, KDOa-2,3Galb-1,4GlcNacb-1,3Galb-1,4Glc, 3'-KDO-3-fucosyllactose, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal, 3'-sialyl-2'-fucosyllactose, 6'-sialyl-2'-fucosyllactose, 6'-sialyl-3-fucosyllactose, Neu5Ac-a2,6-(Neu5Ac-a2,3-)Gal-b1,4-Glc, 3'-sialyl-3-fucosyllactosamine, Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc, 6'-sialyllacto-N-disaccharide, 3'-sialyllacto-N-disaccharide, Neu5Ac-a2,6-(GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,3-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, 6’-KDO-lactose, 6’-KDO-lactosamine, 6’-KDO-lactose-N-disaccharide, KDO-lactose-N-trisaccharide, KDO-lactose-N-tetrasaccharide, KDO-lactose-N-tetrasaccharide, and combinations thereof.

[0356] 41. The method according to any one of embodiments 39 or 40, wherein the culture or incubation medium comprises one or more precursors for the production of the negatively charged, preferably sialylated oligosaccharides.

[0357] 42. The method according to any one of embodiments 39 to 41, wherein the culture or incubation medium contains:

[0358] - at least one carbon source selected from the list consisting of, consisting of or consisting essentially of monosaccharides; disaccharides; oligosaccharides; polysaccharides; polyols; glycerol; complex media including molasses, corn steep liquor, peptone, tryptone or yeast extract; glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose, malt- oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn steep liquor, high fructose syrup, acetate, citrate, lactate and pyruvate, and / or

[0359] - at least one compound selected from the list consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid, CMP-sialic acid, CMP-Neu5Ac and CMP-KDO.

[0360] 43. The method according to any one of embodiments 39 to 42, wherein the culture or incubation medium:

[0361] - is a chemically defined medium, and / or

[0362] - is a basal salt medium comprising sulfate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt and / or selenium salts.

[0363] 44. The method according to any one of embodiments 39 to 43, comprising at least one of the following steps:

[0364] i) using a culture or incubation medium comprising at least one precursor and / or acceptor;

[0365] ii) feeding at least one precursor and / or acceptor to the culture or incubation medium in a reactor or incubator, wherein the total volume of the reactor or incubator ranges from 250 mL to 10,000 m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture or incubation medium prior to the feeding of the precursor and / or acceptor feed;

[0366] iii) adding at least one precursor and / or acceptor feed to the culture or incubation medium in a reactor or incubator, wherein the total volume of the reactor or incubator ranges from 250 mL to 10,000 m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times, the volume of the culture or incubation medium prior to the addition of the precursor and / or acceptor feed, and wherein preferably the pH of the precursor and / or acceptor feed is set between 2.0 and 10.0, and wherein preferably the temperature of the precursor and / or acceptor feed is maintained between 20 °C and 80 °C.

[0367] iv) adding at least one precursor and / or acceptor feed to the culture or incubation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days, by a precursor and / or acceptor feed solution;

[0368] v) adding at least one precursor and / or acceptor feed to the culture or incubation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days, by a precursor and / or acceptor feed solution, and wherein the concentration of the precursor and / or acceptor feed solution is 50 g / L, 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 the precursor and / or acceptor feed solution is set between 2.0 and 10.0, and wherein preferably the temperature of the precursor and / or acceptor feed solution is maintained between 20 °C and 80 °C;

[0369] The method is such that the concentration of the negatively charged, preferably sialylated, oligosaccharide in the final volume of the culture or incubation is at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L,

[0370] wherein preferably the precursor is selected from the list comprising sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose, and wherein preferably the acceptor is lactose.

[0371] 45. The method according to any one of embodiments 39 to 44, comprising at least one of the following steps:

[0372] i) using a cultivation or incubation medium comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of precursors per liter of initial reactor or incubator volume, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m3(cubic meters);

[0373] ii) using a cultivation or incubation medium comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of acceptors per liter of initial reactor or incubator volume, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m3(cubic meters);

[0374] iii) adding to the cultivation or incubation medium in the reactor or incubator a precursor feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of precursors per liter of initial reactor or incubator volume, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m3(cubic meters), preferably in a continuous manner, and preferably such that the final volume of the cultivation or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the cultivation or incubation medium before the addition of the precursor feed;

[0375] iv) adding to the cultivation or incubation medium in the reactor or incubator a recipient feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of acceptors per liter of initial reactor or incubator volume, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m3(cubic meters), preferably in a continuous manner, and preferably such that the final volume of the cultivation or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the cultivation or incubation medium before the addition of the recipient feed;

[0376] v) adding to the culture or incubation medium a precursor feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of precursor per liter of initial reactor or incubator volume, wherein the reactor or incubator total volume ranges from 250 mL to 10,000 m3(cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture or incubation medium prior to the addition of the precursor feed, and wherein preferably the pH of the precursor feed is set between 2.0 and 10.0, and wherein preferably the temperature of the precursor feed is maintained between 20 °C and 80 °C;

[0377] vi) adding to the culture or incubation medium a recipient feed comprising at least 50 g, more preferably at least 75 g, more preferably at least 100 g, more preferably at least 120 g, more preferably at least 150 g of recipient per liter of initial reactor or incubator volume, wherein the reactor or incubator total volume ranges from 250 mL to 10,000 m3(cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture or incubation medium prior to the addition of the recipient feed, and wherein preferably the pH of the recipient feed is set between 2.0 and 10.0, and wherein preferably the temperature of the recipient feed is maintained between 20 °C and 80 °C;

[0378] vii) adding precursor and / or recipient feed to the culture or incubation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days, by a precursor and / or recipient feed solution;

[0379] viii) adding a precursor feed to the cultivation or incubation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days by a precursor feed solution, and wherein the concentration of the precursor feed solution is 50 g / L, 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 the precursor feed solution is set between 2.0 and 10.0, and wherein preferably the temperature of the precursor feed solution is kept between 20 °C and 80 °C;

[0380] ix) adding a recipient feed to the cultivation or incubation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days by a recipient feed solution, and wherein the concentration of the recipient feed solution is 50 g / L, 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 the recipient feed solution is set between 2.0 and 10.0, and preferably the temperature of the recipient feed solution is kept between 20 °C and 80 °C;

[0381] The method is such that the concentration of the negatively charged, preferably sialylated, oligosaccharides in the final volume of the cultivation or incubation is at least 50 g / L, preferably at least 75 g / L, more preferably at least 90 g / L, more preferably at least 100 g / L, more preferably at least 125 g / L, more preferably at least 150 g / L, more preferably at least 175 g / L, more preferably at least 200 g / L,

[0382] wherein preferably the precursor is selected from the list comprising sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose, and wherein preferably the recipient is lactose.

[0383] 46. The method according to any one of embodiments 39 to 45, wherein the cells produce 30 g / L or more of the negatively charged, preferably sialylated oligosaccharide throughout the broth and / or supernatant, and / or wherein the purity of the negatively charged, preferably sialylated oligosaccharide throughout the broth and / or supernatant is at least 80%, the purity being measured based on the total amount of the negatively charged, preferably sialylated oligosaccharide and its precursors produced by the cells throughout the broth and / or supernatant, respectively.

[0384] 47. The method according to any one of embodiments 39 to 46, wherein the cells produce a mixture of two or more oligosaccharides, wherein at least one of the two or more oligosaccharides is the negatively charged, preferably sialylated oligosaccharide.

[0385] 48. The method according to any one of embodiments 39 to 47, wherein the cells produce a mixture of one or more disaccharides and one or more oligosaccharides, wherein at least one of the one or more oligosaccharides is the negatively charged, preferably sialylated oligosaccharide.

[0386] 49. The method according to any one of embodiments 39 to 48, wherein the cells produce a mixture comprising the negatively charged, preferably sialylated oligosaccharide and at least one uncharged (neutral) oligosaccharide.

[0387] 50. The method according to any one of embodiments 39 to 49, wherein the cells produce a mixture comprising: 1) the negatively charged, preferably sialylated oligosaccharide, and 2) a disaccharide and / or an uncharged (neutral) oligosaccharide.

[0388] 51. The method according to any one of embodiments 39 to 50, wherein the negatively charged, preferably sialylated oligosaccharide is recovered, more preferably purified, from the culture or incubation medium and / or the cells.

[0389] 52. The method according to any one of embodiments 39 to 51, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated charcoal or carbon treatment, non-ionic surfactant treatment, enzymatic hydrolysis, tangential flow high performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and / or gel filtration, ligand exchange chromatography, electrodialysis.

[0390] 53. The method according to any one of embodiments 39 to 52, wherein the purification comprises at least one of the following steps: use of activated charcoal or carbon, use of charcoal, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange, temperature adjustment, pH adjustment, pH adjustment by basic or acidic solutions, use of alcohols, use of aqueous-alcoholic mixtures, crystallization, evaporation, precipitation, drying, spray drying, freeze drying, spray freeze drying, freeze spray drying, belt drying, belt drying, vacuum belt drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying or vacuum roller drying.

[0391] 54. Use of the cell according to any one of embodiments 1 to 38 for the production of a negatively charged, preferably sialylated oligosaccharide.

[0392] 55. Use of the method according to any one of embodiments 39 to 53 for the production of a negatively charged, preferably sialylated oligosaccharide.

[0393] The application will be described in further detail in the examples. The following examples serve to further illustrate and clarify the application, without limiting it.

[0394] Example

[0395] Example 1. Materials and methods

[0396] A. Escherichia coli

[0397] Culture medium and cultivation

[0398] Luria Broth (LB) medium consists of 1% tryptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). The minimal medium used for cultivation of experiments in 96-well plates or shake flasks contains 2.00 g / L NH4CI, 5.00 g / L (NH4)2S04, 2.993 g / L KH2P04, 7.315 g / L K2HP04, 8.372 g / L MOPS, 0.5 g / L NaCI, 0.5 g / L MgS04·7H20, 30 g / L sucrose or 30 g / L glycerol, 1 ml / L vitamin solution, 100 μΐ / L molybdate solution and 1 mL / L selenium solution. As precursors and / or acceptors for sugar synthesis, compounds such as galactose, glucose, fructose, fucose, lactose, LacNAc, LNB, co-factors and sialic acid can be added to the medium. The minimal medium is set to a pH of 7 with 1 M KOH. The vitamin solution consists of 3.6 g / L FeCI2·4H20 (ferrous chloride tetrahydrate), 5.0 g / L CaCI2·2H20 (calcium chloride dihydrate), 1.3 g / L MnCI2·2H20 (manganese chloride dihydrate), 0.38 g / L CuCI2·2H20 (copper chloride dihydrate), 0.5 g / L CoCI2·6H20 (cobalt dichloride hexahydrate), 0.94 g / L ZnCI2, 0.0311 g / L H3B04, 0.4 g / L Na2EDTA·2H20 (ethylenediaminetetraacetic acid disodium dihydrate) and 1.01 g / L thiamine hydrochloride. The molybdate solution contains 0.967 g / L NaMo04·2H20 (sodium molybdate dihydrate). The selenium solution contains 42 g / L Se02. The fermentation minimal medium contains 6.75 g / L NH4CI, 1.25 g / L (NH4)2S04, 2.93 g / L KH2P04 and 7.31 g / L KH2P04, 0.5 g / L NaCI, 0.5 g / L MgS04·7H20 (magnesium sulfate heptahydrate), 30 g / L sucrose or 30 g / L glycerol, 1 mL / L vitamin solution, 100 μΐ / L molybdate solution and 1 mL / L selenium solution, which are composed as described above. According to the examples, 0.30 g / L sialic acid, 0.30 g / L GlcNAc, 20 g / L lactose, 20 g / L LacNAc, 20 g / L LNB, 20 g / L LN3, 20 g / L LNT and / or 20 g / L LNnT are additionally added to the medium.Complex medium was sterilized by autoclaving (121 °C, 21 min), minimal medium was sterilized by filtration (0.22 pm Sartorius). If necessary, the selectivity of the medium can be increased by the addition of antibiotics: e.g. chloramphenicol (20 mg / L), carbenicillin (100 mg / L), spectinomycin (40 mg / L) and / or kanamycin (50 mg / L).

[0399] Pre-cultures for 96-well microtiter plate experiments started in cryotubes in 150 pL LB and were incubated at 37 °C in an orbital shaker at 800 rpm overnight. This culture was inoculated into a 96-well square microtiter plate, where 400 pL minimal medium was diluted 400-fold. These final 96-well culture plates were then incubated at 37 °C in an orbital shaker at 800 rpm for 72 h, the time can be shorter or longer. To measure the sugar concentration at the end of the cultivation experiment, the culture broth was boiled for 15 min at 60 °C and then the cells were harvested by centrifugation, a whole broth sample (= average of intracellular and extracellular sugar concentration) was taken from each well.

[0400] In addition, the cultures were diluted to measure the optical density at 600 nm. The cellular performance index or CPI was determined by dividing the oligosaccharide concentration by the biomass (expressed in relative percentage compared to the reference strain). The biomass was empirically determined to be about 1 / 3 of the optical density measured at 600 nm.

[0401] Pre-cultures for bioreactors started with a whole 1 mL cryotube of a strain, inoculated into 250 mL or 500 mL minimal medium in 1 L or 2.5 L shake flasks and incubated at 37 °C in an orbital shaker at 200 rpm for 24 h. Then inoculated into a 5 L bioreactor (250 mL inoculum in 2 L batch medium); the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). The cultivation conditions were set to 37 °C, maximum agitation; the pressure gas flow rate depends on the strain and the bioreactor. The pH was controlled at 6.8 using 0.5 M H2SO4 and 20% NH4OH. The off-gas was cooled. When foam appeared during the fermentation, a 10% silicone antifoam solution was added.

[0402] Plasmid

[0403] The pKD46 (Red helper plasmid, ampicillin resistance), pKD3 (contains the FRT-flanked chloramphenicol resistance (cat) gene), pKD4 (contains the FRT-flanked kanamycin resistance (kan) gene) and pCP20 (expresses FLP recombinase activity) plasmids were all obtained from Prof. R. Cunin (Vrije Universiteit Brussel, Belgium, 2007). The pET28b(+) vector was obtained from Millipore and was adapted for Golden Gate cloning. Plasmids were maintained in host E. coli DH5alpha (F - , phi80dlacZAM15, A(lacZYA-argF)U169, deoR, recAl, endAl, hsdR17(rk - , mk + ), phoA, supE44, lambda - , thi-1, gyrA96, relAl).

[0404] Strains and mutations

[0405] E. coli K12 MG1655 [l-, F-, rph-1] was obtained from the E. coli Genetic Stock Center, USA in March 2007, CGSC strain number: 7740. Gene disruptions, gene introductions and gene replacements were all performed using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). All constitutive promoter, UTR and terminator sequences were derived 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 synthesized by Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and adjusted for codon usage using the tools provided by the suppliers. All strains were kept in cryotubes at -80°C (LB culture mixed 1:1 with 70% glycerol overnight).

[0406] In embodiments of GDP-fucose production, the mutant strain is derived from E. coli K12 MG1655, comprising a knockout of the wcaJ gene of E. coli and a genomic knockin of a constitutive transcriptional unit comprising a sucrose transporter, such as CscB from E. coli W (UniProt ID E0IXR1); a fructokinase, such as Frk derived from Zymomonas mobilis (UniProt ID Q03417); and a sucrose phosphorphorylase, such as BaSP derived from Bifidobacterium adolescentis (UniProt ID A0ZZH6). Production of GDP-fucose in the mutant E. coli strain can be further optimized by genomic knockout of one or more of the E. coli genes including glgC, agp, pfkA, pfkB, pgi, arcA, iclR, pgi, and Ion, as described in WO2016075243 and WO2012007481. Production of GDP-fucose can additionally be optimized including a genomic knockin of a constitutive transcriptional unit of a mannose-6-phosphate isomerase (e.g., manA from E. coli (UniProt ID P00946)), a phosphomannomutase (e.g., manB from E. coli (UniProt ID P24175)), a mannose-1-phosphate guanylyltransferase (e.g., manC from E. coli (UniProt ID P24174)), a GDP-mannose 4,6-lyase (e.g., gmd from E. coli (UniProt ID P0AC88)), and a GDP-L-fucose synthase (e.g., fcl from E. coli (UniProt ID P32055)). Production of GDP-fucose can also be achieved by a genomic knockout of the E. coli fucK and fucI genes, and a genomic knockin of a constitutive transcriptional unit comprising a fucose permease (e.g., fucP from E. coli (UniProt ID P11551)) and a bifunctional enzyme with fucose kinase / fucose-1-phosphate guanylyltransferase activity (e.g., fkp from Bacteroides fragilis (UniProt ID SUV40286.1)). All mutant strains can be additionally modified by a genomic knockout of the E. coli LacZ, LacY, and LacA genes, and a genomic knockin of a constitutive transcriptional unit of a lactose permease (e.g., E. coli LacY (UniProt ID P02920)).For the production of fucosylated sugars, the mutant GDP-fucose producing strain is additionally modified with an expression plasmid comprising a constitutive transcriptional unit of a fucosyltransferase, for example, a constitutive transcriptional unit of the alpha-1,2-fucosyltransferase HpFutC from Helicobacter pylori (UniProt ID Q9X435) to produce 2’-fucosyllactose (2’FL), or a constitutive transcriptional unit of the alpha-1,3-fucosyltransferase HpFucT from Helicobacter pylori (UniProt ID O30511) to produce 3-fucosyllactose (3-FL), or a constitutive transcriptional unit of both the alpha-1,2-fucosyltransferase HpFutC from Helicobacter pylori (UniProt ID Q9X435) and the alpha-1,3-fucosyltransferase HpFucT from Helicobacter pylori (UniProt ID O30511) to produce difucosyllactose (DiFL).

[0407] In embodiments for the production of lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc), the mutant strain is derived from E. coli K12 MG1655 and is modified by knock-out of the E. coli lacZ, lacY, lacA and nagB genes, and genome knock-in of a constitutive transcriptional unit for a lactose permease (e.g., E. coli LacY (UniProt ID P02920)) and a galactoside β-1,3-N-acetylglucosaminyltransferase (e.g., lgtA from Neisseria meningitidis (UniProt ID Q9JXQ6)). In embodiments for the production of LN3-derived oligosaccharides such as lacto-N-tetraose (LNT, Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc), the mutant LN3-producing strain is further modified by genome knock-in or delivery of a constitutive transcriptional unit to the strain by an expression plasmid for a N-acetylglucosamine β-1,3-galactosyltransferase (e.g., wbgO from E. coli O55:H7 (Uniprot ID D3QY14)). In embodiments for the production of LN3-derived oligosaccharides such as lacto-N-neotetraose (LNnT, Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc), the mutant LN3-producing strain is further modified by genome knock-in or delivery of a constitutive transcriptional unit to the strain by an expression plasmid for a N-acetylglucosamine β-1,4-galactosyltransferase (e.g., LgtB from Neisseria meningitidis (Uniprot ID Q51116, sequence version 02, December 1, 2000)). In the mutant E. coli strain, production of LN3, LNT and / or LNnT can be further optimized by genome knock-out of E. coli genes including one or more of galT, ushA, ldhA and agp. The mutant LN3, LNT and / or LNnT-producing strain can also be optionally modified by genome knock-in of a constitutive transcriptional unit for L-glutamine-D-fructose-6-phosphate aminotransferase (e.g., E. coli glmS (UniProt ID P17169, sequence version 04, January 23, 2007)) or a mutant glmS*54 from E. coli with SEQ ID NO 09 (which differs from wild-type E. coli glmS with UniProt ID P17169 by A39T, R250C and G472S mutations as described in Deng et al. (Biochimie 88, 419-29 (2006)) to enhance production of UDP-GlcNAc.The mutant E. coli strain can also optionally be engineered by genome knock-in of a constitutive transcriptional unit containing UDP-glucose-4-epimerase (e.g., galE from E. coli (UniProt ID P09147), phosphoglucomutase (e.g., glmM from E. coli (UniProt ID P31120, sequence version 03, January 23, 2007)) and N-acetylglucosamine-1 -phosphate uridyltransferase / glucosamine-1 -phosphate acetyltransferase (e.g., glmU from E. coli (UniProt ID P0ACC7)). The mutant LN3, LNT and / or LNnT producing E. coli strain can also optionally be engineered by genome knock-in of a constitutive transcriptional unit containing sucrose transporter (e.g., CscB from E. coli W (UniProt ID E0IXR1), fructokinase (e.g., Frk from Zymomonas mobilis (UniProt ID Q03417)) and sucrose phosphorylase (e.g., BaSP from Bifidobacterium adolescentis (UniProt ID A0ZZH6)) to accommodate growth on sucrose.

[0408] In an embodiment of sialic acid production, the mutant strain is derived from E. coli K12 MG1655 comprising a genome knock-in of a constitutive transcriptional unit containing a hydrolytic UDP-N-acetylglucosamine 2-epimerase, e.g., NeuC from Campylobacter jejuni (UniProt ID Q93MP8) or any one of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 08; and an N- ac neuraminic acid synthase, e.g., NeuB from Neisseria meningitidis (UniProt ID E0NCD4).

[0409] Alternatively and / or additionally, sialic acid production can be obtained by genome knock-in of a constitutive transcriptional unit containing one or more copies of a glucosamine 6-phosphate N-acetyltransferase (e.g., GNA1 from Saccharomyces cerevisiae (UniProt ID P43577)), an N-acetylglucosamine 2-epimerase (e.g., AGE from Bacteroides ovatus (UniProt ID A7LVG6)) and an N-ac neuraminic acid synthase (e.g., NeuB from Neisseria meningitidis (UniProt ID E0NCD4)).

[0410] Alternatively and / or additionally, the production of sialic acid can be obtained by genome knock-in of a constitutive transcriptional unit comprising a phosphoglucosamine mutase (e.g., glmM from Escherichia coli (UniProt ID P31120, sequence version 03, January 23, 2007)), an N-acetylglucosamine-1 -phosphate uridyltransferase / glucosamine-1 -phosphate acetyltransferase (e.g., glmU from Escherichia coli (UniProt ID P0ACC7)), a hydrolytic UDP-N-acetylglucosamine 2-epimerase (e.g., NeuC from Campylobacter jejuni (UniProt ID Q93MP8)), or any one of SEQ ID NOs 01, 02, 03, 04, 05, 06, 07 or 08, and an N- acuneuraminic acid synthase (e.g., NeuB from Neisseria meningitidis (UniProt ID E0NCD4)).

[0411] Alternatively and / or additionally, the production of sialic acid can be obtained by genome knock-in of a constitutive transcriptional unit comprising a bifunctional UDP-GlcNAc 2-epimerase / N-acetylmannosamine kinase (e.g., from Mus musculus (strain C57BL / 6J) (UniProt ID Q91WG8)), an N-acylneuraminate-9-phosphate synthase (e.g., from Pseudomonas sp. UW4 (UniProt ID K9NPH9)) and an N-acylneuraminate-9-phosphatase (e.g., from Bacteroides thetaiotaomicron (UniProt ID Q8A712)).

[0412] The production of sialic acid can be further optimized in a mutant E. coli strain by genome knockout of E. coli genes including any one or more of nagA, nagB, nagC, nagD, nagE, nanA, nanE, nanK, manX, manY, and manZ, as described in WO18122225, and / or genome knockout of E. coli genes including any one or more of nanT, poxB, ldhA, adhE, aldB, pflA, pflC, ybiY, ackA, and / or pta, and genome knock-in of a constitutive transcriptional unit comprising one or more copies of L-glutamine-D-fructose-6-phosphate aminotransferase (e.g., E. coli glmS (UniProt ID P17169, sequence version 04, January 23, 2007) or mutant glmS*54 from E. coli with SEQ ID NO 09, which differs from wild-type E. coli glmS with UniProt ID P17169 by A39T, R250C, and G472S mutations, as described in Deng et al. (Biochimie 88, 419-29 (2006)); preferably a phosphatase, such as any of the E. coli genes including 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, ScDOG1 from Saccharomyces cerevisiae, or BsAraL from Bacillus subtilis, as described in WO18122225; and acetyl-CoA synthetase (e.g., acs from E. coli (UniProt ID P27550)).

[0413] For the production of sialylated oligosaccharides, the sialic ac...

Claims

1. A cell comprising a pathway for producing negatively charged, preferably sialylated, oligosaccharides, characterized in that, The cells are genetically engineered to have, express, and / or overexpress a hydrolytic UDP-N-acetyl-D-glucosamine-2-epiisomerase, wherein the hydrolytic UDP-N-acetyl-D-glucosamine-2-epiisomerase comprises an amino acid sequence containing a conserved motif having SEQ ID NO:12 and has hydrolytic UDP-N-acetyl-D-glucosamine-2-epiisomerase activity.

2. The cell of claim 1, wherein the hydrolytic UDP-N-acetyl-D-glucosamine-2-epiisomerase comprises, consists of, or is substantially composed of the following amino acid sequence: The amino acid sequence is at least 80% identical to any of the amino acid sequences shown in SEQ ID NO: 01, 02, 03, 04, 05, 06, 07 or 08 in a segment of at least 150 amino acid residues and / or at least 200 amino acid residues.

3. The cell according to any one of claims 1 or 2, wherein the hydrolytic UDP-N-acetyl-D-glucosamine-2-episomerase comprises an amino acid sequence, said amino acid sequence: - Contains IPR structural fields selected from a list including IPR003331, IPR020004, and IPR029767, as defined in InterPro 90.0 released on August 4, 2022. - Contains the PF02350 motif as defined by PFAM 32.0, released in September 2018. - Contains the cd03786 motif defined by CDD v3.17 released in September 2016. - Contains the panther structure field selected from a list including PTHR43174 and PTHR43174:SF3 as defined in PANTHER 17.0 released on February 23, 2022. - In a segment of at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 amino acid residues, it is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, or at least 99% identical to any amino acid sequence shown in SEQ ID NO: 01, 02, 03, 04, 05, 06, 07, or 08. - Identical to at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, or at least 99% of any full-length amino acid sequence shown in SEQ ID NO: 01, 02, 03, 04, 05, 06, 07, or 08, and / or - As shown in any one of SEQ ID NO: 01, 02, 03, 04, 05, 06, 07 or 08.

4. The cell according to any one of the preceding claims, wherein the hydrolytic UDP-N-acetyl-D-glucosamine-2-episomerase is an nnaA polypeptide.

5. The cell according to any one of the preceding claims, wherein the cell comprises a nucleic acid molecule, the nucleic acid molecule comprising a polynucleotide sequence encoding the hydrolytic UDP-N-acetyl-D-glucosamine-2-epiisomerase.

6. The cell according to claim 5, wherein the nucleic acid molecule: - Operablely linked to a control sequence recognized by the cell, and the nucleic acid molecule further i) integrates into the genome of the cell and / or ii) is presented to the cell on a vector, and / or - It is foreign to the cells in question.

7. The cell according to any one of the preceding claims, wherein the pathway for producing the negatively charged, preferably sialylated oligosaccharide is a sialylation pathway, and / or the cell is genetically engineered to include a sialylation pathway, and / or the cell includes a sialylation pathway, wherein the sialylation pathway has been genetically engineered.

8. The cell according to any one of the preceding claims, wherein the cell: - Further includes pathways selected from the list that include, consist of, or are substantially composed of the following pathways: fucosylation pathway, galactosylation pathway, N-acetylglucosylation pathway, N-acetylgalactosylation pathway, mannosylation pathway, and N-acetylmannosylation pathway. - Genetically engineered to include at least one pathway selected from the list of pathways that are composed of, consist of, or substantially consist of the following pathways: fucosylation pathway, galactosylation pathway, N-acetylglucosylation pathway, N-acetylgalactosylation pathway, mannosylation pathway, and N-acetylmannosylation pathway. - Contains at least one pathway selected from the list comprising, consisting of, or substantially consisting of the following pathways: fucosylation pathway, galactosylation pathway, N-acetylglucosylation pathway, N-acetylgalactosylation pathway, mannosylation pathway, and N-acetylmannosylation pathway, wherein at least one of said pathways has been genetically engineered. - Having, expressing, and / or overexpressing at least one glycosyltransferase selected from the list comprising, consisting of, or substantially consisting of the following glycosyltransferases: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosaminetransferase, N-acetylgalactosaminetransferase, N-acetylmannosaminetransferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosaminetransferase, N-hydroxyacetylneuraminidase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-azolosamine transaminase, UDP-N-acetylglucosamineenolpyruvate transferase, and fucosaminetransferase. - Capable of producing and / or producing one or more nucleotide-activated sugars, - Genetically engineered for the production of one or more of the aforementioned nucleotide activated sugars. - Contains pathways for synthesizing nucleotide activated sugars selected from the list including, composed of, or substantially composed of the following nucleotide activated sugars: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetymannosamine (UDP-ManNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), GDP-mannose (GDP-Man), GDP-fucose (GDP-Fuc), UDP-glucuronic acid, UDP-galacturonic acid, UDP-2-acetamido-2,6-dideoxy-L-arabinose-4-hexose, UDP-2-acetamido-2,6-dideoxy-L-xylose-4-hexose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L-mannose), dTDP-N-acetylmfucosamine, UDP-N-acetylmuramic acid (UDP-L-FucNAc or UDP-2-acetamido-2,6-dideoxy-L-galactose), UDP-N-acetamido-L-pneumoglycosamine (UDP-L-PneNAc or UDP-2-acetamido-2,6-dideoxy-L-tarose), UDP-N-acetylmuramic acid, UDP-N-acetamido-L-quinolamine (UDP-L-QuiNAc or UDP-2-acetamido-2,6-dideoxy-L-glucose), C MP-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-hydroxyacetylneuraminic acid (CMP-Neu5Gc), CMP-2-keto-3-deoxymannose-octylulose acid (CMP-KDO), GDP-rhamnose and UDP-xylose, - Possesses, expresses, and / or overexpresses one or more genes selected from the list including, composed of, or substantially composed of the following genes: mannose-6-phosphate isomerase, phosphogmannose mutase, mannose-1-phosphate guanylate transferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylate transferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine-2-epi-isomerase, UDP-N-acetylglucosamine-2-epi-isomerase, N-acetylglucosamine-6-phosphate-2-epi-isomerase, UDP-GlcNAc 2-Epoisomerase / Kinetic Kinase, Glucosamine-6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, Phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate uridineyltransferase, Glucosamine-1-phosphate acetyltransferase, Bifunctional N-acetylglucosamine-1-phosphate uridineyltransferase / Glucosamine-1-phosphate acetyltransferase, Sialic Acid Synthase, N-acetylneuraminic Acid Lyase, N-acylneuraminic Acid-9-phosphate Synthase, N-acylneuraminic Acid-9-phosphate Phosphatase, Sialic Acid Transporter, CMP Kinase CMP-sialate synthase, D-arabinose-5-phosphate isomerase, KDO-8P synthase, KDO-8-phosphate phosphatase, CMP-KDO synthase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridine transferase, UDP-glucose-4-epimerase, glucose-1-phosphate uridine transferase, phosphogluconomutase, UDP-N-acetylglucosamine-4-epimerase, N-acetylgalactosamine kinase, and UDP-N-acetylgalactosamine pyrophosphorylase. - Capable of producing and / or generating N-acetylmnosamine (ManNAc), sialic acid residues and / or N-acetylglucosamine (GlcNAc), wherein the sialic acid residues are selected from the list comprising, consisting of, or substantially consisting of: Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymannose-octylulose acid (KDO). - Genetically engineered to produce ManNAc, sialic acid residues, and / or GlcNAc. - Capable of generating, and / or generating, the negatively charged, preferably sialylated oligosaccharide from one or more precursors. - Capable of producing and / or producing the negatively charged, preferably sialylated, oligosaccharides from lactose. - Capable of generating and / or generating at least one or more precursors for generating the negatively charged, preferably sialylated, oligosaccharides. - Capable of generating and / or generating all precursors for the production of the negatively charged, preferably sialylated, oligosaccharides. - Genetically engineered precursors for producing at least one or more of the negatively charged, preferably sialylated, oligosaccharides. - Able to produce and / or produce lactose, - Genetically engineered precursors for producing all the negatively charged, preferably sialylated, oligosaccharides. - Capable of producing and / or generating phosphoenolpyruvate (PEP), - Compared to unmodified progenitor cells, cells modified to enhance the synthesis and / or supply of phosphoenolpyruvate (PEP) - Including modifications to reduce acetic acid production compared to unmodified progenitor cells. - Having, expressing, and / or overexpressing at least one gene selected from the list including, composed of, or substantially composed of the following genes: genes encoding disulfide isomerases, thiol oxidases, or molecular chaperones. - A catabolistic pathway comprising at least partially inactivated selected monosaccharides, disaccharides, or oligosaccharides, wherein the monosaccharides, disaccharides, or oligosaccharides are involved in and / or required for the synthesis of the negatively charged, preferably sialylated, oligosaccharides, and / or - Possesses, expresses, and / or overexpresses membrane transport proteins.

9. The cell of claim 8, wherein at least one of the one or more precursors is internalized into the cell via one or more membrane transport proteins.

10. The cell according to any one of the preceding claims, wherein the function of one or more genes involved in one or more reduction pathways in the cell is reduced or knocked out.

11. The cell of claim 10, wherein one or more genes involved in one or more reduction pathways are deactivated by inserting, deleting and / or modifying one or more nucleotides in one or more polynucleotide sequences selected from promoter sequences, ribosome binding sites, untranslated regions, coding sequences and transcription terminator sequences.

12. The cell according to any one of the preceding claims, wherein the negatively charged oligosaccharide is: - A sialylated oligosaccharide having at least one sialic acid residue selected from the list comprising, consisting of, or substantially consisting of the following substances: Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc; and 2-keto-3-deoxymannose-octylulose acid (KDO). - Oligosaccharides, selected from the list comprising, consisting of, or substantially consisting of: negatively charged lactoses, sialylated lactoses, negatively charged mammalian lactoses (MMOs), sialylated MMOs, negatively charged human lactoses (HMOs), sialylated HMOs, O-antigens, oligosaccharide repeating units present in capsular polysaccharides, peptidoglycans, amino sugars, Lewis-type antigen oligosaccharides, negatively charged animal oligosaccharides, sialylated animal oligosaccharides, and negatively charged animal oligosaccharides selected from the list consisting of N-glycans and O-glycans. Animal oligosaccharides, sialylated animal oligosaccharides selected from the list consisting of N-glycans and O-glycans, negatively charged plant oligosaccharides, sialylated plant oligosaccharides, negatively charged plant oligosaccharides selected from the list consisting of N-glycans and O-glycans, sialylated oligosaccharides, negatively charged oligosaccharides containing N-acetyllactosamine, sialylated oligosaccharides containing N-acetyllactosamine, negatively charged oligosaccharides containing lactose-N-biose, sialylated oligosaccharides containing lactose-N-biose, and / or - Selected from a list including the following substances: 3'-sialyl lactose (3'SL), 6'-sialyl lactose (6'SL), 3'-sialyl lactosamine, 3,6-disialyl lactose, 8,3-disialyl lactose, sialylated lactose-N-trisaccharide, sialyl lactose-N-tetrasaccharide a (LSTa), KDOα-2,3Galβ-1,3GlcNAcβ-1,3Galβ-1,4Glc, sialyl lactose-N-tetrasaccharide b (LSTb), sialyl lactose-N-tetrasaccharide c (LSTc), sialyl lactose-N-tetrasaccharide d (LSTd), diasialactose-N-tetrasaccharide, diasialactose-N-neotetrasaccharide, monosialactose-N-hexasaccharide, diasialactose-N-hexasaccharide I, diasialactose-N-hexasaccharide II, monosialactose-N-neohexasaccharide I, monosialactose-N-neohexasaccharide II, diasialactose-N-neohexasaccharide, 3'-sialic acid-3-fucosylated lactose, fucoidanilactose-N-hexasaccharide, diasialactofucosylated lactose-N-neohexasaccharide, monofucosylated monosialactose-N-octasaccharide (sialic acid Lea), sialactose-N-fucohexasaccharide II Disialyllactose-N-fucopentose II, monofucosyldisialyllactose-N-tetrasaccharide, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal, Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'-KDO-lactose-N-disaccharide, 3'-KDO-6'-sialic acid lactose, 3'KDO-8-sialic acid lactose, KDO-2,3Galβ-1,3GalNacβ-1,3Gal α-1,4Gal, β-1,4Gal, KDO, α-2,3Gal, β-1,4Glc, Nac, β-1,3Gal, β-1,4Glc, 3'-KDO-3-fucosyllactose, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,3-Glc, Nac-b1,3-Gal, 3'-sialic acid-2'-fucosyllactose, 6'-sialic acid-2'-fucosyllactose, 6'-sialic acid-3-fucosyllactose, Neu5Ac-a2,6-(Neu5Ac-a2,3-)Gal- b1,4-Glc, 3'-sialyl-3-fucosylated lactosamine, Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc, 6'-sialyl-lactose-N-disaccharide, 3'-sialyl-lactose-N-disaccharide, Neu5Ac-a2,6-(GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc、Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3-)Gal-b1,4-Glc、Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc、Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc、Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc、Neu5Ac-a2,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc、Neu5Ac-a2,6-(Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc、Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc、Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc-b1,3-Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc、Neu5Ac-a2,6-(Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc、Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc、Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3)-Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,3-GlcNAc-b1,3)-Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2, 3-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3)-Gal- b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5 Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-G lcNAc-b1,3)-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4 -GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3)-Ga l-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, 6'-KDO-lactose, 6'-KDO-lactosamine, 6'-KDO-lactose-N-disaccharide, KDO-lactose-N-trisaccharide, KDO-lactose-N-tetrasaccharide, KDO-lactose-N-tetrasaccharide, and combinations thereof.

13. The cell according to any one of the preceding claims, wherein the cell is selected from a list consisting of prokaryotic cells and eukaryotic cells.

14. The cell according to any one of the preceding claims, wherein the cell produces 30 g / L or more of the negatively charged, preferably sialylated oligosaccharides in the whole broth and / or supernatant, and / or wherein the purity of the negatively charged, preferably sialylated oligosaccharides in the whole broth and / or supernatant is at least 80%, the purity being measured based on the total amount of the negatively charged, preferably sialylated oligosaccharides and their precursors produced by the cell in the whole broth and / or supernatant, respectively.

15. The cell according to any one of the preceding claims, wherein the cell is stably cultured in a culture or incubation medium.

16. The cell according to any one of the preceding claims, wherein the cell is capable of producing and / or producing a mixture of two or more oligosaccharides, wherein at least one of the two or more oligosaccharides is the negatively charged, preferably sialylated, oligosaccharide.

17. The cell according to any one of the preceding claims, wherein the cell is capable of producing and / or producing a mixture of one or more disaccharides and one or more oligosaccharides, wherein at least one of the one or more oligosaccharides is the negatively charged, preferably sialylated, oligosaccharide.

18. The cell according to any one of the preceding claims, wherein the cell is capable of producing and / or producing a mixture comprising the negatively charged, preferably sialylated, oligosaccharide and at least one uncharged (neutral) oligosaccharide.

19. The cell according to any one of the preceding claims, wherein the cell is capable of producing and / or producing a mixture comprising: 1) the negatively charged, preferably sialylated oligosaccharide, and 2) disaccharides and / or uncharged (neutral) oligosaccharides.

20. A method for producing negatively charged, preferably sialylated, oligosaccharides, the method comprising: i. Cells according to any one of the preceding claims are cultured and / or incubated in a culture and / or incubation medium under conditions that allow for the production of the negatively charged, preferably sialylated, oligosaccharides. ii. Optionally, the negatively charged, preferably sialylated, oligosaccharide is then isolated and / or purified from the culture and / or incubation.

21. The method according to claim 20, wherein the negatively charged oligosaccharide is: - A sialylated oligosaccharide having at least one sialic acid residue selected from the list comprising, consisting of, or substantially consisting of the following substances: Neu4Ac; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu4,5,7,9Ac4; Neu5,7,8,9Ac4; Neu4,5,7,8,9Ac5; Neu5Gc and 2-keto-3-deoxymannose-octylulose acid (KDO). - Oligosaccharides, selected from the list comprising, consisting of, or substantially consisting of: negatively charged lactoses, sialylated lactoses, negatively charged mammalian lactoses (MMOs), sialylated MMOs, negatively charged human lactoses (HMOs), sialylated HMOs, O-antigens, oligosaccharide repeating units present in capsular polysaccharides, peptidoglycans, amino sugars, Lewis-type antigen oligosaccharides, negatively charged animal oligosaccharides, sialylated animal oligosaccharides, and negatively charged animal oligosaccharides selected from the list consisting of N-glycans and O-glycans. Animal oligosaccharides, sialylated animal oligosaccharides selected from the list consisting of N-glycans and O-glycans, negatively charged plant oligosaccharides, sialylated plant oligosaccharides, negatively charged plant oligosaccharides selected from the list consisting of N-glycans and O-glycans, sialylated oligosaccharides, negatively charged oligosaccharides containing N-acetyllactosamine, sialylated oligosaccharides containing N-acetyllactosamine, negatively charged oligosaccharides containing lactose-N-biose, sialylated oligosaccharides containing lactose-N-biose, and / or - Selected from a list including the following substances: 3'-sialyl lactose (3'SL), 6'-sialyl lactose (6'SL), 3'-sialyl lactosamine, 3,6-disialyl lactose, 8,3-disialyl lactose, sialylated lactose-N-trisaccharide, sialyl lactose-N-tetrasaccharide a (LSTa), KDOα-2,3Galβ-1,3GlcNAcβ-1,3Galβ-1,4Glc, sialyl lactose-N-tetrasaccharide b (LSTb), sialyl lactose-N-tetrasaccharide c (LSTc), sialyl lactose-N-tetrasaccharide d (LSTd), diasialactose-N-tetrasaccharide, diasialactose-N-neotetrasaccharide, monosialactose-N-hexasaccharide, diasialactose-N-hexasaccharide I, diasialactose-N-hexasaccharide II, monosialactose-N-neohexasaccharide I, monosialactose-N-neohexasaccharide II, diasialactose-N-neohexasaccharide, 3'-sialic acid-3-fucosylated lactose, fucoidanilactose-N-hexasaccharide, diasialactofucosylated lactose-N-neohexasaccharide, monofucosylated monosialactose-N-octasaccharide (sialic acid Lea), sialactose-N-fucohexasaccharide II Disialyllactose-N-fucopentose II, monofucosyldisialyllactose-N-tetrasaccharide, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal, Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal, 3'-KDO-lactose, 3'-KDO-lactosamine, 3'-KDO-lactose-N-disaccharide, 3'-KDO-6'-sialic acid lactose, 3'KDO-8-sialic acid lactose, KDO-2,3Galβ-1,3GalNacβ-1,3Gal α-1,4Gal, β-1,4Gal, KDO, α-2,3Gal, β-1,4Glc, Nac, β-1,3Gal, β-1,4Glc, 3'-KDO-3-fucosyllactose, Neu5Ac-a2,8-Neu5Ac-a2,3-Gal-b1,3-Glc, Nac-b1,3-Gal, 3'-sialic acid-2'-fucosyllactose, 6'-sialic acid-2'-fucosyllactose, 6'-sialic acid-3-fucosyllactose, Neu5Ac-a2,6-(Neu5Ac-a2,3-)Gal- b1,4-Glc, 3'-sialyl-3-fucosylated lactosamine, Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc, 6'-sialyl-lactose-N-disaccharide, 3'-sialyl-lactose-N-disaccharide, Neu5Ac-a2,6-(GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-(Fuc-a1,3-)GlcNAc-b1,3-)Gal-b1,4-Glc、Neu5Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3-)Gal-b1,4-Glc、Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc、Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc、Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc、Neu5Ac-a2,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc、Neu5Ac-a2,6-(Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc、Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc、Fuc-a1,4-(Neu5Ac-a2,3-Gal-b1,3-)GlcNAc-b1,3-Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-(Fuc-a1,3-)Glc、Neu5Ac-a2,6-(Neu5Ac-a2,6-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc、Neu5Ac-a2,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc、Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,3-GlcNAc-b1,3)-Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,3-GlcNAc-b1,3)-Gal-b1,4-Glc、Neu5Ac-a2,6-(Neu5Ac-a2, 3-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3)-Gal- b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,3)-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5 Ac-a2,6-(Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,6-(Gal-b1,4-G lcNAc-b1,3)-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4 -GlcNAc-b1,3-Gal-b1,4-Glc, Neu5Ac-a2,6-(Neu5Ac-a2,6-(Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3)-Ga l-b1,4-GlcNAc-b1,3)-Gal-b1,4-Glc, Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc, 6'-KDO-lactose, 6'-KDO-lactosamine, 6'-KDO-lactose-N-disaccharide, KDO-lactose-N-trisaccharide, KDO-lactose-N-tetrasaccharide, KDO-lactose-N-tetrasaccharide, and combinations thereof.

22. The method according to any one of claims 20 or 21, wherein the culture or incubation medium is: - Contains one or more precursors for generating the negatively charged, preferably sialylated, oligosaccharide. - Contains at least one carbon source selected from the list of substances included, composed of, or substantially composed of: monosaccharides; disaccharides; oligosaccharides; polysaccharides; polyols; glycerol; complex culture media including molasses, corn steep liquor, peptone, tryptone, or yeast extract; glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose, maltodextrin, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn steep liquor, high fructose syrup, acetate, citrate, lactate, and pyruvate. - Contains at least one compound selected from the list of substances consisting of: lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid, CMP-sialic acid, CMP-Neu5Ac, and CMP-KDO. - Culture media with defined chemical composition, and / or - It is a basal salt culture medium containing sulfates, phosphates, chlorides, ammonium salts, calcium salts, magnesium salts, sodium salts, potassium salts, iron salts, copper salts, zinc salts, manganese salts, cobalt salts and / or selenium salts.

23. The method according to any one of claims 20 to 22, wherein the method comprises at least one of the following steps: i) Use a culture or incubation medium containing at least one precursor and / or receptor; ii) Add at least one precursor and / or acceptor feed to a culture or incubation medium in a reactor or incubator, wherein the total volume of the reactor or incubator ranges from 250 mL to 10,000 m³, preferably added continuously, and preferably such that the final volume of the culture or incubation medium is no more than three times, no more than two times, and / or less than two times the volume of the culture or incubation medium before the addition of the precursor and / or acceptor feed; iii) Adding at least one precursor and / or acceptor feed to a culture or incubation medium in a reactor or incubator, wherein the total volume of the reactor or incubator ranges from 250 mL to 10,000 m³, preferably in a continuous manner, and preferably such that the final volume of the culture or incubation medium is no more than three times, no more than two times, and / or less than two times the volume of the culture or incubation medium before the addition of the precursor and / or acceptor feed, and wherein preferably, the pH of the precursor and / or acceptor feed is set between 2.0 and 10.0 and / or the temperature of the precursor and / or acceptor feed is maintained between 20°C and 80°C. iv) At least one precursor and / or receptor feed solution is continuously added to the culture or incubation medium over 1, 2, 3, 4 and 5 days. v) Continuously adding at least one precursor and / or receptor feed solution to the culture or incubation medium over days 1, 2, 3, 4, and 5, wherein the concentration of the precursor and / or receptor feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L, or 600 g / L. g / L; and / or the pH of the precursor and / or receptor supplement solution is set between 2.0 and 10.0, and / or the temperature of the precursor and / or receptor supplement solution is maintained between 20°C and 80°C; The method ensures that, in the final volume of the culture or incubation, the concentration of the negatively charged, preferably sialylated, oligosaccharide is at least 50 g / L, at least 75 g / L, at least 90 g / L, at least 100 g / L, at least 125 g / L, at least 150 g / L, at least 175 g / L, and / or at least 200 g / L. Preferably, the precursor is selected from a list including the following substances: sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose; and / or the receptor is lactose.

24. The method according to any one of claims 20 to 23, wherein the method comprises at least one of the following steps: i) Using a culture or incubation medium containing at least 50 g, at least 75 g, at least 100 g, at least 120 g and / or at least 150 g of precursor per liter of initial reactor or incubator volume, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m³. ii) Use a culture or incubation medium containing at least 50 g, at least 75 g, at least 100 g, at least 120 g and / or at least 150 g of the recipient per liter of initial reactor or incubator volume, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m³. iii) Adding a precursor feed containing at least 50 g, at least 75 g, at least 100 g, at least 120 g and / or at least 150 g of the precursor per liter of initial reactor or incubator volume to the culture or incubator medium in a reactor or incubator, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m³, preferably added continuously, and preferably such that the final volume of the culture or incubator medium is no more than three times, no more than two times and / or less than two times the volume of the culture or incubator medium before the addition of the precursor feed; iv) Adding a receptor feeder containing at least 50 g, at least 75 g, at least 100 g, at least 120 g and / or at least 150 g of receptor per liter of initial reactor or incubator volume to the culture or incubator medium in the reactor or incubator, wherein the reactor or incubator volume ranges from 250 mL to 10,000 m³, preferably added continuously, and preferably such that the final volume of the culture or incubator medium is no more than three times, no more than two times and / or less than two times the volume of the culture or incubator medium before the addition of the receptor feeder; v) Adding a precursor feed containing at least 50 g, at least 75 g, at least 100 g, at least 120 g and / or at least 150 g of the precursor per liter of initial reactor or incubator volume to the culture or incubator, wherein the total volume of the reactor or incubator ranges from 250 mL to 10,000 m³, preferably added continuously, and preferably such that the final volume of the culture or incubator is no more than three times, no more than two times and / or less than two times the volume of the culture or incubator before the addition of the precursor feed, and wherein preferably, the pH of the precursor feed is set between 2.0 and 10.0 and / or the temperature of the precursor feed is maintained between 20°C and 80°C; vi) Adding a receptor feeder containing at least 50 g, at least 75 g, at least 100 g, at least 120 g, and / or at least 150 g of receptor per liter of initial reactor or incubator volume to the culture or incubator, wherein the total volume of the reactor or incubator ranges from 250 mL to 10,000 m³, preferably added continuously, and preferably such that the final volume of the culture or incubator is no more than three times, no more than two times, and / or less than two times the volume of the culture or incubator before the addition of the receptor feeder, and wherein preferably, the pH of the receptor feeder is set between 2.0 and 10.0 and / or the temperature of the receptor feeder is maintained between 20°C and 80°C; vii) The precursor and / or receptor feed solution is continuously added to the culture or incubation medium over 1, 2, 3, 4 and 5 days. viii) A precursor feed solution is continuously added to the culture or incubation medium over 1, 2, 3, 4, and 5 days, wherein the concentration of the precursor feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L, or 600 g / L; and preferably, the pH of the precursor feed solution is set between 2.0 and 10.0 and / or the temperature of the precursor feed solution is maintained between 20°C and 80°C. ix) Receptor feed is continuously added to the culture or incubation medium via a receptor feed solution over 1, 2, 3, 4, and 5 days, wherein the concentration of the receptor feed solution is 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, 300 g / L, 325 g / L, 350 g / L, 375 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L, or 600 g / L; and preferably, the pH of the receptor feed solution is set between 2.0 and 10.0 and / or the temperature of the receptor feed solution is maintained between 20°C and 80°C; The method ensures that, in the final volume of culture or incubation, the concentration of the negatively charged, preferably sialylated, oligosaccharide is at least 50 g / L, at least 75 g / L, at least 90 g / L, at least 100 g / L, at least 125 g / L, at least 150 g / L, at least 175 g / L, and / or at least 200 g / L. Preferably, the precursor is selected from a list including the following substances: sialic acid, CMP-sialic acid, CMP-Neu5Ac, CMP-KDO, glucose, galactose and UDP-galactose; and / or the receptor is lactose.

25. The method according to any one of claims 20 to 24, wherein the cells produce 30 g / L or more of the negatively charged, preferably sialylated oligosaccharides in the whole broth and / or supernatant, and / or wherein the purity of the negatively charged, preferably sialylated oligosaccharides in the whole broth and / or supernatant is at least 80%, the purity being determined based on the total amount of the negatively charged, preferably sialylated oligosaccharides and their precursors produced by the cells in the whole broth and / or supernatant, respectively.

26. The method according to any one of claims 20 to 25, wherein the cell produces a mixture of two or more oligosaccharides, wherein at least one of the two or more oligosaccharides is the negatively charged, preferably sialylated, oligosaccharide.

27. The method according to any one of claims 20 to 26, wherein the cell produces a mixture of one or more disaccharides and one or more oligosaccharides, wherein at least one of the one or more oligosaccharides is the negatively charged, preferably sialylated, oligosaccharide.

28. The method according to any one of claims 20 to 27, wherein the cells produce a mixture comprising the negatively charged, preferably sialylated, oligosaccharide and at least one uncharged (neutral) oligosaccharide.

29. The method according to any one of claims 20 to 28, wherein the cells produce a mixture comprising: 1) the negatively charged, preferably sialylated oligosaccharide, and 2) disaccharides and / or uncharged (neutral) oligosaccharides.

30. The method according to any one of claims 20 to 29, wherein the negatively charged, preferably sialylated, oligosaccharide is recovered from the culture or incubation medium and / or the cells, and / or wherein the negatively charged, preferably sialylated, oligosaccharide is purified.

31. The method according to any one of claims 20 to 30, wherein the separation comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated carbon or carbon treatment, nonionic surfactant treatment, enzymatic hydrolysis, tangential flow high efficiency filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and / or gel filtration, ligand exchange chromatography, and electrodialysis.

32. The method according to any one of claims 20 to 31, wherein the purification comprises at least one of the following steps: using activated carbon or carbon, using charcoal, nanofiltration, ultrafiltration, electrophoresis, enzyme treatment or ion exchange, temperature adjustment, pH adjustment, pH adjustment by alkaline or acidic solution, using alcohols, using a water-alcohol mixture, crystallization, evaporation, precipitation, drying, spray drying, freeze drying, spray freeze drying, freeze spray drying, strip drying, belt drying, vacuum strip drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying or vacuum roller drying.

33. Use of the cells according to any one of claims 1 to 19 in the production of negatively charged, preferably sialylated, oligosaccharides.

34. Use of the method according to any one of claims 20 to 32 in the production of negatively charged, preferably sialylated, oligosaccharides.

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