CN116472346A - Production of sialylated oligosaccharide mixtures by cells - Google Patents

Abstract

The invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the invention is in the technical field of culture or fermentation of metabolically engineered cells. The present invention describes a cell metabolically engineered for the production of a mixture of at least three different sialylated oligosaccharides. Furthermore, the present invention provides a method for producing a mixture of at least three different sialylated oligosaccharides by means of a cell and purifying at least one of said sialylated oligosaccharides from the culture.

Description

Production of sialylated oligosaccharide mixtures by cells

The invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the invention is in the technical field of culture or fermentation of metabolically engineered cells. The present invention describes a cell metabolically engineered for the production of a mixture of at least three different sialylated (sialylated) oligosaccharides. Furthermore, the present invention provides a method for producing a mixture of at least three different sialylated oligosaccharides by means of a cell and purifying at least one of said sialylated oligosaccharides from the culture.

Background

Oligosaccharides, which are often present in the form of carbohydrate binding to proteins and lipids, are involved in many life phenomena such as differentiation, development and biological recognition processes associated with fertilization, embryogenesis, inflammation, cancer metastasis and the occurrence and progression of host pathogen adhesion. Oligosaccharides may also be present in non-conjugated glycan forms in body fluids and human milk, where the oligosaccharides also modulate important developmental and immune processes (Bode, early hum Dev.1-4 (2015); reill et al, nat. Rev. Nephrol.15, 346-366 (2019); varki, glycobiology 27,3-49 (2017)). There is great scientific and commercial interest in oligosaccharide mixtures due to the broad spectrum of functions of oligosaccharides. However, the availability of oligosaccharide mixtures is limited because production relies on chemical or chemoenzymatic synthesis or on purification from natural sources, such as animal milk. Chemical synthesis methods are laborious and time-consuming and are difficult to scale up due to the large number of steps involved. Enzymatic pathways using glycosyltransferases (glycosyltransferases) offer a number of advantages over chemical synthesis. Glycosyltransferases catalyze the transfer of sugar moieties from an activated nucleotide-sugar donor to a sugar or non-sugar acceptor (Coutinho et al J.mol.biol.328 (2003) 307-317). These glycosyltransferases are a source of oligosaccharides for the biotechnologies to synthesize and are used in (chemical) enzymatic pathways as well as in cell-based production systems. However, the stereospecificity and regioselectivity of glycosyltransferases remain a challenge to be addressed. In addition, the chemoenzymatic pathway requires in situ regeneration of the nucleotide-sugar donor. Cellular production of oligosaccharides requires tight control of the space-time availability of nucleotide-sugar donors in the vicinity of complementary glycosyltransferases at sufficient levels. Because of these difficulties, current methods generally allow for the synthesis of single oligosaccharides rather than mixtures of oligosaccharides.

It is an object of the present invention to provide a tool and a method by means of which an oligosaccharide mixture comprising at least three different sialylated oligosaccharides can be produced in an efficient, time-and cost-efficient manner and if necessary in a continuous process by means of cells, preferably single cells, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different sialylated mammalian milk oligosaccharides.

This and other objects are achieved according to the present invention by providing a cell for producing an oligosaccharide mixture and a method for producing an oligosaccharide mixture comprising at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different sialylated mammalian milk oligosaccharides, wherein the cell is genetically modified for producing the sialylated oligosaccharides.

Disclosure of Invention

Surprisingly, it has now been found that it is possible to produce an oligosaccharide mixture comprising at least three different sialylated oligosaccharides by means of a single cell, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different sialylated mammalian milk oligosaccharides. The present invention provides a metabolically engineered cell for producing an oligosaccharide mixture and a method for producing an oligosaccharide mixture comprising at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide. The method comprises the following steps: providing a cell that exhibits a glycosyltransferase that is a sialyltransferase and is capable of synthesizing CMP-N-acetylneuraminic acid (CMP-Neu 5 Ac) and that exhibits at least one additional glycosyltransferase and is capable of synthesizing one or more nucleotide-sugars that are donors for the additional glycosyltransferase, and culturing the cell under conditions that allow the production of the oligosaccharide mixture. The invention also provides a method for separating at least one, preferably all of said oligosaccharides from an oligosaccharide mixture. Furthermore, the present invention provides a cell metabolically engineered for the production of an oligosaccharide mixture comprising at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different sialylated mammalian milk oligosaccharides.

Definition of the definition

The words used in this specification to describe the invention and its various specific examples should be understood not only in the sense of their commonly defined meanings, but to include by definition in this specification structure, material or acts beyond the scope of the commonly defined meanings. Thus, if an element can be understood in the context of this specification as including more than one meaning, then its use in a claim must be understood as being generic to all possible meanings supported by the specification and by the word itself.

The various embodiments of the invention and aspects of the embodiments disclosed herein should be understood not only in the order and circumstance specifically described in the present description, but also to include any order and any combination thereof. Whenever the situation requires, all words used in the singular are to be considered to include the plural and vice versa. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the laboratory procedures, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization in cell culture described herein are those well known and commonly employed in the art, laboratory procedures, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization. Nucleic acid and peptide synthesis was performed using standard techniques. Generally, the purification steps are performed according to manufacturer's instructions.

In this specification, specific examples of the invention have been disclosed, and although specific terms are employed, they are used in a descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims. It must be understood that the illustrated specific examples have been set forth only for the purposes of example and that it should not be taken as limiting the invention. It will be apparent to one having ordinary skill in the art that changes, other embodiments, modifications, details, and uses can be made in light of the text and spirit of the invention herein and within the scope of the invention, which is limited only by the claims as interpreted according to the patent law including the doctrine of equivalents. In the following claims, reference signs are provided for the sole purpose of description and are not intended to imply any particular order for the steps to be performed.

In this document and in its claims, the verb "to comprise" and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Throughout this application, the verb "comprise" may be replaced by "consisting of" to con "or" consisting essentially of "to consistessentially of", and vice versa. In addition, the verb "consist of" may be replaced with "consisting essentially of" meaning that the composition as defined herein may comprise additional component(s) in addition to the specifically identified component(s) that do not alter the unique features of the present invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the presence of more than one element unless the context clearly requires that there be one and only one element. The indefinite article "a" or "an" therefore generally means "at least one (at least one). Throughout this application, unless explicitly stated otherwise, the articles "a" and "an" are preferably replaced by "at least two (at least two)," more preferably "at least three (at least four)," even more preferably "at least five (at least five)," even more preferably "at least six (at least six)," most preferably "at least seven (at least seven)," and "at least three (at least six)," respectively.

Specific examples as identified herein may be combined together unless otherwise indicated. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The priority applications, including the entire contents of EP20190198, EP20190200, EP20190202, EP20190204 and EP20190205, are also incorporated herein by reference to the same extent as if specifically and individually indicated to be incorporated by reference.

According to the present invention, the term "polynucleotide" refers generally to any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA; DNA which is a single-stranded and double-stranded region or a mixture of single-stranded, double-stranded and triple-stranded regions; single-stranded and double-stranded RNAs; and RNA that is a mixture of single and double stranded regions; hybrid molecules comprising DNA and RNA that may be single-stranded or more typically double-stranded or triple-stranded regions, or mixtures of single-stranded and double-stranded regions. In addition, as used herein, the term "polynucleotide" refers to a triple region comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only regions having some of the molecules. One of the molecules of the triple-helical region is often an oligonucleotide. As used herein, the term "polynucleotide" also includes DNA or RNA as described above that contains one or more modified bases. Thus, DNA or RNA having a backbone modified for stability or for other reasons is a "polynucleotide" according to the present invention. In addition, DNA or RNA comprising unusual bases (such as inosine) or modified bases (such as tritylated bases) is understood to be covered by the term "polynucleotide". It will be appreciated that a variety of modifications have been made to DNA and RNA for a number of useful purposes known to those of ordinary skill in the art. The term "polynucleotide" as used herein encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as chemical forms of DNA and RNA that are characteristic of viruses and cells, including, for example, simple and complex cells. The term "polynucleotide" also encompasses short polynucleotides commonly referred to as oligonucleotides.

A "polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptides" refer to both short chains (commonly referred to as peptides, oligopeptides and oligomers) and long chains (commonly referred to as proteins). The polypeptide may contain amino acids other than those encoded by the 20 genes. "Polypeptides" include polypeptides modified by natural processes such as processing and other post-translational modifications, and by chemical modification techniques. Such modifications are well described in the basic text and in more detailed monographs, as well as in numerous research documents, and are well known to those of ordinary skill in the art. The same type of modification may be present to the same or different extents at several sites in a given polypeptide. In addition, a given polypeptide may contain multiple types of modifications. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Modifications include, for example: acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavins, covalent attachment of a blood matrix moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of inositol phosphatidate, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid attachment, sulfation, gamma-carboxylation of a glutamate residue, hydroxylation, ADP-ribosylation, selenization, transfer RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. The polypeptide may be branched or cyclic with or without branching. Cyclic, branched and branched chain loop-like polypeptides may be produced by post-translational natural processes and may also be prepared by entirely synthetic methods.

As used herein, the term "polynucleotide encoding a polypeptide (polynucleotide encoding a polypeptide)" encompasses a polynucleotide comprising a sequence encoding a polypeptide of the invention. The term also encompasses polynucleotides that include a single contiguous or non-contiguous region (e.g., interspersed with either an integrated phage or an inserted sequence or an edit) encoding a polypeptide, as well as other regions that may also contain coding and/or non-coding sequences.

"isolated" means altered from the natural state "artificial (by the hand ofman)", i.e., if it exists in nature, it is altered or removed from its original environment or both. For example, a polynucleotide or polypeptide naturally occurring in a living organism is not "isolated", but the same polynucleotide or polypeptide is "isolated" as it is isolated from coexisting materials in its natural state, as that term is used herein. Similarly, the term "synthetic" sequence as used herein means any sequence that has been synthetically produced and not directly isolated from a natural source. The term "synthetic" as used herein means any synthetically produced sequence and not isolated directly from natural sources.

As used herein with reference to a cell or host cell, the terms "recombinant" or "transgene" or "metabolically engineered (metabolically engineered) or" genetically modified (genetically modified ") are used interchangeably and indicate that a cell replicates a heterologous nucleic acid or exhibits a peptide or protein encoded by a heterologous nucleic acid (i.e., a sequence that is" foreign to the cell (foreign to said cell) "or" foreign to the location or environment in the cell (foreign to said location or environment in said 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. Metabolically engineered or recombinant or transgenic cells may contain genes not found in the native (non-recombinant) form of the cell. Recombinant cells may also contain genes found in cells in their native form, where the genes are modified by artificial means and reintroduced into the cells. The term also encompasses cells that contain nucleic acid endogenous to the cell, which nucleic acid has been modified or whose expression or activity has been modified without removing the nucleic acid from the cell; such modifications include gene replacement and promoter replacement; site-specific mutation; modification obtained by the related art. Accordingly, a "recombinant polypeptide (recombinant polypeptide)" is a polypeptide produced by a recombinant cell. As used herein, a "heterologous sequence (heterologous sequence)" or "heterologous nucleic acid (heterologous nucleic acid)" is a sequence or nucleic acid that originates from a source that is foreign to a particular cell (e.g., from a different species), or that is modified from its original form or from a position in the genome if from the same source. Thus, a heterologous nucleic acid operably linked to a promoter is from a source other than that from which the promoter was derived, or if from the same source, is modified from its original form or from a position in the genome. Heterologous sequences can be introduced stably into the genome of the host microbial cell, for example by transfection, conjugation or transduction, wherein the technique is applied depending on the cell and the sequence to be introduced. Various techniques are known to those of ordinary skill in the art and are disclosed, for example, in Sambrook et al, molecular Cloning: a Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (1989). The term "mutant" cell or microorganism as used in the context of the present invention refers to a genetically modified cell or microorganism.

Within the context of the present invention, the term "endogenous" refers to any polynucleotide, polypeptide or protein sequence that is a natural part of a cell and that is present at its natural location in the chromosome of the cell and whose control of expression has not been altered compared to the natural control mechanism acting on its expression. The term "exogenous" refers to any polynucleotide, polypeptide, or protein sequence that originates from outside the cell under study and is not derived from the natural part of the cell or is not present at its natural location in the chromosome or plastid of the cell.

When used in reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme, the term "heterologous" refers to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is derived from or is derived from a source external to the host organism species. In contrast, the use of "homologous" polynucleotides, genes, nucleic acids, polypeptides or enzymes herein indicates polynucleotides, genes, nucleic acids, polypeptides or enzymes derived from a host organism species. When referring to a gene regulatory sequence or helper nucleic acid sequence (e.g., promoter, 5 'non-translational region, 3' non-translational region, poly-a addition sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genomic homology region, recombination site, etc.) for maintaining or manipulating a gene sequence, it is intended that the regulatory sequence or helper sequence is not naturally associated with the gene with which it is juxtaposed in a construct, genome, chromosome or episome. Thus, a promoter operably linked to a gene that is not operably linked to the promoter in its natural state (i.e., in the form of the genome of a non-genetically engineered organism) is referred to herein as a "heterologous promoter (heterologous promoter), even though the promoter may be derived from the same species (or in some cases, from the same organism) as the gene to which it is linked.

The term "modified activity (modified activity) of a protein or enzyme is related to the change in the activity of the protein or enzyme compared to the wild-type (i.e., native) activity of the protein or enzyme. The modified activity may be an elimination, attenuation, reduction or delay of the protein or enzyme compared to the wild-type activity of the protein or enzyme, but may also be an acceleration or enhancement of the protein or enzyme compared to the wild-type activity of the protein or enzyme. The modified activity of a protein or enzyme is obtained by the modified expression of the protein or enzyme or by the expression of a modified (i.e. mutated) form of the protein or enzyme. The modified activity of the enzyme is further related to the modification of the apparent Mie constant (apparent Michaelis constant) Km and/or apparent maximum speed (Vmax) of the enzyme.

The term "modified expression (modified expression) of a gene" is a change in expression as compared to the wild-type expression of the gene at any stage of the production process of the encoded protein. The modified expression is lower or higher than wild-type, wherein the term "higher expression (higher expression) is also defined as" overexpression "of the gene in the case of an endogenous gene, or as" expression "in the case of a heterologous gene that is not present in the wild-type strain. Lower or reduced expression is obtained by means of techniques commonly known to those of ordinary skill in the art, such as using siRNA, crispR, crispRi, riboswitch, recombinant engineering, homologous recombination, ssDNA mutagenesis, RNAi, miRNA, asRNA, mutant genes, knockout genes, transposon mutagenesis, etc., for altering genes in a manner that makes them less capable (i.e., statistically significantly "less capable" compared to functional wild-type genes) or completely incapable (such as knockout genes) of producing a functional end product. The term "riboswitch" as used herein is defined as a portion of messenger RNA that folds into an intricate structure that blocks expression by interfering with translation. Binding of effector molecules induces conformational changes, allowing post-transcriptional regulatory manifestations. Subsequent alteration of the relevant gene in such a way that lower expression is obtained as described above may also be achieved by altering transcription units, promoters, untranslated regions, ribosome binding sites, the sequence of the summer-darwinol (Shine Dalgarno) or transcription terminators. Lower or reduced expression may be obtained, for example, by mutating one or more base pairs in the promoter sequence or completely altering the promoter sequence to a sustained promoter having a lower intensity of expression compared to the wild type or to an inducible promoter causing regulated expression or to an repressible promoter causing regulated expression. Overexpression or expression is obtained by means of techniques commonly known to those of ordinary skill in the art, such as the use of artificial transcription factors, redesign of promoter sequences, ribosome engineering, introduction or reintroduction of expression modules at the true chromatin, the use of high copy number plastids, wherein the gene is part of an "expression cassette (expression cassette) which is a sequence in which promoter sequences, untranslated region sequences (containing ribosome binding sequences, summer dagalol or Kozak sequences), coding sequences and optionally translational terminators are present and which causes expression of functionally active proteins. This is either persistent or regulatory.

The term "persistence (constitutive expression)" is defined as the absence of transcription factors other than subunits of RNA polymerase (e.g., as sigma) under certain growth conditions ⁷⁰ 、σ ⁵⁴ Is a bacterial sigma factor or a related sigma factor; and yeast granulesten RNA polymerase specific factor MTF 1) co-associated with RNA polymerase core enzyme. Non-limiting examples of such transcription factors are CRP, lacI, arcA, cra, iclR in E.coli, or Aft2p, crz1p, skn7 in Saccharomyces cerevisiae (Saccharomyces cerevisiae), or DeoR, gntR, fur in Bacillus subtilis. These transcription factors bind to specific sequences and may block or enhance performance under certain growth conditions. RNA polymerase is a catalytic mechanism for the synthesis of RNA from a DNA template. RNA polymerase binds to specific sequences to initiate transcription, for example via sigma factor in a prokaryotic host or via MTF1 in yeast. Persistence is manifested in providing a constant amount of performance without the need for induction or inhibition.

The term "expression by means of a natural inducer (expression by a natural inducer)" is defined as the facultative or regulated expression of a gene that is expressed only under certain natural conditions of the host (e.g. the organism is in delivery or during lactation), as a response to environmental changes (e.g. including but not limited to hormones, heat, cold, light, oxidative or osmotic stress/signaling), or depending on the location of the developmental stage or the cell cycle of the host cell, including but not limited to apoptosis and autophagy.

The term "control sequence" refers to a sequence recognized by a cellular transcription and translation system that allows transcription and translation of a polynucleotide sequence into a polypeptide. Such DNA sequences are thus necessary to represent operably linked coding sequences in a particular cell or organism. Such control sequences may be, but are not limited to, promoter sequences, ribosome binding sequences, summer-darcino sequences, kezhak sequences, transcription terminator sequences. Suitable control sequences for prokaryotes include, for example, promoters, optional control sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers. If the DNA of the presequence or secretory leader is presented as a preprotein that participates in the secretion of the polypeptide, it may be operably linked to the DNA of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or operably linked to a coding sequence if the ribosome binding site affects the transcription of the sequence; or operably linked to a coding sequence if the ribosome binding site is positioned so as to facilitate translation. The control sequences may additionally be controlled via external chemicals such as, but not limited to, IPTG, arabinose, lactose, allo-lactose, rhamnose or fucose (fucose) via inducible promoters or via genetic circuits that induce or inhibit transcription or translation of the polynucleotide into a polypeptide.

In general, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of secretory leader sequences, contiguous and in reading phase. However, the enhancers do not have to be continuous.

The term "wild type" refers to a genetic or phenotypic condition that is commonly known when it exists in nature.

The term "modified expression of a protein (modified expression of a protein)" as used herein refers to i) higher expression or overexpression of an endogenous protein, ii) expression of a heterologous protein, or iii) expression and/or overexpression of a variant protein with higher activity as compared to the wild-type (i.e., native) protein.

As used herein, the term "mammary cell" generally refers to a mammary epithelial cell, a mammary epithelial luminal cell, or a mammalian epithelial alveolar cell, or any combination thereof. As used herein, the term "breast-like cell" generally refers to a cell having a phenotype/genotype similar to (or substantially similar to) a native breast cell but derived from a non-breast cell source. Such breast-like cells may be engineered to remove at least one undesirable genetic component and/or include at least one predetermined genetic construct specific for the breast cell. Non-limiting examples of breast-like cells may include breast epithelial-like cells, breast epithelial luminal-like cells, non-breast cells that exhibit one or more characteristics of cells of the breast cell lineage, or any combination thereof. Other non-limiting examples of breast-like cells may include cells having a phenotype similar (or substantially similar) to that of natural breast cells, or more particularly similar (or substantially similar) to that of natural breast epithelial cells. Cells having a phenotype or exhibiting at least one characteristic similar to (or substantially similar to) a native mammary gland cell or mammary gland epithelial cell may comprise cells exhibiting a native or having been engineered to be capable of exhibiting at least one milk component (e.g. derived from a mammary gland cell lineage or a non-mammary gland cell lineage).

As used herein, the term "non-mammary cells" may generally include cells of any non-mammary lineage. In the context of the present invention, a non-mammary cell may be any mammalian cell that is capable of being engineered to express at least one milk component. Non-limiting examples of such non-breast cells include hepatocytes, blood cells, kidney cells, umbilical cord blood cells, epithelial cells, epidermal cells, myocytes, fibroblasts, mesenchymal cells, or any combination thereof. In some cases, molecular biology and genome editing techniques can be engineered to simultaneously eliminate, silence, or attenuate innumerable genes.

Throughout this application, unless explicitly stated otherwise, the expressions "capable of the use of the verb > (capable of. of the verb >) and" capable of the use of the verb > (capable to. of the verb >) are preferably replaced with the active language of the verb, and vice versa. For example, the expression "capable of rendering (capable of expressing)" is preferably replaced with "rendering (express"), and vice versa, i.e., "rendering" is preferably replaced with "capable of rendering".

As used herein, the term "variant" is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential characteristics. A typical variant of a polynucleotide differs from another reference polynucleotide in nucleotide sequence. The change in nucleotide sequence of the variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. As discussed below, nucleotide changes may cause amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Typical variants of a polypeptide differ in amino acid sequence from another reference polypeptide. In general, the differences are limited such that the sequence of the reference polypeptide is very similar to the sequence of the variant as a whole and is consistent in many regions. Variants may differ from the reference polypeptide in amino acid sequence by one or more substitutions, additions, deletions in any combination. The substituted or inserted amino acid residues may or may not be residues encoded by the genetic code. Variants of the polynucleotide or polypeptide may be naturally occurring, such as a dual gene variant, or they may be variants that are not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutation-inducing techniques, by direct synthesis, and by other recombinant methods known to those of ordinary skill in the art.

As used herein, the term "derivative" of a polypeptide is a polypeptide that may contain deletions, additions or substitutions of amino acid residues within the amino acid sequence of the polypeptide, but which causes a silent change, thereby producing a functionally equivalent polypeptide. Amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; planar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamic acid; positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Within the context of the present invention, a derivative polypeptide as used herein refers to a polypeptide that is capable of exhibiting substantially similar activity to the original polypeptide in vitro and/or in vivo, as determined by any of a variety of criteria, including, but not limited to, enzymatic activity, and which may be differentially modified during or after translation. Furthermore, non-classical amino acids or chemical amino acid analogs can be introduced into the original polypeptide sequence in substitution or addition.

In some embodiments, the invention encompasses the preparation of functional variants by modifying the structure of an enzyme as used in the invention. Variants may be generated by amino acid substitutions, deletions, additions or combinations thereof. For example, it is reasonably expected that independent substitution of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitution of amino acids with structurally related amino acids (e.g., conservative mutations) will not have a significant impact on the biological activity of the resulting molecule. Conservative substitutions are substitutions that occur within a family of side chain related amino acids. Whether a change in the amino acid sequence of a polypeptide of the invention produces a functional homolog can be readily determined by assessing the ability of the variant polypeptide to produce a response in a cell in a manner similar to the wild-type polypeptide.

The term "functional homolog (functional homolog)" as used herein describes those molecules that have sequence similarity (in other words, homology) and also share at least one functional feature such as biochemical activity (Altenhoff et al, PLoS comp. Biol.8 (2012) e 1002514). Functional homologs will typically produce similar, but not necessarily identical, features to the same extent. Functionally homologous proteins produce the same characteristics, wherein the quantitative measurement produced by one homolog is at least 10% of the other; more typically, at least 20%, between about 30% and about 40% of the quantitative measurement produced from the original molecule; such as between about 50% and about 60%; between about 70% and about 80%; or between about 90% and about 95%; between about 98% and about 100%, or greater than 100%. Thus, in the case of a molecule having enzymatic activity, the functional homologue will have the percentage of enzymatic activity listed above compared to the original enzyme. In the case where the molecule is a DNA binding molecule (e.g., a polypeptide), the homolog will have the above listed percentages of binding affinity as measured by weight of the binding molecule as compared to the original molecule.

Functional homologs and reference polypeptides may be naturally occurring polypeptides, and sequence similarity may be due to convergent or divergent evolutionary events. Functional homologs are sometimes referred to as heterologous homologs, where "heterologous homolog (ortholog)" refers to a homologous gene or protein that is a functional equivalent of a gene or protein referenced in another species.

Heterologous homologs are homologous genes in different species generated by vertical transfer of a single gene of the final common ancestor, where the genes and their primary functions are conserved. Homologous genes are genes inherited in two species from a common ancestor.

The term "heterologous homolog" when used with reference to an amino acid or nucleotide/nucleic acid sequence from a given species refers to the same amino acid or nucleotide/nucleic acid sequence from a different species. It is understood that two sequences are heterologous homologs of each other when they are derived from a common ancestral sequence via linear transfer, and/or are otherwise closely related in terms of both their sequences and their biological functions. Heterologous homologs will typically have a high degree of sequence identity but may not (and typically will not) share 100% sequence identity.

Homologous genes are homologous genes generated by gene replication events. Homologous genes typically belong to the same species, but this is not required. The homologs can be split into intrahomologs (in-paralogs) (pairs of homologs that occur after a speciation event) and extrahomologs (out-paralogs) (pairs of homologs that occur prior to a speciation event). Between species, an off-homohomolog is a pair of homologs that exist between two organisms due to replication prior to speciation. Within a species, an exohomolog is a pair of homologs that are present in the same organism but whose replication event occurs after speciation. The homologues typically have the same or similar functions.

Functional homologs can be identified by analyzing nucleotide and polypeptide sequence alignments. For example, performing a query on a library of nucleotide or polypeptide sequences can identify homologs of related polypeptides, such as biomass-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, or membrane proteins. Sequence analysis may involve BLAST, recombinant BLAST or PSI-BLAST analysis of non-redundant databases using amino acid sequences of biomass-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, or membrane transporters, respectively, as reference sequences. In some cases, the amino acid sequence is deduced from the nucleotide sequence. Typically, those polypeptides in the database that have more than 40% sequence identity are candidates for further evaluation as biomass-modulating polypeptides, glycosyltransferases, proteins involved in nucleotide-activated sugar synthesis, or membrane proteins, respectively. Amino acid sequence similarity allows conservative amino acid substitutions, such as substitution of one hydrophobic residue with another hydrophobic residue or substitution of one polar residue with another polar residue or substitution of one acidic amino acid with another acidic amino acid or substitution of one basic amino acid with another basic amino acid, and the like. Preferably, by conservative substitutions, we mean a combination such as glycine substituted by alanine and vice versa; valine, iso leucine and leucine are replaced by methionine and vice versa; aspartic acid is substituted by glutamic acid and vice versa; asparagine is substituted with glutamyl amino acid and vice versa; serine is substituted by threonine and vice versa; the lysine is replaced by arginine and vice versa; cysteine is substituted by methionine and vice versa; and phenylalanine and tyrosine are substituted by tryptophan and vice versa. If desired, a manual inspection of such candidates may be performed in order to limit the number of candidates to be further evaluated. Manual testing may be performed by selecting those candidates that appear to have domains (e.g., conserved functional domains) present in the productivity-modulating polypeptide.

By polynucleotide, a "Fragment" is meant any portion of a pure (clone) or polynucleotide molecule, particularly a portion of a polynucleotide that retains the functional characteristics of a full-length polynucleotide molecule. Useful fragments include oligonucleotides and polynucleotides useful in hybridization or amplification techniques or for modulating replication, transcription or translation. A "polynucleotide fragment (polynucleotide fragment)" refers to any subsequence of a polynucleotide SEQ ID NO (or genbank No.), which typically comprises or consists of at least about 9, 10, 11, 12 consecutive nucleotides of any of the polynucleotide sequences provided herein, e.g., at least about 30 nucleotides or at least about 50 nucleotides. Exemplary fragments may additionally or alternatively comprise, consist essentially of, or consist of a region comprising a conserved family domain encoding a polypeptide. Exemplary fragments may additionally or alternatively include fragments comprising conserved domains of polypeptides. Thus, a fragment of a polynucleotide SEQ ID NO (or Genbank NO.) preferably means a nucleotide sequence comprising or consisting of the polynucleotide SEQ ID NO (or Genbank NO.), wherein NO more than 200, 150, 100, 50 or 25 consecutive nucleotides, preferably NO more than 50 consecutive nucleotides, are deleted, and the nucleotide sequence retains useful functional characteristics (e.g., activity) of a full-length polynucleotide molecule that can be assessed by a person of ordinary skill in the art by routine experimentation. Alternatively, a fragment of a polynucleotide SEQ ID NO (or Genbank No.) preferably means a nucleotide sequence comprising or consisting of a quantity of consecutive nucleotides from the polynucleotide SEQ ID NO (or Genbank No.), and wherein the quantity of consecutive nucleotides is at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 100%, preferably at least 80%, more preferably at least 87%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 97% of the total length of the polynucleotide(s) has functional properties such as that of a functional full length molecule. Thus, a fragment of a polynucleotide SEQ ID NO (or Genbank No.), preferably means a nucleotide sequence comprising or consisting of the polynucleotide SEQ ID NO (or Genbank No.), wherein an amount of consecutive nucleotides is deleted and wherein the amount is not more than 50.0%, 40.0%, 30.0%, preferably not more than 20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, more preferably not more than 15%, even more preferably not more than 10%, even more preferably not more than 5%, most preferably not more than 2.5% of the full length of the polynucleotide SEQ ID NO (or Genbank No.), and wherein the fragment retains the functional characteristics of a conventional polynucleotide in the art, such as the full length of a useful molecule (e.g. the full length of a molecule).

Throughout this application, the polynucleotide sequence may be represented by SEQ ID NO or alternatively by GenBank NO. Thus, unless explicitly stated otherwise, the terms "polynucleotide SEQ ID NO (polynucleotide SEQ ID NO)" and "polynucleotide GenBank No. (polynucleotide GenBank No.)" are used interchangeably.

Fragments may additionally or alternatively comprise subsequences of polypeptides and protein molecules, or subsequences of polypeptides. In some cases, a fragment or domain is a subsequence of a polypeptide that performs at least one biological function of the intact polypeptide in substantially the same manner as the intact polypeptide, preferably to a similar extent. "subsequence of a polypeptide (subsequence of the polypeptide)" as defined herein refers to a sequence of consecutive amino acid residues derived from a polypeptide. For example, a polypeptide fragment may comprise a recognizable structural motif or functional domain, such as a DNA binding site or domain that binds to a DNA promoter region, an activation domain, or a protein-protein interaction domain, and may initiate transcription. The size of the fragment may vary from as few as 3 amino acid residues to the full length of the complete polypeptide, e.g., at least about 20 amino acid residues in length, e.g., at least about 30 amino acid residues in length. Thus, a fragment of a polypeptide SEQ ID NO (or UniProt ID or Genbank No.) preferably means a polypeptide sequence comprising or consisting of the polypeptide SEQ ID NO (or UniProt ID or Genbank No.), wherein NO more than 80, 60, 50, 40, 30, 20 or 15 consecutive amino acid residues, preferably NO more than 40 consecutive amino acid residues, are deleted, and which performs at least one biological function of the complete polypeptide in substantially the same manner as the complete polypeptide can be routinely assessed by a person of ordinary skill in the art, preferably to a similar or greater extent. Alternatively, a fragment of a polypeptide SEQ ID NO (or unit prot ID or Genbank No.) preferably means a polypeptide sequence comprising or consisting of a quantity of consecutive amino acid residues from the polypeptide SEQ ID NO, and wherein the quantity of consecutive amino acid residues is at least 50.0%, 60.0%, 70.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.5%, 100%, preferably at least 80%, more preferably at least 87%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97%, and which has the function of a polypeptide that is normally performed in a manner similar to that of a polypeptide of the general art by the general knowledge of the general full extent of the polypeptide or of the polypeptide of which it is a polypeptide of the full-length. Thus, a fragment of a polypeptide SEQ ID NO (or UniProt ID or Genbank No.) preferably means a polypeptide sequence comprising or consisting of a sequence of said polypeptide SEQ ID NO (or UniProt ID or Genbank No.), wherein an amount of consecutive amino acid residues is deleted and wherein the amount is not more than 50.0%, 40.0%, 30.0%, preferably not more than 20.0%, 15.0%, 10.0%, 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, more preferably not more than 15%, even more preferably not more than 10%, even more preferably not more than 5%, most preferably not more than 2.5% of the full length of said polypeptide SEQ ID NO (or UniProt ID or Genbank No.), and which has the function of at least one polypeptide as assessed by a general knowledge of the general routine skill in the art of the polypeptide in a substantial full length.

Throughout this application, the polypeptide sequence may be represented by SEQ ID NO or alternatively by UniProt ID or GenBank NO. Thus, unless explicitly stated otherwise, the terms "polypeptide SEQ ID NO (polypeptide SEQ ID NO)" and "polypeptide UniProt ID (polypeptide UniProt ID)" and "polypeptide GenBank No. (polypeptide GenBank No.), are used interchangeably.

Preferably, a fragment of a polypeptide is a functional fragment derived from a polypeptide, preferably having at least one property or activity of the polypeptide to a similar or greater extent. Functional fragments may, for example, comprise functional or conserved domains of polypeptides. It will be appreciated that the polypeptide or fragment thereof may have conservative amino acid substitutions that have substantially no effect on the activity of the polypeptide. By conservative substitution, it is meant that one hydrophobic amino acid is substituted with another hydrophobic amino acid or one polar amino acid is substituted with another polar amino acid or one acidic amino acid is substituted with another acidic amino acid or one basic amino acid is substituted with another basic amino acid, etc. Preferably, by conservative substitutions, we mean a combination such as glycine substituted by alanine and vice versa; valine, iso leucine and leucine are replaced by methionine and vice versa; aspartic acid is substituted by glutamic acid and vice versa; asparagine is substituted with glutamyl amino acid and vice versa; serine is substituted by threonine and vice versa; the lysine is replaced by arginine and vice versa; cysteine is substituted by methionine and vice versa; and phenylalanine and tyrosine are substituted by tryptophan and vice versa. The domains may be characterized, for example, by Pfam (El-Gebali et al, nucleic Acids Res.47 (2019) D427-D432) or by the name of a conserved domain library (Conserved Domain Database; CDD) (https:// www.ncbi.nlm.nih.gov/CDD) (Lu et al, nucleic Acids Res.48 (2020) D265-D268). The contents of each repository are fixed and unchanged at each release. When the contents of a particular database change, the particular database receives a new release version with a new release date. All release versions of each database and their corresponding release dates, as well as the particular content noted for such particular release dates, are available and known to those of ordinary skill in the art. The PFAM database (https:// PFAM. Xfam. Org /) as used herein is the Pfam version 33.1 published on month 6 and 11 of 2020. The protein sequence information and functional information may be provided by comprehensive resources of the protein sequence and labeling data, such as, for example, the universal protein resource (UniProt) (www.uniprot.org) (Nucleic Acids res.2021, 49 (D1), D480-D489). UniProt contains a specialized and well-managed protein database called UniProt knowledge base (UniProtKB), as well as the UniProt reference sequence set (UniRef) and UniProt archive (UniParc). The UniProt identifier (UniProt ID) is unique to each protein present in the database. As used herein, the UniProt ID is the UniProt ID in the UniProt database version of day 05, 5, 2021. Proteins without UniProt ID are referred to herein using the respective Genbank accession numbers (Genbank No.) as found in the NIH gene sequence database at 5 months 05 of 2021 (https:// www.ncbi.nlm.nih.gov/Genbank /) (Nucleic Acids res.2013, 41 (D1), D36-D42) version.

The term "glycosyltransferase" as used herein refers to an enzyme capable of catalyzing the transfer of a sugar moiety from an activated donor molecule to a specific acceptor molecule, thereby forming a glycosidic bond. The oligosaccharides thus synthesized may be of a linear type or a branched type and may contain a plurality of monosaccharide building blocks. Glycosyltransferases have been described as classified into different sequence-based families using nucleotide diphosphate-sugars, nucleotide monophosphate-sugars, and phosphosugars and related proteins (Campbell et al, biochem. J.326, 929-939 (1997)) and are available on the CAzy (carbohydrate active enzyme) website (www.cazy.org).

As used herein, glycosyltransferases may be selected from a list including, but not limited to: fucosyltransferases (e.g., alpha-1, 2-fucosyltransferases, alpha-1, 3/1, 4-fucosyltransferases, alpha-1, 6-fucosyltransferases), sialyltransferases (sialyltransferases) (e.g., alpha-2, 3-sialyltransferases, alpha-2, 6-sialyltransferases, alpha-2, 8-sialyltransferases), galactosyltransferases (e.g., beta-1, 3-galactosyltransferases, beta-1, 4-galactosyltransferases, alpha-1, 3-galactosyltransferases, alpha-1, 4-galactosyltransferases), N-acetylglucoseaminotransferases (e.g., beta-1, 3-N-acetylglucoseaminotransferases) beta-1, 6-N-acetylglucosaminyl transferase), N-acetylgalactosamine aminotransferases (e.g., alpha-1, 3-N-acetylgalactosamine aminotransferases, beta-1, 3-N-acetylgalactosamine aminotransferases), glucosyltransferases, mannosyl transferases, N-acetylmannosyl aminotransferases, xylosyltransferases, glucuronide transferases, galacturonan transferases, glucosaminyl transferases, N-glycolyl neuraminidases, rhamnosyl transferases, N-acetylrhamnosyl transferases, UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-A Zhuo Tangan (altrosamine) transamidase, UDP-N-acetylglucosamine enolpyruvoyl transferase (UDP-N-acetylglucosamine enolpyruvyl transferase) and fucose aminotransferase.

Fucosyltransferases are glycosyltransferases that transfer fucose residues (Fuc) from a GDP-fucose (GDP-Fuc) donor to a glycan acceptor. Fucosyltransferases include alpha-1, 2-fucosyltransferases, alpha-1, 3-fucosyltransferases, alpha-1, 4-fucosyltransferases, and alpha-1, 6-fucosyltransferases, which catalyze the transfer of Fuc residues from GDP-Fuc to a glycan receptor via an alpha-glycosidic linkage. Fucosyltransferases may be found in, but are not limited to, the GT10, GT11, GT23, GT65 and GT68CAZy families. Sialyltransferases are glycosyltransferases that transfer sialic acid groups (e.g., neu5Ac or Neu5 Gc) from a donor (e.g., CMP-Neu5Ac or CMP-Neu5 Gc) to a glycan receptor. Sialyltransferases include alpha-2, 3-sialyltransferases, alpha-2, 6-sialyltransferases, and alpha-2, 8-sialyltransferases, which catalyze the transfer of sialyl groups via alpha-glycosidic linkages to glycan receptors. Sialyltransferases may be found in, but are not limited to, the GT29, GT42, GT80 and GT97CAZy families. Galactosyltransferases are glycosyltransferases that transfer galactosyl (Gal) from a UDP-galactose (UDP-Gal) donor to a glycan acceptor. Galactosyltransferases include beta-1, 3-galactosyltransferases, beta-1, 4-galactosyltransferases, alpha-1, 3-galactosyltransferases, and alpha-1, 4-galactosyltransferases, which transfer Gal residues from UDP-Gal to glycan receptors via alpha-or beta-glycosidic linkages. Galactosyltransferases may be found in, but are not limited to, the GT2, GT6, GT8, GT25 and GT92CAZy families. Glucosyltransferases are glycosyltransferases that transfer a glucosyl group (Glc) from a UDP-glucose (UDP-Glc) donor to a glycan acceptor. Glucosyltransferases include alpha-glucosyltransferases, beta-1, 2-glucosyltransferases, beta-1, 3-glucosyltransferases, and beta-1, 4-glucosyltransferases, which transfer Glc residues from UDP-Glc to glycan receptors via alpha-or beta-glycosidic linkages. Glucosyltransferases may be found in, but are not limited to, the GT1, GT4 and GT25 CAZy families. Mannosyl transferase is a glycosyltransferase that transfers mannosyl (Man) from a GDP-mannose (GDP-Man) donor to a glycan acceptor. Mannosyltransferases include alpha-1, 2-mannosyltransferases, alpha-1, 3-mannosyltransferases and alpha-1, 6-mannosyltransferases, which transfer Man residues from GDP-Man to glycan receptors via alpha-glycosidic bonds. Mannosyltransferases can be found in, but are not limited to, the GT22, GT39, GT62 and GT69 CAZy families. N-acetylglucosamine transferase is a glycosyltransferase that transfers N-acetylglucosamine (GlcNAc) from a UDP-N-acetylglucosamine (UDP-GlcNAc) donor to a glycan acceptor. N-acetylglucosaminyl transferases may be found in, but are not limited to, the GT2 and GT4 CAZy families.

N-acetylgalactosamine transferase is a glycosyltransferase transferring N-acetylgalactosamine (GalNAc) from UDP-N-acetylgalactosamine (UDP-GalNAc) donor to glycan acceptor. N-acetylgalactosamine transferase can be found in, but is not limited to, the GT7, GT12 and GT27 CAZy families. N-acetylmannosaminotransferases are glycosyltransferases that transfer N-acetylmannosamino (ManNAc) from UDP-N-acetylmannosamine (UDP-ManNAc) donors to glycan acceptors. Xylosyltransferases are glycosyltransferases that transfer xylose residues (Xy 1) from UDP-xylose (UDP-Xy 1) donors to glycan acceptors. The xylosyltransferases may be found in, but are not limited to, the GT61 and GT77 CAZy families. Glucuronyl transferase is a glycosyltransferase that transfers glucuronate from a UDP-glucuronate donor to a glycan acceptor via an alpha-or beta-glycosidic linkage. Glucuronyl transferase can be found, but is not limited to, the GT4, GT43 and GT93 CAZy families.

Galacturonate transferase is a glycosyltransferase that transfers galacturonate from a UDP-galacturonate donor to a glycan acceptor. N-glycolyl neuraminidase is a glycosyltransferase that transfers N-glycolyl neuraminidase (Neu 5 Gc) from a CMP-Neu5Gc donor to a glycan acceptor. Rhamnosyl transferase is a glycosyltransferase that transfers a rhamnose residue from a GDP-rhamnose donor onto a glycan acceptor. Rhamnosyltransferases may be found in, but are not limited to, the GT1, GT2 and GT102 CAZy families. N-acetylrhamnosyl transferase is a glycosyltransferase that transfers an N-acetylrhamnose amine (rhamnosamine) residue from a UDP-N-acetyl-L-rhamnose amine donor onto a glycan acceptor. UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-altrose amine transferase is a glycosyltransferase using UDP-2-acetamido-2, 6-dideoxy-L-arabino-4-hexulose for biosynthesis of pseudo-amino acid (pseudo-amino acid), which is a sialic acid sugar used to modify flagellin. UDP-N-acetylglucosamine enolpyruvate transferase (murA) is a glycosyltransferase that transfers enolpyruvyl from phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UDPAG) to form UDP-N-acetylglucosamine enolpyruvate. Fucosyl aminotransferase is a glycosyltransferase that transfers an N-acetylfucosylamine residue from a dTDP-N-acetylfucosylamine or UDP-N-acetylfucosylamine donor onto a glycan acceptor.

The terms "nucleotide-sugar", "nucleotide-activated sugar" or "activated sugar" are used interchangeably herein and refer to an activated form of a monosaccharide. Activation sheetExamples of sugars include, but are not limited to, UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-fucose (GDP-Fuc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2, 6-dideoxy-L-arabinose-4-hexanoate, UDP-2-acetamido-2, 6-dideoxy-L-lyxo-4-hexanoate, UDP-N-acetyl-L-rhamnose amine (UDP-L-RhaNAc or UDP-2-acetamido-2, 6-dideoxy-L-mannosamine), dTDP-N-acetylfucose amine (UDP-L-FucNAc or UDP-2, 6-dideoxy-L-lyxol-4-hexulose, UDP-N-acetylmannosamine (UDP-N-GlcNAc) or UDP-2-dideoxy-L-mannosamine, UDP-N-acetylmannosamine (UDP-N-Acetylmannosamine) (UDP-2-N-Acetamine) or UDP-N-diacetyl-2-aminolyxol-5-aminosugar (UDP-N-Glc-NAc) UDP-N-acetyl muramic acid (acetylmuramic acid), UDP-N-acetyl-L-isorhamnoamine (UDP-L-QuiNAc or UDP-2-acetamido-2, 6-dideoxy-L-glucose), GDP-L-isorhamno, CMP-N-glycolyl neuraminic acid (CMP-Neu 5 Ge), CMP-Neu4Ac, CMP-Neu5Ac9N ₃ 、CMP-Neu4，5Ac ₂ 、CMP-Neu5，7Ac ₂ 、CMP-Neu5，9Ac ₂ 、CMP-Neu5，7(8，9)Ac ₂ CMP-N-glycolyl neuraminic acid (CMP-Neu 5 Gc), UDP-glucuronate, UDP-galacturonate, GDP-rhamnose or UDP-xylose. Nucleotide-sugars act as glycosyl donors in glycosylation reactions. The glycosylation reaction is a reaction catalyzed by glycosyltransferases.

As the term is used herein and as is generally understood in the art of the present day, "Oligosaccharide" refers to a sugar polymer that contains a small amount, typically three to twenty simple sugars (i.e., monosaccharides). Monosaccharides as used herein are reducing sugars. The oligosaccharides may be reducing or non-reducing sugars and have a reducing or non-reducing end. The reducing sugar is any sugar that is capable of reducing another compound and itself oxidized, i.e., the carbonyl carbon of the sugar is oxidized to the carboxyl group. The oligosaccharides as used in the present invention may be of linear structure or may comprise branches. The bond between two sugar units (e.g., glycosidic bond, galactosidic bond, glucosidic bond, etc.) can be represented as, for example, 1,4, 1- > 4, or (1-4), and are used interchangeably herein. For example, the terms "Gal-b 1, 4-Glc", "β -Gal- (1- > 4) -Glc", "Galβ1-4-Glc" and "Gal-b (1-4) -Glc" have the same meaning, i.e., the β -glycosidic linkage of carbon-1 of galactose (Gal) to carbon-4 of glucose (Glc). Each monosaccharide may be in a cyclic form (e.g., furanose in the form of furanose). Linkages between individual monosaccharide units may 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. The oligosaccharides may contain alpha-glycosidic linkages and beta-glycosidic linkages or may contain only beta-glycosidic linkages. Preferably, the oligosaccharides as described herein contain monosaccharides selected from the list as used herein below. Examples of oligosaccharides include, but are not limited to, lewis-type (Lewis-type) antigen oligosaccharides, mammalian milk oligosaccharides, and human milk oligosaccharides. As used herein, "milk-N-disaccharide (LNB) -based oligosaccharides (LNB) -based oligosaccharide" refers to oligosaccharides as defined herein that contain an LNB at their reducing end. As used herein, "N-acetyllactosamine (LacNAc) -based oligosaccharide (LacNAc (N-acetyllactosamine) -based oligosaccharide)" refers to an oligosaccharide as defined herein which contains LacNAc at its reducing end.

The term "monosaccharide" as used herein refers to a sugar that is not decomposable by hydrolysis into simpler sugars, classified by aldoses or ketoses, and contains one or more hydroxyl groups per molecule. Monosaccharides are sugars that contain only one simple sugar. Examples of monosaccharides include hexose, D-glucose furanose, D-galactofuranose, L-galactofuranose, D-mannose, D-Allopyranose (allopyriranose), L-Zhuo Pai furanose, D-Gu Luopai furanose (Gulopyrose), L-Ai Dupai furanose (Idopyranose), D-Talopyranose (Talopyrose), D-ribofuranose, D-Arabinofuranose (Arabinofuranose), D-arabinopyranose, L-Arabinofuranose, L-arabinopyranose, D-xylopipranose, D-leno Su Pai furanose D-Erythrofuranose (Erythrofuranose), D-threose furanose (Throfuranose), heptose, L-glycerol-D-mannose-heptopyranose (LDmanHep), D-glycerol-D-mannose-heptopyranose, (DDmanHep), 6-deoxy-L-Zhuo Pai furanose, 6-deoxy-D-Gu Luopai furanose, 6-deoxy-D-Talopyranose, 6-deoxy-D-galactopyranose, 6-deoxy-L-galactopyranose, 6-deoxy-D-mannopyranose, 6-deoxy-D-glucopyranose, 2-deoxy-D-arabinose-hexose, 2-deoxy-D-erythro-pentose, 2, 6-dideoxy-D-arabinose-hexose, 3, 6-dideoxy-L-arabinose-hexose, 3, 6-dideoxy-D-xylose-hexose, 3, 6-dideoxy-D-ribose-hexose, 2, 6-dideoxy-D-ribose-hexose, 3, 6-dideoxy-L-xylose-hexose, 2-amino-2-deoxy-D-glucose-hexose, 2-amino-2-deoxy-D-galactopyranose 2-amino-2-deoxy-D-mannopyranose, 2-amino-2-deoxy-D-allopyranose, 2-amino-2-deoxy-L-Zhuo Pai-furanose, 2-amino-2-deoxy-D-Gu Luopai-furanose, 2-amino-2-deoxy-L-Ai Dupai-furanose, 2-amino-2-deoxy-D-talopyranose, 2-acetamido-2-deoxy-D-glucopyranose, 2-acetamido-2-deoxy-D-galactopyranose, 2-acetamido-2-deoxy-D-mannopyranose, and pharmaceutical compositions containing the same, 2-acetamido-2-deoxy-D-allopyranose, 2-acetamido-2-deoxy-L-A Zhuo Pai-furanose, 2-acetamido-2-deoxy-D-Gu Luopai-furanose, 2-acetamido-2-deoxy-L-Ai Dupai-furanose, 2-acetamido-2-deoxy-D-talopyranose, 2-acetamido-2, 6-dideoxy-D-galactopyranose, 2-acetamido-2, 6-dideoxy-L-mannopyranose, 2-acetamido-2, 6-dideoxy-D-glucopyranose 2-acetamido-2, 6-dideoxy-L-ar Zhuo Pai-furanose, 2-acetamido-2, 6-dideoxy-D-talopyranose, D-glucuronate, D-galacturonate, D-mannopyranonic acid, D-allopyranonic acid, L-ar Zhuo Pai-furanonic acid, D-Gu Luopai-furanonic acid, L-Gu Luopai-furanonic acid, L-Ai Dupai-furanonic acid, D-talopyranonic acid, sialic acid, 5-amino-3, 5-dideoxy-D-glycero-D-galactose-nono-2-ketonic acid, 5-acetamido-3, 5-dideoxy-D-glycero-D-galacto-non-2-one sugar acid, 5-hydroxyacetylamido-3, 5-dideoxy-D-glycero-D-galacto-non-2-one sugar acid, erythritol, arabitol, xylitol, ribitol, glucitol, galactitol, mannitol, D-ribo-hexo-2-one-pipyranose, D-arabino-hexo-2-one-furanose (D-fructofuranose), D-arabino-hexo-2-one-pipyranose, L-xylo-hexo-2-one pipyranose D-lyxose-hexose-2-ketopipyranose, D-threose-pent-2-one pipyranose, D-android-hept-2-one pipyranose, 3-C- (hydroxymethyl) -D-erythrofuranose, 2,4, 6-trideoxy-2, 4-diamino-D-grape pipyranose, 6-deoxy-3-O-methyl-D-glucose, 3-O-methyl-D-rhamnose, 2, 6-dideoxy-3-methyl-D-ribose-hexose, 2-amino-3-O- [ (R) -1-carboxyethyl ] -2-deoxy-D-grape pipyranose, 2-acetamido-3-O- [ (R) -carboxyethyl ] -2-deoxy-D-glucose, 2-hydroxyacetylamido-3-O- [ (R) -1-carboxyethyl ] -2-deoxy-D-glucose, 3-deoxy-D-lyxose-hepto-2-ketopipyranonic acid, 3-deoxy-D-manno-oct-2-ketopipyranonic acid, 3-deoxy-D-glycero-galactose-non-2-ketopipopyranonic acid, 5, 7-diamino-3, 5,7, 9-tetradeoxy-L-glycero-L-manno-non-2-ketopipyranonic acid 5, 7-diamino-3, 5,7, 9-tetradeoxy-L-glycero-L-azepino-non-2-one thiopyranonic acid, 5, 7-diamino-3, 5,7, 9-tetradeoxy-D-glycero-galactose-non-2-one thiopyranonic acid, 5, 7-diamino-3, 5,7, 9-tetradeoxy-D-glycero-D-talo-non-2-one thiopyranonic acid, glucose, galactose, N-acetylglucosamine, glucosamine, mannose, xylose, N-acetylmannosamine, N-acetylneuraminic acid, N-glycolyl neuraminic acid, N-acetyl galactosamine, fucose, rhamnose, glucuronic acid, gluconic acid, fructose, and polyols.

The term polyol means an alcohol containing a plurality of hydroxyl groups. For example, glycerol, sorbitol or mannitol.

As used herein, the term "disaccharide (disaccharide)" refers to a sugar composed of two monosaccharide units. Examples of disaccharides include lactose (Gal-b 1, 4-Glc), milk-N-disaccharide (Gal-b 1, 3-GlcNAc), N-acetyllactosamine (Gal-b 1, 4-GlcNAc), lacDiNAc (GalNAc-b 1, 4-GlcNAc), N-acetylgalactosamine glucose (GalNAc-b 1, 4-Glc), neu5Ac-a2,3-Gal, neu5Ac-a2,6-Gal and pyranofuloyl- (1-4) -N-hydroxyacetyl neuraminic acid (Fuc- (1-4) -Neu5 Ge).

As used herein, "mammalian milk oligosaccharides (mammalian milk oligosaccharide)" (MMO) refers to oligosaccharides such as (but not limited to) the following: milk-N-triose II, 3-fucosyllactose, 2' -fucosyllactose, 6-fucosyllactose, 2', 3-difucosyllactose, 2', 2-disaccharide lactose, 3, 4-disaccharide lactose, 6' -sialyllactose, 3, 6-disialyllactose, 6' -disialyllactose, 8, 3-disialyllactose, 3, 6-disialyllacto-N-tetraose, lacto-disaccharide, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N-tetraose d, sialyllacto-N-tetraose I, lacto-N-disaccharide-hexaose II, lacto-N-hexaose, lacto-N-neohexaose, p-fucopentaose-N-hexaose 62, lacto-N-fucopentaose I, sialyl-N-hexaose, sialyl-fucopentaose I, sialog-N-hexaose III, sialyl-lacto-N-tetraose and sialyl-hexaose I, sialyl lacto-N-neohexasaccharide II, difucosyl-p-lacto-N-hexasaccharide, difucosyl lacto-N-hexasaccharide a, difucosyl lacto-N-hexasaccharide c, galactosylated polyglucose, fucosylated (fucosylated) oligosaccharides, neutral oligosaccharides and/or sialylated oligosaccharides. Mammalian Milk Oligosaccharides (MMO) include oligosaccharides present in milk found at any stage during lactation, including colostrum from humans (i.e. human milk oligosaccharides or HMOs) and mammals including, but not limited to, cows (Bos Taurus), sheep (Ovisaries), goats (Capra aegagrus hircus)), bactrian camels (Camelus bactrianus)), horses (european wild horses (Equus ferus caballus)), pigs (susca) dogs (subsca subspecies (Canis lupus familiaris)), dogs (domestic canine subspecies (Canis lupus familiaris)), shrimp brown bear (ezo brown bear) (japanese brown bear (Ursus arctos yesoensis)), polar bear (Ursus maritimus), japanese black bear (Ursus thibetanus japonicus)), striped ferrets (striped beaks (Mephitis mephitis)), crown seal (Cystophora cristata)), asian elephants (elephloem, african beast (24) and other giant laboratory rats (24)), kangaroo (24), bottle (nude mice), etc, common pouch foxes (broomcorn (Trichosurus Vulpecula)), koala (Phascolarctos cinereus)), east pouch ferrets (tripod pouch shrew (Dasyurus viverrinus)), duckbill (Ornithorhynchus anatinus)). Human Milk Oligosaccharides (HMOs) are also known as human milk oligosaccharides that are chemically identical to human milk oligosaccharides found in human breast milk, but are produced biotechnologically (e.g., using cell-free systems or cells and organisms including bacteria, fungi, yeast, plants, animals or protozoa, preferably genetically engineered cells and organisms). Human milk-conforming oligosaccharides are sold under the name HiMO.

As used herein, "lactose-based Mammalian Milk Oligosaccharides (MMO)" refers to MMO as defined herein, which contain lactose at its reducing end.

As used herein, the term "Lewis-type antigen" includes the following oligosaccharides: an H1 antigen which is fucα1-2galβ1-3GlcNAc, or briefly 2' flnb; lewis (or Lea) which is the trisaccharide Galβ1-3[ Fucα1-4] GlcNAc, or in short 4-FLNB; lewis b (or Leb), which is the tetrasaccharide Fucα1-2Galβ1-3[ Fucα1-4] GlcNAc, or simply DiF-LNB; sialyl lewis (or sialyl Lea), which is 5-acetylneuraminic- (2-3) -galactosyl- (1-3) - (pyranofucosyl- (1-4)) -N-acetylglucosamine, or abbreviated neu5acα2-3Gal β1-3[ fucα1-4] glcnac; an H2 antigen which is fucα1-2galβ1-4GlcNAc, or otherwise known as 2 'fucosyl-N-acetyl-lactosamine, in short 2' flacnac; lewis x (or Lex), which is the trisaccharide Galβ1-4[ Fucα1-3] GlcNAc, or alternatively referred to as 3-fucosyl-N-acetyl-lactosamine, briefly 3-FLacNAc; lewis (or Ley), which is the tetrasaccharide Fucα1-2Galβ1-4[ Fucα1-3] GlcNAc; and sialyl Lewis x (or sialyl Lex), which is 5-acetylneuraminic- (2-3) -galactosyl- (1-4) - (pyranofucosyl- (1-3)) -N-acetylglucosamine, or abbreviated as Neu5 Ac. Alpha.2-3 Gal. Beta.1-4 [ Fucα1-3] GlcNAc.

The terms "LNB" and "Lacto-N-disaccharide (Lact-N-biose)" are used interchangeably and refer to disaccharide Gal-b1,3-GlcNAc. The terms "LNB" and "Lacto-N-disaccharide (Lact-N-biose)" are used interchangeably and refer to disaccharide Gal-b1,3-GlcNAc.

The term "LacNAc" and "N-acetyllactosamine" are used interchangeably and refer to disaccharide Gal-b1,4-GlcNAc.

As used herein, "sialylated oligosaccharide (sialylated oligosaccharide)" is understood to be a charged sialic acid containing oligosaccharide (i.e., an oligosaccharide having sialic acid residues). It has acidic properties. Sialylated oligosaccharides contain at least one sialyl monosaccharide unit, such as, for example (but not limited to), neu5Ac and Neu5Gc. The sialylated oligosaccharides are sugar structures comprising at least three monosaccharide subunits connected to each other via glycosidic linkages, wherein at least one of the monosaccharide subunits is a sialic acid. The sialylated oligosaccharide may contain more than one sialic acid residue, for example two, three or more sialic acid residues. The sialic acid may be attached to other monosaccharide subunits comprising galactose, glcNAc, sialic acid via an a-glycosidic bond comprising an a-2, 3 bond, a-2, 6 bond. Some examples are 3-SL (3 '-sialyllactose or 3' -SL or Neu5Ac-a2,3-Gal-b1, 4-Glc); 3' -sialyllactosamine; 6-SL (6 '-sialyllactose or 6' -SL or Neu5Ac-a2,6-Gal-b1, 4-Glc); 6' -sialyllactosamine; oligosaccharides comprising 6 '-sialyllactose, 3, 6-disialyllactose (Neu 5Ac-a2,3- (Neu 5Ac-a2, 6) -Gal-b1, 4-Glc), 6' -disialyllactose (Neu 5Ac-a2,6-Gal-b1,4- (Neu 5Ac-a2, 6) -Glc), 8, 3-disialyllactose (Neu 5Ac-a2,8-Neu5Ac-a2,3-Gal-b1, 4-Glc), SGG hexose (neu5acα -2,3galβ -1,3galnacβ -1,3galα -1,4galβ -1, 4gal), sialyltetrasaccharide (neu5acα -2,3galβ -1,4glcnac β -14 GlcNAc), pentasaccharide LSTD (neu5acα -2,3galβ -1,4glcnac β -1,3galβ -1, 4glc), sialylated lacto-N-triose, sialylated lacto-N-tetrasaccharide, sialyl lacto-N-neotetrasaccharide, monosialyl lacto-N-hexose, disialyl lacto-N-neohexose I, monosialyl lacto-N-neohexose II, disialyl lacto-N-neohexose, disialyl lacto-N-tetrasaccharide, disialyl lacto-N-hexose II, sialyl lacto-N-tetrasaccharide, disialyl-lacto-N-hexose I, disialyl-N-neo-hexose I, disialyl lacto-N-neo-N-hexose I, disialyl-N-hexose, sialyl milk-N-neotetraose d, 3' -sialyl-3-fucosyllactose, disialyl monosialyl milk-N-neohexaose, monosialyl Shan Tuoye acid based milk-N-octaose (sialyl Lea), sialyl milk-N-fucose II, disialyl milk-N-fucose II, monosialyl disialyl milk-N-tetraose; and oligosaccharides with one or several sialic acid residues, including (but not limited to): an oligosaccharide moiety selected from the group consisting of gangliosides of GM3 (3' sialyllactose, neu5Ac alpha-2, 3gal beta-4 Glc); and oligosaccharides comprising GM3 motif, GD3neu5ac α -2,8neu5ac α -2,3galβ -1,4glc GT3 (neu5ac α -2,8neu5ac α -2,3galβ -1,4 glc); GM2GalNAc beta-1, 4 (Neu 5Ac alpha-2, 3) Gal beta-1, 4Glc, GM1Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 3) Gal beta-1, 4Glc, GD1aNeu5Ac alpha-2, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 3) Galbeta-1, 4Glc, GT1aNeu5Ac alpha-2, 8Neu5Ac alpha-2, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 3) Galbeta-1, 4Glc, 2GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3) Galbeta-1, 4Glc, 2GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3) Galbeta-1, 4Glc, GT1, 4 (Neu 5Ac alpha-2, 8) Gal beta-1, 8Neu5Ac alpha-2, 3) Galbeta-1, 4 Glc. Galβ -1,3GalNAcβ -1,4 (Neu 5Ac α -2,8Neu5Ac α 2, 3) Galβ -1,4Glc, GT1b Neu5Ac α -2,3Galβ -1,3GalNAcβ -1,4 (Neu 5Ac α -2,8Neu5Ac α 2, 3) Galβ -1,4Glc, GQ1b Neu5Ac α -2,8Neu5Ac α -2,3Galβ -1,3Gal β -1,4 (Neu 5Ac α -2,8Neu5Ac α 2, 3) Galβ -1,4Glc GT1c Galβ -1,3GalNAcβ -1,4 (Neu 5Ac α -2,8Neu5Ac α 2, 3) Galβ -1,4Glc, GQ1c Neu5Ac α -2,3Galβ -1,3GalNAcβ -1,4 (Neu 5Ac α -2,8Neu5Ac α 2, 3) Galβ -1,4Glc, GP1c Neu5Ac α -2,8Neu5Ac alpha-2, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3) Gal beta-1, 4Glc, GD1a Neu5Ac alpha-2, 3Gal beta-1, 3 (Neu 5Ac alpha-2, 6) GalNAc beta-1, 4Gal beta-1, 4Glc, fucosyl-GM 1 Fuc alpha-1, 2Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 3) Gal beta-1, 4Glc; it can be extended to the production of the corresponding gangliosides by reacting the above oligosaccharide moiety with or synthesizing the above oligosaccharide on a ceramide.

As used herein, the terms "alpha-2, 3-sialyltransferase" (alpha-2, 3-sialyltransferase), "," alpha 2,3sialyltransferase "(alpha 2,3 sialyltransferase),", "3-sialyltransferase" (3-sialyltransferase), "," alpha-2,3-sialyltransferase "(alpha-2, 3-sialyltransferase),", "alpha 2,3 sialyltransferase" (alpha 2,3 sialyltransferase), "," 3sialyltransferase (3 sialyltransferase), "," 3-ST ", or" 3ST "are used interchangeably and refer to glycosyltransferases that catalyze the transfer of sialic acid from a donor CMP-Neu5Ac to an acceptor molecule in the alpha-2, 3-linkage. The terms "3 ' sialyllactose", "3 ' -sialyllactose", "α -2,3 sialyllactose", "α 2,3 sialyllactose", "α -2, 3-sialyllactose", "α -2,3 sialyllactose", "α 2,3 sialyllactose", "3 SL" or "3 ' SL" are used interchangeably and refer to products obtained by catalyzing the transfer of sialic acid groups from CMP-Neu5Ac to lactose in the α -2, 3-linkage. As used herein, the terms "alpha-2, 6-sialyltransferase" (alpha-2, 6-sialyltransferase), "," alpha 2,6 sialyltransferase "(alpha 2,6 sialyltransferase),", "6-sialyltransferase" (6-sialyltransferase), "," alpha-2,6-sialyltransferase "(alpha-2, 6-sialyltransferase),", "alpha 2,6 sialyltransferase" (alpha 2,6 sialyltransferase), "," 6 sialyltransferase (6 sialyltransferase), "," 6-ST ", or" 6ST "are used interchangeably and refer to glycosyltransferases that catalyze the transfer of sialic acid from a donor CMP-Neu5Ac to an acceptor molecule in the alpha-2, 6-linkage. The terms "6 ' sialyllactose", "6 ' -sialyllactose", "alpha-2, 6-sialyllactose", "alpha 2,6 sialyllactose", "alpha-2, 6-sialyllactose", "alpha 2,6 sialyllactose", "6 SL" or "6 ' SL" are used interchangeably and refer to products obtained by catalyzing the transfer of sialic acid from CMP-Neu5Ac to lactose in the alpha-2, 6-linkage.

As used herein, the terms "alpha-2, 8-sialyltransferase" (alpha-2, 8-sialyltransferase), "," alpha 2,8 sialyltransferase "(alpha 2,8 sialyltransferase),", "8-sialyltransferase" (8-sialyltransferase), "," alpha-2,8-sialyltransferase "(alpha-2, 8-sialyltransferase),", "alpha 2,8 sialyltransferase" (alpha 2,8 sialyltransferase), "," 8 sialyltransferase (8 sialyltransferase), "," 8-ST ", or" 8ST "are used interchangeably and refer to glycosyltransferases that catalyze the transfer of sialic acid from a donor CMP-Neu5Ac to an acceptor in the alpha-2, 8-linkage.

As used herein and as generally understood in the art of the present state of the art, "fucosylated oligosaccharide (fucosylated oligosaccharide)" is an oligosaccharide bearing fucose residues. Such fucosylated oligosaccharides are sugar structures comprising at least three monosaccharide subunits connected to each other via glycosidic bonds, wherein at least one of the monosaccharide subunits is a fucose. The fucosylated oligosaccharide may contain more than one fucose residue, for example two, three or more fucose residues. The fucosylated oligosaccharides may be neutral oligosaccharides or charged oligosaccharides, e.g. also comprising sialic acid structures. Fucose may be linked to other monosaccharide subunits comprising glucose, galactose, glcNAc via a-glycosidic bond comprising a-1, 2, a-1, 3, a-1, 4, a-1, 6 bond.

Examples include 2' -fucosyllactose (2 ' FL), 3-fucosyllactose (3 FL), 4-fucosyllactose (4 FL), 6-fucosyllactose (6 FL), difucosyllactose (diFL), lactodifucose (lactodifucose; LDFT), milk-N-fucopentaose I (LNFP I), milk-N-fucopentaose II (LNFP II), milk-N-fucopentaose III (LNFP III), milk-N-fucopentaose V (LNFP V), milk-N-fucopentaose VI (LNFP VI), milk-N-neofucopentaose I, milk-N-disaccharide fucohexaose I (LDFH I), milk-N-disaccharide hexaose II (LDFH II), monocyclofucose milk-N-hexaose III (MFLNH III), difucosyl milk-N-hexaose (DFLNHa), difucosyl-milk-N-neohexaose, 3' -sialyl-3-fuconofucosyl milk-N-neohexaose, monocyclofucosyl milk-N-octasaccharide (sialyl Lea), sialyl milk-N-fucohexaose II, sialylmilk-N-fucohexaose II, and sialyltetradec-N-sialyltetrasaccharide.

As used herein, the terms "alpha-1, 2-fucosyltransferase" (alpha-1, 2-fucosyltransferase), "alpha 1, 2-fucosyltransferase" (alpha 1,2 fucosyltransferase), "2-fucosyltransferase" (2-fucosyltransferase), "alpha-1, 2-fucosyltransferase" (alpha-1, 2-fucosyltransferase), "alpha 1, 2-fucosyltransferase" (alpha 1,2 fucosyltransferase), "2 fucosyltransferase (2 fucosyltransferase), and" 2-FT "are used interchangeably and refer to glycosyltransferases that catalyze the transfer of fucose from donor GDP-L-fucose to acceptor molecules in the alpha-1, 2-linkage. The term "2 'fucosyllactose" as used in the present invention, "2' -fucosyllactose (2 '-fucosyllactose) alpha-1, 2-fucosyllactose, alpha-1,2 fucosyllactose, alpha-1, 2-fucosyllactose alpha-1, 2-fucosyllactose" alpha 1,2 fucosyllactose (alpha 1,2 fucosyllactose) "," Gal beta-4 (Fuc alpha 1-2) Glc "," 2FL "or" 2' FL "are used interchangeably and refer to products obtained by catalyzing the transfer of fucose residues from GDP-L-fucose to lactose in the alpha-1, 2-linkage by an alpha-1, 2-fucosyltransferase. The term "difucosyllactose", "di-fucoidan", "lactodifucosyltetrasaccharide", "2 ', 3-difucosylactose", "2 ',3 difucosylactose (2 ',3 difucosyllactose)," α -2',3-fucosyllactose (α -2', 3-fucostylose), "α 2',3-fucosyllactose (fucα1-2Gal β1-4 (fucα1-3) Glc," DFLac "," 2',3 diFL "," DFL "," diFL ", or" diFL "are used interchangeably.

As used herein, the terms "alpha-1, 3-fucosyltransferase" (alpha-1, 3-fucosyltransferase), "," alpha 1,3 fucosyltransferase "(alpha 1,3 fucosyltransferase),", "alpha-1, 3-fucosyltransferase" (alpha-1, 3-fucosyltransferase), "," alpha 1,3-fucosyltransferase "(alpha 1,3 fucosyltransferase),", "3 fucosyltransferase (3 fucosyltransferase),", "3-FT" or "3 FT" are used interchangeably and refer to glycosyltransferases that catalyze the transfer of fucose from donor GDP-L-fucose to acceptor molecules in the alpha-1, 3-linkage. The terms "3-fucosyllactose", "alpha-1, 3-fucosyllactose", "alpha 1,3 fucosyllactose", "alpha-1, 3-fucosyllactose", "alpha 1,3 fucosyllactose", "Gal beta-4 (Fuc alpha 1-3) Glc", "3 FL" or "3-FL" as used in the present invention are used interchangeably and refer to products obtained by catalyzing the transfer of fucose residues from GDP-L-fucose to lactose in the alpha-1, 3-linkage.

As used herein and as generally understood in the current art, "neutral oligosaccharides (neutral oligosaccharide)" are oligosaccharides that do not have a negative charge derived from a carboxylic acid group. Examples of such neutral oligosaccharides are 2' -fucosyllactose (2 ' FL), 3-fucosyllactose (3 FL), 2', 3-difucosyllactose (diFL), milk-N-triose II, milk-N-tetraose, milk-N-neotetraose, milk-N-fucopentaose I, milk-N-neofucopentaose I, milk-N-fucopentaose II, milk-N-fucopentaose III, milk-N-fucopentaose V, milk-N-fucopentaose VI, milk-N-neofucopentaose V, milk-N-difucosohexaose I, milk-N-difucosohexaose II, 6' -galactosyllactose, 3' -galactosyllactose, milk-N-hexaose, milk-N-neohexaose, para-milk-N-neohexaose, difucosyl-milk-N-hexaose and difucosyl-milk-N-neohexaose.

As used herein, the antigen of the human ABO blood group system is an oligosaccharide. Such antigens of the human ABO blood group system are not limited to human structures. The structure involves the A determinants GalNAc- α1,3 (Fuc- α1, 2) -Gal-, B determinants Gal- αl,3 (Fuc- α1, 2) -Gal-and H determinants Fuc- α1, 2-Gal-present on disaccharide core structures comprising Gal- β1,3-GlcNAc, gal- β1,4-GlcNAc, gal- β1,3-GalNAc and Gal- β1, 4-GlcNAc.

As used herein, the terms "LNT II", "LNT-II", "LN 3", "milk-N-triose II", "milk-N-triose (lacto-N-triose), or" milk-N-triose (lacto-N-triose) or "GlcNAcβ1-3Galβ1-4 Glc" are used interchangeably.

The terms "LNT", "lacto-N-tetraose" or "Galβ1-3GlcNAcβ1-3Galβ1-4 Glc" as used in the present invention are used interchangeably.

The terms "LNnT", "lacto-N-neotetraose", "neo-LNT" or "Galβ1-4GlcNAcβ1-3Galβ1-4 Glc" as used in the present invention are used interchangeably.

The terms "LSTa", "LS-tetrasaccharide a (LS-Tetrasaccharide a)", "Sialyl-lacto-N-tetrasaccharide a (Sialyl-lacto-N-tetrasaccharide a)", "Sialyl-lacto-N-tetrasaccharide a (Sialyl-N-tetrasaccharide a", or "Neu 5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc" as used in the present invention are used interchangeably.

The terms "LSTb", "LS-tetrasaccharide b (LS-Tetrasaccharide b)", "Sialyl-lacto-N-tetrasaccharide b (Sialyl-lacto-N-tetrasaccharide b)", "Sialyl-lacto-N-tetrasaccharide b (Sialyl-N-tetrasaccharide b)" or "Gal-b 1,3- (Neu 5Ac-a2, 6) -GlcNAc-b1,3-Gal-b1, 4-Glc" as used herein are used interchangeably.

The terms "LSTc", "LS-tetrasaccharide c (LS-Tetrasaccharide c)", "Sialyl-lacto-N-tetrasaccharide c (Sialyl-lacto-N-tetraose c)", "Sialyl-lacto-N-neotetraose c (Sialyl-lacto-N-neotetraose c)", or "Neu 5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1, 4-Glc" as used herein are used interchangeably.

The terms "LSTd", "LS-tetrasaccharide d (LS-Tetrasaccharide d)", "Sialyl-lacto-N-tetrasaccharide d (Sialyl-lacto-N-tetraose d)", "Sialyl-lacto-N-tetrasaccharide d (Sialyl-lacto-N-tetraose)", "Sialyl-lacto-N-neotetraose d (Sialyl lacto-N-neotetraose) or" Neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc "as used herein are used interchangeably.

The terms "DSLNnT" and "disialylacto-N-neotetraose" are used interchangeably and refer to Neu5Ac-a2,6- [ Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1,4-Glc.

The terms "DSLNT" and "disialylacto-N-tetraose" are used interchangeably and refer to Neu5Ac-a2,6- [ Neu5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3] -Gal-b1,4-Glc. The terms "LNFP-I", "lacto-N-fucopentaose I", "LNFP I", "LNF I OH type I determinant (LNF I OH type I determinant)", "LNF I", "LNF 1", and "H blood group antigen pentasaccharide type 1 (Blood group H antigen pentaose type 1)" are used interchangeably and refer to Fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc.

The terms "GalNAc-LNFP-I" and "blood group A antigen hexose type I (blood group A antigen hexaose type I)" are used interchangeably and refer to GalNAc-a1,3- (Fuc-a 1, 2) -Gal-b1,3-GlcNAc-b1,3-Gal-b1,4-Glc.

The term "LNFP-II" and "lacto-N-fucopentaose II" are used interchangeably and refer to Gal-b1,3- (Fuc-a 1, 4) -GlcNAc-b1,3-Gal-b1,4-Glc.

The term "LNFP-III" and "lacto-N-fucopentaose III" are used interchangeably and refer to Gal-b1,4- (Fuc-a 1, 3) -GlcNAc-b1,3-Gal-b1,4-Glc.

The term "LNFP-V" and "lacto-N-fucopentaose V" are used interchangeably and refer to Gal-b1,3-GlcNAc-b1,3-Gal-b1,4- (Fuc-a 1, 3) -Glc.

The terms "LNFP-VI", "LNnFP V" and "lacto-N-neofucopentaose V (lacto-N-neofucopentaose V)" are used interchangeably and refer to Gal-b1,4-GlcNAc-b1,3-Gal-b1,4- (Fuc-a 1, 3) -Glc.

The term "LNnFP I" and "Lacto-N-neofucopentaose I (Lactobacillus-N-neofucopentaose I)" are used interchangeably and refer to Fuc-a1,2-Gal-b1,4-GlcNAc-b1,3-Gal-b1,4-Glc.

The terms "LNDFH I", "Lacto-N-difiuorohexaose (L-N-difiuorohexaose I)", "LNDFH-I", "LDFH I", "Le" ^b Lactose and Lewis-b hexasaccharide are used interchangeably and refer to Fuc-a1,2-Gal-b1,3- [ Fuc-a1,4]-GlcNAc-b1，3-Gal-b1，4-Glc。

The terms "LNDFH II", "Lacto-N-difiuorohexaose II", "Lewis a-Lewis x" and "LDFH II" are used interchangeably and refer to Fuc-a1,4- (Gal-b 1, 3) -GlcNAc-b1,3-Gal-b1,4- (Fuc-a 1, 3) -Glc.

The terms "LNnDFH", "Lacto-N-neodifuchaxaose" and "Lewis x hexaose" are used interchangeably and refer to Gal-b1,4- (Fuc-a 1, 3) -GlcNAc-b1,3-Gal-b1,4- (Fuc-a 1, 3) -Glc.

The terms "alpha-tetrasaccharide" and "A-tetrasaccharide" are used interchangeably and refer to GalNAc-a1,3- (Fuc-a 1, 2) -Gal-b1,4-Glc.

As used herein, "fucosylation pathway (fucosylation pathway)" is a biochemical pathway consisting of: enzymes and their respective genes, mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-phosphate guanyl transferase, GDP-mannose 4, 6-dehydratase, GDP-L-fucose synthase and/or recycling pathway L-fucose kinase/GDP-fucose pyrophosphorylase, and fucosyltransferases producing alpha 1,2, alpha 1,3, alpha 1,4 and/or alpha 1,6 fucosylated oligosaccharides.

"sialylation pathway (sialylation pathway)" is a biochemical pathway consisting of: enzymes and their respective genes, L-glutamylamine-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucomutase, 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 kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, sialic acid synthase, N-acetylneuraminic acid dissociase, N-acyl neuraminic acid-9-phosphate synthase, sialic acid-9-phosphate synthase and/or sialyl-CMP, and sialyltransferases that produce α2,3, α2,6 and/or α2,8 sialylated oligosaccharides.

As used herein, "galactosylation pathway (galactosylation pathway)" is a biochemical pathway consisting of: enzymes and their respective genes, galactose-1-epimerase, galactokinase, glucokinase, galactose-1-phosphate uridyltransferase, UDP-glucose 4-epimerase, glucose-1-phosphate uridyltransferase and/or glucose phosphomutase, and galactosyltransferases that produce alpha or beta binding galactose on the 2,3, 4 and/or 6 hydroxyl groups of oligosaccharides.

As used herein, "N-acetylglucosamine carbohydrate pathway (N-acetylglucosamine carbohydrate pathway)" is a biochemical pathway consisting of: enzymes and their respective genes, L-glutamyl-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, glucosamine phosphate mutase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate N-acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase and/or glucosamine-1-phosphate acetyltransferase, and alpha or beta binding N-acetylglucosamine glycosyltransferase on the 3, 4 and/or 6 hydroxyl groups of oligosaccharides.

As used herein, "N-acetylgalactose amination pathway (N-acetylgalactosaminylation pathway)" is a biochemical pathway comprising at least one of an enzyme and its respective gene selected from the list comprising: l-glutamylamino acid-D-fructose-6-phosphate aminotransferase, phosphoglucosamine mutase, 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 glycosyltransferase that produces GalNAc modified compounds comprising mono-, di-or oligosaccharides having alpha or beta binding N-acetylgalactosamine on the mono-, di-or oligosaccharides.

As used herein, "mannosylation pathway (mannosylation pathway)" is a biochemical pathway comprising at least one of an enzyme and its respective gene selected from the list comprising: mannose-6-phosphate isomerase, phosphomannose mutase, and/or mannose-1-phosphate guanylate transferase, and glycosyltransferases that produce mannosylated compounds comprising a mono-, di-, or oligosaccharide having an alpha or beta binding mannose on the mono-, di-, or oligosaccharide.

As used herein, the "N-acetylmannosylation pathway (N-acetylmannosaminylation pathway)" is a biochemical pathway comprising at least one of an enzyme and its respective gene selected from the list comprising: L-glutamyl-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, UDP-GlcNAc 2-epimerase and/or ManNAc kinase, and glycosyltransferase that produces a ManNAc modified compound comprising the mono-, di-or oligosaccharide having an alpha or beta binding to N-acetylmannosamine on the mono-, di-or oligosaccharide.

The terms "mannose-6-phosphate isomerase (mannase-6-phosphate isomerase)", "phosphomannose isomerase (phosphomannose isomerase)", "mannose phosphoisomerase (mannose phosphate isomerase)", "phosphohexose isomerase (phosphohexose)", "phosphomannose isomerase (phosphomanoisomerase)", "phosphomannose-isomerase" (phosphomanose), phosphohexose mutase (phosphomanose synthase) "," D-mannose-6-phosphate ketol-isomerase "(D-mannase-6-phosphomana) are used interchangeably and refer to enzymes that catalyze the reversible conversion of D-fructose 6-phosphate to D-mannose 6-phosphate.

The terms "phosphomannomutase" (phosphomannomutase), "mannomutase (mannose phosphomutase)", "phosphomannomutase (phosphomannose mutase)", "D-mannose 1,6-phosphomutase (D-mannomutase 1, 6-phosphomulatase) and" manB "are used interchangeably and refer to enzymes that catalyze the reversible conversion of D-mannose 6-phosphate to D-mannose 1-phosphate.

The terms "mannose-1-guanylate acylase (mannase-1-phosphate guanylyltransferase)", "GTP-mannose-1-guanylate acylase (GTP-mannase-1-phosphate guanylyltransferase)", "mannosyl phosphate isomerase-guanosine 5 '-diphosphate-D-mannose pyrophosphatase (phosphomannose isomerase-guanosine 5' -biphosphole-D-mannose pyrophosphorylase; PIM-GMP)", "GDP-mannose pyrophosphatase (GDP-mannose pyrophosphorylase)", "guanosine 5 '-diphosphate-D-mannose pyrophosphatase (guanosine 5' -biphospho-D-mannose pyrophosphorylase)", "guanosine diphosphate mannose pyrophosphatase (guanosine diphosphomannose pyrophosphorylase)", "guanosine triphosphate-mannose 1-guanylate pyrophosphatase (guanosine triphosphate-mannase 1-phosphate guanylyltransferase)", "mannosyl 1-phosphate pyrophosphatase (GTP-triphosphate) phosphate guanylyltransferase-D-guanosine triphosphate)", and interchangeable phosphates are used for converting mannose to mannose and mannose (GDP-3-D-37).

The terms "GDP-mannose 4, 6-dehydratase" (GDP-mannase 4, 6-dehydratase), "guanosine 5'-diphosphate-D-mannose oxidoreductase (guanosine 5' -biphosphite-D-mannose oxidoreductase)," guanosine diphosphate mannose oxidoreductase (guanosine diphosphomannose oxidoreductase), "guanosine diphosphate mannose4,6-dehydratase (guanosine diphosphomannose, 6-dehydratase)," GDP-D-mannose dehydratase (GDP-D-mannose dehydratase), "GDP-D-mannose 4,6-dehydratase (GDP-D-mannase 4, 6-dehydratase)," GDP-mannose 4, 6-hydrogen-dissociaase (GDP-mannase 4, 6-hydrolase), "GDP-mannose 4, 6-hydrogen-dissociaase (GDP-4-dehydrogenase) and" GDP-6-mannose-6-dehydrogenase "(GDP-4, 6-dehydrogenase) are used interchangeably to form GDP-D-mannose and to form GDP-mannose-6-D-mannose and to be used interchangeably with GDP-D-mannose 4, 6-dehydrogenase (GDP-D-dehydrogenase).

The term "GDP-L-fucose synthase" (GDP-L-fucoidan), "GDP-4-keto-6-deoxy-D-mannose-3, 5-epi-isomerase-4-reductase" (GDP-4-keyo-6-deoxy-D-mannase-3, 5-epi-4-reduction) and "GDP-L-fucose": NADP+4-oxidoreductase (3, 5-epimerization) (GDP-L-fuse: NADP+4-oxidase (3, 5-epothily)) "and" fcl "are used interchangeably and refer to an enzyme that forms the second step in the biosynthesis of GDP-fucose.

The terms "L-fucoskinase/GDP-fucose pyrophosphorylase (L-fucokinase/GDP-fucose pyrophosphorylase)", "L-fucose kinase/L-fucose-1-P guanylase (L-fucokinase/L-fucose-1-P guanylyltransferase)", "GDP-fucose pyrophosphorylase (GDP-fucose pyrophosphorylase)", "GDP-L-fucose pyrophosphorylase (GDP-L-fucose pyrophosphorylase)", and "fkp" are used interchangeably and refer to enzymes that catalyze the conversion of L-fucose-1-phosphate to GDP-fucose using GTP.

The terms "L-glutamine-D-fructose-6-phosphate aminotransferase (L-glutamine-D-fructo-6-phosphate aminotransferase)", "glutamyl-fructose-6-phosphate aminotransferase (isomerization)", "D-fructose-6-phosphate transaminase (isomerization))", "hexose-phosphate aminotransferase (hexosaminitransferase)", "glucosamine-6-phosphate isomerase" (forming a glutamate) (glucamine-6-phosphate isomerase (glucamine-forming) "," glucamine-fructose-6-phosphate aminotransferase (isomerization) "," glucamine-6-phosphate transaminase (isomerization) "," D-fructose-6-phosphate aminotransferase (D-fructo-6-phosphate amidotransferase) "," hexose-6-phosphate aminotransferase (gfu-6-92) "," D-6-phosphate aminotransferase (6-92) ", and" glucose-6-phosphate synthase (35A-92) ", and" glucose-6-phosphate synthase "are used interchangeably.

The terms "glucosamine-6-P deaminase" (glucosamine-6-P deaminase), "glucosamine-6-phosphate deaminase" (glucosamine-6-phosphate deaminase), "GlcN 6P deaminase" (GlcN 6P deaminase), "glucosamine-6-phosphate isomerase (glucosamine-6-phosphate isomerase)," glmD "and" nagB "are used interchangeably and refer to enzymes that catalyze the reversible isomerization-deamination of glucosamine-6-phosphate (GlcN 6P) to form fructose-6-phosphate and ammonium ions.

The terms "phosphoglucosamine mutase (phosphoglucosamine mutase)" and "glmM" are used interchangeably and refer to an enzyme that catalyzes the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate. Phosphoglucamine mutase can also catalyze the formation of glucose-6-P from glucose-1-P, albeit at a 1400-fold lower rate.

The terms "N-acetylglucosamine-6-P deacetylase (N-acetylglucosamine-6-P deacetylase)", "N-acetylglucosamine-6-phosphate deacetylase (N-acetylglucosamine-6-phosphate deacetylase)", and "nagA" are used interchangeably and refer to enzymes that catalyze the hydrolysis of the N-acetyl group of N-acetylglucosamine-6-phosphate (GlcNAc-6-P) to produce glucosamine-6-phosphate (GlcN 6P) and acetate.

N-acyl-glucosamine 2-epimerase is an enzyme that catalyzes the reaction of N-acyl-D-glucosamine = N-acyl-D-mannosamine. Alternative names for this enzyme include N-acetylglucosamine 2-epimerase, N-acetyl-D-glucosamine 2-epimerase, glcNAc 2-epimerase, N-acyl-D-glucosamine 2-epimerase and N-acetylglucosamine epimerase.

UDP-N-acetylglucosamine 2-epimerase is an enzyme that catalyzes the reaction of N-acetyl-D-glucosamine = N-acetylmannosamine. Alternative names for this enzyme include UDP-N-acyl glucosamine 2-epimerase, UDP-GlcNAc-2-epimerase and UDP-N-acetyl-D-glucosamine 2-epimerase.

N-acetyl-mannosamine-6-phosphate 2-epimerase is an enzyme that catalyzes the reaction of N-acetyl-D-glucosamine 6-phosphate = N-acetyl-D-mannosamine 6-phosphate.

The bifunctional UDP-GlcNAc 2-epi-isomerase/kinase is a bifunctional enzyme that catalyzes the reaction UDP-N-acetyl-D-glucosamine=n-acetyl-D-mannosamine and the reaction N-acetyl-D-mannosamine+atp=adp+n-acetyl-D-mannosamine 6-phosphate.

Glucosamine 6-phosphate N-acetyltransferase is an enzyme that catalyzes the transfer of acetyl from acetyl-CoA to D-glucosamine-6-phosphate, thereby producing free CoA and N-acetyl-D-glucosamine 6-phosphate. Alternative names include aminodeoxyglucosamine phosphoacetyl transferase, D-glucosamine-6-PN-acetyl transferase, glucosamine 6-phosphoacetyl transferase, glucosamine 6-phosphate N-acetyl transferase, glucosamine 6-phosphoacetyl transferase, N-acetyl glucosamine-6-phosphate synthase, phosphoglucamine acetyl enzyme, phosphoglucamine N-acetyl phosphatase, phosphoglucamine transacetylase, GNA and GNA1.

The term "N-acetylglucosamine-6-phosphate phosphatase (N-acetylglucosamine-6-phosphate phosphatase)" refers to an enzyme that dephosphorylates N-acetylglucosamine-6-phosphate (GlcNAc-6-P) to thereby synthesize N-acetylglucosamine (GlcNAc).

The term "N-acetylmannosamine-6-phosphate phosphatase (N-acetylmannosamine-6-phosphate phosphatase)" refers to an enzyme that dephosphorylates N-acetylmannosamine-6-phosphate (ManNAc-6P) to N-acetylmannosamine (ManNAc).

The terms "N-acetylmannosamine-6-phosphate 2-epimerase" (N-acetylmannosamine-6-phosphate 2-epomerase), "ManNAc-6-P isomerase" (ManNAc-6-P-isomerase), "ManNAc-6-P2-epimerase" (ManNAc-6-P2-epomerase), "N-acetylglucosamine-6P 2-epimerase" (N-acetylglucosamine-6P 2-epomerase), "nanE" are used interchangeably and refer to enzymes that convert ManNAc-6-P to N-acetylglucosamine-6-phosphate (GlcNAc-6-P).

The terms "acetylglucosamine phosphate mutase (phosphoacetylglucosamine mutase)", "acetylglucosamine phosphate mutase (acetylglucosamine phosphomutase)", "acetylaminodeoxyglucosamine phosphate mutase (acetylaminodeoxyglucose phosphomutase)", "phospho-N-acetylglucosamine mutase (phospho-N-acetylglucosamine mutase)", and "N-acetyl-D-glucosamine 1, 6-phosphate mutase (N-acetyl-D-glucosamine 1, 6-phosphomutase)" are used interchangeably and refer to enzymes that catalyze the conversion of N-acetyl-glucosamine 1-phosphate to N-acetylglucosamine 6-phosphate.

The term "N-acetylglucosamine 1-phosphate uridyltransferase (N-acetylglucosamine 1-phosphate uridylyltransferase)", "N-acetylglucosamine 1-phosphate uridyltransferase (N-acetylglucosamine 1-phosphate transferase)", "UDP-N-acetylglucosamine bisphosphatase (UDP-N-acetylglucosamine diphosphorylase)", "UDP-N-acetylglucosamine pyrophosphorylase (UDP-N-acetylglucosamine pyrophosphorylase)", "uridine bisphosphate acetylglucosamine pyrophosphorylase (uridine diphosphoacetylglucosamine pyrophosphorylase)", "UTP": 2-acetamido-2-deoxy-alpha-D-glucose-1-phosphate uridylyltransferase (UTP: 2-acetamido-2-deoxy-alpha-D-glucose-1-phosphate uridylyltransferase), UDP-GlcNAc pyrophosphorylase (UDP-GlcNAc pyrophosphorylase), glu uridylyltransferase (GlmU uridylyltransferase), acetylglucosamine 1-phosphate uridylyltransferase (Acetylglucamine 1-phosphate uridylyltransferase), UDP-Acetylglucosamine pyrophosphorylase (UDP-Acetylglucosamine pyrophosphorylase), UDP-diphosphouride-N-Acetylglucosamine pyrophosphorylase (uridine diphosphate-N-Acetylglucosamine pyrophosphorylase), uridine diphosphate Acetylglucosamine phosphorylase (uridine diphosphoacetylglucosamine phosphorylase) and acetyl glucosamine 1-phosphate uridylyltransferase (Acetylglucosamine 1-phosphate uridylyltransferase) may be used to catalyze the transfer of GlcNAc-5-uridine from UDP-N-Acetylglucosamine (UDP-5-Acetylglucosamine) to tri-Acetylglucosamine phosphate by the aid of GlcNAc-5-Acetylglucosamine pyrophosphorylase (UDP-Acetylglucosamine pyrophosphorylase).

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

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

As used herein, the terms "Neunac synthase", "N-acetylneuraminic acid synthase (N-acetylneuraminic acid synthase)", "N-acetylneuraminic acid synthase (N-acetylneuraminate synthase)", "sialic acid synthase (sialic acid synthase)", "NeuAc synthase (NeuAc synthase)", "NeuB 1)", "NeuNAc synthase (NeuNAc synthase)", "NANA condensation enzyme (NANA condensing enzyme)", "N-acetylneuraminic acid dissociating enzyme synthase (N-acetylneuraminate lyase synthase)", "N-acetylneuraminic acid condensation enzyme (N-acetylneuraminic acid condensing enzyme)", and refer to enzymes capable of using phosphoenolpyruvate (PEP) in a reaction to synthesize sialic acid from N-acetylmannosamine (ManNAc).

The terms "N-acetylneuraminic acid dissociating enzyme (N-acetylneuraminate lyase)", "Neu 5Ac dissociating enzyme (Neu 5Ac lyase)", "N-acetylneuraminic acid acetolysis dissociating enzyme (N-acetylneuraminate pyruvate-lyase)", "N-acetylneuraminic acid aldolase" (N-acetylneuraminic acid aldolase) "," NALase "," sialylase (sialylase) "," sialylase (sialic acid aldolase) "," sialylase (sialic acid lyase) ", and" na "are used interchangeably and refer to enzymes that degrade N-acetylneuraminic acid salts to N-acetylmannosamine (ManNAc) and pyruvate.

As used herein, the terms "N-acyl neuraminic acid-9-phosphate synthase (N-acyl neuraminic acid-9-phosphate synthase)", "N-acyl neuraminic acid-9-phosphate synthase (N-acyl neuraminic acid-9-phosphate synthetase)", "NANA synthase (NANA synthase)", "NANAS", "NANS", "NmeNANAS", "N-acetyl neuraminic acid acetone acid dissociation enzyme (pyruvate phosphorylation)" (N-acetylneuraminate pyruvate-lyase (pyruvic acid-phosphate)) are used interchangeably and refer to enzymes capable of synthesizing N-acyl neuraminic acid-9-phosphate from N-acetyl mannosamine-6-phosphate (ManNAc-6-phosphate) using phosphoenolpyruvate (PEP) in the reaction.

The term "N-acyl neuraminic acid-9-phosphatase" refers to an enzyme capable of dephosphorylating N-acyl neuraminic acid-9-phosphate to synthesize N-acyl neuraminic acid salt.

As used herein, the terms "CMP-sialic acid synthase (CMP-sialic acid synthase)", "N-acyl neuraminic acid cytidylyltransferase (N-acylneuraminate cytidylyltransferase)", "CMP-sialic acid synthase", "CMP-NeuAc synthase (CMP-NeuAc synthase)", "NeuA", and "CMP-N-acetyl neuraminic acid synthase (CMP-N-acetylneuraminic acid synthase)", are used interchangeably and refer to enzymes capable of synthesizing CMP-N-acetyl neuraminic acid salts from N-acetyl neuraminic acid salts using CTP in a reaction.

The terms "galactose-1-epimerase", "aldolase 1-epimerase", "mutarotase", "aldolase", "galactose mutarotase (galactose mutarotase)", "galactose 1-epimerase" and "D-galactose 1-epimerase (D-galactose 1-epimerase) are used interchangeably and refer to enzymes that catalyze the conversion of beta-D-galactose to alpha-D-galactose.

The terms "galactokinase" (galactokinase), "galactokinase (phosphorylating) (galactokinase (phosphorylating))", and "ATP: d-galactose-1-phosphotransferase (ATP) is used interchangeably and refers to an enzyme that catalyzes the conversion of alpha-D-galactose to alpha-D-galactose 1-phosphate using ATP.

The terms glucokinase and "glucokinase (phosphorylating) (glucokinase (phosphorylating))" are used interchangeably and refer to an enzyme that catalyzes the conversion of D-glucose to D-glucose 6-phosphate using ATP.

The terms "galactose-1-phosphate uridylyltransferase (galactose-1-phosphate uridylyltransferase)", "Gal-1-P uridylyltransferase (Gal-1-P uridylyltransferase)", "UDP-glucose" - -hexose-1-phosphate uridylyltransferase (UDP-glucose- - -hexose-1-phosphate uridylyltransferase) "," uridylyltransferase (uridyl transferas) "," hexose-1-phosphate uridylyltransferase (hexose-1-phosphate uridylyltransferase) "," uridylyltransferase (uridylyltransferase) "," hexose-1-phosphate uridylyltransferase (hexose-1-phosphate uridyltransferase) "," UDP-glucose ": alpha-D-galactose-1-phosphate uridyltransferase (UDP-glucose: alpha-D-galactose-1-phosphate uridylyltransferase), "galB" and "galT" are used interchangeably and refer to an enzyme that catalyzes the reaction D-galactose 1-phosphate + UDP-D-glucose = D-glucose 1-phosphate + UDP-D-galactose.

The terms "UDP-glucose 4-epi-isomerase", "UDP-galactose 4-epi-isomerase", "UDP-glucose-diphosphate-epi-isomerase (uridine diphosphoglucose epimerase)", "galactose vals-converting enzyme (galactose waldensase)", "UDPG-4-epi-isomerase (UDPG-4-epi-isomerase)", "uridine diphosphate-galactose 4-epi-isomerase (uridine diphosphate galactose-epi-ase)", "uridine diphosphate-galactose 4-epi-isomerase (uridine diphospho-galactose-4-epi-ase)", "UDP-glucose-epi-isomerase (UDP-glucose-isomerase)", "4-epi-isomerase (4-epi-ase)", "uridine diphosphate-glucose-4-epi-isomerase (uridine diphosphoglucose-epi-4-ase)", and UDP-glucose-4-epi-isomerase (UDP-glucose-4-epi-4-isomerase) are interchangeable and UDP-glucose-4-epi-isomerase (UDP-glucose-4-glucose-isomerase) and UDP-glucose-4-epi-isomerase (UDP-4-glucose-isomerase) are used interchangeably.

The terms "glucose-1-phosphate uridylyltransferase (glucose-1-phosphate uridylyltransferase)", "UTP- - -glucose-1-phosphate uridylyltransferase (UTP- - -glucose-1-phosphate uridylyltransferase)", "UDP-glucose pyrophosphorylase (UDP glucose pyrophosphorylase)", "UDPG phosphorylase (UDPGphosphorylase)", "UDPG pyrophosphorylase (UDPGpyrophosphorylase)", "uridine 5 '-diphosphate glucose pyrophosphorylase (uridine 5' -diphosphoglucose pyrophosphorylase)", "uridine diphosphate glucose pyrophosphorylase (uridine diphosphoglucose pyrophosphorylase)", "uridine diphosphate-D-glucose pyrophosphorylase (uridine diphosphate-D-glucose pyrophosphorylase)", "uridine-diphosphate glucose pyrophosphorylase (uridine-diphosphate glucose pyrophosphorylase)", and "galU" are used interchangeably and refer to the catalysis of the conversion of glucose-1-UDP to glucose-phosphate.

The terms "phosphoglucomutase (alpha-D-glucose-1, 6-biphosphoryl-dependent)", "phosphoglucomutase (ambiguous) (glucose phosphomutase (ambiguous))", "phosphoglucomutase (ambiguous) (phosphoglucose mutase (ambiguous))") are used interchangeably and refer to enzymes that catalyze the conversion of D-glucose 1-phosphate to D-glucose 6-phosphate.

The terms "UDP-N-acetylglucosamine 4-epimerase", "UDP-acetylglucosamine epimerase (UDP acetylglucosamine epimerase)", "uridine diphosphate acetylglucosamine epimerase (uridine diphosphoacetylglucosamineepimerase)", "uridine diphosphate N-acetylglucosamine 4-epimerase (uridine diphosphate N-acetylglucosamine-4-epimerase)", "uridine 5 '-diphosphate-N-acetylglucosamine 4-epimerase (uridine 5' -biphosphocholine-N-acetylglucosamine-4-epimerase)", "UDP-N-acetyl glucosamine 4-epimerase (UDP-N-acetyl-D-glucosamine 4-epimerase) are used interchangeably and refer to catalyzing the isomerization of UDP-N-acetylglucosamine (UDP-N-acetylglucosamine) to UDP-galactosamine (UDP-Gal).

The terms "N-acetylgalactosamine kinase (N-acetylgalactosamine kinase)", "GALK 2", "GK 2", "GalNAc kinase (GalNAc kinase)", "N-acetylgalactosamine (GalNAc) -1-phosphokinase (N-acetylgalactosamine (GalNAc) -1-phosphokinase)", "ATP: N-acetyl-D-galactosamine 1-phosphate transferase (ATP) is used interchangeably and refers to an enzyme that catalyzes the synthesis of N-acetylgalactosamine 1-phosphate (GalNAc-1-P) from N-acetylgalactosamine (GalNAc) using ATP.

The terms "UDP-N-acetylgalactosamine pyrophosphorylase (UDP-N-acetylgalactosamine pyrophosphorylase)" and "UDP-GalNAc pyrophosphorylase (UDP-GalNAc pyrophosphorylase)" are used interchangeably and refer to enzymes that catalyze the conversion of N-acetylgalactosamine 1-phosphate (GalNAc-1-P) to UDP-N-acetylgalactosamine (UDP-GalNAc) using UTP.

The terms "N-acetylneuraminic acid kinase (N-acetylneuraminate kinase)", "ManNAc kinase (ManNAc kinase)", "N-acetyl-D-mannosamine kinase (N-acetyl-D-mannosamine kinase)", and "nanK" are used interchangeably and refer to enzymes that phosphorylate ManNAc to synthesize N-acetylmannosamine-phosphate (ManNAc-6-P).

The terms "acetyl-CoA synthase (acetyl-coenzyme A synthetase)", "acs", "acetyl-CoA synthase", "acteos", "acetate-CoA synthase", "CoA ligase", "acyl-activating enzyme (acetyl-activating enzyme) and" yfaC "are used interchangeably and refer to enzymes that catalyze the conversion of acetate to acetyl-CoA (actoa) in an ATP-dependent reaction.

The terms "pyruVate dehydrogenase (pyruVate dehydrogenase)", "pyruVate oxidase", "POX", "poxB", and "pyruVate: ubiquinone-8 oxidoreductase (pyruvate) is used interchangeably and refers to an enzyme that catalyzes the oxidative decarboxylation of pyruvate to produce acetate and CO 2.

The terms "lactate dehydrogenase (lactate dehydrogenase)," D-lactate dehydrogenase (D-lactate dehydrogenase), "ldhA", "hslI", "htpH", "D-LDH", "fermented lactate dehydrogenase (fermentative lactate dehydrogenase)," and "D-specific 2-hydroxy acid dehydrogenase (D-specific 2-hydroxyacid dehydrogenase)," are used interchangeably and refer to enzymes that catalyze the conversion of lactate to pyruvate thereby producing NADH.

As used herein, the term "cell productivity index (cellproductivity index; CPI)" refers to the mass of product produced by a cell divided by the mass of cells produced in culture.

The term "purified" refers to a material that is substantially or essentially free of components that interfere with the activity of a biomolecule. With respect to 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 natural state. Typically, the purified sugar, oligosaccharide, protein or nucleic acid of the invention is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% pure, typically at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure, as measured by band intensity based on silver stained gel or other method for determining purity. Purity or homogeneity can be indicated by a variety of means well known in the art, such as polyacrylamide gel electrophoresis of protein or nucleic acid samples, followed by observation after staining. For some purposes, high resolution and the use of HPLC or similar means for purification would be desirable. For oligosaccharides, purity may be determined using methods such as, but not limited to, thin layer chromatography, gas chromatography, NMR, HPLC, capillary electrophoresis, or mass spectrometry.

The term "identity" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, as measured using a sequence comparison algorithm or by visual inspection, for maximum correspondence comparison and alignment. For sequence comparison, one sequence serves as a reference sequence to which the test sequence is compared. When using the sequence comparison algorithm, the test sequence and the reference sequence are input into the computer, the subsequence coordinates are designated as necessary, and the sequence algorithm program parameters are designated. Next, the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. The percent identity can be calculated globally over the full length sequence of the reference sequence, resulting in an overall percent identity score. Alternatively, the percent identity may be calculated within a portion of the sequence of the reference sequence, resulting in a local percent identity score. The use of the full length of the reference sequence in the local sequence alignment results in a percent identity score overall between the test and reference sequences. The percentage identity may be determined using different algorithms such as, for example, BLAST and PSI-BLAST (Altschul et al, 1990,J Mol Biol 215:3, 403-410; altschul et al, 1997,Nucleic Acids Res 25:17, 3389-402), the Clustal Omega method (Sievers et al, 2011, mol. Syst. Biol. 7:539), the MatGAT method (Campanella et al, 2003,BMC Bioinformatics,4:29), or EMBOSS Needle.

The basic local alignment search tool (Basic Local Alignment Search Tool; BLAST) method of alignment is an algorithm provided by the national center for Biotechnology information (National Center for Biotechnology Information; NCBI) to compare sequences using preset parameters. The program compares nucleotide or protein sequences to a sequence database and calculates statistical significance. The Position-specific iterative basic local alignment search tool (Position-Specific Iterative Basic Local Alignment Search Tool; PSI-BLAST) derives a Position-specific scoring matrix (Position-specific scoring matrix; PSSM) or profile from a plurality of sequence alignments of sequences detected above a given score threshold using protein-protein BLAST (BLASTP). The BLAST method can be used for pairwise or multiple sequence alignment. Pairwise sequence alignment is used to identify regions of similarity, which may indicate functional, structural and/or evolutionary relationships between two biological sequences (proteins or nucleic acids). The web page interface of BLAST can be obtained as follows: https: the// blast.ncbi.nlm.nih.gov/blast.cgi.

Clustal Omega (Clustal W) is a multiple sequence alignment program that uses seeded guide trees and HMM distribution-distribution technology (HMM profile-profile technique) to create alignments between three or more sequences. Which produces biologically interesting multiple sequence alignments of divergent sequences. The web interface of Clustal W may be found at https: obtained under// www.ebi.ac.uk/Tools/msa/clustalo. The preset parameters for the multiple sequence alignment and calculation of the percentage of protein sequence identity using the Clustal W method are: enabling a de-alignment of the input sequence: FALSE; enabling an seed class cluster guide tree: TRUE; enabling seed class cluster iteration: TRUE; (number of combined guide tree/HMM) iterations: presetting (0); maximum guide tree iteration: presetting [ -1]; maximum HMM iteration: presetting [ -1]; a command: and (5) comparison.

The matrix global alignment tool (MatGAT) is a computer application that produces a similarity/identity matrix of DNA or protein sequences without the need for pre-alignment of the data. The program uses Myers and Miller global alignment algorithms to make a series of pairwise alignments, calculate similarities and consistency, and then place the results in a distance matrix. The user may specify what type of alignment matrix (e.g., BLOSUM50, BLOSUM62, and PAM 250) to use for their protein sequence checking.

When considering its entire length, EMBOSS Needle (https:// galaxy-iuc. Giluub. Io/embos-5.0-docs/Needle. Html) uses the Needman-Welch global alignment algorithm to find the best alignment (including gaps) of two sequences. The optimal alignment is ensured by exploring all possible alignments and selecting the optimal alignment by a dynamic programming method. The nidman-man algorithm is a member of a class of algorithms that can calculate the best score and alignment in the order of mn steps (where "n" and "m" are the lengths of two sequences). Gap opening penalty (preset 10.0) is the fraction that is deducted when a gap is created. The preset value assumes you use the EBLOSUM62 matrix for protein sequences. Gap extension (preset 0.5) penalty is added to the standard gap penalty for each base or residue in a gap. This is a way to penalize long gaps.

As used herein, a polypeptide having an amino acid sequence that is at least 80% sequence identical to the full length sequence of a reference polypeptide sequence is understood to be a sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91.50%, 92.00%, 92.50%, 93.00%, 93.50%, 94.00%, 94.50%, 95.00%, 95.50%, 96.00%, 96.50%, 97.00%, 97.50%, 98.00%, 98.50%, 99.00%, 99.50%, 99.60%, 99.70%, 99.80%, 99.90%, 100% sequence identical to the full length of the amino acid sequence of the reference polypeptide sequence. Throughout this application, unless explicitly specified otherwise, a polypeptide (or DNA sequence) comprising/consisting of/having an amino acid sequence (or nucleotide sequence) with at least 80% sequence identity to the full length amino acid sequence (or nucleotide sequence) of a reference polypeptide (or nucleotide sequence), typically indicated by SEQ ID NO or UniProt ID or Genbank No., preferably has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% sequence identity to the full length reference sequence.

For the purposes of the present invention, the percent identity is determined using MatGAT2.01 (Campanella et al, 2003,BMC Bioinformatics 4:29). The following preset parameters for the protein were used: (1) vacancy costs exist: 12 and extension: 2; the matrix used in (2) was BLOSUM65. In a preferred embodiment, sequence identity is calculated based on the full length sequence of the specified SEQ ID NO, i.e., the reference sequence, or a portion thereof. Preferably, part thereof means at least 50%, 60%, 70%, 80%, 90% or 95% of the complete reference sequence.

The term "culture" refers to the medium in which the cells are cultured or fermented, the cells themselves, and the oligosaccharides produced by the cells in the whole culture, i.e., produced internally (intra-cellular) and externally (extracellular) of the cells.

The terms "membrane transporter (membrane transporter protein)" and "membrane protein" are used interchangeably and refer to a protein that is part of a cell membrane or interacts with a cell membrane and controls molecular flow and information across a cell. Thus, membrane proteins are involved in transport, whether they are imported into or exported from cells.

Such membrane transporters may be transporters (porters), P-bond hydrolysis driven transporters, β -barren porins (β -Barrel Porin), helper transporters, putative transporters (putative transport protein), and phosphotransfer driven group translocators (translocators) as defined by the transporter class database (Transporter Classification Database) which is operated and managed by Saier Lab Bioinformatics Group via www.tcdb.org and provides for the functionality and phylogenetic classification of membrane transporters. This transport protein class database details the IUBMB approved comprehensive classification system for membrane transport proteins, known as the transport protein class (Transporter Classification; TC) system, the TCDB class search as described herein is defined based on TCDB. Org published as 2019, 6, 17.

The transporters are the collective names of one-way, one-way and one-way transporters using vector-mediated processes (Saier et al, nucleic Acids Res.44 (2016) D372-D379). It belongs to the class of electrochemical potential driven transporters and is also known as a secondary carrier-type promoter. Membrane transporters are included in this category when utilizing carrier-mediated processes to catalyze one-way transporters when individual species are transported by facilitated diffusion or in membrane potential dependent processes (if solutes are charged); when two or more species are transported in opposite directions during tight coupling, not coupled to a direct form of energy other than chemical osmotic energy, then catalyzing the reverse transport of the protein; and/or when two or more species are transported together in the same direction during close coupling, not coupled to a direct energy form other than the chemical osmotic energy, catalyzing the co-transport of proteins that all belong to the secondary carrier (Forrest et al, biochem. Biophys. Acta 1807 (2011) 167-188). Such systems are typically stereospecific. Solute: reverse transport of solutes is a typical feature of secondary carriers. Dynamic association of transporters and enzymes creates functional membrane transport metabolic groups that introduce the matrix, typically obtained from the extracellular compartment, directly into its cellular metabolism (morae and reiitheier, biochem. Biophys.acta 1818 (2012), 2687-2706). Solutes transported through this transport protein system include, but are not limited to, cations, organic anions, inorganic anions, nucleosides, amino acids, polyols, phosphorylated glycolysis intermediates, osmotic agents, and ferrochelatins.

If the membrane transporter hydrolyzes the diphosphate linkage of inorganic pyrophosphate, ATP or another nucleoside triphosphate to drive active absorption and/or extrusion of a solute or solutes, the membrane transporter is included in the class of P-P-linkage hydrolytically driven transporters (Saier et al, nucleic Acids Res.44 (2016) D372-D379). The membrane transporter may or may not be temporarily phosphorylated, but the matrix is not. Matrices transported via class of P-P-bond hydrolysis driven transport proteins include, but are not limited to, cations, heavy metals, beta-glucans, UDP-glucose, lipopolysaccharides, teichoic acids.

Beta-tubulin membrane transporters form transmembrane pores that generally allow solutes to pass through the membrane in an energy-independent manner. The transmembrane portion of these proteins consists of only β strands, forming a β -barrel (Saier et al, nucleic Acids res.44 (2016) D372-D379). These porin proteins are found in the outer membranes of Gram-negative bacteria (Gram-negative bacteria), granosomes, plastids and potentially acid-fast Gram-positive bacteria. Solutes transported through such β -barrel porins include, but are not limited to, nucleosides, raffinose, glucose, β -glucosides, oligosaccharides.

An auxiliary transport protein is defined as a protein that facilitates transport across one or more biological membranes but does not itself directly participate in transport. Such membrane transporters initially function in conjunction with one or more existing transport systems such as, but not limited to, outer membrane factor (outer membrane factor OMF), polysaccharide (PST) transporter, ATP-binding cassette (ATP-binding cassette ABC) transporter. It may provide functions related to energy coupling for transport, structural role in complex formation, providing biogenic or stabilizing functions or regulatory functions (Saier et al, nucleic Acids res.44 (2016) D372-D379). Examples of auxiliary transport proteins include, but are not limited to, the family of polysaccharide copolyenzymes involved in polysaccharide transport, the family of membrane fusion proteins involved in bacteriocin and chemical toxin transport.

Putative transporters comprise families that will be classified elsewhere when the member's transport function is confirmed, or will be eliminated from the transporter classification system if the proposed transport function is demonstrated to be ineffective. Such families include one or more members for which transport functions have been proposed, but evidence of such functions has not been convinced (Saier et al, nucleic Acids Res.44 (2016) D372-D379). Examples of putative transporters falling into this group as under the TCDB system published on month 6 and 17 of 2019 include, but are not limited to, copper transporters.

Phosphotransfer-driven group translocator proteins are also known as bacterial phosphoenolpyruvate: PEP-dependent phosphoryl transfer driven group translocator of the sugar phosphotransferase system (phosphotransferase system; PTS). The reaction product derived from extracellular sugar is cytoplasmic phosphate sugar. Enzymatic components that catalyze the phosphorylation of sugars are superimposed on the transport process during the close coupling process. PTS systems are involved in many different aspects, including regulation and chemotaxis, biofilm formation and pathogenesis (Lengler, J.mol. Microbiol. Biotechnol.25 (2015) 79-93; saier, J.mol. Microbiol. Biotechnol.25 (2015) 73-78). The family of membrane transporters ascribed within phosphotransferase driven group translocates under the TCDB system as published at 6.17 2019 includes PTS systems associated with transport of glucose-glucoside, fructose-mannitol, lactose-N, N' -diacetyl chitobiose- β -glucoside, glucitol, galactitol, mannose-fructose-sorbose, and ascorbate.

The major facilitator superfamily (major facilitator superfamily; MFS) is the superfamily of membrane transporters that catalyze the one-way transport of proteins, solutes: cations (h+, but hardly na+) co-transport proteins and/or solutes: h+ or solute: the solute transports the protein in reverse. Most are 400-600 aminoacyl residues in length and have 12, 14 or occasionally 24 transmembrane alpha-helical wrenches (TMS), as exemplified by Saier Lab Bioinformatics Group (www.tcdb.org )The operational transporter class database.

As used herein, "SET" or "sugar efflux transporter (Sugar Efflux Transporter)" refers to a membrane protein of the SET family, which is a protein having an InterPRO domain IPR004750 and/or is a protein belonging to the eggnog 4.5 family ENOG410 XTE. Authentication of the InterPro domain may be accomplished by using https: the on-line tools on/www.ebi.ac.uk/interpro/or the independent version of the InterProScan (https:// www.ebi.ac.uk/interpro/download. Html) are done using preset values. Identification of orthologous families in eggnognov 4.5 can be performed using either an online version or a stand-alone version of eggNOG-mappervl (http:// eggnogdb. Embl. De/#/app/home).

The term "ferrochelatin (Siderophore)" as used herein refers to secondary metabolites of various microorganisms, which are primarily iron ion-specific chelators. Such molecules have been classified as catecholates (categorites), hydroxamates (hydroxamates), carboxylates, and mixed types. The chelate ferritin is generally synthesized by the non-ribosomal peptide synthase (nonribosomal peptide synthetase; NRPS) dependent pathway or the NRPS independent pathway (NRPS independent pathway; NIS). The most important precursor in the NRPS-dependent chelate ferritin biosynthetic pathway is chorismate (chorismate). The 2,3-DHBA may be formed from chorismate by a three-step reaction catalyzed by isochorismate synthase, isochorismate and 2, 3-dihydroxybenzoate-2, 3-dehydrogenase. The chelate ferritin may also be formed from salicylate which is formed from isochorismate by an isochorismate pyruvate dissociating enzyme. When ornithine is used as a precursor for ferritin, biosynthesis depends on the hydroxylation of ornithine catalyzed by L-ornithine N5-monooxygenase. In the NIS pathway, an important step in the biosynthesis of ferrochelatin is N (6) -hydroxylysine synthase.

Transport proteins are required to export the ferrochelatin outside the cell. Four superfamilies of membrane proteins were identified in this process to this end: major Facilitator Superfamily (MFS); the Multidrug/oligosaccharyl lipid/polysaccharide invertase superfamily (Multidrug/oligosaccharyl-lipid/Polysaccharide Flippase Superfamily; MOP); resistance, nodular, and cell division superfamily (resistance, nodulation and cell division superfamily; RND); ABC superfamily. Generally, genes involved in the export of ferrochelatin are clustered together with the ferrochelatin biosynthesis genes. The term "transferrin export protein (siderophore exporter)" as used herein refers to such transport proteins required to export transferrin outside the cell.

The ATP-binding cassette (ABC) superfamily contains both the uptake and efflux transport systems, and members of these two groups are typically loosely clustered together. Protein-free phosphorylated ATP hydrolyzes to provide energy for transport. There are tens of families in the ABC superfamily, and the families are generally associated with substrate specificity. Members were classified according to class 3.A.1 as defined by the transporter classification database, which operates by Saier Lab Bioinformatics Group available via www.tcdb.org and provides for the functionality and phylogenetic classification of membrane transporters.

The term "enabled efflux" means the transport activity of introducing solutes on the cytoplasmic membrane and/or cell wall. This transport may be achieved by introducing and/or increasing the expression of the transporter as described in the present invention. The term "enhanced efflux" means an improvement in the transport activity of solutes on the cytoplasmic membrane and/or cell wall. Transport of solutes across the cytoplasmic membrane and/or cell wall can be enhanced by introducing and/or increasing the performance of membrane transporters as described herein. "Expression" of a membrane transporter is defined as "overexpression" of a gene encoding the membrane transporter in the case where the gene is an endogenous gene; or "expression" in the case where the gene encoding the membrane transporter is a heterologous gene that is not present in the wild-type strain or cell.

The term "precursor" as used herein refers to a substance that is absorbed or synthesized by cells to specifically produce oligosaccharides according to the invention. In this sense, the precursor may be a receptor as defined herein, but may also be another substance, metabolite, which is first modified intracellular as part of the biochemical synthesis pathway of the oligosaccharide. Examples of such precursors include receptors as defined herein and/or glucose, galactose, fructose, glycerol, sialic acid, fucose, mannose, maltose, sucrose, lactose, dihydroxyacetone, glucosamine, N-acetyl-glucosamine, mannosamine, N-acetyl-mannosamine, galactosamine, N-acetyl galactosamine, phosphorylated sugars such as, for example (but not limited to), glucose-1-phosphate, galactose-1-phosphate, glucose-6-phosphate, fructose-1, 6-biphosphate, mannose-6-phosphate, mannose-1-phosphate, glycerol-3-phosphate, glyceraldehyde-3-phosphate, dihydroxyacetone-phosphate, glucosamine-6-phosphate, N-acetyl-mannosamine-6-phosphate, N-acetyl glucosamine-1-phosphate, N-acetyl-neuraminic acid-9-phosphate, and/or nucleotide-activated sugars as defined herein such as, for example, UDP-glucose, UDP-galactose, UDP-N-acetyl glucosamine, CMP-sialic acid, GDP-mannose, GDP-4-dehydro-6-deoxy-alpha-D-mannose, GDP-fucose. Optionally, the cell is transformed to comprise at least one nucleic acid sequence encoding a protein selected from the group consisting of: lactose transporter, N-acetylneuraminic acid transporter, fucose transporter, transporter of nucleotide-activated sugar, wherein the transporter internalizes into the media to which the precursor is added for oligosaccharide synthesis.

The term "receptor" as used herein refers to a disaccharide or oligosaccharide that may be modified by a glycosyltransferase. Examples of such receptors include lactose, milk-N-disaccharide (LNB), milk-N-triose, milk-N-tetraose (LNT), milk-N-neotetraose (LNnT), N-acetyl-lactosamine (LacNAc), milk-N-pentasaccharide (LNP), milk-N-neopentasaccharide, para-milk-N-pentasaccharide, para-milk-N-neopentasaccharide, milk-N-neopentasaccharide I (lacto-N-novopentaose I), milk-N-hexasaccharide (lacto-N-hexaose; LNH), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (para-lacto-N-hexaose; pLNH), para-lacto-N-heptaose, para-lacto-N-neoheptaose, para-lacto-N-heptaose, lacto-N-octaose (lacto-N-octaose; LNO), lacto-N-neooctaose, iso-lacto-N-octaose, iso-lacto-N-neooctaose, neolacto-N-neooctaose, para-lacto-N-neooctaose, iso-lacto-N-nonaose, neo-N-nonaose, lacto-N-decaose, iso-lacto-N-decaose, neonatal milk-N-decaose, milk-N-neodecaose; galactosyllactose, lactose having 1, 2, 3, 4, 5 or more N-acetyllactosamine units and/or 1, 2, 3, 4, 5 or more lacto-N-disaccharide units and oligosaccharides containing 1 or more N-acetyllactosamine units and/or 1 or more lacto-N-disaccharide units, or intermediates thereof converted into oligosaccharide, fucosylated and sialylated forms.

Throughout this application, unless explicitly stated otherwise, the features "synthesized", "synthesized" and "synthesized" may be used interchangeably with the features "production", "produced" and "production", respectively.

Detailed Description

According to a first aspect, the present invention provides a metabolically engineered cell for producing a mixture comprising at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different mammalian milk oligosaccharides, i.e. a metabolically engineered cell for producing a mixture comprising at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different mammalian milk oligosaccharides. Herein, a single metabolically engineered cell is provided which is capable of expressing, preferably expressing, a glycosyltransferase that is a sialyltransferase, and is capable of synthesizing nucleotide-sugar-CMP-N-acetylneuraminic acid (CMP-Neu 5 Ac), and which exhibits at least one additional glycosyltransferase and is capable of synthesizing one or more sugar-nucleotides that are donors for the additional glycosyltransferase. Throughout the application, unless explicitly stated otherwise, "genetically modified cell (genetically modified cell)" or "metabolically engineered cell (metabolically engineered cell)" preferably means a cell genetically modified or metabolically engineered, respectively, for the production of the mixture comprising at least three different sialylated oligosaccharides according to the invention. In the context of the present invention, at least three different oligosaccharides of the mixture as disclosed herein are preferably not present in the wild-type precursor cells (progenitors) of the metabolically engineered cell.

According to a second aspect, the present invention provides a method for producing a mixture comprising at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different mammalian milk oligosaccharides. The method comprises the following steps:

i) There is provided a cell, preferably a single cell, capable of expressing, preferably expressing, a glycosyltransferase that is a sialyltransferase, and capable of synthesizing a nucleotide-sugar CMP-N-acetylneuraminic acid (CMP-Neu 5 Ac), and which is capable of expressing at least one additional glycosyltransferase, and capable of synthesizing one or more sugar-nucleotides that are donors for the additional glycosyltransferase,

ii) culturing the cell under conditions allowing expression of the glycosyltransferase and synthesis of the nucleotide-sugar such that the cell produces the mixture of at least three different sialylated oligosaccharides,

iii) Preferably, at least one of the oligosaccharides is isolated from the culture, more preferably, all of the oligosaccharides are isolated from the culture.

The sialyltransferase used in the present invention may be any of the sialyltransferases as defined herein.

Within the scope of the present invention, acceptable conditions are understood to be conditions relating to physical or chemical parameters including, but not limited to, temperature, pH, pressure, osmotic pressure, and product/precursor/acceptor concentrations.

In a particular embodiment, the allowable conditions may include a temperature range of 30+/-20 degrees Celsius, a pH range of 7+/-3.

In the context of the present invention, it is understood that the cell produces the oligosaccharide intracellularly. It will be further understood by those of ordinary skill in the art that some or substantially all of the produced oligosaccharides remain intracellular and/or are excreted extracellularly via passive or active transport.

According to the invention, the method for producing a mixture comprising at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, may utilize non-metabolically engineered cells or may utilize metabolically engineered cells, i.e. metabolically engineered cells to produce the mixture comprising at least three different sialylated oligosaccharides.

According to a preferred embodiment of the method and cell of the invention, the metabolically engineered cell is modified by a gene expression module, wherein the expression from any one of the expression modules is sustained or produced by a natural inducer.

The expression module is also referred to as a transcription unit and comprises a polynucleotide for expressing a recombinant gene comprising a coding gene sequence and appropriate transcriptional and/or translational control signals operably linked to the coding gene. The control signal comprises a promoter sequence, a non-translation region, a ribosome binding site, and a terminator sequence. The expression module may contain elements for expressing one single recombinant gene, but may also contain elements for expressing more recombinant genes or may be organized in an operon structure for the integrated expression of two or more recombinant genes. The polynucleotides may be produced by recombinant DNA techniques using techniques well known in the art. Methods of constructing expression modules well known to those of ordinary skill in the art include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. See, for example, the techniques described in: sambrook et al (2001) Molecular Cloning: a laboratory manual, 3 rd edition, cold Spring Harbor Laboratory Press, CSH, new York or Current Protocols in Molecular Biology, john Wiley and Sons, n.y. (1989 and yearly updates).

According to a preferred embodiment of the invention, the cells are modified by one or more expression modules. The expression module may be integrated in the genome of the cell or may be presented to the cell on a vector. The vector may exist in the form of a plasmid, a liposome, a phage, a liposome or a virus stably transformed/transfected into the metabolically engineered cell. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plastids, phages, transgenes, yeast episomes, insert elements, yeast chromosomal elements, viruses, and vectors derived from combinations thereof, such as vectors derived from plastids and phage genetic elements such as cosmids and phagemids. Such vectors may contain selectable markers such as, but not limited to, antibiotic markers, auxotrophic markers, toxin-antitoxin markers, RNA sense/antisense markers. Expression system constructs may contain control regions that regulate and produce expression. In general, in this regard, any system or vector suitable for maintaining, amplifying or expressing polynucleotides and/or expressing polypeptides in a host may be used for expression. Suitable DNA sequences can be inserted into the expression system by any of a variety of well known and customary techniques, such as those described, for example, in Sambrook et al (see above). For recombinant production, the cells may be genetically engineered to incorporate the expression systems of the invention or portions or polynucleotides thereof. Introduction of polynucleotides into cells can be accomplished by a number of standard laboratory manuals, such as those described in Davis et al, basic Methods in Molecular Biology, (1986) and Sambrook et al, 1989.

As used herein, expression module comprises a polynucleotide for expressing at least one recombinant gene. The recombinant gene is involved in the expression of polypeptides that play a role in the synthesis of the oligosaccharide mixture; or the recombinant gene is associated with other pathways in the host cell that are not involved in the synthesis of the mixture of three or more oligosaccharides. The recombinant gene encodes an endogenous protein having a modified expression or activity, preferably the endogenous protein is overexpressed; or the recombinant gene encodes a heterologous protein that is heterogeneously introduced and expressed in the modified cell, preferably over-expressed. Endogenous proteins may have modified expression in cells that also express heterologous proteins.

According to a preferred embodiment of the invention, each of the expression modules is either sustained or generated by a natural inducer. As used herein, sustained performance is understood to be the performance of genes that are transcribed continuously in an organism. Expression by means of a natural inducer is understood to mean the facultative or regulated expression of a gene that is expressed only under certain natural conditions of the host (e.g. the organism is in delivery or during lactation), as a response to environmental changes (including for example but not limited to hormones, heat, cold, light, oxidative or osmotic stress/signalling), or depending on the location of the developmental stage or the cell cycle of the host cell, including but not limited to apoptosis and autophagy.

The present invention provides different types of cells for producing an oligosaccharide mixture comprising three or more sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide having a single metabolically engineered cell. For example, the invention provides a cell, wherein the cell expresses two different glycosyltransferases and the cell synthesizes one single nucleotide-sugar, which is a donor for both of the expressed glycosyltransferases. The invention also provides a cell, wherein the cell expresses three different glycosyltransferases and the cell synthesizes a single nucleotide-sugar, which is a donor for all three expressed glycosyltransferases. The invention also provides a cell, wherein the cell exhibits two different glycosyltransferases and the cell synthesizes two different nucleotide-sugars, wherein a first nucleotide-sugar is a donor for the first glycosyltransferase and a second nucleotide-sugar is a donor for the second glycosyltransferase. The invention also provides a cell, wherein the cell expresses three or more glycosyltransferases and the cell synthesizes one or more different nucleotide-sugars, which are donors for all of the expressed glycosyltransferases.

In the methods and cells described herein, the cells preferably comprise multiple copies of the same coding DNA sequence encoding a protein. In the context of the present invention, the protein may be a glycosyltransferase, a membrane protein or any other protein as disclosed herein. Throughout this application, the feature "multiple" means at least 2, preferably at least 3, more preferably at least 4, even more preferably at least 5.

In a specific example of a method and/or a cell according to the invention, the mixture comprises at least three, preferably at least four, more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different sialylated oligosaccharides.

In additional embodiments of the methods and/or cells according to the invention, the mixture comprises more than one mammalian milk oligosaccharide.

In another embodiment of the method and/or cell according to the invention, at least one of the sialylated oligosaccharides in the mixture is a Mammalian Milk Oligosaccharide (MMO), preferably a lactose-like mammalian milk oligosaccharide, more preferably a Human Milk Oligosaccharide (HMO). In a preferred embodiment, the cells produce more than two mammalian milk oligosaccharides in the produced mixture of at least three different sialylated oligosaccharides. In an even more preferred embodiment, all of the oligosaccharides in the produced mixture of at least three different sialylated oligosaccharides are mammalian milk oligosaccharides.

In another embodiment of the methods and/or cells of the invention, at least one of the oligosaccharides in the mixture is an antigen of the human ABO blood group system. In a specific example, the cells produce an antigen of the human ABO blood group system in the produced mixture of at least three different sialylated oligosaccharides. In a preferred embodiment, the cells produce more than one antigen of the human ABO blood group system in the produced mixture of at least three different sialylated oligosaccharides. In a more preferred embodiment of the method and/or the cell according to the invention, the mixture comprises at least three different antigens of the human ABO blood group system.

In the context of the present invention, the mixture of at least three different sialylated oligosaccharides according to the invention may further comprise neutral oligosaccharides, such as neutral fucosylated oligosaccharides and neutral non-fucosylated oligosaccharides as described herein. Neutral oligosaccharides are non-sialylated oligosaccharides and therefore do not contain acidic monosaccharide subunits. Neutral oligosaccharides comprise uncharged fucosylated oligosaccharides containing one or more fucose subunits in their glycan structure and uncharged nonfucosylated oligosaccharides lacking any fucose subunits. Such neutral oligosaccharides may be, for example, lactose-based oligosaccharides, LNB-based oligosaccharides, lacNAc-based oligosaccharides, galNAc-Glc-based oligosaccharides, and/or GalNAc-GlcNAc-based oligosaccharides as described herein.

In a preferred embodiment of the method and/or the cell according to the invention, the mixture comprises at least three different sialylated oligosaccharides as disclosed herein, and optionally at least one, preferably at least two, more preferably at least three antigens of the human ABO blood group system. In another preferred embodiment of the method and/or cell according to the invention, the mixture comprises at least three different charged oligosaccharides as disclosed herein and optionally at least one, preferably at least two, more preferably at least three, even more preferably at least four different LNB-based oligosaccharides (said LNB-based oligosaccharides being neutral and/or charged, preferably charged, more preferably sialylated), and optionally at least one, preferably at least two, more preferably at least three, even more preferably at least four different LacNAc-based oligosaccharides (said LacNAc-based oligosaccharides being neutral and/or charged, preferably charged, more preferably sialylated).

In another preferred embodiment of the method and/or the cell according to the invention, the mixture comprises at least three, preferably at least four, more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten sialylated Mammalian Milk Oligosaccharides (MMO), preferably lactose-based mammalian milk oligosaccharides, more preferably Human Milk Oligosaccharides (HMO). Throughout the application, unless explicitly stated otherwise, the feature "a mixture comprising at least three different sialylated oligosaccharides (mixture comprising at least three different sialylated oligosaccharides)" is preferably replaced by "a mixture comprising at least three different sialylated MMOs, preferably lactose-based MMOs, more preferably HMOs", also preferably "a mixture comprising at least four different sialylated MMOs, preferably lactose-based MMOs, preferably HMOs (mixture comprising at least four different sialylated MMOs, preferably lactose-based MMOs, more preferably HMOs)" is replaced by "a mixture comprising at least four different sialylated oligosaccharides (mixture comprising at least four different sialylated oligosaccharides)". In the context of the present invention, a mixture of at least three different sialylated mammalian milk oligosaccharides according to a preferred embodiment of the invention may comprise other oligosaccharides, such as mammalian milk oligosaccharides and/or non-mammalian milk oligosaccharides. The other oligosaccharides may be neutral or charged (preferably sialylated) oligosaccharides. Charged oligosaccharides are oligosaccharide structures containing one or more negatively charged monosaccharide subunits, including N-acetylneuraminic acid (Neu 5 Ac), N-glycolylneuraminic acid (Neu 5 Gc), glucuronates and galacturonates, commonly referred to as sialic acid. Charged oligosaccharides are also known as acid oligosaccharides. Throughout the application, the charged oligosaccharides are preferably sialylated oligosaccharides. Throughout the application, the charged oligosaccharides are more preferably sialylated oligosaccharides, except GM3, which are not sialylated ganglioside oligosaccharides (i.e. 3' sialyllactose). Throughout the application, the charged oligosaccharides are even more preferably sialylated oligosaccharides, which are not sialylated ganglioside oligosaccharides. Sialic acid belongs to the family of derivatives of neuraminic acid (5-amino-3, 5-dideoxy-D-glycero-D-galacto-non-2-ketonic acid). Neu5Gc is a derivative of sialic acid formed by hydroxylation of the N-acetyl group at C5 of Neu5 Ac. The other oligosaccharides may be, for example, lactose-based oligosaccharides, LNB-based oligosaccharides and/or LacNAc-based oligosaccharides as described herein. In a preferred embodiment of the method and/or the cell according to the invention, the mixture comprises at least three different sialylated MMO and optionally at least one, preferably at least two, more preferably at least three antigens of the human ABO blood group system as disclosed herein. In another preferred embodiment of the method and/or cell according to the invention, the mixture comprises at least three different charged MMOs as disclosed herein and optionally at least one, preferably at least two, more preferably at least three, even more preferably at least four different LNB-based oligosaccharides (said LNB-based oligosaccharides being neutral and/or charged, preferably charged, more preferably sialylated), and optionally at least one, preferably at least two, more preferably at least three, even more preferably at least four different LacNAc-based oligosaccharides (said LacNAc-based oligosaccharides being neutral and/or charged, preferably charged, more preferably sialylated). In alternative and/or additional preferred embodiments of the method and/or the cell according to the invention, the mammalian milk oligosaccharides constitute at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% of the oligosaccharide mixture according to the invention. In a more preferred embodiment of the method and/or cell according to the invention, all oligosaccharides in the mixture are MMO, preferably lactose-based MMO, more preferably HMO. As already stated herein, the mixture as disclosed herein is preferably the direct result of metabolizing the cell as described herein.

Throughout this application, unless explicitly stated otherwise, the feature "at least one" is preferably replaced with "one" and likewise the feature "at least two" is preferably replaced with "two", etc.

In an optional embodiment of the method and/or the cell according to the invention, the mixture according to the invention further comprises LacdINAc (i.e. GalNAc-bl, 4-GlCNAc) and/or GalNAc-b1, 4-glucose.

In additional and/or alternative embodiments of the method and/or cell according to the invention, the oligosaccharide mixture comprises at least three different sialylated oligosaccharides that differ in the degree of polymerization (degree of polymerization; DP). The degree of polymerization of an oligosaccharide refers to the number of monosaccharide units present in the oligosaccharide structure. As used herein, the degree of polymerization of an oligosaccharide is three (DP 3) or higher, the latter comprising any of 4 (DP 4), 5 (DP 5), 6 (DP 6) or longer. The oligosaccharide mixture as described herein preferably comprises at least three different sialylated oligosaccharides, wherein all oligosaccharides present in the mixture have different degrees of polymerization from each other. For example, the oligosaccharide mixture consists of three sialylated oligosaccharides, wherein the first oligosaccharide is a trisaccharide having a degree of polymerization of 3 (DP 3), the second oligosaccharide is a tetrasaccharide having a degree of polymerization of 4 (DP 4), and the third oligosaccharide is a pentasaccharide having a degree of polymerization of 5 (DP 5).

In a specific example of a method and/or cell according to the invention, the oligosaccharide mixture is composed of at least one neutral oligosaccharide except for three or more sialylated oligosaccharides.

According to one aspect of the methods and/or cells of the invention, the cells produce a mixture comprising four different sialylated oligosaccharides or more than four different sialylated oligosaccharides. In one specific example, such a mixture comprises at least four different oligosaccharides, wherein three of the oligosaccharides have different degrees of polymerization. In one specific example, all of the oligosaccharides in the mixture have different degrees of polymerization as described herein.

According to the method and/or cell of the invention, at least one of said oligosaccharides of the mixture is fucosylated, sialylated, galactosylated, glycosylated, xylosylated, mannosylated, N-acetylglucosamine-containing, N-acetylneuraminic acid-containing, N-glycolylneuraminic acid-containing, N-acetylgalactosamine-containing, rhamnose-containing, glucuronate-containing, galacturonate-containing and/or N-acetylmannosamine-containing.

According to the method and/or cell of the invention, at least one of said sialylated oligosaccharides of the mixture is fucosylated, sialylated, galactosylated, glycosylated, xylosylated, mannosylated, N-acetyl glucosamine containing, N-acetyl neuraminic acid containing, N-glycolyl neuraminic acid containing, N-acetyl galactosamine containing, rhamnose containing, glucuronate containing, galacturonate containing and/or N-acetyl mannosamine containing.

Preferably, the oligosaccharide mixture comprises at least one fucosylated oligosaccharide as defined herein.

Alternatively or additionally, the mixture of oligosaccharides comprises at least one oligosaccharide having 3 or more monosaccharide subunits connected to each other via glycosidic linkages, wherein at least one of the monosaccharide residues is an N-acetylglucosamine (GlcNAc) residue. The oligosaccharide may contain more than one GlcNAc residue, for example two, three or more than three. The oligosaccharides may be neutral oligosaccharides or charged oligosaccharides, e.g. also comprising sialic acid structures. GlcNAc can be present at the reducing end of oligosaccharides. The GlcNAc may also be present at the non-reducing end of the oligosaccharide. The GlcNAc may also be present in oligosaccharide structures. GlcNAc can be linked to other monosaccharide subunits comprising galactose, fucose, neu5Ac, neu5 Gc.

Alternatively or additionally, the oligosaccharide mixture comprises at least one galactosylated oligosaccharide and contains at least one galactose monosaccharide subunit. The galactosylated oligosaccharide is a sugar structure comprising at least three monosaccharide subunits connected to each other via glycosidic linkages, wherein at least one of the monosaccharide subunits is galactose. The galactosylated oligosaccharide may contain more than one galactose residue, for example two, three or more. The galactosylated oligosaccharides may be neutral oligosaccharides or charged oligosaccharides, e.g. also comprising sialic acid structures. Galactose may be linked to other monosaccharide subunits comprising glucose, glcNAc, fucose.

In an additional and/or alternative preferred embodiment of the method and/or cell according to the invention, the oligosaccharide mixture according to the invention comprises sialylated oligosaccharides in a relative abundance of at least 10%, preferably at least 15%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% in the mixture, most preferably all oligosaccharides in the mixture according to the invention are sialylated. It will be appreciated by those of ordinary skill in the art that if the relative abundance of sialylated oligosaccharides in a mixture is defined, the remainder of the oligosaccharides in the mixture are inevitably neutral oligosaccharides. Throughout this application, unless otherwise indicated, the features "oligosaccharides" and "oligosaccharides" are preferably replaced with "one MMO" and "multiple MMOs", respectively, more preferably with "lactose-based MMO" and "multiple lactose-based MMOs", respectively, even more preferably with "one HMO" and "multiple HMOs", respectively.

In additional and/or alternative embodiments of the method and/or cell according to the invention, the oligosaccharide mixture as described herein further comprises neutral oligosaccharides, wherein the relative abundance of said neutral oligosaccharides in the mixture is preferably less than 90%, more preferably less than 80%, even more preferably less than 70%, even more preferably less than 60%, even more preferably less than 50%, even more preferably less than 40%, even more preferably less than 30%, even more preferably less than 20%, even more preferably less than 10%, most preferably all oligosaccharides in the mixture of the invention are charged (preferably sialylated) oligosaccharides.

Thus, in additional and/or alternative embodiments of the method and/or cell according to the invention, the oligosaccharide mixture as described herein consists of charged (preferably sialylated) and neutral oligosaccharides, wherein the relative abundance of said charged (preferably sialylated) oligosaccharides in the mixture is preferably 5-20%, preferably 5-15%, more preferably 10-15%, even more preferably 12-14%, optimally reflecting the relative abundance of charged oligosaccharides in human breast milk and/or colostrum.

In additional and/or alternative embodiments of the methods and/or cells according to the present invention, the oligosaccharide mixture as described herein comprises fucosylated oligosaccharides having a relative abundance in the mixture of at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, most preferably at least 55%. Preferably, the relative abundance of the fucosylated oligosaccharides in the mixture is less than 90%, preferably less than 80%, more preferably less than 70%, even more preferably less than 60%. Thus, the relative abundance of the fucosylated oligosaccharides in the mixture is preferably 10% to 90%, preferably 20% to 80%, more preferably 30% to 60%, even more preferably 40% to 55%, most preferably reflecting the relative abundance of fucosylated oligosaccharides in human breast milk and/or colostrum.

In additional and/or alternative embodiments of the methods and/or cells according to the present invention, the oligosaccharide mixture as described herein further comprises a neutral oligosaccharide selected from neutral fucosylated oligosaccharides and/or neutral non-fucosylated oligosaccharides. In a preferred embodiment of the method and/or cell according to the invention, the neutral oligosaccharide does not comprise a nonfucosylated oligosaccharide. In an alternative preferred embodiment, the neutral oligosaccharide does not comprise a fucosylated oligosaccharide. In a more preferred embodiment, the neutral oligosaccharides comprise fucosylated oligosaccharides and nonfucosylated oligosaccharides. In an even more preferred embodiment, the relative abundance of fucosylated oligosaccharides in the neutral oligosaccharide portion of the mixture is at least 10%, preferably at least 20%, more preferably at least 30%, most preferably at least 35%. Preferably, the relative abundance of fucosylated oligosaccharides in the neutral oligosaccharide portion of the mixture is 10-60%, preferably 20-60%, more preferably 30-60%, even more preferably 30-50%, optimally reflecting the relative abundance of fucosylated oligosaccharides in the neutral oligosaccharide portion of human breast milk and/or colostrum.

In additional and/or alternative embodiments of the methods and/or cells according to the invention, the relative abundance of each oligosaccharide in the mixtures as described herein is at least 5%, preferably at least 10%.

In the context of the present invention, the oligosaccharide mixture as disclosed herein is preferably the direct result of metabolically engineering the cells as described herein. This means that preferably at least one, more preferably at least two, even more preferably at least three, most preferably all of the oligosaccharides in the mixture according to the invention are not produced by the metabolically engineered cell's wild-type precursor cells.

The names of the oligosaccharides as described herein are according to the names and formulas of the oligosaccharides as disclosed by Urshima et al (Trends in Glycoscience and Glycotechnology,2018, volume 30, stage 72, pages SE51-SE 65) and references therein as disclosed in "Prebiotics and Probiotics in human mill. Origins and Functions of Milk-Borne Oligosaccharides and Bacteria", chapters 2 and 3, edit M.McGuire, M.McGuire, L.Bode, elsevier, academic Press, page 506).

In a more preferred embodiment of the method and/or cell according to the invention, the mixture comprises, consists essentially of, or consists of: at least three, preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different neutral sialylated oligosaccharides, preferably selected from the following:

-lactose-based sialylated oligosaccharides, preferably any of the following: 3 '-sialyllactose, 6' -sialyllactose, 3, 6-sialyllactose, 6 '-disialyllactose, 8, 3-disialyllactose, 3' S-2'FL, 6'S-2'FL, 6'S-3-FL, pentasaccharide LSTD (Neu5Ac.alpha. -2, 3Gal.beta. -1,4GlcNAcβ. -1, 3Gal.beta. -1,4 Glc), sialylated lacto-N-trisaccharide, sialylated lacto-N-tetrasaccharide comprising LSTa and LSTb, sialyl-lacto-N-neotetrasaccharide comprising LSTc and LSTd, monosialyllacto-N-hexasaccharide, disialyllacto-N-hexasaccharide I, monosialyllacto-N-neohexasaccharide I monosialyl emulsion-N-neohexasaccharide II, disialyl emulsion-N-neohexasaccharide, disialyl emulsion-N-tetrasaccharide, disialyl emulsion-N-hexasaccharide II, sialyl emulsion-N-tetrasaccharide a, disialyl emulsion-N-hexasaccharide I, sialyl emulsion-N-tetrasaccharide b, 3 '-sialyl-3-fucosyl lactose (3's-3-FL), disialyl monosialyl emulsion-N-neohexasaccharide, sialyl emulsion-N-fucose II, disialyl emulsion-N-fucose II, monosialyl disialyl emulsion-N-tetrasaccharide, FS Gal-LNnH (Gal-a 1,3-Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), DFSGal-LNnH (Gal-a 1,3- [ Fuca1,2] -Gal-b1,4- [ Fuca1,3] -GlcNAc-bl,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), FS-LNnH (Fuca 1,2-Gal-b1,4-GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), MSDF-pair-LNnH (Neu 5Aca2,3-Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,3-Gal-b1,4- [ Fuc-a1,3] -GlcNAc-b1,3-Gal-b1, 4-Glc), GD3 (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3Gal beta-1, 4 Glc), GT3 (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3Gal beta-1, 4 Glc); GM2GalNAc β -1,4 (Neu 5Ac α -2, 3) Galβ -1,4Glc, GM1 (Galβ -1,3GalNAc β -1,4 (Neu 5Ac α -2, 3) Galβ -1,4 Glc), GD1a (Neu 5Ac α -2,3Galβ -1,3GalNAc β -1,4 (Neu 5Ac α -2, 3) Galβ -1,4 Glc), GT1a (Neu 5Ac α -2,8Neu5Ac α -2,3Galβ -1,3GalNAc β -1,4 (Neu 5Ac α -2, 3) Galβ -1,4 Glc), GD2 (Galβ -1,4 (Neu 5Ac α -2,8Neu5Ac α -2, 3) Galβ -1,4 Glc), GT2 (GalNAc β -1,4 (Neu 5Ac α -2, 8) Gal β -1,4 Glc), GT1a (Neu 5Ac α -2, 3) Gal β -1,3Gal 5Ac α -1,4 Glc. GD1b, (galβ -1,3galnacβ -1,4 (neu5acα -2,8neu5acα 2, 3) galβ -1,4 glc), GT1b (neu5acα -2,3galβ -1,3galnacβ -1,4 (neu5acα -2,8neu5acα 2, 3) galβ -1,4 glc), GQ1b (neu5acα -2,8neu5acα -2,3galβ -1,3galnacβ -1,4 (neu5acα -2,8neu5acα 2, 3) galβ -1,4 glc), GT1c (galβ -1,3galnacβ -1,4 (neu5acα -2,8neu5acα -3) galβ -1,4 glc), GQ1, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha 2, 3) Gal beta-1, 4 Glc), GP1c (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha 2, 3) Galbeta-1, 4 Glc), GD1a (Neu 5Ac alpha-2, 3 Galbeta-1, 3 (Neu 5Ac alpha-2, 6) GalNAc beta-1, 4 Galbeta-1, 4 Glc), fucosyl-GM1 (Fuc alpha-1, 2 Galbeta-1, 3GalNAc beta-1, 4 (Neu 5Ac beta-2, 3) Galbeta-1, 4Glc 2 a, 3Gal beta-1, 35 b-2, 35 b-3- [ 1, 35 b-2, 8-Neu5Aca2,3-Gal-b1,4-Glc, neu5Aca2,8-Neu5Gca2,3-Gal-b1,4-Glc, neu5Aca2,8-Neu5Aca2,3-Gal-b1,4-Glc, neu5Gca2,8-Neu5Gca2,3-Gal-b1,4-Glc, neu5Aca2,3-Gal-b1,3- [ Neu5Aca2,6] -Gal-b1,4-Glc, galb1,6- [ Neu5Aca2,3] -Gal-b1,4-Glc, gal-b1,3- [ Neu5Aca2,6] -Gal-b1,4-Glc, neu5Gca2,3-Gal-b1,3-Gal-b1,4-Glc, neu5, 3-Gal-b1, 3-Gal-5 Aca2,3-Gal-b1, 3-Gal-5 Aca-2, 3-Gal-b1, 6] -Gal-b1,4-Glc, galb1,4-GlcNAc-b16- [ Neu5Ac2,3-Gal-b1,3] -Gal-b1,4-Glc, neu5Ac2,6-Gal-b1,4-GlcNAc-b1,6- [ Galb1,3] -Gal-b1,4-Glc, neu5Gca2,3-Gal-b1,4-Glc, neu5Gca2,6-Gal-b1,4-Glc, galMSLNnH (Gala 1,3-Gal-b1,4-GlcNAc-b1,6- [ Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), F-LSTa, F-LSTb, F-LSTc, FS-LNH I, FS-LNH II, FS-LNH III, FS-LNH IV, FS-LNnH I, FS-LNnH II, FS-p-LNnH I, FS-p-LNnH II, DFS-LNH I, DFS-LNH III, DFS-LNH IV, DFS-LNnH, DF-p-LNH sulfate I, DF-p-LNH sulfate II TF-pair-LNH sulfate, neu5GcLNnT, GM2 tetraose, SLNOa, S-LNH I, S-LNH II, S-LNnH I, S-LNnH II, S-pair-LNnH, S-LNH, S-LNnH, S-LNH, S-LN DS-LNH II, S-LNO, FS-LNO I, FS-LNO II, FS-iso-LNO, DFS-iso-LNO I, DFS-iso-LNO II, DFS-LNO I, DFS-NO II, DFS-LNO III, TFS-LNO, TFS-iso-LNO, FDS-LNT I, FDS-LNT II, FDS-LNH I, FDS-LNH II, FDS-LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-neo-LNP I, neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6- [ GlcNAc-b1,3] -Gal-b1,4-Glc, neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc, neu5Ac-a2,6- [ GlcNAc-b1,3] -Gal-b1,4-Glc, gal-b1,3- [ Neu5Gc-a2,6] -Gal-b1,4-Glc, more preferably one or more of the following: 3 '-sialyllactose, 6' -sialyllactose, 3, 6-sialyllactose, 6 '-disialyllactose, 8, 3-disialyllactose, 3' S-2'FL, 6'S-2'FL, 6'S-3-FL, pentasaccharide LSTD (Neu5Ac.alpha. -2, 3Gal.beta. -1,4GlcNAcβ. -1, 3Gal.beta. -1,4 Glc), sialylated lacto-N-trisaccharide, sialylated lacto-N-tetrasaccharide comprising LSTa and LSTb, sialyl-lacto-N-neotetrasaccharide comprising LSTc and LSTd, monosialyllacto-N-hexasaccharide, disialyllacto-N-hexasaccharide I, monosialyllacto-N-neohexasaccharide I monosialyl emulsion-N-neohexasaccharide II, disialyl emulsion-N-neohexasaccharide, disialyl emulsion-N-tetrasaccharide, disialyl emulsion-N-hexasaccharide II, sialyl emulsion-N-tetrasaccharide a, disialyl emulsion-N-hexasaccharide I, sialyl emulsion-N-tetrasaccharide b, 3 '-sialyl-3-fucosyl lactose (3's-3-FL), disialyl monosialyl emulsion-N-neohexasaccharide, sialyl emulsion-N-fucose II, disialyl emulsion-N-fucose II, monosialyl disialyl emulsion-N-tetrasaccharide, FS Gal-LNnH (Gal-a 1,3-Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), DFSGal-LNnH (Gal-a 1,3- [ Fuca1,2] -Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), FS-LNnH (Fuca 1,2-Gal-b1,4-GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), MSDF-pair-LNnH (Neu 5Aca2,3-Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,3-Gal-b1,4- [ Fuc-a1,3] -GlcNAc-b1,3-Gal-b1, 4-Glc), optimally one or more of the following: 3' -sialyllactose, 6' -sialyllactose, 3, 6-sialyllactose, 6' -sialyllactose, 8, 3-disialyllactose, 3's-2' FL, 6'S-2' FL, 6'S-3-FL, pentasaccharide LSTD (neu5ac alpha-2, 3galbeta-1, 4 glcnacbeta-1, 3galbeta-1, 4 glc), sialylated milk-N-trisaccharide, sialylated milk-N-tetrasaccharide, sialylated milk-N-neotetraose, monosialyllacto-N-hexasaccharide, disialyllacto-N-hexasaccharide I, monosialyllacto-N-neohexasaccharide II, disialyllacto-N-neohexasaccharide, disialyllacto-N-tetrasaccharide, disialyllacto-N-hexasaccharide I, sialyllacto-N-tetrasaccharide b, 3' -fucose-N-tetrasialyllacto-N-tetrasaccharide I, sialyllacto-N-tetrasaccharide N-hexasaccharide I, sialyllacto-N-hexasaccharide I, monosialyllacto-N-hexasaccharide I, disialyllacto-3-N-hexasaccharide I; and/or

The LNB-based sialylated oligosaccharides preferably are any of the following: 3 '-sialyl lacto-N-disaccharide (3' slnb), 6 '-sialyl lacto-N-disaccharide (6' slnb), monofucosyl Shan Tuoye acid lacto-N-octasaccharide (sialyl Lea); and/or

LacNAc-based sialylated oligosaccharides, preferably any of the following: 3 '-sialyllactosamine (3' SLacNAc), 6 '-sialyllactosamine (6' SLacNAc), sialyl Lex, neu5Gc-a2,3-Gal-b1,4-GlcNAc.

Preferred mixtures in this case of the invention comprise, consist of or consist essentially of at least three, preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different sialylated oligosaccharides selected from the list comprising: 3 '-sialyllactose, 6' -sialyllactose, 3, 6-sialyllactose, 6 '-disialyllactose, 8, 3-disialyllactose, 3' S-2'FL, 6'S-2'FL, 6'S-3-FL, pentasaccharide LSTD (Neu5Ac.alpha. -2, 3Gal.beta. -1,4GlcNAcβ. -1, 3Gal.beta. -1,4 Glc), sialylated lacto-N-trisaccharide, sialylated lacto-N-tetrasaccharide comprising LSTa and LSTb, sialyl-lacto-N-neotetrasaccharide comprising LSTc and LSTd, monosialyllacto-N-hexasaccharide, disialyllacto-N-hexasaccharide I, monosialyllacto-N-neohexasaccharide I monosialyl emulsion-N-neohexasaccharide II, disialyl emulsion-N-neohexasaccharide, disialyl emulsion-N-tetrasaccharide, disialyl emulsion-N-hexasaccharide II, sialyl emulsion-N-tetrasaccharide a, disialyl emulsion-N-hexasaccharide I, sialyl emulsion-N-tetrasaccharide b, 3 '-sialyl-3-fucosyl lactose (3's-3-FL), disialyl monosialyl emulsion-N-neohexasaccharide, sialyl emulsion-N-fucose II, disialyl emulsion-N-fucose II, monosialyl disialyl emulsion-N-tetrasaccharide, FS Gal-LNnH (Gal-a 1,3-Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), DFSGal-LNnH (Gal-a 1,3- [ Fuca1,2] -Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), FS-LNnH (Fuca 1,2-Gal-b1,4-GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), MSDF-pair-LNnH (Neu 5Aca2,3-Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,3-Gal-b1,4- [ Fuc-a1,3] -GlcNAc-b1,3-Gal-b1, 4-Glc), GD3 (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3Gal beta-1, 4 Glc), GT3 (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3Gal beta-1, 4 Glc); GM2GalNAc β -1,4 (Neu 5Ac α -2, 3) Galβ -1,4Glc, GM1 (Galβ -1,3GalNAc β -1,4 (Neu 5Ac α -2, 3) Galβ -l,4 Glc), GD1a (Neu 5Ac α -2,3Galβ -1,3GalNAc β -1,4 (Neu 5Ac α -2, 3) Galβ -1,4 Glc), GT1a (Neu 5Ac α -2,8Neu5Ac α -2,3Galβ -1,3GalNAc β -1,4 (Neu 5Ac α -2, 3) Galβ -1,4 Glc), GD2 (Galβ -1,4 (Neu 5Ac α -2,8Neu5Ac α -2, 3) Galβ -1,4 Glc), GT2 (GalNAc β -1,4 (Neu 5Ac α -2, 8) Gal β -1,4 Glc), GT1a (Neu 5Ac α -2,8 NAc-1, 4 Glc), (Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha 2, 3) Gal beta-1, 4 Glc), GT1b (Neu 5Ac alpha-2, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha 2, 3) Galbeta-1, 4 Glc), GQ1b (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha 2, 3) Galbeta-1, 4 Glc), GT1c (GalNAc beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3) Gal beta-1, 4 Glc), GQ1c (Neu 5Ac alpha-2, 3) Gal beta-1, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha 2, 3) Gal beta-1, 4 Glc), GP1c (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha 2, 3) Galbeta-1, 4 Glc), GD1a (Neu 5Ac alpha-2, 3 Galbeta-1, 3 (Neu 5Ac alpha-2, 6) GalNAc beta-1, 4 Galbeta-1, 4 Glc), fucosyl-GM1 (Fuc alpha-1, 2 Galbeta-1, 3GalNAc beta-1, 4 (Neu 5Ac beta-2, 3) Galbeta-1, 4Glc 2 a, 3Gal beta-1, 35 b-2, 35 b-3- [ 1, 35 b-2, 8-Neu5Aca2,3-Gal-b1,4-Glc, neu5Aca2,8-Neu5Gca2,3-Gal-b1,4-Glc, neu5Aca2,8-Neu5Aca2,3-Gal-b1,4-Glc, neu5Gca2,8-Neu5Gca2,3-Gal-b1,4-Glc, neu5Aca2,3-Gal-b1,3- [ Neu5Aca2,6] -Gal-b1,4-Glc, galb1,6- [ Neu5Aca2,3] -Gal-b1,4-Glc, gal-b1,3- [ Neu5Aca2,6] -Gal-b1,4-Glc, neu5Gca2,3-Gal-b1,3-Gal-b1,4-Glc, neu5, 3-Gal-b1, 3-Gal-5 Aca2,3-Gal-b1, 3-Gal-5 Aca-2, 3-Gal-b1, 6] -Gal-b1,4-Glc, galb1,4-GlcNAc-b16- [ Neu5Ac2,3-Gal-b1,3] -Gal-b1,4-Glc, neu5Ac2,6-Gal-b1,4-GlcNAc-b1,6- [ Galb1,3] -Gal-b1,4-Glc, neu5Gca2,3-Gal-b1,4-Glc, neu5Gca2,6-Gal-b1,4-Glc, galMSLNnH (Gala 1,3-Gal-b1,4-GlcNAc-b1,6- [ Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), F-LSTa, F-LSTb, F-LSTc, FS-LNH I, FS-LNH II, FS-LNH III, FS-LNH IV, FS-LNnH I, FS-LNnH II, FS-p-LNnH I, FS-p-LNnH II, DFS-LNH I, DFS-LNH III, DFS-LNH IV, DFS-LNnH, DF-p-LNH sulfate I, DF-p-LNH sulfate II TF-pair-LNH sulfate, neu5GcLNnT, GM2 tetraose, SLNOa, S-LNH I, S-LNH II, S-LNnH I, S-LNnH II, S-pair-LNnH, S-LNH, S-LNnH, S-LNH, S-LN DS-LNH II, S-LNO, FS-LNO I, FS-LNO II, FS-iso-LNO, DFS-iso-LNO I, DFS-iso-LNO II, DFS-LNO I, DFS-NO II, DFS-LNO III, TFS-LNO, TFS-iso-LNO, FDS-LNT I, FDS-LNT II, FDS-LNH I, FDS-LNH II, FDS-LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-neo-LNP I, neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6- [ GlcNAc-b1,3] -Gal-b1,4-Glc, neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc, neu5Ac-a2,6- [ GlcNAc-b1,3] -Gal-b1,4-Glc, gal-b1,3 '-sialyl-N-disaccharide (3' SLNB), 6 '-sialyl-N-disaccharide (6' SLNB), sialyl-L-5 Ac-b 2,3 '-sialyl-N-5' sialyl (8 '-sialyl) and 8' -sialyl-N-b 1, 8 '-sialyl-N-sialyl (8' -sialyl) amino acid group, 8 '-sialyl, and 8' -sialyl-N-amino acid.

Preferred mixtures in this context of the invention comprise, consist of or consist essentially of at least three, preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different sialylated oligosaccharides selected from the list comprising: 3 '-sialyllactose, 6' -sialyllactose, 3, 6-sialyllactose, 6 '-disialyllactose, 8, 3-disialyllactose, 3' S-2'FL, 6'S-2'FL, 6'S-3-FL, pentasaccharide LSTD (Neu5Ac.alpha. -2, 3Gal.beta. -1,4GlcNAcβ. -1, 3Gal.beta. -1,4 Glc), sialylated lacto-N-trisaccharide, sialylated lacto-N-tetrasaccharide comprising LSTa and LSTb, sialyl-lacto-N-neotetrasaccharide comprising LSTc and LSTd, monosialyllacto-N-hexasaccharide, disialyllacto-N-hexasaccharide I, monosialyllacto-N-neohexasaccharide I monosialyl emulsion-N-neohexasaccharide II, disialyl emulsion-N-neohexasaccharide, disialyl emulsion-N-tetrasaccharide, disialyl emulsion-N-hexasaccharide II, sialyl emulsion-N-tetrasaccharide a, disialyl emulsion-N-hexasaccharide I, sialyl emulsion-N-tetrasaccharide b, 3 '-sialyl-3-fucosyl lactose (3's-3-FL), disialyl monosialyl emulsion-N-neohexasaccharide, sialyl emulsion-N-fucose II, disialyl emulsion-N-fucose II, monosialyl disialyl emulsion-N-tetrasaccharide, FS Gal-LNnH (Gal-a 1,3-Gal-b1,4- [ Fucal,3] -GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), DFSGal-LNnH (Gal-a 1,3- [ Fuca1,2] -Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), FS-LNnH (Fuca 1,2-Gal-b1,4-GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), MSDF-p-LNnH (Neu 5Aca2,3-Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,3-Gal-b1,4- [ Fuc-a1,3] -GlcNAc-b1,3-Gal-b1, 4-Glc), 3 '-sialyl lacto-N-disaccharide (3' SLNB), 6 '-sialyl lacto-N-disaccharide (6' SLNB), monofucosyl Shan Tuoye acid lacto-N-octasaccharide (sialyl Lea), 3 '-sialyl lactosamine (3' SLacNAc), 6 '-sialyl lactosamine (6' SLacNAc), sialyl Lex and Neu5Gc-a2,3-Gal-b1,4-GlcNAc.

An example of the preferred mixture is a mixture comprising at least three sialylated oligosaccharides selected from the list comprising: 3 '-sialyllactose, 6' -sialyllactose, 3, 6-sialyllactose, 6 '-disialyllactose, 8, 3-disialyllactose, 3' S-2'FL, 6'S-2'FL, 6'S-3-FL, 3 '-sialyl-3-fucosyllactose (3' S-3-FL), sialyl-lacto-N-trisaccharide, sialyl-lacto-N-tetrasaccharide comprising LSTa and LSTb, sialyl-lacto-N-neotetraose comprising LSTc and LSTd, 3 '-sialyl-lacto-N-disaccharide (3' SLNB), 6 '-sialyl-lacto-N-disaccharide (6' SLNB), monofucosyl Shan Tuoye-lacto-N-octasaccharide (sialyl-Lea), 3 '-sialyllactosamine (3' SLacNAc), 6 '-sialyllactosamine (6' SLacNAc) and sialyl Lex.

Exemplary mixtures are described in the specific examples section in this context of the invention.

Optionally, at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different neutral fucosylated oligosaccharides are present in the mixture according to the invention. Preferably, the neutral fucosylated oligosaccharide is preferably selected from:

Lactose-based neutral fucosylated oligosaccharides, preferably any of the following: 2 '-fucosyllactose (2' FL), 3-fucosyllactose (3-FL), 4-fucosyllactose (4 FL), 6-fucosyllactose (6 FL), dif-fucosyllactose (diFL or LDFT), fuc-a1,2-Gal-b1,3-GlcNAc-b1,3- [ Fuc-a1,3- [ Gal-b1,4] -GlcNAc-b1,6] -Gal-b1,4-Glc, milk-N-fuco-pentasaccharide I (LNFP-I; fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc), galNAc-LNFP-I (GalNAc-a 1,3- (Fuc-a 1, 2) -Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-fucopentaose II (LNFP-II; gal-b1,3- (Fuc-a 1, 4) -GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-fucopentaose III (LNFP III; gal-b1,4- (Fuc-a 1, 3) -GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-fucopentaose V (FP-V; gal-b1,3-GlcNAc-b1,3-Gal-b1,4- (c-a, 3) -Fuc-b 1 milk-N-fucopentaose VI (LNFP-VI; gal-b1,4-GlcNAc-b1,3-Gal-b1,4- (Fuc-a 1, 3) -Glc), milk-N-neofucopentaose I (LNnFP I; fuc-a1,2-Gal-b1,4-GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-disaccharide hexasaccharide I (LNDFH I; fuc-a1,2-Gal-b1,3- [ Fuc-a1,4] -GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-disaccharide hexasaccharide II (LNDFH II; fuc-a1,4- (Gal-b 1, 3) -Glc-b 1,3-Gal-b1,4- (c-a 1, 3) -Glc), monosaccharyl hexasaccharide, disaccharide-N-hexasaccharide III, gal-N-hexasaccharide, gal-b1, 3-Gal-hexasaccharide, gal-N-hexasaccharide, gal-b1, 3-Gal-N-hexasaccharide, gal-N-hexasaccharide II (LNDFH 1, 3-b 1,4- (Gal-b 1, 3-Glc-N-b 1,3-Gal-b1, 3-d) or more preferably Gal-N-6-5-fucose: 2 '-fucosyllactose (2' FL), 3-fucosyllactose (3-FL), 4-fucosyllactose (4 FL), 6-fucosyllactose (6 FL), dif-fucosyllactose (diFL or LDFT), fuc-a1,2-Gal-b1,3-GlcNAc-b1,3- [ Fuc-a1,3- [ Gal-b1,4] -GlcNAc-b1,6] -Gal-b1,4-Glc, milk-N-fuco-pentasaccharide I (LNFP-I; fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc), galNAc-LNFP-I (GalNAc-a 1,3- (Fuc-a 1, 2) -Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-fucopentaose II (LNFP-II; gal-b1,3- (Fuc-a 1, 4) -GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-fucopentaose III (LNFP III; gal-b1,4- (Fuc-a 1, 3) -GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-fucopentaose V (FP-V; gal-b1,3-GlcNAc-b1,3-Gal-b1,4- (c-a, 3) -Fuc-b 1 milk-N-fucopentaose VI (LNFP-VI; gal-b1,4-GlcNAc-b1,3-Gal-b1,4- (Fuc-a 1, 3) -Glc), most preferably one or more of the following: 2 '-fucosyllactose (2' FL), 3-fucosyllactose (3-FL), 4-fucosyllactose (4 FL), 6-fucosyllactose (6 FL), dif-or LDFT), gal-LNFP-III, LNDFH III, F-LNH I F-LNH II, F-LNH III, F-LNnH II, F-LNnH I, F-p-LNH II, F-p-LNnH, DF-LNH II, DF-LNH I, DF-LNnH, DF-p-LNH II, DF-p-LNH III, DF-p-LNnH, TF-LNH I TF-LNH II, TF-pair-LNH I, TF-pair-LNH II, TF-pair-LNnH, F-LNO I, F-LNO II, F-LNO III F-LNnO, F-LNnO II, F-iso-LNO, F-iso-LNnO I, F-neo-LNnO, F-p-LNO, DF-iso-LNnO DF-LNO I, DF-LNO II, DF-LNO III, DF-LNnO I, DF-LNnO II, DF-LNnO III, DF-iso-LNO I, DF-iso-LNO II, DF-iso-LNO III, DF-iso-LNO IV, DF-iso-LNO V, DF-iso-LNO VI, DF-iso-LNO VII, DF-pair-LNnO, TF-LNO I, TF-LNO II, TF-LNnO, TF-iso-LNO I, TF-iso-LNO II, TF-iso-LNO III, TF-iso-LNO IV, TF-iso-LNnO, tetra-F-iso-LNO, tetra-F-pair-LNO, penta-F-iso-LNO, F-LND I, F-LND II, DF-LND I, DF-LND II, DF-LND III, DF-LND IV, DF-LND V, DF-LND VI, triF-LND I, triF-LND II, triF-LND III, triF-LND VI, triF-LND VII, tetra-LND I, tetra-F-D II, tetra-LND III, F-LND I, F-LND II, DF-LND, gal-LND 1 (Gal-LND, gal-1), 3-Gal-B1,4-GlcNAc-B1,6- [ Gal-a1,3-Gal-B1,4- [ Fuc-a1,3] -GlcNAc-B1,3] -Gal-B1, 4-Glc), 3-F-cytarose, B-tetraose, B-pentasaccharide, B-hexasaccharide, B-heptasaccharide, DF DGal-LNnT (Gal-a 1,3-Gal-B1,4- [ Fuc-a1,3] -GlcNAc-B1,3-Gal-B1,4- [ Fuc-a1, 3-Glc), TF DGal-LNnH a, TF DGal-LNnH b, DFGal-pair-LNnH; and/or

LNB-based neutral fucosylated oligosaccharides, preferably any of the following: 2' FLNB, 4-FLNB, leb (Fuc-a 1,2-Gal-b1,3- (Fuc-a 1, 4) -GlcNAc); and/or

LacNAc-based neutral fucosylated oligosaccharides, preferably any of the following: 2' FLacNAc, 3-FLacNAc, ley (Fuc-a 1,2-Gal-b1,4- (Fuc-a 1, 3) -GlcNAc).

Preferred mixtures in this context of the invention comprise, consist essentially of, or consist of a mixture of at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different neutral fucosylated oligosaccharides selected from the list comprising: 2 '-fucosyllactose (2' FL), 3-fucosyllactose (3-FL), 4-fucosyllactose (4 FL), 6-fucosyllactose (6 FL), dif-fucosyllactose (diFL or LDFT), fuc-a1,2-Gal-b1,3-GlcNAc-b1,3- [ Fuc-a1,3- [ Gal-b1,4] -GlcNAc-b1,6] -Gal-b1,4-Glc, milk-N-fuco-pentasaccharide I (LNFP-I; fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc), galNAc-LNFP-I (GalNAc-a 1,3- (Fuc-a 1, 2) -Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-fucopentaose II (LNFP-II; gal-b1,3- (Fuc-a 1, 4) -GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-fucopentaose III (LNFP III; gal-b1,4- (Fuc-a 1, 3) -GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-fucopentaose V (FP-V; gal-b1,3-GlcNAc-b1,3-Gal-b1,4- (c-a, 3) -Fuc-b 1 milk-N-fucopentaose VI (LNFP-VI; gal-b1,4-GlcNAc-b1,3-Gal-b1,4- (Fuc-a 1, 3) -Glc), milk-N-neofucopyranose I (LNnFP I; fuc-a1,2-Gal-b1,4-GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-disaccharide hexasaccharide I (LNDFH I; fuc-a1,2-Gal-b1,3- [ Fuc-a1,4] -GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-disaccharide hexasaccharide II (LNDFH II; fuc-a1,4- (Gal-b 1, 3) -GlcNAc-b1,3-Gal-b1,4- (Fuc-a 1, 3) -Glc), monocyclopedic milk-N-hexasaccharide III, difucosyl milk-N-hexasaccharide, difructose-milk-N-neohexasaccharide, LNnDFH (Gal-b 1,4- (c-a 1, 3) -Glc-b 1,3-Gal-b1,4- (Gal-b 1, 3) -Glc-b 1,4- (Gal-a 1, 3) -Glc-b 1,4- (LNDFH-a 1, 3) -Glc-hexasaccharide III, LNF 1, LNF-III, LNF-L-1, LNF-L-B1, 4-GLC-hexasaccharide III F-LNnH I, F-p-LNH II, F-p-LNnH, DF-LNH II, DF-LNH I, DF-LNnH, DF-p-LNH II, DF-p-LNH III, DF-p-LNnH, TF-LNH I, TF-LNH II, TF-p-LNH I, TF-p-LNH II, F-LNO I, F-LNO II, F-LNO III, F-LNnO II, F-iso-LNO I, F-neogenesis-LNnO, F-p-LNO, F-LNO DF-iso-LNnO, DF-LNO I, DF-LNO II, DF-LNO III, DF-LNnO I, DF-LNnO II, DF-LNnO III, DF-iso-LNO I, DF-iso-LNO II, DF-iso-LNO III, DF-iso-LNO IV, DF-iso-LNO V, DF-iso-LNO VI, DF-iso-LNO VII, DF-p-LNnO, TF-LNO I, TF-LNO II, TF-LNnO, TF-iso-LNO I, TF-iso-LNO II, TF-iso-LNO III, TF-iso-LNO IV, TF-iso-LNnO, four-F-iso-LNO, four-F-p-LNO, five-F-iso-LNO, F-LND I, F-LND II, DF-LND I, DF-LND II, DF-LND III, DF-LND IV, DF-LND V, DF-LND VI, triF-LND I, triF-LND II, triF-LND III, triF-LND IV, triF-LND V, triF-LND VI, triF-LND VII, tetra F-LND I, tetra F-LND II, tetra F-LND III, F-LNnD I, F-LNnD II, DF-LNnD, DF-neo-LND, DF-Gal-LNnH (Gal-a 1,3-Gal-B1, 4-NAc-B1, 6- [ Gal-a1,3-Gal-B1,4- [ Fuc-a1,3] -GlcNAc-B1,3] -Gal-B1, 4-Glc), 3-F-isochrotriose, B-tetrasugar, B-pentasugar, B-hexasugar, B-heptasugar, DF DGal-LNnT (Gal-a 1,3-Gal-B1,4- [ Fuc-a1,3] -GlcNAc-B1,3-Gal-B1,4- [ Fuc-a1,3] -Glc), TF DGal-LNnH a, TF DGal-LNnH B, DFGal-pair-LNnH, 2'FLNB, 4-FLNB, leb (Fuc-a 1,2-Gal-B1,3- (Fuc-a 1, 4) -GlcNAc), 2' FLacNAc, 3-FLacNAc and Ley (Fuc-a 1,2-Gal-B1,4- (c-a 1), 3) -GlcNAc.

Exemplary mixtures are described in the specific examples section in this context of the invention.

Optionally, at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different neutral fucosylated oligosaccharides are present in the mixture according to the invention. Preferably, the neutral nonfucosylated oligosaccharides are preferably selected from the group consisting of:

lactose-based neutral nonfucosylated oligosaccharides, preferably any of the following: milk-N-trisaccharide II (LN 3), milk-N-neotetraose (LNnT), milk-N-tetraose (LNT), p-milk-N-neopentasaccharide, p-milk-N-pentasaccharide, p-milk-N-neohexasaccharide, p-milk-N-hexasaccharide, β - (1, 3) galactosyl-p-milk-N-neopentasaccharide, β - (1, 4) galactosyl-p-milk-N-pentasaccharide, gal-a1,4-Gal-b1,4-Glc (Gal-a 1, 4-lactose), β3 '-galactosyl lactose, β6' -galactosyl lactose, gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, gal-a1,4-Gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, gal-b1,3-Galb1,3-Gal-b1,4-Glc, gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1, 4-Glc, galNAc-b1,3-Gal-b1,4-Glc (GalNAc-b 1, 3-Lactuse), gal-b1,3-GalNAc-b1, 3-Lactose, galNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc (globo-N-tetraose), gal-b1,3-GalNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc, galNAc-b1,3-LNT, gal-b1,3-GalNAc-b1,3-LNT, neo-LNT (GlcNAc-b 1,6- [ Gal-b1,3] -Gal-b1, 4-Glc), gal-neo-LNP I (Gal-b 1, 4-NAc-b 1), 6- [ Gal-b1,3-Gal-b1,3] -Gal-b1, 4-Glc), gal-neo-LNP II (Gal-b 1,4-GlcNAc-b1,6- [ Gal-b1,3] -Gal-b1,3-Gal-b1, 4-Glc), gal-neo-LNP III (Gal-b 1,3-Gal-b1,4-GlcNAc-b1,6- [ Gal-b1,3] -Gal-b1, 4-Glc), neo-LNO, galNAc-b1,3-LNnT, gal-b1,3-GalNAc-b1,3-LNnT, LNH, LNnH, iso-LNO, neo-LNnO, LND, iso-LND, galNAc-a1,3-Gal-b1,4-Glc, neo-LNP I, iso-LNT, DGalLNnH, galili pentasaccharide (galipipentasaccharide), more preferably one or more of the following: milk-N-trisaccharide II (LN 3), milk-N-neotetraose (LNnT), milk-N-tetraose (LNT), p-milk-N-neopentasaccharide, p-milk-N-pentasaccharide, p-milk-N-neohexasaccharide, p-milk-N-hexasaccharide, β - (1, 3) galactosyl-p-milk-N-neopentasaccharide, β - (1, 4) galactosyl-p-milk-N-pentasaccharide, gal-a1,4-Gal-b1,4-Glc (Gal-a 1, 4-Lactose), β3 '-galactosyl Lactose, β6' -galactosyl Lactose, galNAc-b1,3-Lactose, balloon-N-tetrasaccharide, optimally one or more of the following: milk-N-trisaccharide II (LN 3), milk-N-neotetraose (LNnT), milk-N-tetraose (LNT), p-milk-N-neopentaose, p-milk-N-pentaose, p-milk-N-neohexaose, p-milk-N-hexaose; and/or

Neutral nonfucosylated oligosaccharides based on LNB; and/or

Neutral nonfucosylated oligosaccharides based on LacNAc, such as, for example, lacDiNAc and poly-LacNAc.

Preferred mixtures in this context of the invention comprise, consist essentially of, or consist of a mixture of at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different neutral nonfucosylated oligosaccharides selected from the list comprising: milk-N-trisaccharide II (LN 3), milk-N-neotetraose (LNnT), milk-N-tetraose (LNT), p-milk-N-neopentasaccharide, p-milk-N-pentasaccharide, p-milk-N-neohexasaccharide, p-milk-N-hexasaccharide, β - (1, 3) galactosyl-p-milk-N-neopentasaccharide, β - (1, 4) galactosyl-p-milk-N-pentasaccharide, gal-a1,4-Gal-b1,4-Glc (Gal-a 1, 4-lactose), β3 '-galactosyl lactose, β6' -galactosyl lactose, gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, gal-a1,4-Gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, gal-b1,3-Galb1,3-Gal-b1,4-Glc, gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1, 4-Glc, galNAc-b1,3-Gal-b1,4-Glc (GalNAc-b 1, 3-Lactuse), gal-b1,3-GalNAc-b1, 3-Lactose, galNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc (ball-N-tetrasaccharide), gal-b1,3-GalNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc, galNAc-b1,3-LNT, gal-bl,3-GalNAc-b1,3-LNT, neo-LNT (GlcNAc-b 1,6- [ Gal-b1,3] -Gal-b1, 4-Glc), gal-neo-LNP I (Gal-b 1,4-GlcNAc-b1,6- [ Gal-b1,3-Gal-b1,3] -Gal-b1, 4-Glc), gal-neo-LNP II (Gal-b 1,4-GlcNAc-b1,6- [ Gal-b1,3] -Gal-b1,3-Gal-b1, 4-Glc), gal-neo-LNP III (Gal-b 1,3-Gal-b1,4-GlcNAc-b1,6- [ Gal-b1,3] -Gal-b1, 4-Glc), neo-LNO, galNAc-b1,3-LNnT, gal-b1,3-GalNAc-b1,3-LNnT, LNH, LNnH, iso-LNO, neo-LNnO, LND, iso-LND, galNAc-a1,3-Gal-b1,4-Glc, neo-LNP I, iso-LNT, DGalLNnH, galili pentasaccharide, lacDiNAc and poly-LacNAc.

Exemplary mixtures are described in the specific examples section in this context of the invention.

In an even more preferred embodiment of the method and/or the cell according to the invention, the mixture according to the invention comprises, essentially consists of or consists of at least three, preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different sialylated oligosaccharides selected from the list comprising: 3 '-sialyllactose, 6' -sialyllactose, 3, 6-sialyllactose, 6 '-disialyllactose, 8, 3-disialyllactose, 3' S-2'FL, 6'S-2'FL, 6'S-3-FL, pentasaccharide LSTD (Neu5Ac.alpha. -2, 3Gal.beta. -1,4GlcNAcβ. -1, 3Gal.beta. -1,4 Glc), sialylated lacto-N-trisaccharide, sialylated lacto-N-tetrasaccharide comprising LSTa and LSTb, sialyl-lacto-N-neotetrasaccharide comprising LSTc and LSTd, monosialyllacto-N-hexasaccharide, disialyllacto-N-hexasaccharide I, monosialyllacto-N-neohexasaccharide I monosialyl emulsion-N-neohexasaccharide II, disialyl emulsion-N-neohexasaccharide, disialyl emulsion-N-tetrasaccharide, disialyl emulsion-N-hexasaccharide II, sialyl emulsion-N-tetrasaccharide a, disialyl emulsion-N-hexasaccharide I, sialyl emulsion-N-tetrasaccharide b, 3 '-sialyl-3-fucosyl lactose (3's-3-FL), disialyl monosialyl emulsion-N-neohexasaccharide, sialyl emulsion-N-fucose II, disialyl emulsion-N-fucose II, monosialyl disialyl emulsion-N-tetrasaccharide, FS Gal-LNnH (Gal-a 1,3-Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), DFSGal-LNnH (Gal-a 1,3- [ Fuca1,2] -Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), FS-LNnH (Fuca 1,2-Gal-b1,4-GlcNAc-b1,6- [ Neu5Aca2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), MSDF-pair-LNnH (Neu 5Aca2,3-Gal-b1,4- [ Fuca1,3] -GlcNAc-b1,3-Gal-b1,4- [ Fuc-a1,3] -GlcNAc-b1,3-Gal-b1, 4-Glc), GD3 (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3Gal beta-1, 4 Glc), GT3 (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3Gal beta-1, 4 Glc); GM2GalNAc β -1,4 (Neu 5Ac α -2, 3) Galβ -1,4Glc, GM1 (Galβ -1,3GalNAc β -1,4 (Neu 5Ac α -2, 3) Galβ -1,4 Glc), GD1a (Neu 5Ac α -2,3Galβ -1,3GalNAc β -1,4 (Neu 5Ac α -2, 3) Galβ -1,4 Glc), GT1a (Neu 5Ac α -2,8Neu5Ac α -2,3Galβ -1,3GalNAc β -1,4 (Neu 5Ac α -2, 3) Galβ -1,4 Glc), GD2 (Galβ -1,4 (Neu 5Ac α -2,8Neu5Ac α -2, 3) Galβ -1,4 Glc), GT2 (GalNAc β -1,4 (Neu 5Ac α -2, 8) Gal β -1,4 Glc), GT1a (Neu 5Ac α -2,8 Gal 5Ac α -1,4 Glc), (Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha 2, 3) Gal beta-1, 4 Glc), GT1b (Neu 5Ac alpha-2, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha 2, 3) Galbeta-1, 4 Glc), GQ1b (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha 2, 3) Galbeta-1, 4 Glc), GT1c (GalNAc beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3) Gal beta-1, 4 Glc), GQ1c (Neu 5Ac alpha-2, 3) Gal beta-1, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha 2, 3) Gal beta-1, 4 Glc), GP1c (Neu 5Ac alpha-2, 8Neu5Ac alpha-2, 3Gal beta-1, 3GalNAc beta-1, 4 (Neu 5Ac alpha-2, 8Neu5Ac alpha 2, 3) Galbeta-1, 4 Glc), GD1a (Neu 5Ac alpha-2, 3 Galbeta-1, 3 (Neu 5Ac alpha-2, 6) GalNAc beta-1, 4 Galbeta-1, 4 Glc), fucosyl-GM1 (Fuc alpha-1, 2 Galbeta-1, 3GalNAc beta-1, 4 (Neu 5Ac beta-2, 3) Galbeta-1, 4Glc 2 a, 3Gal beta-1, 35 b-2, 35 b-3- [ 1, 35 b-2, 8-Neu5Aca2,3-Gal-b1,4-Glc, neu5Aca2,8-Neu5Gca2,3-Gal-b1,4-Glc, neu5Aca2,8-Neu5Aca2,3-Gal-b1,4-Glc, neu5Gca2,8-Neu5Gca2,3-Gal-b1,4-Glc, neu5Aca2,3-Gal-b1,3- [ Neu5Aca2,6] -Gal-b1,4-Glc, galb1,6- [ Neu5Aca2,3] -Gal-b1,4-Glc, gal-b1,3- [ Neu5Aca2,6] -Gal-b1,4-Glc, neu5Gca2,3-Gal-b1,3-Gal-b1,4-Glc, neu5, 3-Gal-b1, 3-Gal-5 Aca2,3-Gal-b1, 3-Gal-5 Aca-2, 3-Gal-b1, 6] -Gal-b1,4-Glc, galb1,4-GlcNAc-b16- [ Neu5Ac2,3-Gal-b1,3] -Gal-b1,4-Glc, neu5Ac2,6-Gal-b1,4-GlcNAc-b1,6- [ Galb1,3] -Gal-b1,4-Glc, neu5Gca2,3-Gal-b1,4-Glc, neu5Gca2,6-Gal-b1,4-Glc, galMSLNnH (Gala 1,3-Gal-b1,4-GlcNAc-b1,6- [ Neu5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3] -Gal-b1, 4-Glc), F-LSTa, F-LSTb, F-LSTc, FS-LNH I, FS-LNH II, FS-LNH III, FS-LNH IV, FS-LNnH I, FS-LNnH II, FS-p-LNnH I, FS-p-LNnH II, DFS-LNH I, DFS-LNH III, DFS-LNH IV, DFS-LNnH, DF-p-LNH sulfate I, DF-p-LNH sulfate II TF-pair-LNH sulfate, neu5GcLNnT, GM2 tetraose, SLNOa, S-LNH I, S-LNH II, S-LNnH I, S-LNnH II, S-pair-LNnH, S-LNH, S-LNnH, S-LNH, S-LN DS-LNH II, S-LNO, FS-LNO I, FS-LNO II, FS-iso-LNO, DFS-iso-LNO I, DFS-iso-LNO II, DFS-LNO I, DFS-NO II, DFS-LNO III, TFS-LNO, TFS-iso-LNO, FDS-LNT I, FDS-LNT II, FDS-LNH I, FDS-LNH II, FDS-LNH III, FDS-LNnH, TS-LNH, SLNnD, FS-neo-LNP I, neu5Ac-a2,3-Gal-b1,4-GlcNAc-b1,6- [ GlcNAc-b1,3] -Gal-b1,4-Glc, neu5Ac-a2,3-GlcNAc-b1,3-Gal-b1,4-Glc, neu5Ac-a2,6- [ GlcNAc-b1,3] -Gal-b1,4-Glc, gal-b1,3- [ Neu5Gc-a2,6] -Gal-b1,4-Glc, 3 '-sialyllacto-N-disaccharide (3' slnb), 6 '-sialyllacto-N-disaccharide (6' slnb), monofucosyl Shan Tuoye-acid lacto-N-octasaccharide (sialyl Lea), 3 '-sialyllactosamine (3' slacnac), 6 '-sialyllactosamine (6' slacnac), sialyl Lex and Neu5Gc-a2,3-Gal-b1,4-GlcNAc, and at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different neutral fucosylated oligosaccharides, preferably selected from the list comprising: 2 '-fucosyllactose (2' FL), 3-fucosyllactose (3-FL), 4-fucosyllactose (4 FL), 6-fucosyllactose (6 FL), dif-fucosyllactose (diFL or LDFT), fuc-a1,2-Gal-b1,3-GlcNAc-b1,3- [ Fuc-a1,3- [ Gal-b1,4] -GlcNAc-b1,6] -Gal-b1,4-Glc, milk-N-fuco-pentasaccharide I (LNFP-I; fuc-a1,2-Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc), galNAc-LNFP-I (GalNAc-a 1,3- (Fuc-a 1, 2) -Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-fucopentaose II (LNFP-II; gal-b1,3- (Fuc-a 1, 4) -GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-fucopentaose III (LNFP III; gal-b1,4- (Fuc-a 1, 3) -GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-fucopentaose V (FP-V; gal-b1,3-GlcNAc-b1,3-Gal-b1,4- (c-a, 3) -Fuc-b 1 milk-N-fucopentaose VI (LNFP-VI; gal-b1,4-GlcNAc-b1,3-Gal-b1,4- (Fuc-a 1, 3) -Glc), milk-N-neofucopyranose I (LNnFP I; fuc-a1,2-Gal-b1,4-GlcNAc-b1,3-Gal-b1, 4-Glc), lacto-N-difiuorohexasaccharide I (LNDFH I; fuc-a1,2-Gal-b1,3- [ Fuc-al,4] -GlcNAc-b1,3-Gal-b1, 4-Glc), milk-N-difucose hexasaccharide II (LNDFH II; fuc-a1,4- (Gal-b 1, 3) -GlcNAc-b1,3-Gal-b1,4- (Fuc-a 1, 3) -Glc), monofucose milk-N-hexasaccharide III, difucose milk-N-hexasaccharide, difucose-milk-N-neohexasaccharide, LNnDFH (Gal-b 1,4- (Fuc-a 1, 3) -GlcNAc-b1,3-Gal-b1,4- (Fuc-a 1, 3) -Glc), A-tetrasaccharide (GalNAc-a 1,3- (c-a 1, 2) -Gal-b1, 4-Glc), gal-DFF, LNF II F-LNnH I, F-p-LNH II, F-p-LNnH, DF-LNH II, DF-LNH I, DF-LNnH, DF-p-LNH II, DF-p-LNH III, DF-p-LNnH, TF-LNH I, TF-LNH II, IF-p-LNH I, IF-p-LNH II, TF-p-LNnH, F-LNO I, F-LNO II, F-LNO III, F-LNnO II, F-iso-LNO I, F-neogenesis-LNnO, F-p-LNO, F-LNO DF-iso-LNnO, DF-LNO I, DF-LNO II, DF-LNO III, DF-LNnO I, DF-LNnO II, DF-LNnO III, DF-iso-LNO I, DF-iso-LNO II, DF-iso-LNO III, DF-iso-LNO IV, DF-iso-LNO V, DF-iso-LNO VI, DF-iso-LNO VII, DF-p-LNnO, TF-LNO I, TF-LNO II, TF-LNnO, TF-iso-LNO I, TF-iso-LNO II, TF-iso-LNO III, TF-iso-LNO IV, TF-iso-LNnO, four-F-iso-LNO, four-F-p-LNO, five-F-iso-LNO, F-LND I, F-LND II, DF-LND I, DF-LND II, DF-LND III, DF-LND IV, DF-LND V, DF-LND VI, triF-LND I, triF-LND II, triF-LND III, triF-LND IV, triF-LND V, triF-LND VI, triF-LND VII, tetra F-LND I, tetra F-LND II, tetra F-LND III, F-LNnD I, F-LNnD II, DF-LNnD, DF-neo-LND, DF-Gal-LNnH (Gal-a 1,3-Gal-B1, 4-NAc-B1, 6- [ Gal-a1,3-Gal-B1,4- [ Fuc-a1,3] -GlcNAc-B1,3] -Gal-B1, 4-Glc), 3-F-isochrotriose, B-tetrasugar, B-pentasugar, B-hexasugar, B-heptasugar, DF DGal-LNnT (Gal-a 1,3-Gal-B1,4- [ Fuc-a1,3] -GlcNAc-B1,3-Gal-B1,4- [ Fuc-a1,3] -Glc), TF DGal-LNnH a, TF DGal-LNnH B, DFGal-pair-LNnH, 2'FLNB, 4-FLNB, leb (Fuc-a 1,2-Gal-B1,3- (Fuc-a 1, 4) -GlcNAc), 2' FLacNAc, 3-FLacNAc and Ley (Fuc-a 1,2-Gal-B1,4- (c-a 1), 3) -GlcNAc, and/or in combination with at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different neutral nonfucosylated oligosaccharides preferably selected from the list comprising: milk-N-trisaccharide II (LN 3), milk-N-neotetraose (LNnT), milk-N-tetraose (LNT), p-milk-N-neopentasaccharide, p-milk-N-pentasaccharide, p-milk-N-neohexasaccharide, p-milk-N-hexasaccharide, β - (1, 3) galactosyl-p-milk-N-neopentasaccharide, β - (1, 4) galactosyl-p-milk-N-pentasaccharide, gal-a1,4-Gal-b1,4-Glc (Gal-a 1, 4-lactose), β3 '-galactosyl lactose, β6' -galactosyl lactose, gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, gal-a1,4-Gal-a1,4-Gal-a1,4-Gal-b1,4-Glc, gal-b1,3-Galb1,3-Gal-b1,4-Glc, gal-b1,3-Gal-b1,3-Gal-b1,3-Gal-b1, 4-Glc, galNAc-b1,3-Gal-b1,4-Glc (GalNAc-b 1, 3-Lactuse), gal-b1,3-GalNAc-b1, 3-Lactose, galNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc (ball-N-tetrasaccharide), gal-b1,3-GalNAc-b1,3-Gal-a1,4-Gal-b1,4-Glc, galNAc-b1,3-LNT, gal-b1,3-GalNAc-b1,3-LNT, neo-LNT (GlcNAc-b 1,6- [ Gal-b1,3] -Gal-b1, 4-Glc), gal-neo-LNP I (Gal-b 1,4-GlcNAc-b1,6- [ Gal-b1,3-Gal-b1,3] -Gal-b1, 4-Glc), gal-neo-LNP II (Gal-b 1,4-GlcNAc-b1,6- [ Gal-b1,3] -Gal-b1,3-Gal-b1, 4-Glc), gal-neo-LNP III (Gal-b 1,3-Gal-b1,4-GlcNAc-b1,6- [ Gal-b1,3] -Gal-b1, 4-Glc), neo-LNO, galNAc-b1,3-LNnT, gal-b1,3-GalNAc-b1,3-LNnT, lacDiNAc, poly-LacNAc, LNH, LNnH, iso-LNO, neo-LNO, LND, iso-LND, galNAc-a1,3-Gal-b1,4-Glc, neo-LNP I, iso-LNT, DGalLNnH, galili pentasaccharide.

Exemplary mixtures in this context comprise, consist of, or consist essentially of: 3 '-sialyllactose, 6' -sialyllactose, 3'S-2' FL, 6'S-2' FL, 6'S-3-FL, 3' -sialyl-3-fucosyllactose (3 'S-3-FL), 2' FL, 3-FL and DiFL. Another exemplary mixture in this context comprises, consists of, or consists essentially of: LN3, LNT, LSTa, 3'sl, 6' sl, LSTb. Another exemplary mixture in this context comprises, consists of, or consists essentially of: LN3, LNnT, LSTc, LSTd, 3'sl and 6' sl. Another exemplary mixture in this context comprises, consists of, or consists essentially of: 2' FL, 3-FL, diFL, LN3, LNT, LNnT, 3' SL, 6' SL, LNFP-I, and LSTc. Another exemplary mixture in this context comprises, consists of, or consists essentially of: 2' FL, 3-FL, diFL, 3' SL, 6' SL, LN3, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LSTa, LSTc, and LSTd. Another exemplary mixture in this context comprises, consists of, or consists essentially of: 2' FL, 3-FL, monofucosyl Shan Tuoye acid based lacto-N-octasaccharide (sialyl Lea), fuc-a1,2-Gal-b1,3- (Fuc-a 1, 4) -GlcNAc (Leb), 3' SL and 6' SL. Another exemplary mixture in this context comprises, consists of, or consists essentially of: 2' FL, 3-FL, 3' SL, 6' SL, sialyl Lex, fuc-a1,2-Gal-b1,4- (Fuc-a 1, 3) -GlcNAc (Ley).

Exemplary mixtures are described in the specific examples section in this context of the invention.

To produce lactose-based oligosaccharides as described herein, and in a specific example of a method and/or cell according to the invention, lactose can be added to the culture so that the cell can be absorbed passively or via active transport; or lactose may be produced by the cell (e.g. after metabolic engineering of the cell for such purposes as known to those of ordinary skill in the art), preferably intracellularly. Lactose, which is preferably comprised in the oligosaccharide mixture according to the invention as described herein, may thus be used as acceptor in the synthesis of mammalian milk oligosaccharides or human milk oligosaccharides (preferably all lactose-based MMOs or HMOs). Lactose producing cells can be obtained by expressing N-acetylglucosamine beta-1, 4-galactosyltransferase and UDP-glucose 4-epi-isomerase. More preferably, the cells are modified for enhanced lactose production. The modification may be any one or more selected from the group comprising: excessive expression of N-acetylglucosamine beta-1, 4-galactosyltransferase and excessive expression of UDP-glucose 4-epi isomerase. Alternatively, cells that use lactose as a receptor in the glycosylation reaction preferably have a transporter for the uptake of lactose from the culture. More preferably, the cells are optimized for lactose absorption. The optimization may be an overexpression of lactose transporter such as lactose permease (permase) from e.g. escherichia coli, kluyveromyces lactis (Kluyveromyces lactis) or lactobacillus casei (Lactobacillus casei) BL 23. Lactose permease is preferably expressed continuously. Lactose may be added at the beginning of the culture, or it may be added when sufficient biomass has been formed during the growth phase of the culture, i.e. the MMO production phase (initiated by adding lactose to the culture) is separated from the growth phase. In a preferred embodiment, lactose is added at the beginning and/or during the cultivation, i.e. the growth phase and the production phase are not separated.

In a preferred embodiment of the method and/or cell according to the invention, lactose killing is counteracted when the cell is grown in an environment where lactose is combined with one or more other carbon sources. In the case of the term "lactose kill", it is meant that the growth of cells in the medium in which lactose is present and another carbon source is hindered. In a preferred embodiment, the cells are genetically modified such that they retain at least 50% of lactose influx without undergoing lactose killing, even at high lactose concentrations, as described in WO 2016/075243. The genetic modification comprises expression and/or overexpression of exogenous and/or endogenous lactose transporter genes by a heterologous promoter that does not produce a lactose killing phenotype, and/or modification of the codon usage of lactose transporter that does not produce a lactose killing phenotype to produce altered expression of the lactose transporter. The content of WO 2016/075243 is hereby incorporated by reference.

To produce LNB-based oligosaccharides as described herein and in additional and/or alternative embodiments of the methods and/or cells according to the invention, LNB (i.e., lacto-N-disaccharide, gal-b1, 3-GlcNAc) can be added to the culture so that the cells can be absorbed passively or via active transport; or the LNB may be produced by the cell (e.g. after metabolic engineering of the cell for such purposes as known to those of ordinary skill in the art), preferably intracellularly. LNBs may thus be used as receptors in the synthesis of LNB-based oligosaccharides (preferably all LNB-based oligosaccharides), which LNBs are preferably comprised in an oligosaccharide mixture according to the invention as described herein. LNB-producing cells can be obtained by expressing an N-acetylglucosamine β -1, 3-galactosyltransferase that can modify GlcNAc (produced in the cell and/or absorbed passively or via active transport) to form the LNB. Preferably, the LNB-producing cell is capable of expressing, preferably expressing, enzymes required for the synthesis of GlcNAc, such as glucosamine 6-phosphate N-acetyltransferase, phosphatase, N-acetylglucosamine beta-1, 3-galactosyltransferase, L-glutamylamino-D-fructose-6-phosphate aminotransferase and UDP-glucose 4-epimerase, preferably glucosamine 6-phosphate N-acetyltransferase and phosphatase, preferably a HAD-like phosphatase, such as any one of the E.coli genes comprising aphA, cof, hisB, otsB, surE, yaed, ycjU, yedP, yfbT, yidA, yigB, yihX, yniC, yrbL, appA, gph, serB, ybhA, ybiV, ybjL, yfb, yieH, yjgL, yjjG, yrfG and Ybiu or PsMupP from Pseudomonas putida, scDOG1 from Saccharomyces cerevisiae and BsAraL from Bacillus subtilis (as described in WO 18122225). Preferably, the cell is metabolically engineered for the production of LNBs. More preferably, the cell is metabolically engineered for enhanced production of LNBs. The cells are preferably modified to express and/or overexpress any one or more of the polypeptides comprising: glucosamine 6-phosphate N-acetyltransferase, phosphatase, N-acetylglucosamine beta-1, 3-galactosyltransferase, L-glutamylD-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epi-isomerase.

Cells that use LNB as a receptor in the glycosylation reaction preferably have a transporter for uptake of LNB from culture. More preferably, the cells are optimized for LNB uptake. The optimisation may be an overexpression of an LNB transporter, such as lactose permease from e.coli, kluyveromyces lactis or lactobacillus casei BL 23. Lactose permease is preferably expressed continuously. The LNB may be added at the beginning of the culture, or it may be added when sufficient biomass has been formed during the growth phase of the culture, i.e. the oligosaccharide production phase (initiated by adding the LNB to the culture) is separated from the growth phase. In a preferred embodiment, the LNB is added at the beginning and/or during the culture, i.e. the growth phase and the production phase are not separated.

To produce LacNAc-based oligosaccharides as described herein and in additional and/or alternative embodiments of the methods and/or cells according to the invention, lacNAc (i.e., N-acetyllactosamine, gal-b1, 4-GlcNAc) may be added to the culture so that the cells may be absorbed passively or via active transport; or LacNAc can be produced by the cell (e.g., after metabolic engineering of the cell for such purposes as known to those of ordinary skill in the art), preferably produced intracellularly. LacNAc can thus be used as acceptor in the synthesis of LacNAc-based oligosaccharides (preferably all LacNAc-based oligosaccharides), which LacNAc is preferably comprised in the oligosaccharide mixture according to the invention as described herein. LacNAc-producing cells can be obtained by expressing N-acetylglucosamine beta-1, 4-galactosyltransferase that can modify GlcNAc (produced in the cell and/or absorbed passively or via active transport) to form LacNAc. Preferably, the LacNAc-producing cells are capable of expressing, preferably expressing, enzymes required for the synthesis of GlcNAc, such as glucosamine 6-phosphate N-acetyltransferase, phosphatase, N-acetylglucosamine beta-1, 4-galactosyltransferase, L-glutamylamino-D-fructose-6-phosphate aminotransferase and UDP-glucose 4-epimerase, preferably glucosamine 6-phosphate N-acetyltransferase and phosphatase (preferably, HAD-type phosphatase). Preferably, the cell is metabolically engineered for the production of LacNAc. More preferably, the cell is metabolically engineered for enhanced LacNAc production. The cells are preferably modified to express and/or overexpress any one or more of the polypeptides comprising: glucosamine 6-phosphate N-acetyltransferase, phosphatase, N-acetylglucosamine beta-1, 4-galactosyltransferase, L-glutamylD-fructose-6-phosphate aminotransferase, and UDP-glucose 4-epi-isomerase.

Cells that use LacNAc as a receptor in the glycosylation reaction preferably have a transporter for absorbing LacNAc from culture. More preferably, the cells are optimized for LacNAc uptake. The optimisation may be an overexpression of an LNB transporter, such as lactose permease from e.coli, kluyveromyces lactis or lactobacillus casei BL 23. Lactose permease is preferably expressed continuously. LacNAc may be added at the beginning of the culture, or it may be added when sufficient biomass has been formed during the growth phase of the culture, i.e. the oligosaccharide production phase (initiated by adding LacNAc to the culture) is separated from the growth phase. In a preferred embodiment, lacNAc is added at the beginning and/or during the cultivation, i.e. the growth phase and the production phase are not separated.

In additional and/or alternative embodiments of the method and/or cell according to the invention, the cell (i) is capable of expressing, preferably expressing, a sialyltransferase, preferably selected from the group consisting of α -2, 3-sialyltransferase, α -2, 6-sialyltransferase and α -2, 8-sialyltransferase, and (ii) is capable of expressing, preferably at least one, preferably at least two, preferably at least three, preferably at least four, even more preferably at least five, even more preferably at least six, and most preferably at least seven additional glycosyltransferases. Preferably selected from the list comprising: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyl transferases, N-acetylgalactosylaminotransferases, N-acetylmannosyl aminotransferases, xylosyltransferases, glucuronide transferases, galacturonan transferase, glucosaminyl transferases, N-glycolyl neuraminidases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-altrose amine transferases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosyl aminotransferases as defined herein.

In a preferred embodiment, the fucosyltransferase is selected from the list comprising: alpha-1, 2-fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-1, 4-fucosyltransferase and alpha-1, 6-fucosyltransferase. In another preferred embodiment, the sialyltransferase is selected from the list comprising: alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase. In another preferred embodiment, the galactosyltransferase is selected from the list comprising: beta-1, 3-galactosyltransferase, N-acetylglucosamine beta-1, 3-galactosyltransferase, beta-1, 4-galactosyltransferase, N-acetylglucosamine beta-1, 4-galactosyltransferase, alpha-1, 3-galactosyltransferase, and alpha-1, 4-galactosyltransferase. In another preferred embodiment, the glucosyltransferase is selected from the list comprising: alpha-glucosyltransferase, beta-1, 2-glucosyltransferase, beta-1, 3-glucosyltransferase and beta-1, 4-glucosyltransferase. In another preferred embodiment, the mannosyltransferase is selected from the list comprising: alpha-1, 2-mannosyltransferase, alpha-1, 3-mannosyltransferase and alpha-1, 6-mannosyltransferase. In another preferred embodiment, the N-acetylglucosaminyl transferase is selected from the list comprising: galactoside beta-1, 3-N-acetylglucosaminyl transferase and beta-1, 6-N-acetylglucosaminyl transferase. In another preferred embodiment, the N-acetylgalactosamine transferase is selected from the list comprising: alpha-1, 3-N-acetylgalactosamine transferase and beta-1, 3-N-acetylgalactosamine transferase.

In another embodiment of the methods and/or cells of the invention, the cells are modified in terms of the performance or activity of at least one, preferably at least two, more preferably all of the glycosyltransferases. In a preferred embodiment, the glycosyltransferase is an endogenous protein of a cell that has been modified to exhibit or be active, preferably the endogenous glycosyltransferase is overexpressed; or the glycosyltransferase is a heterologous protein that is heterogeneously introduced and expressed in the cell, preferably overexpressed. The endogenous glycosyltransferase can have modified expression in cells that also express a heterologous glycosyltransferase.

In another embodiment of the method and/or cell of the invention, at least one, preferably at least two of the glycosyltransferases are fucosyltransferases and the cell is capable of synthesizing GDP-Fuc. GDP-fucose may be provided by enzymes expressed in cells or by cell metabolism. Such cells producing GDP-fucose may exhibit enzymes that convert, for example, fucose added to the cells into GDP-fucose. The enzyme may be, for example, a bifunctional fucose kinase/fucose-1-phosphate guanyl transferase, such as Fkp from Bacteroides fragilis (Bacteroides fragilis), or a combination of a respective fucose kinase together with a respective fucose-1-phosphate guanyl transferase, as known from several species including homo sapiens, boar (Sus scrofa) and brown rat (Rattus norvegicus). In a preferred embodiment of the method and/or the cell of the invention, the cell is capable of expressing at least one, preferably at least two fucosyltransferases selected from the group consisting of: alpha-1, 2-fucosyltransferase, alpha-1, 3/1, 4-fucosyltransferase and alpha-1, 6-fucosyltransferase. Preferably, the fucosyltransferase is selected from the following organisms: such as, for example, helicobacter species, such as, for example, helicobacter pylori (Helicobacter pylori), helicobacter weasel (Helicobacter mustelae); acremonium (Akkermansia) species, such as, for example, acremonium muciniphilum (Akkermansia muciniphila); bacteroides (bacterioides) species such as, for example, bacteroides fragilis, bacteroides vulgaris (Bacteroides vulgatus), bacteroides ovatus (Bacteroides ovatus); coli species such as e.g. e.coli O126, e.coli O55: h7; a Lachnospiraceae (Lachnospiraceae) species; a tannals (Tannerella) species; a Clostridium (Clostridium) species; salmonella (Salmonella) species such as, for example, salmonella enterica (Salmonella enterica), artemisia annua methanotrophic microorganism (Methanosphaerula palustries); a vibrio (butyl vibrio) species; a Prevotella (Prevotella) species; a species of the genus pyrromonas (porphyrimonas), such as for example, pyrromonas catotuber (Porphyromonas catoniae); arabidopsis thaliana (Arabidopsis thaliana); wisdom person; mice (Mus musculus). In a more preferred embodiment of the method and/or cell of the invention, the fucosyltransferase is selected from the list comprising alpha-1, 2-fucosyltransferases and alpha-1, 3/1, 4-fucosyltransferases.

Preferably, the cells are modified to produce GDP-fucose. More preferably, the cells are modified for enhanced GDP-fucose production. The modification may be any one or more selected from the group comprising: UDP-glucose: undecanopentenyl (undecaprenyl) -phosphoglucose-1-phosphate transferase, over-expression of GDP-L-fucose synthase-encoding gene, over-expression of GDP-mannose 4, 6-dehydratase-encoding gene, over-expression of mannose-1-guanylate transferase-encoding gene, over-expression of phosphomannose mutase-encoding gene, and over-expression of mannose-6-phosphate isomerase-encoding gene. Throughout this application, unless explicitly stated otherwise, the features "enhanced" and/or "optimized" production preferably means that modification and/or metabolic engineering introduced into a cell as described herein results in higher yields compared to wild-type precursor cells of the modified cell or metabolically engineered cell. For example, "enhanced GDP-fucose production" preferably means higher intracellular production of GDP-fucose in the modified cells compared to wild-type precursor cells not containing such specific modifications.

Preferably, the cell in this context comprises a fucosylation pathway as described herein.

In another embodiment of the method and/or cell of the invention, at least one, preferably at least two of the glycosyltransferases are sialyltransferases and the cell is capable of synthesizing CMP-Neu5Ac. CMP-Neu5Ac can be provided by enzymes expressed in cells or by cell metabolism. Such cells producing CMP-Neu5Ac may exhibit enzymes that convert sialic acid, e.g., added to the cell, to CMP-Neu5Ac. This enzyme may be a CMP-sialic acid synthetase, such as N-acyl neuraminic acid cytidylyltransferase from several species including Chiren, neisseria meningitidis (Neisseria meningitidis) and Pasteurella multocida (Pasteurella multocida). In a preferred embodiment of the method and/or the cell of the invention, the cell is capable of exhibiting at least one, preferably at least two sialyltransferases selected from the group consisting of alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase. Preferably, the sialyltransferase is selected from the following organisms: such as, for example, pasteurella (Pasteurella) species, such as, for example, pasteurella multocida, dake Ma Bashi bacillus (Pasteurella dagmatis); a Photobacterium species such as, for example, mermaid-light emitting bacterium (Photobacterium damselae), light emitting bacterium JT-ISH-224, phosphorescence light emitting bacterium (Photobacterium phosphoreum), and light emitting bacterium of Leptospira (Photobacterium leiognathi); a species of the genus pyrromonas, such as, for example, pyrromonas cato; streptococcus species such as, for example, streptococcus suis (Streptococcus suis), streptococcus agalactiae (Streptococcus agalactiae), streptococcus viscus (Streptococcus entericus); neisseria meningitidis; campylobacter jejuni (Campylobacter jejuni); haemophilus species (Haemophilus) such as, for example, haemophilus soxhlet (Haemophilus somnus), haemophilus ducreyi (Haemophilus ducreyi), haemophilus parahaemolyticus (Haemophilus parahaemolyticus), haemophilus parasuis (Haemophilus parasuis); vibrio (Vibrio) genus species; an alike bacillus (Alistipes) species, such as, for example, alike bacillus CAG: 268. equibacillus AL-1, saxifraga (Alistines shahii), saxifraga (Alistipes timonensis); actinobacillus species, such as, for example, actinobacillus suis (Actinobacillus suis), actinobacillus capsulatus (Actinobacillus capsulatus); wisdom person; mice. In a more preferred embodiment of the method and/or cell of the invention, the sialyltransferase is selected from the list comprising alpha-2, 3-sialyltransferase and alpha-2, 6-sialyltransferase.

Preferably, the cells are modified to produce CMP-Neu5Ac. More preferably, the cells are modified for enhanced CMP-Neu5Ac production. The modification may be any one or more selected from the group comprising: gene knockout of N-acetylglucosamine-6-phosphate deacetylase, gene knockout of glucosamine-6-phosphate deaminase, overexpression of sialic acid synthase encoding gene, and overexpression of N-acetyl-D-glucosamine-2-epi-isomerase encoding gene. Optionally, the cells are modified to produce GlcNAc and/or UDP-GlcNAc.

Preferably, the cell in this context comprises a sialylation pathway as described herein.

In another embodiment of the methods and/or cells of the invention, at least one, preferably at least two of the additional glycosyltransferases are N-acetylglucosaminyl transferases and the cells are capable of synthesizing UDP-GlcNAc. UDP-GlcNAc may be provided by an enzyme expressed in a cell or by cell metabolism. Such cells producing UDP-GlcNAc may exhibit an enzyme that converts GlcNAc, for example, added to the cell, into UDP-GlcNAc. Such enzymes may be N-acetyl-D-glucosamine kinase, N-acetylglucosamine-6-phosphate deacetylase, phosphoglucosamine mutase, and N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase from several species including Chile, E.coli. Alternatively, the cells may be (preferably metabolically engineered to) express enzymes required for the synthesis of GlcNAc, such as glucosamine 6-phosphate N-acetyltransferase, phosphatase, glycosyltransferase, L-glutamyld-fructose-6-phosphate aminotransferase and UDP-glucose 4-epimerase, preferably glucosamine 6-phosphate N-acetyltransferase and phosphatase (preferably, HAD-type phosphatase). In a preferred embodiment of the method and/or the cell of the invention, the cell is capable of expressing at least one, preferably at least two N-acetylglucosaminyl transferases selected from the group consisting of beta-1, 3-N-acetylglucosaminyl transferase and beta-1, 6-N-acetylglucosaminyl transferase. Preferably, the N-acetylglucosaminyl transferase is selected from the following organisms: such as, for example, neisseria species, such as, for example, neisseria meningitidis, neisseria lactate (Neisseria lactamica), neisseria polysaccharea (Neisseria polysaccharea), neisseria longisseria (Neisseria elongata), neisseria gonorrhoeae (Neisseria gonorrhoeae), neisseria microflava (Neisseria subflava); pasteurella species, such as, for example, up to Ma Bashi bacilli; a new rhizobium (neorthozobium) species, such as, for example, rhizobium capricoum (Neorhizobium galegae); haemophilus species such as, for example, haemophilus parainfluenza (Haemophilus parainfluenzae), haemophilus ducreyi; wisdom person; mice.

Preferably, the cell is modified to produce UDP-GlcNAc. More preferably, the cells are modified for enhanced UDP-GlcNAc production. The modification may be any one or more selected from the group comprising: gene knockout of N-acetylglucosamine-6-phosphate deacetylase, overexpression of L-glutamylD-fructose-6-phosphate aminotransferase, overexpression of phosphoglucomutase, and overexpression of N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase. Optionally, the cell is modified to produce GlcNAc.

Preferably, the cell in this context comprises an N-acetylglucosamine carbohydrate pathway as described herein.

In another embodiment of the method and/or cell of the invention, at least one, preferably at least two of the glycosyltransferases are galactosyltransferases and the cell is capable of synthesizing UDP-Gal. UDP-Gal may be provided by an enzyme expressed in a cell or by cell metabolism. Such cells producing UDP-Gal may exhibit an enzyme that converts, for example, UDP-glucose into UDP-Gal. This enzyme may be, for example, UDP-glucose-4-epimerase GalE as known from several species including Chile, E.coli and brown rat. In a preferred embodiment of the method and/or the cell of the invention, the cell is capable of exhibiting at least one, preferably at least two galactosyltransferases selected from the group consisting of beta-1, 3-galactosyltransferase and beta-1, 4-galactosyltransferase, and/or the cell is capable of exhibiting at least one, preferably at least two galactosyltransferases selected from the group consisting of alpha-1, 3-galactosyltransferase and alpha-1, 4-galactosyltransferase. Preferably, the galactosyltransferase is selected from the following organisms, such as e.g. escherichia coli species, such as e.g. escherichia coli O55: h7, E.coli DEClB, E.coli DEClD; neisseria species, such as, for example, neisseria meningitidis, neisseria lactate, neisseria polysaccharea, neisseria longisseria, neisseria gonorrhoeae, neisseria microflava; a genus of aurora species such as, for example, aurora denitrificans (Kingella denitrificans); brucella (Brucella) species such as, for example, brucella canis (Brucella anis), brucella suis (Brucella suis); salmonella species such as, for example, salmonella enterica, pseudomonas ferrooxidans (Pseudogulbenkiana ferrooxidan), corynebacterium glutamicum (Corynebacterium glutamicum); streptococcus species; arabidopsis thaliana; wisdom person; mice.

Preferably, the cells are modified to produce UDP-Gal. More preferably, the cells are modified for enhanced UDP-Gal production. The modification may be any one or more selected from the group comprising: gene knockout of bifunctional 5' -nucleotidase/UDP-sugar hydrolase encoding gene, gene knockout of galactose-1-phosphate uridylyltransferase encoding gene and overexpression of UDP-glucose-4-epi-isomerase encoding gene.

Preferably, the cell in this context comprises a galactosylation pathway as described herein.

In another embodiment of the method and/or cell of the present invention, at least one, preferably at least two of the glycosyltransferases are N-acetylgalactosamine transferase and the cell is capable of synthesizing UDP-GalNAc. UDP-GalNAc can be provided by an enzyme expressed in a cell or by cell metabolism. Such cells producing UDP-GalNAc may exhibit an enzyme that converts, for example, UDP-glucose into UDP-Gal. This enzyme may be, for example, UDP-glucose-4-epimerase GalE as known from several species including Chile, E.coli and brown rat. In a preferred embodiment of the method and/or the cell of the invention, the cell is capable of exhibiting at least one, preferably at least two N-acetylgalactosamine transferase selected from the group consisting of alpha-1, 3-N-acetylgalactosamine transferase and beta-1, 3-N-acetylgalactosamine transferase. Preferably, the N-acetylgalactosamine transferase is selected from the group consisting of organisms such as, for example, helicobacter species such as, for example, helicobacter ferret; haemophilus species such as, for example, haemophilus influenzae (Haemophilus influenzae); neisseria species, such as, for example, neisseria meningitidis, neisseria lactose, neisseria polysaccharea, neisseria longisseria, neisseria gonorrhoeae, neisseria microflava; rickettsia (Rickettsia) species such as, for example, beckettsia (Rickettsia bellii), rickettsia prazirata (Rickettsia prowazekii), rickettsia japonica (Rickettsia japonica), kang Shili grams of Rickettsia (Rickettsia conorii), feline Rickettsia felis, mosaic Rickettsia (Rickettsia massiliae); wisdom person; mice.

Preferably, the cells are modified to produce UDP-GalNAc. More preferably, the cells are modified for enhanced UDP-GalNAc production. The modification may be any one or more selected from the group comprising: gene knockout of bifunctional 5' -nucleotidase/UDP-sugar hydrolase encoding gene, gene knockout of galactose-1-phosphate uridylyltransferase encoding gene and overexpression of UDP-glucose-4-epi-isomerase encoding gene.

Preferably, the cell in this context comprises an N-acetylgalactose amination pathway as described herein.

Throughout this application, whenever a protein is revealed, for example by reference to SEQ ID NO, i.e. a unique database number (e.g. UNIPROT number) or by reference to a particular organism of origin, that protein embodiment may preferably be replaced by any one of the following embodiments (and thus the protein is considered to be revealed according to all of the following embodiments):

proteins (for example by reference to SEQ ID NO, i.e.unique database numbers (e.g.UNIPOT numbers) or by reference to a specific organism of origin),

a functional homolog, variant or derivative of the protein having at least 80% overall sequence identity to the full length of the protein,

Functional fragments of the proteins and having the same activity, or

Comprising a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity to the full length amino acid sequence of the protein and having the same activity.

For example, when "having the sequence of SEQ ID NO: helicobacter pylori (H.pyri) alpha-1,3-fucosyltransferase (H.pyri alpha-1,3-fucosyltransferase with SEQ ID NO:05), this embodiment is preferably replaced by any one, preferably all, of the following embodiments:

has the sequence of SEQ ID NO: helicobacter pylori alpha-1,3-fucosyltransferase of 05,

comprising a sequence according to SEQ ID NO:05, an alpha-1,3-fucosyltransferase of a polypeptide sequence of 05,

and SEQ ID NO:05 and having at least 80% overall sequence identity to the full length of SEQ ID NO:05, a functional homolog, variant or derivative thereof,

SEQ ID NO:05 and has alpha-1,3-fucosyltransferase activity, or

Comprising a polypeptide comprising a sequence identical to the sequence of SEQ ID NO:05 and having an amino acid sequence having at least 80% sequence identity and having alpha-1,3-fucosyltransferase activity.

In another specific example of the methods and/or cells of the invention, the cells are capable of synthesizing any one of the nucleotide-sugars selected from the list comprising: GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), UDP-2-acetamido-2, 6-dideoxy-L-arabinose-4-hexanone, UDP-2-acetamido-2, 6-dideoxy-L-lyxose-4-hexanone, UDP-N-acetyl-L-rhamnose amine (UDP-L-RhaNAc or UDP-2-acetamido-2, 6-dideoxy-L-mannose) dTDP-N-acetylfucosylamine, UDP-N-acetylfucosylamine (UDP-L-FucNAc or UDP-2-acetamido-2, 6-dideoxy-L-galactose), UDP-N-acetyl-L-neotame amine (UDP-L-PnNAC or UDP-2-acetamido-2, 6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnoamine (UDP-L-QuiNAc or UDP-2-acetamido-2, 6-dideoxy-L-glucose), GDP-L-isorhamnose, CMP-N-glycolylneuraminic acid (CMP-Neu 5 Gc), CMP-Neu4Ac, CMP-Neu5Ac9N ₃ 、CMP-Neu4，5Ac ₂ 、CMP-Neu5，7Ac ₂ 、CMP-Neu5，9Ac ₂ 、CMP-Neu5，7(8，9)Ac ₂ UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose. In a preferred embodiment, the cell is capable of synthesizing two nucleotide-sugars. In a more preferred embodiment, the cell is capable of synthesizing at least three nucleotide activating sugars. In an even more preferred embodiment, the cell is capable of synthesizing at least four nucleotide activating sugars. In a preferred embodiment, the cell is capable of synthesizing at least five nucleotide activating sugars. In another preferred embodiment, the cell is metabolically engineered for the production of nucleotide-sugars. In another preferred embodiment In a cell modified and/or engineered for optimal production of nucleotide-sugars, i.e., enhanced production of nucleotide-sugars as described herein. In a more preferred embodiment, the cell is metabolically engineered for the production of two nucleotide-sugars. In an even more preferred embodiment, the cell is metabolically engineered for the production of three or more nucleotide activating sugars.

In another embodiment of the methods and/or cells of the invention, the cells express one or more polypeptides selected from the list comprising: mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-guanylate transferase, GDP-mannose 4, 6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, GDP-fucose pyrophosphorylase, fucose-1-guanylate transferase, L-glutamylamino-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epi-isomerase, UDP-N-acetylglucosamine 2-epi-isomerase N-acetylmannosamine-6-phosphate 2-epi isomerase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, N-acetylneuraminic acid synthase, N-acetylneuraminic acid dissociase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphate phosphatase, N-acylneuraminic acid cytidylyltransferase, galactose-1-epi isomerase, galactokinase, glucokinase, galactose-1-phosphouridyltransferase, UDP-glucose 4-epi isomerase, glucose-1-phosphouridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epi isomerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase.

In a preferred embodiment of the method and/or the cell according to the invention, the mixture of at least three different sialylated oligosaccharides according to the invention may be produced by providing a cell for producing lactose-based sialylated nonfucosylated oligosaccharides, which cell: 1) Capable of absorbing lactose in culture as described herein or capable of producing lactose upon absorption of glucose by the action of b-1, 4-galactosyltransferase as described herein; and 2) capable of expressing at least one, preferably at least two sialyltransferases as described herein selected from the list comprising alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase; 3) Optionally capable of expressing an N-acetylglucosaminyl transferase, preferably a galactoside beta-1, 3-N-acetylglucosaminyl transferase as described herein; 4) Optionally capable of expressing at least one, preferably at least two galactosyltransferases as described herein selected from the list comprising N-acetylglucosamine β -1, 3-galactosyltransferase, N-acetylglucosamine β -1, 4-galactosyltransferase, α -1, 3-galactosyltransferase, α -1, 4-galactosyltransferase; 5) Optionally capable of expressing at least one, preferably at least two N-acetylgalactosamine transferase as described herein selected from the list comprising α -1, 3-N-acetylgalactosamine transferase and β -1, 3-N-acetylgalactosamine transferase; 6) Capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein, and 7) capable of synthesizing a nucleotide-sugar of each of the glycosyltransferases (if present).

In another and/or additional preferred embodiment of the method and/or cell according to the invention, the mixture of at least three different sialylated oligosaccharides according to the invention may be produced by providing a cell for producing an LNB-based sialylated nonfucosylated oligosaccharide, which cell: 1) Capable of absorbing LNBs in culture as described herein or capable of producing LNBs as described herein; and 2) capable of expressing at least one, preferably at least two sialyltransferases as described herein selected from the list comprising alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase; 3) Capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein, and 4) optionally capable of producing UDP-galactose.

In another and/or additional preferred embodiment of the method and/or the cell according to the invention, the mixture of at least three different sialylated oligosaccharides according to the invention may be produced by providing a cell for producing sialylated nonfucosylated oligosaccharides based on LacNAc, which cell: 1) Capable of absorbing LacNAc in culture as described herein or capable of producing LacNAc as described herein; and 2) capable of expressing at least one, preferably at least two sialyltransferases as described herein selected from the list comprising alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase; 3) Capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein, and 4) optionally capable of producing UDP-galactose.

In another and/or additional preferred embodiment of the method and/or the cell according to the invention, the mixture of at least three different sialylated oligosaccharides according to the invention may be produced by providing a cell for the production of lactose-based sialylated fucosylated oligosaccharides, which cell: 1) Capable of absorbing lactose in culture as described herein or capable of producing lactose upon absorption of glucose by the action of b-1, 4-galactosyltransferase as described herein; and 2) capable of expressing at least one, preferably at least two, fucosyltransferases as described herein selected from the list comprising alpha-1, 2-fucosyltransferases, alpha-1, 3-fucosyltransferases, alpha-1, 4-fucosyltransferases and alpha-1, 6-fucosyltransferases; 3) Capable of expressing at least one, preferably at least two sialyltransferases as described herein selected from the list comprising alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase; 4) Optionally capable of expressing an N-acetylglucosaminyl transferase, preferably a galactoside beta-1, 3-N-acetylglucosaminyl transferase as described herein; 5) Optionally capable of expressing at least one, preferably at least two galactosyltransferases as described herein selected from the list comprising N-acetylglucosamine β -1, 3-galactosyltransferase, N-acetylglucosamine β -1, 4-galactosyltransferase, α -1, 3-galactosyltransferase, α -1, 4-galactosyltransferase; 6) Optionally capable of expressing at least one, preferably at least two N-acetylgalactosamine transferase as described herein selected from the list comprising α -1, 3-N-acetylgalactosamine transferase and β -1, 3-N-acetylgalactosamine transferase; 7) Capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein, 8) capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein; and 9) a nucleotide-sugar capable of synthesizing each of the glycosyltransferases (if present).

In another and/or additional preferred embodiment of the method and/or cell according to the invention, the mixture of at least three different sialylated oligosaccharides according to the invention may be produced by providing a cell for producing an LNB-based sialylated fucosylated oligosaccharide, which cell: 1) Capable of absorbing LNBs in culture as described herein or capable of producing LNBs as described herein; and 2) capable of expressing at least one, preferably at least two, fucosyltransferases as described herein selected from the list comprising alpha-1, 2-fucosyltransferases, alpha-1, 3-fucosyltransferases, alpha-1, 4-fucosyltransferases and alpha-1, 6-fucosyltransferases; 3) Capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein; 4) Capable of expressing at least one, preferably at least two sialyltransferases as described herein selected from the list comprising alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase; 5) Capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein; and 6) optionally, capable of producing UDP-galactose.

In another and/or additional preferred embodiment of the method and/or the cell according to the invention, the mixture of at least three different sialylated oligosaccharides according to the invention may be produced by providing a cell for producing a LacNAc-based sialylated fucosylated oligosaccharide, the cell: 1) Capable of absorbing LacNAc in culture as described herein or capable of producing LacNAc as described herein; and 2) capable of expressing at least one, preferably at least two, fucosyltransferases as described herein selected from the list comprising alpha-1, 2-fucosyltransferases, alpha-1, 3-fucosyltransferases, alpha-1, 4-fucosyltransferases and alpha-1, 6-fucosyltransferases; 3) Capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein; 4) Capable of expressing at least one, preferably at least two sialyltransferases as described herein selected from the list comprising alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase; 5) Capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein; and 6) optionally, UDP-galactose can be produced.

In another and/or additional preferred embodiment of the method and/or cell according to the invention, a cell is provided that is additionally adapted for the production of lactose-based neutral nonfucosylated oligosaccharides, lactose-based neutral fucosylated oligosaccharides, LNB-based neutral nonfucosylated oligosaccharides, LNB-based neutral fucosylated oligosaccharides, lacNAc-based neutral nonfucosylated oligosaccharides and/or LacNAc-based neutral fucosylated oligosaccharides.

A cell adapted for the production of lactose-based neutral nonfucosylated oligosaccharides: 1) Capable of absorbing lactose in culture as described herein or capable of producing lactose upon absorption of glucose by the action of b-1, 4-galactosyltransferase as described herein; and 2) N-acetylglucoseaminotransferase, preferably galactoside beta-1, 3-N-acetylglucoseaminotransferase, as described herein; 3) Optionally capable of expressing at least one, preferably at least two galactosyltransferases as described herein selected from the list comprising: n-acetylglucosamine beta-1, 3-galactosyltransferase, N-acetylglucosamine beta-1, 4-galactosyltransferase, alpha-1, 3-galactosyltransferase, alpha-1, 4-galactosyltransferase; and 4) optionally capable of exhibiting at least one, preferably at least two, N-acetylgalactosamine aminotransferases as described herein selected from the list comprising: alpha-1, 3-N-acetylgalactosamine transferase and beta-1, 3-N-acetylgalactosamine transferase; and 5) a nucleotide-sugar capable of synthesizing each of the glycosyltransferases (if present).

A cell adapted for the production of lactose-based neutral fucosylated oligosaccharides: 1) Capable of absorbing lactose in culture as described herein or capable of producing lactose upon absorption of glucose by the action of b-1, 4-galactosyltransferase as described herein; and 2) capable of expressing at least one, preferably at least two, fucosyltransferases as described herein selected from the list comprising alpha-1, 2-fucosyltransferases, alpha-1, 3-fucosyltransferases, alpha-1, 4-fucosyltransferases and alpha-1, 6-fucosyltransferases; 3) Optionally capable of expressing an N-acetylglucosaminyl transferase, preferably a galactoside beta-1, 3-N-acetylglucosaminyl transferase as described herein; 4) Optionally capable of expressing at least one, preferably at least two galactosyltransferases as described herein selected from the list comprising N-acetylglucosamine β -1, 3-galactosyltransferase, N-acetylglucosamine β -1, 4-galactosyltransferase, α -1, 3-galactosyltransferase, α -1, 4-galactosyltransferase; 5) Optionally capable of expressing at least one, preferably at least two N-acetylgalactosamine aminotransferases as described herein selected from the list comprising: alpha-1, 3-N-acetylgalactosamine transferase and beta-1, 3-N-acetylgalactosamine transferase; 6) Capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein, and 7) capable of synthesizing a nucleotide-sugar of each of the glycosyltransferases (if present).

Cells adapted for production of neutral nonfucosylated oligosaccharides based on LNB are capable of producing LNB as described herein or capable of absorbing LNB in culture as described herein; and can synthesize UDP-Gal.

A cell adapted for producing neutral fucosylated oligosaccharides based on LNB: 1) Capable of absorbing LNBs in culture as described herein or capable of producing LNBs as described herein; and 2) capable of expressing at least one, preferably at least two, fucosyltransferases as described herein selected from the list comprising alpha-1, 2-fucosyltransferases, alpha-1, 3-fucosyltransferases, alpha-1, 4-fucosyltransferases and alpha-1, 6-fucosyltransferases; 3) Capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein; and 4) optionally, capable of producing UDP-galactose.

Cells adapted for producing LacNAc-based neutral nonfucosylated oligosaccharides are capable of producing LacNAc as described herein; and can synthesize UDP-Gal.

Cells adapted for the production of LacNAc-based neutral fucosylated oligosaccharides: 1) Capable of absorbing LacNAc in culture as described herein or capable of producing LacNAc as described herein; and 2) capable of expressing at least one, preferably at least two, fucosyltransferases as described herein selected from the list comprising alpha-1, 2-fucosyltransferases, alpha-1, 3-fucosyltransferases, alpha-1, 4-fucosyltransferases and alpha-1, 6-fucosyltransferases; 3) Capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein; 4) Optionally, UDP-galactose can be produced.

In another more preferred embodiment of the method and/or the cell according to the invention, a mixture of at least three different sialylated oligosaccharides may be produced by providing a cell, said oligosaccharides comprising lactose-based sialylated oligosaccharides, such as for example sialyllactose and sialylated lacto-N-trisaccharide and sialylated lacto-N-tetrasaccharide and/or sialylated lacto-N-neotetrasaccharide and fucosylated oligosaccharides may not be produced by providing a cell, which: 1) Capable of absorbing lactose in culture as described herein or capable of producing lactose upon absorption of glucose by the action of b-1, 4-galactosyltransferase as described herein; and 2) capable of expressing at least one, preferably at least two sialyltransferases as described herein selected from the list comprising alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase; 3) Capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein; 4) N-acetylglucosaminyl transferase, preferably galactoside beta-1, 3-N-acetylglucosaminyl transferase, as described herein can be expressed; 5) Capable of expressing at least one, preferably at least two galactosyltransferases as described herein selected from the list comprising N-acetylglucosamine β -1, 3-galactosyltransferases, N-acetylglucosamine β -1, 4-galactosyltransferases, α -1, 3-galactosyltransferases, α -1, 4-galactosyltransferases; 6) Capable of synthesizing UDP-GlcNAc, preferably the cell has an N-acetylglucose amination pathway as defined herein; 7) Capable of synthesizing UDP-Gal, preferably the cell has a galactosylation pathway as defined herein.

In another more preferred embodiment of the method and/or the cell according to the invention, a mixture of at least three different sialylated oligosaccharides comprising sialylated and neutral lactose-based oligosaccharides (such as e.g. fucosyllactose, sialyllactose, LN3, fucosylated LNT and/or LNnT, sialylated lacto-N-trisaccharide and sialylated lacto-N-tetrasaccharide and/or sialylated lacto-N-neotetrasaccharide) may be produced by providing a cell which: 1) Capable of absorbing lactose in culture as described herein or capable of producing lactose upon absorption of glucose by the action of b-1, 4-galactosyltransferase as described herein; and 2) capable of expressing at least one, preferably at least two sialyltransferases as described herein selected from the list comprising alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase; 3) Capable of synthesizing CMP-sialic acid, preferably the cell has a sialylation pathway as defined herein; 4) Capable of expressing at least one, preferably at least two, fucosyltransferases as described herein selected from the list comprising alpha-1, 2-fucosyltransferases, alpha-1, 3-fucosyltransferases, alpha-1, 4-fucosyltransferases and alpha-1, 6-fucosyltransferases; 5) Capable of synthesizing GDP-fucose, preferably the cell has a fucosylation pathway as defined herein; 6) N-acetylglucosaminyl transferase, preferably galactoside beta-1, 3-N-acetylglucosaminyl transferase, as described herein can be expressed; 7) Capable of expressing at least one, preferably at least two galactosyltransferases as described herein selected from the list comprising N-acetylglucosamine β -1, 3-galactosyltransferases, N-acetylglucosamine β -1, 4-galactosyltransferases, α -1, 3-galactosyltransferases, α -1, 4-galactosyltransferases; 8) Capable of synthesizing UDP-GlcNAc, preferably the cell has an N-acetylglucose amination pathway as defined herein, 9) capable of synthesizing UDP-Gal, preferably the cell has a galactosylation pathway as defined herein.

Exemplary methods and cells according to the invention are described in the examples section. It is emphasized that these embodiments show at least one way of producing a particular mixture. It will be appreciated by those of ordinary skill in the art that if any of the expressed enzymes have the same catalytic activity, preferably to a similar extent, that activity can be readily assessed via routine experimentation, any of the expressed enzymes can be replaced with another enzyme, wherein the activity of the enzyme is compared to the activity of a reference enzyme (e.g., in vitro conversion of a substrate) as disclosed herein.

In a preferred embodiment of the method and/or cell of the invention, any of the oligosaccharides, more preferably all of the oligosaccharides, are translocated to the outside of the cell by passive transport, i.e. without the need to consume energy from the cell by means of an active transport system.

In a preferred embodiment of the method and/or cell of the invention, the cell uses at least one precursor for the production of any one or more of the oligosaccharides. As explained in the definitions disclosed herein, the term "precursor" is to be understood. In a more preferred embodiment, the cells use two or more precursors for the production of any one or more of the oligosaccharides.

In a preferred embodiment of the method of the invention, precursors and/or acceptors are fed into the culture for synthesis of any of the oligosaccharides in the mixture. As explained in the definitions disclosed herein, the term "receptor" is to be understood. In another preferred embodiment of the method, at least two precursors and/or acceptors are fed into the culture for synthesizing any one or more, preferably all, of the oligosaccharides in the mixture. This may be applicable if two or more glycosyltransferases of the same class (e.g. a2, 3-sialyltransferase) are used, which have different affinities (e.g. one sialyltransferase with affinity for lactose and another sialyltransferase with affinity for LNB) for producing a mixture of oligosaccharides according to the invention.

In another specific example of a method and/or cell as described herein, the cell produces a precursor for producing any of the oligosaccharides. In a preferred embodiment, the cells produce one or more precursors for synthesizing the oligosaccharide mixture. In a more preferred embodiment, the cell is modified for optimal production of any of the precursors for synthesis of any of the oligosaccharides.

In a preferred embodiment of the method and/or cell of the invention, at least one precursor for producing any of the oligosaccharides is completely converted into any of the oligosaccharides. In a more preferred embodiment, the cells fully convert any of the precursors to any of the oligosaccharides.

In another preferred embodiment of the method and/or cell of the invention, the cell is further metabolically engineered for use in

i) Modified expression of endogenous membrane proteins and/or

ii) modified activity of endogenous membrane proteins and/or

iii) Expression of homologous membrane proteins and/or

iv) expression of heterologous membrane proteins,

wherein the membrane protein is involved in secretion of any of said oligosaccharides outside the cell. The cell may exhibit one of the membrane proteins involved in secreting any of the oligosaccharides from the cell to the outside of the cell. The cell may also exhibit more than one of the membrane proteins. Any of the membrane proteins may translocate one or more of the oligosaccharides to the outside of the cell. The cell producing a mixture of at least three oligosaccharides may translocate any of the oligosaccharides, the cell comprising a passive diffuser (passive diffusion), a channel membrane protein, a membrane transporter, a membrane carrier protein.

In another preferred embodiment of the method and/or cell of the invention, the cell is further metabolically engineered for use in

i) Modified expression of endogenous membrane proteins and/or

ii) modified activity of endogenous membrane proteins and/or

iii) Expression of homologous membrane proteins and/or

iv) expression of heterologous membrane proteins,

wherein the membrane protein is involved in the uptake of precursors and/or receptors for synthesis of any of the oligosaccharides. The cell may represent one of the membrane proteins that is involved in the uptake of any type of precursor and/or receptor for the synthesis of any of the oligosaccharides. The cell may also exhibit more than one of the membrane proteins involved in the uptake of at least one of the precursors and/or receptors. The cells may be modified for uptake of more than one precursor and/or receptor for synthesis of any of the oligosaccharides. In a preferred embodiment, the cells are modified to absorb all of the desired precursors. In another preferred embodiment, the cells are modified to absorb all receptors.

In a more preferred embodiment of the method and/or cell of the invention, the membrane protein is selected from the list comprising: transporter, P-bond hydrolytically driven transporter, β -tubulin, auxiliary transporter, putative transporter, and phosphotransferase driven group translocator. In even more preferred embodiments of the methods and/or cells of the invention, the transport protein comprises MFS transporter, sugar efflux transporter, and transferrin export protein. In another more preferred embodiment of the methods and/or cells of the invention, the P-P-bond hydrolysis-driven transporter comprises an ABC transporter and a transferrin export protein.

In another preferred embodiment of the method and/or cell of the invention, the membrane protein provides improved production of any one of the oligosaccharides, preferably all of the oligosaccharides. In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, the membrane protein enables the efflux of any of the oligosaccharides, preferably all of the oligosaccharides. In alternative and/or additional preferred embodiments of the methods and/or cells of the invention, the membrane protein provides enhanced efflux of any of the oligosaccharides, preferably all of the oligosaccharides.

In a more preferred embodiment of the method and/or cell of the invention, the cell exhibits a membrane protein belonging to the MFS transporter family, such as, for example, mdfA polypeptides from the MdfA family of multi-drug transporter MdfA from species comprising escherichia coli (UniProt ID P0 AEY), mo Jinsi cronobacter (Cronobacter muytjensii) (UniProt ID A0A2T7ANQ 9), citrobacter young (Citrobacter youngae) (UniProt ID D4BC 23) and Lei Jinsi burg yolker (Yokenella regensburgei) (UniProt ID G9Z5F 4). In another more preferred embodiment of the method and/or cell of the invention, the cell exhibits a membrane protein belonging to the family of sugar efflux transporters such as, for example, setA polypeptides from the SetA family of species comprising escherichia coli (UniProt ID P31675), citrobacter kei (Citrobacter koseri) (UniProt ID A0a078LM 16) and klebsiella pneumoniae (Klebsiella pneumoniae) (UniProt ID A0C4MGS 7). In another more preferred embodiment of the method and/or the cell of the invention, the cell exhibits a membrane protein belonging to the family of transferrin export proteins, such as e.g.E.coli entS (UniProt ID P24077) and E.coli iceT (UniProt ID A0A024L 207). In another more preferred embodiment of the method and/or cell of the invention, the cell exhibits a membrane protein belonging to the family of ABC transporters such as, for example, oppF (UniProt ID P77737) from E.coli, lmrA (UniProt ID A0A1V0NEL 4) from the diacetyl lactic acid variant (Lactococcus lactis subsp. Lacti by. Diacetylactis) of the lactococcus lactis and Blon_2475 (UniProt ID B7GPD 4) from the bifidobacterium infantis (Bifidobacterium longum subsp. Infentis).

In a preferred embodiment of the method and/or cell of the invention, the cell confers enhanced phage resistance. This enhancement of phage resistance may result from reduced expression of the endogenous membrane protein and/or mutation of the gene encoding the endogenous membrane protein. The term "phage insensitivity (phage insensitive)" or "anti-phage resistance" or "anti-phage profile (phage resistant profile)" is understood to mean a bacterial strain that is less sensitive and preferably insensitive to infection and/or killing by phage and/or growth inhibition. As used herein, the term "anti-phage activity" or "resistant to infection by at least one phage (resistant to infection by at least one phage)" refers to an increase in resistance of bacterial cells of the same species to infection by at least one phage family compared to bacterial cells of the same species at the same developmental stage (e.g., culture state) that do not exhibit a functional phage resistance system, as can be determined by, for example, bacterial viability, phage lysogeny, phage genome replication, and phage genome degradation. The phage may be a lytic phage or a temperate (lysogenic) phage. The membrane proteins involved in phage resistance of cells comprise OmpA, ompC, ompF, ompT, btuB, tolC, lamB, fhuA, tonB, fadL, tsx, fepA, yncD, phoE, nfrA and homologs thereof.

In a preferred embodiment of the method and/or cell of the invention, the cell imparts a reduced viscosity. The reduced cell viscosity may be obtained by modified cell wall biosynthesis. Cell wall biosynthesis may be modified, including, for example, reduction or elimination of poly-N-acetyl-glucosamine, intestinal co-antigen, cellulose, colanic acid (colanic acid), core oligosaccharide (core oligosaccharide), osmotically regulated periplasmic and glyceroglycol (glucosyl glucoside), glycan and trehalose (trehalose) synthesis.

According to another embodiment of the method and/or the cell of the invention, the cell is capable of producing phosphoenolpyruvate (PEP). In a preferred embodiment of the method and/or cell of the invention, the cell is modified for enhanced production and/or supply of PEP compared to an unmodified precursor cell.

In a preferred embodiment and as a means for enhanced production and/or supply of PEPs, one or more PEP-dependent, sugar-delivering phosphotransferase systems are disrupted, such as (but not limited to): 1) N-acetyl-D-glucosamine Npi-phosphotransferase (EC 2.7.1.193), which is encoded, for example, by the nagE gene (or the nagABCD cluster) in E.coli or Bacillus species, 2) ManXYZ, which encodes the import of exogenous hexoses (mannose, glucose, glucosamine, fructose, 2-deoxyglucose, mannosamine, N-acetylglucosamine, etc.) and the release of phosphate into the cytoplasm of the enzyme ll Man complex (mannosePTS permease, protein-Npi-histidine-D-mannose phosphotransferase), 3) glucose-specific PTS transporter (encoded, for example, by PtsG/Crr), which absorbs glucose in the cytoplasm and forms glucose-6-phosphate, 4) sucrose-specific PTS transporter, which absorbs sucrose in the cytoplasm and forms sucrose-6-phosphate, 5) fructose-specific PTS transporter (encoded, for example, by the genes fruA and fruB, which encode and the kinase fruK, which absorbs in the first step and forms lactose-6-phosphate in the step 1, and forms lactose-6-phosphate in the step 1-glucose-6-phosphate, and the second step (e.g., 4) which absorbs lactose-6-phosphate in the step 1, and forms lactose-6-phosphate, respectively), 8) Mannitol-specific PTS enzymes that absorb mannitol and/or sorbitol and form mannitol-1-phosphate or sorbitol-6-phosphate, respectively, and 9) trehalose-specific PTS enzymes that absorb trehalose and form trehalose-6-phosphate.

In another and/or additional preferred embodiments and as a means for enhanced production and/or supply of PEP, the complete PTS system is disrupted by disruption of the PtsIH/Crr gene cluster. PtsI (enzyme I) is phosphoenolpyruvate which serves as E.coli K-12: cytoplasmic proteins of the gateway of the sugar phosphotransferase system (PTS sugar). PtsI is one of the two (PtsI and PtsH) sugar non-specific protein components of PTS sugar, which, together with sugar-specific endomembrane permeases, affect a phosphotransferase cascade that causes coupled phosphorylation and transport of various carbohydrate matrices. HPr (histidine-containing protein) is one of two sugar non-specific protein components of PTS sugar. It accepts phosphoryl groups from phosphorylase I (PtsI-P) and then transfers it to the EIIA domain of any of a number of sugar-specific enzymes of PTS sugar (collectively referred to as enzyme II). In reactions requiring PtsH and PtsI, crr or EIIAGlc is phosphorylated by PEP.

In another and/or additional preferred embodiments, the cells are further modified to compensate for the absence of the PTS system of the carbon source by the introduction and/or overexpression of the corresponding permease. Such are, for example, permease or ABC transporters, including but not limited to, transporters that specifically import lactose, such as the transporter encoded by the LacY gene from escherichia coli; a transporter that imports sucrose, such as a transporter encoded by the cscB gene from escherichia coli; glucose-import transporters, such as the transporter encoded by the galP gene from e.coli; a fructose-infused transporter, such as the transporter encoded by the fruI gene from streptococcus mutans (Streptococcus mutans); or sorbitol/mannitol ABC transporter, such as the transporter encoded by the cluster SmoEFGK of rhodobacter sphaeroides (Rhodobacter sphaeroides); trehalose/sucrose/maltose transporter, such as the transporter encoded by the gene cluster ThuEFGK of rhizobium meliloti (Sinorhizobium meliloti); and N-acetylglucosamine/galactose/glucose transporters, such as the transporter encoded by NagP of osnescentella (Shewanella oneidensis). Examples of combinations of PTS deletions with overexpression of alternative transporters are: 1) a deletion of the glucose PTS system (e.g., ptsG gene) is combined with the introduction and/or overexpression of a glucose permease (e.g., galP of glcP), 2) a deletion of the fructose PTS system (e.g., one or more of the fruB, fruA, fruK genes) is combined with the introduction and/or overexpression of a fructose permease (e.g., fruI), 3) a deletion of the lactose PTS system is combined with the introduction and/or overexpression of a lactose permease (e.g., lacY), and/or 4) a deletion of the sucrose PTS system is combined with the introduction and/or overexpression of a sucrose permease (e.g., cscB).

In another preferred embodiment, the cells are modified to compensate for PTS system deletions of the carbon source by introducing a carbohydrate kinase, such as glucokinase (EC 2.7.1.1, EC 2.7.1.2, EC 2.7.1.63), galactokinase (EC 2.7.1.6) and/or fructokinase (EC 2.7.1.3, EC 2.7.1.4). Examples of combinations of PTS deletions with alternative transporter and kinase hypermanifestations are: 1) The deletion of the glucose PTS system (e.g., ptsG gene), the introduction and/or overexpression of glucose-permease (e.g., galP of glcP), the introduction and/or overexpression of glucokinase (e.g., glk), and/or 2) the deletion of the fructose PTS system (e.g., one or more of fruB, fruA, fruK genes), the introduction and/or overexpression of fructose-permease (e.g., fruI), the introduction and/or overexpression of fructokinase (e.g., frk or mak).

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

In a more preferred embodiment, the cells are modified to overexpress any one or more of the polypeptides comprising ppsA from E.coli (UniProt ID P23538), PCK from C.glutamicum (UniProt ID Q6F5A 5), pcka from E.coli (UniProt ID P22259), eda from E.coli (UniProt ID P0A 955), maeA from E.coli (UniProt ID P26616) and maeB from E.coli (UniProt ID P76558).

In another and/or additional preferred embodiment, the cell is modified to exhibit any one or more polypeptides having phosphoenolpyruvate synthase activity, phosphoenolpyruvate carboxykinase activity, oxaloacetate decarboxylase activity, or malate dehydrogenase activity.

In another and/or additional preferred embodiments and as a means for enhanced production and/or supply of PEP, the cells are modified by the deletion of genes encoding phosphoenolpyruvate carboxylase activity and/or pyruvate kinase activity, preferably by the deletion of a gene encoding phosphoenolpyruvate carboxylase, pyruvate carboxylase activity and/or pyruvate kinase activity.

In an illustrative embodiment, the cells are genetically modified by different adaptations such as a combination of overexpression of phosphoenolpyruvate synthase with a deletion of a pyruvate kinase gene, a combination of overexpression of phosphoenolpyruvate synthase with a deletion of a phosphoenolpyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate synthase with a deletion of a pyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate carboxykinase with a deletion of a pyruvate kinase gene, a combination of overexpression of phosphoenolpyruvate carboxykinase with a deletion of a phosphoenolpyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate carboxykinase with a deletion of a pyruvate carboxylase gene, a combination of overexpression of oxaloacetate decarboxylase with a deletion of a pyruvate kinase gene, a combination of overexpression of oxaloacetate decarboxylase with a deletion of a pyruvate carboxylase gene, a combination of overexpression of malate dehydrogenase with a deletion of a pyruvate kinase gene, a combination of overexpression of malate dehydrogenase with a deletion of a phosphoenolpyruvate carboxylase gene, and/or a combination of a deletion of malate dehydrogenase with a deletion of a pyruvate carboxylase gene.

In another illustrative embodiment, the cells are genetically modified by different adaptations such as a combination of overexpression of phosphoenolpyruvate synthase with phosphoenolpyruvate carboxykinase, a combination of overexpression of phosphoenolpyruvate synthase with overexpression of oxaloacetate decarboxylase, a combination of overexpression of phosphoenolpyruvate synthase with overexpression of malate dehydrogenase, a combination of overexpression of phosphoenolpyruvate carboxykinase with overexpression of oxaloacetate decarboxylase, a combination of overexpression of phosphoenolpyruvate carboxykinase with overexpression of malate dehydrogenase, a combination of overexpression of oxaloacetate decarboxylase with overexpression of malate dehydrogenase, a combination of overexpression of phosphoenolpyruvate synthase with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase, a combination of overexpression of phosphoenolpyruvate synthase with overexpression of phosphoenolpyruvate carboxykinase and overexpression of malate dehydrogenase, a combination of overexpression of phosphoenolpyruvate synthase with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate dehydrogenase and the overexpression of malate dehydrogenase, a combination of the overexpression of phosphoenolpyruvate synthase with the overexpression of phosphoenolpyruvate carboxylase and the overexpression of malate dehydrogenase and the overexpression of oxaloacetate dehydrogenase.

In another illustrative embodiment, the cells are genetically modified by different adaptations, such as a combination of overexpression of phosphoenolpyruvate synthase with phosphoenolpyruvate carboxykinase and a deletion of a pyruvate kinase gene, a combination of overexpression of phosphoenolpyruvate synthase with oxaloacetate decarboxylase and a deletion of a pyruvate kinase gene, a combination of overexpression of phosphoenolpyruvate synthase with malate dehydrogenase and a deletion of a pyruvate kinase gene, a combination of overexpression of phosphoenolpyruvate carboxykinase with oxaloacetate decarboxylase and a deletion of a pyruvate kinase gene, a combination of overexpression of phosphoenolpyruvate carboxykinase with a deletion of malate dehydrogenase and a deletion of a pyruvate kinase gene, a combination of overexpression of oxaloacetate decarboxylase with a combination of a overexpression of malate dehydrogenase and a deletion of a pyruvate kinase gene overexpression of phosphoenolpyruvate synthase and overexpression of oxaloacetate decarboxylase and deletion of pyruvate kinase gene, overexpression of phosphoenolpyruvate synthase and overexpression of phosphoenolpyruvate carboxykinase and overexpression of malate dehydrogenase and deletion of pyruvate kinase gene, overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and overexpression of malate dehydrogenase and overexpression of pyruvate kinase gene, overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and overexpression of malate dehydrogenase and deletion of pyruvate kinase gene, the overexpression of phosphoenolpyruvate synthase is combined with the overexpression of oxaloacetate decarboxylase, the overexpression of malate dehydrogenase and the deletion of the pyruvate kinase gene.

In another illustrative embodiment, the cells are genetically modified by different adaptations such as a combination of overexpression of phosphoenolpyruvate synthase with overexpression of phosphoenolpyruvate carboxykinase and deletion of phosphoenolpyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate synthase with overexpression of oxaloacetate decarboxylase and deletion of phosphoenolpyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate synthase with overexpression of malate dehydrogenase and deletion of phosphoenolpyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate carboxykinase with overexpression of oxaloacetate decarboxylase and deletion of phosphoenolpyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate carboxykinase with overexpression of phosphoenolpyruvate carboxykinase and overexpression of phosphoenolpyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate carboxykinase with overexpression of malate dehydrogenase and overexpression of phosphoenolpyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate carboxylase with overexpression of phosphoenolpyruvate carboxylase and overexpression of phosphoenolpyruvate carboxylase gene, a combination of phosphoenolpyruvate synthase with overexpression of phosphoenolpyruvate carboxylase and overexpression of phosphoenolpyruvate carboxylase, and deletion of phosphoenolpyruvate carboxylase gene, a combination of phosphoenolpyruvate carboxylase with overexpression of phosphoenolpyruvate dehydrogenase and of phosphoenolpyruvate carboxylase, the overexpression of phosphoenolpyruvate carboxykinase is combined with the overexpression of oxaloacetate decarboxylase and the overexpression of malate dehydrogenase and the deletion of the phosphoenolpyruvate carboxylase gene, and the overexpression of phosphoenolpyruvate synthase is combined with the overexpression of oxaloacetate decarboxylase and the overexpression of malate dehydrogenase and the deletion of the phosphoenolpyruvate carboxylase gene.

In another illustrative embodiment, the cells are genetically modified by different adaptations, the different adaptations are such as a combination of overexpression of phosphoenolpyruvate synthase with overexpression of phosphoenolpyruvate carboxykinase and deletion of pyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate synthase with overexpression of oxaloacetate decarboxylase and deletion of pyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate synthase with overexpression of malate dehydrogenase and deletion of pyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate carboxykinase with overexpression of oxaloacetate decarboxylase and deletion of pyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate carboxykinase with overexpression of malate dehydrogenase and deletion of pyruvate carboxylase gene, a combination of overexpression of oxaloacetate decarboxylase with overexpression of malate dehydrogenase and deletion of pyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate synthase with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and overexpression of pyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate synthase with overexpression of phosphoenolpyruvate carboxykinase and overexpression of oxaloacetate decarboxylase and the deletion of pyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate carboxylase and the enzyme, and the combination of the overexpression of phosphoenolpyruvate carboxylase and the expression of the enzyme, and the combination of the overexpression of phosphoenolpyruvate carboxylase and the enzyme and the deletion of the enzyme, the overexpression of phosphoenolpyruvate carboxykinase is combined with the overexpression of oxaloacetate decarboxylase and the overexpression of malate dehydrogenase and the deletion of pyruvate carboxylase gene, and the overexpression of phosphoenolpyruvate synthase is combined with the overexpression of oxaloacetate decarboxylase and the overexpression of malate dehydrogenase and the deletion of pyruvate carboxylase gene.

In another exemplary embodiment, the cells are genetically modified by different adaptations such as a combination of overexpression of phosphoenolpyruvate synthase with a deletion of a phosphoenolpyruvate carboxykinase gene and a phosphoenolpyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate synthase with a deletion of a oxaloacetate decarboxylase and a deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate synthase with a overexpression of malate dehydrogenase and a deletion of a pyruvate kinase gene and a phosphoenolpyruvate carboxylase gene, a combination of overexpression of phosphoenolpyruvate carboxykinase with a overexpression of a phosphoenolpyruvate carboxykinase gene and a deletion of a phosphoenolpyruvate carboxylase gene, a combination of overexpression of a phosphoenolpyruvate carboxykinase with a overexpression of a malate dehydrogenase gene and a deletion of a phosphoenolpyruvate carboxylase gene, a combination of a overexpression of a oxaloacetate decarboxylase with a phosphoenolpyruvate dehydrogenase gene and a deletion of a phosphoenolpyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, a deletion of a phosphoenolpyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, a combination of a phosphoenolpyruvate carboxylase gene and a phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase and the overexpression of oxaloacetate decarboxylase and the overexpression of malate dehydrogenase are combined with the deletion of the pyruvate kinase gene and the phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate carboxykinase and the overexpression of oxaloacetate decarboxylase and the deletion of the malate dehydrogenase gene and the phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase and the overexpression of oxaloacetate decarboxylase and the overexpression of malate dehydrogenase are combined with the deletion of the pyruvate kinase gene and the phosphoenolpyruvate carboxylase gene.

In another exemplary embodiment, the cells are genetically modified by different adaptations such as a combination of overexpression of phosphoenolpyruvate synthase and deletion of phosphoenolpyruvate carboxykinase and a combination of the overexpression of pyruvate kinase gene and pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, a combination of the overexpression of phosphoenolpyruvate synthase and the overexpression of oxaloacetate decarboxylase and a combination of the overexpression of pyruvate kinase gene and pyruvate carboxylase gene and the deletion of phosphoenolpyruvate carboxylase gene, a combination of the overexpression of phosphoenolpyruvate synthase and the overexpression of pyruvate kinase gene and the deletion of pyruvate carboxylase gene and phosphoenolpyruvate carboxylase gene, a combination of the overexpression of phosphoenolpyruvate carboxykinase and the overexpression of phosphoenolpyruvate carboxylase gene and the deletion of pyruvate carboxylase gene, a combination of the overexpression of phosphoenolpyruvate carboxykinase and the overexpression of pyruvate carboxylase gene and the deletion of pyruvate carboxylase gene and the gene of pyruvate carboxylase gene and the deletion of pyruvate carboxylase gene and the overexpression of phosphoenolpyruvate carboxylase gene and the combination of the overexpression of phosphoenolpyruvate carboxylase gene and the deletion of the phosphoenolpyruvate carboxylase gene and the combination of the overexpression of phosphoenolpyruvate carboxylase gene and the deletion of the phosphoenolpyruvate carboxylase gene and the phosphoenolpyruvate carboxylase gene, the overexpression of phosphoenolpyruvate synthase and the overexpression of phosphoenolpyruvate carboxykinase and the overexpression of malate dehydrogenase are combined, and the overexpression of phosphoenolpyruvate synthase and the overexpression of phosphoenolpyruvate carboxylase and the overexpression of oxaloacetate decarboxylase are combined, and the overexpression of pyruvate kinase gene and the overexpression of pyruvate carboxylase and the overexpression of malate dehydrogenase are combined, and the overexpression of phosphoenolpyruvate carboxykinase and the overexpression of oxaloacetate decarboxylase and the overexpression of malate dehydrogenase are combined, and the overexpression of pyruvate kinase gene and the overexpression of pyruvate carboxylase gene and the deletion of phosphoenolpyruvate carboxylase gene and the overexpression of malate dehydrogenase are combined, and the overexpression of phosphoenolpyruvate synthase and the overexpression of oxaloacetate decarboxylase and the overexpression of malate dehydrogenase are combined.

According to another preferred embodiment of the method and/or cell of the invention, the cell comprises a modification for reducing acetate production compared to an unmodified precursor cell. The modification may be any one or more selected from the group comprising: the acetyl-coa synthetase is over-expressed, completely or partially knocked out or less functional pyruvate dehydrogenase and completely or partially knocked out or less functional lactate dehydrogenase.

In another specific example of a method and/or a cell of the invention, the cell is modified in terms of the performance or activity of at least one acetyl-coa synthase, such as acs from e.g. escherichia coli, saccharomyces cerevisiae, homo sapiens, mice. In a preferred embodiment, the acetyl-coa synthetase is an endogenous protein of a cell having modified expression or activity, preferably the endogenous acetyl-coa synthetase is expressed transiently; alternatively, the acetyl-coa synthetase is a heterologous protein that is heterogeneously introduced and expressed in the cell, preferably overexpressed. The endogenous acetyl-coa synthase can have a modified expression in cells that also express a heterologous acetyl-coa synthase. In a more preferred embodiment, the cells are modified in terms of the expression or activity of acetyl-CoA synthetase acs (UniProt ID P27550) from E.coli. In another and/or additional preferred embodiment, the cell is modified in terms of the expression or activity of a functional homolog, variant or derivative of acs from E.coli (UniProt ID P27550) that has at least 80% overall sequence identity to the full length of the polypeptide from E.coli (UniProt ID P27550) and has acetyl-CoA synthetase activity.

In alternative and/or additional further embodiments of the methods and/or cells of the invention, the cells are modified in the expression or activity of at least one pyruvate dehydrogenase, such as, for example, from E.coli, saccharomyces cerevisiae, chineses and brown rats. In a preferred embodiment, the cells have been modified to produce at least one protein having reduced function or disabled pyruvate dehydrogenase activity by having at least one pyruvate dehydrogenase encoding gene partially or completely deleted or mutated in a manner generally known to those having ordinary skill in the art. In a more preferred embodiment, the cell has complete gene knockout in the poxB encoding gene, resulting in a cell lacking pyruvate dehydrogenase activity.

In alternative and/or additional other embodiments of the methods and/or cells of the invention, the cells are modified in the expression or activity of at least one lactate dehydrogenase, such as, for example, from E.coli, saccharomyces cerevisiae, chineses and brown rats. In a preferred embodiment, the cells have been modified to produce at least one protein having reduced function or disabled lactate dehydrogenase activity by having at least one lactate dehydrogenase-encoding gene partially or completely deleted or mutated in a manner generally known to those of ordinary skill in the art. In a more preferred embodiment, the cell has complete gene knockout in the ldhA encoding gene, resulting in a cell lacking lactate dehydrogenase activity.

According to another preferred embodiment of the method and/or cell of the invention, the cell comprises reduced or reduced expression and/or eliminates, reduces or delays activity compared to an unmodified precursor cell, any one or more of the following comprising the proteins: beta-galactosidase, galactosido-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine inhibitor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose: undecanopentenyl-phosphoglucose-1-phosphate transferase, L-fucokinase, L-fucose isomerase, N-acetylneuraminic acid dissociating enzyme, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epi isomerase, EIIAB-Man, EHC-Man, EIID-Man, ushA, galactose-1-phosphate uridyltransferase, glucose-1-phosphate adenyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcription inhibitor IclR, lon protease, glucose-specific translocation phosphotransferase IIBC component ptsG, glucose-specific translocation Phosphotransferase (PTS) IIBC component malX, enzyme IIA ^Glc Beta-glucoside specific PTS enzyme II, fructose specific PTS polyphosphorylated protein FruA and FruB, alcohol dehydrogenase and acetaldehydeDehydrogenase, pyruvate-methylate dissociating enzyme, acetate kinase, phosphoryl transferase (phosphoacyltransferase), phosphoacetyl transferase, pyruvate decarboxylase.

According to another preferred embodiment of the method and/or the cell of the invention, the cell comprises a catabolic pathway for a selected monosaccharide, disaccharide or oligosaccharide, which catabolic pathway is at least partially inactive, said monosaccharide, disaccharide or oligosaccharide being involved in and/or required for the production of any of said oligosaccharides from the mixture.

Another embodiment of the invention provides a method and a cell wherein a mixture comprising at least three different sialylated oligosaccharides is in and/or produced by a fungus, yeast, bacteria, insect, animal, plant and protozoan cell as described herein. The cells are selected from a list comprising bacteria, yeasts, protozoa or fungi, or refer to plant or animal cells. The latter bacteria preferably belong to the Proteus (Proteus) or Thielavia (Firmicum) or the Cyanobacteria (Cyanobacteria) or the Deinococcus-Thermus (Deinococcus). The latter bacteria belonging to the Proteobacteria phylum preferably belong to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacteria are preferably related to any strain belonging to the species Escherichia coli, such as but not limited to Escherichia coli B, escherichia coli C, escherichia coli W, escherichia coli K12, escherichia coli Nissel (Nissle). More particularly, the latter term relates to a cultured E.coli strain designated as E.coli K12 strain, which is well adapted to the laboratory environment and which, unlike the wild-type strain, has lost its ability to grow 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. Thus, the invention is particularly directed to a mutated and/or transformed E.coli cell or strain as indicated above, wherein the E.coli strain is a K12 strain. The E.coli K12 strain is more preferably E.coli MG1655. Bacteria belonging to the latter class of the phylum Thick-walled bacteria preferably belong to the class Bacillus (Bacillus), preferably Lactobacillus (Lactobacillus) with members such as Lactobacillus plantarum (Lactobacillus lactis), leuconostoc mesenteroides (Leuconostoc mesenteroides), or Bacillus (Bacilles) with members such as from the genus Bacillus (Bacillus), such as Bacillus subtilis (Bacillus subtilis) or Bacillus amyloliquefaciens (B.amyloliquefaciens). The latter bacteria belonging to the phylum actinomycetes (actinomycetes) preferably belong to the family Corynebacterium (Corynebacterium) with the member Corynebacterium glutamicum or Corynebacterium nonfermentans (C.afermentans) or to the family Streptomyces (Streptomycetaceae) with the member Streptomyces griseus (Streptomyces griseus) or Streptomyces freudenreichii (S.fradiiae). The latter yeasts preferably belong to the phylum ascomycetes (Ascomycota) or Basidiomycetes (Basidiomycota) or Deuteromycetes (Deuteromonas) or Zygomycotes. The latter yeasts preferably belong to the genus Saccharomyces (members with, for example, saccharomyces cerevisiae (S.bayanus), saccharomyces boulardii (S.boulardii)), pichia (Pichia) with, for example, pichia pastoris (Pichia pastoris), pichia anomala (P.anomala), pichia kluyverensis (P.kluyveri), kluyveromyces (Komagataella), hansenula (Hansenula), kluyveromyces (Kluyveromyces) (members with, for example, kluyveromyces lactis (Kluyveromyces lactis), candida (K.marxianus), kluyveromyces (K.thermals), debaryomyces (Debaromyces), yarrowia (Yarrowia) such as, for example, yarrowia lipolytica (Yarrowia lipolytica)) or Candida such as Candida (Trichosporon) (e.g., candida rugosa (Starmerella bombicola)). The latter yeasts are preferably selected from Pichia pastoris (Pichia pastoris), yarrowia lipolytica (Yarrowia lipolitica), saccharomyces cerevisiae and Kluyveromyces lactis (Kluyveromyces lactis). The latter fungi preferably belong to the genus Rhizopus (Rhizopus), dictyostelium (Dictyostelium), penicillium (Penicillium), white fungus (Mucor) or Aspergillus (Aspergillus). Plant cells include cells of flowering and non-flowering plants, as well as algal cells, such as unicellular algae (Chlamydomonas), chlorella (Chlorella), and the like. Preferably, the plant is a tobacco, alfalfa, rice, tomato, cotton, rapeseed, soybean, maize or corn plant. The latter animal cells are preferably derived from non-human mammals (e.g., cattle, buffalo, pigs, sheep, mice, rats), birds (e.g., chickens, ducks, ostriches, turkeys, pheasants), fish (e.g., swordfish, salmon, tuna, sea bass, trout, catfish), invertebrates (e.g., lobsters, crabs, shrimp, clams, oysters, mussels, sea urchins), reptiles (e.g., snakes, crocodiles, turtles), amphibians (e.g., frogs), or insects (e.g., flies, nematodes) or genetically modified cell lines derived from human cells excluding embryonic stem cells. Both human and non-human mammalian cells are preferably selected from the list comprising: epithelial cells such as, for example, mammary epithelial cells, embryonic kidney cells (e.g., HEK293 or HEK 293T cells), fibroblasts, COS cells, chinese hamster ovary (Chinese hamster ovary; CHO) cells, murine myeloma cells such as, for example, N20, SP2/0 or YB2/0 cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, such as described in WO 21067641. The insect cells of the latter are preferably derived from: spodoptera frugiperda (Spodoptera frugiperda) such as, for example, sf9 or Sf21 cells, bombyx mori (Bombyx mori), cabbage looper (Mamestra brassicae), spodoptera frugiperda (Trichoplusia ni) such as, for example, BTI-TN-5B1-4 cells or drosophila melanogaster (Drosophila melanogaster) such as, for example, drosophila S2 cells. The latter protozoan cells are preferably Leishmania tarabica (Leishmania tarentolae) cells.

In a preferred embodiment of the method and/or cell of the invention, the cell is a viable gram-negative bacterium comprising reduced or eliminated synthetic poly-N-acetyl-glucosamine (PNAG), intestinal co-antigen (Enterobacterial Common Antigen; ECA), cellulose, colanic acid, core oligosaccharide, osmoregulation periplasmic glucan (Osmoregulated Periplasmic Glucan; OPG), glyceroglycosides, glycans and/or trehalose as compared to the unmodified precursor cell.

In a more preferred embodiment of the method and/or cell, the reducing or eliminating of synthetic poly-N-acetyl-glucosamine (PNAG), intestinal bacterial common antigen (ECA), cellulose, cola acid, core oligosaccharide, osmoregulation Periplasmic Glucan (OPG), glycerol glucoside, glycan and/or trehalose is provided by a mutation of any one or more glycosyltransferases involved in the synthesis of any one of the poly-N-acetyl-glucosamine (PNAG), intestinal bacterial common antigen (ECA), cellulose, cola acid, core oligosaccharide, osmoregulation Periplasmic Glucan (OPG), glycerol glucoside, glycan and/or trehalose, wherein the mutation provides a deletion or lower expression of any one of the glycosyltransferases. The glycosyltransferase comprises a glycosyltransferase gene encoding a poly-N-acetyl-D-glucosamine synthase subunit, UDP-N-acetylglucosamine-undecyipentenyl-phosphate N-acetylglucosamine phosphate transferase, fuc4NAc (4-acetamido-4, 6-dideoxy-D-galactose) transferase, UDP-N-acetyl-D-mannuronate transferase, a glycosyltransferase gene encoding a cellulose synthase catalytic subunit, a cellulose biosynthetic protein, a cacao biosynthetic glucuronyl transferase, a cacao biosynthetic galactosyltransferase, a cacao biosynthetic fucosyltransferase, UDP-glucose: undecanoenyl-phosphoglucose-1-phosphotransferase, putative kola biosynthetic glycosyltransferase, UDP-glucuronic acid: LPS (HepIII) glycosyltransferase, ADP-heptose-LPS heptose transferase 2, ADP-heptose: LPS heptyltransferase 1, putative ADP-heptose: LPS heptyltransferase 4, lipopolysaccharide core biosynthesis protein, UDP-glucose: (glucosyl) LPS alpha-1, 2-glucosyltransferase, UDP-D-glucose: (glucosyl) LPS alpha-1, 3-glucosyltransferase, UDP-D-galactose: (glucosyl) lipopolysaccharide-1, 6-D-galactosyltransferase, lipopolysaccharide glucosyltransferase I, lipopolysaccharide core heptyltransferase 3, beta-1, 6-galactofuranonyl transferase, undecyipentenyl-phosphate 4-deoxy-4-formylamino-L-arabinosyltransferase, lipid IVA 4-amino-4-deoxy-L-arabinosyltransferase, bacterial terpene alcohol glucosyltransferase, putative family 2 glycosyltransferase, osmoregulation Periplasmic Glucan (OPG) biosynthetic protein G, OPG biosynthetic protein H, glucosyl glycerate phosphorylase, liver glucose synthase, 1, 4-alpha-glucan branching enzyme, 4-alpha-glucan transferase and trehalose-6-phosphate synthase. In an illustrative embodiment, the cell is mutated in any one or more of the glycosyltransferases, including pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcaI, wcaJ, wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA and yaiP, wherein the mutation provides a deletion or lower expression of any one of the glycosyltransferases.

In alternative and/or additional preferred embodiments of the method and/or the cell, the reduced or eliminated synthesis of poly-N-acetyl-glucosamine (PNAG) is provided by over-expression of a carbon storage modulator-encoding gene, deletion of a na+/h+ antiporter modulator-encoding gene, and/or deletion of a sensor histidine kinase-encoding gene.

Microorganisms or cells as used herein can be found in monosaccharides, disaccharides, oligosaccharides, polysaccharides, polyols, glycerol; complex media comprising molasses, corn steep liquor, peptone, tryptone, yeast extract or mixtures thereof (such as, for example, mixed raw materials, preferably mixed monosaccharide raw materials, such as, for example, hydrolyzed sucrose) are grown on as the primary carbon source. The term "complex medium" means a medium in which the exact composition is not defined. The term primarily means the most important carbon source for the formation of related biological products, biomass, carbon dioxide and/or byproducts (such as acids and/or alcohols, such as acetates, lactates and/or ethanol), i.e. 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% of all required carbon are derived from the above indicated carbon sources. In one embodiment of the invention, the carbon source is the sole carbon source of the organism, i.e. 100% of all required carbon is derived from the carbon sources indicated above. Common primary carbon sources include, but are not limited to, glucose, glycerol, fructose, maltose, lactose, arabinose, malt-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, sucrose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn steep liquor, high fructose syrup, acetate, citrate, lactate and pyruvate. The term complex medium means a medium in which the exact composition is not defined. Examples are molasses, corn steep liquor, peptone, tryptone or yeast extract.

In another preferred embodiment, the microorganisms or cells described herein use split metabolism with a production pathway and a biomass pathway as described in WO2012/007481, which is incorporated herein by reference. The organism may, for example, be genetically modified to alter a gene selected from the group consisting of a phosphoglucose isomerase gene, a phosphofructokinase gene, a fructose-6-phosphate aldolase gene, a fructose isomerase gene and/or fructose: the gene of the PEP phosphotransferase gene accumulates fructose-6-phosphate.

According to another embodiment of the method of the invention, the conditions allowing the production of said oligosaccharides in the mixture comprise culturing the cells of the invention using a medium to produce the oligosaccharide mixture, wherein the medium lacks any precursors and/or acceptors for the production of any of said oligosaccharides and is combined with further addition of at least one precursor and/or acceptor feed (acceptors feed) to the medium for the production of any of said oligosaccharides, preferably for the production of all of said oligosaccharides in the mixture.

In a preferred embodiment, the method for producing an oligosaccharide mixture as described herein comprises at least one of the following steps:

i) Using a medium comprising at least one precursor and/or acceptor;

ii) adding at least one precursor and/or acceptor feed to the medium in a reactor, wherein the total reactor volume is between 250mL (milliliter) and 10.000m ³ Preferably in a continuous manner within (cubic meters) and preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium prior to addition of the precursor and/or acceptor feed;

iii) Adding at least one precursor and/or acceptor feed to the medium in a reactor, wherein the total reactor volume is between 250mL (milliliter) and 10.000m ³ Within (cubic meters), preferably in a continuous manner, and preferably such that the cultivation is effectedThe final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium prior to the addition of the precursor and/or acceptor feed, and wherein preferably the pH of the precursor and/or acceptor feed is set between 3 and 7, and wherein preferably the temperature of the precursor and/or acceptor feed is maintained between 20 ℃ and 80 ℃;

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

v) adding at least one precursor and/or acceptor feed to the medium in a continuous manner over the course of 1, 2, 3, 4, 5 days by means of a feed solution, and 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 ℃ and 80 ℃;

the method produces any one of the oligosaccharides in a concentration of at least 50g/L, preferably at least 75g/L, more preferably at least 90g/L, more preferably at least 100g/L, more preferably at least 125g/L, more preferably at least 150g/L, more preferably at least 175g/L, more preferably at least 200g/L in the final culture.

In another and/or additional preferred embodiments, the method for producing an oligosaccharide mixture as described herein comprises at least one of the following steps:

i) Using a medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor volume is between 250mL and 10.000m ³ (cubic meters) range;

ii) adding at least one precursor and/or acceptor to the culture medium in one pulse or in a discontinuous (pulsed) manner, wherein the total reactor volume is between 250mL (milliliter) and 10.000m ³ Preferably, the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium prior to addition of the precursor and/or acceptor feed pulse (cubic meters);

iii) In response to a pulse or in a discontinuous (pulsed) mannerAdding at least one precursor and/or acceptor feed to the medium in a reactor, wherein the total reactor volume is between 250mL (milliliter) and 10.000m ³ Preferably such that the final volume of the medium does not exceed three times, preferably does not exceed two times, more preferably is less than 2 times the volume of the medium prior to the addition of the precursor and/or acceptor feed pulse, and wherein preferably the pH of the precursor and/or acceptor feed pulse is set between 3 and 7, and wherein preferably the temperature of the precursor and/or acceptor feed pulse is maintained between 20 ℃ and 80 ℃;

iv) at least one precursor and/or acceptor feed is added to the culture medium in a discontinuous (pulsed) manner by means of the feed solution over a period 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;

v) adding at least one precursor and/or acceptor feed to the medium in a discontinuous (pulsed) manner by means of a feed solution over a period 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, and wherein preferably the pH of the feed solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is kept between 20 ℃ and 80 ℃;

The method produces any one of the oligosaccharides in a concentration of at least 50g/L, preferably at least 75g/L, more preferably at least 90g/L, more preferably at least 100g/L, more preferably at least 125g/L, more preferably at least 150g/L, more preferably at least 175g/L, more preferably at least 200g/L in the final culture.

In another more preferred embodiment, the method for producing an oligosaccharide mixture as described herein comprises at least one of the following steps:

i) Using a medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor volume is between 250mL and 10.000m ³ (cubic meters) range;

ii) adding to the medium a medium comprising at least 50, preferably at least 75, preferably to at least one liter of initial reactor volumeLactose feed with 100, more preferably at least 120, more preferably at least 150 grams lactose less, wherein the total reactor volume is between 250mL (milliliters) and 10.000m ³ Preferably in a continuous manner within (cubic meters) and preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than 2 times the volume of the medium prior to the addition of the lactose feed;

iii) Adding to the medium a lactose feed comprising at least 50, 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, wherein the reactor volume is between 250mL and 10.000m ³ Preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed three times, preferably does not exceed two times, more preferably is less than 2 times the volume of the medium before the lactose feed is added, and wherein preferably the pH of the lactose feed is set between 3 and 7, and wherein preferably the temperature of the lactose feed is maintained between 20 ℃ and 80 ℃;

iv) adding lactose feed to the medium in a continuous manner by means of feed solution over a period of 1 day, 2 days, 3 days, 4 days, 5 days;

v) adding lactose feed to the medium in a continuous manner by means of a feed solution over a period of 1, 2, 3, 4, 5 days, and wherein the concentration of the lactose feed solution is 50g/L, preferably 75g/L, more preferably 100g/L, more preferably 125g/L, more preferably 150g/L, more preferably 175g/L, more preferably 200g/L, more preferably 225g/L, more preferably 250g/L, more preferably 275g/L, more preferably 300g/L, more preferably 325g/L, more preferably 350g/L, more preferably 375g/L, more preferably 400g/L, more preferably 450g/L, more preferably 500g/L, even more preferably 550g/L, most preferably 600g/L; and 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 ℃ and 80 ℃;

The method produces any one of the oligosaccharides in a concentration of at least 50g/L, preferably at least 75g/L, more preferably at least 90g/L, more preferably at least 100g/L, more preferably at least 125g/L, more preferably at least 150g/L, more preferably at least 175g/L, more preferably at least 200g/L in the final culture.

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

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

In another specific example of the methods described herein, the host cells are cultured for at least about 60, 80, 100, or about 120 hours or in a continuous manner.

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

Preferably, when performing the method as described herein, the first stage of exponential cell growth is provided by adding a carbon source, preferably glucose or sucrose, to the culture medium before adding the precursor, preferably lactose, to the culture in the second stage.

In another preferred embodiment of the method of the invention, the first phase of exponential cell growth is provided by adding a carbon-based matrix, preferably glucose or sucrose, to the medium comprising a precursor, preferably lactose, followed by a second phase in which only the carbon-based matrix, preferably glucose or sucrose, is added to the culture.

In another preferred embodiment of the method of the invention, the first phase of exponential cell growth is provided by adding a carbon-based matrix, preferably glucose or sucrose, to the medium comprising a precursor, preferably lactose, followed by a second phase in which the carbon-based matrix, preferably glucose or sucrose, and the precursor, preferably lactose, are added to the culture.

In an alternative preferred embodiment, in the method as described herein, the precursor has been added together with the carbon-based matrix in the first stage of the exponential growth.

In another preferred embodiment of the method, the medium contains at least one precursor selected from the group comprising: lactose, galactose, fucose, sialic acid, glcNAc, galNAc, milk-N-disaccharide (LNB) and N-acetyllactosamine (LacNAc).

According to the invention, the method as described herein preferably comprises the step of isolating any one or more, preferably all, of the oligosaccharides from the culture.

The term "isolating from the culture (separating from said cultivation)" means that any one of the oligosaccharides, preferably all of the oligosaccharides, is harvested, collected or extracted from the cells and/or the medium in which they are grown.

Any of the oligosaccharides may be isolated from an aqueous medium in which cells are grown in a conventional manner. In the case where the oligosaccharide is still present in the cells producing the oligosaccharide mixture, conventional means for releasing or extracting the oligosaccharide from the cells may be used, such as cell disruption using: high pH, thermal shock, sonic treatment, french press, homogenization, enzymatic hydrolysis, chemical hydrolysis, solvent hydrolysis, detergents, hydrolysis. This preferably involves clarifying the oligosaccharide-containing mixture to remove suspended particles and contaminants, especially cells, cell components, insoluble metabolites and debris resulting from culturing genetically modified cells. In this step, the oligosaccharide-containing mixture may be clarified in a conventional manner. Preferably, the oligosaccharide-containing mixture is clarified by centrifugation, flocculation, decantation and/or filtration. Another step of separating the oligosaccharide from the oligosaccharide-containing mixture preferably involves removing substantially all proteins and peptides, amino acids, RNA and DNA, as well as any endotoxins and glycolipids that may interfere with the subsequent separation steps, from the oligosaccharide-containing mixture, preferably after it has been clarified. In this step, proteins and related impurities may be removed from the oligosaccharide-containing mixture in a conventional manner. Preferably, proteins, salts, byproducts, dyes, endotoxins and other related impurities are removed from the oligosaccharide-containing mixture by ultrafiltration, nanofiltration, biphasic partitioning, reverse osmosis, microfiltration, activated carbon or carbon treatment, treatment with a non-ionic surfactant, enzymatic digestion, tangential flow high performance filtration, tangential flow ultrafiltration, electrophoresis (e.g., using plate (slide) -polyacrylamide or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE)), affinity chromatography (using affinity ligands, including, e.g., DEAE-sepharose, poly-L-lysine and polymyxin-B, endotoxin-selective adsorbent matrices), ion exchange chromatography (such as, but not limited to, cation exchange, anion exchange, mixed bed ion exchange, inside-to-outside ligand binding), hydrophobic interaction chromatography and/or gel filtration (i.e., particle size exclusion chromatography), in particular by chromatography, more particularly by ion exchange chromatography or hydrophobic interaction chromatography or ligand exchange chromatography. In addition to size exclusion chromatography, the protein and related impurities are retained by the chromatographic medium or selected membranes, and the oligosaccharides remain in the oligosaccharide-containing mixture.

In another preferred embodiment, the method as described herein also provides for further purification of any one or more of the oligosaccharides from the oligosaccharide mixture. Further purification of the oligosaccharides may be achieved, for example, by using (activated) charcoal or carbon, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange to remove any residual DNA, proteins, LPS, endotoxins or other impurities. Alcohol (such as ethanol) and hydroalcoholic (aquo) mixtures may also be used. Another purification step is achieved by crystallization, evaporation or precipitation of the product. Another purification step is drying of the produced oligosaccharides, such as spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying (band dry), band drying (beltdry), vacuum band drying, drum (drying), roller (roller) drying, vacuum drum drying or vacuum drum drying.

In an illustrative embodiment, the isolation and purification of at least one, and preferably all, of the oligosaccharides produced is performed in a process comprising the following steps in any order:

a) The culture or clarified version thereof is combined with a polypeptide having a molecular weight cut-off (molecular weight cut-off; MWCO) of 600-3500Da, ensuring retention of the produced oligosaccharides and passing at least a part of the proteins, salts, by-products, dyes and other related impurities,

b) Using the membrane, the retentate from step a) is subjected to a diafiltration process with an aqueous solution of an inorganic electrolyte, followed by diafiltration with pure water as appropriate to remove excess electrolyte,

c) And collecting the oligosaccharide enriched retentate in salt form from cations of the electrolyte.

In an alternative exemplary embodiment, the isolation and purification of at least one, and preferably all, of the oligosaccharides produced is performed in a process comprising the following steps in any order: subjecting the culture or clarified form thereof to two membrane filtration steps using different membranes, wherein

A membrane having a molecular weight cut-off of between about 300 and about 500 daltons (Dalton), an

Another membrane having a molecular weight cut-off of between about 600 and about 800 daltons.

In an alternative exemplary embodiment, the isolation and purification of at least one, preferably all, of the oligosaccharides produced is performed in a process comprising the steps of treating the culture or clarified form thereof with a strong cation exchange resin in the h+ form and a weak anion exchange resin in the free base form, in any order.

In an alternative exemplary embodiment, the isolation and purification of at least one of the produced oligosaccharides is performed in the following manner. A culture comprising the produced oligosaccharides, biomass, media components and contaminants was applied to the following isolation and purification steps:

i) The biomass is isolated from the culture and,

ii) performing a cation exchanger treatment for removing positively charged species,

iii) Anion exchanger treatment is performed for removing negatively charged species,

iv) carrying out a nanofiltration step and/or an electrodialysis step,

wherein a purified solution comprising the produced oligosaccharides is provided with a purity of greater than or equal to 80%. Optionally, the purified solution is dried by any one or more drying steps selected from the list comprising: spray drying, lyophilization, spray freeze drying, freeze spray drying, ribbon drying, belt drying, vacuum ribbon drying, vacuum belt drying, drum drying, vacuum drum drying, and vacuum drum drying.

In an alternative exemplary embodiment, the isolation and purification of at least one, and preferably all, of the oligosaccharides produced is performed in a process comprising the following steps in any order: subjecting the culture to an enzymatic treatment; removing biomass from the culture; ultra-filtration; nano-filtration; and performing column chromatography. Preferably, such column chromatography is a single column or multiple columns. More preferably, the column chromatography step is simulated moving bed chromatography. Such simulated moving bed chromatography preferably comprises i) at least 4 columns, wherein at least one column comprises a weak or strong cation exchange resin; and/or II) four zones I, II, III and IV having different flow rates; and/or iii) a water-containing debonding agent; and/or iv) an operating temperature of 15 to 60 degrees celsius.

In a particular embodiment, the present invention provides the produced oligosaccharides or oligosaccharide mixture that is dried into a powder by any one or more drying steps selected from the list comprising: spray drying, freeze drying, spray freeze drying, freeze spray drying, ribbon drying, belt drying, vacuum ribbon drying, vacuum belt drying, drum drying, vacuum drum drying and vacuum drum drying, wherein the dry powder contains < 15-wt.% water, preferably < 10-wt.% water, more preferably < 7-wt.% water, most preferably < 5-wt.% water.

In a third aspect, the invention provides the use of a metabolically engineered cell as described herein for the production of a mixture comprising at least three different sialylated oligosaccharides.

To identify oligosaccharides in a mixture comprising at least three different sialylated oligosaccharides produced in a cell as described herein, the monomer building block (e.g., monosaccharide or glycan unit composition), the muta-isomerised configuration of the side chains, the presence and position of substituents, the degree of polymerisation/molecular weight and the bonding mode can be identified by standard methods known in the art such as, for example, methylation analysis, reductive cleavage, hydrolysis, gas chromatography-mass spectrometry (gas chromatography-mass spectrometry; GC-MS), matrix assisted laser desorption/ionization-mass spectrometry (Matrix-assisted laser desorption/ionization-mass spectrometry; MALDI-MS), electrospray ionization-mass spectrometry (Electrospray ionization-mass spectrometry; ESI-MS), high Performance Liquid Chromatography (HPLC) with ultraviolet or refractive index detection, high performance anion exchange chromatography (High-Performance Anion-Exchange chromatography with Pulsed Amperometric Detection; hpa ec-PAD) with pulsed amp detection, electrophoresis (capillary electrophoresis; CE), infrared (in; capillary)/nuclear and raman spectroscopy (Nuclear magnetic resonance). The crystal structure may be solved using, for example, solid state NMR, fourier transform infrared (Fourier transform infrared; FT-IR) spectroscopy, and wide-angle X-ray scattering (WAXS). The degree of polymerization (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 sugar, methods such as, for example, acid-catalyzed hydrolysis, high performance liquid chromatography (high performance liquid chromatography; HPLC) or gas-liquid chromatography (gas-liquid chromatography; GLC) (after conversion to aldol acetate) can be used. To determine the glycosidic bond, the saccharide was methylated with methyl iodide and a strong base in DMSO, hydrolyzed, reduced to partially methylated sugar alcohols, acetylated to methylated aldol acetate, and analyzed by GLC/MS (gas-liquid chromatography combined with mass spectrometry). To determine the oligosaccharide sequence, partial polymerization is performed using acids or enzymes to determine the structure. To identify the mutarotamase configuration, the oligosaccharides are subjected to an enzymatic analysis, e.g. contacting them with an enzyme specific for a specific type of linkage, e.g. beta-galactosidase or alpha-glucosidase, etc., and NMR can be used to analyze the product.

Products comprising oligosaccharide mixtures

In some embodiments, the oligosaccharide mixture produced as described herein is incorporated into a food product (e.g., a human food or feed), a dietary supplement, a pharmaceutical ingredient, a cosmetic ingredient, or a pharmaceutical product. In some embodiments, the oligosaccharide mixture is mixed with one or more ingredients suitable for use in food, dietary supplements, pharmaceutical ingredients, cosmetic ingredients, or pharmaceutical products.

In some embodiments, the dietary supplement comprises at least one prebiotic ingredient and/or at least one probiotic ingredient.

"prebiotics (probiotics)" are substances that promote the growth of microorganisms beneficial to the host, particularly microorganisms in the gastrointestinal tract. In some embodiments, the dietary supplement provides a variety of probiotics, including oligosaccharide mixtures produced and/or purified by the processes disclosed in the present specification to promote the growth of one or more beneficial microorganisms. Examples of the prebiotic component of the dietary supplement include other prebiotic molecules (such as HMO) and plant polysaccharides (such as inulin, pectin, b-glucan and xylooligosaccharides). A "probiotic" product typically contains viable microorganisms that are displaced or added to the gastrointestinal microbiota in order to benefit the recipient. Examples of such microorganisms include Lactobacillus species such as Lactobacillus acidophilus (L. Acidophilus) and Lactobacillus bulgaricus (L. Bulgaricum), bifidobacterium species such as Bifidobacterium animalis (B. Animalis), bifidobacterium longum (B. Longum) and Bifidobacterium infantis (B. Infentis) (e.g., bi-26), and Saccharomyces boulardii (Saccharomyces boulardii). In some embodiments, the oligosaccharide mixture produced and/or purified by the process of the specification is orally administered in combination with such microorganisms.

Examples of other ingredients of the dietary supplement include disaccharides (such as lactose), monosaccharides (such as glucose and galactose), thickeners (such as acacia), acidity regulators (such as trisodium citrate), water, skim milk and flavoring agents.

In some embodiments, the oligosaccharide mixture is incorporated into a human infant food product (e.g., an infant formula). Infant formulas are generally manufactured foods for feeding to infants as a complete or partial replacement for human breast milk. In some embodiments, infant formulas are sold in powder form and are prepared by mixing with water for bottle feeding or cup feeding to infants. The composition of infant formulas is typically designed to substantially mimic human breast milk. In some embodiments, the oligosaccharide mixture produced and/or purified by the process in this specification is included in infant formulas to provide nutritional benefits similar to those provided by oligosaccharides in human breast milk. In some embodiments, the oligosaccharide mixture is mixed with one or more ingredients of an infant formula. Examples of infant formula ingredients include skim milk, carbohydrate sources (e.g., lactose), protein sources (e.g., whey protein concentrate and casein), fat sources (e.g., vegetable oils such as palm oil, high oleic safflower oil, canola oil, coconut oil and/or sunflower oil, and fish oils), vitamins (such as vitamins a, bb, bi2, C, and D), minerals (such as potassium citrate, calcium citrate, magnesium chloride, sodium citrate, and calcium phosphate), and possibly Human Milk Oligosaccharides (HMOs). Such HMOs may include, for example, diFL, milk-N-triose II, LNT, LNnT, milk-N-fucopentaose I, milk-N-neofucopentaose, milk-N-fucopentaose II, milk-N-fucopentaose III, milk-N-fucopentaose V, milk-N-neofucopentaose V, milk-N-disaccharide hexaose I, milk-N-disaccharide hexaose II, 6 '-galactosyl lactose, 3' -galactosyl lactose, milk-N-hexaose, and milk-N-neohexaose.

In some embodiments, the one or more infant formula ingredients comprise skim 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 comprise lactose, whey protein concentrate, and/or high oleic safflower oil.

In some embodiments, the concentration of the oligosaccharide mixture in the infant formula is about the same concentration as the concentration of oligosaccharides typically present in human breast milk. In some embodiments, the concentration of each individual oligosaccharide in the mixture of oligosaccharides in the infant formula is about the same concentration as the concentration of oligosaccharides typically present in human breast milk.

In some embodiments, the oligosaccharide mixture is incorporated into a feed formulation, wherein the feed is selected from the list comprising pet food, animal formulas, veterinary products, post-weaning feed, or creep feed.

Each embodiment disclosed in the context of one aspect of the invention is also disclosed in the context of all other aspects of the invention unless explicitly stated otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization in cell culture described above and below are those well known and commonly employed in the art, laboratory procedures, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization in cell culture. Nucleic acid and peptide synthesis was performed using standard techniques.

Generally, the purification steps are performed according to manufacturer's instructions.

Other advantages follow specific embodiments, examples, and accompanying drawings. It goes without saying that the features mentioned above and those yet to be explained below can be used not only in the respectively specified combination but also in other combinations or independently without departing from the scope of the invention.

The invention relates to the following specific examples:

1. a metabolically engineered cell producing a mixture of at least three different sialylated oligosaccharides, wherein the cell

-glycosyltransferases expressed as sialyltransferases, and

capable of synthesizing nucleotide-sugar CMP-N-acetylneuraminic acid (CMP-Neu 5 Ac), and

-expressing at least one additional glycosyltransferase, an

-being capable of synthesizing one or more nucleotide-sugars, wherein said nucleotide-sugar is a donor for the additional glycosyltransferase.

2. The cell of embodiment 1, wherein the cell is modified by a gene expression module, characterized in that expression from any one of the expression modules is sustained or produced by a natural inducer.

3. The cell of any one of embodiments 1 and 2, wherein the cell produces a mixture of charged and neutral oligosaccharides.

4. The cell of any one of embodiments 1-3, wherein the oligosaccharide mixture comprises at least three different oligosaccharides that differ in degree of polymerization.

5. The cell of any one of embodiments 1 to 4, wherein the cell produces four or more different sialylated oligosaccharides.

6. The cell of any one of embodiments 1 to 5, wherein any one of the additional glycosyltransferases is selected from the list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosyltransferase, N-acetylmannosylaminyltransferase, xylosyltransferase, glucuronyltransferase, galacturonate transferase, glucosaminyltransferase, N-glycolylneuraminidase, rhamnosyltransferase.

7. The cell of any one of embodiments 1-6, wherein the cell is modified in the expression or activity of at least one of the glycosyltransferases.

8. The cell of any one of embodiments 1-7, wherein any one of the additional glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-Neu5Ac.

9. The cell of any one of embodiments 1-8, wherein any one of the additional glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-fucose (GDP-Fuc).

10. The cell of any one of embodiments 1-9, wherein any one of the additional glycosyltransferases is an N-acetylglucosamine aminotransferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine (UDP-GlcNAc).

11. The cell of any one of embodiments 1-10, wherein any one of the additional glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose (UDP-Gal).

12. The cell of any one of embodiments 1 to 11, wherein any one of the nucleotide-sugars is selected from the list comprising: GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP-N-glycolylneuraminic acid (CMP-Neu 5 Gc), UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose.

13. The cell of any one of embodiments 1 to 12, wherein the oligosaccharide mixture comprises at least one neutral oligosaccharide in addition to three or more sialylated oligosaccharides.

14. The cell of any one of embodiments 1 to 13, wherein at least one of the sialylated oligosaccharides is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, containing N-acetylglucosamine, containing N-acetylneuraminic acid, containing N-glycolylneuraminic acid, containing N-acetylgalactosamine, containing rhamnose, containing glucuronate, containing galacturonate and/or containing N-acetylmannosamine.

15. The cell of any one of embodiments 1 to 14, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide.

16. The cell of any one of embodiments 1 to 15, wherein the oligosaccharide mixture comprises at least one oligosaccharide comprising an N-acetylglucosamine monosaccharide unit.

17. The cell of any one of embodiments 1 to 16, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide.

18. The cell of any one of embodiments 1-17, wherein the oligosaccharide mixture comprises at least one oligosaccharide that is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains N-acetylglucosamine, contains N-acetylneuraminic acid, contains N-glycolylneuraminic acid, contains N-acetylgalactosamine, contains rhamnose, contains glucuronate, contains galacturonate, and/or contains N-acetylmannosamine.

19. The cell of any one of embodiments 1 to 18, wherein the cell is further genetically modified for use in

i) Modified expression of endogenous membrane proteins and/or

ii) modified activity of endogenous membrane proteins and/or

iii) Expression of homologous membrane proteins and/or

iv) expression of heterologous membrane proteins,

wherein the membrane protein is involved in secretion of any one of said oligosaccharides of said mixture outside the cell.

20. The cell of any one of embodiments 1 to 19, wherein the cell is further genetically modified for use in

i) Modified expression of endogenous membrane proteins and/or

ii) modified activity of endogenous membrane proteins and/or

iii) Expression of homologous membrane proteins and/or

iv) expression of heterologous membrane proteins,

wherein the membrane protein is involved in the uptake of a precursor for synthesis of any of the oligosaccharides.

21. The cell of any one of embodiments 1 to 20, wherein the cell produces a precursor for synthesizing any one of the oligosaccharides.

22. The cell of any one of embodiments 1 to 21, wherein any one of the oligosaccharides is a mammalian milk oligosaccharide.

23. The cell of any one of embodiments 1 to 22, wherein all of the oligosaccharides are mammalian milk oligosaccharides.

24. The cell of any one of embodiments 1 to 21, wherein any one of the oligosaccharides is an antigen of the human ABO blood group system.

25. A method for producing a mixture of at least three different sialylated oligosaccharides by means of a cell, the method comprising the steps of:

i) Providing a cell that (a) exhibits a glycosyltransferase that is a sialyltransferase and is capable of synthesizing nucleotide-sugar CMP-Neu5Ac, and (b) exhibits at least one additional glycosyltransferase, and (c) is capable of synthesizing at least one or more nucleotide-sugar, wherein the nucleotide-sugar is a donor for the additional glycosyltransferase, and

ii) culturing the cell under conditions permitting expression of the glycosyltransferase and synthesis of the nucleotide-sugar, and

iii) Preferably, at least one of the oligosaccharides is isolated from the culture.

26. The method of embodiment 25, wherein the cell is a metabolically engineered cell of any one of embodiments 1-24.

27. The method of any one of embodiments 25 and 26, wherein the cell produces a mixture of charged and neutral oligosaccharides.

28. The method of any one of embodiments 25 to 27, wherein the oligosaccharide mixture comprises at least three different oligosaccharides that differ in degree of polymerization.

29. The method of any one of embodiments 25 to 28, wherein the cell produces four or more different sialylated oligosaccharides.

30. The method of any one of embodiments 25 to 29, wherein any one of the additional glycosyltransferases is selected from the list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosyltransferase, N-acetylmannosylaminyltransferase, xylosyltransferase, glucuronyltransferase, galacturonate transferase, glucosaminyltransferase, N-glycolylneuraminidase, rhamnosyltransferase.

31. The method of any one of embodiments 25-30, wherein any one of the additional glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-N-acetylneuraminic acid (CMP-Neu 5 Ac).

32. The method of any one of embodiments 25-31, wherein any one of the additional glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-fucose (GDP-Fuc).

33. The method of any one of embodiments 25-32, wherein any one of the additional glycosyltransferases is an N-acetylglucosamine aminotransferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine (UDP-GlcNAc).

34. The method of any one of embodiments 25-33, wherein any one of the additional glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose (UDP-Gal).

35. The method of any one of embodiments 25 to 34, wherein any one of the nucleotide-sugars is selected from the list comprising: GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP-N-glycolylneuraminic acid (CMP-Neu 5 Gc), UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose.

36. The method of any one of embodiments 25-35, wherein the oligosaccharide mixture comprises at least one neutral oligosaccharide in addition to three or more sialylated oligosaccharides.

37. The method of any one of embodiments 25-36, wherein at least one of the sialylated oligosaccharides is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains N-acetylglucosamine, contains N-acetylneuraminic acid, contains N-glycolylneuraminic acid, contains N-acetylgalactosamine, contains rhamnose, contains glucuronate, contains galacturonate, and/or contains N-acetylmannosamine.

38. The method of any one of embodiments 25 to 37, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide.

39. The method of any one of embodiments 25 to 38, wherein the oligosaccharide mixture comprises at least one oligosaccharide comprising an N-acetylglucosamine monosaccharide unit.

40. The method of any one of embodiments 25-39, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide.

41. The method of any one of embodiments 25-40, wherein the oligosaccharide mixture comprises at least one oligosaccharide that is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains N-acetylglucosamine, contains N-acetylneuraminic acid, contains N-glycolylneuraminic acid, contains N-acetylgalactosamine, contains rhamnose, contains glucuronate, contains galacturonate, and/or contains N-acetylmannosamine.

42. The method of any one of embodiments 25-41, wherein the cell uses at least one precursor for synthesizing any one or more of the oligosaccharides, preferably the cell uses two or more precursors for synthesizing any one or more of the oligosaccharides.

43. The method of any one of embodiments 25 to 42, wherein the cell produces a precursor for synthesizing any one of the oligosaccharides.

44. The method of any one of embodiments 25 to 43, wherein any one of the oligosaccharides is a mammalian milk oligosaccharide.

45. The method of any one of embodiments 25 to 44, wherein all of the oligosaccharides are mammalian milk oligosaccharides.

46. The method of any one of embodiments 25-43, wherein any one of the oligosaccharides is an antigen of the human ABO blood group system.

47. The method of any one of embodiments 25 to 46, wherein the precursor for synthesizing any one of the oligosaccharides is fully converted to any one of the oligosaccharides.

48. The method of any one of embodiments 25 to 47, wherein the isolating comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, reverse osmosis, microfiltration, activated carbon or carbon treatment, tangential flow high performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.

49. The method of any one of embodiments 25 to 48, further comprising purifying any one of the oligosaccharides from the cell.

50. The method of any one of embodiments 25 to 49, wherein the purifying comprises at least one of the following steps: using activated carbon or carbon, using charcoal, nanofiltration, ultrafiltration or ion exchange, using alcohols, using hydroalcoholic mixtures, crystallization, evaporation, precipitation, drying, spray drying or lyophilization.

51. The cell of any one of embodiments 1 to 24 or the method of any one of embodiments 25 to 50, wherein the cell is selected from the group consisting of a microorganism, a plant or an animal cell, preferably the microorganism is a bacterium, a fungus or a yeast, preferably the plant is a rice, cotton, rapeseed, soybean, maize or corn plant, preferably the animal is an insect, a fish, a bird or a non-human mammal, preferably the animal cell is a mammalian cell line.

52. The cell of any one of embodiments 1 to 24 and 51 or the method of any one of embodiments 25 to 51, wherein the cell is the following: bacteria, preferably E.coli strains, more preferably E.coli strains as K-12 strains, even more preferably E.coli K12 strains as E.coli MG 1655.

53. The cell of any one of embodiments 1 to 24 and 51 or the method of any one of embodiments 25 to 51, wherein the cell is a yeast cell.

54. Use of a cell as in any one of embodiments 1 to 24, 51 to 53 or a method as in any one of embodiments 25 to 53 for the production of a mixture of at least three different sialylated oligosaccharides.

Furthermore, the present invention relates to the following preferred specific examples:

1. a metabolically engineered cell producing a mixture of at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different sialylated mammalian milk oligosaccharides,

wherein the cell

Metabolically engineered for the production of the mixture, and

-glycosyltransferases expressed as sialyltransferases, and

capable of synthesizing nucleotide-sugar CMP-N-acetylneuraminic acid (CMP-Neu 5 Ac), and

-expressing at least one additional glycosyltransferase, an

-being capable of synthesizing one or more nucleotide-sugars, wherein said nucleotide-sugar is a donor for the additional glycosyltransferase.

2. The cell of preferred embodiment 1, wherein the cell is modified by a gene expression module, characterized in that expression from any of the expression modules is sustained or produced by a natural inducer.

3. The cell of any one of preferred embodiments 1 or 2, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein.

4. The cell of any one of preferred embodiments 1 to 3, wherein the cell produces a mixture of charged and neutral oligosaccharides.

5. The cell of any one of preferred embodiments 1 to 4, wherein the mixture comprises, consists of, or consists essentially of: charged and neutral fucosylated and/or nonfucosylated oligosaccharides.

6. The cell of any one of preferred embodiments 1 to 5, wherein the oligosaccharide mixture comprises at least three different sialylated oligosaccharides that differ in the degree of polymerization.

7. The cell of any one of preferred embodiments 1 to 6, wherein the cell produces at least four, preferably at least five, more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different sialylated oligosaccharides.

8. The cell of any one of preferred embodiments 1 to 7, wherein any one of the additional glycosyltransferases is selected from the list comprising: fucosyltransferases, sialyltransferases, galactosyltransferases, glucosyltransferases, mannosyltransferases, N-acetylglucosaminyl transferases, N-acetylgalactosylaminotransferases, N-acetylmannosylamino transferases, xylosyltransferases, glucuronosyltransferases, galacturonan transferase, glucosaminyl transferases, N-glycolylneuraminidases, rhamnosyltransferases, N-acetylrhamnosyltransferases, UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-A Zhuo Tangan (altrosamine) transferases, UDP-N-acetylglucosamine enolpyruvyl transferases and fucosylaminobferases,

Preferably, the fucosyltransferase is selected from the list comprising: alpha-1, 2-fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-1, 4-fucosyltransferase, alpha-1, 6-fucosyltransferase,

preferably, the sialyltransferase is selected from the list comprising: alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase,

preferably, the galactosyltransferase is selected from the list comprising: beta-1, 3-galactosyltransferase, N-acetylglucosamine beta-1, 3-galactosyltransferase, beta-1, 4-galactosyltransferase, N-acetylglucosamine beta-1, 4-galactosyltransferase, alpha-1, 3-galactosyltransferase and alpha-1, 4-galactosyltransferase,

preferably, the glucosyltransferase is selected from the list comprising: alpha-glucosyltransferase, beta-1, 2-glucosyltransferase, beta-1, 3-glucosyltransferase and beta-1, 4-glucosyltransferase,

preferably, the mannosyltransferase is selected from a list comprising: alpha-1, 2-mannosyltransferase, alpha-1, 3-mannosyltransferase and alpha-1, 6-mannosyltransferase,

Preferably, the N-acetylglucosaminyl transferase is selected from the list comprising: galactoside beta-1, 3-N-acetylglucosaminyl transferase and beta-1, 6-N-acetylglucosaminyl transferase,

preferably, the N-acetylgalactosamine transferase is selected from the list comprising: alpha-1, 3-N-acetylgalactosamine transferase and beta-1, 3-N-acetylgalactosamine transferase.

9. The cell of any one of preferred embodiments 1 to 8, wherein the cell is modified in the expression or activity of at least one of the glycosyltransferases.

10. The cell of any one of preferred embodiments 1-9, wherein any one of the additional glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-Neu5Ac.

11. The cell of any one of preferred embodiments 1-10, wherein any one of the additional glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-fucose (GDP-Fuc).

12. The cell of any one of preferred embodiments 1-11, wherein any one of the additional glycosyltransferases is an N-acetylglucosamine aminotransferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine (UDP-GlcNAc).

13. The cell of any one of preferred embodiments 1-12, wherein any one of the additional glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose (UDP-Gal).

14. The cell of any one of preferred embodiments 1-13, wherein any one of the additional glycosyltransferases is an N-acetylgalactosamine transferase and one of the donor nucleotide-sugars is UDP-N-acetylgalactosamine (UDP-GalNAc).

15. The cell of any one of preferred embodiments 1-14, wherein any one of the additional glycosyltransferases is an N-acetylmannosaminotransferase and one of the donor nucleotide-sugars is UDP-N-acetylmannosamine (UDP-ManNAc).

16. The cell of any one of preferred embodiments 1 to 15, wherein any one of the nucleotide-sugars is selected from the list comprising: GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP-N-glycolylneuraminic acid (CMP-Neu 5 Gc), CMP-Neu4Ac, CMP-Neu5Ac9N ₃ 、CMP-Neu4，5Ac ₂ 、CMP-Neu5，7Ac ₂ 、CMP-Neu5，9Ac ₂ 、CMP-Neu5，7(8，9)Ac ₂ UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose, UDP-2-acetamido-2, 6-dideoxy-L-arabinose (arabino) -4-ketohexose, UDP-2-acetamido-2, 6-dideoxy-L-lyxose (1 yxo) -4-ketohexose, UDP-N-acetyl-L-rhamnose amine (UDP-L-RhaNAc or UDP-2-acetamido-2, 6-dideoxy-L-mannose), dTDP-N-acetylfucose amine, UDP-N-acetylfucose amine (UDP-L-FucNAc or UDP-2-acetamido-2, 6-dideoxy-L-galactose), UDP-N-acetyl-L-neotame amine (pnicose) (UDP-L-PneNAC or UDP-2, 6-dideoxy-L-rhamnose), UDP-N-acetylglucosamine (UDP-N-acetylglucosamine), UDP-N-acetylisomannac or UDP-2-acetylisomannac or UDP-isomannide (UDP-2, 6-isomannide).

17. The cell of any one of preferred embodiments 1 to 16, wherein the cell exhibits one or more polypeptides selected from the list comprising: mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-guanylate transferase, GDP-mannose 4, 6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, GDP-fucose pyrophosphorylase, fucose-1-guanylate transferase, L-glutamylamino-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epi-isomerase, UDP-N-acetylglucosamine 2-epi-isomerase N-acetylmannosamine-6-phosphate 2-epi isomerase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, N-acetylneuraminic acid synthase, N-acetylneuraminic acid dissociase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphate phosphatase, N-acylneuraminic acid cytidylyltransferase, galactose-1-epi isomerase, galactokinase, glucokinase, galactose-1-phosphouridyltransferase, UDP-glucose 4-epi isomerase, glucose-1-phosphouridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epi isomerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein the cell is modified in terms of the performance or activity of any of the polypeptides.

18. The cell of any one of preferred embodiments 1 to 17, wherein the cell is capable of synthesizing at least two nucleotide-sugars, preferably at least three nucleotide-sugars, more preferably at least four nucleotide-sugars, even more preferably at least five nucleotide-sugars.

19. The cell of any one of preferred embodiments 1 to 18, wherein the oligosaccharide mixture comprises at least one neutral oligosaccharide in addition to three or more sialylated oligosaccharides.

20. The cell of preferred embodiment 19, wherein the neutral oligosaccharide is selected from the list comprising neutral fucosylated oligosaccharides and neutral non-fucosylated oligosaccharides.

21. The cell of any one of preferred embodiments 1 to 20, wherein at least one of the sialylated oligosaccharides is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, N-acetylglucosamine-containing, N-acetylneuraminic acid-containing, N-glycolylneuraminic acid-containing, N-acetylgalactosamine-containing, rhamnose-containing, glucuronate-containing, galacturonate-containing and/or N-acetylmannosamine-containing.

22. The cell of any one of preferred embodiments 1 to 21, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide.

23. The cell of any one of preferred embodiments 1 to 22, wherein the oligosaccharide mixture comprises at least one oligosaccharide comprising an N-acetylglucosamine monosaccharide unit.

24. The cell of any one of preferred embodiments 1 to 23, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide.

25. The cell of any one of preferred embodiments 1 to 24, wherein the oligosaccharide mixture comprises at least one oligosaccharide which is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains N-acetylglucosamine, contains N-acetylneuraminic acid, contains N-glycolylneuraminic acid, contains N-acetylgalactosamine, contains rhamnose, contains glucuronate, contains galacturonate and/or contains N-acetylmannosamine.

26. The cell of any one of preferred embodiments 1 to 25, wherein the cell uses at least one precursor for producing any one or more of the oligosaccharides, preferably the cell uses two or more precursors for producing any one or more of the oligosaccharides, said precursors being fed from a culture medium into the cell.

27. The cell of any one of preferred embodiments 1 to 26, wherein the cell produces at least one precursor for producing any one of the oligosaccharides.

28. The cell of any one of preferred embodiments 1 to 27, wherein the at least one precursor for producing any one of the oligosaccharides is fully converted to any one of the oligosaccharides.

29. The cell of any one of preferred embodiments 1 to 28, wherein the cell produces the oligosaccharide intracellularly, and wherein a portion or substantially all of the produced oligosaccharide remains intracellular and/or is excreted outside the cell via passive or active transport.

30. The cell of any one of preferred embodiments 1 to 29, wherein the cell is further genetically modified for use in

i) Modified expression of endogenous membrane proteins and/or

ii) modified activity of endogenous membrane proteins and/or

iii) Expression of homologous membrane proteins and/or

iv) expression of heterologous membrane proteins,

wherein the membrane protein is involved in secreting any of said oligosaccharides from the mixture outside the cell, preferably wherein the membrane protein is involved in secreting all of said oligosaccharides from the mixture from the cell.

31. The cell of any one of preferred embodiments 1 to 30, wherein the cell is further genetically modified for use in

i) Modified expression of endogenous membrane proteins and/or

ii) modified activity of endogenous membrane proteins and/or

iii) Expression of homologous membrane proteins and/or

iv) expression of heterologous membrane proteins,

wherein the membrane protein is involved in the absorption of precursors and/or receptors for the synthesis of any of said oligosaccharides in the mixture, preferably wherein the membrane protein is involved in the absorption of all of said desired precursors, more preferably wherein the membrane protein is involved in the absorption of all of said receptors.

32. The cell of any one of preferred embodiments 30 or 31, wherein the membrane protein is selected from the list comprising: transporter (transporter), P-P-bond hydrolysis-driven transporter (transporter), beta-bungee, auxiliary transporter (transporter), putative transporter, phosphotransferase-driven group translocator,

preferably, the transporter comprises an MFS transporter, a sugar efflux transporter, and a transferrin export protein,

preferably, the P-P-bond hydrolytically driven transporter comprises an ABC transporter and a transferrin export protein.

33. The cell of any one of preferred embodiments 30 to 32, wherein the membrane protein provides improved production and/or is capable of achieving and/or enhancing the efflux of any one of said oligosaccharides.

34. The cell of any one of preferred embodiments 1 to 33, wherein the cell resists lactose killing when grown in an environment where lactose is combined with one or more other carbon sources.

35. The cell of any one of the preferred embodiments 1-34, wherein the cell comprises a modification for reducing the production of acetic acid as compared to an unmodified precursor cell.

36. The cell of preferred embodiment 35, wherein the cell comprises any one or more of a protein that reduces or reduces expression and/or eliminates, reduces or delays activity as compared to an unmodified precursor cell comprising: beta-galactosidase, galactosido-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine inhibitor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose: undecanopentenyl-phosphoglucose-1-phosphate transferase, L-fucokinase, L-fucose isomerase, N-acetylneuraminic acid dissociating enzyme, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epi isomerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridyltransferase, glucose-1-phosphate adenyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcription inhibitor IclR, lon protease, glucose-specific translocation phosphotransferase IIBC component ptsG, glucose-specific translocation Phosphotransferase (PTS) IIBC component malX, enzyme IIA ^Glc Beta-glucoside specific PTS enzyme II, fructose specific PTS polyphosphorylated protein FruA and FruB, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate-methyl acid dissociationEnzymes, acetate kinase, phosphoryl transferase, phosphoacetyl transferase, pyruvate decarboxylase.

37. The cell of any one of preferred embodiments 1 to 36, wherein the cell is capable of producing phosphoenolpyruvate (PEP).

38. The cell of any one of preferred embodiments 1 to 37, wherein the cell is modified for enhanced production and/or supply of phosphoenolpyruvate (PEP) compared to an unmodified precursor cell.

39. The cell of any one of preferred embodiments 1 to 38, wherein any one of the oligosaccharides is a mammalian milk oligosaccharide.

40. The cell of any one of preferred embodiments 1 to 39, wherein all of the oligosaccharides are mammalian milk oligosaccharides.

41. The cell of any one of preferred embodiments 1 to 38, wherein any one of the oligosaccharides is an antigen of the human ABO blood group system.

42. A method for producing a mixture of at least three different sialylated oligosaccharides by means of a cell, preferably a single cell, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different sialylated mammalian milk oligosaccharides, the method comprising the steps of:

i) Providing a cell that (a) exhibits a glycosyltransferase that is a sialyltransferase and is capable of synthesizing nucleotide-sugar CMP-Neu5Ac, and (b) exhibits at least one additional glycosyltransferase, and (c) is capable of synthesizing at least one or more nucleotide-sugar, wherein the nucleotide-sugar is a donor for the additional glycosyltransferase, and

ii) culturing the cell under conditions allowing expression of the glycosyltransferase and synthesis of the nucleotide-sugar such that the cell produces the mixture of at least three different sialylated oligosaccharides,

iii) Preferably, at least one of the oligosaccharides is isolated from the culture, more preferably all of the oligosaccharides are isolated from the culture.

43. The method of preferred embodiment 42, wherein the cell is a metabolically engineered cell of any one of embodiments 1-41.

44. The method of preferred embodiment 43, wherein the cell is modified by a gene expression module, characterized in that expression from any of said expression modules is sustained or produced by a natural inducer.

45. The method of any of preferred embodiments 43 or 44, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein.

46. The method of any one of preferred embodiments 42-45, wherein the cells produce a mixture of charged and neutral oligosaccharides.

47. The method of any of the preferred embodiments 42-46, wherein the mixture comprises, consists of, or consists essentially of: charged and neutral fucosylated and/or nonfucosylated oligosaccharides.

48. The method of any one of preferred embodiments 42-47, wherein the oligosaccharide mixture comprises at least three different sialylated oligosaccharides that differ in degree of polymerization.

49. The method of any one of preferred embodiments 42-48, wherein the cell produces at least four, preferably at least five, more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different sialylated oligosaccharides.

50. The method of any one of preferred embodiments 42 to 49, wherein any one of the additional glycosyltransferases is selected from the list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosyltransferase, N-acetylmannosylaminotransferase, xylosyltransferase, glucuronidase, galacturonan transferase, glucosaminotransferase, N-glycolylneuraminidase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-altrose amine transferase, UDP-N-acetylglucosamine enolpyruvyl transferase and fucosylaminotransferase,

Preferably, the fucosyltransferase is selected from the list comprising: alpha-1, 2-fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-1, 4-fucosyltransferase, alpha-1, 6-fucosyltransferase,

preferably, the sialyltransferase is selected from the list comprising: alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase,

preferably, the galactosyltransferase is selected from the list comprising: beta-1, 3-galactosyltransferase, N-acetylglucosamine beta-1, 3-galactosyltransferase, beta-1, 4-galactosyltransferase, N-acetylglucosamine beta-1, 4-galactosyltransferase, alpha-1, 3-galactosyltransferase and alpha-1, 4-galactosyltransferase,

preferably, the glucosyltransferase is selected from the list comprising: alpha-glucosyltransferase, beta-1, 2-glucosyltransferase, beta-1, 3-glucosyltransferase and beta-1, 4-glucosyltransferase,

preferably, the mannosyltransferase is selected from a list comprising: alpha-1, 2-mannosyltransferase, alpha-1, 3-mannosyltransferase and alpha-1, 6-mannosyltransferase,

Preferably, the N-acetylglucosaminyl transferase is selected from the list comprising: galactoside beta-1, 3-N-acetylglucosaminyl transferase and beta-1, 6-N-acetylglucosaminyl transferase,

preferably, the N-acetylgalactosamine transferase is selected from the list comprising: alpha-1, 3-N-acetylgalactosamine transferase and beta-1, 3-N-acetylgalactosamine transferase.

51. The method of any one of preferred embodiments 42-50, wherein the cell is modified in the performance or activity of at least one of the glycosyltransferases.

52. The method of any one of preferred embodiments 42-51, wherein any one of the additional glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-N-acetylneuraminic acid (CMP-Neu 5 Ac).

53. The method of any one of preferred embodiments 42-52, wherein any one of the additional glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-fucose (GDP-Fuc).

54. The method of any one of preferred embodiments 42-53, wherein any one of the additional glycosyltransferases is an N-acetylglucosamine aminotransferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine (UDP-GlcNAc).

55. The method of any one of preferred embodiments 42-54, wherein any one of the additional glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose (UDP-Gal).

56. The method of any one of preferred embodiments 42-55, wherein any one of the additional glycosyltransferases is an N-acetylgalactosamine transferase and one of the donor nucleotide-sugars is UDP-N-acetylgalactosamine (UDP-GalNAc).

57. The method of any one of preferred embodiments 42-56, wherein any one of the additional glycosyltransferases is an N-acetylmannosaminotransferase and one of the donor nucleotide-sugars is UDP-N-acetylmannosamine (UDP-ManNAc).

58. The method of any one of preferred embodiments 42 to 57, wherein any one of the nucleotide-sugars is selected from the list comprising: GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP-N-glycolylneuraminic acid (CMP-Neu 5 Gc), CMP-Neu4Ac, CMP-Neu5Ac9N ₃ 、CMP-Neu4，5Ac ₂ 、CMP-Neu5，7Ac ₂ 、CMP-Neu5，9Ac ₂ 、CMP-Neu5，7(8，9)Ac ₂ UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose, UDP-2-acetamido-2, 6-dideoxy-L-arabinose-4-hexanone, UDP-2-acetylAmino-2, 6-dideoxy-L-lyxose-4-hexulose, UDP-N-acetyl-L-rhamnose amine (UDP-L-RhaNAc or UDP-2-acetamido-2, 6-dideoxy-L-mannose), dTDP-N-acetylfucose amine, UDP-N-acetylfucose amine (UDP-L-FucNAc or UDP-2-acetamido-2, 6-dideoxy-L-galactose), UDP-N-acetyl-L-neotame amine (UDP-L-PneNAC or UDP-2-acetamido-2, 6-dideoxy-L-talose), UDP-N-acetylmuramic acid, UDP-N-acetyl-L-isorhamnose amine (UDP-L-QuiNAc or UDP-2-acetamido-2, 6-dideoxy-L-glucose), and GDP-L-isorhamnose.

59. The method of any one of preferred embodiments 42-58, wherein the cell exhibits one or more polypeptides selected from the list comprising: mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-guanylate transferase, GDP-mannose 4, 6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, GDP-fucose pyrophosphorylase, fucose-1-guanylate transferase, L-glutamylamino-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epi-isomerase, UDP-N-acetylglucosamine 2-epi-isomerase N-acetylmannosamine-6-phosphate 2-epi isomerase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, N-acetylneuraminic acid synthase, N-acetylneuraminic acid dissociase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphate phosphatase, N-acylneuraminic acid cytidylyltransferase, galactose-1-epi isomerase, galactokinase, glucokinase, galactose-1-phosphouridyltransferase, UDP-glucose 4-epi isomerase, glucose-1-phosphouridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epi isomerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein the cell is modified in terms of the performance or activity of any of the polypeptides.

60. The method of any one of preferred embodiments 42 to 59, wherein the cell is capable of synthesizing at least two nucleotide-sugars, preferably at least three nucleotide-sugars, more preferably at least four nucleotide-sugars, even more preferably at least five nucleotide-sugars.

61. The method of any one of preferred embodiments 42-60, wherein the oligosaccharide mixture also comprises at least one neutral oligosaccharide in addition to three or more sialylated oligosaccharides.

62. The method of preferred embodiment 61, wherein the neutral oligosaccharide is selected from the list comprising neutral fucosylated oligosaccharides and neutral nonfucosylated oligosaccharides.

63. The method of any one of preferred embodiments 42-62, wherein at least one of the sialylated oligosaccharides is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, N-acetylglucosamine-containing, N-acetylneuraminic acid-containing, N-glycolylneuraminic acid-containing, N-acetylgalactosamine-containing, rhamnose-containing, glucuronate-containing, galacturonate-containing and/or N-acetylmannosamine-containing.

64. The method of any one of preferred embodiments 42-63, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide.

65. The method of any one of preferred embodiments 42-64, wherein the oligosaccharide mixture comprises at least one oligosaccharide comprising an N-acetylglucosamine monosaccharide unit.

66. The method of any one of preferred embodiments 42-65, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide.

67. The method of any one of preferred embodiments 42-66, wherein the oligosaccharide mixture comprises at least one oligosaccharide which is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, N-acetylglucosamine-containing, N-acetylneuraminic acid-containing, N-glycolylneuraminic acid-containing, N-acetylgalactosamine-containing, rhamnose-containing, glucuronate-containing, galacturonate-containing and/or N-acetylmannosamine-containing.

68. The method of any one of preferred embodiments 42-67, wherein the cell uses at least one precursor for producing any one or more of the oligosaccharides, preferably the cell uses two or more precursors for producing any one or more of the oligosaccharides, said precursors being fed from the culture medium into the cell.

69. The method of any one of preferred embodiments 42-68, wherein the cell produces at least one precursor for producing any one of the oligosaccharides.

70. The method of any one of preferred embodiments 42-69, wherein the at least one precursor for producing any one of the oligosaccharides is fully converted to any one of the oligosaccharides.

71. The method of any one of preferred embodiments 42-70, wherein the cell produces the oligosaccharide intracellularly, and wherein a portion or substantially all of the produced oligosaccharide remains intracellular and/or is excreted outside the cell via passive or active transport.

72. The method of any one of preferred embodiments 42 to 71, wherein the cell is further genetically modified for use in

i) Modified expression of endogenous membrane proteins and/or

ii) modified activity of endogenous membrane proteins and/or

iii) Expression of homologous membrane proteins and/or

iv) expression of heterologous membrane proteins,

wherein the membrane protein is involved in secreting any of said oligosaccharides from the mixture outside the cell, preferably wherein the membrane protein is involved in secreting all of said oligosaccharides from the mixture from the cell.

73. The method of any one of preferred embodiments 42 to 72, wherein the cell is further genetically modified for use in

i) Modified expression of endogenous membrane proteins and/or

ii) modified activity of endogenous membrane proteins and/or

iii) Expression of homologous membrane proteins and/or

iv) expression of heterologous membrane proteins,

wherein the membrane protein is involved in the absorption of precursors and/or receptors for the synthesis of any of said oligosaccharides in the mixture, preferably wherein the membrane protein is involved in the absorption of all of said desired precursors, more preferably wherein the membrane protein is involved in the absorption of all of said receptors.

74. The method of any one of preferred embodiments 72 or 73, wherein the membrane protein is selected from the list comprising: transporter, P-P-bond hydrolytically driven transporter, beta-bungee, auxiliary transporter, putative transporter, phosphotransferase driven group translocator,

preferably, the transporter comprises an MFS transporter, a sugar efflux transporter, and a transferrin export protein,

preferably, the P-P-bond hydrolytically driven transporter comprises an ABC transporter and a transferrin export protein.

75. The method of any one of the preferred embodiments 72-74, wherein the membrane protein provides improved production and/or is capable of achieving and/or enhancing the efflux of any one of the oligosaccharides.

76. The method of any one of preferred embodiments 42-75, wherein the cells resist lactose killing when grown in an environment where lactose is combined with one or more other carbon sources.

77. The method of any one of the preferred embodiments 42-76, wherein the cell comprises a modification for reducing acetate production as compared to an unmodified precursor cell.

78. The method of preferred embodiment 77, wherein the cell comprises any one or more of a protein that reduces or reduces expression and/or eliminates, reduces or delays activity compared to an unmodified precursor cell comprising: beta-galactosidase, galactosido-acetyltransferase, N-acetylglucosamine-6-phosphateDeacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine inhibitor, ribonucleotide monophosphate, EIICBA-Nag, UDP-glucose: undecanopentenyl-phosphoglucose-1-phosphate transferase, L-fucokinase, L-fucose isomerase, N-acetylneuraminic acid dissociating enzyme, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epi isomerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridyltransferase, glucose-1-phosphate adenyltransferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2, glucose-6-phosphate isomerase, aerobic respiration control protein, transcription inhibitor IclR, lon protease, glucose-specific translocation phosphotransferase IIBC component ptsG, glucose-specific translocation Phosphotransferase (PTS) IIBC component malX, enzyme IIA ^Glc Beta-glucoside specific PTS enzyme II, fructose-specific PTS polyphosphorylated transfer protein FruA and FruB, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate-methyl alcohol dissociating enzyme, acetate kinase, phosphoacyltransferase, phosphoacetyltransferase, and pyruvate decarboxylase.

79. The method of any one of preferred embodiments 42-78, wherein the cell is capable of producing phosphoenolpyruvate (PEP).

80. The method of any one of preferred embodiments 42 to 79, wherein the cell is modified for enhanced production and/or supply of phosphoenolpyruvate (PEP) compared to an unmodified precursor cell.

81. The method of any one of preferred embodiments 42-80, wherein any one of the oligosaccharides is a mammalian milk oligosaccharide.

82. The method of any one of preferred embodiments 42-81, wherein all of the oligosaccharides are mammalian milk oligosaccharides.

83. The method of any one of preferred embodiments 42-82, wherein any one of the oligosaccharides is an antigen of the human ABO blood group system.

84. The method of any one of preferred embodiments 42 to 83, wherein the conditions comprise:

-using a medium comprising at least one precursor and/or acceptor for producing any of the oligosaccharides, and/or

-adding to the culture medium at least one precursor and/or acceptor feed for producing any of said oligosaccharides.

85. The method of any one of the preferred embodiments 42-84, comprising at least one of the following steps:

i) Using a medium comprising at least one precursor and/or acceptor;

ii) adding at least one precursor and/or acceptor feed to the medium in a reactor, wherein the total reactor volume is between 250mL (milliliter) and 10.000m ³ Preferably in a continuous manner within (cubic meters) and preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium prior to addition of the precursor and/or acceptor feed;

iii) Adding at least one precursor and/or acceptor feed to the medium in a reactor, wherein the total reactor volume is between 250mL (milliliter) and 10.000m ³ Preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed three times, preferably does not exceed two times, more preferably is less than two times the volume of the medium prior to the addition of the precursor and/or acceptor feed, and wherein preferably the pH of the precursor and/or acceptor feed is set between 3 and 7, and wherein preferably the temperature of the precursor and/or acceptor feed is maintained between 20 ℃ and 80 ℃;

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

v) adding at least one precursor and/or acceptor feed to the medium in a continuous manner over the course of 1, 2, 3, 4, 5 days by means of a feed solution, and 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 ℃ and 80 ℃;

the method produces any one of the oligosaccharides in a concentration of at least 50g/L, preferably at least 75g/L, more preferably at least 90g/L, more preferably at least 100g/L, more preferably at least 125g/L, more preferably at least 150g/L, more preferably at least 175g/L, more preferably at least 200g/L in the final culture.

86. The method of any one of the preferred embodiments 42-84, comprising at least one of the following steps:

i) Using a medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor volume is between 250mL and 10.000m ³ (cubic meters) range;

ii) adding to the medium a lactose feed comprising at least 50, preferably at least 75, preferably at least 100, preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor volume is between 250mL and 10.000m ³ Preferably in a continuous manner, and preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium prior to the addition of the lactose feed;

iii) Adding to the medium a lactose feed comprising at least 50, preferably at least 75, preferably at least 100, preferably at least 120, preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor volume is in the range of 250mL to 10.000m3 (cubic meters), preferably in a continuous manner, and preferably such that the final volume of the medium does not exceed three times, preferably does not exceed two times, preferably is less than two times the volume of the medium before the lactose feed is added, and wherein preferably the pH of the lactose feed is set between 3 and 7, and wherein preferably the temperature of the lactose feed is maintained between 20 ℃ and 80 ℃;

iv) adding lactose feed to the medium in a continuous manner by means of feed solution over a period of 1 day, 2 days, 3 days, 4 days, 5 days;

v) adding lactose feed to the medium in a continuous manner over the course of 1, 2, 3, 4, 5 days by means of a feed solution, and wherein the concentration of the lactose feed solution is 50g/L, preferably 75g/L, more preferably 100g/L, more preferably 125g/L, more preferably 150g/L, more preferably 175g/L, more preferably 200g/L, more preferably 225g/L, more preferably 250g/L, more preferably 275g/L, more preferably 300g/L, more preferably 325g/L, more preferably 350g/L, more preferably 375g/L, more preferably 400g/L, more preferably 450g/L, more preferably 500g/L, even more preferably 550g/L, most preferably 600g/L, and wherein preferably the pH of the feed solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is kept between 20 ℃ and 80 ℃;

The method produces any one of the oligosaccharides in a concentration of at least 50g/L, preferably at least 75g/L, more preferably at least 90g/L, more preferably at least 100g/L, more preferably at least 125g/L, more preferably at least 150g/L, more preferably at least 175g/L, more preferably at least 200g/L in the final culture.

87. The method of preferred embodiment 86, wherein the lactose feed is achieved by adding lactose from the beginning of the culture at a concentration of at least 5mM, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150mM, more preferably at a concentration of > 300 mM.

88. The method of any of preferred embodiments 86 or 87, wherein the lactose feed is achieved by adding lactose to the culture at a concentration such that a lactose concentration of at least 5mM, preferably 10mM or 30mM is obtained throughout the production phase of the culture.

89. The method of any one of the preferred embodiments 42 to 88, wherein the host cells are cultured for at least about 60, 80, 100, or about 120 hours or in a continuous manner.

90. The method of any one of preferred embodiments 42 to 89, wherein the cells are cultured in a medium comprising a carbon source comprising a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, glycerol, a complex medium comprising molasses, corn steep liquor, peptone, tryptone or yeast extract; preferably, wherein the carbon source is selected from the list comprising: glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose, malt-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose, starch, cellulose, hemicellulose, molasses, corn steep liquor, high fructose syrup, acetate, citrate, lactate and pyruvate.

91. The method of any one of preferred embodiments 42-90, wherein the medium contains at least one precursor selected from the group consisting of: lactose, galactose, fucose, glcNAc, galNAc, lacto-N-disaccharide (LNB), N-acetyllactosamine (LacNAc).

92. The method of any one of preferred embodiments 42-91, wherein the first stage of exponential cell growth is provided by adding a carbon-based matrix, preferably glucose or sucrose, to the medium before adding the precursor, preferably lactose, to the medium in the second stage.

93. The method of any one of preferred embodiments 42-92, wherein the first phase of exponential cell growth is provided by adding a carbon-based matrix, preferably glucose or sucrose, to the medium comprising a precursor, preferably lactose, followed by a second phase wherein only carbon-based matrix, preferably glucose or sucrose, is added to the medium.

94. The method of any one of preferred embodiments 42-93, wherein the first stage of exponential cell growth is provided by adding a carbon-based matrix, preferably glucose or sucrose, to the medium comprising a precursor, preferably lactose, followed by a second stage wherein the carbon-based matrix, preferably glucose or sucrose, and the precursor, preferably lactose, are added to the medium.

95. The method of any one of the preferred embodiments 42-94, wherein the separating comprises at least one of the following steps: clarification, ultrafiltration, nanofiltration, biphasic partitioning, reverse osmosis, microfiltration, activated carbon or carbon treatment, treatment with a nonionic surfactant, enzymatic digestion, tangential flow high performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.

96. The method of any one of preferred embodiments 42-95, further comprising purifying any one of the oligosaccharides from the cell.

97. The method of preferred embodiment 96, wherein the purifying comprises at least one of the following steps: using activated carbon or carbon, using charcoal, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange; alcohol is used, hydroalcoholic mixture is used, crystallization, evaporation, precipitation, drying, spray drying, lyophilization, spray freeze drying, freeze spray drying, band drying (band drying), belt drying (belt drying), vacuum band drying, drum drying, vacuum drum drying or vacuum drum drying.

98. The cell according to any one of preferred embodiments 1 to 41 or the method according to any one of preferred embodiments 42 to 97, wherein the cell is a bacterium, a fungus, a yeast, a plant cell, an animal cell or a protozoan cell,

preferably, the bacterium is an Escherichia coli (Escherichia coli) strain, more preferably an Escherichia coli strain as K-12 strain, even more preferably an Escherichia coli K-12 strain is Escherichia coli MG1655,

preferably, the fungus belongs to a genus selected from the group comprising: rhizopus (Rhizopus), reticulus (Dictyostelium), penicillium (Penicillium), white fungus (Mucor) or Aspergillus (Aspergillus),

preferably, the yeast belongs to a genus selected from the group comprising: saccharomyces (Saccharomyces), zygosaccharomyces (Zygosaccharomyces), pichia (Pichia), colt (Komagataella), hansenula (Hansenula), yarrowia (Yarrowia), candida globosa (Starerella), kluyveromyces (Kluyveromyces) or Debaryomyces (Debaromyces),

preferably, the plant cell is an algal cell or is derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soybean, maize or corn plants,

Preferably, the animal cells are derived from non-human mammals, birds, fish, invertebrates, reptiles, amphibians or insects, or are genetically modified cell lines derived from human cells excluding embryonic stem cells, more preferably the human and non-human mammalian cells are epithelial cells, embryonic kidney cells, fibroblasts, COS cells, chinese hamster ovary (Chinese hamster ovary; CHO) cells, murine myeloma cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, more preferably the insect cells are derived from Spodoptera frugiperda (Spodoptera frugiperda), bombyx mori (Bombyxmori), cabbage looper (Mamestra brassicae), trichoplusia ni (Trichoplusia ni) or Drosophila melanogaster (Drosophila melanogaster),

preferably, the protozoan cell is a Leishmania tarabica (Leishmania tarentolae) cell.

99. The cell of preferred embodiment 98 or the method of preferred embodiment 98, wherein the cell is a viable Gram-negative bacterium (Gram-negative bacterium) comprising reduced or eliminated synthetic poly-N-acetyl-glucosamine (PNAG), an intestinal bacterial common antigen (Enterobacterial Common Antigen; ECA), cellulose, cola, core oligosaccharide, osmoregulation periplasmic glucan (Osmoregulated Periplasmic Glucan; OPG), glyceroglycosides, glycans and/or trehalose as compared to the unmodified precursor cell.

100. Use of a cell according to any one of the preferred embodiments 1 to 41, 98, 99 or a method according to any one of the preferred embodiments 42 to 99 for the production of a mixture of at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide.

The invention will be described in more detail in the embodiments and the accompanying drawings in which

FIG. 1 shows a graphical representation of the results of a chromatographic profile obtained from a whole broth sample of LSTc producing E.coli strain S2 (Table 8) exhibiting the a-2, 6-sialyltransferase of the genus Photobacterium (Photobacterium sp.) JT-ISH-224 with SEQ ID NO 25 and analyzing for the presence of oligosaccharides via the Dionex method, as described in example 1. The indicated peaks represent the following glycans (retention times): 1, lactose (5.325 min); 2, LN3 (6.825 min); 3, LNnT (9.625 min); 4 sialic acid (20.359 min); 5, LSTc (30.809 min); 6,6' SL (31.984 min).

FIG. 2 shows a graphical representation of the results of a chromatographic profile obtained for a whole broth sample from E.coli strain S5 (Table 9) producing LSTd, which shows the a-2, 3-sialyltransferase from Pasteurella multocida (P.multocida) with SEQ ID NO22 and the presence of oligosaccharides analyzed via the Dionex method as described in example 1. The indicated peaks represent the following glycans (retention times): 1, the number of the components is 1, lactose (5.334 min); 2, LN3 (6.825 min); 3, LNnT (9.567 min); 4 sialic acid (20.017 min); 5, LSTd (31.709 min); 6,3' SL (33.050 min).

The following examples serve as further illustration and explanation of the invention and are not intended to be limiting.

Examples

Example 1 E.coli materials and methods

Culture medium

Lu Liya the medium (Luria Broth; LB) consists of 1% tryptone (Difco, belgium, emblica) 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR, belgium). The minimal medium in 96-well plates or shake flasks used in the culture experiments contained 2.00g/L NH4Cl, 5.00g/L (NH) ₄ ) 2SO4, 2.993g/L KH2PO4, 7.315g/L K2HPO4, 8.372g/L MOPS, 0.5g/L NaCl, 0.5g/L MgSO4.7H2O, 30g/L sucrose or 30g/L glycerol, 1mL/L vitamin solution, 100. Mu.l/L molybdate solution, and 1mL/L selenium solution. As specified in the respective examples, 0.30g/L sialic acid, 20g/L lactose, 20g/L LacNAc and/or 20g/L LNB were additionally added as precursors to the medium. The minimal medium was set to pH 7 using 1M KOH. The vitamin solution is composed of 3.6g/L FeCl2.4H2O, 5g/L CaCl2.2H2O, 1.3g/L MnCl2.2H2O, 0.38g/L CuCl2.2H2O, 0.5a/L CoCl2.6H2O, 0.94g/L ZnCl2, 0.0311g/L H3BO4, 0.4g/L Na2EDTA.2H2O and 1.01g/L thiamine HCl. The molybdate solution contained 0.967g/L NaMoO4.2H2O. The selenium solution contained 42g/L Seo2.

The minimal medium for fermentation contained 6.75g/L NH4Cl, 1.25g/L (NH 4) 2SO4, 2.93g/L KH2PO4 and 7.31g/L KH2PO4, 0.5g/L NaCl, 0.5g/L MgSO4.7H2O, 30g/L sucrose or 30g/L glycerol, 1mL/L vitamin solution, 100. Mu.L molybdate solution and 1mL/L selenium solution, with the same composition as described above. As specified in the respective examples, 0.30g/L sialic acid, 20g/L lactose, 20g/L LacNAc and/or 20g/L LNB were additionally added as precursors to the medium.

The complex medium was sterilized by high pressure treatment (121 ℃,21 min) and the minimal medium by filtration (0.22 μm Sartorius). The medium was made selective by adding the following antibiotics, if necessary: such as chloramphenicol (20 mg/L), carbocillin (carbicillin) (100 mg/L), spectinomycin (40 mg/L), and/or Kangmycin (50 mg/L).

Plastid body

pKD46 (red helper plasmid, ampicillin (Ampicillin) resistance), pKD3 (containing the FRT-flanking chloramphenicol resistance (cat) gene), pKD4 (containing the FRT-flanking Kang Mei element resistance (kan) gene) and pCP20 (exhibiting FLP recombinase activity) plasmids were obtained from professor r.cunin (Vrije Universiteit Brussel, belgium, 2007). The plastids were maintained in E.coli DH5a (F) ^- 、phi80dlacZAM15、Δ(lacZYA-argF)U169、deoR、recA1、endA1、hsdR17(rk ^- ，mk ⁺ )、phoA、supE44、λ ^- Thi-1, gyrA96, relA 1).

Strains and mutations

Coli K12 MG1655[ lambda ] ^- 、F ^- 、rph-1]Is obtained from the escherichia coli gene reserve center (united states) at month 3 of 2007, CGSC strain number: 7740. gene disruption, gene introduction and gene replacement were carried out using the techniques disclosed in Datsenko and Wanner (PNAS 97 (2000), 6640-6645). This technique is based on antibiotic selection after homologous recombination by lambda red recombinase. Subsequent catalysis by the invertase recombinase ensures removal of the antibiotic selection cassette in the final production strain. Transformants harboring the red helper plasmid pKD46 were grown to OD at 30℃in 10mL of LB medium with ampicillin (100 mg/L) and L-arabinose (10 mM) ₆₀₀ nm is 0.6. The cells were made inductively receptive by washing the cells first with 50mL of ice-cold water and second with 1mL of ice-cold water. Next, the cells were resuspended in 50. Mu.L of ice-cold water. By 50. Mu.L of fineCells and 10-100ng of linear double stranded DNA product by using Gene Pulser ^TM (BioRad) (600Ω,25 μFD and 250 volts). After electroporation, cells were added to 1mL of LB medium incubated for 1h at 37 ℃ and finally spread onto LB agar containing 25mg/L chloramphenicol or 50mg/L Kang Mei elements to select for antibiotic resistant transformants. Selected mutants were verified by PCR with primers upstream and downstream of the modified region and grown on LB agar at 42 ℃ to allow for helper plastid loss. Mutants were tested for ampicillin sensitivity. Linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and derivatives thereof as templates. The primer used has a part of the sequence complementary to the template and another part complementary to the side on which recombination must take place on the chromosomal DNA. For genomic gene knockout, the homologous regions are designed 50-nt upstream and 50-nt downstream of the start and stop codons of the relevant gene. For the genomic gene insertion, the transcription start point (+1) must be considered. The PCR product was purified by PCR, digested with Dpnl, repurified from agarose gel, and suspended in a lysis buffer (5 mM Tris, pH 8.0). Selected mutants were transformed with pCP20 plastids, ampicillin and chloramphenicol resistant plastids, showing temperature-sensitive replication and heat induction of FLP synthesis. An ampicillin resistant transformant was selected at 30 ℃, followed by purification of a few colonies in LB at 42 ℃ and then testing for all antibiotic resistance and FLP helper plastid loss. Gene knockout and gene insertion were checked with control primers.

In one embodiment of GDP-fucose production, the mutant strain is derived from e.coli K12 MG1655 comprising gene knockout of e.coli wcaJ and thyA genes and gene insertion of a persistent transcription unit comprising a sucrose transporter like CscB of e.coli W with SEQ ID NO 01, a fructokinase like Frk derived from zymomonas mobilis (Zymomonas mobilis) with SEQ ID NO 02 and a sucrose phosphorylase like BaSP derived from bifidobacterium adolescentis (Bifidobacterium adolescentis) with SEQ ID NO 03, for example. For the production of fucosylated oligosaccharides, the mutant GDP-fucose producing strain is additionally modified with a expressible body comprising the persistent transcriptional unit of an a-1, 2-fucosyltransferase, such as, for example, hpF utC of helicobacter pylori (H.pyri) with SEQ ID NO 04; and/or a-1, 3-fucosyltransferase, such as HpFACT, for example helicobacter pylori with SEQ ID NO 05, and modified with a persistent transcription unit for a selectable marker, such as, for example, E.coli thyA with SEQ ID NO 06. Sustained transcriptional units of fucosyltransferase genes may also be present in mutant E.coli strains via genomic gene insertion. GDP-fucose production can be further optimized in mutant E.coli strains by means of gene knockout of E.coli genes comprising glgC, agp, pfkA, pfkB, pgi, arcA, iclR, pgi and lon, as described in WO2016075243 and WO 2012007481. GDP-fucose production may additionally be optimized, including genomic gene insertion of the persistent transcription units of mannose-6-phosphate isomerase (such as, e.g., manA of E.coli having SEQ ID NO 07), phosphomannose mutase (such as, e.g., manB of E.coli having SEQ ID NO 08), mannose-1-guanyl phosphate transferase (such as, e.g., manC of E.coli having SEQ ID NO 9), GDP-mannose 4, 6-dehydratase (such as, e.g., gmd of E.coli having SEQ ID NO 10), and GDP-L-fucose synthase (such as, e.g., fcl of E.coli having SEQ ID NO 11). GDP-fucose production can also be obtained by genomic gene knockout of E.coli fucK and fucI genes and genomic gene insertion of a persistent transcription unit containing a fucose permease such as FucP of E.coli with SEQ ID NO 12 and a bifunctional fucose kinase/fucose-1-phosphoglycerate guanyl transferase such as fkp of E.fragilis with SEQ NO ID 13. If mutant strains producing GDP-fucose are intended to produce fucosylated lactose structures, the strains are additionally modified by genomic gene knockout of the E.coli LacZ, lacY and LacA genes and by genomic gene insertion of the continuous transcription unit of lactose permease (e.g.E.coli LacY with SEQ ID NO 14).

Alternatively, and/or in addition, the production of GDP-fucose and/or fucosylation structures may be further optimized in a genetically engineered mutant E.coli strain having a persistent transcription unit comprising a membrane transporter such as, for example, mdfA from Mo Jinsi Cronobacter (Unit Prot ID A0A2T7ANQ 9), mdfA from Citrobacter Young (Unit ID D4BC 23), mdfA from E.coli (Unit Prot ID P0 AEY), mdfA from Lei Jinsi about Ke's bacteria (Unit Prot ID G9Z5F 4), iceT from E.coli (Unit Prot ID A0A024L 207) or iceT from Citrobacter Young (Unit Prot ID D4B8A 6).

In one example of sialic acid production, the mutant strain is derived from E.coli K12 MG1655, which comprises gene knock-out of E.coli nagA and nagB genes and gene insertion of a persistent transcription unit comprising a glucosamine 6-phosphate N-acetyl transferase as GNA1, e.g.Saccharomyces cerevisiae having SEQ ID NO 15, an N-acetyl glucosamine 2-epi-isomerase as AGE, e.g.oval Bacteroides (Bacteroides ovatus) having SEQ ID NO 16, and an N-acetyl neuraminic acid (Neu 5 Ac) synthase as NeuB, e.g.NeuB of Neisseria meningitidis (Neisseria meningitidis) having SEQ ID NO 17. Sialic acid production can be further optimized in mutant E.coli strains with the following: a genomic gene knockout comprising any one or more of the nagC, nagD, nagE, nanA, nanE, nanK, manX, manY and manZ escherichia coli genes (as described in WO 18122225); and/or a genomic gene knockout of an escherichia coli gene comprising any one or more of nanT, poxB, ldhA, adhE, aldB, pflA, pflC, ybiY, ack and/or pta; and a genomic gene insertion of a persistent transcription unit comprising L-glutamyld-fructose-6-phosphate aminotransferase, such as, for example, the mutant glmS 54 of escherichia coli having SEQ ID NO 18 (different from the wild-type escherichia coli glmS protein by means of a39T, R C and G472S mutation); and phosphatases such as, for example, yqaB of any of the escherichia coli having SEQ ID NO 19 or the escherichia coli genes comprising aphA, cof, hisB, otsB, surE, yaed, ycjU, yedP, yfbT, yidA, yigB, yihX, yniC, yrbL, appA, gph, serB, ybhA, ybiV, ybjL, yfb, yieH, yjgL, yjjG, yrfG and YbiU or PsMupP from pseudomonas putida, scDOG1 from saccharomyces cerevisiae and BsAraL from bacillus subtilis (as described in WO 18122225); and acetyl-CoA synthetases such as acs from E.coli (UniProt ID P27550), for example. Sialic acid production can also be obtained by gene knock-out of the nagA and nagB genes of E.coli and by gene insertion of a persistent transcription unit containing a glucosamine phosphate mutase such as glmM of E.coli having SEQ ID NO 31, an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyl transferase such as glmU of E.coli having SEQ ID NO 32, a UDP-N-acetylglucosamine 2-epi-isomerase such as NeuC of E.jejuni having SEQ ID NO 20, and an N-acetylneuraminic acid synthase such as NeuB of Neisseria meningitidis having SEQ ID NO 17, for example. Also in this mutant strain, sialic acid production may be further optimized with genomic gene insertion of a persistent transcription unit comprising L-glutamyld-fructose-6-phosphate aminotransferase as e.g. the mutation glmS 54 of escherichia coli with SEQ ID NO 18; and phosphatases such as, for example, yqaB of any of the escherichia coli having SEQ ID NO 19 or the escherichia coli genes comprising aphA, cof, hisB, otsB, surE, yaed, ycjU, yedP, yfbT, yidA, yigB, yihX, yniC, yrbL, appA, gph, serB, ybhA, ybiV, ybjL, yfb, yieH, yjgL, yjjG, yrfG and YbiU or PsMupP from pseudomonas putida, scDOG1 from saccharomyces cerevisiae and BsAraL from bacillus subtilis (as described in WO 18122225); and acetyl-CoA synthetases such as acs from E.coli (UniProt ID P27550), for example.

Alternatively and/or additionally sialic acid production can be obtained by genomic gene insertion of a persistent transcription unit containing bifunctional UDP-GlcNAc 2-epi-isomerase/N-acetylmannosamine kinase (UniProt ID Q91WG 8) as e.g.N-acyl neuraminic acid-9-phosphate synthase (UniProt ID K9NPH 9) from Pseudomonas UW4 and N-acyl neuraminic acid-9-phosphatase as e.g.from the candidate species magnetotactic genus (Candidatus Magnetomorum) HK-1 (UniProt ID KPA 15328.1) or from Bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron) (UniProt ID Q8A 712).

Alternatively and/or additionally sialic acid production can be obtained by genomic gene insertion of a persistent transcription unit containing a glucosamine phosphate mutase (SEQ ID NO 31) as e.g. glmM from escherichia coli, an N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyl transferase (SEQ ID NO 32) as e.g. glmU from escherichia coli, a bifunctional UDP-GlcNAc 2-epi-isomerase N-acetylmannosamine kinase (UniProt ID Q91WG 8) as e.g. from mice (strain C57 BL/6J), an N-acyl neuraminic acid-9-phosphate synthase (UniProt ID K9NPH 9) as e.g. from pseudomonas UW4, or an N-acyl neuraminic acid-9-phosphatase as e.g. from the candidate species magnetotactic HK-1 (UniProt ID KPA 15328.1) or from bacteroides (prot ID Q8 a) 712.

In the example of sialylated oligosaccharide production, the sialic acid producing strain further requires the expression of a PmultST 3-like polypeptide consisting of amino acid residues 1 to 268 of the NeuA of, for example, pasteurella multocida with SEQ ID NO 21, nmesitST 3 of NeuA with SEQ ID NO 23, pmultST2 of PmultST 70 (GenBank NO. AAK 02592.1) from, for example, pasteurella multocida, beta-galactoα -2, 6-sialyltransferase (UniProt ID O66375) of PdST6 from, for example, mermaid, or a polypeptide consisting of amino acid residues 1 to 268 of the beta-galactosidα -2, 3-sialyltransferase of, for example, SEQ ID NO 22 or of UniProt ID Q9CLP3 with the activity of, or a polypeptide consisting of the beta-galactoα -2, 6-sialyltransferase (UniProt ID O66375) of, for example, beta-galactoα -2, 6-sialyltransferase from, for example, pmultocida 70 (GenBank NO. AAK 02592.1) of P, or a polypeptide consisting of the beta-galactosyltransferase 6- α -2, 6-sialyltransferase of, such as, for example, P2 or a 6-sialyltransferase of, such as P6 from P.P.sp. The persistent transcriptional units of PmNeuA and sialyltransferase can be delivered to the mutant strain via genomic gene insertion or via the expression plasmid. If mutant strains producing sialic acid and CMP-sialic acid are intended to produce sialyllactose structures, the strains are additionally modified by genomic gene knockout of the E.coli LacZ, lacY and LacA genes and by genomic gene insertion of the continuous transcriptional unit of a lactose permease, such as E.coli LacY with SEQ ID NO 14.

Alternatively and/or additionally, sialic acid and/or sialyloligosaccharide production may be further optimized with genomic gene insertion of a persistent transcriptional unit comprising a membrane transporter, such as e.g. a sialic acid transporter, like e.g. nanT (UniProt ID P41036) from e.coli K-12 mg1655, from e.g. e.coli O6: nanT from H1 (UniProt ID Q8FD 59), E.coli O157: nanT of H7 (UniProt ID Q8X9G 8) or nanT from E.alberti (E.albertii) (UniProt ID B1EFH 1); or transport proteins such as, for example, entS (UniProt ID P24077) from E.coli, entS (UniProt ID A0A378GQ 13) from Kluyveromyces ascorbate (Kluyvera ascorbata) or EntS (UniProt ID A0A6Y2K4E 8) from Salmonella arizonae (Salmonella enterica subsp. Arizonae), mdfA (UniProt ID A0A2T7ANQ 9) from Mo Jinsi Cronobacter, mdfA (UniProt ID D4BC 23) from E.Yang citrate, mdfA (UniProt ID P0 AEY) from E.coli, mdfA (UniProt ID G9Z5F 4) from Lei Jinsi about Klebsiella, ICT (UniProt ID A0A 0249L 207) from E.coli, ICT (UniProt ID D4A 6) from E.bergamotialis, mdfA (UniProt ID D4A 6) from E.coli, mdfA (UniProt ID D4BC 8) from E.E.coli, mdfA (UniProt ID P35) from E.coli, or MdfA (UniProt ID 7) from E.coli; or ABC transporters such as, for example, oppF (UniProt ID P77737) from E.coli, lmrA (UniProt ID A0A1V0NEL 4) from the diacetyl lactic acid variant of the lactococcus lactis subspecies lactis or Blon_2475 (UniProt ID B7GPD 4) from the Bifidobacterium longum subspecies infancy.

All mutant strains producing sialic acid, CMP-sialic acid and/or sialylated oligosaccharides can optionally be adapted for growth on sucrose via genomic gene insertion of a persistent transcriptional unit containing a sucrose transporter like CscB of e.coli W with SEQ ID NO 01, a fructokinase like Frk derived e.g. from zymomonas mobilis with SEQ ID NO 02 and a sucrose phosphorylase like BaSP derived e.g. from bifidobacterium adolescentis with SEQ ID NO 03.

In one example of the production of LN3 (GlcNAc-b 1,3-Gal-b1, 4-Glc) and oligosaccharides derived therefrom comprising lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), the mutant strain is derived from E.coli K12MG1655 and is modified by gene knockout of E.coli LacZ and nagB genes, and by gene insertion modification of the galactoglycoside beta-1, 3-N-acetylglucosamintransferase as, for example, lgtA of Neisseria meningitidis having SEQ ID NO 26. For LNT or LNnT production, the mutant strain is further modified by the following persistent transcriptional units, which can be delivered to the strain via genomic gene insertion or from the expressible body, respectively: such as, for example, E.coli O55 with SEQ ID NO 27: n-acetylglucosamine beta-1, 3-galactosyltransferase of WbgO of H7 or N-acetylglucosamine beta-1, 4-galactosyltransferase of LgtB of Neisseria meningitidis having SEQ ID NO 28. Optionally, multiple copies of the galactoside beta-1, 3-N-acetylglucosamine aminotransferase, N-acetylglucosamine beta-1, 3-galactosyltransferase, and/or N-acetylglucosamine beta-1, 4-galactosyltransferase gene may be added to the mutant E.coli strain. In addition, LNT and/or LNnT production may be enhanced by improving UDP-GlcNAc production by gene insertion of one or more genetically modified strains with a sustained transcriptional unit of an L-glutamyld-fructose-6-phosphate aminotransferase, such as, for example, mutant glmS 54 of escherichia coli having SEQ ID NO 18. In addition, the strain may optionally be modified to enhance UDP-galactose production by genomic gene knockout of E.coli ushA, galT, ldhA and agp genes. The mutant E.coli strain may also optionally be adapted for genomic gene insertion with persistent transcription units for use in: UDP-glucose-4-epi-isomerase as galE of E.coli having SEQ ID NO29, glucosamine phosphate mutase as glmM of E.coli having SEQ ID NO 31, N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase as glmU of E.coli having SEQ ID NO 32, for example. Mutant strains may also be suitable for growth on sucrose via gene insertion of a persistent transcription unit containing a sucrose transporter such as CscB of e.coli W having SEQ ID NO 01, a fructokinase such as Frk derived from zymomonas mobilis having SEQ ID NO 02 and a sucrose phosphorylase such as BaSP derived from bifidobacterium adolescentis having SEQ ID NO 03, for example.

Alternatively and/or additionally, the production of LN3, LNT, LNnT and oligosaccharides derived therefrom may be further optimized by genomic gene insertion of a persistent transcription unit comprising a membrane transporter such as, for example, mdfA from Mo Jinsi Cronobacter (UniProt ID A0A2T7ANQ 9), mdfA from Citrobacter bergii (UniProt ID D4BC 23), mdfA from E.coli (UniProt ID P0 AEY), mdfA from Lei Jinsi Butyrosporum (UniProt ID G9Z5F 4), iceT from E.coli (UniProt ID A0A024L 207) or iceT from Citrobacter bergii (UniProt ID D4B8A 6) in a mutant E.coli strain.

Preferably, but not necessarily, glycosyltransferases, proteins involved in nucleotide activated sugar synthesis and/or membrane transporters are fused via the N-and/or C-terminus to a soluble enhancer tag such as, for example, the following: SUMO tags, MBP tags, his, FLAG, strep-II, halo-tags, nusA, thioredoxin, GST and/or Fh8 tags to enhance their solubility (Cost et al, front. Microbiol.2014, https:// doi. Org/10.3389/fmib. 2014.00063; fox et al, protein Sci.2001, 10 (3), 622-630; jia and Jeaon, open biol.2016, 6:160196).

Optionally, the mutant E.coli strain is modified by genomic gene insertion of a continuous transcriptional unit encoding a companion protein such as, for example, dnaK, dnaJ, grpE or GroEL/ES companion protein system (Baney x F., palumbo J.L. (2003) Improving Heterologous Protein Folding via Molecular Chaperone and Foldase Co-Expressio)n. in: vailancourt P.E. (code) E.coli Gene Expression protocols methods in Molecular Biology ^TM Volume 205, humana Press).

Optionally, the mutant E.coli strain is modified to produce a glycosyl minimized E.coli strain comprising a genomic gene knockout of any one or more of the non-essential glycosyltransferase genes comprising pgaC, pgaD, rfe, rffT, rffM, bcsA, bcsB, bcsC, wcaA, wcaC, wcaE, wcaI, wcaJ, wcaL, waaH, waaF, waaC, waaU, waaZ, waaJ, waaO, waaB, waaS, waaG, waaQ, wbbl, arnC, arnT, yfdH, wbbK, opgG, opgH, ycjM, glgA, glgB, malQ, otsA and yaiP.

All sustained promoters, UTRs and terminator sequences are derived from the libraries described by Mutalik et al (Nat. Methods 2013, no.10, 354-360) and Camfray et al (Nucleic Acids Res.2013, 41 (9), 5139-5148): the genes were expressed using the promoters MutalikP5 ("PROM 0005_MutalikP 5") and apFAB82 ("PROM 0050_apFAB 82") as described by Mutalik et al (Nat. Methods 2013, no.10, 354-360), the UTR used comprising GalE_BCD12 ("UTR 0010_GalE BCD12") and GalE_LeuAB ("UTR 0014_GalE_LeuAB") as described by Mutalik et al (Nat. Methods 2013, no.10, 354-360), and the terminator sequence used being ilvGEDA ("0007_ilGEDA") as described by Camfray et al (Nucleic Acids Res.2013, 41 (9), 5139-5148). All genes were sequenced synthetically on a Twist Bioscience (twistbioscience. Com) or IDT (eu. Idtna. Com) and the codon usage was adapted using the tools of the suppliers. The SEQ ID NOS described in the present invention are summarized in Table 1.

All strains were stored in frozen vials at-80℃and (overnight LB cultures were mixed with 70% glycerol at a 1:1 ratio).

Table 1: summary of SEQ ID NOs described in the present invention

Culture conditions

Pre-incubation for the 96-well microtiter plate experiments was started from frozen vials, performed in 150 μLLB, and incubated overnight at 37℃on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96-well square microtiter plate, diluted 400-fold with 400 μl of minimal medium. These final 96-well culture plates were then incubated at 37℃for 72 hours or less or longer on an orbital shaker at 800 rpm. To measure the sugar concentration at the end of the culture experiment, whole broth samples (=average intracellular and extracellular sugar concentrations) were obtained from each well by boiling the broth at 60 ℃ for 15min, followed by brief centrifugation of the cells.

The pre-culture of the bioreactor starts with an entire 1mL frozen vial with a certain strain, is inoculated in 250mL or 500mL minimal medium in 1L or 2.5L shake flasks and incubated at 37 ℃ on an orbital shaker at 200rpm for 24h. Followed by inoculation of a 5L bioreactor (250 mL of inoculum in 2L batch medium); the process is controlled by MFCS control software (Sartorius Stedim Biotech, melsungen, germany). Culture conditions were set to 37 ℃ and maximum agitation; the pressure gas flow rate depends on the strain and the bioreactor. The pH was controlled at 6.8 using 0.5M H2S04 and 20% NH4 OH. The exhaust gas is cooled. When foaming increased during fermentation, a 10% silicone defoamer solution was added.

Optical density

The cell density of the cultures is typically monitored by measuring the optical density at 600nm (Implen Nanophotometer NP, westburg, belgium, or with Spark 10M microplate reader, tecan, switzerland).

Analytical analysis

Standards such as, but not limited to, sucrose, lactose, lacNAc, lacto-N-disaccharide (LNB), fucosylated LacNAc (2 ' FLacNAc, 3-FLacNAc), sialylated LacNAc, (3 ' SLacNAc,6' SLacNAc), fucosylated LNB (2 ' FLNB, 4' FLNB), lacto-N-triose II (LN 3), lacto-N-tetraose (LNT), lacto-N-neo-tetraose (LNnT), LNFP-I, LNFP-II, LNFP-III, LNFP-V, LSTa, LSTc and LSTd are commercially available from Carbosynth (UK), elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with internally manufactured standards.

Neutral oligosaccharides were analyzed on a Waters AcquityH grade UPLC under evaporative light scattering detector (Evaporative Light Scattering Detector; ELSD) or Refractive Index (RI) detection. In a Waters Acquity UPLC BEH Amide column (2.1X100 mm;1.7 μm) column and Acquity UPLC BEH Amide VanGuard column (++>2.1X5 mm) was injected with a 0.7. Mu.L volume of sample. The column temperature was 50 ℃. The mobile phase consisted of a 1/4 water and 3/4 acetonitrile solution to which was added 0.2% triethylamine. The method was isocratic at a flow rate of 0.130 mL/min. The ELS detector has an offset tube temperature of 50 ℃ and an N2 gas pressure of 50psi, a gain of 200 and a data rate of 10pps. The temperature of the RI detector was set to 35 ℃.

Sialylated oligosaccharides were analyzed on a Waters Acquity H grade UPLC under Refractive Index (RI) detection. In a Waters Acquity UPLC BEH Amide column (2.1X100 mm;1.7 μm) was injected with a 0.5 μl volume of the sample. The column temperature was 50 ℃. The mobile phase consisted of a mixture of 70% acetonitrile to which 0.05% pyrrolidine was added, 26% ammonium acetate buffer (150 mM) and 4% methanol. The method was isocratic at a flow rate of 0.150 mL/min. The temperature of the RI detector was set to 35 ℃.

Neutral and sialylated saccharides were analyzed on a Waters Acquity H grade UPLC under Refractive Index (RI) detection. In a Waters Acquity UPLC BEH Amide column (2.1X100 mm;1.7 μm) was injected with a 0.5 μl volume of the sample. The column temperature was 50 ℃. Mobile phase consisted of 72% acetonitrile with 28% acetic acid to which 0.1% triethylamine was addedAmmonium buffer (100 mM). The method was isocratic at a flow rate of 0.260 mL/min. The temperature of the RI detector was set to 35 ℃.

For analysis on a mass spectrometer, a Waters Xevo TQ-MS with electrospray ionization (Electron Spray Ionisation; ESI) was used at a desolvation temperature of 450 ℃, a nitrogen desolvation gas flow rate of 650L/h and a cone voltage of 20V. For all oligosaccharides, the MS was operated in negative mode in selected ion monitoring (selected ion monitoring; SIM). The separation was carried out at 35℃on a Waters Acquity UPLC with a Thermo Hypercarb column (2.1X100 mm;3 μm). A gradient was used in which the eluent a was ultrapure water and 0.1% formic acid and in which the eluent B was acetonitrile and 0.1% formic acid. The oligosaccharides were separated within 55min using the following gradient: from 2% to 12% of the release agent B initially increases within 21min, from 12% to 40% of the release agent B increases a second time within 11min, and from 40% to 100% of the release agent B increases a third time within 5min. As a washing step, 100% of the eluent B was used for 5min. For column equilibration, the initial conditions of 2% of eluent B were restored within 1min and maintained for 12min.

Neutral and sialylated saccharides at low concentrations (below 50 mg/L) were analyzed by pulsed amperometric detection (pulsed amperometric detection; PAD) on a Dionex HPAEC system. A sample of 5. Mu.L was injected over a Dionex CarboPac PA200 column of 4X 250mm and Dionex CarboPac PA200 guard column of 4X 50 mm. The column temperature was set at 30 ℃. A gradient was used wherein the eluent a was deionized water, wherein the eluent B was 200mM sodium hydroxide and wherein the eluent C was 500mM sodium acetate. The oligosaccharides were separated within 60min while maintaining a constant ratio of 25% of the debonding agent B using the following gradient: the initial isocratic step of 75% of the debonding agent A was maintained for 10min, the initial increase of 0% to 4% of the debonding agent C within 8min, the second isocratic step of 71% of the debonding agent A and 4% of the debonding agent C was maintained for 6min, the second increase of 4% to 12% of the debonding agent C within 2.6min, the third isocratic step of 63% of the debonding agent A and 12% of the debonding agent C was maintained for 3.4min, and the third increase of 12% to 48% of the debonding agent C within 5 min. As a washing step, 48% of the eluent C was used for 3min. For column equilibration, the initial conditions of 75% of the eluent a and 0% of the eluent C were restored within 1min and maintained for 11min. The flow rate applied was 0.5mL/min.

Example 2 materials and methods of Saccharomyces cerevisiae

Culture medium

The strains were grown on defined yeast media containing 6.7g/L of amino acid-free yeast nitrogen source base (YNB without AA, difco), 20g/L agar (Difco) (solid culture), 22g/L glucose monohydrate or 20g/L lactose and 0.79g/L CSM or 0.77g/L CSM-Ura, 0.77g/L CSM-Trp or 0.77g/L CSM-His (MP Biomedicals) with synthesis with complete supplementation mix (SD CSM) or CSM omission (drop-out) (SD CSM-Ura, SD CSM-Trp).

Strain

Saccharomyces cerevisiae BY4742, produced BY Brachmann et al (Yeast (1998) 14:115-32), was used and was available from Eurocarf culture collection. All mutant strains were produced by homologous recombination or plastid transformation using the Gietz method (Yeast 11:355-360, 1995).

Plastid body

In one example of GDP-fucose (fucose) production, the yeast expression Plasmid p2a_2μ_Fuc (Chan 2013, plasmid 70,2-17) is used for expression of foreign genes in Saccharomyces cerevisiae. The plastid contains an ampicillin resistance gene and bacterial origin of replication to allow selection and maintenance in E.coli, and 2. Mu. Yeast ori and Ura3 selectable markers for selection and maintenance in yeast. This plastid additionally contains persistent transcriptional units for: lactose permease as LAC12 of kluyveromyces lactis e.g. having SEQ ID NO 30, GDP-mannose 4, 6-dehydratase as gmd of e.coli e.g. having SEQ ID NO 10, and GDP-L-fucose synthase as fcl of e.coli e.g. having SEQ ID NO 11. In another embodiment, yeast expression plastid p2a_2μ_fuc2 can be used as an alternative expression plastid to p2a_2μ_fucplastid comprising the following persistent transcription units immediately following the ampicillin resistance gene, bacterial ori, 2μ yeast ori and Ura3 selectable markers: lactose permease such as LAC12 of kluyveromyces lactis having SEQ ID NO 30, fucose permease such as furp of escherichia coli having SEQ ID NO 12, bifunctional fucose kinase/fucose-1-guanyl phosphate transferase such as fkp of bacteroides fragilis having SEQ ID NO 13, for example. To further produce fucosylated oligosaccharides, p2a_2μ_fuc2 and its variants p2a_2μ_fuc2 additionally contain persistent transcription units for use as e.g. α -1, 2-fucosyltransferases of hputc of helicobacter pylori with SEQ ID NO 04 and/or as e.g. α -1, 3-fucosyltransferases of hputt of helicobacter pylori with SEQ ID NO 05.

In one example of sialic acid production and CMP-sialic acid production, yeast expression plastids are derived from pRS420 plastid series (Christianson et al, 1992,Gene 110:119-122) containing a TRP1 selectable marker and persistent transcriptional units for: l-glutamylamino acid-D-fructose-6-phosphate aminotransferase as, for example, mutant glmS.times.54 of E.coli having SEQ ID NO 18; phosphatases such as, for example, yqaB of any of the escherichia coli having SEQ ID NO 19 or the escherichia coli genes comprising aphA, cof, hisB, otsB, surE, yaed, ycjU, yedP, yfbT, yidA, yigB, yihX, yniC, yrbL, appA, gph, serB, ybhA, ybiV, ybjL, yfb, yieH, yjgL, yjjG, yrfG and YbiU or psuppp from pseudomonas putida, scDOG1 from saccharomyces cerevisiae and BsAraL from bacillus subtilis (as described in WO 18122225); n-acetylglucosamine 2-epimerase of AGEs of Bacteroides ovale, for example having SEQ ID NO 16; n-acetylneuraminic acid synthase of NeuB as NeuB of Neisseria meningitidis having, for example, SEQ ID NO 17; and N-acyl neuraminic acid cytidylyltransferase of NeuA as, for example, pasteurella multocida having SEQ ID NO 21. Optionally, a persistent transcription unit for glucosamine 6-phosphate N-acetyltransferase as GNA1 of Saccharomyces cerevisiae, e.g., with SEQ ID NO 15, is also added. In an example of producing sialylated oligosaccharides, the plastid additionally comprises persistent transcriptional units for: lactose permease such as LAC12 of kluyveromyces lactis having, for example, SEQ ID NO 30; and PmultST 3-like polypeptides as, for example, beta-galactoside alpha-2, 3-sialyltransferase (UniProt ID Q9CLP 3) from PmultST3 of Pasteurella multocida or consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having beta-galactoside alpha-2, 3-sialyltransferase activity as shown in SEQ ID NO 22, nmeiST 3 from Neisseria meningitidis (SEQ ID NO 23) or PmultST2 from Pasteurella multocida strain Pm70 (GenBank NO. AAK 02592.1) such as for example beta-galactoside alpha-2, 6-sialyltransferase (UniProt ID O66375) from P.mermaid light emitting bacteria or P-JT-ISH-224-ST6 (UniProt ID A8QYL 1) from P-JT-ISH-224 from P-ISH-224 or P-JT-ISH-224-ST 6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 with beta-galactoside alpha-2, 6-sialyltransferase activity as SEQ ID NO 25 and/or P-JT-ISH-224-ST 6-like polypeptide consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 with beta-galactoside alpha-2, 6-sialyltransferase activity as SEQ ID NO 24 and/or alpha-2, 8-sialyltransferase (UniProt ID Q64689) as for example mice.

In one example of UDP-galactose production, yeast display plastids are derived from pRS 420-plastid series (Christianson et al, 1992,Gene 110:119-122) containing a HIS3 selectable marker and a persistent transcriptional unit for UDP-glucose 4-epi-isomerase as galE, e.g., E.coli with SEQ ID NO 29. In the example of LN3 and LN3 derived oligosaccharides (e.g., LNT or LNnT) production, the plastid is further modified with a sustained transcriptional unit for use in: lactose permease like LAC12 of kluyveromyces lactis e.g. having SEQ ID NO 30, galactoside beta-1, 3-N-acetylglucosamintransferase like lgtA of neisseria meningitidis e.g. having SEQ ID NO 26, e.g. e.coli O55 having SEQ ID NO 27: n-acetylglucosamine beta-1, 3-galactosyltransferase of WbgO of H7 or N-acetylglucosamine beta-1, 4-galactosyltransferase of lgtB as, for example, neisseria meningitidis having SEQ ID NO 28.

Preferably, but not necessarily, any one or more of the glycosyltransferases, proteins involved in nucleotide activated sugar synthesis and/or membrane transporters are fused N-terminally and/or C-terminally to a SUMOstar tag (e.g. obtained from pYSUMOstar, life Sensors, malvern, PA) to enhance its solubility.

Optionally, the mutant yeast strain is modified with a genomic gene insert encoding a persistent transcriptional unit of a companion protein such as, for example, hsp31, hsp32, hsp33, sno4, kar2, ssb1, sse2, ssa1, ssa2, ssa3, ssa4, ssb2, ecm10, ssc1, ssq1, ssz1, lhs1, hsp82, hsc82, hsp78, hsp104, tcp1, ct4, ct8, ct2, ct3, ct5, ct6, or ct7 (Gong et al, 2009, mol. Syst. Biol.5:275).

The plastids were maintained in E.coli DH 5. Alpha. Host purchased from Invitrogen (F ^- 、phi80dlacZδM15、δ(lacZYA-argF)U169、deoR、recA1、endA1、hsdR17(rk ^- 、mk ⁺ )、phoA、supE44、λ ^- Thi-1, gyrA96, relA 1).

Heterologous and homologous manifestation

The gene to be expressed, whether it is plastid-derived or genome-derived, is synthesized synthetically by one of the following companies: DNA2.0, gen9, IDT or Twist Bioscience. Expression may be further facilitated by optimizing codon usage to that of the expression host. The genes were optimized using the vendor's tools.

Culture conditions

In general, yeast strains were initially grown on SD CSM plates to obtain single colonies. These discs were grown at 30℃for 2-3 days. Beginning with a single colony, the preculture was grown at 30℃overnight at 5mL and shaken at 200 rpm. Subsequent 125mL shake flask experiments were inoculated with 2% of this preculture in 25mL medium. These flasks were incubated at 30℃with orbital shaking at 200 rpm.

Gene expression promoter

The genes were expressed using a synthetic sustained promoter as described by Blazeck (Biotechnology and Bioengineering, volume 109, no. 11, 2012).

Example 3 production of oligosaccharide mixture comprising 3'SL, 6' SL, 3 '-sialylated LacNAc and 6' -sialylated LacNAc Using modified E.coli host

Coli K-12 mg1655 strain modified by gene insertion of a genome for a sustained transcriptional unit of N-acyl neuraminic acid cytidylyltransferase (neuA) of septicemia of SEQ ID NO 21 and containing the gene lacZ of escherichia coli, further transformed with a expressible plasmid containing a sustained transcriptional unit for α -2, 3-sialyltransferase of septicemia of SEQ ID NO 22 and α -2, 6-sialyltransferase of mermaid light emitting bacteria of SEQ ID NO 24. The novel strain was evaluated in a growth experiment against the production of oligosaccharide mixtures comprising 3'sl, 6' sl, 3 '-sialylated LacNAc (3' slacnac) and 6 '-sialylation (6' slacnac) in whole broth samples according to the culture conditions provided in example 1, wherein the medium contained glycerol as carbon source and sialic acid, lactose and LacNAc as precursors.

Example 4 production of oligosaccharide mixtures comprising 3'SL, 6' SL, 3 '-sialylated LacNAc and 6' -sialylated LacNAc Using modified E.coli hosts

An escherichia coli K-12 MG1655 strain modified by genomic gene insertion of a sustained transcriptional unit for an N-acyl neuraminic acid cytidylyltransferase (neuA) of a pasteurella multocida having SEQ ID NO 21, further gene insertion mutation with genomic genes of escherichia coli nagA, nagB and lacZ genes, and of a sustained transcriptional unit for mutation glmS 54 having SEQ ID NO 18 from escherichia coli, GNA1 having SEQ ID NO 15 from saccharomyces cerevisiae, phosphatase yqaB of escherichia coli having SEQ ID NO 19 and LgtB having SEQ ID NO 28 from neisseria meningitidis. In the next step, the novel strain is transformed with a expressible plasmid containing the persistent transcriptional units of an alpha-2, 3-sialyltransferase for Pasteurella multocida having SEQ ID NO 22 and an alpha-2, 6-sialyltransferase for mermaid light emitting bacteria having SEQ ID NO 24. The novel strain was evaluated in a growth experiment against the production of oligosaccharide mixtures comprising 3'sl, 6' sl, 3 '-sialylated LacNAc (3' slacnac) and 6 '-sialylated LacNAc (6' slacnac) in whole broth samples according to the culture conditions provided in example 1, wherein the medium contained glycerol as carbon source and sialic acid and lactose as precursors.

Example 5 production of oligosaccharide mixture comprising 3'SL, 6' SL, 3 '-sialylated LacNAc and 6' -sialylated LacNAc Using modified E.coli host

The E.coli K-12 MG1655 strain modified to produce sialic acid as described in example 1 was further modified with gene knockout of the E.coli lacZ gene and transformed with a expressible plasmid consisting of the persistent transcriptional units of neuA for Pasteurella multocida with SEQ ID NO 21, alpha-2, 3-sialyltransferase for Pasteurella multocida with SEQ ID NO 22 and alpha-2, 6-sialyltransferase for mermaid light emitting bacteria with SEQ ID NO 24. The novel strain was evaluated in a growth experiment against the production of oligosaccharide mixtures comprising 3'sl, 6' sl, 3 '-sialylated LacNAc (3' slacnac) and 6 '-sialylated LacNAc (6' slacnac) in whole broth samples according to the culture conditions provided in example 1, wherein the medium contained glycerol as carbon source and lactose and LacNAc as precursors.

Example 6 production of oligosaccharide mixture comprising 3'SL, 6' SL, 3 '-sialylated LacNAc and 6' -sialylated LacNAc Using modified E.coli host

The E.coli K-12 MG1655 strain modified to produce sialic acid as described in example 1 was further deleted by the genomic gene of E.coli lacZ and the genomic gene insertion mutation for the persistent transcription unit of LgtB with SEQ ID NO 28 from Neisseria meningitidis to produce LacNAc, and transformed by a expressible plasmid containing the persistent transcription units for neuA with Pasteurella with SEQ ID NO 21, alpha-2, 3-sialyltransferase with Pasteurella with SEQ ID NO 22 and alpha-2, 6-sialyltransferase with mermaid light emitting bacteria with SEQ ID NO 24. The novel strain was evaluated in a growth experiment against the production of oligosaccharide mixtures comprising 3'sl, 6' sl, 3 '-sialylated LacNAc (3' slacnac) and 6 '-sialylated LacNAc (6' slacnac) in whole broth samples according to the culture conditions provided in example 1, wherein the medium contained glycerol as carbon source and lactose as precursor.

EXAMPLE 7 production of sialylated LacNAc and Poly-LacNAc structures Using modified E.coli hosts

The E.coli K-12 MG1655 strain modified to produce sialic acid as described in example 1 was further subjected to a genomic gene insertion mutation for the persistent transcription unit of LgtB with SEQ ID NO 28 from Neisseria meningitidis to produce LacNAc, and was transformed with a expressible plasmid containing the persistent transcription units for neuA with Pasteurella multocida with SEQ ID NO 21, alpha-2, 3-sialyltransferase with Pasteurella multocida with SEQ ID NO 22 and alpha-2, 6-sialyltransferase with mermaid light emitting bacteria with SEQ ID NO 24. In a next step, the mutant strain is further transformed with a compatible expression plasmid containing a persistent transcriptional unit for the galactoside beta-1, 3-N-acetylglucosaminyl transferase (LgtA) of Neisseria meningitidis having SEQ ID NO 26. In a growth experiment, the novel strain was evaluated against the production of an oligosaccharide mixture comprising LacNAc, poly LacNAc structures, i.e. (Gal-b 1, 4-GlcNAc) N, constructed by repeating N-acetyllactosamine units beta 1,3 linked to each other according to the culture conditions provided in example 1; and 3 '-sialyl LacNAc, 6' -sialyl LacNAc and sialyl poly LacNAc structures in which Gal residues are sialylated, wherein the medium contains glycerol as a carbon source and wherein no precursor is required to be supplied to the culture.

Example 8 production of oligosaccharide mixture comprising 3'SL, 6' SL, 3 '-sialylated LNB and 6' -sialylated LNB Using modified E.coli host

An escherichia coli K-12 MG1655 strain modified by genomic gene insertion for the persistent transcription unit of neuA of pasteurella multocida having SEQ ID NO 21 and containing gene knockout of the escherichia coli lacZ gene, further transformed with a expressible plasmid containing the persistent transcription unit for the α -2, 3-sialyltransferase of pasteurella multocida having SEQ ID NO 22 and the α -2, 6-sialyltransferase of mermaid light emitting bacteria having SEQ ID NO 24. The novel strain was evaluated in a growth experiment against the production of an oligosaccharide mixture comprising 3'sl, 6' sl, 3 '-sialylated LNB (3' slnb) and 6 '-sialylated LNB (6' slnb) in a whole broth sample according to the culture conditions provided in example 1, wherein the medium contained glycerol as carbon source and sialic acid, lactose and LNB as precursors.

Example 9 production of oligosaccharide mixture comprising 3'SL, 6' SL, 3 '-sialylated LNB and 6' -sialylated LNB Using modified E.coli host

An escherichia coli K-12 MG1655 strain modified by genomic gene insertion of a persistent transcription unit for neuA of pasteurella multocida having SEQ ID NO 21, further deleted with genomic genes of escherichia coli nagA, nagB and lacZ genes and a genomic gene insertion mutation of a persistent transcription unit for mutation glmS 54 having SEQ ID NO 18 from escherichia coli, GNA1 having SEQ ID NO 15 from saccharomyces cerevisiae and for gene insertion mutation from escherichia coli O55: wbgO of H7 with SEQ ID NO 27 to produce LNB. In the next step, the novel strain is transformed with a expressible plasmid containing the persistent transcriptional units of an alpha-2, 3-sialyltransferase for Pasteurella multocida having SEQ ID NO 22 and an alpha-2, 6-sialyltransferase for mermaid light emitting bacteria having SEQ ID NO 24. The novel strain was evaluated in a growth experiment against the production of an oligosaccharide mixture comprising 3'sl, 6' sl, 3 '-sialylated LNB (3' slnb) and 6 '-sialylated LNB (6' slnb) in a whole broth sample according to the culture conditions provided in example 1, wherein the medium contained glycerol as carbon source and sialic acid and lactose as precursors.

Example 10 production of oligosaccharide mixture comprising 3'SL, 6' SL, 3 '-sialylated LNB and 6' -sialylated LNB Using modified E.coli host

The E.coli K-12 MG1655 strain modified to produce sialic acid as described in example 1 was further modified with gene knockout of the E.coli lacZ gene and transformed with a expressible plasmid consisting of the persistent transcriptional units of neuA for Pasteurella multocida with SEQ ID NO 21, alpha-2, 3-sialyltransferase for Pasteurella multocida with SEQ ID NO 22 and alpha-2, 6-sialyltransferase for mermaid light emitting bacteria with SEQ ID NO 24. The novel strain was evaluated in a growth experiment against the production of an oligosaccharide mixture comprising 3'sl, 6' sl, 3 '-sialylated LNB (3' slnb) and 6 '-sialylated LNB (6' slnb) in a whole broth sample according to the culture conditions provided in example 1, wherein the medium contained glycerol as carbon source and lactose and LNB as precursors.

Example 11 production of oligosaccharide mixture comprising 3'SL, 6' SL, 3 '-sialylated LNB and 6' -sialylated LNB Using modified E.coli host

The E.coli K-12 MG1655 strain modified to produce sialic acid as described in example 1 was further deleted for the genomic gene of the E.coli lacZ gene and used for the gene from E.coli O55: h7 has a genetic insertion mutation of the persistent transcription unit of WbgO of SEQ ID NO 27 to produce LNB and is transformed by a expressible plasmid containing the persistent transcription unit for neuA of Pasteurella multocida having SEQ ID NO 21, alpha-2, 3-sialyltransferase of Pasteurella multocida having SEQ ID NO 22 and alpha-2, 6-sialyltransferase of mermaid light emitting bacteria having SEQ ID NO 24. The novel strain was evaluated in a growth experiment against the production of an oligosaccharide mixture comprising 3'sl, 6' sl, 3 '-sialylated LNB (3' slnb) and 6 '-sialylated LNB (6' slnb) in a whole broth sample according to the culture conditions provided in example 1, wherein the medium contained glycerol as carbon source and lactose as precursor.

Example 12 production of oligosaccharide mixture comprising fucosylated and sialylated lactose Structure Using modified E.coli host

The E.coli strain adapted for GDP-fucose production as exemplified in example 1 was further transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression elements for one or two selected fucosyltransferases and wherein the second plasmid contains persistence expression elements for one or two selected sialyltransferases and the N-acyl neuraminic acid cytidylyltransferase (NeuA) of Pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The strain was transformed to represent 1) one fucosyltransferase in combination with two sialyltransferases, 2) two fucosyltransferases in combination with one sialyltransferase, or 3) two fucosyltransferases in combination with two sialyltransferases (table 3). The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose structures as shown in table 3 in whole broth samples according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and sialic acid and lactose as precursors.

Table 2: overview of plastids cloned with persistent transcription units for one or both fucosyltransferase genes or for one or both sialyltransferase genes

Table 3: oligosaccharide production was assessed in a growth experiment in a mutant E.coli strain according to the culture conditions as described in example 1, wherein the medium contained sucrose as carbon source and sialic acid and lactose as precursors.

* See Table 2 for plastid information

Example 13 production of oligosaccharide mixture comprising fucosylated and sialylated lactose Structure Using modified E.coli host

The escherichia coli strains adapted for GDP-fucose production as exemplified in example 1 were further modified for sialic acid production by genomic gene deletion of the escherichia coli genes nag4, nagB, nanA, nanE and nanK and by genomic gene insertion of the persistent transcription unit for mutant glmS 54 of escherichia coli with SEQ ID NO 18, GNA1 of saccharomyces cerevisiae with SEQ ID NO 15, N-acetyl glucosamine 2-epimerase (AGE) of bacteroides ovatus with SEQ ID NO 16 and N-acetyl neuraminic acid synthase (neuB) of neisseria meningitidis with SEQ ID NO 17. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The strain was transformed to represent 1) one fucosyltransferase in combination with two sialyltransferases, 2) two fucosyltransferases in combination with one sialyltransferase, or 3) two fucosyltransferases in combination with two sialyltransferases (table 4). The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose structures as shown in table 4 in whole broth samples, according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 4: oligosaccharide production was assessed in a growth experiment in a mutant E.coli strain according to the culture conditions as described in example 1, wherein the medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

Example 14 production of oligosaccharide mixture comprising fucosylated and sialylated lactose Structure Using modified E.coli host

The E.coli strain adapted for GDP-fucose production as exemplified in example 1 was further modified for sialic acid production by genomic gene excision of E.coli gene nagA, nagB, nanA, nanE and nanK and by genomic gene insertion of the persistent transcription unit for mutant glmS 54 of E.coli with SEQ ID NO 18, UDP-N-acetylglucosamine 2-epimerase (neuC) of Campylobacter jejuni with SEQ ID NO 20 and N-acetylneuraminic acid synthase (neuB) of Neisseria meningitidis with SEQ ID NO 17. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The strain was transformed to represent 1) one fucosyltransferase in combination with two sialyltransferases, 2) two fucosyltransferases in combination with one sialyltransferase, or 3) two fucosyltransferases in combination with two sialyltransferases (table 5). The novel strains were evaluated in a growth experiment against the production of an oligosaccharide mixture comprising fucosylated and sialylated lactose structures as shown in table 5 in a whole broth sample, according to the culture conditions provided in example 1, wherein the culture contained sucrose as carbon source and lactose as precursor.

Table 5: oligosaccharide production was assessed in a growth experiment in a mutant E.coli strain according to the culture conditions as described in example 1, wherein the medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

Example 15 production of oligosaccharide mixtures comprising fucosylated and sialylated lactose Structure Using modified E.coli hosts

The E.coli strain adapted for sialic acid production as exemplified in example 1 was further modified via genomic gene knockout of the E.coli wcaJ gene to increase the intracellular pool of GDP-fucose. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The strain was transformed to represent 1) one fucosyltransferase in combination with two sialyltransferases, 2) two fucosyltransferases in combination with one sialyltransferase, or 3) two fucosyltransferases in combination with two sialyltransferases (table 6). The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose structures as shown in table 6 in whole broth samples, according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 6: oligosaccharide production was assessed in a growth experiment in a mutant E.coli strain according to the culture conditions as described in example 1, wherein the medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

Example 16 production of oligosaccharide mixtures comprising fucosylated and sialylated lactose Structure Using modified E.coli hosts

The E.coli strain adapted for sialic acid production as exemplified in example 1 was further modified to increase the intracellular pool of GDP-fucose via genomic gene knockout of E.coli wcaJ, fucK and fucI genes and genomic gene insertion of the persistence presentation unit for the fucose permease (fucP) of E.coli having SEQ ID NO 12 and the bifunctional fucose kinase/fucose-1-phosphogguanyl transferase of E.fragilis having SEQ NO ID 13 (fkp). In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The strain was transformed to represent 1) one fucosyltransferase in combination with two sialyltransferases, 2) two fucosyltransferases in combination with one sialyltransferase, or 3) two fucosyltransferases in combination with two sialyltransferases (table 7). The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose structures as shown in table 7 in whole broth samples, according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 7: oligosaccharide production was assessed in a growth experiment in a mutant E.coli strain according to the culture conditions as described in example 1, wherein the medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

EXAMPLE 17 production of oligosaccharide mixture comprising sialylated LN3, 3' SL and LSTa Using modified E.coli host

The E.coli strain modified to produce LNT as described in example 1 was further modified by genomic gene knockout of the E.coli lacZ gene and transformed by a expressive plasmid containing a persistent expression cassette for NeuA of P.septicum with SEQ ID NO 21 and alpha-2, 3-sialyltransferase of P.septicum with SEQ ID NO 22. The novel strain was evaluated in a growth experiment against the production of oligosaccharide mixtures comprising LN3, 3 '-sialylated LN3 (Neu 5Ac-a2,3-GlcNAc-b1,3-Gal-b1, 4-Glc), LNT, 3' SL and LSTa according to the culture conditions in the 96-well tray provided in example 1, wherein the medium contained glycerol as carbon source and both sialic acid and lactose as precursors.

EXAMPLE 18 production of oligosaccharide mixture comprising 6' sl, sialylated LN3 and LSTc Using modified E.coli host

The E.coli strain modified to produce LNnT as described in example 1 was further modified by genomic gene knockout of the E.coli lacZ gene and transformed with a expressive plasmid containing a persistent expression cassette for NeuA of Pasteurella multocida having SEQ ID NO 21 and a selected alpha-2, 6-sialyltransferase. In this experiment, the mermaid light emitting bacterium having SEQ ID NO 24 was tested for alpha-2, 6-sialyltransferase and the light emitting bacterium genus JT-ISH-224 having SEQ ID NO 25 for alpha-2, 6-sialyltransferase. Thus, three different strains were produced, each exhibiting a single α -2, 6-sialyltransferase in a specific transcriptional unit. Table 8 shows an overview of transcriptional units for selected a-2, 6-sialyltransferase proteins. The novel strain was evaluated in a 96-well tray in a growth experiment according to the culture conditions provided in example 1, wherein the medium contained glycerol as carbon source and both sialic acid and lactose as precursors. After 72 hours of incubation, the culture broth was collected and the sugar mixture was analyzed as described in example 1. All novel strains produced oligosaccharide mixtures comprising 6' SL, LN3, LNnT and LSTc (Neu 5Ac-a2,6-Gal-b1,4-GlcNAc-b1,3-Gal-b1, 4-Glc). FIG. 1 shows a chromatogram obtained via strain S2 analyzed by the Dionex method as described in example 1. UPLC can also be used to detect the compound 6' -sialylated LN3 (Neu 5Ac-a2,6- (GlcNAc-b 1, 3) -Gal-b1, 4-Glc) in these samples under RI detection as described in example 1. The ratio of 6' SL, LN3, sialylated LN3, LNnT and LSTc produced in the mixture of new strains can be precisely regulated by selecting the expressed alpha-2, 6-sialyltransferases and transcriptional units for expression of these sialyltransferases.

Table 8: summary of mutant E.coli strains exhibiting different a-2, 6-sialyltransferases in specific transcriptional units.

EXAMPLE 19 production of oligosaccharide mixture comprising sialylated LN3, 3' SL and LSTd Using modified E.coli host

The E.coli strain modified to produce LNnT as described in example 1 was further modified by genomic gene knockout of the E.coli lacZ gene and transformed with a expressive plasmid containing a persistent expression cassette for NeuA of Pasteurella multocida having SEQ ID NO 21 and a selected alpha-2, 3-sialyltransferase. In this experiment, the alpha-2, 3-sialyltransferase of Pasteurella multocida having SEQ ID NO 22 and the alpha-2, 3-sialyltransferase of Neisseria meningitidis having SEQ ID NO 23 were tested, the tri-alpha-2, 3-sialyltransferases each being cloned in two different transcriptional units. Thus, four different strains were produced, each exhibiting a single α -2, 3-sialyltransferase in a specific transcriptional unit. Table 9 shows an overview of transcriptional units for selected alpha-2, 3-sialyltransferase proteins. The novel strain was evaluated in a 96-well tray in a growth experiment according to the culture conditions provided in example 1, wherein the medium contained glycerol as carbon source and both sialic acid and lactose as precursors. After 72 hours of incubation, the culture broth was collected and the sugar mixture was analyzed as described in example 1. All novel strains produced oligosaccharide mixtures comprising 3' SL, LN3, LNnT and LSTd (Neu 5Ac-a2,3-Gal-b1,4-GlcNAc-b1,3-Gal-b1, 4-Glc). FIG. 2 shows a chromatogram obtained via strain S5 analyzed by the Dionex method as described in example 1. UPLC can also be used to detect the compound 3' -sialylated LN3 (Neu 5Ac-a2,3-GlcNAc-b1,3-Gal-b1, 4-Glc) in these samples under RI detection as described in example 1. The ratio of 3' SL, LN3, sialylated LN3, LNnT and LSTd produced in the mixture of new strains can be precisely regulated by selecting the expressed alpha-2, 3-sialyltransferases and transcriptional units for expression of these sialyltransferases.

Table 9: summary of mutant E.coli strains exhibiting different alpha-2, 3-sialyltransferases in specific transcriptional units.

EXAMPLE 20 production of oligosaccharide mixture comprising sialylated LN3, 3' SL and LSTa Using modified E.coli host

The E.coli strain modified to produce sialic acid as described in example 1 was further modified by genomic gene insertion of the persistent transcription unit for the galactoside beta-1, 3-N-acetylglucosaminyl transferase (LgtA) of Neisseria meningitidis with SEQ ID NO 26 and E.coli O55 with SEQ ID NO 27 to allow the production of LNT: H7N-acetylglucosamine beta-1, 3-galactosyltransferase (WbgO). In the next step, the novel strain is further modified by genomic gene knockout of the E.coli lacZ gene and transformed by a expressive plasmid having a persistent transcription unit for NeuA of Pasteurella multocida having SEQ ID NO 21 and alpha-2, 3-sialyltransferase of Pasteurella multocida having SEQ ID NO 22. The novel strain was evaluated in a growth experiment against the production of an oligosaccharide mixture comprising LN3, 3 '-sialylated LN3 (Neu 5Ac-a2,3-GlcNAc-b1,3-Gal-b1, 4-Glc), LNT, 3' SL and LSTa, according to the culture conditions provided in example 1, wherein the medium contains sucrose as carbon source and lactose as precursor.

EXAMPLE 21 production of oligosaccharide mixture comprising sialylated LN3, 6' SL and LSTc Using modified E.coli host

The E.coli strain modified to produce sialic acid as described in example 1 was further modified by genomic gene insertion of the persistent transcription unit for LgtA of Neisseria meningitidis with SEQ ID NO 26 and LgtB of Neisseria meningitidis with SEQ ID NO 28 to allow production of LNnT. In the next step, the novel strain is further modified by genomic gene knockout of the E.coli lacZ gene and transformed by a expressive plasmid with a persistent transcription unit for NeuA of Pasteurella septicaemia with SEQ ID NO 21 and an alpha-2, 6-sialyltransferase of the genus P.JT-ISH-224 with SEQ ID NO 25. The novel strain was evaluated in a growth experiment in 96-well plates according to the culture conditions provided in example 1 for the production of oligosaccharide mixtures comprising LN3, 6 '-sialylated LN3 (Neu 5Ac-a2,6- (GlcNAc-b 1, 3) -Gal-b1, 4-Glc), 6' SL, LNnT and LSTc, wherein the medium contained sucrose as carbon source and lactose as precursor.

EXAMPLE 22 production of oligosaccharide mixture comprising sialylated LN3, 3' SL and LSTd Using modified E.coli host

The E.coli strain modified to produce sialic acid as described in example 1 was further modified by genomic gene insertion of the persistent transcription unit for LgtA of Neisseria meningitidis with SEQ ID NO 26 and LgtB of Neisseria meningitidis with SEQ ID NO 28 to allow production of LNnT. In the next step, the novel strain is further modified by genomic gene knockout of the E.coli lacZ gene and transformed by a expressive plasmid having a persistent transcription unit for NeuA of Pasteurella multocida having SEQ ID NO 21 and alpha-2, 3-sialyltransferase of Pasteurella multocida having SEQ ID NO 22. The novel strain was evaluated in a growth experiment against the production of an oligosaccharide mixture comprising LN3, 3 '-sialylated LN3 (Neu 5Ac-a2,3-GlcNAc-b1,3-Gal-b1, 4-Glc), LNnT, 3' SL and LSTd, according to the culture conditions provided in example 1, wherein the medium contains sucrose as carbon source and lactose as precursor.

Example 23 when evaluated in a fed batch fermentation process with glycerol, sialic acid and lactose, an oligosaccharide mixture comprising sialylated LN3, LSTa and 3' SL was produced in the fermentation broth of the mutant E.coli strain

Mutant E.coli strains capable of producing LN3, LNT, 3' SL, and LSTa as described in example 17 were selected for further evaluation during fed-batch fermentation in a 5L bioreactor. Fed-batch fermentation was performed at the bioreactor scale as described in example 1. In these examples, glycerol was used as a carbon source and lactose was added as a precursor to the batch medium. Sialic acid was also added via additional feeds during fed-batch. A sample of conventional broth was taken and the produced sugar was measured as described in example 1. Fermentation broth of selected strains obtained after the fed-batch phase was evaluated for production of an oligosaccharide mixture comprising LN3, 3 '-sialylated LN3 (Neu 5Ac-a2,3-GlcNAc-b1,3-Gal-b1, 4-Glc), LNT, LSTa and 3' SL.

Example 24 when evaluated during fed-batch fermentation with sucrose and lactose, an oligosaccharide mixture comprising sialylated LN3, LSTa and 3' SL was produced in the fermentation broth of the mutant E.coli strain

Mutant E.coli strains capable of producing LN3, LNT, 3' SL, and LSTa as described in example 20 were selected for further evaluation during fed-batch fermentation in a 5L bioreactor. Fed-batch fermentation was performed at the bioreactor scale as described in example 1. In these examples, sucrose was used as a carbon source and lactose was added as a precursor to the batch medium. A sample of conventional broth was taken and the produced sugar was measured as described in example 1. Fermentation broth of selected strains obtained after the fed-batch phase was evaluated for production of an oligosaccharide mixture comprising LN3, 3 '-sialylated LN3 (Neu 5Ac-a2,3-GlcNAc-b1,3-Gal-b1, 4-Glc), LNT, LSTa and 3' SL.

EXAMPLE 25 oligosaccharide mixtures comprising sialylated LN3, para-lacto-N-neohexose, disialylated LNnT, LSTc and 6' SL were produced in the fermentation broth of the mutant E.coli strain when evaluated during fed-batch fermentation with glycerol, sialic acid and lactose

Mutant E.coli strains having persistent transcription units for the alpha-2, 6-sialyltransferase with the genus P.JT-ISH-224 of SEQ ID NO 25 as described in example 18 were selected for further evaluation in a fed-batch fermentation process in a 5L bioreactor. Fed-batch fermentation was performed at the bioreactor scale as described in example 1. In these examples, glycerol was used as a carbon source and lactose was added as a precursor to the batch medium. Sialic acid was also added via additional feeds during fed-batch. A sample of conventional broth was taken and the produced sugar was measured as described in example 1. UPLC analysis showed that the fermentation broth of the selected strain obtained after the batch phase contained lactose, LN3 and LNnT, while the fermentation broth of the selected strain obtained after the fed-batch phase contained an oligosaccharide mixture comprising LN3, 6 '-sialylated LN3 (Neu 5Ac-a2,6- (GlcNAc-b 1, 3) -Gal-b1, 4-Glc), LNnT, LSTc and 6' SL. At the end of the fed-batch, the mixture also contained p-lacto-N-neohexasaccharide (pLNnH), sialylated p-lacto-N-neohexasaccharide and disialylated LNnT, two structures that were not detected in the growth experimental analysis due to limited detection levels and small total production levels.

EXAMPLE 26 production of oligosaccharide mixture comprising sialylated LN3, para-lacto-N-neohexose, disialylated LNnT, LSTc and 6' SL in fermentation broth of mutant E.coli strains when evaluated during fed-batch fermentation with sucrose and lactose

Mutant E.coli strains capable of producing LN3, sialylated LN3, LNnT, 6' SL, and LSTc as described in example 21 were selected for further evaluation in a fed-batch fermentation process in a 5L bioreactor. Fed-batch fermentation was performed at the bioreactor scale as described in example 1. In these examples, sucrose was used as a carbon source and lactose was added as a precursor to the batch medium. A sample of conventional broth was taken and the produced sugar was measured as described in example 1. Fermentation broth of selected strains obtained after the fed-batch phase was evaluated for production of an oligosaccharide mixture comprising LN3, 6 '-sialylated LN3 (Neu 5Ac-a-2,6- (GlcNAc-b-1, 3) -Gal-b-1, 4-Glc), LNnT, LSTc, 6' SL, p-lacto-N-neohexasaccharide, sialylated p-lacto-N-neohexasaccharide and disialylated LNnT.

Example 27 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The escherichia coli strain adapted for GDP-fucose production as described in example 1 was further modified by genomic gene knockout of the escherichia coli nagA and nagB genes and genomic gene insertion of a persistence expression cassette for the mutation glmS 54 of escherichia coli with SEQ ID NO 18, lgtA of neisseria meningitidis with SEQ ID NO 26 and escherichia coli O55 with SEQ ID NO 27: wbgO of H7. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (table 10) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and sialic acid and lactose as precursors.

Table 10: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and sialic acid and lactose as precursors.

* See Table 2 for plastid information

EXAMPLE 28 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The escherichia coli strain adapted for GDP-fucose production as described in example 1 was further modified by genomic gene knockout of the escherichia coli nagA and nagB genes and genomic gene insertion of a persistence expression cassette for the mutation glmS 54 of escherichia coli with SEQ ID NO 18, lgtA of neisseria meningitidis with SEQ ID NO 26 and LgtB of neisseria meningitidis with SEQ ID NO 28. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 1 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (table 11) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and sialic acid and lactose as precursors.

Table 11: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and sialic acid and lactose as precursors.

* See Table 2 for plastid information

Example 29 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The escherichia coli strain adapted for GDP-fucose production as described in example 1 was further modified by genomic gene knockout of escherichia coli nagA, nagB, nanA, nanE and nanK genes and genomic gene insertion of a persistence-expressing cassette for mutation glmS 54 of escherichia coli with SEQ ID NO 18, GNA1 of saccharomyces cerevisiae with SEQ ID NO 15, phosphatase yqaB of escherichia coli with SEQ ID NO19, AGE of oval with SEQ ID NO 16, neuB of neisseria meningitidis with SEQ ID NO 17, lgtA of neisseria meningitidis with SEQ ID NO 26 and escherichia coli O55 with SEQ ID NO 27: wbgO of H7. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (table 12) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 12: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

Example 30 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The escherichia coli strains adapted for GDP-fucose production as described in example 1 were further modified by genomic gene knockout of escherichia coli nagA, nagB, nanA, nanE and nanK genes and genomic gene insertion of a persistence-expressing cassette for mutation glmS 54 of escherichia coli with SEQ ID NO 18, GNA1 of saccharomyces cerevisiae with SEQ ID NO 15, phosphatase yqaB of escherichia coli with SEQ ID NO19, AGE of oval with SEQ ID NO 16, neuB of neisseria meningitidis with SEQ ID NO 17, lgtA of neisseria meningitidis with SEQ ID NO 26 and LgtB of neisseria meningitidis with SEQ ID NO 28. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, lacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNT structures in whole broth samples (Table 13) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 13: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

Example 31 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The escherichia coli strain adapted for GDP-fucose production as described in example 1 was further modified by genomic gene knockout of escherichia coli nagA, nagB, nanA, nanE and nanK genes and genomic gene insertion of a persistence-expressing cassette for mutant glmS 54 of escherichia coli having SEQ ID NO 18, UDP-N-acetylglucosamine 2-epimerase (neuC) of campylobacter jejuni having SEQ ID NO 20, neuB of neisseria meningitidis having SEQ ID NO 17, lgtA of neisseria meningitidis having SEQ ID NO 26 and escherichia coli O55 having SEQ ID NO 27: wbgO of H7. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures as shown in table 14 in whole broth samples according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 14: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

Example 32 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The escherichia coli strain adapted for GDP-fucose production as described in example 1 was further modified by genomic gene knockout of escherichia coli nagA, nagB, nanA, nanE and nanK genes and genomic gene insertion of a persistence-expressing cassette for mutant glmS 54 of escherichia coli with SEQ ID NO 18, neuC of campylobacter jejuni with SEQ ID NO 20, neuB of neisseria meningitidis with SEQ ID NO 17, lgtA of neisseria meningitidis with SEQ ID NO 26 and LgtB of neisseria meningitidis with SEQ ID NO 28. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (table 15) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 15: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

Example 33 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The E.coli strain adapted for sialic acid production as described in example 1 was further modified with a genomic gene knockout of the E.coli wcaJ gene to increase the intracellular pool of GDP-fucose, and with a genomic gene insertion modification of the cassette for the following persistence: lgtA of neisseria meningitidis having SEQ ID NO 26 and escherichia coli O55 having SEQ ID NO 27: wbgO of H7. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (table 16) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 16: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

Example 34 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The E.coli strain adapted for sialic acid production as described in example 1 was further modified by genomic gene knock-out of the E.coli wcaJ gene to increase the intracellular pool of GDP-fucose and the genomic gene insertion of the persistence presentation cassette for LgtA of Neisseria meningitidis with SEQ ID NO 26 and LgtB of Neisseria meningitidis with SEQ ID NO 28. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. Novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, lacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 17) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 17: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

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* See Table 2 for plastid information

Example 35 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The E.coli strain adapted for sialic acid production as described in example 1 was further modified by genomic gene knockout of E.coli wcaJ, fucK and fucI genes and by genomic gene insertion of a persistence expression cassette for the fucose permease (furp) of E.coli having SEQ ID NO 12, the bifunctional fucose kinase/fucose-1-phosphate guanylate transferase (fkp) of E.fragilis having SEQ ID NO 13, lgtA of Neisseria meningitidis having SEQ NO ID 26 and E.coli O55 having SEQ ID NO 27: wbgO of H7. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (table 18) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 18: in a growth experiment, the production of an oligosaccharide mixture comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of an E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

Example 36 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The E.coli strain adapted for sialic acid production as described in example 1 was further modified by genomic gene knockout of E.coli wcaJ, fucK and fucI genes and by genomic gene insertion of a persistence expression cassette for furP of E.coli having SEQ ID NO 12, fkp of Bacteroides fragilis having SEQ ID NO 13, lgtA of Neisseria meningitidis having SEQ ID NO 26 and LgtB of Neisseria meningitidis having SEQ ID NO 28. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. Novel strains were evaluated in a growth experiment according to the culture conditions provided in example 1 for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, lacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (table 19), wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 19: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

Example 37 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The escherichia coli strain adapted for GDP-fucose production as described in example 1 was further modified by genomic gene knockout of the escherichia coli nagA, nagB, ushA and galT genes and genomic gene insertion of a persistence expression cassette for galE of escherichia coli having SEQ ID NO 29, mutant glmS 54 of escherichia coli having SEQ ID NO 18, GNA1 of saccharomyces cerevisiae having SEQ ID NO 15, phosphatase yqaB of escherichia coli having SEQ ID NO 19, lgtA of neisseria meningitidis having SEQ ID NO 26 and escherichia coli O55 having SEQ ID NO 27: wbgO of H7. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (table 20) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and sialic acid and lactose as precursors.

Table 20: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and sialic acid and lactose as precursors.

* See Table 2 for plastid information

Example 38 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The escherichia coli strain adapted for GDP-fucose production as described in example 1 was further modified by genomic gene knockout of escherichia coli nagA, nagB, ushA and galT genes and genomic gene insertion of a persistence expression cassette for galE of escherichia coli having SEQ ID NO 29, mutant glmS 54 of escherichia coli having SEQ ID NO 18, GNA1 of saccharomyces cerevisiae having SEQ ID NO 15, phosphatase yqaB of escherichia coli having SEQ ID NO 19, lgtA of neisseria meningitidis having SEQ ID NO 26 and LgtB of neisseria meningitidis having SEQ ID NO 28. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. Novel strains were evaluated in a growth experiment according to the culture conditions provided in example 1 for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, lacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (table 21), wherein the cultures contained sucrose as carbon source and sialic acid and lactose as precursors.

Table 21: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and sialic acid and lactose as precursors.

* See Table 2 for plastid information

Example 39 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The escherichia coli strain adapted for GDP-fucose production as described in example 1 was further modified by genomic gene knockout of escherichia coli nagA, nagB, nanA, nanE, nanK, ushA and galT genes and genomic gene insertion of a persistence-expressing cassette for galE of escherichia coli having SEQ ID NO 29, mutant glmS 54 of escherichia coli having SEQ ID NO 18, GNA1 of saccharomyces cerevisiae having SEQ ID NO 15, phosphatase yqaB of escherichia coli having SEQ ID NO 19, N-acetylglucosamine 2-epimerase (AGE) of oval neisseria having SEQ ID NO 16, neuB of neisseria meningitidis having SEQ ID NO 17, lgtA of neisseria meningitidis having SEQ ID NO 26 and escherichia coli O55 having SEQ ID NO 27: wbgO of H7. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (table 22) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 22: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

EXAMPLE 40 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The escherichia coli strains adapted for GDP-fucose production as described in example 1 were further modified by genomic gene knockout of escherichia coli nagA, nagB, nanA, nanE, nanK, ush and galT genes and genomic gene insertion of a persistence-expressing cassette for galE of escherichia coli having SEQ ID NO 29, mutant glmS 54 of escherichia coli having SEQ ID NO 18, GNA1 of saccharomyces cerevisiae having SEQ ID NO 15, phosphatase yqaB of escherichia coli having SEQ ID NO 19, AGE of oval having SEQ ID NO 16, neuB of neisseria meningitidis having SEQ ID NO 17, lgtA of neisseria meningitidis having SEQ ID NO 26 and LgtB of neisseria meningitidis having SEQ ID NO 28. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, lacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 23) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 23: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

Example 41 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The escherichia coli strain adapted for GDP-fucose production as described in example 1 was further modified by genomic gene knockout of escherichia coli nagA, nagB, nanA, nanE, nanK, ushA and galT genes and genomic gene insertion of a persistence expression cassette for galE of escherichia coli having SEQ ID NO 29, mutant glmS 54 of escherichia coli having SEQ ID NO 18, neuC of campylobacter jejuni having SEQ ID NO 20, neuB of neisseria meningitidis having SEQ ID NO 17, lgtA of neisseria meningitidis having SEQ ID NO 26 and escherichia coli O55 having SEQ ID NO 27: wbgO of H7. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. When evaluated in a growth experiment according to the culture conditions provided in example 1, the novel strains were evaluated for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (table 24), wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 24: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

EXAMPLE 42 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The escherichia coli strain adapted for GDP-fucose production as described in example 1 was further modified by genomic gene knockout of escherichia coli nagA, nagB, nanA, nanE, nanK, ushA and galT genes and genomic gene insertion of a persistence-expressing cassette for galE of escherichia coli having SEQ ID NO 29, mutant glmS 54 of escherichia coli having SEQ ID NO 18, neuC of campylobacter jejuni having SEQ ID NO 20, neuB of neisseria meningitidis having SEQ ID NO 17, lgtA of neisseria meningitidis having SEQ ID NO 26 and LgtB of neisseria meningitidis having SEQ ID NO 28. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (table 25) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 25: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

EXAMPLE 43 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The E.coli strain adapted for sialic acid production as described in example 1 was further modified with the genomic gene knockout of E.coli wcaJ, ushA and galT genes and the genomic gene insertion of the persistence expression cassette for galE of E.coli having SEQ ID NO 29, lgtA of Neisseria meningitidis having SEQ ID NO 26 and E.coli O55 having SEQ ID NO 27: wbgO of H7. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (table 26) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 26: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

EXAMPLE 44 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The E.coli strain adapted for sialic acid production as described in example 1 was further modified with the genomic gene knockout of E.coli wcaJ, ushA and galT genes and the genomic gene insertion of the persistence presentation cassette for galE of E.coli having SEQ ID NO 29, lgtA of N.meningitidis having SEQ ID NO 26 and LgtB of N.meningitidis having SEQ ID NO 28. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. Novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, lacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 27) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 27: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

EXAMPLE 45 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The E.coli strain adapted for sialic acid production as described in example 1 was further modified by genomic gene knockout of E.coli wcaJ, fucK, fucI, ushA and galT genes and by genomic gene insertion of a persistence-expressing cassette for galE of E.coli having SEQ ID NO 29, furP of E.coli having SEQ ID NO 12, fkp of Bacteroides fragilis having SEQ ID NO 13, lgtA of Neisseria meningitidis having SEQ ID NO 26 and E.coli O55 having SEQ ID NO 27: wbgO of H7. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. The novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LNB, fucosylated and sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures in whole broth samples (table 28) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 28: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

EXAMPLE 46 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified E.coli host

The E.coli strain adapted for sialic acid production as described in example 1 was further modified by genomic gene knockout of E.coli wcaJ, fucK, fucI, ushA and galT genes and by genomic gene insertion of a persistence-expressing cassette for galE of E.coli having SEQ ID NO 29, furP of E.coli having SEQ ID NO 12, fkp of Bacteroides fragilis having SEQ ID NO 13, lgtA of Neisseria meningitidis having SEQ ID NO 26 and LgtB of Neisseria meningitidis having SEQ ID NO 28. In a next step, the novel strain is transformed with two compatible expression plasmids, wherein the first plasmid contains persistence expression units for one or two selected fucosyltransferases, and wherein the second plasmid contains persistence expression units for one or two selected sialyltransferases and NeuA of pasteurella multocida having SEQ ID NO 21. Table 2 presents an overview of the six plastids used. Novel strains were evaluated in a growth experiment against the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, lacNAc, fucosylated and sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures in whole broth samples (Table 29) according to the culture conditions provided in example 1, wherein the cultures contained sucrose as carbon source and lactose as precursor.

Table 29: in a growth experiment, the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in a whole culture broth of a mutant E.coli strain was evaluated according to the culture conditions as described in example 1, wherein the culture medium contained sucrose as carbon source and lactose as precursor.

* See Table 2 for plastid information

Example 47 production of oligosaccharide mixtures comprising fucosylated and sialylated lactose Structure Using modified Saccharomyces cerevisiae hosts

The saccharomyces cerevisiae strain was adapted for production of GDP-fucose and CMP-sialic acid and expression of one or more fucosyltransferases and one or more sialyltransferases as described in example 2 with a first yeast expression plasmid (variant of p2a_2μ_fuc) comprising persistent transcription units for: LAC12 of kluyveromyces lactis having SEQ ID NO 30, gmd of escherichia coli having SEQ ID NO 10, fcl of escherichia coli having SEQ ID NO 11, and one or two selected fucosyltransferases; and the second yeast expression plasmid comprises persistent transcriptional units for: mutant glmS 54 of escherichia coli having SEQ ID NO 18, phosphatase yqaB of escherichia coli having SEQ ID NO 19, AGE of bacteroides ovatus having SEQ ID NO 16, neuB of neisseria meningitidis having SEQ ID NO 17, neuA of pasteurella multocida having SEQ ID NO 21, and one or two selected sialyltransferases. Table 30 shows the fucosyltransferases and sialyltransferases selected in the plastids cloned in this experiment. The strain was transformed to represent 1) one fucosyltransferase in combination with two sialyltransferases, 2) two fucosyltransferases in combination with one sialyltransferase, or 3) two fucosyltransferases in combination with two sialyltransferases (table 31). Mutant yeast strains were evaluated in a growth experiment for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose structures (as shown in table 31) using SD CSM-Ura-Trp omitting medium comprising lactose as precursor according to the culture conditions in example 2.

Table 30: overview of plastids cloned with persistent transcription units for one or both fucosyltransferase genes or for one or both sialyltransferase genes

Table 31: the production of oligosaccharide mixtures was assessed by means of mutant s.cerevisiae strains exhibiting selected fucosyltransferase and sialyltransferase genes when cultivated in SD CSM-Ura-Trp omission medium comprising lactose as precursor.

* See table 30 for plastid information

EXAMPLE 48 production of oligosaccharide mixture comprising 3 '-sialylated LN3, LNB, 3' SL and LSTa Using modified Saccharomyces cerevisiae host

The saccharomyces cerevisiae strains were adapted for CMP-sialic acid and LNT production and expression of β -galactoside α -2, 3-sialyltransferase as described in example 2 with a first yeast expression plasmid comprising persistent transcription units for: LAC12 of kluyveromyces lactis with SEQ ID NO 30, mutant glmS 54 of escherichia coli with SEQ ID NO 18, phosphatase yqaB of escherichia coli with SEQ ID NO 19, AGE of bacteroides ovatus with SEQ ID NO 16, neuB of neisseria meningitidis with SEQ ID NO 17, neuA of pasteurella multocida with SEQ ID NO 21, beta-galactosida-2, 3-sialyltransferase of pasteurella multocida with SEQ ID NO 22; and the second yeast expression plasmid comprises persistent transcriptional units for: galE of escherichia coli having SEQ ID NO 29, lgtA of neisseria meningitidis having SEQ ID NO 26, escherichia coli O55 having SEQ ID NO 27: wbgO of H7. Mutant yeast strains were evaluated in a growth experiment for the production of oligosaccharide mixtures comprising LN3, 3 '-sialylated LN3, LNT, LNB, sialylated LNB, 3' sl and LSTa using SD CSM-Trp-His omitting medium comprising lactose as precursor according to the culture conditions in example 2.

EXAMPLE 49 production of oligosaccharide mixture comprising 6 '-sialylated LN3, lacNAc, 6' SL and LSTc Using modified Saccharomyces cerevisiae host

The saccharomyces cerevisiae strains were adapted for CMP-sialic acid and LNnT production and expression of β -galactoside α -2, 6-sialyltransferase as described in example 2 with a first yeast expression plasmid comprising persistent transcription units for: LAC12 of kluyveromyces lactis with SEQ ID NO 30, mutant glmS 54 of escherichia coli with SEQ ID NO 18, phosphatase yqaB of escherichia coli with SEQ ID NO 19, AGE of bacteroides ovatus with SEQ ID NO 16, neuB of neisseria meningitidis with SEQ ID NO 17, neuA of pasteurella multocida with SEQ ID NO 21 and β -galactosida-2, 6-sialyltransferase of mermaid light emitting bacteria with SEQ ID NO 24 and the second yeast expression bodies comprise persistent transcriptional units for: coli galE with SEQ ID NO 29, lgtA with neisseria meningitidis with SEQ ID NO 26, lgtB with neisseria meningitidis with SEQ ID NO 28. Mutant yeast strains were evaluated in a growth experiment for the production of oligosaccharide mixtures comprising LN3, 6 '-sialylated LN3, LNnT, lacNAc, sialylated LacNAc, 6' sl and LSTc using SD CSM-Trp-His omitting medium comprising lactose as precursor according to the culture conditions in example 2.

EXAMPLE 50 production of oligosaccharide mixture comprising 3 '-sialylated LN3, lacNAc, 3' SL and LSTd Using modified Saccharomyces cerevisiae host

The saccharomyces cerevisiae strains were adapted for CMP-sialic acid and LNnT production and expression of β -galactoside α -2, 3-sialyltransferase as described in example 2 with a first yeast expression plasmid comprising persistent transcription units for: LAC12 of kluyveromyces lactis with SEQ ID NO 30, mutant glmS 54 of escherichia coli with SEQ ID NO 18, phosphatase yqaB of escherichia coli with SEQ ID NO 19, AGE of bacteroides ovatus with SEQ ID NO 16, neuB of neisseria meningitidis with SEQ ID NO 17, neuA of pasteurella multocida with SEQ ID NO 21 and β -galactosida-2, 3-sialyltransferase of pasteurella multocida with SEQ ID NO 22 and the second yeast expression bodies comprise persistent transcription units for: coli galE with SEQ ID NO 29, lgtA with neisseria meningitidis with SEQ ID NO 26, lgtB with neisseria meningitidis with SEQ ID NO 28. Mutant yeast strains were evaluated in a growth experiment for the production of oligosaccharide mixtures comprising LN3, 3 '-sialylated LN3, LNnT, lacNAc, sialylated LacNAc, 3' sl and LSTd using SD CSM-Trp-His omitting medium comprising lactose as precursor according to the culture conditions in example 2.

Example 51 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified Saccharomyces cerevisiae host

The saccharomyces cerevisiae strain was adapted for production of GDP-fucose, CMP-sialic acid and LNT and expression of selected fucosyltransferases and sialyltransferases with a first yeast expression plasmid (variant of p2a_2μ_fuc) comprising persistent transcription units for: LAC12 of kluyveromyces lactis having SEQ ID NO 30, gmd of escherichia coli having SEQ ID NO 10, fcl of escherichia coli having SEQ ID NO 11 and one or two selected fucosyltransferases (see table 30), and the second yeast expression vector comprises persistent transcriptional units for: mutant glmS 54 of escherichia coli having SEQ ID NO 18, phosphatase yqaB of escherichia coli having SEQ ID NO 19, AGE of bacteroides ovatus having SEQ ID NO 16, neuB of neisseria meningitidis having SEQ ID NO 17, neuA of pasteurella multocida having SEQ ID NO 21 and one or two selected sialyltransferases (see table 30), and the third yeast expression vector comprising persistent transcriptional units for: galE of escherichia coli having SEQ ID NO 29, lgtA of neisseria meningitidis having SEQ ID NO 26, escherichia coli O55 having SEQ ID NO 27: wbgO of H7. Mutant yeast strains were evaluated in a growth experiment for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, LNB, sialylated LNB, LN3, sialylated LN3, LNT and fucosylated and sialylated LNT structures (table 32) using SD CSM-Ura-Trp-His omitting medium comprising lactose as precursor according to the culture conditions in example 2.

Table 32: the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides in whole culture broth of mutant s.cerevisiae strains which exhibit selected fucosyltransferase and sialyltransferase genes when cultivated in SD CSM-Ura-Trp-His omitting medium comprising lactose as a precursor was evaluated.

* See table 30 for plastid information

Example 52 production of oligosaccharide mixture comprising fucosylated and sialylated oligosaccharide Structure Using modified Saccharomyces cerevisiae host

The saccharomyces cerevisiae strain was adapted for production of GDP-fucose, CMP-sialic acid, and LNnT and expression of selected fucosyltransferases and sialyltransferases with a first yeast expression plasmid (variant of p2a_2μ_fuc) comprising persistent transcription units for: LAC12 of kluyveromyces lactis having SEQ ID NO30, gmd of escherichia coli having SEQ ID NO 10, fcl of escherichia coli having SEQ ID NO 11 and one or two selected fucosyltransferases (see table 30), and the second yeast expression vector comprises persistent transcriptional units for: mutant glmS 54 of escherichia coli having SEQ ID NO 18, phosphatase yqaB of escherichia coli having SEQ ID NO 19, AGE of bacteroides ovatus having SEQ ID NO 16, neuB of neisseria meningitidis having SEQ ID NO 17, neuA of pasteurella multocida having SEQ ID NO 21 and one or two selected sialyltransferases (see table 30), and the third yeast expression vector comprising persistent transcriptional units for: coli galE with SEQ ID NO 29, lgtA with neisseria meningitidis with SEQ ID NO 26, lgtB with neisseria meningitidis with SEQ ID NO 28. Mutant yeast strains were evaluated in a growth experiment for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose, lacNAc, sialylated LacNAc, LN3, sialylated LN3, LNnT and fucosylated and sialylated LNnT structures (table 33) using SD CSM-Ura-Trp-His omitting medium comprising lactose as a precursor according to the culture conditions in example 2.

Table 33: the production of oligosaccharide mixtures comprising tri-, tetra-and penta-oligosaccharides, which mixtures were detectable in whole culture broth of mutant s.cerevisiae strains which exhibited the selected fucosyltransferase and sialyltransferase genes when cultured in SD CSM-Ura-Trp-His omitting medium comprising lactose as a precursor, was evaluated.

* See table 30 for plastid information

Example 53 production of oligosaccharide mixtures comprising fucosylated and sialylated oligosaccharide structures Using modified Saccharomyces cerevisiae hosts

Saccharomyces cerevisiae strains were adapted for GDP-fucose and CMP-sialic acid production and expression of one or more fucosyltransferases and one or more sialyltransferases with a yeast artificial chromosome (yeastartificial chromosome; YAC) comprising persistent transcription units for: LAC12 of kluyveromyces lactis with SEQ ID NO 30, gmd of escherichia coli with SEQ ID NO 10, fcl of escherichia coli with SEQ ID NO 11, mutant glmS 54 of escherichia coli with SEQ ID NO 18, phosphatase yqaB of escherichia coli with SEQ ID NO 19, AGE of bacteroides ovatus with SEQ ID NO 16, neuB of neisseria meningitidis with SEQ ID NO 17, neuA of pasteurella multocida with SEQ ID NO 21, one or two selected fucosyltransferases and one or two selected sialyltransferases. Table 34 shows the selected fucosyltransferases and sialyltransferases in the YACs produced in this experiment. Mutant yeast strains were evaluated in a growth experiment for the production of oligosaccharide mixtures comprising fucosylated and sialylated lactose structures (as shown in table 35) using SD CSM medium comprising lactose as precursor according to the culture conditions in example 2.

Table 34: overview of cloned fucosyltransferase genes and sialyltransferase genes in different Yeast Artificial chromosomes

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Table 35: the production of oligosaccharide mixtures in whole culture broth of mutant s.cerevisiae strains which exhibit selected fucosyltransferase and sialyltransferase genes from yeast artificial chromosomes when cultured in SD CSM medium containing lactose as a precursor was evaluated.

* See table 34 for an overview of YACs

Example 54 materials and methods for Bacillus subtilis

Culture medium

Two different media were used, namely enriched Lu Liya medium (LB) and minimal medium for shake flasks (minimal medium for shake flask; MMsf). Minimal medium uses trace element mixtures.

The trace element mixture consisted of 0.735g/L CaCl2.2H2O, 0.1g/L MnCl2.2H2O, 0.033g/L CuCl2.2H2O, 0.06g/L CoCl2.6H2O, 0.17g/L ZnCl2, 0.0311g/L H3BO4, 0.4g/L Na2EDTA.2H2O, and 0.06g/L Na2MoO 4. The ferric citrate solution contained 0.135g/L FeCl3.6H2O, 1g/L sodium citrate (Hoch 1973 PMC 1212887).

Lu Liya the medium (Luria Broth; LB) consists of 1% tryptone (Difco, belgium, emblica) 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR, belgium). Lu Liya the media agar (Luria broth agar; LBA) disc consisted of LB medium with the addition of 12g/L agar (Difco, belgium, embobond, belgium).

Minimal medium for shake flasks (MMsf) experiments contained 2.00g/L (NH 4) 2SO4, 7.5g/L KH2PO4, 17.5g/L K2HPO4, 1.25g/L sodium citrate, 0.25g/L MgSO4.7H2O, 0.05g/L tryptophan, 10 up to 30g/L glucose or another carbon source including, but not limited to, fructose, maltose, sucrose, glycerol, and maltotriose as specified in the examples, 10ml/L trace element mixture, and 10ml/L ferric citrate solution. The medium was set to pH 7 with 1M KOH. Lactose, LNB or LacNAc may be added depending on the experiment.

The complex medium (e.g.LB) is sterilized by high pressure treatment (121 ℃, 21') and the minimal medium is sterilized by filtration (0.22 μm Sartorius). If necessary, the medium is made selective by the addition of antibiotics, such as, for example, zeocin (20 mg/L).

Strains, plastids and mutations

Bacillus subtilis 168, available from the Bacillus gene storage center (Ohio, USA), U.S.A.

Plastids with gene deletion via Cre/lox were constructed as described by Yan et al (Appl. & environm. Microbioal, month 8, 2008, pages 5556-5562). Gene disruption is performed via homologous recombination with linear DNA and via electroporation transformation, as described by Xue et al (J. Microb. Meth.34 (1999) 183-191). Methods of gene knockout are described by Liu et al (Metab. Engine.24 (2014) 61-69). This method uses 1000bp homology upstream and downstream of the target gene.

An integration vector as described by Popp et al (sci.rep., 2017,7, 15158) is used as a representation vector and can be used further for genome integration if necessary. Suitable promoters for expression may be derived from the parts repository (iGem): sequence id: bba _k143012, bba _k823000, bba _k823002 or Bba _k82303. Cloning can be performed using gibbon Assembly (Gibson Assembly), gold gate Assembly (Golden Gate Assembly), cliva Assembly, LCR, or restriction engagement (restriction ligation).

In one example of the production of lactose-based oligosaccharides, a mutant strain of bacillus subtilis is produced to contain a gene encoding a lactose input (importer), such as e.coli lacY with SEQ ID NO 14. In one embodiment of 2' FL, 3FL and/or difL production, an alpha-1, 2-and/or alpha-1, 3-fucosyltransferase expression construct is additionally added to the strain. In one example of LN3 production, a persistent transcriptional unit comprising a galactoside beta-1, 3-N-acetylglucosaminyl transferase, such as lgtA (SEQ ID NO 26) from Neisseria meningitidis, for example, is additionally added to the strain. In one example of LNT production, the strain producing LN3 is further modified with a persistent transcriptional unit comprising an N-acetylglucosamine beta-1, 3-galactosyltransferase, such as, for example, wbgO (SEQ ID NO 27) from E.coli O55: H7. In one example of LNnT production, the strain producing LN3 is further modified with a persistent transcriptional unit comprising an N-acetylglucosamine beta-1, 4-galactosyltransferase, such as lgtB (SEQ ID NO 28) from Neisseria meningitidis, for example.

In one embodiment of sialic acid production, mutant Bacillus subtilis strains are produced by over-expressing a fructose-6-P-aminotransferase, such as native fructose-6-P-aminotransferase (UniProt ID P0CI 73), to enhance intracellular glucosamine-6-phosphate pools. In addition, the enzymatic activities of the genes nagA, nagB and gamA are disrupted by gene knockout and are overexpressed on the genome, such as, for example, glucosamine-6-P-aminotransferase (SEQ ID NO 15) of Saccharomyces cerevisiae, N-acetylglucosamine-2-epimerase (SEQ ID NO 16) such as, for example, from Bacteroides ovale, and N-acetylneuraminic acid synthase (SEQ ID NO 17) such as, for example, from Neisseria meningitidis. To allow sialylated oligosaccharide production, the sialic acid producing strain is further modified with a sustained transcriptional unit comprising one or more copies of the N-acyl neuraminic acid cytidylyltransferase (SEQ ID NO 21) as e.g.NeuA enzyme from Pasteurella multocida and the beta-galactosidase alpha-2, 3-sialyltransferase (UniProt ID Q9CLP 3) as e.g.PmultST 3 from Pasteurella multocida, or the amino acid residues 1 to 268 of UniProt ID Q9CLP3 with beta-galactosidase activity (SEQ ID NO 22) or the NmeniST3 (SEQ ID NO 23) from Neisseria meningitidis or the beta-galactosidase alpha-2, 6-sialyltransferase (beta-6-sialyltransferase) as e.g.Pd6 from Prot.Q 9CLP3 or the beta-sialyltransferase (SEQ ID NO 22) from Neisseria meningitidis or the NmeniST3 (SEQ ID NO 23) from Pasteurella multocida subspecies Pasteurensis strain Pm70 (AAK02592.1), or the amino acid residues 6-sialyltransferase (beta-6 alpha-6-sialyltransferase) as e.6 from Prot 9CLP3 with beta-sialyltransferase activity (UniProt 6, beta-6-sialyltransferase (1) or the amino acid residues 1 to 35H 35 from UniProt 6-sialyltransferase (1) with beta-sialyltransferase activity, P-JT-ISH-224-ST 6-like polypeptide (SEQ ID NO 25) consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 of 6-sialyltransferase activity and/or alpha-2, 8-sialyltransferase (UniProt ID Q64689) as, for example, a mouse.

Heterologous and homologous manifestation

The gene to be expressed, whether it is plastid-derived or genome-derived, is synthesized synthetically by one of the following companies: DNA2.0, gen9, twist Biosciences or IDT.

Expression may be further facilitated by optimizing codon usage to that of the expression host. The genes were optimized using the vendor's tools.

Culture conditions

Pre-culture of 96 well microtiter plate experiments was performed starting from frozen vials or single colonies starting from LB plates in 150. Mu.L LB and incubated overnight at 37℃on an orbital shaker at 800 rpm. This culture was used as inoculum for 96-well square microtiter plates, diluted 400-fold with 400 μl of MMsf medium. Each strain was grown as a biological replicate in multiple wells of a 96-well plate. These final 96-well culture plates were then incubated at 37℃for 72 hours or less or longer on an orbital shaker at 800 rpm. At the end of the culture experiment, samples were taken from each well to measure the supernatant concentration (extracellular sugar concentration, after 5min of short centrifugation of the cells), or (=whole broth concentration, intracellular and extracellular sugar concentrations as defined herein) by boiling the broth at 90 ℃ for 15min or 60min at 60 ℃ before short centrifugation of the cells.

In addition, the cultures were diluted to measure the optical density at 600 nm. The cell efficiency index or CPI was determined by dividing the oligosaccharide concentration by the biomass (in relative percentage compared to the reference strain). Approximately 1/3 of the optical density of the biomass measured at 600nm was empirically determined.

EXAMPLE 55 production of oligosaccharide mixture comprising 2' FL, 3-FL, diFL, 3' SL, 6' SL, 3' S-2' FL, 3' S-3-FL, 6'S-2' FL, 6'S-3-FL Using modified Bacillus subtilis host

Bacillus subtilis strain was modified by genomic gene knockout of nagA, nagB, glmS and gamA genes and genomic gene insertion of the following persistent transcription units as described in example 54: lactose permease (LacY) of E.coli having SEQ ID NO 14, sucrose transporter (CscB) of E.coli W (SEQ ID NO 01), fructokinase (Frk) from Z.mobilis (SEQ ID NO 02), sucrose phosphorylase (BaSP) from Bifidobacterium adolescentis (SEQ ID NO 03), native fructose-6-P-aminotransferase (UniProt ID P0CI 73), glucosamine 6-phosphate N-acetyltransferase GNA1 (SEQ ID NO 15) from Saccharomyces cerevisiae, mutant L-glutamylate-D-fructose-6-phosphate aminotransferase (glmS 54) from E.coli (SEQ ID NO 18); phosphatases, such as, for example, phosphatases selected from the group consisting of the E.coli genes comprising aphA, cof, hisB, otsB, surE, yaed, ycjU, yedP, yfbT, yidA, yigB, yihX, yniC, yqaB, yrbL, appA, gph, serB, ybhA, ybiV, ybjL, yfb, yieH, yjgL, yjjG, yrfG and YbiU or PsMupP from Pseudomonas putida, scDOG1 from Saccharomyces cerevisiae or BsAraL from Bacillus subtilis (as described in WO 18122225); n-acetylglucosamine 2-epimerase (AGE) (SEQ ID NO 16) from Bacteroides ovalis, N-acetylneuraminic acid synthase (SEQ ID NO 17) from Neisseria meningitidis, and N-acyl neuraminic acid cytidylyltransferase NeuA (SEQ ID NO 21) from Pasteurella multocida. In the next step, the strain is transformed with a expression plasmid comprising persistent transcription units for: three copies of PmultST 3-like polypeptide consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 having β -galactoside α -2, 3-sialyltransferase activity as set forth in SEQ ID NO 23; and three copies of PdST 6-like polypeptide consisting of amino acid residues 108 to 497 of UniProt ID O66375 having β -galactoside α -2, 6-sialyltransferase activity (SEQ ID NO 24). In a further step, the mutant strain is transformed with a second compatible expression plasmid comprising persistent transcriptional units for the α -1, 2-fucosyltransferase HpFUTC having SEQ ID NO 04 and the α -1, 3-fucosyltransferase HpFUCT having SEQ ID NO 05. Novel strains were evaluated in a growth experiment on MMsf medium containing lactose for production of 2' FL, 3-FL, diFL, 3' sl, 6' sl, 3's-2' FL, 3's-3-FL, 6'S-2' FL, 6'S-3-FL according to the culture conditions provided in example 54. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC.

EXAMPLE 56 production of oligosaccharide mixture comprising 3' SL, LN3, LNT, sialylated LN3 and LSTa Using modified Bacillus subtilis host

The bacillus subtilis strain was modified for LN3 production and growth on sucrose by genomic gene knockout of nagA, nagB, glmS and gamA genes and genomic gene insertion comprising a sustained transcriptional unit encoding the following genes as described in example 54: lactose permease from E.coli (LacY) (SEQ ID NO 14), native fructose-6-P-aminotransferase (UniProt ID P0CI 73), galactoside beta-1, 3-N-acetylglucosamintransferase from Neisseria meningitidis LgtA (SEQ ID NO 26), sucrose transporter from E.coli W (CscB) (SEQ ID NO 01), fructokinase from Z.mobilis (Frk) (SEQ ID NO 02) and sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) (SEQ ID NO 03). In the next step of the process, the process is carried out, the mutant strain is further comprised of from E.coli O55: genomic gene insertion modification of the persistent transcriptional unit of the N-acetylglucosamine β -1, 3-galactosyltransferase WbgO (SEQ ID NO 27) of H7 to produce LNT. The mutant bacillus subtilis strain is further modified by genomic gene insertion of a persistent transcription unit comprising glucosamine 6-phosphate N-acetyltransferase GNA1 (SEQ ID NO 15) from saccharomyces cerevisiae; two copies of mutant L-glutamyld-fructose-6-phosphate aminotransferase (gmS x 54) (SEQ ID NO 18) from escherichia coli; phosphatases, such as, for example, phosphatases selected from the group consisting of the E.coli genes comprising aphA, cof, hisB, otsB, surE, yaed, ycjU, yedP, yfbT, yidA, yigB, yihX, yniC, yqaB, yrbL, appA, gph, serB, ybhA, ybiV, ybjL, yfb, yieH, yjgL, yjjG, yrfG and YbiU or PsMupP from Pseudomonas putida, scDOG1 from Saccharomyces cerevisiae or BsAraL from Bacillus subtilis (as described in WO 18122225); n-acetylglucosamine 2-epimerase (AGE) (SEQ ID NO 16) from Bacteroides ovalis, N-acetylneuraminic acid synthase (SEQ ID NO 17) from Neisseria meningitidis, N-acyl neuraminic acid cytidylyltransferase NeuA (SEQ ID NO 21) from Pasteurella multocida; and three copies of a PmultST 3-like polypeptide (as set forth in SEQ ID NO 22) consisting of amino acid residues 1 to 268 of UniProt ID Q9CLP3 with β -galactosidase α -2, 3-sialyltransferase activity. The novel strains were evaluated in a growth experiment on MMsf medium containing lactose as precursor for the production of a mixture comprising 3' sl, LN3, sialylated LN3, LNT and LSTa (Neu 5Ac-a2,3-Gal-b1,3-GlcNAc-b1,3-Gal-b1, 4-Glc) according to the culture conditions provided in example 54. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC.

EXAMPLE 57 materials and methods of Corynebacterium glutamicum

Culture medium

Two different media were used, namely enriched tryptone-yeast extract (TY) medium and minimal medium for shake flasks (minimal medium for shake flask; MMsf). Minimal medium 1000x stock trace element mixtures were used.

The trace element mixture consisted of 10g/L CaCl2, 10g/L FeSO4.7H2O, 10g/L MnSO4.H2O, 1g/L ZnSO4.7H2O, 0.2g/L CuSO4, 0.02g/L NiCl2.6H2O, 0.2g/L biotin (pH 7.0) and 0.03g/L protocatechuic acid.

Minimal medium (MMsf) experiments for shake flasks contained 20g/L (NH 4) 2SO4, 5g/L urea, 1g/L KH2PO4, 1g/L K2HPO4, 0.25g/L MgSO4.7H2O, 42g/L MOPS, 10 up to 30g/L glucose or another carbon source including, but not limited to, fructose, maltose, sucrose, glycerol and maltotriose as specified in the examples, 1ml/L trace element mixture. Lactose, LNB and/or LacNAc may be added to the medium depending on the experiment.

TY medium consisted of 1.6% tryptone (Difco, emblica Bodheim Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR, belgium). TY agar (TYA) discs consisted of TY medium with the addition of 12g/L agar (Difco, emblica Bodheim, belgium).

The complex medium (e.g.TY) is sterilized by high pressure treatment (121 ℃, 21') and the minimal medium by filtration (0.22 μm Sartorius). If necessary, the medium is made selective by the addition of antibiotics, such as Kang Mei (kanamycin), ampicillin.

Strains and mutations

Corynebacterium glutamicum ATCC 13032 is available from the American type culture Collection (American Type Culture Collection).

Integrative plastid vectors based on the Cre/loxP technology as described by Suzuki et al (appl. Microbiol. Biotechnol., 4, 67 (2) 2005: 225-33) and thermosensitive shuttle vectors as described by Okibe et al (Journal of Microbiological Methods 85, 2011, 155-163) were constructed for gene deletion, mutation and insertion. Suitable promoters for expression of the (heterologous) gene may be derived from YIm et al (Biotechnol. Bioeng., 11 months 2013, 110 (11): 2959-69). The colonization may be performed using gibbon assembly, gold gate assembly, cliva assembly, LCR, or restriction binding.

In one example of the production of lactose-based oligosaccharides, mutant strains of corynebacterium glutamicum are produced to contain a gene encoding a lactose input (importer), such as e.g. E.coli lacY with SEQ ID NO 14. In one embodiment of 2' FL, 3FL and/or difL production, an alpha-1, 2-and/or alpha-1, 3-fucosyltransferase expression construct is additionally added to the strain.

In one example of LN3 production, a persistent transcriptional unit comprising a galactoside beta-1, 3-N-acetylglucosaminyl transferase, such as lgtA (SEQ ID NO 26) from Neisseria meningitidis, for example, is additionally added to the strain. In one example of LNT production, the strain producing LN3 is further modified with a persistent transcriptional unit comprising an N-acetylglucosamine beta-1, 3-galactosyltransferase, such as, for example, wbgO (SEQ ID NO 27) from E.coli O55: H7. In one example of LNnT production, the strain producing LN3 is further modified with a persistent transcriptional unit comprising an N-acetylglucosamine beta-1, 4-galactosyltransferase, such as lgtB (SEQ ID NO 28) from Neisseria meningitidis, for example.

In one embodiment of sialic acid production, mutant C.glutamicum strains are produced by over-expressing a fructose-6-P-aminotransferase, such as native fructose-6-P-aminotransferase (UniProt ID Q8NND 3), to enhance the intracellular glucosamine-6-phosphate pool. In addition, the enzymatic activities of the genes nagA, nagB and gamA are disrupted by gene knockout and are overexpressed on the genome, such as, for example, glucosamine-6-P-aminotransferase (SEQ ID NO 15) of Saccharomyces cerevisiae, N-acetylglucosamine-2-epimerase (SEQ ID NO 16) such as, for example, from Bacteroides ovale, and N-acetylneuraminic acid synthase (SEQ ID NO 17) such as, for example, from Neisseria meningitidis. To allow sialylated oligosaccharide production, the sialic acid producing strain is further modified with a sustained transcriptional unit comprising one or more copies of the N-acyl neuraminic acid cytidylyltransferase (SEQ ID NO 21) as e.g.NeuA enzyme from Pasteurella multocida and the beta-galactosidase alpha-2, 3-sialyltransferase (UniProt ID Q9CLP 3) as e.g.PmultST 3 from Pasteurella multocida, or the amino acid residues 1 to 268 of UniProt ID Q9CLP3 with beta-galactosidase activity (SEQ ID NO 22) or the NmeniST3 (SEQ ID NO 23) from Neisseria meningitidis or the beta-galactosidase alpha-2, 6-sialyltransferase (beta-6-sialyltransferase) as e.g.Pd6 from Prot.Q 9CLP3 or the beta-sialyltransferase (SEQ ID NO 22) from Neisseria meningitidis or the NmeniST3 (SEQ ID NO 23) from Pasteurella multocida subspecies Pm70 (AAK 02592.1) or the beta-galactosidase alpha-2, 6 alpha-sialyltransferase (UniProt Q9CLP 3) from Prot.3 or the amino acid residues 6-sialyltransferase (alpha-6-75) from Prot 6P 6 or the human sialyltransferase (UniProc 6 alpha-3-YP 8) with beta-sialyltransferase activity (SEQ ID NO 2 or the enzyme alpha-3 from UniProP 6-Ala. 35), P-JT-ISH-224-ST 6-like polypeptide (SEQ ID NO 25) consisting of amino acid residues 18 to 514 of UniProt ID A8QYL1 of 6-sialyltransferase activity and/or alpha-2, 8-sialyltransferase (UniProt ID Q64689) as, for example, a mouse.

Heterologous and homologous manifestation

The gene to be expressed, whether it is plastid-derived or genome-derived, is synthesized synthetically by one of the following companies: DNA2.0, gen9, twist Biosciences or IDT.

Expression may be further facilitated by optimizing codon usage to that of the expression host. The genes were optimized using the vendor's tools.

Culture conditions

Pre-culture of 96 well microtiter plate experiments was performed in 150. Mu.L TY starting from frozen vials or single colonies starting from TY plates and incubated overnight at 37℃on an orbital shaker at 800 rpm. This culture was used as inoculum for 96-well square microtiter plates, diluted 400-fold with 400 μl of MMsf medium. Each strain was grown as a biological replicate in multiple wells of a 96-well plate. These final 96-well culture plates were then incubated at 37℃for 72 hours or less or longer on an orbital shaker at 800 rpm. At the end of the culture experiment, samples were taken from each well to measure the supernatant concentration (extracellular sugar concentration, after 5min of brief centrifugation of the cells), or by boiling the culture at 60 ℃ for 15min prior to brief centrifugation of the cells (=whole culture concentration, intracellular and extracellular sugar concentrations, as defined herein).

In addition, the cultures were diluted to measure the optical density at 600 nm. The cell efficiency index or CPI was determined by dividing the measured oligosaccharide concentration in the complete broth by the biomass (in relative percentage compared to the reference strain). Approximately 1/3 of the optical density of the biomass measured at 600nm was empirically determined.

EXAMPLE 58 production of oligosaccharide mixtures comprising LN3, sialylated LN3, 6' SL, LNnT and LSTc Using modified Corynebacterium glutamicum hosts

The corynebacterium glutamicum strains were modified for LN3 production and growth on sucrose by genomic gene knockout of the ldh, cgl2645, nagB, gamA and nagA genes encoding lactose permease (LacY) (SEQ ID NO 14), native fructose-6-P-aminotransferase (UniProt ID Q8NND 3), galactoside beta-1, 3-N-acetylglucoseaminotransferase LgtA (SEQ ID NO 26) from neisseria meningitidis, sucrose transporter (CscB) (SEQ ID NO 01) from escherichia coli W, fructokinase (Frk) (SEQ ID NO 02) from zymomonas mobilis and sucrose phosphorylase (BaSP) (SEQ ID NO 03) from bifidobacterium, and by genomic gene insertion of a sustained transcriptional unit comprising genes as described in example 57. In the next step, the mutant strain is further modified by genomic gene insertion comprising a sustained transcriptional unit of N-acetylglucosamine β -1, 4-galactosyltransferase LgtB (SEQ ID NO 28) from neisseria meningitidis to produce LNnT. In the next step, the mutant strain is further modified by genomic gene insertion comprising the following persistent transcriptional units to produce sialic acid: native fructose-6-P-aminotransferase (UniProt ID Q8NND 3), glucosamine-6-P-aminotransferase (SEQ ID NO 15) from Saccharomyces cerevisiae, acetylglucosamine acetyl glucosamine-2-epimerase (SEQ ID NO 16) from Bacteroides ovalis, and N-acetylneuraminic acid synthase (SEQ ID NO 17) from Neisseria meningitidis. In the next step, the novel strain is transformed with a expressisome comprising persistent transcription units for the NeuA enzyme from Pasteurella multocida (SEQ ID NO 21) and the beta-galactosidase alpha-2, 6-sialyltransferase PdST6 from mermaid light emitting bacteria (UniProt ID O66375). Novel strains were evaluated in a growth experiment on MMsf medium containing lactose for the production of oligosaccharide mixtures comprising LN3, 6 '-sialylated LN3 (Neu 5Ac-a2,6- (GlcNAc-b 1, 3) -Gal-b1, 4-Glc), 6' sl, LNnT and LSTc according to the culture conditions provided in example 57. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC.

EXAMPLE 59 production of oligosaccharide mixture comprising 3'SL, 6' SL, LNB, 3 '-sialylated LNB and 6' -sialylated LNB Using modified Corynebacterium glutamicum host

The corynebacterium glutamicum strain was modified as described in example 57 by means of genomic gene knockout of the ldh, cgl2645, nagB, gamA and nagA genes and genomic gene insertion comprising the continuous transcriptional unit of genes encoding lactose permease (LacY) from E.coli (SEQ ID NO 14), O55 from E.coli: wbgO with SEQ ID NO 27, galE with SEQ ID NO 29 from E.coli, native fructose-6-P-aminotransferase (UniProt ID Q8NND 3), glmS 54 with SEQ ID NO 18, glucosamine-6-P-aminotransferase (SEQ ID NO 15) from Saccharomyces cerevisiae, N-acetylglucosamine-2-epimerase (SEQ ID NO 16) from Bacteroides ovale, N-acetylneuraminic acid synthase (SEQ ID NO 17) from Neisseria meningitidis. In the next step, the novel strain is transformed with a expressisome comprising persistent transcription units for the NeuA enzyme from pasteurella multocida (SEQ ID NO 21), the β -galactoside α -2, 3-sialyltransferase PmultST3 from pasteurella multocida (UniProt ID Q9CLP 3) and the β -galactoside α -2, 6-sialyltransferase PdST6 from mermaid light emitting bacteria (UniProt ID O66375). The novel strains were evaluated in a growth experiment on MMsf medium comprising lactose and glucose for the production of oligosaccharide mixtures comprising 3'sl, 6' sl, LNB, 3 '-sialylated LNB (3' slnb) and 6 '-sialylated LNB (6' slnb) according to the culture conditions provided in example 57. After 72 hours of incubation, the culture broth was collected and analyzed for sugars on UPLC.

Example 60 materials and methods of Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

Culture medium

Chlamydomonas reinhardtii cells were cultured in Tris-acetate-phosphate (TAP) medium (pH 7.0). TAP medium was used with 1000 Xstock Hutner's trace element mixture. Hutner's trace element mixture consisted of 50g/L Na2EDTA.H2O (Titriplex III), 22g/L ZnSO4.7H2O, 11.4g/L H BO3, 5g/L MnCl2.4H2O, 5g/L FeSO4.7H2O, 1.6g/L CoCl2.6H2O, 1.6g/L CuSO4.5H2O and 1.1g/L (NH 4) 6MoO 3.

TAP medium contained 2.42g/L tris (hydroxymethyl) aminomethane, 25mg/L salt stock solution, 0.108g/L K HPO4, 0.054g/L KH2PO4, and 1.0mL/L glacial acetic acid. The salt stock solution consisted of 15g/L NH4CL, 4g/L MgSO4.7H2O and 2g/L CaCl2.2H2O. As precursors and/or acceptors for sugar synthesis, compounds such as galactose, glucose, fructose, fucose, lactose, lacNAc, LNB may be added. The medium is sterilized by high pressure treatment (121 ℃, 21'). For stock cultures on slant agar, TAP medium containing 1% agar (with purified high intensity, 1000g/cm 2) was used.

Strains, plastids and mutations

Chlamydomonas reinhardtii wild type strain 21gr (CC-1690, wild type, mt+), 6145C (CC-1691, wild type, mt-), CC-125 (137C, wild type, mt+), CC-124 (137C, wild type, mt-) are available from the Chlamydomonas resource center (Chlamydomonas Resource Center) of university of Minnesota (University of Minnesota, U.S. A.) (https:// www.chlamycollection.org).

The expressive body is derived from pSI103, as available from the Chlamydomonas resource center. The colonization may be performed using gibbon assembly, gold gate assembly, cliva assembly, LCR, or restriction binding. Suitable promoters for expression of the (heterologous) gene may be derived, for example, from Scandon et al (Algal Res.2016, 15:135-142). Targeted genetic modifications, such as gene knockout or gene replacement, can be made using the Crispr-Cas technique as described, for example, by Jiang et al (Eukaryotic Cell 2014, 13 (11): 1465-1469).

Transformation by electroporation was performed as described by Wang et al (Biosci.Rep.2019, 39:BSR2018210). Cells were grown in liquid TAP medium under constant aeration and continuous light at a light intensity of 8000Lx until cell densities reached 1.0-2.0x107 cells/ml. Next, cells were inoculated into fresh liquid TAP medium at a concentration of 1.0X106 cells/ml and grown under continuous light for 18-20 hours until the cell density reached 4.0X106 cells/ml. Next, cells were collected by centrifugation at 1250g for 5min at room temperature, washed and resuspended and frozen for 10min with pre-chilled liquid TAP medium (Sigma, usa) containing 60mM sorbitol. Next, 250. Mu.L of the cell suspension (corresponding to 5.0X107 cells) was placed in a pre-cooled 0.4cm electroporation cuvette (400 ng/mL) with 100ng plastid DNA. Electroporation was performed using a BTXECM830 electroporation device (1575 Ω,50 μfd) with 6 500V pulses each having a pulse length of 4ms and a pulse interval of 100 ms. Immediately after electroporation, the cuvette was placed on ice for 10min. Finally, the cell suspension was transferred to a 50mL conical centrifuge tube containing 10mL of freshly prepared liquid TAP medium with 60mM sorbitol, which was allowed to recover overnight under dim light by slow shaking. After overnight recovery, cells were re-harvested and inoculated with starch embedding onto a selective 1.5% (w/v) agar-TAP tray containing either ampicillin (100 mg/L) or chloramphenicol (100 mg/L). The trays were then incubated at 23+ -0.5℃under continuous illumination with a light intensity of 8000 Lx. Cells were analyzed after 5-7 days.

In one embodiment for the production of UDP-galactose, chlamydomonas reinhardtii cells are modified with a transcriptional unit comprising a gene encoding a galactokinase (KIN, uniProt ID Q9SEE 5) such as Arabidopsis thaliana and a UDP-sugar pyrophosphorylase (UniProt ID Q9C5I 1) such as USP from Arabidopsis thaliana (A. Thaliana), for example.

In one example of LN3 production, a persistent transcriptional unit comprising a galactoside beta-1, 3-N-acetylglucosaminyl transferase, such as lgtA (SEQ ID NO 26) from Neisseria meningitidis, for example, is additionally added to the strain. In one example of LNT production, the strain producing LN3 is further modified with a persistent transcriptional unit comprising an N-acetylglucosamine beta-1, 3-galactosyltransferase, such as, for example, wbgO (SEQ ID NO 27) from E.coli O55: H7. In one example of LNnT production, the strain producing LN3 is further modified with a persistent transcriptional unit comprising an N-acetylglucosamine beta-1, 4-galactosyltransferase, such as lgtB (SEQ ID NO 28) from Neisseria meningitidis, for example.

In one embodiment of the production of GDP-fucose, chlamydomonas reinhardtii cells are modified with transcription units for GDP-fucose synthase (GER 1, uniProt ID O49213) as, for example, from Arabidopsis thaliana.

In one embodiment of fucosylation, the Chlamydomonas reinhardtii cells may be modified with a expressive body comprising a persistent transcriptional unit for use with an alpha-1, 2-fucosyltransferase (SEQ ID NO 04) such as HpFatc from helicobacter pylori and/or an alpha-1, 3-fucosyltransferase (SEQ ID NO 05) such as HpFact from helicobacter pylori, for example.

In one embodiment of the CMP-sialic acid synthesis, chlamydomonas reinhardtii cells are modified with persistent transcription units for use as UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UniProt ID Q9Y 223) from the GNE of homo sapiens or mutated forms of the human GNE polypeptide comprising the R263L mutation, as N-acyl neuraminic acid-9-phosphate synthase (UniProt ID Q9NR 45) from the NANS of homo sapiens for example, and as N-acyl neuraminic acid cytidylyltransferase (UniProt ID Q8 NFW) from the CMAS of homo sapiens for example. In one embodiment of the production of sialylated oligosaccharides, the Chlamydomonas reinhardtii cells are modified by CMP-sialic acid transporter proteins such as CST (UniProt ID Q61420) from mice and Golgi-localized sialyltransferases from species such as e.g. homo sapiens, mice, brown rats.

Heterologous and homologous manifestation

The gene to be expressed, whether it is plastid-derived or genome-derived, is synthesized synthetically by one of the following companies: DNA2.0, gen9, twist Biosciences or IDT.

Expression may be further facilitated by optimizing codon usage to that of the expression host. The genes were optimized using the vendor's tools.

Culture conditions

Chlamydomonas reinhardtii cells were cultured in selective TAP-agar plates at 23+/-0.5℃at a light intensity of 8000Lx under 14/10h light/dark cycles. Cells were analyzed after 5 to 7 days of culture.

With respect to high density cultures, cells may be cultured in closed systems as described by Chen et al (bioresour. Technology.2011, 102:71-81) and Johnson et al (biotechnol. Prog.2018, 34:811-827) such as, for example, vertical or horizontal tube photobioreactors, stirred tank photobioreactors, or slab photobioreactors.

Example 61 production of oligosaccharide mixtures comprising sialylated LNB and sialylated LacNAc structures in mutant Chlamydomonas reinhardtii cells

Genomic gene insertion of a sustained transcriptional unit comprising a mutated form of UDP-N-acetylglucosamine-2-epi isomerase/N-acetylmannosamine kinase GNE from homo sapiens (UniProt ID Q9Y 223), N-acylneuraminic acid-9-phosphate synthase NANS (UniProt ID Q9NR 45) from homo sapiens and N-acylneuraminic acid cytidylyltransferase CMAS (UniProt ID Q8 NFW) from homo sapiens different from a native polypeptide with an R263L mutation was engineered for the production of CMP-sialic acid as described in example 60. In the next step, the cells were modified by genomic gene insertion of a persistent transcription unit comprising CMP-sialic acid transporter CST (UniProt ID Q61420) from mice, α -2, 3-sialyltransferase (UniProt ID P61943 and E9PSJ 1) from brown mice, and α -2, 6-sialyltransferase (UniProt ID P13721) from brown mice. In the final step, the cells are transformed with a genomic gene insert comprising the persistent transcriptional unit of the Arabidopsis gene encoding galactokinase (KIN, uniProt ID Q9SEE 5) and UDP-sugar pyrophosphorylase (USP) (UniProt ID Q9C5I 1), and E.coli O55 with SEQ ID NO 27: H7N-acetylglucosamine beta-1, 3-galactosyltransferase WbgO and N-acetylglucosamine beta-1, 4-galactosyltransferase LgtB of Neisseria meningitidis having SEQ ID NO 28. The novel strain was evaluated in a culture experiment against the production of an oligosaccharide mixture comprising 3 '-sialyllactose-N-disaccharide (3' slnb), 6 '-sialyllactose-N-disaccharide (6' slnb), 3 '-sialyllactosamine (3' slacnac) and 6 '-sialyllactosamine (6' slacnac) according to the culture conditions provided in example 60 on a TAP agar plate comprising galactose, glucose and N-acetylglucosamine as precursors. After 5 days of incubation, cells were collected and analyzed for sugar production on UPLC.

Example 62 materials and methods for animal cells

Isolation of mesenchymal Stem cells from adipose tissue of different mammals

Fresh adipose tissue is obtained from slaughterhouses (e.g., cattle, pigs, sheep, chickens, ducks, catfish, snakes, frogs) or liposuction surgery (e.g., in the case of humans, after signing informed consent) and is kept in phosphate buffered saline supplemented with antibiotics. Enzymatic digestion of adipose tissue was performed followed by centrifugation to isolate mesenchymal stem cells. The isolated intermediate leaf stem cells were transferred to a cell culture flask and grown under standard growth conditions (e.g., 37 ℃, 5% co 2). The initial medium included DMEM-F12, RPMI and alpha-MEM medium (supplemented with 15% fetal bovine serum) and 1% antibiotics. Subsequently after the first pass, the medium was replaced with medium supplemented with 10% fbs (fetal bovine serum). For example, ahmad and Shakoori (2013,Stem Cell Regen Med.9 (2): 29-36), which are incorporated by reference herein in their entirety for all purposes, describe certain variations of the methods described in this example.

Isolation of mesenchymal Stem cells from milk

This example illustrates the isolation of mesenchymal stem cells from milk collected under sterile conditions from a human or any other mammal, such as described herein. Equal volumes of phosphate buffered saline were added to the diluted milk, followed by centrifugation for 20min. The cell pellet was washed three times with phosphate buffered saline and cells were inoculated in cell culture flasks under standard culture conditions in DMEM-F12, RPMI and alpha-MEM medium supplemented with 10% fetal bovine serum and 1% antibiotics. For example, hassiotou et al (2012,Stem Cells.30 (10): 2164-2174), which is incorporated by reference herein in its entirety for all purposes, describe certain variations of the methods described in this embodiment.

Differentiation of Stem cells Using 2D and 3D culture System

The isolated mesenchymal cells can differentiate into mammary-like epithelial cells 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 enterprises, 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 by reference herein in its entirety for all purposes.

For 2D culture, the isolated cells were initially inoculated in a culture dish in growth medium supplemented with 10ng/ml of epithelial growth factor and 5pg/ml of insulin. At confluence, cells were fed with growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100U/ml penicillin, 100ug/ml streptomycin) and 5pg/ml insulin for 48h. To induce differentiation, cells were fed with complete growth medium containing 5pg/ml insulin, 1pg/ml cortisol, 0.65ng/ml triiodothyroxine, 100nM dexamethasone and 1pg/ml prolactin. After 24h, serum was removed from the complete induction medium.

For 3D culture, the isolated cells were trypsinized and cultured in matrigel, hyaluronic acid or ultra low adhesion surface culture plates for six days, and induced differentiation and lactate by addition of growth medium supplemented with 10ng/ml epithelial growth factor and 5pg/ml insulin. At confluence, cells were fed with growth medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (100U/ml penicillin, 100ug/ml streptomycin) and 5pg/ml insulin for 48h. To induce differentiation, cells were fed with complete growth medium containing 5pg/ml insulin, 1pg/ml cortisol, 0.65ng/ml triiodothyroxine, 100nM dexamethasone and 1pg/ml prolactin. After 24h, serum was removed from the complete induction medium.

Method for producing mammary gland-like cells

Mammalian cells were induced for pluripotency by reprogramming with viral vectors encoding Oct4, sox2, klf4 and c-Myc. The resulting reprogrammed cells were then cultured in Mammocult medium (obtained from Stem Cell Technologies) or mammary gland cell enrichment medium (DMEM, 3% fbs, estrogens, progesterone, heparin, hydrocortisone, insulin, EGF) to render them mammary gland-like cells from which expression of the selected milk component could be induced. Alternatively, epigenetic remodeling is performed using a remodeling system such as CRISPR/Cas9 to activate related selection genes, such as casein, a-lactalbumin to be sustained, to allow expression of their respective proteins, and/or down-regulate and/or gene knock-out selected endogenous genes, as described, for example, in WO21067641, which is incorporated herein by reference in its entirety for all purposes.

Culturing

The complete growth medium included high glucose DMEM/F12, 10% FBS, 1% NEAA, 1% pen/strep, 1% ITS-X, 1%F-Glu, 10ng/ml EGF and 5pg/ml hydrocortisone. Complete lactation medium included high glucose DMEM/F12, 1% NEAA, 1% pen/strep, 1% ITS-X, 1%F-Glu, 10ng/ml EGF, 5pg/ml hydrocortisone, and 1pg/ml prolactin (5 ug/ml in Hyuh 1991). Cells were seeded at a density of 20,000 cells per square centimeter on collagen-coated flasks in complete growth medium and left to stand in complete growth medium for adhesion and expansion for 48 hours, after which the medium was changed to complete lactation medium. After exposure to lactation medium, cells began to differentiate and stopped growing. Within about one week, cells begin to secrete milk products such as milk fat, lactose, casein and whey into the culture medium. The desired concentration of the lactation medium can be achieved by concentration or dilution by ultrafiltration. The desired salt balance of the lactation medium can be achieved by dialysis, e.g. to remove undesired metabolites from the medium. The hormones and other growth factors used may be selectively extracted by resin purification, e.g., using nickel resin to remove His tagged growth factors, to further reduce the level of contaminants in the lactate product.

EXAMPLE 63 evaluation of LacNAc, sialylated LacNAc and sialyl-Lewis x production in non-mammary adult Stem cells

Isolated mesenchymal cells and reprogrammed breast-like cells as described in example 62 were modified via CRISPR-CAS to overexpress β -1, 4-galactosyltransferase 4B4GalT4 from homo sapiens (UniProt ID O60513), GDP-fucose synthase GFUS from homo sapiens (UniProt ID Q13630), galactosidα -1, 3-fucosyltransferase FUT3 from homo sapiens (UniProt ID P21217), N-acyl neuraminic acid cytidylyltransferase from mice (UniProt ID Q99KK 2) and CMP-N-acetylneuraminic acid- β -1, 4-galactosidα -2, 3-sialyltransferase ST3GAL3 from homo sapiens (UniProt ID Q11203) and α -2, 6-sialyltransferase from brown rats (prot ID P13721). For the host, all genes introduced into the cell are codon optimized. Cells were seeded at a density of 20,000 cells per square centimeter on collagen-coated flasks in complete growth medium and left to stand in complete growth medium for adhesion and expansion for 48 hours, after which the medium was changed to complete lactation medium for about 7 days. After culturing as described in example 62, cells were subjected to UPLC to analyze production of LacNAc, 3 '-sialylated LacNAc, 6' -sialylated LacNAc, and sialic acid-lewis x.

Claims (100)

1. A metabolically engineered cell producing a mixture of at least three different sialylated (sialylated) oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different sialylated mammalian milk oligosaccharides,

wherein the cell

Metabolically engineered for use in producing the mixture, and

glycosyltransferase (glycosyltransferase) expressed as sialyltransferase, and

capable of synthesizing nucleotide-sugar CMP-N-acetylneuraminic acid (CMP-Neu 5 Ac), and

exhibiting at least one additional glycosyltransferase, an

One or more nucleotide-sugars can be synthesized, wherein the nucleotide-sugar is a donor for the additional glycosyltransferase.

2. The cell of claim 1, wherein the cell is modified by a gene expression module, characterized in that expression from any one of the expression modules is sustained or produced by a natural inducer.

3. The cell of any one of claims 1 or 2, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein.

4. A cell according to any one of claims 1 to 3, wherein the cell produces a mixture of charged and neutral oligosaccharides.

5. The cell of any one of claims 1 to 4, wherein the mixture comprises, consists of, or consists essentially of: charged and neutral fucosylated (fucosylated) and/or nonfucosylated oligosaccharides.

6. The cell of any one of claims 1 to 5, wherein the oligosaccharide mixture comprises at least three different sialylated oligosaccharides that differ in the degree of polymerization.

7. The cell of any one of claims 1 to 6, wherein the cell produces at least four, preferably at least five, more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different sialylated oligosaccharides.

8. The cell of any one of claims 1 to 7, wherein any one of the additional glycosyltransferases is selected from the list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosamine transferase, N-acetylmannosylaminotransferase, xylosyltransferase (xylosyltransferase), glucuronidase, galacturonase, glucosaminotransferase, N-glycolylneuraminidase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-Zhuo Tangan (altrosamine) transferase, UDP-N-acetylglucosamine enolacetone acyltransferase (UDP-N-acetylglucosamine enolpyruvyl transferase) and fucosyltransferase,

Preferably, the fucosyltransferase is selected from the list comprising: alpha-1, 2-fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-1, 4-fucosyltransferase, alpha-1, 6-fucosyltransferase,

preferably, the sialyltransferase is selected from the list comprising: alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase,

preferably, the galactosyltransferase is preferably selected from the list comprising: beta-1, 3-galactosyltransferase, N-acetylglucosamine beta-1, 3-galactosyltransferase, beta-1, 4-galactosyltransferase, N-acetylglucosamine beta-1, 4-galactosyltransferase, alpha-1, 3-galactosyltransferase and alpha-1, 4-galactosyltransferase,

preferably, the glucosyltransferase is selected from the list comprising: alpha-glucosyltransferase, beta-1, 2-glucosyltransferase, beta-1, 3-glucosyltransferase and beta-1, 4-glucosyltransferase,

preferably, the mannosyltransferase is selected from the list comprising: alpha-1, 2-mannosyltransferase, alpha-1, 3-mannosyltransferase and alpha-1, 6-mannosyltransferase,

preferably, the N-acetylglucosaminyl transferase is selected from the list comprising: galactoside beta-1, 3-N-acetylglucosaminyl transferase and beta-1, 6-N-acetylglucosaminyl transferase,

Preferably, the N-acetylgalactosamine transferase is selected from the list comprising: alpha-1, 3-N-acetylgalactosamine transferase and beta-1, 3-N-acetylgalactosamine transferase.

9. The cell of any one of claims 1 to 8, wherein the cell is modified in the expression or activity of at least one of said glycosyltransferases.

10. The cell of any one of claims 1 to 9, wherein any one of the additional glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-Neu5Ac.

11. The cell of any one of claims 1 to 10, wherein any one of the additional glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-fucose (GDP-Fuc).

12. The cell of any one of claims 1 to 11, wherein any one of the additional glycosyltransferases is an N-acetylglucosamine aminotransferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine (UDP-GlcNAc).

13. The cell of any one of claims 1 to 12, wherein any one of the additional glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose (UDP-Gal).

14. The cell of any one of claims 1 to 13, wherein any one of the additional glycosyltransferases is an N-acetylgalactosamine transferase and one of the donor nucleotide-sugars is UDP-N-acetylgalactosamine (UDP-GalNAc).

15. The cell of any one of claims 1 to 14, wherein any one of the additional glycosyltransferases is an N-acetylmannosyl aminotransferase and one of the donor nucleotide-sugars is UDP-N-acetylmannosamine (UDP-ManNAc).

16. The cell of any one of claims 1 to 15, wherein any one of the nucleotide-sugars is selected from a list comprising: GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP-N-hydroxyacetylneuraminic acid (CMP-Neu 5 Gc), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7 (8, 9) Ac2, UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose, UDP-2-acetamido-2, 6-dideoxy-L-arabinose (aro) -4-hexanone, UDP-2-acetamido-2, 6-dideoxy-L-lyxo-butan-4-e, UDP-N-acetylmannosamine (UDP-N-acetylmannosamine) or UDP-6-acetylmannosamine (UDP-N-acetylmannosamine) (UDP-N-6-acetylmannosamine or UDP-acetylmannosamine (UDP-N-6-acetylmannosamine) UDP-N-acetyl-L-neotame (pneumamine) (UDP-L-Pnenac or UDP-2-acetamido-2, 6-dideoxy-L-talose), UDP-N-acetyl muramic acid (acetylmuramic acid), UDP-N-acetyl-L-isorhamnoamine (quinovosamine) (UDP-L-QuiNAc or UDP-2-acetamido-2, 6-dideoxy-L-glucose), GDP-L-isorhamnose.

17. The cell of any one of claims 1 to 16, wherein the cell expresses one or more polypeptides selected from the list comprising: mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-guanyl phosphate transferase, GDP-mannose 4, 6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, GDP-fucose pyrophosphorylase, fucose-1-guanyl phosphate transferase, L-glutamylamino acid-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epi-isomerase, UDP-N-acetylglucosamine 2-epi-isomerase N-acetylmannosamine-6-phosphate 2-epi isomerase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, N-acetylneuraminic acid synthase, N-acetylneuraminic acid dissociase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphate phosphatase, N-acylneuraminic acid cytidylyltransferase, galactose-1-epi isomerase, galactokinase, glucokinase, galactose-1-phosphouridyltransferase, UDP-glucose 4-epi isomerase, glucose-1-phosphouridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epi isomerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein the cell is modified in terms of the performance or activity of any of the polypeptides.

18. The cell of any one of claims 1 to 17, wherein the cell is capable of synthesizing at least two nucleotide-sugars, preferably at least three nucleotide-sugars, more preferably at least four nucleotide-sugars, even more preferably at least five nucleotide-sugars.

19. The cell of any one of claims 1 to 18, wherein the oligosaccharide mixture comprises at least one neutral oligosaccharide in addition to three or more sialylated oligosaccharides.

20. The cell of claim 19, wherein the neutral oligosaccharide is selected from the list comprising neutral fucosylated oligosaccharides and neutral non-fucosylated oligosaccharides.

21. The cell of any one of claims 1 to 20, wherein at least one of the sialylated oligosaccharides is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, containing N-acetylglucosamine, containing N-acetylneuraminic acid, containing N-glycolylneuraminic acid, containing N-acetylgalactosamine, containing rhamnose, containing glucuronate, containing galacturonate and/or containing N-acetylmannosamine.

22. The cell of any one of claims 1 to 21, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide.

23. The cell of any one of claims 1 to 22, wherein the oligosaccharide mixture comprises at least one oligosaccharide comprising an N-acetylglucosamine monosaccharide unit.

24. The cell of any one of claims 1 to 23, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide.

25. The cell of any one of claims 1 to 24, wherein the oligosaccharide mixture comprises at least one oligosaccharide which is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains N-acetylglucosamine, contains N-acetylneuraminic acid, contains N-glycolylneuraminic acid, contains N-acetylgalactosamine, contains rhamnose, contains glucuronate, contains galacturonate and/or contains N-acetylmannosamine.

26. The cell of any one of claims 1 to 25, wherein the cell uses at least one precursor for producing any one or more of the oligosaccharides, preferably the cell uses two or more precursors for producing any one or more of the oligosaccharides, said precursors being fed into the cell from a culture medium.

27. The cell of any one of claims 1 to 26, wherein the cell produces at least one precursor for producing any one of the oligosaccharides.

28. The cell of any one of claims 1 to 27, wherein the at least one precursor for producing any one of the oligosaccharides is fully converted to any one of the oligosaccharides.

29. The cell of any one of claims 1 to 28, wherein the cell produces the oligosaccharide intracellularly, and wherein a portion or substantially all of the produced oligosaccharide remains intracellular and/or is excreted outside the cell via passive or active transport.

30. The cell of any one of claims 1 to 29, wherein the cell is further genetically modified for use in

i) Modified expression of endogenous membrane proteins and/or

ii) modified activity of endogenous membrane proteins and/or

iii) Expression of homologous membrane proteins and/or

iv) expression of heterologous membrane proteins,

wherein the membrane protein is involved in secreting any of said oligosaccharides from the mixture outside the cell, preferably wherein the membrane protein is involved in secreting all of said oligosaccharides from the mixture from the cell.

31. The cell of any one of claims 1 to 30, wherein the cell is further genetically modified for use in

i) Modified expression of endogenous membrane proteins and/or

ii) modified activity of endogenous membrane proteins and/or

iii) Expression of homologous membrane proteins and/or

iv) expression of heterologous membrane proteins,

wherein the membrane protein is involved in the absorption of precursors and/or receptors for the synthesis of any of said oligosaccharides in the mixture, preferably wherein the membrane protein is involved in the absorption of all of said desired precursors, more preferably wherein the membrane protein is involved in the absorption of all of said receptors.

32. The cell of any one of claims 30 or 31, wherein the membrane protein is selected from the list comprising: transporter (transporter), P-P-bond hydrolysis-driven transporter (transporter), beta-bungee, auxiliary transporter (transport protein), putative transporter (putative transport protein), phosphotransferase-driven group translocator (transporter),

preferably, the transporter comprises an MFS transporter, a sugar efflux transporter, and a transferrin export protein,

preferably, the P-bond hydrolytically driven transporter comprises an ABC transporter and a transferrin export protein (siderophore exporter).

33. The cell of any one of claims 30 to 32, wherein the membrane protein provides improved production and/or is capable of achieving and/or enhancing the efflux of any one of said oligosaccharides.

34. The cell of any one of claims 1 to 33, wherein the cell is resistant to lactose killing when grown in an environment where lactose is combined with one or more other carbon sources.

35. The cell of any one of claims 1 to 34, wherein the cell comprises a modification for reducing the production of acetic acid compared to an unmodified precursor cell (progenitor).

36. The cell of claim 35, wherein the cell comprises any one or more of a protein that reduces or reduces expression and/or eliminates, reduces or delays activity as compared to an unmodified precursor cell comprising: beta-galactosidase, galactosido-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine inhibitor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose: undecanopentenyl (undecaptenyl) -phosphoglucose-1-phosphotransferase, L-fucokinase, L-fucose isomerase, N-acetylneuraminic acid dissociating enzyme, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epi isomerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphouridyltransferase, glucose-1-phosphoadenylacyl transferase, glucose-1-phosphatase, ATP-dependent 6-phosphofructokinase isozyme 1, ATP-dependent 6-phosphofructokinase isozyme 2 glucose-6-phosphate isomerase, aerobic respiration control protein, transcription inhibitor IclR, lon protease, glucose-specific translocation phosphotransferase IIBC component ptsG, glucose-specific translocation Phosphotransferase (PTS) IIBC component malX, enzyme IIAGlc, beta-glucoside specific PTS enzyme II, fructose-specific PTS polyphosphorylated transfer protein FruA and FruB, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate-methylase dissociation enzyme, acetate kinase, phosphoryltransferase (phosphoacetyl transferase), phosphoacetyl transferase, pyruvate decarboxylase.

37. The cell of any one of claims 1 to 36, wherein the cell is capable of producing phosphoenolpyruvate (PEP).

38. The cell of any one of claims 1 to 37, wherein the cell is modified for enhanced production and/or supply of phosphoenolpyruvate (PEP) compared to an unmodified precursor cell.

39. The cell of any one of claims 1 to 38, wherein any one of the oligosaccharides is a mammalian milk oligosaccharide.

40. The cell of any one of claims 1 to 39, wherein all of the oligosaccharides are mammalian milk oligosaccharides.

41. The cell of any one of claims 1 to 38, wherein any one of the oligosaccharides is an antigen of the human ABO blood group system.

42. A method for producing a mixture of at least three different sialylated oligosaccharides by means of a cell, preferably a single cell, wherein the mixture comprises more than one mammalian milk oligosaccharide, preferably wherein the mixture comprises at least three different sialylated mammalian milk oligosaccharides, the method comprising the steps of:

i) Providing a cell that (a) exhibits a glycosyltransferase that is a sialyltransferase and is capable of synthesizing nucleotide-sugar CMP-Neu5Ac, and (b) exhibits at least one additional glycosyltransferase, and (c) is capable of synthesizing at least one or more nucleotide-sugar, wherein the nucleotide-sugar is a donor for the additional glycosyltransferase, and

ii) culturing the cell under conditions allowing expression of the glycosyltransferase and synthesis of the nucleotide-sugar such that the cell produces the mixture of at least three different sialylated oligosaccharides,

iii) Preferably, at least one of the oligosaccharides is isolated from the culture, more preferably all of the oligosaccharides are isolated from the culture.

43. The method of claim 42, wherein the cell is a metabolically engineered cell of any one of embodiments 1-41.

44. The method of claim 43, wherein the cells are modified by a gene expression module, wherein expression from any of the expression modules is sustained or produced by a natural inducer.

45. The method of claim 43 or 44, wherein the cell comprises multiple copies of the same coding DNA sequence encoding a protein.

46. The method of any one of claims 42 to 45, wherein the cells produce a mixture of charged and neutral oligosaccharides.

47. The method of any one of claims 42 to 46, wherein the mixture comprises, consists of, or consists essentially of: charged and neutral fucosylated and/or nonfucosylated oligosaccharides.

48. The method of any one of claims 42 to 47, wherein the oligosaccharide mixture comprises at least three different sialylated oligosaccharides that differ in degree of polymerization.

49. The method of any one of claims 42 to 48, wherein the cell produces at least four, preferably at least five, more preferably at least six, most preferably at least seven, at least eight, at least nine, at least ten different sialylated oligosaccharides.

50. The method of any one of claims 42 to 49, wherein any one of the additional glycosyltransferases is selected from the list comprising: fucosyltransferase, sialyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, N-acetylglucosaminyltransferase, N-acetylgalactosyltransferase, N-acetylmannosylaminotransferase, xylosyltransferase, glucuronidase, galacturonan transferase, glucosaminotransferase, N-glycolylneuraminidase, rhamnosyltransferase, N-acetylrhamnosyltransferase, UDP-4-amino-4, 6-dideoxy-N-acetyl-beta-L-altrose amine transferase, UDP-N-acetylglucosamine enolpyruvyl transferase and fucosylaminotransferase,

Preferably, the fucosyltransferase is selected from the list comprising: alpha-1, 2-fucosyltransferase, alpha-1, 3-fucosyltransferase, alpha-1, 4-fucosyltransferase, alpha-1, 6-fucosyltransferase,

preferably, the sialyltransferase is selected from the list comprising: alpha-2, 3-sialyltransferase, alpha-2, 6-sialyltransferase and alpha-2, 8-sialyltransferase,

preferably, the galactosyltransferase is preferably selected from the list comprising: beta-1, 3-galactosyltransferase, N-acetylglucosamine beta-1, 3-galactosyltransferase, beta-1, 4-galactosyltransferase, N-acetylglucosamine beta-1, 4-galactosyltransferase, alpha-1, 3-galactosyltransferase and alpha-1, 4-galactosyltransferase,

preferably, the glucosyltransferase is selected from the list comprising: alpha-glucosyltransferase, beta-1, 2-glucosyltransferase, beta-1, 3-glucosyltransferase and beta-1, 4-glucosyltransferase,

preferably, the mannosyltransferase is selected from the list comprising: alpha-1, 2-mannosyltransferase, alpha-1, 3-mannosyltransferase and alpha-1, 6-mannosyltransferase,

preferably, the N-acetylglucosaminyl transferase is selected from the list comprising: galactoside beta-1, 3-N-acetylglucosaminyl transferase and beta-1, 6-N-acetylglucosaminyl transferase,

Preferably, the N-acetylgalactosamine transferase is selected from the list comprising: alpha-1, 3-N-acetylgalactosamine transferase and beta-1, 3-N-acetylgalactosamine transferase.

51. The method of any one of claims 42 to 50, wherein the cell is modified in the expression or activity of at least one of said glycosyltransferases.

52. The method of any one of claims 42-51, wherein any one of the additional glycosyltransferases is a sialyltransferase and one of the donor nucleotide-sugars is CMP-N-acetylneuraminic acid (CMP-Neu 5 Ac).

53. The method of any one of claims 42-52, wherein any one of the additional glycosyltransferases is a fucosyltransferase and one of the donor nucleotide-sugars is GDP-fucose (GDP-Fuc).

54. The method of any one of claims 42 to 53, wherein any one of the additional glycosyltransferases is an N-acetylglucosamine aminotransferase and one of the donor nucleotide-sugars is UDP-N-acetylglucosamine (UDP-GlcNAc).

55. The method of any one of claims 42-54, wherein any one of the additional glycosyltransferases is a galactosyltransferase and one of the donor nucleotide-sugars is UDP-galactose (UDP-Gal).

56. The method of any one of claims 42 to 55, wherein any one of the additional glycosyltransferases is an N-acetylgalactosamine transferase and one of the donor nucleotide-sugars is UDP-N-acetylgalactosamine (UDP-GalNAc).

57. The method of any one of claims 42 to 56, wherein any one of the additional glycosyltransferases is an N-acetylmannosyl aminotransferase and one of the donor nucleotide-sugars is UDP-N-acetylmannosamine (UDP-ManNAc).

58. The method of any one of claims 42 to 57, wherein any one of the nucleotide-sugars is selected from a list comprising: GDP-Fuc, CMP-Neu5Ac, UDP-GlcNAc, UDP-Gal, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylmannosamine (UDP-ManNAc), GDP-mannose (GDP-Man), UDP-glucose (UDP-Glc), CMP-N-glycolylneuraminic acid (CMP-Neu 5 Gc), CMP-Neu4Ac, CMP-Neu5Ac9N3, CMP-Neu4,5Ac2, CMP-Neu5,7Ac2, CMP-Neu5,9Ac2, CMP-Neu5,7 (8, 9) Ac2, UDP-glucuronate, UDP-galacturonate, GDP-rhamnose, UDP-xylose, UDP-2-acetamido-2, 6-dideoxy-L-arabino-4-hexose, UDP-2-acetamido-2, 6-dideoxy-L-lyxol-hexanone, CMP-Neu4Ac, 5Ac2, CMP-Neu5,7 (8, 9) Ac2, UDP-glucuronide, UDP-D-mannosamine, UDP-2-N-acetylmannosamine, UDP-N-acetylmannosamine or UDP-N-acetylmannosamine UDP-N-acetyl-L-neotame (UDP-L-PnenAC or UDP-2-acetamido-2, 6-dideoxy-L-talose), UDP-N-acetyl muramic acid, UDP-N-acetyl-L-isorhamnoamine (UDP-L-QuiNAc or UDP-2-acetamido-2, 6-dideoxy-L-glucose), GDP-L-isorhamno.

59. The method of any one of claims 42 to 58, wherein the cell expresses one or more polypeptides selected from the list comprising: mannose-6-phosphate isomerase, phosphomannomutase, mannose-1-guanyl phosphate transferase, GDP-mannose 4, 6-dehydratase, GDP-L-fucose synthase, fucose permease, fucokinase, GDP-fucose pyrophosphorylase, fucose-1-guanyl phosphate transferase, L-glutamylamino acid-D-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, phosphoglucamine mutase, N-acetylglucosamine-6-phosphate deacetylase, N-acylglucosamine 2-epi-isomerase, UDP-N-acetylglucosamine 2-epi-isomerase N-acetylmannosamine-6-phosphate 2-epi isomerase, glucosamine 6-phosphate N-acetyltransferase, N-acetylglucosamine-6-phosphate phosphatase, N-acetylmannosamine kinase, phosphoacetylglucosamine mutase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyltransferase, N-acetylneuraminic acid synthase, N-acetylneuraminic acid dissociase, N-acylneuraminic acid-9-phosphate synthase, N-acylneuraminic acid-9-phosphate phosphatase, N-acylneuraminic acid cytidylyltransferase, galactose-1-epi isomerase, galactokinase, glucokinase, galactose-1-phosphouridyltransferase, UDP-glucose 4-epi isomerase, glucose-1-phosphouridyltransferase, phosphoglucomutase, UDP-N-acetylglucosamine 4-epi isomerase, N-acetylgalactosamine kinase and UDP-N-acetylgalactosamine pyrophosphorylase, preferably wherein the cell is modified in terms of the performance or activity of any of the polypeptides.

60. The method of any one of claims 42 to 59, wherein the cell is capable of synthesizing at least two nucleotide-sugars, preferably at least three nucleotide-sugars, more preferably at least four nucleotide-sugars, even more preferably at least five nucleotide-sugars.

61. The method of any one of claims 42 to 60, wherein the oligosaccharide mixture also comprises at least one neutral oligosaccharide in addition to three or more sialylated oligosaccharides.

62. The method of claim 61, wherein the neutral oligosaccharide is selected from the list comprising neutral fucosylated oligosaccharides and neutral non-fucosylated oligosaccharides.

63. The method of any one of claims 42 to 62, wherein at least one of the sialylated oligosaccharides is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, containing N-acetylglucosamine, containing N-acetylneuraminic acid, containing N-glycolylneuraminic acid, containing N-acetylgalactosamine, containing rhamnose, containing glucuronate, containing galacturonate and/or containing N-acetylmannosamine.

64. The method of any one of claims 42 to 63, wherein the oligosaccharide mixture comprises at least one fucosylated oligosaccharide.

65. The method of any one of claims 42 to 64, wherein the oligosaccharide mixture comprises at least one oligosaccharide comprising an N-acetylglucosamine monosaccharide unit.

66. The method of any one of claims 42 to 65, wherein the oligosaccharide mixture comprises at least one galactosylated oligosaccharide.

67. The method of any one of claims 42 to 66, wherein the oligosaccharide mixture comprises at least one oligosaccharide which is fucosylated, galactosylated, glucosylated, xylosylated, mannosylated, contains N-acetylglucosamine, contains N-acetylneuraminic acid, contains N-glycolylneuraminic acid, contains N-acetylgalactosamine, contains rhamnose, contains glucuronate, contains galacturonate and/or contains N-acetylmannosamine.

68. The method of any one of claims 42 to 67, wherein the cell uses at least one precursor for producing any one or more of the oligosaccharides, preferably the cell uses two or more precursors for producing any one or more of the oligosaccharides, said precursors being fed from the culture medium into the cell.

69. The method of any one of claims 42 to 68, wherein the cell produces at least one precursor for producing any one of said oligosaccharides.

70. The method of any one of claims 42 to 69, wherein the at least one precursor for producing any one of the oligosaccharides is fully converted to any one of the oligosaccharides.

71. The method of any one of claims 42 to 70, wherein the cell produces the oligosaccharide intracellularly, and wherein a portion or substantially all of the produced oligosaccharide remains intracellular and/or is excreted outside the cell via passive or active transport.

72. The method of any one of claims 42 to 71, wherein the cell is further genetically modified for use in

i) Modified expression of endogenous membrane proteins and/or

ii) modified activity of endogenous membrane proteins and/or

iii) Expression of homologous membrane proteins and/or

iv) expression of heterologous membrane proteins,

wherein the membrane protein is involved in secreting any of said oligosaccharides from the mixture outside the cell, preferably wherein the membrane protein is involved in secreting all of said oligosaccharides from the mixture from the cell.

73. The method of any one of claims 42 to 72, wherein the cell is further genetically modified for use in

i) Modified expression of endogenous membrane proteins and/or

ii) modified activity of endogenous membrane proteins and/or

iii) Expression of homologous membrane proteins and/or

iv) expression of heterologous membrane proteins,

wherein the membrane protein is involved in the absorption of precursors and/or receptors for the synthesis of any of said oligosaccharides in the mixture, preferably wherein the membrane protein is involved in the absorption of all of said desired precursors, more preferably wherein the membrane protein is involved in the absorption of all of said receptors.

74. The method of any one of claims 72 or 73, wherein the membrane protein is selected from the list comprising: transporter, P-P-bond hydrolytically driven transporter, beta-bungee, auxiliary transporter, putative transporter, phosphotransferase driven group translocator,

preferably, the transporter comprises an MFS transporter, a sugar efflux transporter, and a transferrin export protein,

preferably, the P-P-bond hydrolytically driven transporter comprises an ABC transporter and a transferrin export protein.

75. The method of any one of claims 72 to 74, wherein the membrane protein provides improved production and/or is capable of achieving and/or enhancing the efflux of any one of said oligosaccharides.

76. The method of any one of claims 42 to 75, wherein the cells resist lactose killing when grown in an environment where lactose is combined with one or more other carbon sources.

77. The method of any one of claims 42 to 76, wherein the cell comprises a modification for reducing acetate production as compared to an unmodified precursor cell.

78. The method of claim 77A method, wherein the cell comprises any one or more of a protein that reduces or reduces expression and/or eliminates, reduces or delays activity as compared to an unmodified precursor cell comprising: beta-galactosidase, galactosido-acetyltransferase, N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase, N-acetylglucosamine inhibitor, ribonucleotide monophosphatase, EIICBA-Nag, UDP-glucose undecylenyl-phosphoglucose-1-phosphate transferase, L-fucokinase, L-fucose isomerase, N-acetylneuraminidase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate 2-epi isomerase, EIIAB-Man, EIIC-Man, EIID-Man, ushA, galactose-1-phosphate uridylyltransferase, glucose-1-phosphate atpase, ATP-dependent 6-phosphate fructokinase isoenzyme 1, dependent 6-phosphate fructokinase isoenzyme 2, glucose-6-phosphate BC isomerase, aerobic control protein, transcription factor, IIR-specific translocation enzyme, IIA, IIC-specific translocation enzyme, PTS-specific translocation enzyme group IIA, and PTS-specific translocation enzyme group IIA (IIA-specific translocation enzyme) ^Glc Beta-glucoside specific PTS enzyme II, fructose-specific PTS polyphosphorylated transfer protein FruA and FruB, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate-methyl alcohol dissociating enzyme, acetate kinase, phosphoacyltransferase, phosphoacetyltransferase, and pyruvate decarboxylase.

79. The method of any one of claims 42 to 78, wherein the cell is capable of producing phosphoenolpyruvate (PEP).

80. The method of any one of claims 42 to 79, wherein the cell is modified for enhanced production and/or supply of phosphoenolpyruvate (PEP) compared to an unmodified precursor cell.

81. The method of any one of claims 42 to 80, wherein any one of the oligosaccharides is mammalian milk oligosaccharides.

82. The method of any one of claims 42 to 81, wherein all of the oligosaccharides are mammalian milk oligosaccharides.

83. The method of any one of claims 42 to 82, wherein any one of the oligosaccharides is an antigen of the human ABO blood group system.

84. The method of any one of claims 42 to 83, wherein the conditions comprise:

using a medium comprising at least one precursor and/or receptor for the production of any of the oligosaccharides, and/or

At least one precursor and/or acceptor feed(s) for producing any of the oligosaccharides is added to the medium.

85. The method of any one of claims 42 to 84, comprising at least one of the following steps:

i) Using a medium comprising at least one precursor and/or acceptor;

ii) adding at least one precursor and/or acceptor feed to the medium in a reactor, wherein the total reactor volume is between 250mL (milliliter) and 10.000m ³ Preferably in a continuous manner within (cubic meters) and preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium prior to addition of the precursor and/or acceptor feed;

iii) Adding at least one precursor and/or acceptor feed to the medium in a reactor, wherein the total reactor volume is between 250mL (milliliter) and 10.000m ³ Preferably in a continuous manner within (cubic meters), and preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium prior to the addition of the precursor and/or acceptor feed, and wherein preferably the pH of the precursor and/or acceptor feed is set to between 3 and 7, and wherein preferably the precursor and/or acceptor feed The temperature is kept between 20 ℃ and 80 ℃;

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

v) adding at least one precursor and/or acceptor feed to the medium in a continuous manner over the course of 1, 2, 3, 4, 5 days by means of a feed solution, and 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 ℃ and 80 ℃;

the method produces any one of the oligosaccharides in a concentration of at least 50g/L, preferably at least 75g/L, more preferably at least 90g/L, more preferably at least 100g/L, more preferably at least 125g/L, more preferably at least 150g/L, more preferably at least 175g/L, more preferably at least 200g/L in the final culture.

86. The method of any one of claims 42 to 84, comprising at least one of the following steps:

i) Using a medium comprising at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor volume is between 250mL and 10.000m ³ (cubic meters) range;

ii) adding to the medium a lactose feed comprising at least 50, preferably at least 75, preferably at least 100, preferably at least 120, more preferably at least 150 grams of lactose per liter of initial reactor volume, wherein the reactor volume is between 250mL and 10.000m ³ Preferably in a continuous manner, and preferably such that the final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium prior to the addition of the lactose feed;

iii) Adding to the medium a lactose feed comprising at least 50, 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, wherein the reactor volume is between 250mL and 10.000m ³ Within (cubic meters), preferably in a continuous manner, and preferably such that the cultivation is effectedThe final volume of the medium is no more than three times, preferably no more than two times, more preferably less than two times the volume of the medium prior to the addition of the lactose feed, and wherein preferably the pH of the lactose feed is set between 3 and 7, and wherein preferably the temperature of the lactose feed is maintained between 20 ℃ and 80 ℃;

iv) adding lactose feed to the medium in a continuous manner by means of feed solution over a period of 1 day, 2 days, 3 days, 4 days, 5 days;

v) adding lactose feed to the medium in a continuous manner over the course of 1, 2, 3, 4, 5 days by means of a feed solution, and wherein the concentration of the lactose feed solution is 50g/L, preferably 75g/L, more preferably 100g/L, more preferably 125g/L, more preferably 150g/L, more preferably 175g/L, more preferably 200g/L, more preferably 225g/L, more preferably 250g/L, more preferably 275g/L, more preferably 300g/L, more preferably 325g/L, more preferably 350g/L, more preferably 375g/L, more preferably 400g/L, more preferably 450g/L, more preferably 500g/L, even more preferably 550g/L, most preferably 600g/L, and wherein preferably the pH of the feed solution is set between 3 and 7, and wherein preferably the temperature of the feed solution is kept between 20 ℃ and 80 ℃;

the method produces any one of the oligosaccharides in a concentration of at least 50g/L, preferably at least 75g/L, more preferably at least 90g/L, more preferably at least 100g/L, more preferably at least 125g/L, more preferably at least 150g/L, more preferably at least 175g/L, more preferably at least 200g/L in the final culture.

87. The method of claim 86, wherein the lactose feed is achieved by adding lactose from the beginning of the culture at a concentration of at least 5mM, preferably at a concentration of 30, 40, 50, 60, 70, 80, 90, 100, 150mM, more preferably at a concentration of >300 mM.

88. The method of any one of claims 86 or 87, wherein the lactose feed is achieved by adding lactose to the medium at a concentration such that a lactose concentration of at least 5mM, preferably 10mM or 30mM is obtained throughout the production phase of the culture.

89. The method of any one of claims 42 to 88, wherein the host cells are cultured for at least about 60, 80, 100, or about 120 hours or in a continuous manner.

90. The method of any one of claims 42 to 89, wherein the cell is cultured in a medium comprising a carbon source comprising monosaccharides, disaccharides, oligosaccharides, polysaccharides, polyols, glycerol, a complex medium comprising molasses, corn steep liquor, peptone, tryptone or yeast extract; preferably, wherein the carbon source is selected from the list comprising: glucose, glycerol, fructose, sucrose, maltose, lactose, arabinose (arabinose), malt-oligosaccharides, maltotriose, sorbitol, xylose, rhamnose, galactose, mannose, methanol, ethanol, trehalose (trehalose), starch, cellulose, hemicellulose, molasses, corn steep liquor, high fructose syrup, acetate, citrate, lactate and pyruvate.

91. The method of any one of claims 42 to 90, wherein the medium contains at least one precursor selected from the group comprising: lactose, galactose, fucose, glcNAc, galNAc, lacto-N-disaccharide (LNB), N-acetyllactosamine (LacNAc).

92. The method of any one of claims 42 to 91, wherein the first stage of exponential cell growth is provided by adding a carbon-based matrix, preferably glucose or sucrose, to the medium before adding the precursor, preferably lactose, to the medium in the second stage.

93. The method of any one of claims 42 to 92, wherein the first stage of exponential cell growth is provided by adding a carbon-based matrix, preferably glucose or sucrose, to the medium comprising a precursor, preferably lactose, followed by a second stage wherein only carbon-based matrix, preferably glucose or sucrose, is added to the medium.

94. The method of any one of claims 42 to 93, wherein the first stage of exponential cell growth is provided by adding a carbon-based matrix, preferably glucose or sucrose, to the medium comprising a precursor, preferably lactose, followed by a second stage wherein the carbon-based matrix, preferably glucose or sucrose, and the precursor, preferably lactose, are added to the medium.

95. The method of any one of claims 42 to 94, wherein the separating comprises at least one of: clarification, ultrafiltration, nanofiltration, biphasic partitioning, reverse osmosis, microfiltration, activated carbon or carbon treatment, treatment with a nonionic surfactant, enzymatic digestion, tangential flow high performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration, ligand exchange chromatography.

96. The method of any one of claims 42 to 95, further comprising purifying any one of the oligosaccharides from the cell.

97. The method of claim 96, wherein the purifying comprises at least one of: using activated carbon or carbon, using charcoal, nanofiltration, ultrafiltration, electrophoresis, enzymatic treatment or ion exchange, using alcohol, using a mixture of water and alcohol, crystallization, evaporation, precipitation, drying, spray drying, lyophilization (freeze drying), spray freeze drying (spray freeze drying), freeze spray drying, band drying, vacuum band drying, drum drying, roller drying, vacuum drum drying or vacuum drum drying.

98. The cell of any one of claims 1 to 41 or the method of any one of claims 42 to 97, wherein the cell is a bacterium, fungus, yeast, plant cell, animal cell or protozoan cell,

preferably, the bacterium is an Escherichia coli (Escherichia coli) strain, more preferably an Escherichia coli strain as a K-12 strain, even more preferably an Escherichia coli K-12 strain is Escherichia coli MG1655,

preferably, the fungus belongs to a genus selected from the group comprising: rhizopus (Rhizopus), reticulus (Dictyostelium), penicillium (Penicillium), white fungus (Mucor) or Aspergillus (Aspergillus),

preferably, the yeast belongs to a genus selected from the group comprising: saccharomyces (Saccharomyces), zygosaccharomyces (Zygosaccharomyces), pichia (Pichia), colt (Komagataella), hansenula (Hansenula), yarrowia (Yarrowia), candida globosa (Starerella), kluyveromyces (Kluyveromyces) or Debaryomyces (Debaromyces),

preferably, the plant cell is an algal cell or derived from tobacco, alfalfa, rice, tomato, cotton, rapeseed, soybean, maize or corn plants,

Preferably, the animal cells are derived from non-human mammals, birds, fish, invertebrates, reptiles, amphibians or insects, or are genetically modified cell lines derived from human cells excluding embryonic stem cells, more preferably the human and non-human mammalian cells are epithelial cells, embryonic kidney cells, fibroblasts, COS cells, chinese hamster ovary (Chinese hamster ovary; CHO) cells, murine myeloma cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, more preferably the insect cells are derived from Spodoptera frugiperda (Spodoptera frugiperda), bombyx mori (Bombyxmori), cabbage looper (Mamestra brassicae), trichoplusia ni (Trichoplusia ni) or Drosophila melanogaster (Drosophila melanogaster),

preferably, the protozoan cell is a Leishmania tarabica (Leishmania tarentolae) cell.

99. The cell of claim 98 or the method of claim 98, wherein the cell is a viable Gram-negative bacterium (Gram-negative bacterium) comprising reduced or eliminated synthetic poly-N-acetyl-glucosamine (PNAG), intestinal co-antigen (Enterobacterial Common Antigen; ECA), cellulose, colanic acid, core oligosaccharide (core oligosaccharide), osmotically regulated periplasmic dextran (Osmoregulated Periplasmic Glucan; OPG), glycerol glucoside (glycol), glycans and/or trehalose, as compared to the unmodified precursor cell.

100. Use of a cell according to any one of claims 1 to 41, 98, 99 or a method according to any one of claims 42 to 99 for producing a mixture of at least three different sialylated oligosaccharides, wherein the mixture comprises more than one mammalian milk oligosaccharide.