Production of disaccharides and / or milk oligosaccharides from cells with reduced UDP-GlcNAc synthesis

Abstract

The invention is in the technical field of synthetic biology, metabolic engineering and cell culture. The present invention provides cells for the production of disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) wherein the synthesis of UDP-N-acetylglucosamine in the cells is made poorer in function. The invention further provides the use of said cells in culture or incubation. Also described are methods for producing disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) and purifying the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) by using the cells.

Description

Technical Field

[0001] The present invention is in the technical field of synthetic biology, metabolic engineering and cell culture. The present invention provides cells for producing disaccharides and / or oligosaccharides (e.g., milk oligosaccharides), wherein the synthesis of UDP-N-acetylglucosamine in the cells is functionally impaired. The present invention further provides the use of the cells in culture or incubation. The present invention also describes methods for producing disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) and purifying the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) using the cells. Background Art

[0002] Disaccharides and oligosaccharides are extremely diverse in their chemical structure and are composed of a wide variety of monosaccharides (e.g., glucose, galactose, N-acetylglucosamine, xylose, rhamnose, fucose, mannose, N-acetylneuraminic acid, N-acetylgalactosamine, galactosamine, glucosamine, glucuronic acid, and galacturonic acid). Disaccharides and oligosaccharides are widely distributed in all living organisms and play an important role in a variety of physiological and pathological processes (e.g., cell migration, signal transduction, cell-cell adhesion, inflammation, and immune responses). Economical production of these disaccharides and oligosaccharides is crucial to fully benefit from their biological advantages. An important group of oligosaccharides includes milk oligosaccharides, such as mammalian milk oligosaccharides (MMOs) and human milk oligosaccharides (HMOs), found in mammalian and human milk, respectively. A wide variety of synthetic methods have been developed, ranging from extraction to chemical synthesis to enzymatic synthesis. These methods are currently the least used, while biotechnological fermentation production is now being pursued and commercialized. Methods for producing disaccharides and / or oligosaccharides are well known to those skilled in the art (e.g., as described in Faijes et al. (2019), US2010120096A, JP2013201913, WO2022 / 034067).

[0003] N-acetylglucosamine (GlcNAc) is an important monosaccharide that is used in the synthesis of disaccharides and / or oligosaccharides in its free form as well as in its phosphorylated form (i.e., GlcNAc-6-phosphate) and in its activated form, UDP-GlcNAc. The availability of the balance of these different forms of GlcNAc often determines the efficiency of the disaccharide and / or oligosaccharide synthesis reaction in the cell. Summary of the Invention SUMMARY OF THE INVENTION

[0005] The object of the present invention is to provide instrument and method, can produce disaccharide and / or oligosaccharide (for example, milk oligosaccharide) by means of described instrument and method, preferably with efficient, timely and cost-effective mode, and described instrument and method produce a large amount of desired disaccharide and / or oligosaccharide (for example, milk oligosaccharide).According to the present invention, this and other targets are realized by being provided for the method and cell of producing disaccharide and / or oligosaccharide (for example, milk oligosaccharide).The present invention also provides the method for purifying described disaccharide and / or oligosaccharide (for example, milk oligosaccharide).Further, the invention provides as described in this article carry out genetic modification and wherein make the synthetic cell of UDP-GlcNAc deteriorate in function aspect.The present invention also provides by the purified disaccharide and / or oligosaccharide (for example, milk oligosaccharide) of process mentioned above.By reading the present invention, the further benefit of teaching of the present invention will be obvious for those skilled in the art.

[0006] definition

[0007] The words used in this specification to describe the present invention and its various embodiments should be understood not only in their generally defined meanings, but also to include structures, materials, or actions that extend beyond the generally defined meanings through specific definitions in this specification. Therefore, if an element in the context of this specification can be understood to include more than one meaning, its use in the claims must be understood to be general to all possible meanings supported by this specification and by the word itself.

[0008] The various aspects and embodiments of the present invention disclosed herein should be understood not only in the order and scenario specifically described in this specification, but also in any order and any combination thereof. Each embodiment identified in this article can be combined together, unless otherwise stated. All publications, patents and patent applications mentioned in this specification are incorporated herein by reference, to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference in its entirety. Unless otherwise specifically stated, all words used in the singular should be considered to include the plural, and vice versa. Unless otherwise defined, all technical and scientific terms used in this article generally have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used in this article and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described in this article 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 carried out according to the manufacturer's instructions.

[0009] In this specification, embodiments of the present invention have been disclosed, and although specific terms are employed, these terms are used in a descriptive sense only and not for limiting purposes, and the scope of the invention is set forth in the claims below. It must be understood that the illustrative embodiment is set forth for illustrative purposes only, and it should not be considered as limiting the invention. It will be apparent to those skilled in the art that changes, other embodiments, improvements, details and uses may be made consistent with the letter and spirit of the disclosure herein and within the scope of the disclosure, and the scope of the disclosure is limited only by the claims and interpreted in accordance with patent law, including the doctrine of equivalents. In the claims below, the reference characters used to indicate claim steps are provided for convenience of description only and are not intended to imply any particular order for performing the steps, unless otherwise specifically stated.

[0010] Throughout the application, unless otherwise expressly stated, the feature "synthesize" is used interchangeably with the feature "produce". Throughout the application, unless otherwise expressly stated, the expression "can ..." is preferably replaced by the active voice of the verb, and vice versa. For example, the expression "can express" is preferably replaced by "express", and vice versa, i.e., "express" is preferably replaced by "can express". In this document and in its claims, the verbs "comprise", "have" and "comprises" and their variations are used in their non-restrictive sense to mean that the items following the word are included, but items not specifically mentioned are not excluded. Throughout the application, the verb "comprise (comprising)" can be replaced by "consisting of" or "consisting essentially of", and vice versa. In addition, the verb "consisting of" can be replaced by "consisting essentially of", which means that the composition defined in this document may contain additional components other than those specifically identified, and the additional components do not change the unique characteristics of the invention. In this document and in its claims, unless otherwise specifically stated, the verbs "comprise," "have," and "include" and their conjugations may be replaced with "consisting of" (and their conjugations) or "consisting essentially of" (and their conjugations), and vice versa. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that only one of the element be present. Thus, the indefinite article "a" or "an" often means "at least one."

[0011] Throughout this document and in the claims, unless expressly stated otherwise, the articles "a" and "an" are preferably replaced by "at least one," more preferably by "at least two," more preferably by "at least three," more preferably by "at least four," more preferably by "at least five," more preferably by "at least six," and most preferably by "at least two." When used in connection with a numerical value (e.g., "about 10") or in connection with a range (e.g., "about x to about y"), the word "about" or "approximately" preferably means that the value or range is interpreted as being as accurate as the method used to measure it. If no margin of error is specifically specified, the expression "about" or "approximately" when used in connection with a numerical value is to be interpreted as having the same rounding as the given value. Throughout this document and in the claims thereof, unless expressly stated otherwise, the expression "x to y" (where x and y represent numerical values) refers to a numerical range with x being the lower limit of the range and y being the upper limit of the range. Herein, x and y are also included in the range.

[0012] According to the present invention, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. It should be understood that DNA or RNA containing rare bases (e.g., inosine) or modified bases (e.g., tritylated bases) are encompassed by the term "polynucleotide". It will be appreciated that DNA and RNA have been subjected to a very wide variety of modifications that serve many useful purposes known to those skilled in the art. As employed herein, the term "polynucleotide" encompasses such chemically modified, enzymatically modified, or metabolically modified forms of polynucleotides, as well as chemical forms of DNA and RNA characteristic of viruses and cells (including, for example, simple and complex cells). The term "polynucleotide" also encompasses short polynucleotides that are often referred to as oligonucleotides.

[0013] "Polypeptide" refers to any peptide or protein comprising two or more amino acids linked to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains (commonly referred to as peptides, oligopeptides and oligomers) and longer chains (commonly referred to as proteins). Polypeptides may contain other amino acids in addition to the 20 genetically encoded amino acids. "Polypeptides" include those modified by natural processes (e.g., processing and other post-translational modifications), but also include those modified by chemical modification techniques well known to technicians. The same type of modification can be present at several sites in a given polypeptide to the same or varying extent. Further, a given polypeptide can contain many types of modifications. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Polypeptides can be branched or cyclic, with or without branches. Cyclic, branched, or branched annular polypeptides can be produced by natural post-translational processes and can also be prepared by completely synthetic methods.

[0014] As used herein, the term "polynucleotide encoding a polypeptide" encompasses oligonucleotides comprising a sequence encoding a polypeptide of the present invention. The term also encompasses polynucleotides comprising a single continuous region or discontinuous regions (e.g., interrupted by integrated phage or insertion sequences or editing) encoding the polypeptide, together with additional regions that may also contain coding and / or non-coding sequences.

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

[0016] "Recombinant" means genetically modified DNA that is produced by transplanting or splicing genes from one species into the cells of a host organism of a different species. Such DNA becomes part of the host's genetic makeup and is replicated.

[0017] The terms "recombinant" or "transgenic" or "metabolically engineered" or "genetically engineered" as used herein with respect to cells or host cells are used interchangeably and indicate that the cell replicates a heterologous nucleic acid or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence that is "foreign to the cell" or "foreign to the location or environment in the cell"). Such cells are described as being transformed with at least one heterologous or exogenous gene, or as being transformed by the introduction of at least one heterologous or exogenous gene. Recombinant or metabolically engineered or genetically engineered or transgenic cells may contain genes that are not found in the native (non-recombinant) form of the cell. Recombinant cells may also contain genes found in the native form of the cell, wherein the genes have been modified by artificial means and reintroduced into the cell. The term also encompasses cells comprising 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 those obtained by gene replacement, promoter replacement, site-specific mutation, CrispR, riboswitch, 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 cells that have been modified by removing a nucleic acid endogenous to the cell (e.g., knocking out a gene) using well-known techniques common to those skilled in the art.

[0018] As used herein, a "heterologous sequence" or "heterologous nucleic acid" is a sequence or nucleic acid that is derived from a source foreign to a particular cell (e.g., from a different species) or, if derived from the same source, has been modified from its original form or position in the genome. Thus, a heterologous nucleic acid operably linked to a promoter is derived from a source different from the source from which the promoter is derived or, if derived from the same source, has been modified from its original form or position in the genome. A heterologous sequence can be stably introduced into the genome of a host cell, for example, by transfection, transformation, conjugation, or transduction, whereby techniques that will depend on the cell and the sequence to be introduced can be applied. Various techniques are known to those skilled in the art and are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). The terms "mutant" or "modified" cell, as used within the context of the present invention, refers to a genetically modified cell.

[0019] Within the context of the present disclosure, the term "endogenous" refers to any polynucleotide, polypeptide, or protein sequence that is a natural part of a cell and occurs at its natural location in the cell's chromosomes, and whose expression control is unchanged compared to the natural control mechanisms that act on its expression. The term "exogenous" refers to any polynucleotide, polypeptide, or protein sequence that originates from outside the cell in question and is not a natural part of the cell, or that does not occur at its natural location in the cell's chromosomes or on a plasmid.

[0020] When used in relation to polynucleotides, genes, nucleic acids, polypeptides or enzymes, the term "heterologous" refers to polynucleotides, genes, nucleic acids, polypeptides or enzymes from or derived from other sources except the host organism. In contrast, "homologous" polynucleotides, genes, nucleic acids, polypeptides or enzymes are used herein to represent polynucleotides, genes, nucleic acids, polypeptides or enzymes derived from the host organism. When referring to gene regulatory sequences or auxiliary nucleic acid sequences (e.g., promoters, 5' untranslated regions, 3' untranslated regions, poly A addition sequences, intron sequences, splice sites, ribosome bind sites, internal ribosome entry sequences, genome homology regions, recombination sites etc.) for maintaining or manipulating gene sequences, "heterologous" means that the regulatory sequences or auxiliary sequences are not naturally associated with the genes to which the regulatory sequences or auxiliary nucleic acid sequences are juxtaposed in constructs, genomes, chromosomes or episomes. Thus, a promoter that is operably linked to a gene to which it is not operably linked in its native state (i.e., in the genome of a non-genetically modified organism) is referred to herein as a "heterologous promoter," even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked.

[0021] The term "modified expression" of a gene relates to a change in expression compared to the wild-type expression of the gene in any stage of the production process of a desired disaccharide and / or oligosaccharide (e.g., a desired milk oligosaccharide). The modified expression is lower or higher expression compared to the wild-type, wherein the term "higher expression" is also defined as "overexpression" of the gene in the case of an endogenous gene, or is defined as "expression" in the case of a heterologous gene not present in a wild-type strain. Lower expression is obtained by means of well-known techniques common to those skilled in the art (e.g., using siRNA, CrispR, CrispRi, riboswitches, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutant genes, knockout genes, transposon mutagenesis, etc.), which are used to change genes in such a way that the gene is "less able" (i.e., statistically significantly "less able" compared to a functional wild-type gene) or is completely unable (e.g., knocked-out genes) to produce a functional end product. As used herein, the term "riboswitch" is defined as a portion of a messenger RNA that folds into a complex structure that blocks expression by interfering translation. The binding of effector molecules induces conformational changes, thereby allowing post-transcriptional regulation of expression. In addition to changing the target gene as described above to obtain lower expression, lower expression can also be obtained by changing the transcription unit, promoter, untranslated region, ribosome binding site, Shine Dalgarno sequence or transcription terminator. Lower expression or reduced expression can, for example, be obtained in the following manner: one or more base pairs in the promoter sequence are mutated, or the promoter sequence is completely changed into a constitutive promoter with lower expression intensity than the wild type or an inducible promoter that causes regulated expression or a repressible promoter that causes regulated expression. Overexpression or expression is obtained by means of well-known techniques common to those skilled in the art (e.g., use of artificial transcription factors, de novo design of promoter sequences, ribosome engineering, introduction or reintroduction of expression modules at euchromatin, use of high copy number plasmids), wherein the gene is part of an "expression cassette", which refers to any sequence in which a promoter sequence, an untranslated region sequence (including a ribosome binding sequence, a Shine Dalgarno sequence or a Kozak sequence), a coding sequence and optionally a transcription terminator are present and results in the expression of a functionally active protein. The expression is constitutive or conditional or regulated or tunable.

[0022] The term "constitutive expression" is defined as the absence of transcription factors other than subunits of RNA polymerase (e.g., bacterial sigma factors such as sigma) under certain growth conditions. 70 , σ 54Or the related σ-factor, and yeast mitochondrial RNA polymerase specific factor MTF1, which is co-associated with the RNA polymerase core enzyme) to regulate expression. Non-limiting examples of such transcription factors are CRP, LacI, ArcA, Cra, IclR in Escherichia coli (E. coli), or Aft2p, Crz1p, Skn7 in Saccharomyces cerevisiae, or DeoR, GntR, Fur in Bacillus subtilis (B. subtilis). These transcription factors bind to specific sequences and can block or enhance expression under certain growth conditions. RNA polymerase is a catalytic machine for synthesizing RNA from a DNA template. RNA polymerase binds to a specific DNA sequence to initiate transcription, for example, via the σ factor in a prokaryotic host or via MTF1 in yeast. Constitutive expression provides a constant expression level without the need for induction or repression.

[0023] The term "regulated expression" is defined as expression that is regulated under certain growth conditions by transcription factors other than subunits of RNA polymerase (e.g., bacterial sigma factors). Examples of such transcription factors are described above. Typically, expression regulation is achieved with the aid of inducers such as, but not limited to, IPTG, arabinose, rhamnose, fucose, allolactose, or pH shifts or temperature shifts or carbon depletion or substrate or generated products.

[0024] The term "control sequence" refers to a sequence recognized by the cellular transcription and translation machinery to allow transcription and translation of a polynucleotide sequence into a polypeptide. Thus, such DNA sequences are necessary for expression of an operably linked coding sequence in a particular host cell, cell, or organism. Such control sequences may include, but are not limited to, promoter sequences, ribosome binding sequences, Shine Dalgarno sequences, Kozak sequences, and transcription terminator sequences. Suitable control sequences for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. DNA for a presequence or secretory leader can be operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer can be operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site can be operably linked to a coding sequence if it is positioned so as to facilitate translation. Furthermore, the control sequences can be controlled by external chemicals (such as, but not limited to, IPTG, arabinose, lactose, allolactose, rhamnose, or fucose) via inducible promoters or via genetic circuits that induce or repress transcription or translation of the polynucleotide into a polypeptide.

[0025] Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.

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

[0027] As used herein, the term "modified expression of a protein" refers to: i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein, iii) expression and / or overexpression of a variant protein having higher activity than the wild-type (i.e., native in the expression host) protein, iv) reduced expression of an endogenous protein, or v) expression and / or overexpression of a variant protein having reduced activity compared to the wild-type (i.e., native in the expression host) protein. Preferably, as used herein, the term "modified expression of a protein" refers to: i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein, or iii) expression and / or overexpression of a variant protein having higher activity than the wild-type (i.e., native in the expression host) protein.

[0028] The term "modified activity" of a protein relates to a non-native activity of the protein at any stage of the production process of a desired disaccharide and / or oligosaccharide (e.g., a desired milk oligosaccharide). As used herein with respect to the activity of a protein, the term "non-native" indicates that the protein has been modified to have an activity that is abolished, impaired, reduced, delayed, higher, accelerated, or improved compared to the native activity of the protein. The modified activity of a protein can be obtained by modified expression of the protein or by expression of a modified (i.e., mutant) form of the protein. The mutant form of the protein can be obtained by expression of a mutant form of a gene encoding the protein (which, for example, comprises a deletion, insertion, and / or mutation of one or more nucleotides compared to the native gene sequence). The mutant form of a gene can be obtained by techniques well known to those skilled in the art (e.g., but not limited to, site-specific mutations; CrispR; riboswitches; recombineering; ssDNA mutagenesis; transposon mutagenesis).

[0029] As used herein with respect to a cell that produces a disaccharide, the term "non-naturally occurring" indicates that the disaccharide i) is not produced in nature, or ii) when produced in nature, is not produced by the cell in the same amount; and that the cell has been genetically engineered to produce the disaccharide or to have increased production of the disaccharide.

[0030] As used herein in relation to cells that produce oligosaccharides (e.g., milk oligosaccharides), the term "non-naturally occurring" indicates that the oligosaccharide i) is not produced in nature, or ii) when produced in nature, is not produced by the cell in the same amount; and that the cell has been genetically engineered to be able to produce the oligosaccharide (more particularly the milk oligosaccharide) or to have a higher production of the oligosaccharide (more particularly the milk oligosaccharide).

[0031] As used herein in relation to cells that produce disaccharides and milk-oligosaccharides, the term "non-native" indicates that the disaccharides and milk-oligosaccharides i) are not produced in nature, or ii) when produced in nature, are not produced by the cell in the same amount; and that the cell has been genetically engineered to be able to produce the disaccharides and milk-oligosaccharides or to have increased production of the disaccharides and milk-oligosaccharides.

[0032] As the term is used herein, a "variant" is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs from another reference polynucleotide in nucleotide sequence. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs from another reference polypeptide in amino acid sequence. Typically, the differences are limited, so that the sequences of the reference polypeptide and the variant are generally very similar and, in many regions, are identical. A variant and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. A variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or it may be an unknown naturally occurring variant. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0033] In some embodiments, the present invention contemplates preparing variants by modifying the structure of the enzymes used in the present invention. Variants can be generated by amino acid substitutions, deletions, additions, or a combination thereof.

[0034] With respect to polynucleotides, a "fragment" refers to a clone or any portion of a polynucleotide molecule, particularly a portion of a polynucleotide.

[0035] Throughout this application, the sequence of a polynucleotide may be represented by a SEQ ID NO or, alternatively, by a Gene ID (Maglott et al. (2011) Nucl. Acids Res. 39, Issue suppl_1, D52-D57) or a GenBank NO (https: / / www.ncbi.nlm.nih.gov / genbank / ). Therefore, the terms "polynucleotide SEQ ID NO," "polynucleotide Gene ID," and "polynucleotide GenBank NO" may be used interchangeably unless expressly stated otherwise.

[0036] Throughout the application, the sequence of a polypeptide may be represented by a SEQ ID NO or, alternatively, by a UniProt ID. Therefore, the terms "polypeptide SEQ ID NO" and "polypeptide UniProt ID" may be used interchangeably unless expressly stated otherwise.

[0037] Domains can be characterized, for example, by the following tools: Pfam (El-Gebali et al., Nucleic Acids Res. 47 (2019) D427-D432), IPR (InterPro Domain) (http: / / ebi.ac.uk / interpro) (Mitchell et al., Nucleic Acids Res. 47 (2019) D351-D360), Conserved Domain Database (CDD) nomenclature (https: / / www.ncbi.nlm.nih.gov / cdd) (Lu et al., Nucleic Acids Res. 48 (2020) D265-D268) or 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). Protein or polypeptide sequence information and functional information can be provided by a wide range of resources about protein sequences and annotation data, such as the Universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49 (D1), D480-D489). UniProt contains an expert and richly managed protein database called UniProt Knowledgebase (UniProtKB), together with UniProt Reference Clusters (UniRef) and UniProt Archive (UniParc). The UniProt identifier (UniProt ID) is unique for each protein present in the database. Throughout the application, the sequence of the polypeptide is represented by SEQ ID NO or UniProt ID. Unless otherwise stated, the UniProt ID of the protein described corresponds to its sequence version 01 present in the UniProt database (www.uniprot.org) version 2021_03 and accessed on June 9, 2021.

[0038] InterPro provides functional analysis of proteins by classifying them into families and predicting structural domains and important sites. To classify proteins in this way, InterPro uses predictive models, called signatures, provided by several different databases (called member databases) that make up the InterPro federation. The protein signatures from these member databases are combined into a single searchable resource, where their individual strengths are harnessed to create a powerful integrated database and diagnostic tool.

[0039] Those skilled in the art will understand that for the databases used herein, including InterPro 90.0 (released on August 4, 2022) and eggNOG 5.0 (released in 2019), the content of each database is fixed at each release and will not change. When the content of a particular database is changed, a new release version with a new release date is accepted for that particular database. All release versions of each database with their corresponding release dates and the specific content annotated at these specific release dates are available and known to those skilled in the art.

[0040] As used herein, the term "monosaccharide" refers to a sugar that cannot be broken down into simpler sugars by hydrolysis, is classified as an aldose, ketose, deoxysugar, deoxyaminosugar, uronic acid, aldonic acid, ketoaldonic acid, aldaric acid, or sugar alcohol, and contains one or more hydroxyl groups per molecule. A monosaccharide is a sugar that contains only one simple sugar.

[0041] As used herein, the term "phosphorylated monosaccharide" refers to a phosphorylated monosaccharide. 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.

[0042] The terms "activated monosaccharide," "nucleotide-activating sugar," "nucleotide-sugar," "activated sugar," "nucleoside," or "nucleotide donor" are used interchangeably herein 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-arabino-4-hexulose, UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetamido-2,6-dideoxy-L- Nucleotide sugars include UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-phospho-L-glucose (UDP-L-PneNAC or UDP-2-acetylamino-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnose (UDP-L-QuiNAc or UDP-2-acetylamino-2,6-dideoxy-L-glucose), GDP-L-isorhamnose, CMP-sialic acid (CMP-Neu5Ac or CMP-N-acetylneuraminic acid), GDP-fucose (GDP-Fuc), GDP-rhamnose, and UDP-xylose. Nucleotide sugars serve as glycosyl donors in glycosylation reactions. Glycosylation reactions are reactions catalyzed by glycosyltransferases.

[0043] As used herein, the term "glycosyltransferase" refers to an enzyme that catalyzes the transfer of a sugar moiety from a donor to a specific acceptor, thereby forming a glycosidic bond. The donor can be a precursor as defined herein. Glycosyltransferases and related proteins that use nucleotide diphosphate-sugar, nucleotide monophosphate-sugar, and sugar phosphate have been described and classified into different sequence-based families (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, consisting of, or consisting essentially of a fucosyltransferase, a sialyltransferase, a galactosyltransferase, a glucosyltransferase, a mannosyltransferase, an N-acetylglucosamine transferase, an N-acetylgalactosamine transferase, an N-acetylmannosamine transferase, a xylosyltransferase, a glucuronyltransferase, a galacturonyltransferase, a glucosamine transferase, an N-glycolylneuraminic acid transferase, a rhamnosyltransferase, an N-acetylrhamnosyltransferase, a UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, a UDP-N-acetylglucosamine enolpyruvyltransferase, and a fucosamine transferase.

[0044] As used herein, the term "disaccharide" refers to a sugar polymer comprising two simple sugars (i.e., monosaccharides). Examples of disaccharides include lactose (Gal-β1,4-Glc), lacto-N-biose (LNB, Gal-β1,3-GlcNAc), N-acetyllactosamine (LacNAc, Gal-β1,4-GlcNAc), LacDiNAc (GalNAc-β1,4-GlcNAc), N-acetylgalactosaminylglucose (GalNAc-β1,4-Glc), Neu5Ac-α2,3-Gal, Neu5Ac-α2,6-Gal, and 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). Disaccharides include charged disaccharides that carry a negative charge, such as sialylated disaccharides, such as Neu5Ac-α2,3-Gal, Neu5Ac-α2,6-Gal, and Fuc-(1-4)-Neu5Gc, and uncharged (i.e., neutral) disaccharides, such as lactose, LNB, LacNAc, sucrose, maltose, and melibiose.

[0045] As used herein and as generally understood in the prior art, the term "oligosaccharide" refers to a sugar polymer comprising a small number, typically three to twenty, preferably three to ten simple sugars, i.e., monosaccharides. The oligosaccharides used in the present invention can be linear structures or can contain branches. The bond between two sugar units (e.g., glycosidic bond, galactosidic bond, glucosidic bond, etc.) can be expressed as, for example, 1,4, 1->4, or (1-4), and are 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", "Galβ1-4-Glc", "Gal-b(1-4)-Glc" and "Gal-β(1-4)-Glc" have the same meaning, i.e., a β-glycosidic bond connecting carbon-1 of galactose (Gal) to carbon-4 of glucose (Glc). Each monosaccharide can be in a cyclic form (e.g., a pyranose or furanose form). The bonds between individual monosaccharide units can include α1->2, α1->3, α1->4, α1->6, α2->1, α2->3, α2->4, α2->6, β1->2, β1->3, β1->4, β1->6, β2->1, β2->3, β2->4, and β2->6. Oligosaccharides can contain both α- and β-glycosidic bonds, or can contain only α-glycosidic bonds or only β-glycosidic bonds. The term "polysaccharide" refers to a compound composed of a large number, typically more than twenty, of monosaccharides connected by glycosidic bonds.Examples of oligosaccharides include, but are not limited to, Lewis-type antigenic oligosaccharides, milk oligosaccharides, mammalian (including human) milk oligosaccharides, O-antigens, enterobacterial common antigens (ECA), glycan chains present in lipopolysaccharides (LPS), oligosaccharide repeats present in capsular polysaccharides, peptidoglycans (PG), amino sugars, antigens of the human ABO blood group system, 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, oligosaccharides containing N-acetylglucosamine, oligosaccharides containing lacto-N-biose, oligosaccharides containing N-acetyllactosamine, sialylated oligosaccharides containing N-acetylglucosamine, neutral (uncharged) oligosaccharides containing N-acetylglucosamine, negatively charged oligosaccharides containing N-acetylglucosamine, fucosylated oligosaccharides containing N-acetylglucosamine, non-fucosylated oligosaccharides containing N-acetylglucosamine, sialylated oligosaccharides containing lacto-N-biose, Sugars, neutral (uncharged) oligosaccharides comprising lacto-N-biose, negatively charged oligosaccharides comprising lacto-N-biose, fucosylated oligosaccharides comprising lacto-N-biose, non-fucosylated oligosaccharides comprising lacto-N-biose, sialylated oligosaccharides comprising N-acetyllactosamine, neutral (uncharged) oligosaccharides comprising N-acetyllactosamine, negatively charged oligosaccharides comprising N-acetyllactosamine, fucosylated oligosaccharides comprising N-acetyllactosamine, non-fucosylated oligosaccharides comprising N-acetyllactosamine, chitosan , oligosaccharides including chitosan, heparosan, chondroitin sulfate, glycosaminoglycan oligosaccharides, heparin, heparan sulfate, dermatan sulfate, acetylated hyaluronan, hyaluronic acid, keratan sulfate, erlose (Glc-α1,4-Glc-α1,2-Fru), lactococcal-N-triose II (GlcNAc-β1,3-Gal-β1,4-Fru), lactococcal-N-tetraose, lactococcal-N-neotetraose and globotriose.

[0046] The terms "negatively charged oligosaccharide" or "acidic oligosaccharide" are used interchangeably and refer to oligosaccharides having a negative charge. In a preferred embodiment, the negatively charged oligosaccharide is a sialylated oligosaccharide. As used herein, "sialylated oligosaccharide" is understood to be an oligosaccharide comprising negatively charged sialic acid, i.e., an oligosaccharide having one or more sialic acid residues. It has an acidic nature. Some examples are 3'SL (3'-sialyllactose, Neu5Ac-α2,3-Gal-β1,4-Glc), 3'-sialyllactosamine, 6'SL (6' sialyllactose, Neu5Ac-α2,6-Gal-β1,4-Glc), 8'SL (8' sialyllactose, Neu5Ac-α2,8-Gal-β1,4-Glc), 3,6-disialyllactose (Neu5Ac-α2,3-(Neu5Ac-α2,6)-Gal-β1,4-Glc), 6, 6'-disialyl lactose (Neu5Ac-α2,6-Gal-β1,4-(Neu5Ac-α2,6)-Glc), 8,3-disialyl lactose (Neu5Ac-α2,8-Neu5Ac-α2,3-Gal-β1,4-Glc), 6'-sialyl lactosamine, 3'-sialyl lactosamine, oligosaccharides containing 6'sialyl lactose, SGG hexose (Neu5Acα-2,3Galβ-1,3GalNacβ-1,3Galα-1,4Galβ-1,4Gal ), sialylated tetraose, sialylated pentose, sialylated lacto-N-triose, sialylated lacto-N-tetraose, sialylated lacto-N-neotetraose, LSTc(Neu5Ac-α2,6-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc), LSTd(Neu5Ac-α2,3-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc), monosialyl lacto-N-hexose, disialyl lacto-N-hexose I, monosialyl lacto-N-neohexose I, monosialoyl lacto-N-neohexose II, disialoyl lacto-N-neohexose, disialoyl lacto-N-tetraose, disialoyl lacto-N-hexose II, sialyl lacto-N-tetraose a (LSTa, Neu5Ac-α2,3-Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc), disialoyl lacto-N-hexose I, sialyl lacto-N-tetraose b (LSTb, Gal-β1,3-(Neu5Ac-α2,6)-GlcNAc-β1,3-Gal-β1,4-Glc4-Glc), 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, monofucosylmonosialyllacto-N-octose (sialyl Lea), sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, monofucosyldisialyllacto-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-6'sialyllactose, 3'-KDO-8'sialyllactosamine, 3'-KDO-10'sialyllactosamine, 3'-KDO-20'sialyllactosamine, 3'-KDO-30'sialyllactosamine, 3'-KDO-40'sialyllactosamine, 3'-KDO-5'sialyllactosamine, 3'-KDO-6'sialyllactosamine, 3'-KDO-8'sialyllactosamine, 3'-KDO-10 ... Acyl lactose, 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, 3'-sialyl Acyl-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 and oligosaccharides carrying one or more sialic acid residues, including but not limited to oligosaccharide moieties of gangliosides selected from the following: GM3 (3' sialyllactose, Neu5Acα-2,3Galβ-4Glc) and oligosaccharides containing the GM3 motif, GD3 (Neu5Acα-2,8Neu5Acα-2,3Galβ-1,4Glc), GT3 (Neu5Acα-2,8Neu5Acα-2,8Neu5Acα-2,3Galβ-1,4Glc), GM2 (GalNAcβ-1,4(Neu5Acα-2,3 )Galβ-1,4Glc), GM1(Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc), GD1a(Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc), G T1a(Neu5Acα-2,8Neu5Acα-2,3Galβ-1,3GalNAcβ-1,4(Neu5Acα-2,3)Galβ-1,4Glc), GD2(GalNAcβ-1,4(Neu5Acα-2,8Neu5Acα2,3)Galβ-1,4Glc), GT2,

[0047] (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

[0048] (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-2,8Neu5Acα 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 which can be extended to produce the corresponding gangliosides by reacting the above oligosaccharide moieties with ceramide or synthesizing the above oligosaccharides on ceramide.

[0049] "Charged oligosaccharides" are oligosaccharide structures that contain one or more negatively charged monosaccharide subunits, including N-acetylneuraminic acid (Neu5Ac), commonly known as sialic acid, N-glycolylneuraminic acid (Neu5Gc), glucuronic acid, galacturonic acid, and 2-keto-3-deoxymannooctonic acid (KDO). Charged oligosaccharides are also known as acidic oligosaccharides. In contrast, neutral (uncharged) oligosaccharides are non-sialylated oligosaccharides and, therefore, do not contain acidic monosaccharide subunits. Neutral oligosaccharides include uncharged fucosylated oligosaccharides that contain one or more fucose subunits in their glycan structure, as well as uncharged non-fucosylated oligosaccharides that lack any fucose subunits. Other examples of charged oligosaccharides are sulfated chitosan and deacetylated chitosan.

[0050] As used herein, the terms "neutral oligosaccharide" and "uncharged oligosaccharide" are used interchangeably and, as generally understood in the art, refer to oligosaccharides that do not have a negative charge from a carboxylic acid group. 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, GlcNAcβ1-3Galβ1-4Glc), lacto-N-tetraose (LNT, Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentose I, lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentose I, lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentose II ... I, lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentose II, lacto-N-neotetraose (LNnT, Fucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose 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, monofucosyl emulsion- N-hexose-III, difucosyllactose-N-hexose (a), 6'-galactosyllactose, 3'-galactosyllactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose, difucosyllacto-N-hexose, difucosyllacto-N-neohexose, trifucosyllacto-N-hexose, a1,3-galactosyl-3-fucosyllactose, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)Glc, GalNAc-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)Glc3-)Glc, 2-fucosyllactulose, 3-fucosyl-N-acetyllactosamine, 2'-fucosyl-N-acetyllactosamine, difucosyl-N-acetyllactosamine, 4-fucosyllacto-N-biose, 2'-fucosyllacto-N-biose, difucosyllacto-N-biose and GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, Glc NAc-b1,6-(GlcNAc-b1,3-)Gal-b1,4-Glc, lacto-N-pentose (LN5), lacto-N-neopentose, p-lacto-N-pentose, p-lacto-N-neopentose, lacto-N-neopentose I, lacto-N-heptose (LN7), lacto-N-neoheptose, p-lacto-N-neoheptose, p-lacto-N-heptose, lacto-N-octose (LNO), lacto-N-neooctose, isolactose N-octose, lacto-N-octose, allolacto-N-neooctose, neo-lacto-N-neooctose, lacto-N-neooctose (pLNnO), allolacto-N-nonose, neo-lacto-N-nonose, lacto-N-nonose (LN9), lacto-N-decose, allolacto-N-decose, neo-lacto-N-decose, lacto-N-neodecose, lacto-N-neodecose, lacto-N-neodecose (pLNnD), a1,3-galactosyl lacto-N-neotetraose Sugar, GlcNAc-b1,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc, GlcNAc-b1,6-(Gal-b1,4-Gl cNAc-b1,3-)Gal-b1,4-Glc and GlcNAc-b1,6-(Gal-b1,3-GlcNAc-b1,3-)Gal-b1,4-Glc. ,

[0051] As used herein and as generally understood in the prior art, "fucosylated oligosaccharides" are oligosaccharides carrying a fucose residue. Such fucosylated oligosaccharides are sugar structures comprising at least three monosaccharide subunits interconnected via glycosidic bonds, wherein at least one of the monosaccharide subunits is fucose. Fucosylated oligosaccharides may comprise more than one fucose residue, for example two, three or more. Fucosylated oligosaccharides may be neutral oligosaccharides or charged oligosaccharides, which, for example, also comprise a sialic acid structure. Fucose may be linked to other monosaccharide subunits (including glucose, galactose, GlcNAc) via α-glycosidic bonds (including α-1,2, α-1,3, α-1,4, α-1,6 bonds).Examples include 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), difucosyllactose (diFL), lacto-N-fucopentose I (LNFP I), Gal-a1,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (Gal-LNFP I), GalNAc-a1,3-(Fuc-a1,2-)Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc (GalNAc-LNFP I), lacto-N-fucopentose II (LNFP II), lacto-N-fucopentose III (LNFP III), lacto-N-fucopentose V (LNFP V), lacto-N-fucopentose VI (LNFP VI), lacto-N-neofucopentose I, lacto-N-difucohexose I (LDFH I), lacto-N-difucohexose II (LDFH II), monofucosyl lacto-N-hexose III (MFLNH III), difucosyllactose-N-hexose (a), difucosyllactose-N-hexose, difucosyl-lacto-N-neohexose, trifucosyllactose-N-hexose, a1,3-galactosyl-3-fucosyllactose, Gal-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)Glc, GalNAc-a1,3-(Fuc-a1,2-)Gal-b1,4-(Fuc-a1,3-)Glc, 2-fucosyllactulose, 3-fucosyl-N-acetyllactosamine, 2'-fucosyl-N-acetyllactosamine, difucosyl-N-acetyllactosamine, 4-fucose sialyllacto-N-biose, 2'-fucosyllacto-N-biose, difucosyllacto-N-biose and GlcNAc-b1,3-Gal-b1,4-(Fuc-a1,3-)Glc, 3'-sialyl-2'-fucosyllactose, 6'-sialyl-2'-fucosyllactose, 6'-sialyl-3-fucosyllactose, 3'-sialyl-3-fucosyllactose, disialylmonofucosyllacto-N-neohexose, monofucosylmonosialyllacto-N-octose (sialyl Lea), sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, monofucosyldisialyllacto-N-tetraose.

[0052] Mammalian milk oligosaccharides include oligosaccharides present in milk (including colostrum) found at any stage during lactation from humans and mammals including, but not limited to, cattle (Bos taurus), sheep (Ovisaries), goats (Capra aegagrus hircus), Bactrian camels (Camelus bactrianus), horses (Equus feruscaballus), pigs (Sus scropha), dogs (Canis lupus familiaris), Hokkaido brown bears (Ursus arctosyesoensis), polar bears (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephants (Loxodonta africana), giant anteaters (Myrmecophaga tridactyla), bottlenose dolphins (Tursiops truncates), minke whales (Balaenoptera acutorostrata), Eugene's wallaby (Macropuseugenii), giant red kangaroo (Macropus rufus), fox kangaroo (Trichosurus vulpecula), koala (Phascolarctos cinereus), eastern quoll (Dasyurus viverrinus), platypus (Ornithorhynchus anatinus).As used herein, "mammalian milk oligosaccharides" or MMOs refer to oligosaccharides such as, but not limited to, those: 3-fucosyllactose, 2'-fucosyllactose, 6-fucosyllactose, 2',3-difucosyllactose, 2',2-difucosyllactose, 3,4-difucosyllactose, 6'-sialylactose, 3'-sialylactose, 3,6-disialylactose, 6,6'-disialylactose, 8,3-disialylactose, 3,6-disialyllacto-N-tetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentose II, lacto-N-fucopentose I, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, sialyllacto-N-tetraose C, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentose II, lacto-N-fucopentose I, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, sialyllacto-N-tetraose C, lacto-N-tetraose, lacto-N-fucopentose Sialyl lacto-N-tetraose b, sialyl lacto-N-tetraose a, lacto-N-difucohexose I, lacto-N-difucohexose II, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, monofucosylmonosialyl lacto-N-tetraose c, monofucosyl p-lacto-N-hexose, monofucosyl lacto-N-hexose III, isofucosylated lacto-N- Hexose III, isomeric fucosylated lacto-N-hexose I, sialyl lacto-N-hexose, sialyl lacto-N-neohexose II, difucosyl-p-lacto-N-hexose, difucosyl lacto-N-hexose, difucosyl lacto-N-hexose a, difucosyl lacto-N-hexose c, galactosylated chitosan, fucosylated oligosaccharides, neutral oligosaccharides and / or sialylated oligosaccharides.

[0053] The term "human milk oligosaccharides" or "HMOs" refers to oligosaccharides found in human breast milk (including premature human milk, colostrum and term human milk). HMOs include fucosylated oligosaccharides, non-fucosylated neutral oligosaccharides and sialylated oligosaccharides. Examples of HMOs include 3-fucosyllactose, 2'-fucosyllactose, 2',3-difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, LN3, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentose II, lacto-N-fucopentose I, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentose II, lacto-N-fucopentose I, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, sialyllactose, lacto-N-fucopentose VI ... Sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, difucosyllacto-N-tetraose, lacto-N-hexose, lacto-N-difucohexose I, lacto-N-difucohexose II, disialyllacto-N-tetraose, fucosyllacto-N-hexose, difucosyllacto-N-hexose, fucodisialyllacto-N-hexose, disialyllacto-N-hexose.

[0054] The terms "N-acetylglucosamine 1-phosphate uridyltransferase", "N-acetylglucosamine-1-phosphate uridyltransferase", "UDP-N-acetylglucosamine diphosphorylase", "UDP-N-acetylglucosamine pyrophosphorylase", "uridine diphosphate acetylglucosamine pyrophosphorylase", "UTP:2-acetylamino-2-deoxy-α-D-glucose-1-phosphate uridyltransferase", "UDP-GlcNAc pyrophosphorylase", "GlmU uridyltransferase", "acetylglucosamine 1-phosphate uridyltransferase" and "UDP-GlcNAc pyrophosphorylase" are used interchangeably. The terms "uridine diphosphate uridyltransferase," "UDP-acetylglucosamine pyrophosphorylase," "uridine diphosphate-N-acetylglucosamine pyrophosphorylase," "uridine diphosphate acetylglucosamine phosphorylase," and "acetylglucosamine 1-phosphate uridyltransferase" are used interchangeably and refer to an enzyme that catalyzes the conversion of N-acetylglucosamine 1-phosphate (GlcNAc-1-P) to UDP-N-acetylglucosamine (UDP-GlcNAc) by transferring uridine 5-monophosphate from uridine 5-triphosphate (UTP).

[0055] The term "glucosamine-1-phosphate acetyltransferase" refers to an enzyme that catalyzes the transfer of an acetyl group from acetyl-CoA to glucosamine-1-phosphate (GlcN-1-P) to produce N-acetylglucosamine-1-phosphate (GlcNAc-1-P).

[0056] The term "glmU" refers to a bifunctional enzyme that has both N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase activities and catalyzes two sequential reactions in the de novo biosynthetic pathway for UDP-GlcNAc. The C-terminal domain catalyzes the transfer of an acetyl group from acetyl-CoA to GlcN-1-P to produce GlcNAc-1-P, which is converted to UDP-GlcNAc via transfer of uridine 5-monophosphate, a reaction catalyzed by the N-terminal domain.

[0057] As used herein, the term "bifunctional N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase" refers to a polypeptide comprising both N-acetylglucosamine-1-phosphate uridyltransferase activity and glucosamine-1-phosphate acetyltransferase activity.

[0058] The terms "L-glutamine-D-fructose-6-phosphate aminotransferase," "glutamine-fructose-6-phosphate transaminase (isomerizing)," "hexose phosphate aminotransferase," "glucosamine-6-phosphate isomerase (glutamine forming)," "glutamine-fructose-6-phosphate transaminase (isomerizing)," "D-fructose-6-phosphate aminotransferase," "fructose-6-phosphate aminotransferase," "glucosamine phosphate isomerase," "glucosamine 6-phosphate synthase," "GlcN6P synthase," "GFA," "glms," "glmS," and "glmS*54" are used interchangeably and refer to an enzyme that catalyzes the conversion of D-fructose-6-phosphate to D-glucosamine-6-phosphate using L-glutamine.

[0059] As used herein, the term "pathway for producing disaccharides" is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of disaccharides as defined herein. The pathway for producing disaccharides may include, but is not limited to, pathways involving the synthesis of nucleotide-activated sugars and the transfer of the nucleotide-activated sugars to an acceptor to produce the disaccharides of the present invention. As used herein, the term "pathway for producing oligosaccharides" is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of oligosaccharides as defined herein. The pathway for producing oligosaccharides (e.g., milk oligosaccharides) may include, but is not limited to, pathways involving the synthesis of nucleotide-activated sugars and the transfer of the nucleotide-activated sugars to an acceptor to produce the oligosaccharides of the present invention (e.g., milk oligosaccharides). As used herein, the term "pathway for producing disaccharides and milk oligosaccharides" is a biochemical pathway consisting of the enzymes and their respective genes involved in the synthesis of disaccharides and milk oligosaccharides as defined herein. The pathways for producing disaccharides and milk oligosaccharides may include, but are not limited to, pathways involved in synthesizing nucleotide activated sugars and transferring the nucleotide activated sugars to acceptors to produce the disaccharides and milk oligosaccharides of the present invention. Examples of such pathways include, but are not limited to, fucosylation pathways, sialylation pathways, galactosylation pathways, N-acetylglucosaminylation pathways, N-acetylgalactosaminylation pathways, mannosylation pathways, and N-acetylmannosaminylation pathways.

[0060] The pathway for producing a disaccharide may include a pathway for synthesizing and / or importing a cofactor used in the pathway for producing a disaccharide.

[0061] The pathway for producing oligosaccharides (eg, milk oligosaccharides) may include pathways for the synthesis and / or import of cofactors used in the pathway for producing oligosaccharides (eg, milk oligosaccharides).

[0062] The pathway for producing disaccharides and milk-oligosaccharides may include pathways for the synthesis and / or import of cofactors used in the pathway for producing disaccharides and milk-oligosaccharides.

[0063] The term "purified" refers to a material that is substantially or essentially free of components that interfere with the activity of a biomolecule. For cells, carbohydrates, nucleic acids, and polypeptides, the term "purified" refers to a material that is substantially or essentially free of components that normally accompany the material (as found in its native state). Typically, the purified carbohydrates, oligosaccharides, proteins, or nucleic acids of the invention are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% pure, and usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure, as measured by band intensity on a silver-stained gel or other method for determining purity. Purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein or nucleic acid sample followed by visualization after staining. For some purposes, high resolution will be required and HPLC or similar means of purification will be employed. 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. Further herein, the terms "contaminants" and "impurities" preferably refer to particles, cells, cellular components, metabolites, cell debris, proteins, peptides, amino acids, nucleic acids, glycolipids, and / or endotoxins that may be present in an aqueous medium (e.g., a culture or incubation medium).

[0064] As used herein, the term "clarification" refers to the act of treating an aqueous medium (e.g., a culture, incubation medium) to remove suspended particles and contaminants from the production process, such as cells, cellular components, insoluble metabolites and debris, which may interfere with the final purification of the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides). Such treatment may be carried out in a conventional manner by centrifugation, flocculation, flocculation with optional sonication, gravity filtration, microfiltration, foam separation or vacuum filtration (e.g., through ceramic filters, which may include Celite TM filter aid) to carry out.

[0065] The term "culture" refers to the culture medium in which cells are cultured or fermented, the cells themselves, and the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) or oligosaccharide mixtures (e.g., milk oligosaccharide mixtures) produced by the cells in the whole culture fluid, i.e., inside (intracellularly) and outside (extracellularly) of the cells. As used herein, the terms "culture medium" and "incubation medium" are used interchangeably and refer to the medium in which cells are cultured.

[0066] The term "incubation" refers to a mixture in which disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) or a mixture of oligosaccharides (e.g., milk oligosaccharides) are produced. The mixture may include one or more enzymes, one or more precursors, and one or more acceptors (present in a buffer solution) as defined herein, and incubated at a certain temperature for a certain period of time so as to enable the production of i) disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) or ii) a mixture of oligosaccharides (e.g., milk oligosaccharides) by catalysis of the one or more enzymes using the one or more precursors and the one or more acceptors in the mixture. The mixture may also include: i) cells obtained after culture or incubation, optionally subjecting the cells to cell lysis; ii) a buffer solution or a culture or incubation medium in which cells are cultured or fermented; and iii) disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) or a mixture of oligosaccharides (e.g., milk oligosaccharides) produced by cells in a full culture solution, i.e., inside (intracellularly) and outside (extracellularly) of the cells. The incubation may also be culture as defined herein.

[0067] The terms "reactor" and "incubator" refer to containers filled with the culture or incubation. Examples of reactors or incubators include, but are not limited to, microfluidic devices, well plates, tubes, shake flasks, fermenters, bioreactors, process vessels, cell culture incubators, and CO2 incubators.

[0068] As used herein, the term "cell productivity index (CPI)" refers to the mass of disaccharides produced by cells divided by the mass of cells produced in culture. The term "cell productivity index (CPI)" also refers to the mass of oligosaccharides (e.g., milk oligosaccharides) produced by cells divided by the mass of cells produced in culture. The term "CPI" as used herein will also be understood as the mass of the mixture of disaccharides and milk oligosaccharides produced by cells divided by the mass of cells produced in culture. The term "CPI" as used herein will also be understood as the mass of the oligosaccharide mixture (e.g., milk oligosaccharide mixture) produced by cells divided by the mass of cells produced in culture.

[0069] As used in this article, term " breast cell " generally refers to mammalian mammary epithelial cell, mammalian mammary epithelial luminal cell or mammalian epithelial acinar cell, or its any combination.As used in this article, term " breast-like cell " generally refers to the mammalian cell with the phenotype / genotype similar to natural mammalian mammary cell (or substantially similar) but derived from mammalian non-breast cell source.Such mammalian mammary sample cell can be transformed to remove at least one undesirable genetic component and / or include at least one predetermined genetic construct typical for mammalian mammary cell.The non-limiting example of mammalian mammary sample cell can include: mammalian mammary epithelial cell, mammalian mammary epithelial luminal cell, the mammalian non-breast cell of one or more characteristics of the cell showing mammalian mammary cell lineage, or its any combination.The further non-limiting example of mammalian mammary sample cell can include the phenotype similar to natural mammalian mammary cell (or substantially similar), or more particularly the mammalian cell with the phenotype similar to natural mammalian mammary epithelial cell (or substantially similar). Mammalian cells having a phenotype similar to (or substantially similar to) natural mammalian mammary cells or mammalian mammary epithelial cells or exhibiting at least one feature similar to (or substantially similar to) natural mammalian mammary cells or mammalian mammary epithelial cells can include mammalian cells (e.g., derived from mammary cell lineages or non-mammary cell lineages) that naturally exhibit or have been modified to express at least one milk component. As used herein, the term "non-mammary cells" can generally include any mammalian cell of non-mammary lineages. In the context of the present invention, non-mammary cells can be any mammalian cell that can be modified to express at least one milk component. Non-limiting examples of such non-mammary cells include: hepatocytes, blood cells, kidney cells, umbilical cord blood cells, epithelial cells, epidermal cells, muscle cells, fibroblasts, mesenchymal cells, or any combination thereof. In some cases, molecular biology and genome editing techniques can be modified to eliminate, silence, or weaken numerous genes simultaneously.

[0070] As used in this article, the term "precursor" refers to a substance that is absorbed by a cell or synthesized for the specific production of a disaccharide and / or oligosaccharide (e.g., milk oligosaccharide) or oligosaccharide mixture (e.g., milk oligosaccharide mixture) according to the present invention. In this sense, a precursor can be an acceptor as defined herein, but can also be another substance, metabolite, cofactor that is first modified within a cell as part of the biochemical synthesis pathway of a disaccharide and / or oligosaccharide (e.g., milk oligosaccharide) or oligosaccharide mixture (e.g., milk oligosaccharide mixture). As used in this article, the term "precursor" will also be understood to be a chemical compound that participates in incubation or an enzymatic reaction to produce another compound (e.g., an intermediate or acceptor, as defined in this article) (as part of the metabolic pathway of a disaccharide and / or oligosaccharide (e.g., milk oligosaccharide) or oligosaccharide mixture (e.g., milk oligosaccharide mixture)). As used herein, the term "precursor" shall also be understood as a donor that is used by a glycosyltransferase to modify an acceptor as defined herein with a sugar moiety in a glycosidic bond as part of a metabolic pathway for a disaccharide and / or oligosaccharide (e.g., a milk oligosaccharide) or a mixture of oligosaccharides (e.g., a milk oligosaccharide mixture). Examples of such precursors include an acceptor as defined herein, and / or dihydroxyacetone, glucosamine, N-acetylglucosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, galactosyl lactose, a phosphorylated sugar or sugar phosphate, such as, but not limited to, glucose-1-phosphate, galactose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, mannose-6-phosphate, mannose-1-phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone-phosphate , glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylmannosamine-6-phosphate, N-acetylglucosamine-1-phosphate, N-acetylneuraminic acid-9-phosphate and nucleotide activating sugars, such as nucleotide diphosphate-sugars and nucleotide monophosphate-sugars as defined herein, such as UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, CMP-sialic acid, GDP-mannose, GDP-4-dehydro-6-deoxy-α-D-mannose, GDP-fucose.

[0071] Optionally, the cells used to produce the sugars (e.g., disaccharides and / or milk-oligosaccharides) are transformed to contain and express at least one nucleic acid sequence encoding a protein selected from the group consisting of a lactose transporter, an N-acetylneuraminic acid transporter, a fucose transporter, a glucose transporter, a galactose transporter, a transporter for nucleotide-activated sugars, wherein the transporter internalizes precursors added to the medium for the synthesis of the sugars (e.g., disaccharides and / or milk-oligosaccharides) of the present invention.

[0072] As used herein, the term "acceptor" 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, maltose, sucrose, lactose, lactulose, lactobionic acid (LBA), lacto-N-triose, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-pentose (LNP), lacto-N-neopentose, p-lacto-N-pentose, p-lacto-N-neopentose, Lacto-N-neopentose I, Lacto-N-hexose (LNH), Lacto-N-neohexose (LNnH), p-Lacto-N-neohexose (pLNnH), p-Lacto-N-hexose (pLNH), Lacto-N-heptose, Lacto-N-neoheptose, p-Lacto-N-neoheptose, p-Lacto-N-heptose, Lacto-N-octose (LNO), Lacto-N-neooctose, Allolacto-N-octose, p-Lacto-N-octose saccharides, allolacto-N-neooctose, neolacto-N-neooctose, paralacto-N-neooctose, allolacto-N-nonose, neolacto-N-nonose, lacto-N-nonose, lacto-N-nonose, lacto-N-decose, allolacto-N-decose, neolacto-N-decose, lacto-N-neodecanose, and oligosaccharides comprising one or more N-acetyllactosamine units and / or one or more lacto-N-biose units, or intermediates thereof, fucosylated and sialylated forms thereof, ceramides, N-acylated sphingoids, glucosylceramides, lactosylceramides, lactosylceramides, sphingosine, phytosphingosine, sphingosine synthons, peptide backbones having β-GlcNAc-Asn residues, glycoproteins having terminal GlcNAc and Gal residues, immunoglobulins. Detailed Description of the Invention

[0074] According to a first aspect, the present invention provides a cell capable of synthesizing and / or synthesizing UDP-N-acetylglucosamine (UDP-GlcNAc) and genetically engineered for the production of disaccharides and / or oligosaccharides (e.g., milk oligosaccharides), wherein the cell comprises a pathway for the production of said disaccharides and / or oligosaccharides (e.g., milk oligosaccharides), characterized in that UDP-GlcNAc synthesis in the cell is functionally impaired.

[0075] According to a second aspect, the present invention provides a method for producing disaccharides and / or oligosaccharides (e.g., milk oligosaccharides), wherein the method comprises culturing and / or incubating the cells described herein in a culture and / or incubation medium under conditions allowing the production of disaccharides and / or oligosaccharides (e.g., milk oligosaccharides).

[0076] In the context of the present invention, permissive conditions are understood as conditions concerning physical or chemical parameters including, but not limited to, temperature, pH, pressure, osmotic pressure and product / precursor / acceptor concentration.

[0077] In a particular embodiment, the permissive conditions may include a temperature range of 30 + / - 20 degrees Celsius, and a pH range of 7 + / - 3.

[0078] In a preferred embodiment, the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) are separated from the culturing and / or incubation.In another and / or additional preferred embodiment, the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) are purified.

[0079] In a particular embodiment of the methods and / or cells of the present invention, the cells are capable of synthesizing and / or synthesizing UDP-GlcNAc. UDP-GlcNAc can be provided by an enzyme expressed in the cell or by the metabolism of the cell. UDP-GlcNAc is formed from fructose-6-phosphate (Fru-6-P) in the four-step form of the Leloir pathway. In eukaryotic cells, four sequential reactions include: (a) conversion of fructose-6-phosphate (Fru-6-P) to glucosamine-6-phosphate (GlcN-6-P); (b) acetylation of GlcN-6-P to GlcNAc-6-P; (c) isomerization of GlcNAc-6-P to GlcNAc-1-P; and (d) uridylylation of GlcNAc-1-P to give UDPGlcNAc. In prokaryotes, the first and last steps are essentially the same as in eukaryotes, but GlcN-6-P is first isomerized to give GlcN-1-P, which is then N-acetylated. Each step of the eukaryotic pathway is catalyzed by a separate enzyme, while in bacteria there are only three enzyme proteins involved in UDP-GlcNAc biosynthesis. These enzymes can be any one or more of the following: N-acetyl-D-glucosamine kinase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, and N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase.

[0080] In a preferred embodiment of the method and / or cell, the cell synthesizes UDP-GlcNAc. In a more preferred embodiment, the cell comprises a pathway for producing UDP-GlcNAc. The pathway for producing UDP-GlcNAc is composed of enzymes involved in the synthesis of UDP-GlcNAc and their respective genes. The enzymes involved in the synthesis of UDP-GlcNAc include, but are not limited to, bifunctional N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, and glucosamine-1-phosphate acetyltransferase. The synthesis of UDP-GlcNAc can utilize one or more cofactors. Examples of cofactors include, but are not limited to, Mg, Mg, MgCl ... 2+ 、Co 2+ 、Mn2 + , Ca 2+ 、Zn 2+ 、Ni 2+ and FAD. The pathway for producing UDP-GlcNAc may include pathways for synthesizing and / or importing cofactors used in the pathway for producing UDP-GlcNAc. In other words, the cells of the present invention may include pathways for producing and / or importing Mg. 2+ 、Co 2+ 、Mn 2+ , Ca 2+ 、Zn 2+ 、Ni 2+ and FAD. Alternatively, the cell of the present invention does not synthesize the necessary cofactors for the synthesis of UDP-GlcNAc, but has all other enzymes necessary for the synthesis of UDP-GlcNAc. The cell can synthesize UDP-GlcNAc after being supplemented with one or more cofactors.

[0081] In a further particular embodiment, the UDP-GlcNAc synthesis in the cell is made to deteriorate in terms of function. In this article, the cell wherein the UDP-GlcNAc synthesis deteriorates in terms of function will be understood as: the cell has a lower UDP-GlcNAc production compared to the cell wherein the UDP-GlcNAc synthesis does not deteriorate in terms of function. According to the present invention, the synthesis of UDP-GlcNAc is made to deteriorate in terms of function in the cell by making one or more genes involved in the approach for the production of UDP-GlcNAc deteriorate in terms of function. Alternatively and / or additionally, the synthesis of UDP-GlcNAc is made to deteriorate in terms of function in the cell by making one or more genes involved in the approach for the production of and / or the input of one or more cofactors used in the synthesis of UDP-GlcNAc deteriorate in terms of function. Preferably, the synthesis of UDP-GlcNAc is made to deteriorate in terms of function in the cell by making one or more genes involved in the approach for the production of and / or the input of one or more cofactors selected from the following lists: Mg 2+ 、Co 2+ 、Mn 2+ , Ca 2+ 、Zn 2+ 、Ni 2+With FAD.Making gene variation in function will be understood as making gene less able (that is, statistically significantly " less able " or fully unable (for example, the gene of knocking out) compared to functional wild-type gene) to produce functional final product.Gene variation in function can be made by means of common well-known technology for technicians, for example, by any one or more of the insertion, disappearance and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the following lists: promoter sequence, ribosome binding site, non-translated region, coding sequence and transcription terminator sequence of the gene, so that the gene less able to produce functional final product.Methods such as siRNA, CrispR, CrispRi, riboswitch, recombineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutant gene and transposon mutagenesis can be used at this. Alternatively and / or additionally, the synthesis of UDP-GlcNAc is functionally impaired in the cell by replacing the natural pathway for UDP-GlcNAc production present in the cell with another pathway for UDP-GlcNAc production that gives lower UDP-GlcNAc production compared to the natural UDP-GlcNAc production pathway of the cell.

[0082] In a preferred embodiment of the methods and / or cells of the present invention, UDP-GlcNAc synthesis is achieved by expressing at least one gene selected from the list consisting of genes encoding a bifunctional N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, and glucosamine-1-phosphate acetyltransferase, and the at least one gene is functionally impaired as described herein. In other words, UDP-GlcNAc synthesis is achieved by expressing at least one gene selected from the list consisting of genes encoding a bifunctional N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, and glucosamine-1-phosphate acetyltransferase, and the at least one gene is functionally impaired as described herein. In another preferred embodiment of the method and / or cell of the invention, at least one gene involved in the synthesis and / or import of a cofactor involved in the synthesis of UDP-GlcNAc is functionally impaired or knocked out, preferably said cofactor is selected from the list comprising: Mg 2+ 、Co 2+ 、Mn 2+ , Ca 2+ 、Zn 2+ 、Ni 2+In another embodiment, the invention provides the method for the synthesis of UDP-GlcNAc and / or FAD.In a preferred embodiment, make at least one of the described genes involved in the synthesis of UDP-GlcNAc and / or the synthesis of the cofactor involving UDP-GlcNAc and / or the input involved in functional variation, this is carried out by inserting, disappearing and / or modifying one or more Nucleotide in the polynucleotide sequence selected from the following list comprising: promoter sequence, ribosome bind site, non-translational region, encoding sequence and transcription terminator sequence of described at least one gene.It will be understood in this article that by making at least one of the described genes deteriorate functionally in the cell, the described cell is made to have the UDP-GlcNAc that deteriorates functionally synthetic.As a result, described cell produces less UDP-GlcNAc than the cell that wherein does not make one of described genes deteriorate functionally.However, term " less UDP-GlcNAc " should not be interpreted as comprising 0g / L UDP-GlcNAc, because cell no longer survived at that time. In a more preferred embodiment, UDP-GlcNAc synthesis is obtained by expressing at least two genes selected from the list consisting of genes encoding a bifunctional N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, and glucosamine-1-phosphate acetyltransferase, and i) at least one of the at least two genes is functionally impaired, ii) at least two of the at least two genes are functionally impaired, or iii) all of the at least two genes are functionally impaired, as described herein.

[0083] In another and / or additional more preferred embodiment of the methods and / or cells of the invention, said at least one gene encodes an enzyme, wherein said enzyme is selected from the enzyme class selected from the list comprising EC:2.7.7.23, EC:2.3.1.157 and EC:5.4.2.3.

[0084] In another and / or additional more preferred embodiment of the methods and / or cells of the invention, the at least one gene encodes an enzyme, wherein the enzyme comprises a polypeptide sequence comprising an IPR domain selected from the list consisting of IPR001451, IPR002618, IPR005175, IPR005835, IPR005843, IPR005844, IPR005882, IPR011004, IPR016055, IPR016066, IPR016657, IPR018357, IPR023915, IPR025877, IPR029044, IPR036900 and IPR038009 as defined by InterPro 90.0 released on August 4, 2022.

[0085] In another and / or additional more preferred embodiment of the methods and / or cells of the invention, the at least one gene encodes an enzyme, wherein the enzyme comprises a polypeptide sequence comprising a PFAM domain selected from the list consisting of PF00132, PF00408, PF00465, PF00483, PF01070, PF01565, PF01704, PF02878, PF02879, PF02880, PF03479, PF04030, PF05199, PF12146, PF12804, PF13562 and PF14602 as defined by InterPro 90.0 released on August 4, 2022.

[0086] In another and / or additional more preferred embodiment of the methods and / or cells of the invention, the at least one gene encodes an enzyme, wherein the enzyme comprises a polypeptide sequence comprising a conserved protein domain selected from the list consisting of: cd03086 and cd03353 as defined by InterPro 90.0 released on August 4, 2022.

[0087] In another and / or additional more preferred embodiment of the methods and / or cells of the invention, the at least one gene encodes an enzyme, wherein the enzyme is part of the NOG family selected from the list comprising: COG1109 and COG4284 as defined by eggNOG5.0 released in 2019.

[0088] In another and / or additional more preferred embodiment of the method and / or cell of the invention, said at least one gene encodes an enzyme, wherein said enzyme uses a cofactor selected from the list comprising: Mg 2+ 、Co 2+ 、Mn2+ , Ca 2+ 、Zn 2+ 、Ni 2+ and FAD.

[0089] In a more preferred embodiment, the at least one gene encodes an enzyme, wherein the enzyme is selected from enzyme class EC: 2.3.1.157; comprising a polypeptide sequence comprising the IPR domains IPR001451, IPR005175, IPR005882, IPR011004, IPR018357, IPR025877, IPR029044 and IPR038009 as defined by InterPro 90.0 released on August 4, 2022; comprising a polypeptide sequence comprising the IPR domains IPR001451, IPR005175, IPR005882, IPR011004, IPR018357, IPR025877, IPR029044 and IPR038009 as defined by InterPro 90.0 released on August 4, 2022. PFAM domains PF00132, PF00465, PF00483, PF01070, PF01565, PF03479, PF04030, PF05199, PF12804, PF13562, and PF14602 as defined by InterPro 90.0, released on August 4, 2022; comprising a polypeptide sequence comprising a conserved protein domain cd03353 as defined by InterPro 90.0, released on August 4, 2022; and a portion of NOG family COG4284 as defined by eggNOG5.0, released in 2019; and using a polypeptide sequence comprising Mg 2+ 、Co 2+ 、Mn 2+ , Ca 2+ and Zn 2+ Select the cofactor from the list.

[0090] In another more preferred embodiment, the at least one gene encodes an enzyme, wherein the enzyme is selected from enzyme class EC: 5.4.2.3; comprises a polypeptide sequence comprising IPR domains IPR005843, IPR005844, IPR016055, IPR016066, IPR016657 and IPR036900 as defined by InterPro 90.0 released on August 4, 2022; comprises a polypeptide sequence comprising PFAM domains PF00408, PF02878, PF02879 and PF02880 as defined by InterPro 90.0 released on August 4, 2022; comprises a polypeptide sequence comprising 90.0; and part of the NOG family COG1109 defined by eggNOG5.0 released in 2019; and using proteins from the Mg 2+ 、Ni 2+ and FAD from the list of selected cofactors.

[0091] In another more preferred embodiment, the at least one gene encodes an enzyme, wherein the enzyme is selected from enzyme class EC: 2.7.7.23; comprising a polypeptide sequence comprising an IPR domain IPR001451, IPR002618, IPR005175, IPR005835, IPR005882, IPR011004, IPR018357, IPR023915, IPR025877, IPR029044, IPR038009 as defined by InterPro 90.0 released on August 4, 2022; comprising a polypeptide sequence comprising an IPR domain IPR001451, IPR002618, IPR005175, IPR005835, IPR005882, IPR011004, IPR018357, IPR023915, IPR025877, IPR029044, IPR038009 as defined by InterPro 90.0 released on August 4, 2022 PFAM domains PF00132, PF00483, PF01704, PF03479, PF12146, PF12804, PF13562, and PF14602 as defined by InterPro 90.0, released on August 4, 2022; comprising a polypeptide sequence comprising a conserved protein domain cd03353 as defined by InterPro 90.0, released on August 4, 2022; and a portion of NOG family COG4284 as defined by eggNOG5.0, released in 2019; and using a polypeptide sequence comprising Mg 2+ 、Co 2+ 、Mn 2+ , Ca 2+ 、Zn 2+ Select the cofactor from the list.

[0092] In an additional and / or alternative particular embodiment, the cell is genetically modified for the production of disaccharides, wherein the cell comprises a pathway for the production of the disaccharides. In a preferred embodiment, the cell is genetically modified for the production of two or more disaccharides. In another and / or additional preferred embodiment, the cell is genetically modified for the production of the enhancing of disaccharides, the enhanced uptake of one or more precursors and / or acceptors employed in the synthesis of disaccharides, the better outflow of disaccharides, the reduction of by-products (for example, acid), the increased availability of cofactors (for example, ATP, NADP, NADPH), and / or the better metabolic flux of any one of sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N-acetylgalactosaminylation, mannosylation and / or N-acetylmannosaminylation pathways present in the cell.

[0093] In an additional and / or alternative particular embodiment, described cell carries out genetic engineering for the production of oligosaccharides (for example, milk oligosaccharides), wherein said cell comprises the approach for the production of described oligosaccharides.In a preferred embodiment, described cell carries out genetic engineering for the production of two or more oligosaccharides (for example, two or more milk oligosaccharides).In another and / or additional preferred embodiment, described cell carries out genetic engineering for the production of the enhancing of oligosaccharides (for example, milk oligosaccharides), the uptake of the enhancing of one or more precursors and / or acceptors employed in the synthesis of oligosaccharides (for example, milk oligosaccharides), the better outflow of oligosaccharides (for example, milk oligosaccharides), the generation of the reduction of by-product (for example, acid), the increase availability of cofactor (for example, ATP, NADP, NADPH), and / or the better metabolic flux by any one in the sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N-acetylgalactosaminylation, mannosylation and / or N-acetylmannose aminylation pathway present in described cell.

[0094] In an additional and / or alternative particular embodiment, the cell is genetically engineered to produce disaccharides and milk oligosaccharides, wherein the cell comprises a pathway for producing the disaccharides and milk oligosaccharides. In a preferred embodiment, the cell is genetically engineered to produce one disaccharide and one milk oligosaccharide. In another preferred embodiment, the cell is genetically engineered to produce a) two or more disaccharides and b) one milk oligosaccharide. In another preferred embodiment, the cell is genetically engineered to produce a) one disaccharide and b) two or more milk oligosaccharides. In another preferred embodiment, the cell is genetically engineered to produce a) two or more disaccharides and b) two or more milk oligosaccharides.

[0095] In another and / or additional preferred embodiment, the cell is genetically engineered for enhanced production of disaccharides and milk oligosaccharides, enhanced uptake of one or more precursors and / or acceptors used in the synthesis of disaccharides and milk oligosaccharides, better efflux of disaccharides and milk oligosaccharides, reduced production of by-products (e.g., acids), increased availability of cofactors (e.g., ATP, NADP, NADPH), and / or better metabolic flux through any of the sialylation, fucosylation, galactosylation, N-acetylglucosaminylation, N-acetylgalactosaminylation, mannosylation and / or N-acetylmannosaminylation pathways present in the cell.

[0096] In a preferred aspect of the methods and / or cells of the invention, the genetically engineered cells are modified with gene expression modules, wherein expression from any one of the expression modules is constitutive or tunable.

[0097] The expression module is also referred to as a transcription unit and comprises a polynucleotide for expressing a recombinant gene, which comprises a coding gene sequence and suitable transcription and / or translation control signals operably connected to the coding sequence. The control signals include a promoter sequence, an untranslated region, a ribosome binding site, and a terminator sequence. The expression module can include elements for expressing a single recombinant gene but can also include elements for expressing multiple recombinant genes, or can be organized into an operon structure for the integrated expression of two or more recombinant genes. The polynucleotide can be produced by recombinant DNA technology using techniques well known in the art. It is well known to those skilled in the art that methods for constructing expression modules include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. See, for example, Sambrook et al., (2001) Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, CSH, New York or the technology described in Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989 and annual updates).

[0098] According to a preferred aspect of the present invention, the cell is modified with one or more expression modules. The expression module can be integrated into the genome of the cell or can be presented to the cell on a carrier. The carrier can exist in the form of plasmid, clay, phage, liposome or virus to be stably transformed / transfected into the cell through metabolic modification. This type of carrier especially includes chromosomal, additional and virus-derived carriers, such as those derived from bacterial plasmids, phages, transposons, yeast episomes, insertion elements, yeast chromosome elements, viruses, and carriers derived from their combinations, such as those derived from plasmid and phage genetic elements (such as clay and phagemids). These carriers can include selective markers, such as but not limited to antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA sense / antisense markers. The expression system construct can include regulation and control and cause the control region of expression. Generally, any system or carrier suitable for maintaining, propagating or expressing polynucleotides and / or expressed polypeptides in a host can be used for expression in this respect. The appropriate DNA sequence can be inserted into the expression system by any of a variety of well-known and conventional techniques, for example, those described in Sambrook et al. (see above). For recombinant production, cells can be genetically engineered to incorporate the expression system of the present invention, or portions thereof, or polynucleotides. Introduction of polynucleotides into cells can be accomplished by methods described in many standard laboratory manuals, for example, Sambrook et al., 1989 (see above).

[0099] 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 disaccharides and / or oligosaccharides (e.g., milk oligosaccharides); or the recombinant gene is associated with other pathways in the cell that are not involved in the synthesis of disaccharides and / or oligosaccharides (e.g., milk oligosaccharides). The recombinant gene encodes an endogenous protein with modified expression or activity, preferably overexpressed; or the recombinant gene encodes a heterologous protein that is heterogenously introduced and expressed (preferably overexpressed) in the modified cell. The endogenous protein may have modified expression in a cell that also expresses a heterologous protein.

[0100] According to a preferred aspect of the invention, the expression of each of said expression modules is constitutive or tunable, as defined herein.

[0101] In a preferred embodiment of the method and / or cell of the present invention, the pathway for producing disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) is selected from the list comprising, consisting of, or essentially consisting of: a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway, and an N-acetylmannosaminylation pathway. In a more preferred embodiment, the cell is genetically modified to comprise at least one of the pathways. In an even more preferred embodiment, the cell comprises at least one of the pathways, wherein at least one of the pathways has been genetically modified.

[0102] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises a fucosylation pathway. In a more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise a fucosylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been metabolically engineered to comprise a fucosylation pathway, wherein any one or more of the genes selected from a list comprising, consisting of, or consisting essentially of the following have modified and / or enhanced expression: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, and fucosyltransferase.

[0103] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises a sialylation pathway. In a more preferred embodiment, the cell is metabolically engineered to comprise a sialylation pathway. In another more preferred embodiment, the cell has been metabolically engineered to comprise a sialylation pathway, wherein any one or more of the genes selected from a list comprising, consisting of, or substantially consisting of the following have modified and / or enhanced expression: 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, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, sialic acid transporter, CMP kinase, CMP-sialic acid synthase, and sialyltransferase.

[0104] 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 more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise a galactosylation pathway, wherein any one or more of the genes selected from a list comprising, consisting of, or substantially consisting of the following have modified and / or enhanced expression: galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase, and galactosyltransferase.

[0105] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises an "N-acetylglucosaminylation" pathway. In a more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise an N-acetylglucosaminylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been metabolically engineered to comprise an N-acetylglucosaminylation pathway, wherein any one or more of the genes selected from a list comprising, consisting of, or consisting essentially of the following have modified and / or enhanced expression: L-glutamine-D-fructose-6-phosphate aminotransferase, 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 a glycosyltransferase that transfers GlcNAc.

[0106] 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 more preferred additional and / or alternative embodiment, the cell has been metabolically engineered to comprise an N-acetylgalactosaminylation pathway, wherein any one or more of the genes selected from the list comprising, consisting of, or consisting essentially of: L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine 1-phosphate 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-transferases has modified and / or enhanced expression.

[0107] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises a "mannosylation" pathway. In a more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise a mannosylation pathway. In another even more preferred additional and / or alternative embodiment, the cell has been metabolically engineered to comprise a mannosylation pathway in which any one or more of the genes selected from the list comprising, consisting of, or consisting essentially of mannose-6-phosphate isomerase, phosphomannose mutase, and / or mannose-1-phosphate guanylyltransferase and mannosyltransferase have modified and / or enhanced expression.

[0108] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises an "N-acetylmannosamination" pathway. In a more preferred additional and / or alternative embodiment, the cell is metabolically engineered to comprise an N-acetylmannosamination pathway. In another more preferred additional and / or alternative embodiment, the cell has been metabolically engineered to comprise an N-acetylmanosylation pathway, wherein any one or more of the genes selected from the list comprising, consisting of, or consisting essentially of: L-glutamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucosamine mutase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-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 ManNAc-transferring glycosyltransferases have modified and / or enhanced expression.

[0109] In another and / or additional preferred embodiment of the methods and / or cells of the invention, the cell comprises one or more pathways for monosaccharide synthesis. The pathways for monosaccharide synthesis comprise, consist of or consist essentially of enzymes such as carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, 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 any one or more nucleoside mono- or diphosphates (e.g., UMP and UDP, respectively), and enzymes involved in the synthesis of phosphoenolpyruvate (PEP).

[0110] In another and / or additional preferred embodiment of the method and / or cell of the present invention, the cell comprises one or more pathways for the synthesis of phosphorylated monosaccharides. The pathways for the synthesis of phosphorylated monosaccharides comprise, consist of, or consist essentially of enzymes involved in the synthesis of one or more monosaccharides, one or more nucleoside mono-, di-, and / or triphosphates, and enzymes involved in the synthesis of phosphoenolpyruvate (PEP), such as, but not limited to, PEP synthases, carboxylases, decarboxylases, isomerases, epimerases, reductases, enolases, phosphorylases, carboxykinases, kinases, phosphatases, aldolases, hydrolases, and dehydrogenases. In another and / or additional preferred embodiment of the method and / or cell of the present invention, the cell comprises one or more pathways for the synthesis of one or more nucleotide activated sugars. The pathway for nucleotide activated sugar synthesis comprises, consists of, or consists essentially of enzymes such as PEP synthase, carboxylase, decarboxylase, isomerase, epimerase, reductase, enolase, phosphorylase, carboxykinase, kinase, phosphatase, aldolase, hydrolase, dehydrogenase, mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP- fructose 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-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphate phosphatase, CMP-sialic acid synthase, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase, phosphoglucomutase and / or N-acetylglucosamine-1-phosphate uridyltransferase.

[0111] 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 essentially consisting of: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosaminetransferase, N-acetylgalactosaminetransferase, N-acetylmannosaminetransferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosaminetransferase, N-glycolylneuraminic acid transferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucosamine enolpyruvyltransferase and fucosamine transferase.

[0112] In a more preferred embodiment of the method and / or cell of the invention, the fucosyltransferase is selected from the list comprising, consisting of or consisting essentially of α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,4-fucosyltransferase and α-1,6-fucosyltransferase. In an alternative and / or further more preferred embodiment of the method and / or cell of the invention, the sialyltransferase is selected from the list comprising, consisting of or consisting essentially of α-2,3-sialyltransferase, α-2,6-sialyltransferase and α-2,8-sialyltransferase. In an alternative and / or further more preferred embodiment of the method and / or cell of the invention, the galactosyltransferase is selected from the list comprising, consisting of or consisting essentially of: β-1,3-galactosyltransferase, N-acetylglucosamine β-1,3-galactosyltransferase, β-1,4-galactosyltransferase, N-acetylglucosamine β-1,4-galactosyltransferase, α-1,3-galactosyltransferase and α-1,4-galactosyltransferase. In an alternative and / or further more preferred embodiment of the method and / or cell of the invention, the glucosyltransferase is selected from the list comprising, consisting of or consisting essentially of: α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase and β-1,4-glucosyltransferase. In an alternative and / or further more preferred embodiment of the methods and / or cells of the present invention, the mannosyltransferase is selected from the list comprising, consisting of, or consisting essentially of: alpha-1,2-mannosyltransferase, alpha-1,3-mannosyltransferase, and alpha-1,6-mannosyltransferase. In an alternative and / or further more preferred embodiment of the methods and / or cells of the present invention, the N-acetylglucosamine transferase is selected from the list comprising, consisting of, or consisting essentially of: galactoside beta-1,3-N-acetylglucosamine transferase, and beta-1,6-N-acetylglucosamine transferase. In an alternative and / or further more preferred embodiment of the methods and / or cells of the present invention, the N-acetylgalactosamine transferase is selected from the list comprising, consisting of, or consisting essentially of: alpha-1,3-N-acetylgalactosamine transferase.

[0113] In an alternative and / or further more preferred embodiment of the methods and / or cells of the invention, the cell is modified in terms of the expression or activity of at least one of the glycosyltransferases. In a preferred embodiment, the glycosyltransferase is an endogenous protein of the cell having modified expression or activity, preferably the endogenous glycosyltransferase is overexpressed; alternatively, the glycosyltransferase is a heterologous protein that is heterologously introduced and expressed (preferably overexpressed) in the cell. The endogenous glycosyltransferase may have modified expression in a cell that also expresses a heterologous glycosyltransferase.

[0114] In another and / or additional preferred embodiment of the method and / or cell, the cell is capable of producing, preferably produces, one or more nucleotide activating sugars, preferably the cell is genetically engineered for the production of one or more of said nucleotide activating sugars. Herein, the one or more nucleotide activating sugars are 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-acetylamino-2,6-dideoxy--L-arabino-4-hexulose, UDP-2-acetylamino-2,6-dideoxy--L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6-dideoxy-L-mannose), dTDP -N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-l-phospho-succinyl-L-glucosamine (UDP-L-PneNAc or UDP-2-acetylamino-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnosamine (UDP-L-QuiNAc or UDP-2 -acetylamino-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-Neu5,7(8,9)Ac2, CMP-Neu5Gc, GDP-rhamnose, and UDP-xylose.

[0115] In another and / or additional preferred embodiment of the method and / or cell, the cell comprises a pathway for synthesizing 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-acetylamino-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetylamino-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6 -dideoxy-L-mannose), dTDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-l-phospho-succinyl-L-olactose (UDP-L-PneNAC or UDP-2-acetylamino-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnosamine (UDP-L-QuiN Ac or UDP-2-acetylamino-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-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), GDP-rhamnose, and UDP-xylose.

[0116] Cell employed in this article is optionally genetically modified to express the de novo synthesis of UDP-GlcNAc.UDP-GlcNAc can be provided by the enzyme expressed in cell or by the metabolism of cell.This type of cell that produces UDP-GlcNAc can express the enzyme that for example GlcNAc (it is to be added into cell) is converted into UDP-GlcNAc.These enzymes can be for comprising the following, being made up of the following or being made up of the following list substantially any one or more: N-acetyl-D-glucosamine kinase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase and difunctional N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, it is from several species, comprises Homo sapiens (Homo sapiens), Escherichia coli.Preferably, described cell is modified to produce UDP-GlcNAc.

[0117] Additionally or alternatively, the cells used in this article are optionally genetically modified to express the de novo synthesis of CMP-Neu5Ac. CMP-Neu5Ac can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cells producing CMP-Neu5Ac can express an enzyme that converts, for example, sialic acid into CMP-Neu5Ac. The enzyme can be a CMP-sialic acid synthetase, such as N-acylneuraminic acid cytidylyltransferase from several 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 produce CMP-Neu5Ac for enhanced. The modification may be any one or more selected from the group consisting of, comprising, or essentially consisting of: knockout of N-acetylglucosamine-6-phosphate deacetylase, knockout of glucosamine-6-phosphate deaminase, overexpression of CMP-sialic acid synthetase, and overexpression of a gene encoding N-acetyl-D-glucosamine-2-epimerase.

[0118] Additionally or alternatively, the cells used in this article are optionally genetically modified to express the de novo synthesis of GDP-fucose. GDP-fucose can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cells producing GDP-fucose can express an enzyme that, for example, fucose (which is to be added to the cell) is converted into GDP-fucose. The enzyme can be, for example, a bifunctional fucokinase / fucose-1-phosphate guanylyltransferase, such as the Fkp from Bacteroides fragilis, or a separate fucokinase together with a separate fucose-1-phosphate guanylyltransferase, as known from several species, including Homo sapiens, wild boar (Sus scrofa) and Rattus norvegicus (Rattus norvegicus). Preferably, the cell is modified to produce GDP-fucose. More preferably, the cell is modified to produce GDP-fucose for enhanced. The modification can be any one or more selected from the list comprising, consisting of, or essentially consisting of: knockout of a gene encoding UDP-glucose:undecylenyl phosphate glucose-1-phosphotransferase, overexpression of a gene encoding GDP-L-fucose synthase, overexpression of a gene encoding GDP-mannose 4,6-dehydratase, overexpression of a gene encoding mannose-1-phosphate guanylyltransferase, overexpression of a gene encoding phosphomannose mutase, and overexpression of a gene encoding mannose-6-phosphate isomerase.

[0119] Additionally or alternatively, the cells used in this article are optionally genetically modified to express the de novo synthesis of UDP-Gal. UDP-Gal can be provided by an enzyme expressed in the cell or by the metabolism of the cell. Such cells producing UDP-Gal can express an enzyme that, for example, UDP-glucose is converted into UDP-Gal. This enzyme can be, for example, UDP-glucose-4-epimerase GalE, as known from several 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 produce UDP-Gal for enhanced production. The modification can be any one or more selected from a list comprising, consisting of, or essentially consisting of the following: knockout of a bifunctional 5'-nucleotidase / UDP-sugar hydrolase encoding gene, knockout of a galactose-1-phosphate uridyltransferase encoding gene, and overexpression of a UDP-glucose-4-epimerase encoding gene.

[0120] Additionally or alternatively, the cell used in this article is optionally genetically modified to express the de novo synthesis of UDP-GalNAc.UDP-GalNAc can be synthesized from UDP-GlcNAc by the effect of one-step reaction, wherein using UDP-N-acetylglucosamine 4-epimerase, for example from the wbgU of class Shigelloides, from the gne of Yersinia enterocolitica or from the wbpP of Pseudomonas aeruginosa serotype O6.Preferably, the cell is modified to produce UDP-GalNAc.More preferably, the cell is modified to produce for the UDP-GalNAc of enhancing.

[0121] Additionally or alternatively, the cells used in this article are optionally genetically modified to express the de novo synthesis of UDP-ManNAc. UDP-ManNAc can be directly synthesized from UDP-GlcNAc via the epimerization reaction performed by UDP-GlcNAc 2-epimerase (e.g., cap5P from Staphylococcus aureus (Staphylococcus aureus), RWE from Escherichia coli, Cps19fK from Streptococcus pneumoniae (S.pneumoniae) and RfbC from Salmonella enterica (S.enterica). Preferably, the cells are modified to produce UDP-ManNAc. More preferably, the cells are modified to produce UDP-ManNAc for enhanced.

[0122] 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 essentially consisting of: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, 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, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N- Acylneuraminic acid-9-phosphatase, 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.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, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GlcNAc 2-epimerase / kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, 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.

[0123] In a particular embodiment of the method and / or cell, the cell is genetically engineered to produce disaccharides and / or oligosaccharides (e.g., milk oligosaccharides), wherein the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) are selected from the list consisting of or consisting essentially of: lactobiose; mammalian milk biose; human milk biose; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); mammalian milk oligosaccharides; human milk oligosaccharides; neutral (uncharged) milk oligosaccharides; negatively charged, preferably sialylated milk oligosaccharides; fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; Animal milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine; milk oligosaccharides containing N-acetylglucosamine; milk oligosaccharides containing N-acetyllactosamine; milk oligosaccharides containing fucosylated N-acetyllactosamine; milk oligosaccharides containing sialylated N-acetyllactosamine; milk oligosaccharides containing milk-N-biose; milk oligosaccharides containing fucosylated milk-N-biose; and milk oligosaccharides containing sialylated milk-N-biose.

[0124] In a more preferred embodiment, the oligosaccharide is a mammalian milk oligosaccharide (MMO) as described herein. In another more preferred embodiment, the oligosaccharide is a human milk oligosaccharide (HMO) as described herein. In another more preferred embodiment, the fucosylated milk oligosaccharide is selected from the list comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lactose-N-hexose and difucosyl-lactose-N-neohexose. In another more preferred embodiment, the sialylated lacto-oligosaccharide is selected from the list comprising: 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N -hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II and monofucosyldisialyllacto-N-tetraose. In another more preferred embodiment, the neutral (uncharged) milk oligosaccharide containing N-acetylglucosamine is selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose.

[0125] In a more preferred embodiment, the oligosaccharide (e.g., milk oligosaccharide) is selected from the list comprising, consisting of, or consisting essentially of: 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose I I, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lact-N-hexose, difucosyl-lact-N-neohexose, lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto- N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose, 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialoyllacto-N-tetraose, disialoyllacto-N-neotetraose, monosialyllacto-N-hexose, disialoyllacto-N-neotetraose, Sialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II and monofucosyldisialyllacto-N-tetraose.

[0126] In the context of the present invention, the oligosaccharide is preferably in free form, ie the oligosaccharide does not comprise any protecting groups.

[0127] In another and / or additional preferred embodiment of the method and / or cell of the present invention, the cell is capable of producing from one or more precursors defined herein, preferably producing the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) from one or more precursors defined herein. In a more preferred embodiment, the precursor is lactose. Preferably, the one or more precursors are fed to the cell from a culture medium or a 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 even more preferred embodiment, the cell is genetically engineered to produce at least one of the one or more precursors. In an even more preferred embodiment, the cell is genetically engineered to produce all of the one or more precursors. In another even more preferred embodiment, at least one of the one or more precursors is internalized into the cell via one or more membrane proteins. In another preferred embodiment, the precursors used by the cell to produce the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) are completely converted into the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides).

[0128] In another and / or additional preferred embodiment of the method of the present invention and / or cell, the cell is further genetically modified to have, preferably express, glutamine-fructose-6-phosphate aminotransferase. In a more preferred embodiment, the cell is further genetically modified to overexpress glutamine-fructose-6-phosphate aminotransferase.

[0129] In another more preferred embodiment, the glutamine-fructose-6-phosphate aminotransferase has glutamine-fructose-6-phosphate aminotransferase activity and is selected from enzyme class EC: 2.6.1.16.

[0130] In another and / or additional more preferred embodiment, the glutamine-fructose-6-phosphate aminotransferase has glutamine-fructose-6-phosphate aminotransferase activity and comprises a polypeptide sequence comprising an IPR domain selected from the list consisting of or essentially consisting of IPR001347, IPR005855, IPR017932, IPR029055, IPR035466, IPR035490, IPR036291, IPR046348 and IPR047084 as defined by InterPro 90.0 released on August 4, 2022.

[0131] In another and / or additional more preferred embodiment, the glutamine-fructose-6-phosphate aminotransferase has glutamine-fructose-6-phosphate aminotransferase activity and comprises a polypeptide sequence comprising a PFAM domain selected from the list consisting of or essentially consisting of: PF00310, PF01380, PF01408, PF13230, PF13537 and PF13580 as defined by InterPro 90.0 released on August 4, 2022.

[0132] In another and / or additional more preferred embodiment, the glutamine-fructose-6-phosphate aminotransferase has glutamine-fructose-6-phosphate aminotransferase activity and comprises a polypeptide sequence comprising a conserved protein domain selected from the list consisting of or essentially consisting of: cd00714, cd05007, cd05008, cd05009, cd05013 and cd05710 as defined by InterPro 90.0 released on August 4, 2022.

[0133] In another and / or additionally more preferred embodiment, the glutamine-fructose-6-phosphate aminotransferase has glutamine-fructose-6-phosphate aminotransferase activity and is part of NOG family COG0449 defined by eggNOG5.0 released in 2019.

[0134] In another and / or additional preferred embodiment of the method and / or cell of the present invention, the cell comprises a nucleic acid molecule comprising a polynucleotide sequence encoding the glutamine-fructose-6-phosphate aminotransferase described herein. In a more preferred embodiment, the nucleic acid molecule is operably linked to a control sequence recognized by the cell, and the nucleic acid molecule is further i) integrated into the genome of the cell and / or ii) presented to the cell on a vector. In another and / or additional more preferred embodiment, the nucleic acid molecule is foreign to the cell.

[0135] In another and / or additional preferred embodiment of the methods and / or cells of the invention, the cells are modified for enhanced synthesis and / or supply of phosphoenolpyruvate (PEP).

[0136] In another and / or additional preferred embodiment of the methods and / or cells of the invention, the cells are further modified for reduced degradation of acetyl-CoA and / or its main precursor pyruvate.

[0137] In a preferred embodiment, the oligosaccharide of the present invention is produced by cells cultivated in cell culture. Within the context of the present invention, the cell culture comprises the in vitro and / or in vitro culture of the cell. In another and / or other more preferred embodiment, the cell culture is a fermentation. Alternatively and / or in another more preferred embodiment, the cell is cultivated or incubated among the reactor defined in this article. Alternatively and / or in another more preferred embodiment, the cell is cultivated or incubated among the incubator defined in this article.

[0138] In another and / or additional preferred embodiment, the cell is cultivated in a culture medium comprising a carbon source comprising, consisting of, or essentially consisting of: monosaccharides, disaccharides, oligosaccharides, polysaccharides, polyols, glycerol, a complex medium (which comprises molasses, corn steep liquor, peptone, tryptone, or yeast extract). Preferably, the carbon source is selected from a list comprising, consisting of, or essentially consisting of: glucose, N-acetylglucosamine (GlcNAc), glycerol, fructose, sucrose, maltose, lactose, arabinose, maltooligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn steep liquor, high fructose syrup, acetate, citrate, lactate, and pyruvate. In a more preferred embodiment, the culture medium comprises at least one carbon source selected from the list consisting of glucose, fructose, sucrose, and glycerol. In another and / or other preferred embodiment, the culture or incubation medium comprises at least one compound selected from the list consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid and CMP-sialic acid. In another and / or other more preferred embodiment, the culture or incubation medium is a medium with a known chemical composition. In another and / or other preferred embodiment, the culture or incubation medium is a minimal salt medium comprising sulfate, phosphate, chloride, ammonium, calcium, magnesium, sodium, potassium, iron, copper, zinc, manganese, cobalt and / or selenium. In another and / or other preferred embodiment, the culture or incubation medium comprises one or more precursors for the production of the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides). In a more preferred embodiment, the culture or incubation medium comprises one or more cofactors selected from the list comprising, consisting of or essentially consisting of: Mg 2 + 、Co 2+ 、Mn 2+ , Ca 2+ 、Zn 2+ 、Ni 2+ and FAD.

[0139] In another and / or additional preferred embodiment of the method of the present invention, the method for producing a disaccharide as described herein comprises at least one of the following steps:

[0140] i) adding at least one precursor and / or acceptor feed to a culture medium or a culture medium in a reactor, wherein the total reactor volume is from 250 mL (milliliters) to 10,000 mL 3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture medium or culture medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture medium or culture medium before the addition of the precursor and / or acceptor feed;

[0141] ii) adding at least one precursor and / or acceptor feed to the culture medium or the culture medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days with the aid of a feed solution;

[0142] iii) adding at least one precursor and / or acceptor feed to the culture medium or the culture medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days with the aid of a feed solution, wherein preferably the pH of the feed solution is set between 3 and 7; and wherein preferably the temperature of the feed solution is maintained between 20° C. and 80° C.;

[0143] The method results in a disaccharide wherein the concentration in the final volume of the culture medium or culture medium is at least 30 g / L.

[0144] In another and / or additional preferred embodiment of the method of the present invention, the method for producing a disaccharide as described herein comprises at least one of the following steps:

[0145] i) adding at least one precursor and / or acceptor to the culture medium in one pulse or in a discontinuous (pulsated) manner, wherein the total reactor volume is from 250 mL (milliliters) to 10,000 mL 3 (cubic meters), preferably such that the final volume of the culture medium or culturing medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture medium or culturing medium before the addition of the precursor and / or acceptor feed pulse;

[0146] ii) adding at least one precursor and / or acceptor feed to the culture medium or the culture medium in a discontinuous (pulsatile) manner with the aid of a feed solution over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days;

[0147] iii) adding at least one precursor and / or acceptor feed to the culture medium or the culture medium in a discontinuous (pulsating) manner with the aid of a feed solution over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, wherein preferably the pH of the feed solution is set between 3 and 7; and wherein preferably the temperature of the feed solution is maintained between 20° C. and 80° C.;

[0148] The method results in a disaccharide wherein the concentration in the final volume of the culture medium or culture medium is at least 30 g / L.

[0149] In a further more preferred embodiment, the method for producing a disaccharide as described herein comprises at least one of the following steps:

[0150] i) adding a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume to the culture medium or the culture medium, wherein the total reactor volume is from 250 mL (milliliters) to 10,000 mL 3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture medium or culture medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture medium or culture medium before the addition of the lactose feed;

[0151] ii) adding a lactose feed to the culture medium or the culture medium in a continuous manner with the aid of a feed solution during 1 day, 2 days, 3 days, 4 days, 5 days;

[0152] iii) adding a lactose feed to the culture medium or cultivation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days with the aid of a feed solution, 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, still 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 wherein preferably, the temperature of the feed solution is maintained between 20° C. and 80° C.;

[0153] The method results in a disaccharide wherein the concentration in the final volume of the culture medium or culture medium is at least 30 g / L.

[0154] In another and / or additional preferred embodiment of the method of the present invention, the method for producing the oligosaccharides described herein (e.g., milk oligosaccharides) comprises at least one of the following steps:

[0155] i) adding at least one precursor and / or acceptor feed to a culture medium or a culture medium in a reactor, wherein the total reactor volume is from 250 mL (milliliters) to 10,000 mL 3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture medium or culture medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture medium or culture medium before the addition of the precursor and / or acceptor feed;

[0156] ii) adding at least one precursor and / or acceptor feed to the culture medium or the culture medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days with the aid of a feed solution;

[0157] iii) adding at least one precursor and / or acceptor feed to the culture medium or the culture medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days with the aid of a feed solution, wherein preferably the pH of the feed solution is set between 3 and 7; and wherein preferably the temperature of the feed solution is maintained between 20° C. and 80° C.;

[0158] The method results in oligosaccharides (eg, milk oligosaccharides) wherein the concentration in the final volume of the culture medium or culture medium is at least 30 g / L.

[0159] In another and / or additional preferred embodiment of the method of the present invention, the method for producing the oligosaccharides described herein (e.g., milk oligosaccharides) comprises at least one of the following steps:

[0160] i) adding at least one precursor and / or acceptor to the culture medium in one pulse or in a discontinuous (pulsated) manner, wherein the total reactor volume is from 250 mL (milliliters) to 10,000 mL 3 (cubic meters), preferably such that the final volume of the culture medium or culturing medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the culture medium or culturing medium before the addition of the precursor and / or acceptor feed pulse;

[0161] ii) adding at least one precursor and / or acceptor feed to the culture medium or the culture medium in a discontinuous (pulsatile) manner with the aid of a feed solution over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days;

[0162] iii) adding at least one precursor and / or acceptor feed to the culture medium or the culture medium in a discontinuous (pulsating) manner with the aid of a feed solution over the course of 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, wherein preferably the pH of the feed solution is set between 3 and 7; and wherein preferably the temperature of the feed solution is maintained between 20° C. and 80° C.;

[0163] The method results in oligosaccharides (eg, milk oligosaccharides) wherein the concentration in the final volume of the culture medium or culture medium is at least 30 g / L.

[0164] In a further more preferred embodiment, the method for producing the oligosaccharides described herein (e.g., milk oligosaccharides) comprises at least one of the following steps:

[0165] i) adding a lactose feed comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume to the culture medium or the culture medium, wherein the total reactor volume is from 250 mL (milliliters) to 10,000 mL 3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the culture medium or culture medium is not more than three times, preferably not more than two times, more preferably less than two times the volume of the culture medium or culture medium before the addition of the lactose feed;

[0166] ii) adding a lactose feed to the culture medium or the culture medium in a continuous manner with the aid of a feed solution during 1 day, 2 days, 3 days, 4 days, 5 days;

[0167] iii) adding a lactose feed to the culture medium or cultivation medium in a continuous manner over the course of 1 day, 2 days, 3 days, 4 days, 5 days with the aid of a feed solution, 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, still 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 wherein preferably, the temperature of the feed solution is maintained between 20° C. and 80° C.;

[0168] The method results in oligosaccharides (eg, milk oligosaccharides) wherein the concentration in the final volume of the culture medium or culture medium is at least 30 g / L.

[0169] Preferably, the lactose feeding is achieved by adding lactose from the start of the culture 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.

[0170] In another aspect, the lactose feeding is achieved by adding lactose to the culture medium in such a concentration 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.

[0171] In a further embodiment of the methods described herein, the cells are cultured for at least about 60, 80, 100, or about 120 hours, or in a continuous manner.

[0172] In a preferred embodiment, the carbon source, preferably sucrose, is provided in the culture medium or culture medium for 3 or more days, preferably up to 7 days; and / or at least 100, advantageously at least 105, more advantageously at least 110, even more advantageously at least 120 g of sucrose per liter of initial culture volume is provided in the culture medium or culture medium in a continuous manner, so that the final volume of the culture medium or culture medium is no more than three times, advantageously no more than two times, more advantageously less than two times the volume of the culture medium or culture medium before culturing.

[0173] Preferably, when performing the methods described herein, a first phase of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the culture medium or culture medium, followed by addition of lactose to the culture medium or culture medium in a second phase.

[0174] In an alternative preferred embodiment, in the methods described herein, lactose is already added during the first phase of exponential growth together with the carbon substrate.

[0175] In another and / or additional preferred embodiment of the method and / or cell of the present invention, the cell comprises an at least partially inactivated catabolic pathway for selected mono-, di- or oligosaccharides that are involved in and / or required for the synthesis of said disaccharides and / or oligosaccharides (e.g. milk oligosaccharides).

[0176] In another and / or additional preferred embodiment of the methods and / or cells of the present invention, the cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably selected from the group 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.

[0177] The latter bacterium preferably belongs to the phylum Proteobacteria or Firmicutes or Cyanobacteria or Deinococcus-Thermus or Actinobacteria. The latter bacterium belonging to the phylum Proteobacteria preferably belongs to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacterium preferably relates to any strain belonging to the species Escherichia coli, such as but not limited to E. coli B, E. coli C, E. coli W, E. coli K12, E. coli Nissle. More particularly, the latter term relates to a cultured strain of E. coli - designated as the E. coli K12 strain - which is well adapted to the laboratory environment and, unlike the wild-type strain, has lost its ability to flourish in the intestine. Well-known examples of E. coli K12 strains are K12 wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Therefore, the present invention particularly relates to mutated and / or transformed E. coli cells or strains as indicated above, wherein the E. coli strain is a K12 strain. More preferably, the E. coli K12 strain is E. coli MG1655. The latter bacterium belonging to the phylum Firmicutes preferably belongs to the Bacillus, preferably the order Lactobacilliales, which has members such as Lactobacillus lactis, Leuconostoc mesenteroides, or the order Bacillales, which has members such as from the genus Bacillus, such as Bacillus subtilis or B. amyloliquefaciens. The latter bacterium belonging to the phylum Actinobacteria preferably belongs to the family Corynebacteriaceae, which has members Corynebacterium glutamicum or C. afermentans, or to the family Streptomycetaceae, which has members Streptomyces griseus or S. fradiae. The latter bacterium belonging to the phylum Proteobacteria preferably belongs to the family Vibrionaceae, which has as its member Vibrio natriegens.The latter yeast preferably belongs to the phylum Ascomycota or Basidiomycota or Deuteromycota or Zygomycetes. The latter yeast preferably belongs to the genus Saccharomyces (with members such as Saccharomyces cerevisiae, S. bayanus, S. boulardii), Zygosaccharomyces, Pichia (with members such as Pichia pastoris, P. anomala, P. kluyveri), Komagataella, Hansenula, Kluyveromyces (with members such as Kluyveromyces lactis lactis, K. marxianus, K. thermotolerans, Debaromyces, Candida, Schizosaccharomyces, Schwanniomyces, Torulaspora, Yarrowia (e.g., Yarrowia lipolytica), or Starmerella (e.g., Starmerella bombicola). The latter yeast is preferably selected from the group consisting of 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 preferably belongs to the genus Rhizopus, Dictyostelium, Penicillium, Mucor, or Aspergillus. Plant cells include cells of flowering plants and non-flowering plants, as well as algae cells, such as Chlamydomonas and Chlorella. Preferably, the plant is tobacco, rose, alfalfa, rice, tomato, cotton, oilseed rape, soybean, maize, or corn. 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., cow, bison, pig, sheep, mouse, rat, primate (e.g., chimpanzee, orangutan, gorilla, monkey (e.g., Old World monkey, New World monkey), lemur), dog, cat, rabbit, horse, cow, goat, steer, deer, musk deer, bovine, whale, dolphin, hippopotamus, elephant, rhino, giraffe, zebra, lion, cheetah, tiger, panda, red panda, otter), bird (e.g., chicken, duck, ostrich, turkey, pheasant), fish (e.g., swordfish, salmon, tuna, sea bass, trout, catfish), invertebrate (e.g., lobster, crab, shrimp, clams, oysters, mussels, sea urchin), reptile (e.g., snake, alligator, turtle), amphibian (e.g., frog), or insect (e.g., fly, nematode), or is a genetically modified cell line derived from a human cell (excluding embryonic stem cells). Both human and non-human mammalian cells are preferably selected from the list comprising: epithelial cells, such as mammary epithelial cells, embryonic kidney cells (e.g. HEK293 or HEK 293T cells), fibroblasts, COS cells, Chinese hamster ovary (CHO) cells, murine myeloma cells, such as N20, SP2 / 0 or YB2 / 0 cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, such as those described in WO21067641, preferably mesenchymal stem cells or derivatives thereof described in WO21067641, lactocytes derived from mammalian induced pluripotent stem cells (preferably human induced pluripotent stem cells), lactocytes as part of a mammary glandular organoid, postpartum mammary epithelial cells, polarized mammary cells, preferably polarized mammary cells selected from the group consisting of viable primary mammary epithelial cells, viable mammary myoepithelial cells, viable mammary progenitor cells, viable immortalized mammary epithelial cells, viable immortalized mammary myoepithelial cells, viable immortalized mammary progenitor cells, non-mammary adult stem cells or derivatives thereof, as well known to those skilled in the art, such as those described in WO21067641. 2021 / 219634, WO 2022 / 054053, WO 2021 / 141762, WO 2021 / 142241, WO 2021 / 067641 and WO 2021 / 242866.The latter insect cell is 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 cell is preferably a Leishmania tarentolae cell.

[0178] 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 gene LacY or LAC12, respectively.

[0179] In another and / or additional preferred embodiment of the methods and / or cells of the invention, the cells produce 30 g / L or more of the disaccharide in the whole broth and / or supernatant, and / or wherein the disaccharide in the whole broth and / or supernatant has a purity of at least 80%, measured based on the total amount of the disaccharide and its precursors produced by the cells in the whole broth and / or supernatant, respectively. In a more preferred embodiment, the cells produce 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 g / L, 72g / L, 73g / L, 74g / L, 75g / L, 76g / L, 77g / L, 78g / L, 79g / L, 80g / L, 81g / L, 82g / L, 83g / L, 84g / L, 85g / L or more than 85g / L of the disaccharide.

[0180] In another and / or additional preferred embodiment of the methods and / or cells of the invention, the cells produce 30 g / L or more of the oligosaccharides (e.g., milk oligosaccharides) in the whole broth and / or supernatant, and / or wherein the oligosaccharides (e.g., milk oligosaccharides) in the whole broth and / or supernatant have a purity of at least 80%, measured based on the total amount of oligosaccharides (e.g., milk oligosaccharides) and their precursors produced by the cells in the whole broth and / or supernatant, respectively. In a more preferred embodiment, the cells produce 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, g / L, 84g / L, 85g / L or more than 85g / L of such oligosaccharides (e.g., milk oligosaccharides).

[0181] In another and / or additional preferred embodiment of the methods and / or cells of the invention, the functionally impaired synthesis of UDP-GlcNAc confers unaffected and / or enhanced i) disaccharide and / or oligosaccharide (e.g., milk oligosaccharide) formation, ii) productivity, iii) biomass production, iv) cell growth, and / or v) yield of produced disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) relative to corresponding unmodified or unengineered cells.

[0182] In another aspect of the invention, the cell produces disaccharides and / or oligosaccharides as described herein (eg, milk oligosaccharides).In a preferred embodiment of the method and / or cell of the invention, the cell produces a mixture of oligosaccharides, such as a mixture of milk oligosaccharides.

[0183] In another aspect of the invention, the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) produced by the cells of the invention are recovered from the culture or incubation medium and / or the cells. In a preferred embodiment, the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) are purified.

[0184] The term "separated from the culture or incubation" means that the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) are harvested, collected, or recovered from the medium of cells and / or their growth. The disaccharides and oligosaccharides (e.g., milk oligosaccharides) can be separated from the aqueous culture medium or culture medium in which cells are grown in a conventional manner. In the case where the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) are still present in the cells producing the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides), conventional methods can be used to release or extract the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) from the cells, such as by cell destruction using high pH, ​​heat shock, ultrasonic treatment, French press, homogenate, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergent, hydrolysis, etc. The culture medium or culture medium and / or cell extract can then be used together and separately to further separate the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides). This preferably involves clarifying the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) to remove suspended particles and contaminants, particularly cells, cellular components, insoluble metabolites and debris produced by culturing genetically engineered cells. In this step, the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) can be clarified in a conventional manner. Preferably, the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) are clarified by centrifugation, flocculation, decantation and / or filtration. The second step of separating the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) preferably involves removing substantially all final remaining proteins, peptides, amino acids, RNA, DNA, endotoxins and glycolipids from the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) that can interfere with the subsequent separation step, preferably after it / they have been clarified. In this step, remaining proteins and related impurities can be removed from the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) in a conventional manner. Preferably, residual proteins, salts, by-products, color, endotoxins and other related impurities are removed from the di- and / or oligosaccharides (e.g., milk oligosaccharides) by ultrafiltration, nanofiltration, two-phase partitioning, reverse osmosis, microfiltration, activated carbon or carbon treatment, treatment with non-ionic surfactants, enzymatic digestion, tangential flow high performance filtration, tangential flow ultrafiltration, electrophoresis (e.g., using plate-polyacrylamide or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands including, for example, DEAE-Sepharose, poly-L-lysine and polymyxin-B, endotoxin-selective adsorption matrices), ion exchange chromatography (e.g., but not limited to, cation exchange, anion exchange, mixed bed ion exchange, inside-out ligand attachment), hydrophobic interaction chromatography and / or gel filtration (i.e., size exclusion chromatography), in particular by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography. With the exception of size exclusion chromatography, remaining protein and related impurities are retained by the chromatography medium or membrane of choice.

[0185] In a further preferred embodiment, the method described in this article also provides further purification of disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) of the present invention. The further purification of the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) can be completed, for example, by using (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzyme treatment or ion exchange to remove any remaining DNA, protein, LPS, endotoxin or other impurities. Alcohols, such as ethanol, and aqueous alcohol mixtures can also be used. Another purification step is completed by crystallization, evaporation or precipitation of the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides). Another purification step is drying, for example spray drying or freeze drying the produced disaccharides and / or oligosaccharides (e.g., milk oligosaccharides).

[0186] In an exemplary embodiment, the separation and purification of the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) is carried out in a process comprising the following steps in any order:

[0187] a) contacting the culture or a clarified form thereof with a nanofiltration membrane having a molecular weight cut-off (MWCO) of 600-3500 Da, thereby ensuring the retention of the produced disaccharides and / or oligosaccharides (e.g. milk oligosaccharides) and allowing at least a portion of the proteins, salts, by-products, colors and other related impurities to pass through;

[0188] b) subjecting the retentate from step a) to a diafiltration process using said membrane with an aqueous solution of an inorganic electrolyte, followed by optional diafiltration with pure water to remove excess electrolyte,

[0189] c) and collecting the retentate enriched in said disaccharides and / or oligosaccharides (eg milk oligosaccharides) in the form of salts of cations from said electrolyte.

[0190] In an alternative exemplary embodiment, the separation and purification of the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) is performed in a process comprising the following steps in any order: subjecting the culture or a clarified form thereof to two membrane filtration steps using different membranes, wherein

[0191] - a membrane having a molecular weight cut-off of about 300 to about 500 Daltons, and

[0192] - Another membrane has a molecular weight cut-off of about 600 to about 800 Daltons.

[0193] In an alternative exemplary embodiment, the separation and purification of the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) is carried out in a process comprising the following steps, in any order: treating the culture or a clarified form thereof with a strong cation exchange resin in H+-form and a weak anion exchange resin in free base form.

[0194] In an alternative exemplary embodiment, the separation and purification of the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) is performed as follows: a culture comprising i) produced disaccharides and / or oligosaccharides (e.g., milk oligosaccharides), 2) biomass, 3) medium components, and 4) contaminants (wherein the purity of the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) produced in the culture is <80%) is subjected to the following purification steps:

[0195] i) separating the biomass from the culture,

[0196] ii) cation exchanger treatment to remove positively charged materials,

[0197] iii) anion exchanger treatment to remove negatively charged materials,

[0198] iv) a nanofiltration step and / or an electrodialysis step,

[0199] A purified solution is provided, comprising the produced disaccharides and / or oligosaccharides (eg milk-oligosaccharides) at a purity greater than or equal to 80%. Optionally, the purified solution is spray-dried.

[0200] In an alternative exemplary embodiment, the separation and purification of the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) is carried out in a process comprising the following steps in any order: enzymatic treatment of the culture; removal of biomass from the culture; ultrafiltration; nanofiltration; and a column chromatography step. Preferably, such column chromatography is a single column or multiple columns. Further 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 or strong cation exchange resin; and / or ii) four zones I, II, III, and IV with different flow rates; and / or iii) an eluent comprising water; and / or iv) an operating temperature of 15 to 60 degrees Celsius.

[0201] In a particular embodiment, the present invention provides the produced disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) spray-dried to a powder, wherein the spray-dried powder comprises <15 wt% water, preferably <10 wt% water, more preferably <7 wt% water, most preferably <5 wt% water.

[0202] To identify the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) described herein, monomer building blocks (e.g., monosaccharide or polysaccharide unit composition), side chain anomeric configuration, the presence and position of substituent groups, degree of polymerization / molecular weight, and linkage patterns can be identified by standard methods known in the art such as those listed below: methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas chromatography-mass spectrometry), MALDI-MS (matrix-assisted laser desorption / ionization-mass spectrometry), ESI-MS (electrospray ionization-mass spectrometry), HPLC (high performance liquid chromatography with ultraviolet or refractive index detection), HPAEC-PAD (high performance anion exchange chromatography with pulsed amperometric detection), CE (capillary electrophoresis), IR (infrared) / Raman spectroscopy, and NMR (nuclear magnetic resonance) spectroscopy. Crystal structures can be solved using, for example, 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, for example, by viscometry and SEC (SEC-HPLC, high performance size exclusion chromatography). To identify the monomeric components of the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides), methods such as acid-catalyzed hydrolysis, HPLC (high performance liquid chromatography) or GLC (gas liquid chromatography) (after conversion to sugar alcohol acetate) can be used. To determine glycosidic bonds, the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) are methylated with methyl iodide and a strong base in DMSO, hydrolyzed, reduced to partially methylated sugar alcohols, acetylated to methylated sugar alcohol acetates, and analyzed by GLC / MS (gas liquid chromatography-mass spectrometry). To determine the glycan sequence, partial depolymerization is performed using acids or enzymes to determine the structure. To identify the anomeric configuration, the disaccharide and / or oligosaccharide (e.g., milk oligosaccharide) is subjected to enzymatic analysis, for example, by contacting it with an enzyme specific for a particular type of linkage (e.g., β-galactosidase or α-glucosidase, etc.), and the products can be analyzed using NMR.

[0203] In another aspect, the present invention provides the use of a cell as described herein for producing disaccharides and / or oligosaccharides (e.g., milk oligosaccharides), wherein the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) are selected from the list consisting of or consisting essentially of: lactobiose; mammalian milk biose; human milk biose; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (uncharged) milk oligosaccharides; negatively charged, preferably sialylated milk oligosaccharides; mammalian milk oligosaccharides (MMO); human milk oligosaccharides (HMO); neutral (uncharged) milk oligosaccharides; fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated milk oligosaccharides; acidified mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; fucosylated milk oligosaccharides selected from the list consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neo Fucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated milk oligosaccharides selected from the list consisting of 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LS Td), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialylacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialylacto-N-neohexose I, monosialylacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine;Neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine, preferably selected from the list comprising lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; milk oligosaccharides containing N-acetylglucosamine; milk oligosaccharides containing N-acetyllactosamine; milk oligosaccharides containing fucosylated N-acetyllactosamine; milk oligosaccharides containing sialylated N-acetyllactosamine; milk oligosaccharides containing lacto-N-biose; milk oligosaccharides containing fucosylated lacto-N-biose; and milk oligosaccharides containing sialylated lacto-N-biose.

[0204] In another aspect, the present invention provides the use of the methods described herein for producing disaccharides and / or oligosaccharides (e.g., milk oligosaccharides), wherein the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) are selected from the list consisting of or consisting essentially of: lactobiose; mammalian milk biose; human milk biose; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (uncharged) milk oligosaccharides; negatively charged, preferably sialylated milk oligosaccharides; mammalian milk oligosaccharides (MMO); human milk oligosaccharides (HMO); neutral (uncharged) milk oligosaccharides; fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated milk oligosaccharides; sialyl milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; fucosylated milk oligosaccharides selected from the list consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N- Neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated milk oligosaccharides selected from the list consisting of 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), sialyllacto-N-tetraose d (LSTe ... STd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialylacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialylacto-N-neohexose I, monosialylacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) oligosaccharides containing N-acetylglucosamine;Neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine selected from the list consisting of lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; milk oligosaccharides containing N-acetylglucosamine; milk oligosaccharides containing N-acetyllactosamine; milk oligosaccharides containing fucosylated N-acetyllactosamine; milk oligosaccharides containing sialylated N-acetyllactosamine; milk oligosaccharides containing lacto-N-biose; milk oligosaccharides containing fucosylated lacto-N-biose; and milk oligosaccharides containing sialylated lacto-N-biose.

[0205] In another aspect, the present invention provides a purified disaccharide and / or purified oligosaccharide (e.g., milk oligosaccharide) as described herein, or a purified oligosaccharide mixture (e.g., a purified milk oligosaccharide mixture), a purified disaccharide mixture, or a purified mixture of one or more disaccharides and one or more milk oligosaccharides for use in medicine, preferably for use in the prevention or treatment of gastrointestinal disorders.

[0206] In another aspect, the invention provides the purposes of the purified disaccharides and / or oligosaccharides (for example, milk oligosaccharides) obtained by the method described herein in food or feed preparations, in dietary supplements, in cosmetic ingredients or in pharmaceutical ingredients. In some embodiments, the disaccharides and / or oligosaccharides (for example, milk oligosaccharides) are mixed with one or more ingredients suitable for food, feed, dietary supplements, pharmaceutical ingredients, cosmetic ingredients or medicines. The purified disaccharides and / or oligosaccharides (for example, milk oligosaccharides) can be used for manufacturing preparations, as food additives, prebiotics, symbiotics, for the supplementation of infant food, adult food, young animal feed, adult animal feed, or as therapeutically or pharmaceutically active compounds, or used in cosmetic applications. In another aspect, the invention provides the purposes of the disaccharides and / or milk oligosaccharides described herein as additives in food, preferably as additives in human food and / or pet food, more preferably as additives in human infant food. In the context of the present invention, the food is a human food, preferably a baby food, a human infant food and / or an infant formula or an infant supplement, and the feed is a pet food, an animal milk replacer, a veterinary product, a veterinary feed supplement, a nutritional supplement, a weaning feed or a creep feed.

[0207] In another preferred embodiment, a preparation further comprising at least one probiotic microorganism is provided. In another preferred embodiment of the present invention, the preparation is a nutritional composition. In a more preferred embodiment, the preparation is a medical preparation, a dietary supplement, a dairy drink or an infant formula. "Prebiotics" are substances that promote the growth of microorganisms (particularly microorganisms in the gastrointestinal tract) that are beneficial to the host. In some embodiments, dietary supplements provide a variety of prebiotics, including disaccharides and / or oligosaccharides (e.g., milk oligosaccharides), which are prebiotics purified by the methods disclosed in this specification, to promote the growth of one or more beneficial microorganisms. Examples of prebiotic ingredients for dietary supplements include other prebiotic molecules (e.g., HMOs) and plant polysaccharides (e.g., inulin, pectin, b-glucans and xylo-oligosaccharides). "Prebiotics" products typically include live microorganisms that replace or are added to the gastrointestinal microbial flora for the benefit of the recipient. Examples of such microorganisms include Lactobacillus species (e.g., Lactobacillus acidophilus and Lactobacillus bulgaricus), Bifidobacterium species (e.g., B. animalis, B. longum, and B. infantis (e.g., Bi-26), and Saccharomyces boulardii. In some embodiments, the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) produced and / or purified by the methods of the present specification are orally administered in combination with such microorganisms. Examples of further ingredients for dietary supplements include oligosaccharides (e.g., 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyl lactose, 6'-sialyl lactose), disaccharides (e.g., lactose), monosaccharides (e.g., glucose, galactose, L-fucose, sialic acid, glucosamine, and N-acetylglucosamine), thickeners (e.g., gum arabic), acidity regulators (e.g., trisodium citrate), water, skim milk, and flavorings.

[0208] In some embodiments, the disaccharide and / or oligosaccharide (for example, milk oligosaccharide) purified by the method described herein is incorporated into people's infant food (for example, infant formula).Infant formula is generally for feeding artificial food to babies as a complete or partial substitute for human breast milk.In some embodiments, infant formula is sold with powder, and is ready to be bottled or cup-fed to babies by mixing with water.The composition of infant formula is typically designed to simulate human breast milk in general.In some embodiments, the disaccharide and / or oligosaccharide (for example, milk oligosaccharide) purified by the method described herein is included in infant formula, to provide those similar nutritional benefits provided by the disaccharide and / or oligosaccharide in human breast milk.In some embodiments, the purified disaccharide and / or oligosaccharide (for example, milk oligosaccharide) is mixed with one or more components of infant formula. Examples of infant formula ingredients include fat-free 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 (e.g., vitamins A, B6, B12, C, and D), minerals (e.g., potassium citrate, calcium citrate, magnesium chloride, sodium chloride, sodium citrate, and calcium phosphate), and possibly human milk oligosaccharides (HMOs). In some embodiments, the one or more infant formula ingredients include fat-free milk, a carbohydrate source, a protein source, a fat source, and / or vitamins and minerals. In some embodiments, the one or more infant formula ingredients include lactose, whey protein concentrate, and / or high oleic safflower oil. In some embodiments, the concentration of the disaccharides and / or oligosaccharides (e.g., milk oligosaccharides) in the infant formula is approximately the same concentration as that typically found in human breast milk. In some embodiments, di- and / or oligosaccharides (eg, milk oligosaccharides) purified by the methods described herein are added to the infant formula at a concentration that is approximately the same as the concentration at which these compounds are typically found in human breast milk.

[0209] Unless otherwise defined, all technical and scientific terms used in this article generally have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used in this article and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described above and below are those well known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, purification steps are carried out according to the manufacturer's instructions.

[0210] Further advantages can be derived from the specific embodiments and examples. It goes without saying that the features mentioned above and those still to be explained below can be used not only in the respectively specified combination, but also in other combinations or on their own without departing from the scope of the present invention.

[0211] Furthermore, the present invention relates to the following particular embodiments:

[0212] 1. A cell capable of synthesizing, preferably synthesizing, UDP-N-acetylglucosamine (UDP-GlcNAc), said cell comprising a pathway for producing oligosaccharides, said cell being genetically modified for the production of said oligosaccharides, characterized in that said UDP-GlcNAc synthesis in said cell is functionally impaired.

[0213] 2. The cell according to embodiment 1, wherein the pathway for producing oligosaccharides is selected from the list consisting of a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway and an N-acetylmannosaminylation pathway, 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.

[0214] 3. The cell according to any one of embodiments 1 or 2, wherein the cell:

[0215] - has, preferably expresses, more preferably overexpresses, one or more glycosyltransferases selected from the list consisting of fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosamine transferase, N-acetylgalactosamine transferase, N-acetylmannosamine transferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosamine transferase, N-glycolylneuraminic acid transferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucosamine enolpyruvyltransferase and fucosamine transferase, and / or

[0216] - capable of producing, preferably producing, one or more nucleotide activating sugars, preferably the cell is genetically engineered for the production of one or more of said nucleotide activating sugars.

[0217] 4. The cell according to any one of the preceding embodiments, wherein the cell comprises a pathway for synthesizing a nucleotide activated sugar selected from the list consisting 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-acetylamino-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetylamino-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6-dideoxy-L-mannose), d TDP-N-acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-phospho-L-galactose (UDP-L-PneNAc or UDP-2-acetylamino-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnosamine (UDP-L-QuiNAc or UDP- 2-acetylamino-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-Neu5,7(8,9)Ac2, CMP-N-glycolylneuraminic acid (CMP-Neu5Gc), GDP-rhamnose, and UDP-xylose.

[0218] 5. The cell according to any one of the preceding embodiments, wherein the cell has, preferably expresses, one or more genes selected from the list consisting of: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, 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, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, 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, more preferably overexpressing one or more genes selected from the list comprising: mannose-6-phosphate isomerase, phosphomannosemutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GlcNAc 2-epimerase / kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, 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.

[0219] 6. The cell of any one of the preceding embodiments, wherein the UDP-GlcNAc synthesis comprises at least one gene selected from the list consisting of genes encoding a bifunctional N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, and glucosamine-1-phosphate acetyltransferase, and wherein the at least one gene is functionally impaired.

[0220] 7. The cell of embodiment 6, wherein the at least one gene encodes an enzyme, wherein the enzyme:

[0221] - an enzyme class selected from the list consisting of EC: 2.7.7.23, EC: 2.3.1.157 and EC: 5.4.2.3,

[0222] comprising a polypeptide sequence comprising an IPR domain selected from the list consisting of IPR001451, IPR002618, IPR005175, IPR005835, IPR005843, IPR005844, IPR005882, IPR011004, IPR016055, IPR016066, IPR016657, IPR018357, IPR023915, IPR025877, IPR029044, IPR036900 and IPR038009 as defined by InterPro 90.0 released on August 4, 2022,

[0223] comprising a polypeptide sequence comprising a PFAM domain selected from the list consisting of PF00132, PF00408, PF00465, PF00483, PF01070, PF01565, PF01704, PF02878, PF02879, PF02880, PF03479, PF04030, PF05199, PF12146, PF12804, PF13562, and PF14602 as defined by InterPro 90.0 released on August 4, 2022,

[0224] - comprising a polypeptide sequence comprising a conserved protein domain selected from the list consisting of cd03086 and cd03353 as defined by InterPro 90.0 released on August 4, 2022,

[0225] - is part of a NOG family selected from a list consisting of: COG1109, COG4284, and / or

[0226] - Using a cofactor selected from the list including: Mg 2+ 、Co 2+ 、Mn 2+ , Ca 2+ 、Zn 2+ 、Ni 2+ and FAD.

[0227] 8. The cell according to any one of the preceding embodiments, wherein at least one gene involved in the synthesis and / or import of the following cofactors is rendered functionally impaired: a cofactor involved in the synthesis of UDP-GlcNAc, preferably said cofactor is selected from the group consisting of Mg 2+ 、Co 2+ 、Mn 2+ , Ca2+ 、Zn 2+ 、Ni 2+ and a list of FADs.

[0228] 9. The cell of any one of embodiments 6 to 8, wherein the at least one gene is functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list comprising: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the at least one gene.

[0229] 10. The cell according to any one of the preceding embodiments, wherein the oligosaccharide is selected from the list comprising: neutral (uncharged) oligosaccharides; negatively charged, preferably sialylated oligosaccharides; milk oligosaccharides, preferably mammalian milk oligosaccharides (MMOs), more preferably human milk oligosaccharides (HMOs); sialylated milk oligosaccharides; neutral (uncharged) milk oligosaccharides; fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) Human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigens (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigenic oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, which are preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, which are preferably selected from the group consisting of N-glycans and O-glycans; fucosylated oligosaccharides, which are preferably selected from the group consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3- difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated oligosaccharides, preferably selected from the group comprising 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllactose-N-tetraose a (LSTa), sialyllactose-N-tetraose b (LSTb), sialyllactose -N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose;Neutral (uncharged) oligosaccharides containing N-acetylglucosamine, preferably selected from the group consisting of lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; non-fucosylated neutral (uncharged) oligosaccharides; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate.

[0230] 11. The cell according to any one of the preceding embodiments, wherein the cell is capable of producing the oligosaccharide from one or more precursors, preferably producing the oligosaccharide from one or more precursors, preferably the precursor is lactose.

[0231] 12. The cell according to embodiment 11, 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.

[0232] 13. The cell according to any one of embodiments 11 or 12, 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.

[0233] 14. The cell of any one of embodiments 11 to 13, wherein at least one of the one or more precursors is internalized into the cell via one or more membrane proteins.

[0234] 15. The cell according to any one of the preceding embodiments, wherein the cell is further genetically engineered to have, preferably express, more preferably overexpress, glutamine-fructose-6-phosphate aminotransferase.

[0235] 16. The cell of embodiment 15, wherein the glutamine-fructose-6-phosphate aminotransferase has glutamine-fructose-6-phosphate aminotransferase activity and:

[0236] - is selected from enzyme class EC: 2.6.1.16,

[0237] comprising a polypeptide sequence comprising an IPR domain selected from the list consisting of IPR001347, IPR005855, IPR017932, IPR029055, IPR035466, IPR035490, IPR036291, IPR046348 and IPR047084 as defined by InterPro 90.0 released on August 4, 2022,

[0238] - comprising a polypeptide sequence comprising a PFAM domain selected from the list consisting of PF00310, PF01380, PF01408, PF13230, PF13537 and PF13580 as defined by InterPro 90.0 released on August 4, 2022,

[0239] - comprising a polypeptide sequence comprising a conserved protein domain selected from the list consisting of cd00714, cd05007, cd05008, cd05009, cd05013 and cd05710 as defined by InterPro 90.0 released on 4 August 2022, and / or

[0240] - is part of the NOG family COG0449 defined by eggNOG5.0 released in 2019.

[0241] 17. The cell of any one of embodiments 15 or 16, wherein the cell comprises a nucleic acid molecule comprising a polynucleotide sequence encoding the glutamine-fructose-6-sulfate aminotransferase.

[0242] 18. The cell of embodiment 17, wherein the nucleic acid molecule is operably linked to a control sequence recognized by the cell, and the nucleic acid molecule is further i) integrated into the genome of the cell and / or ii) presented to the cell on a vector.

[0243] 19. The cell of any one of embodiments 17 or 18, wherein the nucleic acid molecule is foreign to the cell.

[0244] 20. The cell according to any one of the preceding embodiments, wherein the cell is modified for enhanced synthesis and / or supply of phosphoenolpyruvate (PEP).

[0245] 21. The cell of any one of the preceding embodiments, wherein the cell is further modified for reduced degradation of acetyl-CoA and / or its primary precursor pyruvate.

[0246] 22. The cell of any one of the preceding embodiments, wherein the cell comprises an at least partially inactivated catabolic pathway for a selected mono-, di- or oligosaccharide involved in and / or required for the synthesis of the oligosaccharide.

[0247] 23. The cell according to any one of the preceding embodiments, wherein the cell is a bacterium, a fungus, a yeast, a plant cell, an animal cell or a protozoan cell,

[0248] - Preferably, the bacterium belongs to a phylum selected from the group consisting of Proteobacteria, Firmicutes, Cyanobacteria, Deinococcus-Thermus and Actinobacteria; more preferably, the bacterium belongs to a family selected from the group consisting of Enterobacteriaceae, Bacillaceae, Lactobacillus, Corynebacterium and Vibrio; even more preferably, the bacterium is selected from the list consisting of Escherichia coli strains, Bacillus subtilis strains, Vibrio natriuresis strains; even more preferably, the Escherichia coli strain is K12 strain; most preferably, the Escherichia coli K12 strain is Escherichia coli MG1655,

[0249] - preferably, the fungus belongs to a genus selected from the group consisting of Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus,

[0250] - preferably, the yeast belongs to a genus selected from the group consisting of Saccharomyces, Zygosaccharomyces, Pichia, Komagata, Hansenula, Yarrowia, Stamosaccharomyces, Kluyveromyces, Debaryomyces, Candida, Schizosaccharomyces, Schwannella or Torulospora; more preferably, the yeast is selected from the group consisting of Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Pichia methanolica, Pichia stipitis, Candida boidinii, Schizosaccharomyces pombe, Schwannella occidentalis, Torulospora delavirdii, Yarrowia lipolytica, Zygosaccharomyces rouxii and Zygosaccharomyces bailii,

[0251] - Preferably, the plant cell is an algae cell or is derived from tobacco, alfalfa, rice, tomato, cotton, oilseed rape, soybean, maize or corn plants,

[0252] -Preferably, the animal cell is derived from an insect, amphibian, reptile, invertebrate, fish, bird or mammalian cell (excluding human embryonic stem cells); more preferably, the mammalian cell is selected from the list comprising the following: epithelial cells, embryonic kidney cells, fibroblasts, COS cells, Chinese hamster ovary (CHO) cells, mouse myeloma cells, NIH-3T3 cells, mammary cells derived from mammalian induced pluripotent stem cells, more preferably, the mammalian induced pluripotent stem cells are human induced pluripotent stem cells, postpartum mammary epithelial cells, polarized mammary cells; more preferably, the polarized mammary cells are selected from the group comprising living primary mammary epithelial cells, living mammary myoepithelial cells, living mammary progenitor cells, living immortalized mammary epithelial cells, living immortalized mammary myoepithelial cells, living immortalized mammary progenitor cells, non-mammary adult stem cells or derivatives thereof; more preferably, the insect cell is derived from Spodoptera frugiperda, Bombyx mori, Spodoptera brassicae, Trichoplusia ni or Drosophila melanogaster,

[0253] - Preferably, the protozoan cell is a Leishmania cell.

[0254] 24. The cell according to any one of the preceding embodiments, wherein the cell is a bacterium, a fungus, a yeast, a plant cell, an animal cell, or a protozoan cell.

[0255] 25. The cell according to any one of the preceding embodiments, wherein the cell is Escherichia coli or yeast having a lactose permease positive phenotype, preferably wherein the lactose permease is encoded by the gene LacY or LAC12, respectively.

[0256] 26. A method for producing oligosaccharides, the method comprising:

[0257] i. cultivating and / or incubating the cell according to any one of the preceding embodiments in a culture and / or incubation medium under conditions allowing the production of said oligosaccharide,

[0258] ii. Preferably, said oligosaccharides are separated from said culturing and / or incubating.

[0259] 27. The method of embodiment 26, wherein the culture or incubation medium comprises one or more precursors for producing the oligosaccharide.

[0260] 28. The method according to any one of embodiments 26 or 27, wherein the culture medium comprises at least one carbon source selected from the group consisting of glucose, fructose, sucrose and glycerol.

[0261] 29. The method according to any one of embodiments 26 to 28, wherein the culture or incubation medium comprises at least one compound selected from the group consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid and CMP-sialic acid.

[0262] 30. The method of any one of embodiments 26 to 29, wherein the cells produce 30 g / L or more of the oligosaccharides in the whole broth and / or supernatant, and / or wherein the oligosaccharides in the whole broth and / or supernatant have a purity of at least 80%, measured based on the total amount of oligosaccharides and their precursors produced by the cells in the whole broth and / or supernatant, respectively.

[0263] 31. The method of any one of embodiments 26 to 30, wherein functionally impaired synthesis of UDP-GlcNAc confers unaffected and / or enhanced i) oligosaccharide formation, ii) productivity, iii) biomass production, iv) cell growth, and / or v) yield of produced oligosaccharides relative to corresponding unmodified cells.

[0264] 32. The method according to any one of embodiments 26 to 31, wherein the oligosaccharide is recovered from the culture or incubation medium and / or the cells, more preferably purified.

[0265] 33. Use of a cell according to any one of embodiments 1 to 25 for producing an oligosaccharide, wherein the oligosaccharide is selected from the list comprising: a neutral (uncharged) oligosaccharide; a negatively charged, preferably sialylated oligosaccharide; a milk oligosaccharide, preferably a mammalian milk oligosaccharide (MMO), more preferably a human milk oligosaccharide (HMO); a sialylated milk oligosaccharide; a neutral (uncharged) milk oligosaccharide; a fucosylated milk oligosaccharide; a non-fucosylated neutral (uncharged) milk oligosaccharide; a sialylated mammalian milk oligosaccharide; a neutral (uncharged) mammalian milk oligosaccharide; a fucosylated mammalian milk oligosaccharide; a non-fucosylated neutral (uncharged) mammalian milk oligosaccharide; a sialylated human milk oligosaccharide; a neutral (uncharged) mammalian milk oligosaccharide; a fucosylated mammalian milk oligosaccharide; a non-fucosylated neutral (uncharged) mammalian milk oligosaccharide; a neutral neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigens (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigenic oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; fucosylated oligosaccharides, preferably selected from the group comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL) , 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated oligosaccharides, which are preferably selected from the group comprising: 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllactose Lacto-N-tetraose c (LSTc), Lacto-N-tetraose d (LSTd), disialoyl lacto-N-tetraose, disialoyl lacto-N-neotetraose, monosialoyl lacto-N-hexose, disialoyl lacto-N-hexose I, disialoyl lacto-N-hexose II, monosialoyl lacto-N-neohexose I, monosialoyl lacto-N-neohexose II, disialoyl lacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialoyl lacto-N-hexose, disialoyl monofucosyllacto-N-neohexose, sialoyl lacto-N-fucohexose II, disialoyl lacto-N-fucopentose II, and monofucosyldisialoyl lacto-N-tetraose;Neutral (uncharged) oligosaccharides containing N-acetylglucosamine, preferably selected from the group consisting of lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; non-fucosylated neutral (uncharged) oligosaccharides; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate.

[0266] 34. Use of the method according to any one of embodiments 26 to 32 for the production of oligosaccharides, wherein the oligosaccharides are selected from the list comprising: neutral (uncharged) oligosaccharides; negatively charged, preferably sialylated oligosaccharides; milk oligosaccharides, preferably mammalian milk oligosaccharides (MMO), more preferably human milk oligosaccharides (HMO); sialylated milk oligosaccharides; neutral (uncharged) milk oligosaccharides; fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; Neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; O-antigens; enterobacterial common antigens (ECA); oligosaccharide repeats present in capsular polysaccharides; peptidoglycans; amino sugars; Lewis-type antigenic oligosaccharides; antigens of the human ABO blood group system; animal oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; plant oligosaccharides, preferably selected from the group consisting of N-glycans and O-glycans; fucosylated oligosaccharides, preferably selected from the group comprising 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL) , 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated oligosaccharides, which are preferably selected from the group comprising: 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllactose Lacto-N-tetraose c (LSTc), Lacto-N-tetraose d (LSTd), disialoyl lacto-N-tetraose, disialoyl lacto-N-neotetraose, monosialoyl lacto-N-hexose, disialoyl lacto-N-hexose I, disialoyl lacto-N-hexose II, monosialoyl lacto-N-neohexose I, monosialoyl lacto-N-neohexose II, disialoyl lacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialoyl lacto-N-hexose, disialoyl monofucosyllacto-N-neohexose, sialoyl lacto-N-fucohexose II, disialoyl lacto-N-fucopentose II, and monofucosyldisialoyl lacto-N-tetraose;Neutral (uncharged) oligosaccharides containing N-acetylglucosamine, preferably selected from the group consisting of lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, p-lacto-N-neohexose; oligosaccharides containing N-acetylglucosamine; oligosaccharides containing N-acetyllactosamine; oligosaccharides containing lacto-N-biose; non-fucosylated neutral (uncharged) oligosaccharides; chitosan; oligosaccharides containing chitosan; heparosan; chondroitin sulfate; glycosaminoglycan oligosaccharides; heparin; heparan sulfate; dermatan sulfate; hyaluronan; hyaluronic acid; and keratan sulfate.

[0267] More particularly, the present invention relates to the following preferred particular embodiments:

[0268] 1. A cell capable of synthesizing and / or synthesizing UDP-N-acetylglucosamine (UDP-GlcNAc), said cell comprising a pathway for producing disaccharides and / or milk-oligosaccharides, said cell being genetically modified for the production of said disaccharides and / or milk-oligosaccharides, characterized in that said UDP-GlcNAc synthesis in said cell is functionally impaired.

[0269] 2. The cell according to preferred embodiment 1, wherein:

[0270] - said pathway for producing disaccharides and / or milk oligosaccharides is selected from the list consisting of or consisting essentially of: a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway and an N-acetylmannosaminylation pathway, and / or

[0271] - said pathway for producing disaccharides and / or milk-oligosaccharides is selected from the list consisting of or consisting essentially of: a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway and an N-acetylmannosaminylation pathway, and wherein said cell is genetically engineered to comprise at least one of said pathways, and / or said cell comprises at least one of said pathways, wherein at least one of said pathways has been genetically engineered.

[0272] 3. The cell according to any one of preferred embodiments 1 or 2, wherein the cell:

[0273] - having, expressing and / or overexpressing one or more glycosyltransferases selected from the list consisting of or consisting essentially of: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosamine transferase, N-acetylgalactosamine transferase, N-acetylmannosamine transferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosamine transferase, N-glycolylneuraminic acid transferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucosamine enolpyruvyltransferase and fucosamine transferase,

[0274] - is capable of producing and / or produces one or more nucleotide activating sugars,

[0275] - genetically engineered to produce one or more nucleotide activated sugars,

[0276] - comprising a pathway for synthesizing a nucleotide-activated sugar selected from the list 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-acetylamino-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetylamino-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6-dideoxy-L-mannose), dTDP-N- Acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-l-phospho-glucosamine (UDP-L-PneNAc or UDP-2-acetylamino-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnosamine (UDP-L-QuiNAc or UDP-2-acetylamino-2,6-dideoxy-L-talose), CMP-Neu5Ac, CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-Neu5Gc, GDP-rhamnose, and UDP-xylose,

[0277] - having and / or expressing one or more genes selected from the list consisting of or consisting essentially of: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, 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, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, 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, more preferably overexpressing one or more genes selected from the list comprising: mannose-6-phosphate isomerase, phosphomannosemutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GlcNAc 2-epimerase / kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, 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, and / or,

[0278] comprising at least partially inactivated catabolic pathways for selected monosaccharides, disaccharides or oligosaccharides which are involved and / or required for the synthesis of said disaccharides and / or milk-oligosaccharides.

[0279] 4. The cell according to any one of the preceding preferred embodiments, wherein the UDP-GlcNAc synthesis comprises at least one gene selected from the list consisting of or essentially consisting of: genes encoding a bifunctional N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, and glucosamine-1-phosphate acetyltransferase, and wherein the at least one gene is functionally impaired.

[0280] 5. The cell according to preferred embodiment 4, wherein the at least one gene encodes an enzyme, wherein the enzyme:

[0281] - an enzyme class selected from the list consisting of or consisting essentially of EC: 2.7.7.23, EC: 2.3.1.157 and EC: 5.4.2.3,

[0282] comprising a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of IPR001451, IPR002618, IPR005175, IPR005835, IPR005843, IPR005844, IPR005882, IPR011004, IPR016055, IPR016066, IPR016657, IPR018357, IPR023915, IPR025877, IPR029044, IPR036900 and IPR038009 as defined by InterPro 90.0 released on August 4, 2022,

[0283] comprising a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of PF00132, PF00408, PF00465, PF00483, PF01070, PF01565, PF01704, PF02878, PF02879, PF02880, PF03479, PF04030, PF05199, PF12146, PF12804, PF13562, and PF14602 as defined by InterPro 90.0, released on August 4, 2022,

[0284] - comprising a polypeptide sequence comprising a conserved protein domain selected from the list consisting of or consisting essentially of: cd03086 and cd03353 as defined by InterPro 90.0 released on August 4, 2022,

[0285] - is part of a NOG family selected from the list consisting of or consisting essentially of: COG1109 and COG4284 as defined by eggNOG5.0 released in 2019, and / or

[0286] - using a cofactor selected from the list consisting of or consisting essentially of: Mg 2+ 、Co 2+ 、Mn 2+ , Ca 2+ 、Zn 2+ 、Ni 2+ and FAD.

[0287] 6. The cell according to any of the preceding preferred embodiments, wherein at least one gene involved in the synthesis and / or import of the following cofactors is rendered functionally impaired:

[0288] - cofactors involved in the synthesis of UDP-GlcNAc, and / or

[0289] - a cofactor involved in the synthesis of UDP-GlcNAc and selected from the list consisting of or consisting essentially of: Mg 2+ 、Co 2+ 、Mn 2+ , Ca 2+ 、Zn 2+ 、Ni 2+ and FAD.

[0290] 7. The cell according to any one of preferred embodiments 4 to 6, wherein the at least one gene is functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list comprising: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the at least one gene.

[0291] 8. A cell according to any one of the preceding preferred embodiments, wherein the disaccharide and / or milk oligosaccharide is selected from the list consisting of or essentially consisting of: lactobiose; mammalian milk disaccharide; human milk disaccharide; N-acetyllactosamine (LacNAc); milk-N-biose (LNB); neutral (uncharged) milk oligosaccharide; negatively charged milk oligosaccharide; sialylated milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); fucosylated milk oligosaccharide; non-fucosylated neutral (uncharged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (uncharged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; fucosylated milk oligosaccharides selected from the list consisting of: 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N- N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated milk oligosaccharides selected from the list consisting of 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto- N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine;Neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine selected from the following list: lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, and p-lacto-N-neohexose; milk oligosaccharides containing N-acetylglucosamine; milk oligosaccharides containing N-acetyllactosamine; milk oligosaccharides containing fucosylated N-acetyllactosamine; milk oligosaccharides containing sialylated N-acetyllactosamine; milk oligosaccharides containing lacto-N-biose; milk oligosaccharides containing fucosylated lacto-N-biose; and milk oligosaccharides containing sialylated lacto-N-biose.

[0292] 9. The cell according to any one of the preceding preferred embodiments, wherein the cell:

[0293] - is capable of producing and / or produces said disaccharides and / or milk-oligosaccharides from one or more precursors,

[0294] - is capable of producing from lactose and / or produces said disaccharides and / or milk oligosaccharides from lactose,

[0295] - capable of producing and / or produces at least one precursor for the production of said disaccharides and / or milk-oligosaccharides,

[0296] - is capable of producing and / or produces all precursors for the production of said disaccharides and / or milk-oligosaccharides,

[0297] - genetically engineered to produce at least one precursor for the production of said disaccharides and / or milk-oligosaccharides, and / or

[0298] - genetically engineered to produce all precursors for the production of said disaccharides and / or milk-oligosaccharides.

[0299] 10. The cell of preferred embodiment 9, wherein at least one of the one or more precursors is internalized into the cell via one or more membrane proteins.

[0300] 11. The cell according to any one of the preceding preferred embodiments, wherein the cell is further genetically engineered to possess, express and / or overexpress glutamine-fructose-6-phosphate aminotransferase.

[0301] 12. The cell according to preferred embodiment 11, wherein the glutamine-fructose-6-phosphate aminotransferase has glutamine-fructose-6-phosphate aminotransferase activity, and:

[0302] - is selected from enzyme class EC: 2.6.1.16,

[0303] comprising a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of IPR001347, IPR005855, IPR017932, IPR029055, IPR035466, IPR035490, IPR036291, IPR046348 and IPR047084 as defined by InterPro 90.0 released on August 4, 2022,

[0304] - comprising a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of: PF00310, PF01380, PF01408, PF13230, PF13537 and PF13580 as defined by InterPro 90.0 released on August 4, 2022,

[0305] - comprising a polypeptide sequence comprising a conserved protein domain selected from the list consisting of or consisting essentially of: cd00714, cd05007, cd05008, cd05009, cd05013 and cd05710 as defined by InterPro 90.0 released on August 4, 2022, and / or

[0306] - is part of the NOG family COG0449 defined by eggNOG5.0 released in 2019.

[0307] 13. The cell according to any one of preferred embodiments 11 or 12, wherein the cell comprises a nucleic acid molecule comprising a polynucleotide sequence encoding the glutamine-fructose-6-sulfate aminotransferase, and wherein the nucleic acid molecule:

[0308] - is operably linked to a control sequence recognized by the cell, the nucleic acid molecule further i) integrated into the genome of the cell and / or ii) presented to the cell on a vector, and / or

[0309] - is foreign to the cell.

[0310] 14. The cell according to any one of the preceding preferred embodiments, wherein the cell:

[0311] - modified for enhanced synthesis and / or supply of phosphoenolpyruvate (PEP), and / or

[0312] - further modified to reduce the degradation of acetyl-CoA and / or its main precursor pyruvate.

[0313] 15. The cell according to any one of the preceding preferred embodiments, wherein the cell is:

[0314] - bacteria, fungi, yeast, plant cells, animal cells or protozoan cells,

[0315] - Escherichia coli (E. coli) or yeast with a lactose permease-positive phenotype, and / or

[0316] - Escherichia coli or yeast having a lactose permease-positive phenotype, wherein the lactose permease is encoded by the gene LacY or LAC12, respectively.

[0317] 16. A method for producing disaccharides and / or milk oligosaccharides, the method comprising:

[0318] i. cultivating and / or incubating the cell according to any one of the preceding preferred embodiments in a culture and / or incubation medium under conditions allowing the production of said disaccharides and / or milk oligosaccharides, and / or

[0319] ii. separating the disaccharide and / or milk-oligosaccharide from the cultivation and / or incubation.

[0320] 17. The method according to preferred embodiment 16, wherein:

[0321] - said culture or incubation medium comprises one or more precursors for the production of said disaccharides and / or milk-oligosaccharides,

[0322] - the culture medium comprises at least one carbon source selected from the group consisting of glucose, fructose, sucrose and glycerol, and / or

[0323] - the culture or incubation medium comprises at least one compound selected from the list consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid and CMP-sialic acid.

[0324] 18. The method according to any one of preferred embodiments 16 or 17, wherein the cells produce 30 g / L or more of the disaccharides and / or milk-oligosaccharides in the whole broth and / or supernatant, and / or wherein the disaccharides and / or milk-oligosaccharides in the whole broth and / or supernatant have a purity of at least 80%, measured based on the total amount of disaccharides and / or milk-oligosaccharides and their precursors produced by the cells in the whole broth and / or supernatant, respectively.

[0325] 19. The method according to any one of preferred embodiments 16 to 18, wherein the functionally impaired synthesis of UDP-GlcNAc confers unaffected and / or enhanced i) disaccharide and / or milk-oligosaccharide formation, ii) productivity, iii) biomass production, iv) cell growth, and / or v) yield of produced disaccharides and / or milk-oligosaccharides relative to corresponding non-engineered cells.

[0326] 20. The method according to any one of preferred embodiments 16 to 19, wherein:

[0327] - recovering said disaccharide and / or milk-oligosaccharide from said culture or incubation medium and / or said cells, and / or

[0328] - Purification of said disaccharides and / or milk-oligosaccharides.

[0329] 21. Use of a cell according to any one of preferred embodiments 1 to 15 for producing disaccharides and / or milk oligosaccharides, wherein the disaccharides and / or milk oligosaccharides are selected from the list consisting of or essentially consisting of: lactobiose; mammalian milk biose; human milk biose; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (uncharged) milk oligosaccharides; negatively charged milk oligosaccharides; sialylated milk oligosaccharides; mammalian milk oligosaccharides (MMO); human milk oligosaccharides (HMO); fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) Mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; fucosylated milk oligosaccharides selected from the list consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose lacto-N-neohexose; sialylated lacto-oligosaccharides selected from the group consisting of 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose d (LSTd), lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated lacto-oligosaccharides selected from the group consisting of 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose d (LSTd), lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated lacto-oligosaccharides selected from the group consisting of 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N lacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine;Neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine selected from the following list: lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, and p-lacto-N-neohexose; milk oligosaccharides containing N-acetylglucosamine; milk oligosaccharides containing N-acetyllactosamine; milk oligosaccharides containing fucosylated N-acetyllactosamine; milk oligosaccharides containing sialylated N-acetyllactosamine; milk oligosaccharides containing lacto-N-biose; milk oligosaccharides containing fucosylated lacto-N-biose; and milk oligosaccharides containing sialylated lacto-N-biose.

[0330] 22. Use of the method according to any one of preferred embodiments 16 to 20 for the production of disaccharides and / or milk oligosaccharides, wherein the disaccharides and / or milk oligosaccharides are selected from the list consisting of or essentially consisting of: lactobiose; mammalian milk disaccharides; human milk disaccharides; N-acetyllactosamine (LacNAc); milk-N-biose (LNB); neutral (uncharged) milk oligosaccharides; negatively charged milk oligosaccharides; sialylated milk oligosaccharides; mammalian milk oligosaccharides (MMO); human milk oligosaccharides (HMO); fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) milk oligosaccharides ) mammalian milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; fucosylated milk oligosaccharides selected from the list consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fuco Pentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated milk oligosaccharides selected from the list comprising 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose d (LSTd), s ... lacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine;Neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine selected from the following list: lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, and p-lacto-N-neohexose; milk oligosaccharides containing N-acetylglucosamine; milk oligosaccharides containing N-acetyllactosamine; milk oligosaccharides containing fucosylated N-acetyllactosamine; milk oligosaccharides containing sialylated N-acetyllactosamine; milk oligosaccharides containing lacto-N-biose; milk oligosaccharides containing fucosylated lacto-N-biose; and milk oligosaccharides containing sialylated lacto-N-biose.

[0331] The present invention will be described in more detail in the examples.The following examples will serve as further illustration and clarification of the present invention and are not intended to be limiting. Example

[0332] Example 1. Materials and methods

[0333] A. Escherichia coli

[0334] Culture medium and cultivation

[0335] Luria Broth (LB) medium consisted of 1% tryptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco), and 0.5% sodium chloride (VWR, Leuven, Belgium). The minimal medium used in 96-well plate or shake flask culture experiments contained 2.00 g / L NH4Cl, 5.00 g / L (NH4)2SO4, 2.993 g / L KH2PO4, 7.315 g / L K2HPO4, 8.372 g / L MOPS, 0.5 g / L NaCl, 0.5 g / L MgSO4.7H2O, 30 g / L sucrose or 30 g / L glycerol, 1 ml / L vitamin solution, 100 μl / L molybdate solution, and 1 mL / L selenium solution. As precursors and / or acceptors for sugar (e.g., disaccharides and / or milk oligosaccharides) synthesis, compounds such as galactose, glucose, fructose, fucose, lactose, cofactors can be added to the culture medium. Minimal medium is set to a pH of 7 with 1M KOH. The vitamin solution is composed of 3.6g / L FeCl2.4H2O, 5.0g / L CaCl2.2H2O, 1.3g / L MnCl2.2H2O, 0.38g / L CuCl2.2H2O, 0.5g / L CoCl2.6H2O, 0.94g / L ZnCl2, 0.0311g / L H3BO4, 0.4g / L Na2EDTA.2H2O, and 1.01g / L thiamine hydrochloride. The molybdate solution contains 0.967g / L NaMoO4.2H2O. The selenium solution contains 42g / L SeO2. The minimal medium that is used for fermentation comprises 6.75g / LNH4Cl, 1.25g / L (NH4)2SO4, 2.93g / L KH2PO4 and 7.31g / L KH2PO4, 0.5g / L NaCl, 0.5g / LMgSO4.7H2O, 30g / L sucrose or 30g / L glycerol, 1mL / L vitamin solution, 100 μ L / L molybdate solution and 1mL / L selenium solution, has the identical composition described above.As specifically indicated in each example, 20g / L lactose and / or 20g / L glucose are added to the substratum in addition.Compound medium is sterilized by autoclaving (121 ℃, 21 minutes), and minimal medium is sterilized by filtration (0.22 μ m Sartorius). When necessary, the culture medium was made selective by adding antibiotics: for example, chloramphenicol (20 mg / L), carbenicillin (100 mg / L), spectinomycin (40 mg / L) and / or kanamycin (50 mg / L). Precultures for 96-well microtiter plate experiments were started from frozen vials in 150 μL LB and incubated overnight at 37° C. on an orbital shaker at 800 rpm.With this culture, be used as the inoculum for 96-hole square microtiter plate (having 400 μ L minimal medium) by diluting 400x.Then, with these final 96-hole culture plates, under 37 ℃ on fixed orbital shaking table with 800rpm incubation 72 hours, or shorter, or longer.In order to measure sugar concentration when the culture experiment finishes, by before making the cell spin centrifugal, culturing fluid was boiled at 60 ℃ and took full culturing fluid sample (mean value of=intracellular and extracellular sugar concentration) from each hole in 15 minutes.Start the pre-culture thing that is used for bioreactor from the whole 1mL freezing bottle of a certain bacterial strain, be inoculated in the minimal medium that is in 250mL or 500mL in 1L or 2.5L shaking flask, and with it, under 37 ℃ on fixed orbital shaking table with 200rpm incubation 24 hours. A 5-L bioreactor was then inoculated (250 mL inoculum in 2 L batch medium); the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). Culture conditions were set at 37°C and maximum agitation; the pressure gas flow rate depended on the strain and bioreactor. The pH was controlled at 6.8 using 0.5 M H2SO4 and 20% NH4OH. The exhaust gas was cooled. If foaming occurred during fermentation, a 10% silicone antifoam solution was added.

[0336] Strains and mutations

[0337] Escherichia coli K12 MG1655[λ - ,F -,rph-1] was obtained from Coli Genetic Stock Center (US), CGSC strain #: 7740 in March 2007. Gene disruption, gene introduction and gene replacement were performed by using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). All constitutive promoter, UTR and terminator sequences were derived from 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 synthesized and ordered at Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com) and codon usage was adjusted using the supplier's tools. All strains were stored at -80°C in cryovials (overnight LB cultures mixed 1:1 with 70% glycerol).

[0338] In one example for GDP-fucose production, the mutant strain is derived from E. coli K12 MG1655, which comprises a knockout of the E. coli wcaJ gene and a genomic knockin of a constitutive transcription unit comprising a sucrose transporter, such as CscB from E. coli W (UniProt ID E0IXR1); a fructokinase, such as Frk from Zymomonas mobilis (UniProt ID Q03417); and a sucrose phosphorylase, such as BaSP from Bifidobacterium adolescentis (UniProt ID DAOZZH6). In the mutant E. coli strain, GDP-fucose production can be further optimized by genomic knockout of any one or more of the E. coli genes including glgC, agp, pfkA, pfkB, pgi, arcA, iclR, pgi and lon, as described in WO2016075243 and WO2012007481. GDP-fucose production can be additionally optimized, including genomic knock-in of constitutive transcription units for: mannose-6-phosphate isomerase, e.g., manA from E. coli (UniProt ID P00946); phosphomannose mutase, e.g., manB from E. coli (UniProt ID P24175); mannose-1-phosphate guanylyltransferase, e.g., manC from E. coli (UniProt ID P24174); GDP-mannose 4,6-dehydratase, e.g., gmd from E. coli (UniProt ID P0AC88); and GDP-L-fucose synthase, e.g., fcl from E. coli (UniProt ID P32055). GDP-fucose production can also be obtained by genomic knockout of the E. coli fucK and fucI genes and genomic knockin of a constitutive transcription unit comprising a fucose permease, such as fucP from E. coli (UniProt ID P11551); and a bifunctional enzyme with fucokinase / fucose-1-phosphate guanylyltransferase activity, such as fkp from Bacteroides fragilis (UniProt ID SUV40286.1). All mutant strains can be additionally modified with genomic knockout of the E. coli LacZ, LacY and LacA genes and with genomic knockin of a constitutive transcription unit for a lactose permease, such as E. coli LacY (UniProt ID P02920).To produce fucosylated sugars, the mutant GDP-fucose producing strain is additionally modified with an expression plasmid containing constitutive transcription units for: a fucosyltransferase, such as α-1,2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435), to produce 2'-fucosyllactose (2'FL); or α-1,3-fucosyltransferase HpFucT from H. pylori (UniProt ID DO30511), to produce 3-fucosyllactose (3-FL); or α-1,2-fucosyltransferase HpFutC from H. pylori (UniProt ID Q9X435) and α-1,3-fucosyltransferase HpFucT from H. pylori (UniProt ID O30511) to produce difucosyllactose (DiFL).

[0339] In one example for the production of lacto-N-triose (LN3, GlcNAc-β1,3-Gal-β1,4-Glc), the mutant strain is derived from Escherichia coli K12 MG1655 and is modified with knockouts of the E. coli lacZ, lacY, lacA, and nagB genes and with genomic knock-ins of constitutive transcription units for: a lactose permease, such as E. coli LacY (UniProt ID P02920); and a galactoside β-1,3-N-acetylglucosaminyltransferase, such as lgtA from Neisseria meningitidis (UniProt ID Q9JXQ6). In one example 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 with a constitutive transcription unit for N-acetylglucosamine β-1,3-galactosyltransferase (e.g., wbgO from Escherichia coli O55:H7 (Uniprot ID D3QY14)) delivered to the strain via genomic knock-in or from an expression plasmid. In one example 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 with a constitutive transcription unit for N-acetylglucosamine β-1,4-galactosyltransferase (e.g., LgtB from Neisseria meningitidis (UniprotID Q51116, sequence version 02, December 1, 2000)) delivered to the strain via genomic knock-in or from an expression plasmid. In the mutant E. coli strain, LN3, LNT, and / or LNnT production can be further optimized using genomic knockouts of any one or more E. coli genes including galT, ushA, ldhA, and agp. Optionally, the mutant LN3, LNT and / or LNnT producing strains can also be modified with a genomic knock-in of a constitutive transcription unit of an L-glutamine-D-fructose-6-phosphate aminotransferase, such as E. coli glmS (UniProt ID P17169, sequence version 04, January 23, 2007) or mutant glmS*54 from E. coli (having SEQ ID NO: 01 and differing from wild-type E. coli glmS having UniProt ID P17169 by the A39T, R250C and G472S mutations, as described by Deng et al. (Biochimie 88, 419-29 (2006)) for enhanced UDP-GlcNAc production.Optionally, the mutant E. coli strain can also be adapted with genomic knock-in of the constitutive transcription units for UDP-glucose-4-epimerase, such as galE from E. coli (UniProtID P09147), phosphoglucosamine mutase, such as glmM from E. coli (UniProtID P31120, sequence version 03, January 23, 2007), and N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, such as glmU from E. coli (UniProtID P0ACC7). Optionally, the mutant LN3, LNT and / or LNnT producing E. coli strain can also be adapted to grow on sucrose via genomic knock-in of constitutive transcription units comprising a sucrose transporter, such as CscB from E. coli W (UniProt ID E0IXR1); a fructokinase, such as Frk from Zymomonas mobilis (UniProt ID Q03417); and a sucrose phosphorylase, such as BaSP from Bifidobacterium adolescentis (UniProt ID A0ZZH6).

[0340] In one example for sialic acid production, the mutant strain is derived from Escherichia coli K12 MG1655 containing a genomic knock-in of constitutive transcription units comprising one or more copies of glucosamine 6-phosphate N-acetyltransferase, such as GNA1 from Saccharomyces cerevisiae (UniProt ID P43577); N-acetylglucosamine 2-epimerase, such as AGE from Bacteroides ovatus (UniProt ID A7LVG6); and N-acetylneuraminic acid synthase, such as NeuB from Neisseria meningitidis (UniProt ID EONCD4).

[0341] Alternatively and / or additionally, sialic acid production can be obtained by genomic knock-in of constitutive transcription units comprising a UDP-N-acetylglucosamine 2-epimerase, such as NeuC from Campylobacter jejuni (C. jejuni) (UniProt ID Q93MP8); and an N-acetylneuraminic acid synthase, such as NeuB from Neisseria meningitidis (UniProt ID E0NCD4).

[0342] Alternatively and / or additionally, sialic acid production can be obtained by genomic knock-in of constitutive transcription units comprising a phosphoglucosamine mutase, e.g., glmM from Escherichia coli (UniProt ID DP31120, sequence version 03, January 23, 2007); an N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, e.g., glmU from Escherichia coli (UniProt ID POACC7); a UDP-N-acetylglucosamine 2-epimerase, e.g., NeuC from Campylobacter jejuni (UniProt ID Q93MP8); and an N-acetylneuraminic acid synthase, e.g., NeuB from Neisseria meningitidis (UniProt ID EONCD4).

[0343] Alternatively and / or additionally, sialic acid production can be obtained by genomic knock-in of a constitutive transcription unit comprising a bifunctional UDP-GlcNAc 2-epimerase / N-acetylmannosamine kinase, e.g., from mouse (strain C57BL / 6J) (UniProt ID Q91WG8); an N-acylneuraminic acid-9-phosphate synthase, e.g., from Pseudomonas sp. UW4 (UniProtID K9NPH9); and an N-acylneuraminic acid-9-phosphatase, e.g., from Bacteroides thetaiotaomicron (UniProtID Q8A712).

[0344] In the mutant E. coli strains, sialic acid production can be further optimized with genomic knockouts of E. coli genes comprising any one or more of nagA, nagB, nagC, nagD, nagE, nanA, nanE, nanK, manX, manY and manZ (as described in WO18122225) and / or genomic knockouts of E. coli genes comprising any one or more of nanT, poxB, ldhA, adhE, aldB, pflA, pflC, ybiY, ackA and / or pta, and with genomic knockin of a constitutive transcription unit comprising one or more copies of L-glutamine-D-fructose-6-phosphate aminotransferase, such as E. coli glmS (UniProt ID P17169, sequence version 04, January 23, 2007) or mutant glmS*54 from E. coli having SEQ ID NO: 01 and having UniProt The wild-type E. coli glmS of IDP17169 differs in A39T, R250C and G472S mutations as described by Deng et al. (Biochimie 88, 419-29 (2006)); preferably, a phosphatase, such as any one 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 a phosphatase from Pseudomonas putida. putida), ScDOG1 from Saccharomyces cerevisiae, or BsAraL from Bacillus subtilis, as described in WO18122225; and acetyl-CoA synthetase, such as acs from Escherichia coli (UniProt ID P27550).

[0345] For sialylated oligosaccharide production, the sialic acid producing strain is further modified to express an N-acylneuraminic acid cytidylyltransferase, such as the NeuA enzyme from Pasteurella multocida (UniProt ID A0A849CI62), and to express a sialyltransferase, such as the α-2,3-sialyltransferase PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having β-galactoside α-2,3-sialyltransferase activity (SEQ ID NO: 02), or the α-2,6-sialyltransferase PdST6 from Photobacterium damselae (UniProt ID O66375) or a PmultST3-like polypeptide having β-galactoside α-2,6-sialyltransferase activity (SEQ ID NO: 03). The PdST6-sample polypeptide (SEQ ID NO:03) of amino acid residue 108 to 497 composition of O66375. The constitutive transcription unit of N-acylneuraminic acid cytidylyltransferase and sialyltransferase can be knocked into or delivered to described mutant strain via expression plasmid via genome.If the mutant strain of described generation sialic acid and CMP-sialic acid is intended to make sialylated lactose structure, then with the genome knockout of intestinal bacteria LacZ, LacY and LacA gene and with about lactose permease for example the genome knocking into of the constitutive transcription unit of intestinal bacteria LacY (UniProt ID P02920) come modify described bacterial strain in addition. All mutant strains producing sialic acid, CMP-sialic acid and / or sialylated sugars can optionally be adapted to grow on sucrose via genomic knock-in of constitutive transcription units comprising a sucrose transporter, such as CscB from Escherichia coli W (UniProt ID E0IXR1); a fructokinase, such as Frk from Zymomonas mobilis (UniProt ID Q03417); and a sucrose phosphorylase, such as BaSP from Bifidobacterium adolescentis (UniProt ID A0ZZH6).

[0346] In another example, the mutant E. coli strain adapted for LNT production described herein can be further modified with one or more copies of a glucosamine 6-phosphate N-acetyltransferase, such as GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), an N-acetylglucosamine 2-epimerase, such as AGE from Bacteroides ovatus (UniProt ID A7LVG6), and an N-acetylneuraminic acid synthase, such as NeuB from Neisseria meningitidis (UniProt ID E0NCD4); and an expression plasmid comprising constitutive expression cassettes for: N-acylneuraminic acid cytidylyltransferase (NeuA) from Pasteurella multocida (UniProt ID A0A849CI62), and 1) an α-2,3-sialyltransferase PmultST3 from Pasteurella multocida (UniProt ID A0A849CI63). Q9CLP3) or a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 with β-galactoside α-2,3-sialyltransferase activity (SEQ ID NO: 02), or 2) an α-2,6-sialyltransferase (PdST6) from Photorhabdus mermaidae (UniProt ID 066375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID 066375 with β-galactoside α-2,6-sialyltransferase activity (SEQ ID NO: NO:03), to produce 1) LSTa (Neu5Ac-α2,3-Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc) or 2) LSTb (Gal-β1,3-(Neu5Ac-α2,6)-GlcNAc-β1,3-Gal-β1,4-Glc), respectively.In another example, the mutant E. coli strain adapted for LNnT production described herein can be further modified with one or more copies of a glucosamine 6-phosphate N-acetyltransferase, such as GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), an N-acetylglucosamine 2-epimerase, such as AGE from Bacteroides ovatus (UniProt ID A7LVG6), and an N-acetylneuraminic acid synthase, such as NeuB from Neisseria meningitidis (UniProt ID E0NCD4); and an expression plasmid comprising constitutive expression cassettes for: N-acylneuraminic acid cytidylyltransferase (NeuA) from Pasteurella multocida (UniProt ID A0A849CI62), and 1) an α-2,3-sialyltransferase PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having β-galactoside α-2,3-sialyltransferase activity (SEQ ID NO: 02), or 2) an α-2,6-sialyltransferase (PdST6) from Photorhabdus mermaidae (UniProtID O66375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProtID O66375 having β-galactoside α-2,6-sialyltransferase activity (SEQ ID NO: NO:03), to produce 1) LSTd (Neu5Ac-α2,3-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc) or 2) LSTc (Neu5Ac-α2,6-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc), respectively.

[0347] B. Saccharomyces cerevisiae

[0348] Culture medium and cultivation

[0349] Strains are grown on synthetic defined yeast medium (SD CSM) or CSM drop-out (SD CSM-Ura, SD CSM-Trp, SD CSM-His) with a complete supplement mixture containing 6.7 g / L Yeast Nitrogen Base (YNB w / o AA, Difco), 20 g / L agar (Difco) (solid culture), 22 g / L glucose monohydrate or 20 g / L lactose, and 0.79 g / L CSM or 0.77 g / L CSM-Ura, 0.77 g / L CSM-Trp or 0.77 g / L CSM-His (MP Biomedicals). Compounds such as galactose, glucose, fructose, fucose, lactose, cofactors can be added to the culture medium as precursors and / or acceptors for sugar (e.g., disaccharides and / or lacto-oligosaccharides) synthesis. Typically, yeast strains are initially grown on SD CSM plates to obtain single colonies. The plates were grown at 30°C for 2-3 days. From a single colony, a preculture was grown overnight at 30°C in 5 mL with shaking at 200 rpm. Subsequently, 2% of this preculture was used to inoculate 125 mL shake flasks in 25 mL of culture medium. The shake flasks were incubated at 30°C with orbital shaking at 200 rpm.

[0350] Strains, plasmids, and mutations

[0351] Saccharomyces cerevisiae BY4742, created by Brachmann et al. (Yeast (1998) 14: 115-32), available at the Euroscarf culture collection, was used. All mutant strains were created by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11: 355-360, 1995). Genes were expressed using synthetic constitutive promoters, such as those described by, for example, Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012), Redden and Alper (Nat. Commun. 2015, 6, 7810), Liu et al. (Microb. Cell Fact. 2020, 19, 38), Xu et al. (Microb. Cell Fact. 2021, 20, 148), and Lee et al. (ACS Synth. Biol. 2015, 4 (9), 975-986).

[0352] In an example for producing GDP-fucose, yeast expression plasmids such as p2a_2μ_Fuc (Chan 2013, Plasmid 70, 2-17) can be used to express foreign genes in Saccharomyces cerevisiae. The plasmid contains an ampicillin resistance gene and a bacterial replication origin (to allow selection and maintenance in E. coli) as well as 2μ yeast ori and Ura3 selection markers (for selection and maintenance in yeast). The plasmid is further modified with constitutive transcription units for the following: lactose permease, such as LAC12 from Kluyveromyces lactis (UniProt ID P07921); GDP-mannose 4,6-dehydratase, such as gmd from E. coli (UniProt ID P0AC88); and GDP-L-fucose synthase, such as fcl from E. coli (UniProt ID P32055). Yeast expression plasmid p2a_2μ_Fuc2 can be used as an alternative expression plasmid to plasmid p2a_2μ_Fuc, which contains constitutive transcription units for the following: lactose permease, such as LAC12 from Kluyveromyces lactis (UniProt ID P07921); fucose permease, such as fucP from Escherichia coli (UniProt ID P11551); and a bifunctional enzyme with fucokinase / fucose-1-phosphate guanylyltransferase activity, such as fkp from Bacteroides fragilis (UniProt ID SUV40286.1). To further produce fucosylated sugars, p2a_2μ_Fuc and its variant p2a_2μ_Fuc2 additionally contain constitutive transcription units for fucosyltransferase.

[0353] In one example for producing UDP-galactose, a yeast expression plasmid can be derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122), which contains a HIS3 selection marker and a constitutive transcription unit for a UDP-glucose-4-epimerase, such as galE from Escherichia coli (UniProt ID P09147). This plasmid can be further modified with constitutive transcription units for a lactose permease, such as LAC12 from Kluyveromyces lactis (UniProt ID P07921), and a galactoside β-1,3-N-acetylglucosaminyltransferase activity, such as lgtA from Neisseria meningitidis (UniProt ID Q9JXQ6). In one example for further producing LN3-derived oligosaccharides such as LNT, the mutant LN3-producing strain is further modified with a constitutive transcription unit for N-acetylglucosamine β-1,3-galactosyltransferase (e.g., WbgO from Escherichia coli O55:H7 (Uniprot ID D3QY14)). In one example for producing 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 with a constitutive transcription unit for N-acetylglucosamine β-1,4-galactosyltransferase (e.g., LgtB from Neisseria meningitidis (Uniprot ID Q51116, sequence version 02, December 1, 2000)).

[0354] In one example for the production of sialic acid and CMP-sialic acid, the yeast expression plasmid is derived from the pRS420-plasmid series (Christianson et al., 1992, Gene 110: 119-122), which contains a TRP1 selection marker and constitutive transcription units for: L-glutamine-D-fructose-6-phosphate aminotransferase, such as E. coli glmS (UniProtID P17169, sequence version 04 (January 23, 2007)) or mutant glmS*54 from E. coli (which has SEQ ID NO: 01 and differs from wild-type E. coli glmS with UniProt ID P17169 by the A39T, R250C and G472S mutations, as described by Deng et al. (Biochimie 88, 419-29 (2006))); phosphatase, such as SurE from E. coli (UniProtID

[0015] The present invention also includes a constitutive transcription unit for a glucosamine 6-phosphate N-acetyltransferase, such as GNA1 from Saccharomyces cerevisiae (UniProt ID P43577).

[0015] The present invention also includes a constitutive transcription unit for a siderophore transporter, such as entS from Escherichia coli (UniProt ID P24077, sequence version 02 (November 1, 1997)).In one example for producing sialylated sugars, the plasmid further comprises constitutive transcription units for a lactose permease, such as LAC12 from Kluyveromyces lactis (UniProt ID P07921); and a sialyltransferase, such as an α-2,3-sialyltransferase, such as α-2,3-sialyltransferase PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide having β-galactoside α-2,3-sialyltransferase activity consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 (SEQ ID NO: 02), or an α-2,6-sialyltransferase, such as α-2,6-sialyltransferase (PdST6) from Photorhabditis elegans (UniProt ID 066375) or a PdST6-like polypeptide having β-galactoside α-2,6-sialyltransferase activity consisting of amino acid residues 108 to 497 of UniProtID 066375 (SEQ ID NO: 03).

[0355] Preferably, but not necessarily, any one or more of the glycosyltransferases and / or proteins involved in nucleotide activated sugar synthesis are fused at the N-terminus and / or C-terminus to a SUMOstar tag (e.g., obtained from pYSUMOstar, Life Sensors, Malvern, PA) to enhance their solubility.

[0356] Optionally, the mutant yeast strain is modified with a genomic knock-in of a constitutive transcription unit encoding a chaperone protein, such as Hsp31, Hsp32, Hsp33, Sno4, Kar2, Ssb1, Sse1, Sse2, Ssa1, Ssa2, Ssa3, Ssa4, Ssb2, Ecm10, Ssc1, Ssq1, Ssz1, Lhs1, Hsp82, Hsc82, Hsp78, Hsp104, Tcp1, Cct4, Cct8, Cct2, Cct3, Cct5, Cct6, or Cct7. Plasmids were maintained in the host E. coli DH5α (F - ,phi80dlacZdeltaM15,delta(lacZYA-argF)U169,deoR,recA1,endA1,hsdR17(rk - ,mk + ),phoA,supE44,lambda - ,thi-1,gyrA96,relA1).

[0357] C. Bacillus subtilis

[0358] Culture medium and cultivation

[0359] Two media were used to culture Bacillus subtilis: complex media such as rich Luria Broth (LB) and minimal media for shake flask cultures. LB media consisted of 1% tryptone (Difco), 0.5% yeast extract (Difco), and 0.5% sodium chloride (VWR). Luria Broth agar (LBA) plates consisted of LB media supplemented with 12 g / L agar (Difco). Minimal media contained 2.00 g / L (NH4)2SO4, 7.5 g / L KH2PO4, 17.5 g / L K2HPO4, 1.25 g / L sodium citrate, 0.25 g / L MgSO4.7H2O, 0.05 g / L tryptophan, 10 to 30 g / L glucose (or another carbon source, including but not limited to fructose, maltose, sucrose, glycerol, and maltotriose), 10 mL / L trace element mix, and 10 mL / L ferric citrate solution. The culture medium was set to a pH of 7 with 1 M KOH. As precursors and / or acceptors for sugar (e.g., disaccharides and / or lacto-oligosaccharides) synthesis, compounds such as galactose, glucose, fructose, fucose, lactose, cofactors may be added to the culture medium. The trace element mixture consisted of 0.735 g / L CaCl2.2H2O, 0.1 g / L MnCl2.2H2O, 0.033 g / L CuCl2.2H2O, 0.06 g / L CoCl2.6H2O, 0.17 g / L ZnCl2, 0.0311 g / L H3BO4, 0.4 g / L Na2EDTA.2H2O, and 0.06 g / L Na2MoO4. The ferric citrate solution contained 0.135 g / L FeCl3.6H2O, 1 g / L sodium citrate (Hoch 1973 PMC 1212887). The compound culture medium (for example LB) is sterilized by autoclaving (121 ℃, 21 minutes), and the minimal culture medium is sterilized by filtration (0.22 μm Sartorius). When needed, substratum is made to have selectivity by adding antibiotic. Initially, the substratum is grown on LB agar to obtain a single bacterium colony. These flat plates are grown overnight at 37 ℃. From a single bacterium colony, pre-culture is grown overnight in 5mL at 37 ℃, wherein shaken with 200rpm. Subsequently, in a 25mL substratum, a 125mL shake flask experiment is inoculated with 2% of this pre-culture. These shake flasks are incubated 72 hours at 37 ℃, or shorter, or longer, wherein with a fixed track shaking of 200rpm. At the end of the incubation experiment, samples were taken to measure the supernatant concentration (extracellular sugar concentration after spinning down the cells for 5 minutes) or by boiling the culture broth at 90°C for 15 minutes or at 60°C for 60 minutes before spinning down the cells (=total broth concentration, i.e., intracellular and extracellular sugar concentrations).

[0360] Strains, plasmids, and mutations

[0361] Using Bacillus subtilis 168, it is available at the Bacillus Genetic Stock Center (Bacillus Genetic Stock Center) (Ohio, USA). As described by Yan et al. (Appl & Environm.Microbial., September 2008, pp. 5556-5562), a plasmid for gene deletion via Cre / lox is constructed. Gene disruption is carried out by homologous recombination with linear DNA and transformation via electroporation, as described by Xue et al. (J.microb.Meth.34 (1999) 183-191). The method for gene knockout is described by Liu et al. (Metab.Engine.24 (2014) 61-69). The integration vector described by Popp et al. (Sci.Rep., 2017, 7, 15158) is used as an expression vector and, if necessary, can be further used for genomic integration. Suitable promoters for expression can be derived from the partial repository (iGem): sequence id: BBa_K143012, BBa_K823000, BBa_K823002 or BBa_K823003. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

[0362] In one example for producing lactose-based oligosaccharides, a Bacillus subtilis mutant strain was created to contain genes encoding a lactose importer (e.g., E. coli lacY with UniProt ID P02920). In one example for producing LN3, the Bacillus subtilis strain was modified with a genomic knock-in containing a constitutive transcription unit for a lactose importer (e.g., E. coli lacY with UniProt ID P02920) and a galactoside β-1,3-N-acetylglucosamine transferase (e.g., LgtA from Neisseria meningitidis (UniProt ID Q9JXQ6)). For LNT production, the LN3-producing strain was further modified with a constitutive transcription unit for an N-acetylglucosamine β-1,3-galactosyltransferase (e.g., WbgO from E. coli O55:H7 (UniProt ID D3QY14)). For LNnT production, the LN3-producing strain is further modified with a constitutive transcription unit for N-acetylglucosamine β-1,4-galactosyltransferase (e.g., LgtB from Neisseria meningitidis (UniProt ID Q51116, sequence version 02, December 1, 2000). N-acetylglucosamine β-1,3-galactosyltransferase and N-acetylglucosamine β-1,4-galactosyltransferase can be delivered to the strain via genomic knock-in or from an expression plasmid. To produce fucosylated sugars, the Bacillus subtilis strain is modified with a constitutive transcription unit for a fucosyltransferase. In one example for sialic acid production, a mutant Bacillus subtilis strain was created by overexpressing a fructose-6-P-aminotransferase, such as the native fructose-6-P-aminotransferase glmS (UniProt ID DP0CI73), to enhance the intracellular glucosamine-6-phosphate pool. Furthermore, the enzymatic activities of the genes nagA, nagB, and gamA were disrupted by genetic knockout, and glucosamine-6-P-aminotransferase (e.g., GNA1 from Saccharomyces cerevisiae (UniProt ID P43577)), N-acetylglucosamine 2-epimerase (e.g., from Bacteroides ovatus (UniProt ID A7LVG6)), and N-acetylneuraminic acid synthase (e.g., from Neisseria meningitidis (UniProt ID EONCD4)) were overexpressed genomically.To allow for the production of sialylated sugars, the sialic acid producing strain is further modified with a constitutive transcription unit comprising an N-acylneuraminic acid cytidylyltransferase, such as NeuA from Pasteurella multocida (UniProt ID A0A849CI62); and a sialyltransferase, such as an α-2,3-sialyltransferase, such as α-2,3-sialyltransferase PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide having β-galactoside α-2,3-sialyltransferase activity consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 (SEQ ID NO: 02), or an α-2,6-sialyltransferase, such as α-2,6-sialyltransferase (PdST6) from Photorhabditis elegans (UniProt ID The mutant strains are modified by knocking in a genomic transcriptome comprising the following constitutive transcription units: sucrose transporter (CscB) from Escherichia coli W (UniProt ID 0IXR1), fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417), and sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (UniProt ID A0ZZH6). Optionally, a constitutive transcription unit for an iron carrier transporter, such as entS from Escherichia coli (UniProt ID P24077, sequence version 02 (November 1, 1997), is also added.

[0363] D. Corynebacterium glutamicum

[0364] Culture medium and cultivation

[0365] Use two different substratums, i.e., complex medium such as enriching tryptone yeast extract (TY) substratum and minimal medium (MMsf) for shaking flask. Minimal medium uses 1000x reserve trace element mixture. Trace element mixture is by 10g / L CaCl , 10g / L FeSO .7H 2 O, 10g / L MnSO .H 2 O, 1g / L ZnSO .7H 2 O, 0.2g / L CuSO , 0.02g / L NiCl .6H 2 O, 0.2g / L biotin (pH 7) and 0.03g / L protocatechuic acid composition. Minimal medium (MMsf) for shake flask experiments contained 20 g / L (NH4)2SO4, 5 g / L urea, 1 g / L KH2PO4, 1 g / L K2HPO4, 0.25 g / L MgSO4.7H2O, 42 g / L MOPS, from 10 up to 30 g / L glucose or another carbon source (including but not limited to fructose, maltose, sucrose, glycerol and maltotriose) (when specifically specified in the examples) and 1 mL / L trace element mixture. As precursors and / or acceptors for sugar (e.g., disaccharides and / or lacto-oligosaccharides) synthesis, compounds such as galactose, glucose, fructose, fucose, lactose, cofactors can be added to the culture medium. TY medium consists of 1.6% tryptone (Difco, Erembodegem, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR.Leuven, Belgium). TY agar (TYA) flat board is made up of the TY substratum that is added with 12g / L agar (Difco, Erembodegem, Belgium).Compound medium (such as TY) is sterilized by autoclaving (121 ℃, 21 minutes), and minimal medium is sterilized by filtration (0.22 μm Sartorius).When needed, make substratum have selectivity by adding antibiotic.From freezing bottle or from the single bacterium colony of TY flat board in 6mL TY, start preculture, and it is incubated overnight with 200rpm on fixed orbit shaking table at 37 ℃.Subsequently, in 25mL MMsf substratum, inoculate 125mL shake flask experiment with 2% this preculture.By these shake flasks at 37 ℃ incubation 72 hours, or shorter, or longer, wherein have the fixed orbit shaking of 200rpm. At the end of the incubation experiment, samples were taken to measure the supernatant concentration (extracellular sugar concentration after spinning down the cells for 5 minutes) or by boiling the culture broth at 90°C for 15 minutes or at 60°C for 60 minutes before spinning down the cells (=total broth concentration, i.e., intracellular and extracellular sugar concentrations).

[0366] Strains and mutations

[0367] Use Corynebacterium glutamicum, it is available (ATCC 13032) at American Type Culture Collection.By using by the Cre / loxP technology described by people such as Suzuki (Appl.Microbiol.Biotechnol., 2005Apr, 67 (2): 225-33) prepare integration plasmid vector, and construct by the temperature-sensitive shuttle vector described by people such as Okibe (Journal of Microbiological Methods 85,2011,155-163) for gene deletion, sudden change and insertion.The promotor suitable for (heterologous) gene expression can be derived from people such as Yim (Biotechnol.Bioeng., 2013Nov, 110 (11): 2959-69).Clone can be carried out by using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction connection. In one example for the generation of oligosaccharides based on lactose, create Corynebacterium glutamicum mutant bacterial strain to comprise the gene of coding lactose importer (for example, with the intestinal bacteria lacY of UniProtID P02920).In one example for the generation of LN3, with comprising lactose importer (for example, with the intestinal bacteria lacY of UniProt ID P02920) and galactoside β-1, the genome of the constitutive expression unit of 3-N-acetylglucosamine transferase (for example, from the LgtA (UniProtIDQ9JXQ6) of Neisseria meningitidis) knocks into and modifies described Corynebacterium glutamicum bacterial strain.For LNT, produce, with about N-acetylglucosamine β-1, the constitutive transcription unit of 3-galactosyl transferase (for example, from the WbgO (UniProtID D3QY14) of intestinal bacteria O55:H7) further modifies the bacterial strain of described generation LN3. For LNnT production, the strain producing LN3 is further modified with a constitutive transcription unit for N-acetylglucosamine β-1,4-galactosyltransferase (e.g., LgtB from Neisseria meningitidis (UniProt IDQ51116, sequence version 02, December 1, 2000). N-acetylglucosamine β-1,3-galactosyltransferase and N-acetylglucosamine β-1,4-galactosyltransferase can be knocked into the genome or delivered to the strain from an expression plasmid. To further produce fucosylated sugars, the mutant C. glutamicum strain is further modified with a constitutive transcription unit for a fucosyltransferase.In one example for sialic acid production, a mutant Corynebacterium glutamicum strain was created by overexpressing a fructose-6-P-aminotransferase, for example the native fructose-6-P-aminotransferase glmS (UniProt ID Q8NND3, sequence version 03, January 23, 2007), to enhance the intracellular glucosamine-6-phosphate pool. Furthermore, the enzymatic activities of the genes nagA, nagB, and gamA were disrupted by genetic knockout, and glucosamine-6-P-aminotransferase (e.g., GNA1 from Saccharomyces cerevisiae (UniProt ID P43577)), N-acetylglucosamine 2-epimerase (e.g., from Bacteroides ovatus (UniProt ID A7LVG6)), and N-acetylneuraminic acid synthase (e.g., from Neisseria meningitidis (UniProt ID 10NCD4)) were overexpressed on the genome. To allow for the production of sialylated sugars, the sialic acid producing strain is further modified with a constitutive transcription unit comprising an N-acylneuraminic acid cytidylyltransferase, such as NeuA from Pasteurella multocida (UniProt ID A0A849CI62); and a sialyltransferase, such as an α-2,3-sialyltransferase, such as α-2,3-sialyltransferase PmultST3 from Pasteurella multocida (UniProt ID Q9CLP3) or a PmultST3-like polypeptide having β-galactoside α-2,3-sialyltransferase activity consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 (SEQ ID NO: 02), or an α-2,6-sialyltransferase, such as α-2,6-sialyltransferase (PdST6) from Photorhabditis elegans (UniProt ID The mutant strains can be modified with a genomic knock-in of the constitutive transcription units of Escherichia coli W (UniProt ID E0IXR1), a fructokinase (Frk) from Zymomonas mobilis (UniProt ID Q03417), and a sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (UniProt ID A0ZZH6) for growth on sucrose.

[0368] E. Chlamydomonas reinhardtii

[0369] Culture medium and cultivation

[0370] Chlamydomonas reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7). TAP medium used a 1000x stock Hutner trace element mixture. The Hutner trace element mixture consisted of 50 g / L Na2EDTA.H2O (Titriplex III), 22 g / L ZnSO4.7H2O, 11.4 g / L H3BO3, 5 g / L MnCl2.4H2O, 5 g / L FeSO4.7H2O, 1.6 g / L CoCl2.6H2O, 1.6 g / L CuSO4.5H2O, and 1.1 g / L (NH4)6MoO3. TAP medium contained 2.42 g / L Tris (tris(hydroxymethyl)aminomethane), 25 mg / L salt stock solution, 0.108 g / L K2HPO4, 0.054 g / L KH2PO4, and 1.0 mL / L glacial acetic acid. The salt stock solution consists of 15 g / L NH4Cl, 4 g / L MgSO4.7H2O and 2 g / L CaCl2.2H2O. As precursors and / or acceptors for the synthesis of sugars (e.g., disaccharides and / or lacto-oligosaccharides), compounds such as galactose, glucose, fructose, fucose, lactose, cofactors can be added. The culture medium is sterilized by autoclaving (121°C, 21 minutes). For the stock culture on agar slant, TAP medium (with purified high strength, 1000 g / cm2) containing 1% agar is used. 2 ).

[0371] Chlamydomonas reinhardtii cells were cultured on selective TAP-agar plates at 23 + / - 0.5 ° C. with a 14 / 10 hour light / dark cycle (with a light intensity of 8000 Lx). Cells were analyzed after 5 to 7 days of culture. For high-density cultures, cells can be cultured in closed systems such as vertical or horizontal tube photobioreactors, stirred tank photobioreactors, or flat-plate photobioreactors, as described by Chen et al. (Bioresour. Technol. 2011, 102: 71-81) and Johnson et al. (Biotechnol. Prog. 2018, 34: 811-827).

[0372] Strains, plasmids, and mutations

[0373] Chlamydomonas reinhardtii wild type strains 21gr (CC-1690, wild type, mt+), 6145C (CC-1691, wild type, mt-), CC-125 (137c, wild type, mt+), CC-124 (137c, wild type, mt-), available from the Chlamydomonas Resource Center (https: / / www.chlamycollection.org) (University of Minnesota, USA) were used. The expression plasmid was derived from pSI103, available from the Chlamydomonas Resource Center. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR, or restriction ligation. Suitable promoters for (heterologous) gene expression can be derived from, for example, Scranton et al. (Algal Res. 2016, 15: 135-142). Targeted gene modification (e.g., gene knockout or gene replacement) can be performed using, for example, the Crispr-Cas technology described by Jiang et al. (Eukaryotic Cell 2014, 13(11): 1465-1469). Transformation via electroporation can be performed as described by Wang et al. (Biosci. Rep. 2019, 39: BSR2018210) and as described, for example, in WO22034067 or in WO22034069.

[0374] In one example for the production of sialic acid and CMP-sialic acid, the mutant strain is derived from Chlamydomonas reinhardtii and modified as described, for example, in WO22034067. In one example for the production of sialylated oligosaccharides, Chlamydomonas reinhardtii cells are modified with a CMP-sialic acid transporter (e.g., CST from mouse (Mus musculus) (UniProtID Q61420)) and a Golgi-localized sialyltransferase selected from species such as Homo sapiens, Mouse, Rattus norvegicus. In one example for the synthesis of GDP-fucose, the mutant strain is derived from Chlamydomonas reinhardtii and modified as described, for example, in WO22034067. In one example for fucosylation, Chlamydomonas reinhardtii cells can be modified with an expression plasmid containing constitutive transcription units for α-1,2-fucosyltransferase and / or α-1,3-fucosyltransferase. In one example for UDP-galactose synthesis, the mutant strain is derived from Chlamydomonas reinhardtii and modified as described, for example, in WO 22034067. In one example for LN3 production, the mutant strain is derived from Chlamydomonas reinhardtii and modified as described, for example, in WO 22034067 to contain a transcription unit for a galactoside β-1,3-N-acetylglucosaminyltransferase, such as LgtA from Neisseria meningitidis (UniProtID Q9JXQ6). In one example for LNT or LNnT production, the LN3-producing strain is further modified with a constitutive transcription unit comprising an N-acetylglucosamine β-1,3-galactosyltransferase (e.g., WbgO from Escherichia coli O55:H7 (Uniprot ID DD3QY14)) or an N-acetylglucosamine β-1,4-galactosyltransferase (e.g., LgtB from Neisseria meningitidis (Uniprot ID Q51116, sequence version 02, December 1, 2000), respectively. In one example for producing one or more fucosylated uncharged oligosaccharides, a Chlamydomonas reinhardtii strain is modified for production of GDP-fucose, UDP-galactose, LN3, LNT, and / or LNnT as described herein, and for expression of one or more compatible fucosyltransferases.In one example for producing one or more sialylated oligosaccharides such as LSTa and LSTb, a Chlamydomonas reinhardtii strain is modified to produce CMP-sialic acid, UDP-galactose, LN3, and LNT as described herein, and to express one or more compatible sialyltransferases, such as an α-2,3-sialyltransferase, such as the α-2,3-sialyltransferase PmultST3 from Pasteurella multocida (UniProtID Q9CLP3) or a PmultST3-like polypeptide having β-galactoside α-2,3-sialyltransferase activity consisting of amino acid residues 1 to 268 of UniProtID Q9CLP3 (SEQ ID NO: 02), or an α-2,6-sialyltransferase, such as the α-2,6-sialyltransferase PdST6 from Photorhabditis mermanni (UniProtID Q9CLP3). 066375) or a PdST6-like polypeptide consisting of amino acid residues 108 to 497 of UniProtID 066375 having β-galactoside α-2,6-sialyltransferase activity (SEQ ID NO: 03). In one example for producing one or more sialylated oligosaccharides such as LSTc and LSTd, a Chlamydomonas reinhardtii strain is modified for production of CMP-sialic acid, UDP-galactose, LN3, and LNnT as described herein, and for expression of one or more compatible sialyltransferases.

[0375] F. Animal cells

[0376] Isolation of mesenchymal stem cells from adipose tissue of different animals

[0377] Fresh adipose tissue is obtained from slaughterhouses (e.g., cattle, pigs, sheep, chickens, ducks, catfish, snakes, frogs) or liposuction (e.g., in the case of humans, after informed consent) and maintained in phosphate-buffered saline supplemented with antibiotics. Enzymatic digestion of the adipose tissue is performed, followed by centrifugation to isolate mesenchymal stem cells. The isolated mesenchymal stem cells are transferred to cell culture flasks and grown under standard growth conditions (e.g., 37° C., 5% CO 2 ). The initial culture medium includes DMEM-F12, RPMI, and α-MEM culture media (supplemented with 15% fetal bovine serum), and 1% antibiotics. Subsequently, after the first passage, the culture medium is replaced with a culture medium supplemented with 10% FBS (fetal bovine serum). For example, Ahmad and Shakoori (2013, Stem Cell Regen. Med. 9(2): 29-36) (which is incorporated herein by reference in its entirety for all purposes) describe certain variations of the methods described herein in this example.

[0378] Isolation of mesenchymal stem cells from milk

[0379] This example illustrates the isolation of mesenchymal stem cells from milk collected under aseptic conditions from humans or any other mammals (e.g., as described herein). An equal volume of phosphate-buffered saline was added to the diluted milk, followed by centrifugation for 20 minutes. The cell pellet was washed three times with phosphate-buffered saline, and the cells were seeded in cell culture flasks under standard culture conditions in DMEM-F12, RPMI, and α-MEM culture media supplemented with 10% fetal bovine serum and 1% antibiotics. For example, Hassiotou et al. (2012, Stem Cells. 30(10): 2164-2174) (which is incorporated herein by reference in its entirety for all purposes) describes certain variations of the methods described herein in this example.

[0380] Stem cell differentiation using 2D and 3D culture systems

[0381] Mesenchymal cells isolated from adipose tissue of different animals or from milk as described above can be differentiated into mammary epithelial and luminal cells in 2D and 3D culture systems. See, for example, Huynh et al., 1991, Exp Cell Res. 197(2): 191-199; Gibson et al., 1991, In Vitro Cell Dev Biol Anim. 27(7): 585-594; Blatchford et al., 1999, Animal Cell Technology': Basic & Applied Aspects, Springer, Dordrecht. 141-145; Williams et al., 2009, Breast Cancer Res 11(3): 26-43; and Arevalo et al., 2015, Am J Physiol Cell Physiol. 310(5): C348-C356, each of which is incorporated herein by reference in its entirety for all purposes.

[0382] For 2D cultivation, initially the cells separated are seeded in the growth medium that is supplemented with 10ng / mL epidermal growth factor and 5pg / mL insulin in culture plate.When converging, with the growth medium that is supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100U / mL penicillin, 100ug / mL streptomycin) and 5pg / mL insulin feeder cells 48 hours.In order to induce differentiation, with the complete growth medium feeder cells that comprises 5pg / mL insulin, 1pg / mL hydrocortisone, 0.65ng / mL triiodothyronine, 100nM dexamethasone and 1pg / mL prolactin.After 24 hours, from complete induction medium, remove serum.

[0383] For 3D cultivation, the cells separated are carried out trypsinization, and it is cultivated six days in Matrigel, hyaluronic acid or ultra-low attachment surface culture plate, and induces it to differentiate and lactate by adding the growth medium that is supplemented with 10ng / mL epidermal growth factor and 5pg / mL insulin.When converging, with the growth medium that is supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100U / mL penicillin, 100ug / mL streptomycin) and 5pg / mL insulin feeder cells 48 hours.In order to induce differentiation, with the complete growth medium feeder cells that comprises 5pg / mL insulin, 1pg / mL hydrocortisone, 0.65ng / mL triiodothyronine, 100nM dexamethasone and 1pg / mL prolactin.After 24 hours, from complete induction medium, remove serum.

[0384] Method for preparing mammary-like cells

[0385] In the next step, cells are brought to inducible pluripotency by being reprogrammed with the viral vector encoding Oct4, Sox2, Klf4 and c-Myc. Then, the reprogrammed cells of gained are cultured in Mammocult culture medium (available from StemCell Technologies) or mammary cell enrichment culture medium (DMEM, 3% FBS, estrogen, progesterone, heparin, hydrocortisone, insulin, EGF) to make it become mammary gland sample, from which the expression of selected milk components can be induced. Alternatively, epigenetics is remodeled by using remodeling system such as CRISPR / Cas9, to activate selected target gene (for example, casein, α-lactalbumin) and become constitutively opened, thereby allowing the expression of its respective protein, and / or lowering and / or knocking out selected endogenous gene, as described in WO21067641 (it is incorporated herein by reference in its entirety for all purposes). In one example for producing one or more oligosaccharides, isolated mesenchymal cells reprogrammed into mammary-like cells are modified via CRISPR-CAS as described, for example, in WO22034067, WO22034070, and WO22034075.

[0386] nourish

[0387] Complete growth medium contains high glucose DMEM / F12, 10% FBS, 1% NEAA, 1% pen / strep, 1% ITS-X, 1% F-Glu, 10 ng / mL EGF, and 5 pg / mL hydrocortisone. Complete lactation medium contains high glucose DMEM / F12, 1% NEAA, 1% pen / strep, 1% ITS-X, 1% F-Glu, 10 ng / mL EGF, 5 pg / mL hydrocortisone, and 1 pg / mL prolactin (5 ug / mL, in Hyunh 1991). Cells are plated at 20,000 cells / cm 2 The cells are seeded at a density of 100 μg / mL onto a collagen-coated bottle in complete growth medium and allowed to adhere and expand in complete growth medium for 48 hours, after which the medium is converted to complete lactation medium. After exposure to the lactation medium, the cells begin to differentiate and stop growing. In approximately one week, the cells begin to secrete lactation products such as milk fat, lactose, casein, and whey into the culture medium. The desired concentration of the lactation medium can be obtained by concentrating or diluting (by ultrafiltration). The desired salt balance of the lactation medium can be obtained by dialysis (for example, to remove unwanted metabolites from the culture medium). The hormones and other growth factors used can be selectively extracted by resin purification, for example, using nickel resin to remove growth factors with His tags to further reduce the contaminant levels in the lactation products.

[0388] G. Optical density

[0389] The cell density of the cultures was monitored regularly by measuring the optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium, or with Spark 10M microplate reader, Tecan, Switzerland). The maximum growth rate (mumax) was calculated based on the observed optical density at 600 nm using the R package grofit.

[0390] H. Growth rate / velocity measurements

[0391] The maximum growth rate (μMax) was calculated based on the observed optical density at 600 nm by using the R package grofit.

[0392] I. Heterologous and homologous expression

[0393] The gene to be expressed, whether it comes from a plasmid or from the genome, is synthesized synthetically by one of the following companies: IDT or Twist Bioscience. The proteins described in this disclosure are summarized in Table 1. Unless otherwise indicated, the UniProt ID of the described protein corresponds to its sequence version 01 as present in the UniProt database version release 2021_03 on June 9, 2021. Expression can be further promoted by optimizing codon usage to that of the expression host. The supplier's tools were used to optimize the genes. Table 1. Overview of proteins with corresponding SEQ ID NO or UniProt ID described in this disclosure (sequence version 01, UniProt database 2021_03 on June 9, 2021)

[0394]

[0395] ■Sequence version 02 (February 1, 2005) present in the UniProt database 2021_03 on June 9, 2021;

[0396] ■■Sequence version 03 (February 10, 2009) present in the UniProt database 2021_03 on June 9, 2021;

[0397] ■■■Sequence version 03 (July 5, 2005) present in the UniProt database 2021_03 on June 9, 2021;

[0398] *Sequence version 02 (November 1, 1997) present in the UniProt database 2021_03 on June 9, 2021;

[0399] **Sequence version 03 (January 23, 2007) present in the UniProt database 2021_03 on June 9, 2021;

[0400] ***Sequence version 03 (December 2, 2020) present in the UniProt database 2021_03 on June 9, 2021;

[0401] ****Sequence version 03 (January 23, 2007) present in the UniProt database 2021_03 on June 9, 2021;

[0402] *****Sequence version 02 (January 23, 2007) present in the UniProt database 2021_03 on June 9, 2021;

[0403] °Sequence version 04 (January 23, 2007) present in the UniProt database 2021_03 on June 9, 2021;

[0404] °°Sequence version 02 (December 1, 2000) present in the UniProt database 2021_03 on June 9, 2021;

[0405] °°°Sequence version 03 (July 19, 2003) present in the UniProt database 2021_03 on June 9, 2021;

[0406] °°°°Sequence version 03 (October 13, 2009) present in the UniProt database 2021_03 on June 9, 2021;

[0407] °°°°°Sequence version 02 (February 1, 2005) present in the UniProt database 2021_03 on June 9, 2021;

[0408] ∞Sequence version 02 (April 7, 2021) present in the UniProt database 2021_03 on June 9, 2021;

[0409] ∞∞Sequence version 04 (June 2, 2021) present in UniProt database 2021_03 on June 9, 2021.

[0410] J. Analytical Analysis

[0411] Standards such as, but not limited to, sucrose, lactose, 3'SL, 6'SL, lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), LNFP-I, LNFP-II, LNFP-III, LNFP-V, LSTa, LSTc, and LSTd were purchased from Carbosynth (UK), Elicityl (France), and IsoSep (Sweden). Other compounds were analyzed using standards prepared in-house.

[0412] Neutral oligosaccharides were analyzed on a Waters Acquity H-class UPLC with evaporative light scattering detector (ELSD) or refractive index (RI) detection. A volume of 0.7 μL of sample was injected onto an Acquity UPLC BEH Amide VanGuard column ( Waters Acquity UPLC BEH Amide column (2.1x100mm) 1.7 μm). The column temperature was 50°C. The mobile phase consisted of 1 / 4 water and 3 The ELSD detector had a drift tube temperature of 50°C, an N2 gas pressure of 50 psi, a gain of 200, and a data rate of 10 pps. The RI detector temperature was set at 35°C.

[0413] Sialyl oligosaccharides were analyzed on a Waters Acquity H-class UPLC with refractive index (RI) detection. A volume of 0.5 μL of sample was injected onto a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm; The column temperature was 50°C. The mobile phase consisted of a mixture of 70% acetonitrile, 26% ammonium acetate buffer (150 mM), and 4% methanol to which 0.05% pyrrolidine was added. The method was isocratic with a flow rate of 0.150 mL / min. The temperature of the RI detector was set at 35°C.

[0414] Both neutral and sialylated sugars were analyzed on a Waters Acquity H-class UPLC with refractive index (RI) detection. A volume of 0.5 μL of sample was injected onto a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm; The column temperature was 50° C. The mobile phase consisted of a mixture of 72% acetonitrile and 28% ammonium acetate buffer (100 mM) to which 0.1% triethylamine was added. The method was isocratic with a flow rate of 0.260 mL / min. The temperature of the RI detector was set at 35° C.

[0415] For analysis on a mass spectrometer, a Waters Xevo TQ-MS with electrospray ionization (ESI) was used, with a desolvation temperature of 450° C., a nitrogen desolvation gas flow rate of 650 L / h, and a cone voltage of 20 V. The MS was operated in negative mode with selected ion monitoring (SIM) for all oligosaccharides. Separation was performed on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1×100 mm; 3 μm) at 35° C. A gradient was used in which eluent A was ultrapure water with 0.1% formic acid and eluent B was acetonitrile with 0.1% formic acid. The oligosaccharides were separated over 55 minutes using the following gradient: an initial increase in eluent B from 2 to 12% in 21 minutes, a second increase in eluent B from 12 to 40% in 11 minutes, and a third increase in eluent B from 40 to 100% in 5 minutes. As a washing step, 100% eluent B was used for 5 minutes. For column equilibration, the initial conditions of 2% eluent B were restored within 1 minute and maintained for 12 minutes.

[0416] Neutral sugars and sialylated sugars at low concentrations (less than 50 mg / L) were analyzed on a Dionex HPAEC system with pulsed amperometric detection (PAD). A volume of 5 μL of sample was injected onto a Dionex CarboPac PA200 column 4x250 mm with a Dionex CarboPac PA200guard column 4x50 mm. The column temperature was set to 30° C. A gradient was used where eluent A was deionized water, eluent B was 200 mM sodium hydroxide, and eluent C was 500 mM sodium acetate. Oligosaccharides were separated over 60 minutes while maintaining a constant proportion of 25% eluent B using the following gradient: an initial isocratic step (75% eluent A) with a hold of 10 minutes, an initial increase from 0 to 4% eluent C over 8 minutes, a second isocratic step (71% eluent A and 4% eluent C) with a hold of 6 minutes, a second increase from 4 to 12% eluent C over 2.6 minutes, a third isocratic step (63% eluent A and 12% eluent C) with a hold of 3.4 minutes, and a third increase from 12 to 48% eluent C over 5 minutes. As a wash step, 48% eluent C was used for 3 minutes. For column equilibration, the initial conditions of 75% eluent A and 0% eluent C were restored over 1 minute and held for 11 minutes. The applied flow rate was 0.5 mL / min.

[0417] Lactobionic acid is analyzed on the Dionex HPAEC system with pulsed current detection (PAD). The volume of 5 μL sample is injected on the Dionex CarboPac PA01 post 2x250mm with Dionex CarboPac PA01 guard post 4x50mm. Column temperature is set to 30 ℃. Use gradient, wherein eluent A is ultrapure water, eluent B is 200mM sodium hydroxide, and eluent C is 500mM sodium acetate. The total gradient time is 41 minutes, and in the first 7 minutes, starts with 50% B and 5% C. During the next 18 minutes, the concentration slowly changes to 50% B and 40% C at 25 minutes, followed by a rinsing step with 100% C for 7 minutes. For column balance, the initial condition with 50% B and 5% C is recovered within 9 minutes. The flow rate applied is 0.25mL / minute.

[0418] Example 2. Evaluation of sialic acid and 6'SL production using a modified E. coli host in which the glmU gene was functionally impaired

[0419] The E. coli K-12 MG1655 strain was modified for sialic acid production by genomic knock-in of a constitutive transcription unit comprising mutant glmS*54 from E. coli (SEQ ID NO: 01) (which differs from wild-type E. coli glmS with UniProt ID P17169 by the A39T, R250C, and G472S mutations as described by Deng et al. (Biochimie 88, 419-29 (2006)), glucosamine 6-phosphate N-acetyltransferase GNA1 from Saccharomyces cerevisiae (UniProt ID P43577), N-acylglucosamine 2-epimerase AGE from Bacteroides ovatus (UniProt ID A7LVG6), and N-acetylneuraminic acid synthase NeuB from Neisseria meningitidis (UniProt ID 10NCD4). To allow the production of 6'SL, the strain was further modified with constitutive transcription units for the N-acylneuraminic acid cytidylyltransferase NeuA from Campylobacter jejuni (UniProtID Q93MP7) and NeuA from Haemophilus influenzae (SEQ ID NO: 04) and two constitutive transcription units for the expression of a PdST6-like polypeptide (SEQ ID NO: 03) consisting of amino acid residues 108 to 497 of UniProtID O66375 and having β-galactoside α-2,6-sialyltransferase activity. Further, the strain was modified with a genomic knock-in for overexpression of the acetyl-CoA synthetase acs from Escherichia coli (UniProt IDP27550) and a genomic knock-out of the O-antigen gene cluster (thereby deleting all genes between wbbK and wcaN, including wbbK and wcaN). Next, the strain was modified as described in Example 1 for growth on sucrose. The mutant strain A thus obtained was then further engineered to create five new strains (B, C, D, E, and F) having a glmU gene (UniProt ID P0ACC7) that was functionally impaired compared to the native glmU gene present in strain A. Each strain was created by a different genomic knock-in comprising a constitutive transcription unit of the native glmU gene from E. coli (UniProt ID P0ACC7), introduced into the genome of E. coli cells at a foreign location, i.e., at the insN locus, together with different promoter (P), untranslated region (UTR), and terminator (T) sequences (Table 3), and subsequent genomic knock-out of the native glmU gene from E. coli (locus 3,913,830<-3,915,200).According to the culture conditions provided in Example 1, the new strains were evaluated for sialic acid and 6'SL production in a growth experiment, wherein the strains were cultured in a minimal medium with 30 g / L sucrose and 20 g / L lactose. The strains were grown in 96-well plates with four biological replicates. After 72 hours of incubation, the culture fluids were harvested and analyzed for sugars on UPLC. For each strain with a specific glmU transcription unit, the measured sialic acid concentration, the sum of sialylated molecules (sialic acid and 6'SL), and the biomass concentration were averaged over all biological replicates and then standardized to the average sialic acid concentration, sum of sialylated molecules, and biomass concentration of a reference strain A with the same genetic makeup but lacking any modification to the native glmU gene. This experiment showed that each knockdown of glmU in the five strains resulted in higher sialic acid and sialylated molecules (sialic acid and 6'SL) production compared to the reference strain A (Table 2). Surprisingly, this experiment also showed that each knockdown of glmU in the five strains resulted in lower biomass production in all five strains B, C, D, E and F compared to the reference strain A (Table 2), leading to higher CPI for sialic acid and for sialylated molecules.

[0420] Table 2. Relative production (%) of sialic acid (SA), relative production (%) of total sialylated molecules (SA + 6'SL), relative production (%) of biomass, CPI (%) of SA, and CPI (%) of total sialylated molecules (SA + 6'SL) of modified E. coli strains B, C, D, E, and F expressing a knockdown transcription unit of E. coli glmU integrated at the insN locus and having a genomic knockout of the native glmU gene, compared to a genetically engineered reference strain A lacking its native glmU gene. The strains were evaluated in growth experiments according to the culture conditions provided in Example 1, wherein the culture medium contained 30 g / L sucrose and 20 g / L lactose.

[0421]

[0422] *For details on the promoter (P), untranslated region (UTR), and terminator (T) sequences used to express the E. coli glmU gene (UniProt ID P0ACC7), see Table 3

[0423] Table 3. Promoter, untranslated region (UTR) and terminator sequences for expression of the glmU enzyme from E. coli (UniprotID POACC7) from the knockdown transcription units integrated into the mutant E. coli strains B, C, D, E, F, H, I and J given in Tables 2 and 4.

[0424]

[0425] Example 3. Evaluation of sialic acid and 6'SL production using a modified E. coli host in which the glmU gene was functionally disabled and E. coli glmS (UniProt ID P17169) was overexpressed

[0426] The mutant E. coli strain F described in Example 2 was further modified to overexpress its native E. coli glmS gene (UniProtID P17169) by integrating a strong constitutive promoter sequence at the native glmU locus (locus 3,913,830 <-3,915,200) 5' of the E. coli glmS gene. This created three new strains, each expressing E. coli glmS from a different strong constitutive promoter sequence (Table 4). The new bacterial strains were further transformed with an expression plasmid containing constitutive transcription units for the following: N-acylneuraminic acid cytidylyltransferase neuA (UniProt ID A0A849CI62) from Pasteurella multocida, a PdST6-like polypeptide (SEQ ID NO:03) consisting of amino acid residues 108 to 497 of UniProt ID 066375 with β-galactoside α-2,6-sialyltransferase activity, and an iron carrier transporter entS (UniProt ID P24077) from Escherichia coli, resulting in bacterial strains H, I, and J (Table 4). Bacterial strain F was also transformed with the expression plasmid, resulting in reference strain G (Table 4). All bacterial strains were evaluated for sialic acid and 6'SL production in a growth experiment according to the culture conditions provided in Example 1, wherein the bacterial strains were cultivated in a minimal medium with 30 g / L sucrose and 20 g / L lactose. The bacterial strains were grown in 96-well plates with four biological replicates. After 72 hours of incubation, the culture fluids were harvested and analyzed for sugars on UPLC. For each strain with a specific constitutive promoter sequence that controls the overexpression of E. coli glmS, the measured sialic acid concentration, the sum of sialylated molecules (sialic acid and 6'SL), and the biomass concentration were averaged across all biological replicates and then normalized to the average sialic acid concentration, sum of sialylated molecules, and biomass concentration of a reference strain lacking a constitutive promoter in front of glmS. This experiment showed that expression of E. coli glmS from different strong constitutive promoter sequences resulted in higher sialic acid and sialylated molecules (sialic acid and 6'SL) production compared to reference strain G (Table 4). No effect was seen for biomass production.

[0427] Table 4. Relative production of sialic acid (%), total sialylated molecules (sialic acid + 6'SL, %), and biomass (%) by modified E. coli strains H, I, and J expressing a knockdown transcription unit of E. coli glmU (i.e., P111-UTR64-glmU-T10, Table 2) and overexpressing the native glmS gene (UniProtID P17169) from a strong constitutive promoter sequence, compared to a genetically engineered reference strain G lacking its native glmS gene. The strains were evaluated in growth experiments according to the culture conditions provided in Example 1, wherein the medium contained 30 g / L sucrose and 20 g / L lactose.

[0428]

[0429] *For details on the promoter (P), untranslated region (UTR), and terminator (T) sequences used to express the E. coli glmU gene (UniProt ID P0ACC7), see Table 3

[0430] Example 4. Evaluation of modified E. coli hosts for the production of sialic acid and 6'SL when evaluated in fed-batch fermentations with sucrose and lactose

[0431] The mutant E. coli strains G, H and I described in Example 3 were selected for further evaluation in fed-batch fermentation. Fed-batch fermentation was carried out on a bioreactor scale as described in Example 1. Sucrose was used as a carbon source, and lactose was added to the batch culture medium. During the fed-batch period, sucrose was added via another feed. In contrast to the culture experiments described herein and in which a final sample was taken only at the end of the culture (i.e., 72 hours described herein), broth samples were regularly taken out at several time points during the fermentation pr...

Claims

1. A cell capable of synthesizing and / or synthesizing UDP-N-acetylglucosamine (UDP-GlcNAc), said cell comprising a pathway for producing disaccharides and / or milk oligosaccharides, said cell being genetically engineered for the production of said disaccharides and / or milk oligosaccharides, characterized in that The UDP-GlcNAc synthesis in the cell is rendered functionally impaired.

2. The cell according to claim 1, wherein: - said pathway for producing disaccharides and / or milk oligosaccharides is selected from the list consisting of or consisting essentially of: a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway and an N-acetylmannosaminylation pathway, and / or - said pathway for producing disaccharides and / or milk-oligosaccharides is selected from the list consisting of or consisting essentially of: a fucosylation pathway, a sialylation pathway, a galactosylation pathway, an N-acetylglucosaminylation pathway, an N-acetylgalactosaminylation pathway, a mannosylation pathway and an N-acetylmannosaminylation pathway, and wherein said cell is genetically engineered to comprise at least one of said pathways, and / or said cell comprises at least one of said pathways, wherein at least one of said pathways has been genetically engineered.

3. The cell according to any one of claims 1 or 2, wherein the cell: - having, expressing and / or overexpressing one or more glycosyltransferases selected from the list consisting of or consisting essentially of: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosamine transferase, N-acetylgalactosamine transferase, N-acetylmannosamine transferase, xylosyltransferase, glucuronyltransferase, galacturonyltransferase, glucosamine transferase, N-glycolylneuraminic acid transferase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine transaminase, UDP-N-acetylglucosamine enolpyruvyltransferase and fucosamine transferase, - is capable of producing and / or produces one or more nucleotide activating sugars, - genetically engineered to produce one or more nucleotide activated sugars, - comprising a pathway for synthesizing a nucleotide-activated sugar selected from the list 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-acetylamino-2,6-dideoxy-L-arabino-4-hexulose, UDP-2-acetylamino-2,6-dideoxy-L-lyxo-4-hexulose, UDP-N-acetyl-L-rhamnosamine (UDP-L-RhaNAc or UDP-2-acetylamino-2,6-dideoxy-L-mannose), dTDP-N- Acetylfucosamine, UDP-N-acetylfucosamine (UDP-L-FucNAc or UDP-2-acetylamino-2,6-dideoxy-L-galactose), UDP-N-acetyl-L-l-phospho-glucosamine (UDP-L-PneNAc or UDP-2-acetylamino-2,6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnosamine (UDP-L-QuiNAc or UDP-2-acetylamino-2,6-dideoxy-L-talose), CMP-Neu5Ac, CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7(8,9)Ac2, CMP-Neu5Gc, GDP-rhamnose, and UDP-xylose, - having and / or expressing one or more genes selected from the list consisting of or consisting essentially of: mannose-6-phosphate isomerase, phosphomannose mutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, 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, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, 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, more preferably overexpressing one or more genes selected from the list comprising: mannose-6-phosphate isomerase, phosphomannosemutase, mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, fucose-1-phosphate guanylyltransferase, L-glutamine-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, N-acetylglucosamine-6-P deacetylase, N-acylglucosamine 2-epimerase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine-6-phosphate 2-epimerase, UDP-GlcNAc 2-epimerase / kinase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, phosphoacetylglucosamine mutase, Neu5Ac synthase, N-acetylneuraminic acid lyase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphatase, 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, and / or, comprising at least partially inactivated catabolic pathways for selected monosaccharides, disaccharides or oligosaccharides which are involved and / or required for the synthesis of said disaccharides and / or milk-oligosaccharides.

4. The cell of any one of the preceding claims, wherein the UDP-GlcNAc synthesis has at least one gene selected from the list consisting of or essentially consisting of: genes encoding a bifunctional N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, and glucosamine-1-phosphate acetyltransferase, and wherein the at least one gene is functionally impaired.

5. The cell of claim 4, wherein the at least one gene encodes an enzyme, wherein the enzyme: - an enzyme class selected from the list consisting of or consisting essentially of EC: 2.7.7.23, EC: 2.3.1.157 and EC: 5.4.2.3, comprising a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of IPR001451, IPR002618, IPR005175, IPR005835, IPR005843, IPR005844, IPR005882, IPR011004, IPR016055, IPR016066, IPR016657, IPR018357, IPR023915, IPR025877, IPR029044, IPR036900 and IPR038009 as defined by InterPro 90.0 released on August 4, 2022, comprising a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of PF00132, PF00408, PF00465, PF00483, PF01070, PF01565, PF01704, PF02878, PF02879, PF02880, PF03479, PF04030, PF05199, PF12146, PF12804, PF13562, and PF14602 as defined by InterPro 90.0, released on August 4, 2022, - comprising a polypeptide sequence comprising a conserved protein domain selected from the list consisting of or consisting essentially of: cd03086 and cd03353 as defined by InterPro 90.0 released on August 4, 2022, - is part of a NOG family selected from the list consisting of or consisting essentially of: COG1109 and COG4284 as defined by eggNOG5.0 released in 2019, and / or - using a cofactor selected from the list consisting of or consisting essentially of: Mg 2+ 、Co 2+ 、Mn 2+ , Ca 2+ 、Zn 2+ 、Ni 2+ and FAD.

6. The cell according to any one of the preceding claims, wherein at least one gene involved in the synthesis and / or import of the following cofactors is rendered functionally impaired: - cofactors involved in the synthesis of UDP-GlcNAc, and / or - a cofactor involved in the synthesis of UDP-GlcNAc and selected from the list consisting of or consisting essentially of: Mg 2+ 、Co 2+ 、Mn 2+ , Ca 2+ 、Zn 2+ 、Ni 2+ and FAD.

7. The cell according to any one of claims 4 to 6, wherein the at least one gene is functionally impaired by insertion, deletion and / or modification of one or more nucleotides in one or more polynucleotide sequences selected from the list comprising: a promoter sequence, a ribosome binding site, an untranslated region, a coding sequence and a transcription terminator sequence of the at least one gene.

8. The cell according to any one of the preceding claims, wherein the disaccharide and / or milk oligosaccharide is selected from the list consisting of or consisting essentially of: lactobiose; mammalian milk disaccharide; human milk disaccharide; N-acetyllactosamine (LacNAc); lacto-N-biose (LNB); neutral (uncharged) milk oligosaccharide; negatively charged milk oligosaccharide; sialylated milk oligosaccharide; mammalian milk oligosaccharide (MMO); human milk oligosaccharide (HMO); fucosylated milk oligosaccharide; non-fucosylated neutral (uncharged) milk oligosaccharide; sialylated mammalian milk oligosaccharide; neutral (uncharged) mammalian milk oligosaccharide; fucosylated mammalian milk oligosaccharide Animal milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; fucosylated milk oligosaccharides selected from the list comprising: 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose Pentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated milk oligosaccharides selected from the list consisting of 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto- N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II, and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine;Neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine selected from the following list: lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, and p-lacto-N-neohexose; milk oligosaccharides containing N-acetylglucosamine; milk oligosaccharides containing N-acetyllactosamine; milk oligosaccharides containing fucosylated N-acetyllactosamine; milk oligosaccharides containing sialylated N-acetyllactosamine; milk oligosaccharides containing lacto-N-biose; milk oligosaccharides containing fucosylated lacto-N-biose; and milk oligosaccharides containing sialylated lacto-N-biose.

9. The cell of any one of the preceding claims, wherein the cell: - is capable of producing and / or produces said disaccharides and / or milk-oligosaccharides from one or more precursors, - is capable of producing from lactose and / or produces said disaccharides and / or milk oligosaccharides from lactose, - capable of producing and / or produces at least one precursor for the production of said disaccharides and / or milk-oligosaccharides, - is capable of producing and / or produces all precursors for the production of said disaccharides and / or milk-oligosaccharides, - genetically engineered to produce at least one precursor for the production of said disaccharides and / or milk-oligosaccharides, and / or - genetically engineered to produce all precursors for the production of said disaccharides and / or milk-oligosaccharides.

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

11. The cell according to any one of the preceding claims, wherein the cell is further genetically engineered to possess, express and / or overexpress glutamine-fructose-6-phosphate aminotransferase.

12. The cell of claim 11, wherein the glutamine-fructose-6-phosphate aminotransferase has glutamine-fructose-6-phosphate aminotransferase activity, and: - is selected from enzyme class EC: 2.6.1.16, comprising a polypeptide sequence comprising an IPR domain selected from the list consisting of or consisting essentially of IPR001347, IPR005855, IPR017932, IPR029055, IPR035466, IPR035490, IPR036291, IPR046348 and IPR047084 as defined by InterPro 90.0 released on August 4, 2022, - comprising a polypeptide sequence comprising a PFAM domain selected from the list consisting of or consisting essentially of: PF00310, PF01380, PF01408, PF13230, PF13537 and PF13580 as defined by InterPro 90.0 released on August 4, 2022, - comprising a polypeptide sequence comprising a conserved protein domain selected from the list consisting of or consisting essentially of: cd00714, cd05007, cd05008, cd05009, cd05013 and cd05710 as defined by InterPro 90.0 released on August 4, 2022, and / or - is part of the NOG family COG0449 defined by eggNOG5.0 released in 2019.

13. The cell of any one of claims 11 or 12, wherein the cell comprises a nucleic acid molecule comprising a polynucleotide sequence encoding the glutamine-fructose-6-sulfate aminotransferase, and wherein the nucleic acid molecule: - is operably linked to a control sequence recognized by the cell, the nucleic acid molecule further i) integrated into the genome of the cell and / or ii) presented to the cell on a vector, and / or - is foreign to the cell.

14. The cell of any one of the preceding claims, wherein the cell: - modified for enhanced synthesis and / or supply of phosphoenolpyruvate (PEP), and / or - further modified to reduce the degradation of acetyl-CoA and / or its main precursor pyruvate.

15. The cell of any one of the preceding claims, wherein the cell is: - bacteria, fungi, yeast, plant cells, animal cells or protozoan cells, - Escherichia coli (E. coli) or yeast with a lactose permease-positive phenotype, and / or - Escherichia coli or yeast having a lactose permease-positive phenotype, wherein the lactose permease is encoded by the gene LacY or LAC12, respectively.

16. A method for producing disaccharides and / or milk oligosaccharides, the method comprising: iii. Cultivating and / or incubating the cell according to any one of the preceding claims in a culture and / or incubation medium under conditions allowing the production of said disaccharides and / or milk oligosaccharides, and / or iv. separating the disaccharide and / or milk-oligosaccharide from the cultivation and / or incubation.

17. The method according to claim 16, wherein: - said culture or incubation medium comprises one or more precursors for the production of said disaccharides and / or milk-oligosaccharides, - the culture medium comprises at least one carbon source selected from the group consisting of glucose, fructose, sucrose and glycerol, and / or - the culture or incubation medium comprises at least one compound selected from the list consisting of lactose, galactose, glucose, UDP-galactose (UDP-Gal), sialic acid and CMP-sialic acid.

18. The method of any one of claims 16 or 17, wherein the cells produce 30 g / L or more of the disaccharides and / or milk-oligosaccharides in the whole broth and / or supernatant, and / or wherein the disaccharides and / or milk-oligosaccharides in the whole broth and / or supernatant have a purity of at least 80%, measured based on the total amount of disaccharides and / or milk-oligosaccharides and their precursors produced by the cells in the whole broth and / or supernatant, respectively.

19. The method according to any one of claims 16 to 18, wherein the functionally impaired synthesis of UDP-GlcNAc confers unaffected and / or enhanced i) disaccharide and / or milk-oligosaccharide formation, ii) productivity, iii) biomass production, iv) cell growth, and / or v) yield of the produced disaccharides and / or milk-oligosaccharides relative to corresponding non-engineered cells.

20. The method according to any one of claims 16 to 19, wherein: - recovering said disaccharide and / or milk-oligosaccharide from said culture or incubation medium and / or said cells, and / or - Purification of said disaccharides and / or milk-oligosaccharides.

21. Use of a cell according to any one of claims 1 to 15 for the production of disaccharides and / or milk oligosaccharides, wherein the disaccharides and / or milk oligosaccharides are selected from the list consisting of or essentially consisting of: lactobiose; mammalian milk disaccharides; human milk disaccharides; N-acetyllactosamine (LacNAc); milk-N-biose (LNB); neutral (uncharged) milk oligosaccharides; negatively charged milk oligosaccharides; sialylated milk oligosaccharides; mammalian milk oligosaccharides (MMO); human milk oligosaccharides (HMO); fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) mammalian milk oligosaccharides Animal milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; fucosylated milk oligosaccharides selected from the list comprising: 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose II, lacto-N-fucopentose III, lacto-N-fucopentose V, lacto-N-fucopentose VI, lacto-N-neofucopentose V, lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated milk oligosaccharides selected from the list comprising 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose d (LSTd), di-s ... Acyl lacto-N-tetraose, disialyl lacto-N-neotetraose, monosialyl lacto-N-hexose, disialyl lacto-N-hexose I, disialyl lacto-N-hexose II, monosialyl lacto-N-neohexose I, monosialyl lacto-N-neohexose II, disialyl lacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyl lacto-N-hexose, disialyl monofucosyllacto-N-neohexose, sialyl lacto-N-fucohexose II, disialyl lacto-N-fucopentose II and monofucosyldisialyl lacto-N-tetraose; neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine;Neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine selected from the following list: lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose, and p-lacto-N-neohexose; milk oligosaccharides containing N-acetylglucosamine; milk oligosaccharides containing N-acetyllactosamine; milk oligosaccharides containing fucosylated N-acetyllactosamine; milk oligosaccharides containing sialylated N-acetyllactosamine; milk oligosaccharides containing lacto-N-biose; milk oligosaccharides containing fucosylated lacto-N-biose; and milk oligosaccharides containing sialylated lacto-N-biose.

22. Use of the method according to any one of claims 16 to 20 for producing disaccharides and / or milk oligosaccharides, wherein the disaccharides and / or milk oligosaccharides are selected from the list consisting of or essentially consisting of: lactobiose; mammalian milk disaccharides; human milk disaccharides; N-acetyllactosamine (LacNAc); milk-N-biose (LNB); neutral (uncharged) milk oligosaccharides; negatively charged milk oligosaccharides; sialylated milk oligosaccharides; mammalian milk oligosaccharides (MMO); human milk oligosaccharides (HMO); fucosylated milk oligosaccharides; non-fucosylated neutral (uncharged) milk oligosaccharides; sialylated mammalian milk oligosaccharides; neutral (uncharged) milk oligosaccharides; milk oligosaccharides; fucosylated mammalian milk oligosaccharides; non-fucosylated neutral (uncharged) mammalian milk oligosaccharides; sialylated human milk oligosaccharides; neutral (uncharged) human milk oligosaccharides; fucosylated human milk oligosaccharides; non-fucosylated neutral (uncharged) human milk oligosaccharides; fucosylated milk oligosaccharides selected from the list consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 4-fucosyllactose (4FL), 6-fucosyllactose (6FL), 2',3-difucosyllactose (diFL), lacto-N-fucopentose I, lacto-N-neofucopentose I, lacto-N-fucopentose lacto-N-neohexose; sialylated lacto-oligosaccharides selected from the group consisting of 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose d (LSTd), lacto-N-difucohexose I, lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated lacto-oligosaccharides selected from the group consisting of 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N-tetraose d (LSTd), lacto-N-difucohexose II, difucosyl-lacto-N-hexose and difucosyl-lacto-N-neohexose; sialylated lacto-oligosaccharides selected from the group consisting of 3' sialyllactose (3'SL), 6' sialyllactose (6'SL), sialyllacto-N-tetraose a (LSTa), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), sialyllacto-N-tetraose d (LSTd), disialyllacto-N lacto-N-tetraose, disialyllacto-N-neotetraose, monosialyllacto-N-hexose, disialyllacto-N-hexose I, disialyllacto-N-hexose II, monosialyllacto-N-neohexose I, monosialyllacto-N-neohexose II, disialyllacto-N-neohexose, 3'-sialyl-3-fucosyllactose, fucodisialyllacto-N-hexose, disialylmonofucosyllacto-N-neohexose, sialyllacto-N-fucohexose II, disialyllacto-N-fucopentose II and monofucosyldisialyllacto-N-tetraose; neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine;Neutral (uncharged) milk oligosaccharides containing N-acetylglucosamine selected from the following list: lacto-N-triose II (LN3), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 6'-galactosyl lactose, 3'-galactosyl lactose, lacto-N-hexose, lacto-N-neohexose, p-lacto-N-hexose and p-lacto-N-neohexose; milk oligosaccharides containing N-acetylglucosamine; milk oligosaccharides containing N-acetyllactosamine; milk oligosaccharides containing fucosylated N-acetyllactosamine; milk oligosaccharides containing sialylated N-acetyllactosamine; Milk oligosaccharides comprising lacto-N-biose; milk oligosaccharides comprising fucosylated lacto-N-biose; and milk oligosaccharides comprising sialylated lacto-N-biose.

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